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The Chromatography and Sample Preparation Terminology Guide
Ronald E. Majors and John Hinshaw
n 2002, LCGC provided readers of the more commonly accepted terms with a gas chromatography (GC) (3). Still, there are many terms in com- I glossary to organize the myriad mon usage that are not in alignment terms used in gas chromatography with the IUPAC definitions and that (1). Likewise, in 2008, the third glos- nomenclature will be covered here as sary of common and not-so-common well. terms and “buzz words” for reference This terminology guide is not in- to high performance liquid chromatog- tended to be an in-depth listing or raphy (HPLC) columns and column highly theoretical coverage. For ex- technology was published (2). It is ample, we have elected not to cover time for an update because new terms many of the myriad terms used in have arisen or, in some cases, their instrumentation, detection, data han- original meanings have expanded or dling, and validation associated with changed. Because there are a num- chromatographic analysis but have ber of terms common to both GC chosen to use terms that may be en- and LC, we decided to combine the countered in everyday laboratory work glossaries into one large listing. In around columns, injection techniques, addition, with new sample prepara- phases, method development, sample tion technologies also making their preparation tasks, and general usage. appearance, expanding the glossary to The listing should be helpful to those include terminology specific to sample just starting in chromatography but it preparation was another goal. Finally, can also serve as a refresher for long- ion chromatography (IC), which some time users in the field. feel is a subset of LC, does have some of its own nomenclature and so those References terms are included here. (1) J.V. Hinshaw, LCGC 20(11), 1034–1040 We stick to the conventions of the (2002). International Union of Pure and Ap- (2) R.E. Majors and P.W. Carr, LCGC 26(2),118–168 (2008). plied Chemistry (IUPAC) in their “No- (3) L.S. Ettre, “Nomenclature for Chroma- menclature for Chromatography” that tography” in Pure and Appl. Chem. 65(4), provides guidance and changes in some 819–872 (1993). 6 Terminology Guide October 2013 www.chromatographyonline.com
Absorption: The process of retention in 96-well collection plate: A fixed-size which the solute partitions into a liquid- polyethylene rectangular plate (127.8 like coating. mm × 85.5 mm): consisting of an array Accelerated solvent extraction (ASE): of 8 × 12 (96) small “test tubes” called Trade name for a pressurized fluid ex- wells; volumes of wells range from 0.5 traction system introduced by Dionex to 2 mL. and now sold by Thermo Fisher Scien- 96-well filtration plate: A fixed-size tific; see pressurized fluid extraction polyethylene rectangular plate (127.8 for details of technique. mm × 85.5 mm) consisting of an array Active flow technology: A concept of 8 × 12 (96) of small filter tubes (vol- that incorporates two types of column umes range from 0.5 to 2 mL); a mem- designs: curtain flow technology means brane filter placed at the bottom of the segmenting the flow at the injection end well is used to filter liquid samples; of the column to ensure the analyte sees sometimes a prefilter is placed above the middle of the packed bed where it is the membrane filter to prevent clogging not disturbed by wall effects; parallel seg- with particulate samples. mented flow at the column outlet selects 96-well plate: A small rectangular just the middle portion of the flow profile plastic plate consisting of 96 individual resulting in improved efficiency without wells that are basically small-volume test the presence of wall effects, giving the tubes arranged in an 8 × 12 well pat- best overall column efficiency; a special tern; used for liquid handling and other endfitting design is used to sample the such requirements. center of the parabolic flow profile. 96-well solid-phase extraction plate: Active sampling: In active gas sam- A small rectangular plastic plate con- pling, a pump is used to push the sample sisting of 96 individual flow-through through a mass flow controller and into SPE wells arranged in an 8 × 12 array the canister. Additional sample can be that have top and bottom frits to con- collected, relative to the amount that tain solid particles of sorbent or resin to can be collected by passive sampling, perform SPE on a miniaturized scale; by pressurizing the canister with sample. generally 1 mg to 0.2 g of packing is Commonly the sample is pressurized to placed into the well, which can have 103 kPa (15 psig), effectively doubling a volume of up to 2 mL; used for au- the sample volume. tomated SPE with xyz liquid handling Active site: A reactive or strongly at- systems or customized workstations. tracting site on the surface of a chro- matographic packing that may bind A analytes or cause peak tailing; some- A solvent: Usually the weaker solvent in times mobile phase additives (such as a a binary eluent or gradient elution sepa- competing base) can negate the effects ration. In reversed-phase liquid chroma- of active sites. tography (LC), the A solvent typically is Activity: In adsorption chromatography, water or a water-rich mixture. the relative strength of the surface of the A term: The first term in the van Deem- packing. For silica gel, the more avail- ter equation. See eddy dispersion term able the silanol groups, the more active and van Deemter equation. the surface. Activity can be controlled Want fast, high-resolution separations, plus a range of conventional and unique selectivities to meet all your needs? Thermo Scientifi c™ Accucore™ HPLC and nanoLC columns feature solid core particles, are based on Core Enhanced Technology™ and offer speed and selectivity combined.
magentablackcyanyellow36613100330_2536560.pgs 09.14.2013 01:02 ADVANSTAR_PDF/X-1a 8 Terminology Guide October 2013 www.chromatographyonline.com by the addition of water or other polar Adsorption isotherm: In adsorption, a modifier that hydrogen-bonds to the plot of the equilibrium concentration of active sites thereby reducing the surface sample in the mobile phase per unit vol- activity; can also refer to biological ac- ume versus the concentration in the sta- tivity of a biomolecule. tionary phase per unit weight. The shape Additive: A substance added to the of the adsorption isotherm can determine mobile phase to improve the separation the chromatographic behavior of the sol- or detection characteristics; examples ute such as tailing, fronting, or overload. would be a competing base to negate Aerogel: A packing prepared when the the effects of silanols, a chelating agent dispersing agent is removed from a gel to block metal sites, or addition of a system without collapsing the gel struc- UV-absorbing compound to perform ture. Silica gels and glass beads used for indirect photometric detection. SEC are examples of aerogels that can Adsorbent: Packing used in adsorption retain their structures even at the high chromatography. Silica gel and alumina pressure used in HPLC. See xerogels. are the most frequently used adsorbents in Affinity chromatography: A tech- chromatography and sample preparation. nique in which a biospecific adsorbent Adsorption: A process of retention in is prepared by coupling a specific li- which the interactions between the gand (such as an enzyme, antigen, or solute and the surface of an adsorbent hormone) for the macromolecule of dominate. The forces can be strong interest to a solid support (or carrier). forces (for example, hydrogen bonds) or This immobilized ligand will interact weak (van der Waals forces). For silica only with molecules that can selectively gel, the silanol group is the driving force bind to it. Molecules that will not bind for adsorption and any solute functional are eluted unretained. The retained group which can interact with this compound can later be released in a group can be retained on silica. The purified state. Affinity chromatogra- term adsorption places emphasis on the phy is normally practiced as an “on–off” surface versus penetration or embedding separation technique. in the stationary phase coated or bonded Agarose: High-molecular-weight poly- to a surface. saccharide used as a separation medium Adsorption chromatography: One of in biochromatography. It is used in bead the basic separation and SPE modes that form and often used in gel filtration chro- relies on the adsorption process to ef- matography using aqueous mobile phases. fect a separation. Silica gel and alumina Alkoxysilane: A reactant used for the are the most frequently used normal- preparation of chemically bonded phase adsorbents in LC. Molecules phases. It will react with silica gel as fol- are retained by the interaction of their lows : R3SiOR + ≡SiOH → ≡Si–OSiR3 polar function groups with the surface + ROH where R is an alkyl group. functional groups (for example, silanols Alumina: A normal-phase adsorbent of silica). Carbon is also used as an ad- used in adsorption chromatography. sorbent in a reversed-phase LC mode. Aluminium oxide (Al2O3) is a porous Porous polymer, carbonaceous, and adsorbent which is available with a molecular sieve packings in GC exhibit slightly basic surface; neutral and acidic adsorptive properties as well. modifications can also be made. Basic www.chromatographyonline.com Terminology Guide October 2013 9 alumina can have advantages over silica, added to a sample before injection to cut which is considered to have an acidic down on interactions between analytes surface; alumina is seldom used as an that are unstable or behave poorly in HPLC column packing in practice. In the GC flow path on active sites; the GC applications, alumina will separate protectorants are chosen so that they low molecular-weight gases. do not interfere with the analysis of the Amino phase: A propylamino phase compounds of interest yet prevent these used in normal-phase chromatography. compounds from interacting with the It is a somewhat reactive phase for any active sites in the flow path; these pro- solute molecule (for example, aldehydes) tectorants are not generally required for or mobile phase additive that can react LC and LC–MS. with amines. The amino phase has found Analytical column: A chromatogra- some applications as a weak anion ex- phy column used for qualitative and changer and for the separation of carbo- quantitative analysis; a typical analyti- hydrates using a water–acetonitrile mo- cal column for LC will be 50–250 cm bile phase. It is a relatively unstable phase. × 4.6 mm, but columns with smaller Amperes full-scale (AFS): Extent of the diameters (down to 0.05 mm i.d.) can maximum detector output, for detectors also be considered as analytical col- utilizing an electrometer. umns. GC analytical columns range Amperometric detection: Electro- in length from 1 m to as much as 60 chemical detection applying a constant m, with inner diameters ranging from potential to the working electrode. Mea- less than 100 µm up to 2 mm. Station- sured current from oxidation or reduc- ary phases can be coated or bonded tion is proportional to the sample con- onto the interior of the tubing; packed centration. Very selective and sensitive GC columns are generally wider and method. Works with electrode reactions shorter and are less frequently used not changing the electrode surface (for nowadays. Chromatography columns example, cyanide, nitrite, thiosulfate, can be constructed of stainless steel, phenols). Only approximately 10% of glass, glass-lined stainless steel, PEEK, the analyte is oxidized or reduced. May fused silica, and other metallic and be used standalone as well as in series or nonmetallic materials. parallel to other detectors. Anion exchange: The ion-exchange Ampholyte: A substance that carries procedure used for the separation of an- both positive and negative charges (they ions. Synthetic resins, bonded phase sili- are amphoteric). Examples: amino acids, cas, and other metal oxides are available proteins. for this mode. A typical anion-exchange Amphoteric ion-exchange resin: Ion- functional group is tetraalkylammo- exchange resins that have both positive nium, making a strong anion exchanger. and negative ionic groups. These resins are An amino group on a bonded stationary most useful for ion retardation where all phase would be an example of a weak ionic materials can be removed from solu- anion exchanger. tion since the anionic and cationic func- Argentation SPE: The incorporation tionalities coexist on the same material. of a silver salt into the SPE stationary Analyte protectorant: In GC, a chemi- phase will help in retaining compounds cal compound or compounds that are with olefinic bonds. Normally used in 10 Terminology Guide October 2013 www.chromatographyonline.com organic solvents to maximize charge- transfer interactions. Array 96-well plate: A 96-well SPE 1.0 plate where the 96 individual wells are removable from the base plate; such a wa = A + B 0.5 hp setup allows users to place different BA types and amounts of SPE sorbents 0.1 0.0 t t t into various configurations in each of 32 1 36 p 40 2 44 48 the 96-wells. This type of 96-well plate Normalized peak height Time (s) has also been referred to as a flexible 96- Figure 1: Example of a tailing peak. (Mod- well plate configuration. ified with permission from reference 3.) Asymmetry: Factor describing the shape of a chromatographic peak. The- Average particle size (dp): The aver- ory assumes a Gaussian shape and that age particle size of the packing in the peaks are symmetrical. A quantitative column. A 5-µm LC column would be measure is the peak asymmetry factor, packed with particles with a definite which is the ratio of the distance from particle size distribution because pack- the peak apex to the back side of the ings are never monodisperse. Particle chromatography curve over the distance sizes in GC usually are expressed in from the peak apex to the front side of terms of mesh size distribution; 80–100 the chromatography curve at 10% of the mesh is a commonly used particle range. peak height. Various other measures of See particle size distribution. asymmetry are in common use, espe- cially the USP method. See also Foley– B Dorsey equation. B solvent: Usually the stronger solvent in Asymmetry factor: A factor that de- a binary eluent or gradient separation. In notes band shape; calculated from the reversed-phase LC, typically the organic chromatographic peak by dropping a modifier or modifier-rich binary mixture perpendicular at the peak apex and with water. a horizontal line at 10% of the peak B term: The second term of the van height; at the intersection the distance Deemter equation; the first term of the to the tail of the peak along the hori- Golay equation. See longitudinal dif- zontal line (distance B) divided by the fusion, molecular diffusion term, van distance along the horizontal line to Deemter equation, Golay equation. the front of the peak (distance A); Back extraction: Used in liquid–liquid this ratio is the peak asymmetry fac- extraction to perform an additional ex- tor; for a symmetrical peak the value traction to further purify a sample; ini- is one; for a fronting peak the value is tially the extraction may take place with less than one; for a tailing peak, the an aqueous solvent buffered at a high pH value is greater than one; the higher and an immiscible organic solvent; after the value the less symmetrical the peak the initial extraction takes place and in- is; values greater than 2 are generally terferences are removed, then by having unacceptable. another aqueous solution at a low pH, Atmosphere (atm): A unit of pressure. 1 one can back-extract the analyte into the atm = 101.325 kPa or 14.6959 psi. organic layer based on the analyte now www.chromatographyonline.com Terminology Guide October 2013 11 being in a neutral form. An example tion of the chromatographic band as it would be for the cleanup of an acidic moves down the column. The peak is substance containing –COOH groups; injected as a narrow slug and ideally at high pH the carboxyl would be ionized each separated component would elute and prefer the aqueous layer and impu- as a narrow slug of pure compound if rities may migrate to the organic phase not for the process of band broadening. and discarded; then the pH of the aque- The measure of band broadening is the ous layer can be adjusted to a low value. peak dispersion, σ, or more correctly N, Now the carboxyl group is in an union- the number of theoretical plates in the ized form and readily extracted into the column. Sometimes called band disper- organic layer as a purified substance. sion or band spreading. Backflushing: Useful in chromatogra- Band width: See peak width at base and phy to remove compounds that are held peak width at half-height. strongly at the head of a column. By re- Baseline: The baseline is the line drawn versing the flow at the conclusion of a by the recording device representing the run, analytes trapped at the head of the signal from the detector when only mo- column can be flushed from the column bile phase is passing through, in the ab- entrance because they have a shorter dis- sence of any solutes. It also represents the tance to travel; sometimes a strong solvent point from which calculations are often in LC or elevated temperatures in GC made on peaks to determine peak area will be needed to move them along. A or peak height. valve or fluidic device is used to effect the Baseline drift: Term for any regular change of mobile-phase flow direction. change occurring in baseline signal Backflushing can be used for analysis of from an LC or GC detector; it may these compounds or merely to remove arise from changes in flow rate of the them from the column. mobile phase or from stationary phase Back-pressure regulator: In LC, a de- bleed and may trend in a positive or vice placed on-line after the detector to negative direction. Baseline drift oc- maintain a positive pressure on the flow curs over a longer period of time than cell minimizing solvent outgassing prob- baseline noise. lems in the detector. In GC, the term Baseline noise: Irregular variations usually refers to a carrier-gas regulator in (short term) in the chromatographic the split vent line that maintains a con- baseline as a result of electrical noise or stant pressure at the inlet as split flows temperature fluctuations, outgassing in change. the flow cell, or poorly mixed mobile- Bakeout: The process of removing con- phase solvents. taminants from a column by operation Bed volume: See column volume. at elevated temperatures, which should BEH: Bridged ethyl hybrid; an inorganic– not exceed the maximum column tem- organic HPLC particle; has higher pH perature. limits than silica gel. Band: Refers to the chromatographic BET method: A method for measuring peak as it moves along and is eluted from surface area developed by Bruner, Em- the column. mett, and Teller (BET) that uses nitro- Band broadening: The process of in- gen adsorption–condensation in pores at crease in width and concomitant dilu- liquid nitrogen temperature. Pore volume 12 Terminology Guide October 2013 www.chromatographyonline.com
Tangents drawn to the in ection points
1.000
σ 0.882
In ection points
wi 0.607 wi = 2σ
wh 0.500 wh = 2.355σ
3σ Normalized peak height 0.324
0.134 4σ
0.044 5σ
wb = 4σ
Figure 2: Widths of a Gaussian peak at various heights as a function of the standard deviation σs) of the peak. (Modified with permission from reference 2.) and pore size distribution can also be ob- Binary mobile phase: Mobile phase con- tained from BET calculations. sisting of two solvents or buffers (or one Bidentate silane: A specific type of of each). bonded phase in which a short hydrocar- Bind–elute : In SPE, the normal mode of bon bridge connects two silicon atoms in operation where upon loading the sample a silane that is bounded to the surface onto a conditioned sorbent or resin, the through two siloxane groups. analytes of interest are retained (bound) Bimodal: In SEC, can be a porous while interferences and perhaps some of packing material that has two distinct the matrix is not retained by the pack- pore sizes or pore size distributions; in ing; after a wash step to remove some of ion-exchange or HILIC chromatog- the undesired sample components, the raphy or sample preparation can be a elution step uses a strong solvent to elute packing material that has two types the analytes of interest in a small volume. of functionalities (for example, cation Biocompatible: A term to indicate that exchange and reversed phase; cation the column or instrument component and anion) on one packing; in some will not irreversibly or strongly adsorb cases, mixed beds consisting of two or deactivate biomolecules, such as pro- different packings in one column can teins. Frequently means metal-free or be bimodal. ceramic surfaces and components. Thermo Scientifi c™ SOLA cartridges and plates revolutionize SPE. This fi rst fritless SPE product provides greater reproducibility with cleaner, more consistent extracts. Achieve greater throughput and improve your bottom line without changing your workfl ow. It’s time to simplify by doing more with less.
• Join the revolution at thermoscientifi c.com/sola-spe © 2011 Thermo Fisher Scientifi c Inc. All rights reserved. © 2011 Thermo Fisher Scientifi
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Blank: More correctly named method Breakthrough volume: The volume at blank. A blank prepared to represent which a particular solute pumped con- the matrix as closely as possible. The tinuously through a column will begin method blank is prepared and analyzed to be eluted. It is related to the column exactly like the field samples. Purpose: volume plus the retention factor of the Assess contamination introduced dur- solute. It is useful to determine the ing sample preparation activities. total sample capacity of the column Bleed: Loss of material from a column for a particular solute. or septum due to high-temperature BTEX: Refers to benzene, toluene, eth- operation. May result in ghost peaks ylbenzene, and xylenes analysis. plus increased detector baseline offset Buffer: A solution that maintains con- and noise; in extreme cases, bleeding stant pH by resisting changes in pH as chemicals from the stationary phase a result of dilution or addition of small may build up on detector surfaces. amounts of acids and bases. Blending: Refers to the process of mak- Buffer capacity: A quantitative mea- ing a heterogeneous sample into a more sure of the potential of a buffer solu- consistent and uniform sample by some tion (defined as the number of equiva- type of blending operation; the most lents of strong acid or base to cause a popular type of blender is the mechani- one unit change in the pH of 1 L of cal blender that chops a semisoft mate- a buffer solution) or simply the abil- rial into smaller parts. ity of a buffer to withstand injections Bonded phase: A stationary phase that of a buffered sample solution without has been chemically bonded to the a change in mobile-phase pH; capac- inner wall of an open-tubular (capil- ity determined by pH, buffer pKa, and lary) column or to the support particles. buffer concentration. In LC, the substrate is usually a silica Buffer strength: See ionic strength. gel particle or other base material. Bonded-phase chromatography: The C most popular mode in LC, in which a C4, C8, C18: Refers to the alkyl chain phase chemically bonded to a support length of a reversed bonded phase. is used for the separation. The most C term: The interphase mass transfer popular support for bonded-phase term of the van Deemter and Golay chromatography is microparticulate equations. silica gel and the most popular type of Canister collection: A stainless steel bonded phase is the organosilane, such vessel designed to hold vacuum to as octadecyl (for reversed-phase chro- less than 1.3 Pa (10 mTorr) or pres- matography). Approximately 70% of sure to 275 kPa (40 psig). Canisters all HPLC is carried out on chemically are available in a range of volumes: bonded phases. 400 mL, 1.0 L, 3.0 L, 6.0 L, and 15 Bonded-phase concentration: See L. The size of canister used usually coverage. depends on the concentration of the Boxcar chromatography: See column analytes in the sample, the sampling switching; alternate name. time, the flow rate, and the sample Breakthrough capacity: See break- volume required for the sampling pe- through volume. riod. Typically, smaller canisters are www.chromatographyonline.com Terminology Guide October 2013 15 used for more concentrated samples, Capillary tubing: Tubing to connect such as soil gas collection, 3-L and various parts of the chromatograph in 6-L canisters are used to obtain in- order to direct flow to the proper place. tegrated (TWA) ambient air samples Most capillary tubing used in HPLC is at sampling times of up to 24 h, and less than 0.020 in. in internal diameter. large 15-L canisters are used for refer- The smallest useful internal diameter is ence standards. Sampling time will be about 0.004 in. limited by the combination of canis- Capping: Same as endcapping. ter size and the flow rate at which the Carbon load: For a bonded-phase sample is to be collected. silica, term usually used to describe Capacity: See sample capacity. the surface coverage or the degree to Capacity factor (k’): Deprecated name which the available silanols on the for retention factor. column packing’s surface have reacted Capillary column: Refers to chro- and been replaced with the bonded matography columns of small inner phase; the higher the carbon load, the diameter, ostensibly small enough to lower number of residual silanols. The display a capillary effect with liquids. carbon load is normally expressed as The diameter below which a column is % carbon (for example, 12% carbon). considered “capillary” is poorly defined. In reversed-phase LC, the higher the See open-tubular column, capillary carbon load, the greater the analyte LC. retention. Capillary column, packed: A capillary Carrier: A term most often used in af- column that is packed with stationary- finity chromatography; refers to the phase particles. In GC, 1/16-in. o.d. by support that is used to attach the ac- 1-mm i.d. columns are common. tive ligand, usually by a covalent bond. Capillary electrochromatography Can also refer to the support in other (CEC): A hybrid technique where chromatography modes, such as LLC. capillary columns are packed with Carrier gas: Term for the gaseous mo- chromatographic sorbents and elec- bile phase in GC. troosmotic flow moves mobile phase Cartridge: Generally refers to the con- through the column rather than pres- tainer used in SPE or filtration; a car- sure; the technique has the surface- tridge may be as simple as a medical- mediated selectivity potential of HPLC grade syringe barrel that is filled with and the high efficiency of CE. packing contained at both ends by frits; Capillary GC: See open-tubular col- it can also be a molded device or even umn. a stainless steel device that contains Capillary LC: Generally refers to similar sorts of packing material. In HPLC carried out in a fused-silica or SPE, the device is also referred to as other type of capillary column; most an SPE tube. of the time the dimensions are in the Cartridge column: A column type sub-0.5-mm i.d. range. Has also been that has no endfittings and is held in a called micro LC. cartridge holder. The column consists Capillary micellar electrochromatog- of a tube and the packing is contained raphy (CMEC): The CEC version of by frits in each end of the tube. Car- MEKC. tridges are easy to change and are less 16 Terminology Guide October 2013 www.chromatographyonline.com expensive and more convenient than ample, C8, C18). Specifically excludes conventional columns with endfittings. the short chains typical methyl, isopro- Cation-exchange chromatography: pyl, and sec-butyl groups also attached The form of ion-exchange chroma- to the silane. tography that uses resins or packings Channeling: Occurs when voids cre- with functional groups that can sepa- ated in the packing material cause mo- rate cations. An example of a strong bile phase and accompanying solutes cation functional group would be a to move more rapidly than the average sulfonic acid; a weak cation-exchange flow velocity allowing band broadening functional group would be a carbox- to occur. The voids are created by poor ylic acid. packing or erosion of the packed bed. Centrifugation: Centrifugation is Charged aerosol detection (CAD): a process that involves the use of the The effluent from the LC column is centrifugal force for the sedimenta- nebulized and then vaporized in a tion of mixtures with a centrifuge (see heated drift tube, which results in a centrifuge). This process is used to cloud of analyte particles; these par- separate two immiscible liquids. More- ticles are charged and then the current dense components of the mixture mi- from the charged particle flux is mea- grate away from the axis of the centri- sured. The ELSD technique measures fuge, while less-dense components of the light scattering properties of the the mixture migrate toward the axis. aerosol particles. CAD is more sensi- Chemists and biologists may increase tive and gives a more linear response the effective gravitational force on a test than ELSD; it is also a universal detec- tube so as to more rapidly and com- tion method. pletely cause the precipitate (“pellet”) Check valve: A device inserted into a to gather on the bottom of the tube. moving fluid stream that allows flow of The remaining solution is properly the stream in only one direction; most called the “supernate” or “supernatant often used on the inlet and outlet sides liquid.” The supernatant liquid is then of an HPLC pump. either quickly decanted from the tube Chelating resin: Chelating resin con- without disturbing the precipitate, or tains functional groups that will inter- withdrawn with a Pasteur pipette. act with cationic species (for example, Centrifuge: A centrifuge is a piece metals such as copper, iron, heavy of equipment, generally driven by an metal ions); useful for concentrating electric motor (some older models were trace quantities or for separation. spun by hand), that puts an object in Chemical filtration: A liquid sample rotation around a fixed axis, applying is passed through a packing material a force perpendicular to the axis (see (for example, adsorbent, ion exchange, centrifugation). and so forth) that selectively interacts Certify: Specific SPE products for with one or more compounds within drugs of abuse isolation and analysis. the sample and acts as a chemical way Chain length: The length of carbon to remove and purify the liquid sample. chain in the hydrocarbon portion of a Regular filtration does not involve any reversed-phase packing. It is expressed chemical interaction but merely re- as the number of carbon atoms (for ex- moves particulates. www.chromatographyonline.com Terminology Guide October 2013 17
Chemical suppression: Remove back- Chiral stationary phase (CSP): A sta- ground conductivity by ion exchange. tionary phase that is designed to sepa- Converts the eluent into a low- or non- rate enantiomeric compounds. The conducting component (for example, phase can be coated or bonded to solid carbonate into carbonic acid, hydrox- supports, created in situ on the surface ide into water, or nitric acid into water). of the solid support, or can include sur- Anion analysis: the counter cation face cavities that allow specific interac- (for example, sodium) is replaced by tions with one enantiomeric form. the proton (H+). Cation analysis: the Chlorosilane: A chemical reagent used counter anion (for example, nitrate) is to prepare siloxane bonded phases; re- replaced by hydroxide (OH-). The mea- activity changes from a monochlorosi- sured signal is the corresponding acid lane < dichlorosilane < trichlorosilane; or base of the anion or cation respec- the alkyl portion (for example, octa- tively. The dissociation of these acids decyl, octyl, and so forth) will dictate and bases influences the signal. All sup- the hydrophobicity of the resultant pressor devices work on this principle. bonded phase; alkoxysilanes can be The typical background conductivity used but are less reactive. after suppression is <20 µS/cm. Chopping: The process of mechanically Chemisorption: Sorption due to a cutting a sample into smaller parts. chemical reaction with the packing. Chromatogram: A plot of detector Most such interactions are irreversible. signal output versus time or elution Usually occurs on packings with reac- volume during the chromatographic tive functional groups such as silanol or process. bonded amino phases. Chemisorption Chromatograph: (n) A device used to is common with metal oxide phases implement a chromatographic separa- that have strong Lewis acid sites. tion; (v) the act of separation by chro- Chip format: A miniaturization tech- matography. nique where small channels on a glass, Chromatographic conditions: Those polymer or other type of matrix are chromatographic method experimen- used instead of large bore columns, tal parameters that describe how an capillaries, and so forth. The result- analysis was performed. Sufficient ing format is greatly reduced in size information must be presented so that compared to conventional chromato- the analysis can be duplicated for veri- graphic instruments; advantages are fication purposes. the reduction in sample, solvent, and Classification: The process of sizing so forth; can be more easily coupled to column packing particles; generally, in detection techniques (for example, MS HPLC a small particle size distribution and MS-MS) where smaller amounts provides better efficiency and a greater of mobile phase can lead to sensitivity permeability because of the absence of enhancements and reduction in ion fines. Classification can be performed suppression. by sedimentation, elutriation, and Chiral recognition: The ability of a using centrifugal air classifiers. chiral stationary phase to interact dif- Co-ion: An ion of the same sign of ferently with two enantiomers leading charge as the ionic groups making up to their chromatographic separation. the stationary phase. 18 Terminology Guide October 2013 www.chromatographyonline.com
Coating efficiency (CE, UTE, UTE%): Column outlet flow rate, corrected
A metric for evaluating column quality. (Fa): In GC, the column outlet flow The minimum theoretical plate height rate corrected from column tempera- divided by the observed plate height: ture and outlet pressure to room tem-
CE = Hmin/H perature and pressure, for example, Cold injection: An injection that takes the flow rate as measured by a flow place at temperatures below the final meter. Difficult to measure directly oven temperature, usually at or below for narrow-bore open-tubular col- the solvent boiling point. umns, the flow rate can be calcu- Column: The tube and stationary phase lated from the average carrier-gas through which mobile phase flows re- linear velocity, pressure drop, temper- sulting in a chromatographic separa- atures, and the column dimensions: – 2 tion. Fa = (u πdc T0)/(4jTc). Such calcula- Column chromatography: A ny tions are the basis of electronic pres- form of chromatography that uses sure control. a column, tube, or plate to hold the Column overload: If one exceeds the stationary phase. Open-column chro- sample capacity (or loading capacity) of matography, HPLC, and open-tubu- a column, peaks will become distorted lar capillary gas chromatography are and may be difficult to measure and all forms of column chromatography. to achieve reproducible chromatogra- Most often refers to open-column phy from run to run. One can measure chromatography used for prepara- column capacity by running a break- tive work. through study (see breakthrough vol- Column dead time: See hold-up time. ume). Column equilibration: To provide re- Column packing: The solid material, producible results, a column should be usually a porous solid with or without equilibrated with the surrounding envi- a chemically interactive surface, placed ronment be it a temperature condition, inside or on the walls of the column mobile phase equilibrium, pressure used to differentially retain analytes; condition, and so forth; in GC, it is also referred to as the stationary important that the temperature of the phase; common packings include un- column be stabilized after a tempera- bonded and bonded silica, resins, in- ture programmed run and in LC, the organic–organic hybrids, graphitized column must be returned to its original carbon, porous polymers, and molecu- conditions before another gradient is lar sieves. run. Column performance: Denotes the
Column inner diameter (dc): The column efficiency. See theoretical inner diameter of an uncoated chro- plate. matography column. Column plate number: Denotes the Column length (L): The length of the column efficiency. See theoretical analytical chromatography column plate. used to perform the chromatographic Column suppressor: Initial setup. separation. Distinct from the length Packed ion-exchanger columns were of a precolumn (LC) or retention gap used for chemical suppression. Draw- (GC) connected in series. backs: require regeneration, changes www.chromatographyonline.com Terminology Guide October 2013 19 of the selectivity throughout the of two columns connected in series by usage. a retention modulation device, thereby Column switching: The use of mul- generating much higher resolution than tiple columns connected by switching with any single column. valves to effect better chromatographic Comprehensive two-dimensional separations or for sample cleanup. Frac- chromatography: Two-dimensional tions from a primary column can be chromatography applied to every frac- switched to two or more secondary tion. See two-dimensional chroma- columns which in turn can be further tography. diverted to additional columns or to Compressibility correction factor (j): the detector (or detectors); sometimes Due to gas compressibility, the carrier referred to as multidimensional chro- gas expands and its velocity increases matography. as it proceeds along a GC column from Column temperature (T ): c Tempera- the inlet pressure pi to the outlet pres- ture of the column. A uniform tem- sure po. The carrier gas compressibility perature across the column usually is correction factor corrects the carrier desirable; however, GC separations gas velocity at the outlet of a GC col- also may be performed with a moving umn to the average carrier gas velocity: temperature gradient along the column j = 3(P 2 – 1)/2(P 3 – 1), where P is the length. column pressure drop: P = pi/po Column volume (Vc): The volume of Concentration: The process of increas- the unpacked, uncoated column: Vc ing the strength or density of a diluted 2 = AcL = πrc L, where Ac and L are the sample; a more concentrated sample cross-sectional area of the tube and the will be easier to measure; concentration tube length, respectively. can be accomplished by a wide variety Cool-down time: Length of time re- of sample preparation techniques such quired to cool a GC oven from the final as evaporation, adsorption, diffusion, oven temperature to the initial oven and so forth. temperature. Shorter cool-down times Conditioning (SPE): This is gener- allow a greater number of analyses to ally considered to be the first step in be performed in a given time period. SPE; the stationary phase must first Competing base: In reversed-phase be put into a chemical or physical LC, addition of a small basic com- state that it can accept the sample pound such as triethylamine or di- solution loaded in the second SPE methyloctylamine at 25–50 mM step; a conditioning solvent is passed concentration to the mobile phase to through the SPE stationary phase inhibit basic analytes from interact- where it will solvate the phase so that ing with residual silanols; works by it will more easily sorb the sample of law of mass action because the con- interest; for a reversed-phase SPE car- centration of competing base is much tridge, methanol or acetonitrile serves greater than that of the analyte. See as a conditioning solvent; sometimes also additive. the excess conditioning solvent must Comprehensive GC (GC×GC): Two- be removed but the packing shouldn’t dimensional technique in which all be allowed to dry out because that compounds experience the selectivity may affect the “conditioned” phase. 20 Terminology Guide October 2013 www.chromatographyonline.com
Conductivity: Conductivity is the in- method used by analytical chemists herent parameter of all ions. Therefore to reduce the sample size of a powder it is the signal for measuring the chro- without creating a systematic bias. The matogram. It may be measured directly technique involves pouring the sample or after chemical and sequential sup- so that it takes on a conical shape, and pression respectively. As conductivity is then flattening it out into a cake. The strongly dependent on the temperature cake is then divided into quarters; (around 2 %/°C), a thorough isolation the two quarters that sit opposite one and thermal stabilization of the detec- another are discarded, and the other tor block is recommended. The mea- two are combined and constitute the sured conductivity of a solution is given reduced sample. The same process is by the sum of the “single ion conduc- continued until an appropriate sample tivities.” The single ion conductivity is size remains. Analyses are made with a linear function of the concentration respect to the sample left behind. of the ion and its equivalent conduc- Continuous liquid–liquid extraction: tivity. At very low concentrations or at Useful when the KD value is very low constant ion strength the equivalent or the required sample volume is very conductivity is constant. The mea- large when multiple extractions are im- sured signal in nonsuppressed IC is practical; also if the extraction is slow, proportional to the concentration and a long time may be required for equi- the difference of eluent and sample ion: librium to be established; in continu- As the ionic strength of the solution is ous LLE, fresh solvent is continually constant (stoichiometric ion exchange) recycled through the aqueous sample; also the equivalent conductivities are continuous extractors are available for constant. This leads to very linear cali- heavier-than-water and lighter-than- bration curves. The measured signal water solvents. in suppressed IC is proportional to the Controlled surface porosity support: sum of the equivalent conductivities Same as porous layer bead and pel- of the analyte ion and the counterion licular stationary phase. (which has been introduced by suppres- Cool on-column injection: Cool on- sion). As only the sample ion and its column injection is a technique of in- counterion is adding to the measured troducing a sample as a liquid directly conductivity after suppression, the total into a GC column; this lack of prior concentration is changing during the vaporization offers the following advan- peak. Therefore the equivalent conduc- tages: It eliminates sample discrimina- tivities are no longer constant. This is tion; it eliminates sample alteration; one reason for the inherent nonlinear- and it provides high analytical preci- ity of the calibration curves in sup- sion. However, there are some special pressed IC. requirements: It requires relatively Coning and quartering: A sample size clean samples; real samples are often reduction technique where a portion of too concentrated for on-column injec- free-flowing solid material (powder) is tion and must be diluted; and peak systematically divided into quadrants splitting or peak distortion can occur to achieve a statistically representative due to differing polarities of solvent, sample. Coning and quartering is a stationary phase, and solutes. www.chromatographyonline.com Terminology Guide October 2013 21
Core–shell: See superficially porous HPLC and CE (MEKC) by a partition- particles (SPPs). ing mechanism. Coulometric detector: Same as am- Cross-linked phase: A stationary perometric detection, but with a con- phase that includes cross-linked poly- version rate of 100%. All the analyte mer chains. Usually it is also bonded is oxidized or reduced at the working to the column inner wall. See bonded electrode. The response is larger than phase. with amperometric detection. But on Crosslinking: For resins, during the the other hand also the baseline noise process of copolymerization to form is larger. Therefore the detection limits a three-dimensional matrix a difunc- are almost the same. tional monomer is added to form cross- Counterion: In an ion-exchange pro- linkages between adjacent polymer cess, the ion in solution used to displace chains. The degree of cross-linking the ion of interest from the ionic site. is determined by the amount of this In ion pairing, it is the ion of opposite monomer added to the reaction. For charge added to the mobile phase to example, divinylbenzene is a typical form a neutral ion pair in solution. cross-linking agent for the production Coupled columns: A form of column of polystyrene ion-exchange resins. The switching that uses a primary column swelling and diffusion characteristics connected to two secondary columns of a resin are governed by its degree of via a selector valve. Fractions from col- cross-linking. umn one can be selectively transferred Crushing: Tungsten carbide variable to columns two and three for addi- jaw crushers for reducing the size of tional separation to occur. The term is large, extremely hard, brittle samples. also used to describe two or more col- Curtain flow technology: Curtain umns connected in series to provide an flow technology refers to the process increased number of plates. of injection of sample across a radial Coverage: Refers to the amount of cross section of an HPLC column to bonded phase on a silica support in ensure the analyte sees the middle por- bonded phase chromatography. Cov- tion of the packed bed and not the wall erage is usually described in µmol/m2 where flow effects may be different; the or in terms of %C (w/w). technique is coupled with a parallel seg- Crash plate: Refers to the process of mented flow fitting at the column out- precipitating protein from plasma by let to select just the middle portion of the addition of a miscible organic sol- the flow profile resulting in improved vent such as acetonitrile; when a 96- efficiency without the presence of wall well flow-through or fixed-well plate is effects. used for this process, it is referred to as Cutting: Cutting mills can reduce soft- crashing and the plate a crash plate. to-medium hard materials (diameter < Critical micelle concentration (CMC): 100 mm). The concentration of an ionic surfac- Cyano phase: A chemically bonded tant above which a micelle is formed phase that terminates with the -CN by aggregation; micelles added to the functional group; it can be used in mobile phase are used to improve the normal-phase chromatography as separation of nonionic substances in a moderate polarity sorbent and in 22 Terminology Guide October 2013 www.chromatographyonline.com reversed-phase chromatography as a Degassing: The process of removing short chain bonded phase. dissolved gas from the mobile phase Cyclodextrins: Cyclic oligomers of prior or during use. Dissolved gas that several D-(+)-glucopyranose units used may come out of solution in the detec- in chiral HPLC and CE separations; tor cell can cause baseline spikes and popular ones are named α-, β-, and noise. Dissolved air can affect certain γ-CDs; they have a truncated cone detectors, such as electrochemical (by shape, a relatively hydrophobic cavity, reaction) or fluorescence (by quench- and primary and secondary hydroxyl ing). Dissolved gases can also cause groups at their end; separate on basis pumps to lose prime. Degassing is car- of differential inclusion of enantio- ried out by heating the solvent or by mers; modified CDs with derivatized vacuum (in a vacuum flask), or on-line hydroxyl groups are also used for se- using evacuation of a tube made from a lectivity modification. gas-permeable substance such as PTFE, or by helium sparging. D Denaturing HPLC: Use of reversed- Data acquisition rate: A term refer- phase HPLC to investigate genetic ring to the rate of sampling of a de- mutations by the investigation of DNA tector output. To characterize a chro- base pairs. matographic peak at least 20–30 data Derivatization: A technique used in points must be collected. The data chemistry that transforms a chemical acquisition rate, usually measured in compound into a product (the reac- hertz, defines how many data points tion’s derivate) of similar chemical per second are collected while the peak structure, called a derivative. Gener- is moving through the detector. For fast ally, a specific functional group of chromatography, the data acquisition the compound participates in the de- rate must be sufficiently rapid to char- rivatization reaction and transforms acterize a narrow peak. Modern detec- the compound into one with a differ- tors have data rates up to 200 Hz; also ent reactivity, solubility, boiling point known as data rate and sampling rate. (volatility), melting point, aggregate See detector time constant. state, or chemical composition. The Dead volume: Dead volume is extra resulting new chemical properties can volume experienced by solutes as they be used for quantification (for example, pass through a chromatographic sys- UV or fluorescence detection) or better tem, in particular any unswept vol- separation properties. ume exposed to the mobile phase flow. Desalting: Technique where low- Excessive dead volume causes addi- molecular-weight salts and other com- tional peak broadening. Related to the pounds can be removed from nonionic hold-up volume, which is the volume and high-molecular-weight com- of mobile phase necessary to elute an pounds. The use of a reversed-phase unretained compound. See hold-up packing to retain sample compounds volume. by hydrophobic effects yet allow salts Deep-well plate: A 96-well plate ca- to pass through unretained would be pable of handling up to 2 mL of liquid an example of desalting. The use of an volume per well. SEC column to exclude large molecules www.chromatographyonline.com Terminology Guide October 2013 23 and retain lower-molecular-weight salts layer of material that contains holes is another example. Desalting using of various sizes, or pores. Smaller dialysis is commonly used in protein solutes and fluid pass through the purification. membrane, but the membrane Desorption: The process in chroma- blocks the passage of larger sub- tography where a molecule residing on stances (for example, red blood cells, the surface of a packing material or on large proteins). It is a technique used another solid surface (for example, col- in biological sample prep to desalt umn wall or frit) or stationary phase biological fluids. moves from the surface into the mobile Diatomaceous earth: Also known as phase. diatomite or kieselguhr, it is a natu- Detector response time: Time for a rally occurring, soft, siliceous sedi- detector to respond to ~90% of the in- mentary rock that is easily crumbled coming solute amount. The response into a fine white to off-white powder. time is generally taken as 2–4 times the Diatomaceous earth consists of fos- time constant. See also detector time silized remains of diatoms, a type of constant. hard-shelled algae. It has a particle Detector time constant (τ): The time size ranging from less than 3 µm to for a detector to respond to 1/e = 63.2% more than 1 mm, but typically 10– of an instantaneous change in solute 200 µm. Depending on the granular- amount. In general the detector time ity, this powder can have an abrasive constant should be less than 10% of feel, similar to pumice powder, and is the peak width at half-height. Excessive very light as a result of its high poros- detector dead volume, slowly respond- ity. The typical chemical composition ing electronics, digital data acquisition of oven-dried diatomaceous earth is speeds, and signal filtering strongly in- 80–90% silica, with 2–4% alumina fluence detector response times. Too- (attributed mostly to clay minerals) slow detector response times cause peak and 0.5–2% iron oxide; highly puri- tailing, loss of peak height and detect- fied diatomaceous earth is used as a ability, plus loss of peak resolution for support for chromatography and for closely adjacent peaks. supported liquid–liquid extraction. Dextran: Polydextran-based packing Diethylaminoethyl (DEAE): A popu- material primarily used for low pres- lar weak anion-exchange functionality sure biochromatography; an example typically attached to cellulose or Sep- would be Sephadex (Amersham Phar- harose (GE Healthcare); used for the macia Biotech). separation of biomolecules. Dialysis: Dialysis works on the prin- Diethylene glycol succinate (DEGS): ciples of the diffusion of solutes and A GC stationary phase. ultrafiltration of fluid across a semi- Diffusion coefficient (DM or DG; DS permeable membrane. Diffusion is or DL): A fundamental parameter of a property of substances in water; a molecule in the liquid or gaseous substances in water tend to move mobile phase (DM or DG) or in the from an area of high concentration liquid stationary phase (DS or DL) that to an area of low concentration. A expresses the degree of free mobility semipermeable membrane is a thin of the molecule in solution. Expressed 24 Terminology Guide October 2013 www.chromatographyonline.com in cm2/s, the diffusion coefficient is Dimethylchlorosilane (DMCS): Some- dependent on molecular weight of the times used for silanizing glass GC parts solute, temperature, solvent viscosity, such as inlet liners and endcapping sil- and molar volume of the solute. A ica-based HPLC bonded phases. Dis- typical value of a small molecule (100 posable presilanized inlet liners are a Da) in a liquid phase at room tem- preferable alternative that avoid use and perature is on the order of 10-5 cm2/s. storage of this hazardous reagent. Gaseous solutes in helium carrier gas Diode-array detection (DAD): Each at 120 °C have diffusion coefficients wavelength of the UV and visible range that are several orders of magnitude of the light is measured with an indi- higher, around 0.4 cm2/s. vidual diode. The optical resolution of Digestion: The process of treating an the detector is defined by the number insoluble chemical compound with a of diodes used (for example, 844 di- reactive substance (for example, for odes: optical resolution = 1.4 nm). inorganic compounds it might be a Diol phase: A hydrophilic phase use- strong acid; for a biological compound ful in both normal and reversed phase. it might be an enzyme) that will break It consists of a diol structure (two -OH it down or disintegrate the compound groups on adjacent carbon atoms in into a more soluble form that can be an aliphatic chain). In normal-phase further treated or analyzed. work, it is less polar than silica and in Digital chromatography: The pro- reversed phase work has been used for cess of solid-phase extraction (SPE) is the separation of proteins and poly- sometimes referred to as digital chro- peptides. matography. A substance is either on Direct injection: Sample enters the the SPE stationary phase or is off the inlet and is swept into the column by stationary phase during its retention carrier gas flow. No sample splitting and elution; in chromatography we or venting occurs during or after the are often trying to resolve closely re- injection. lated substances by exploiting subtle Direct sampling: A method of sample differences in retention in more of an collection where a sample is taken di- analog separation mode; in terms of k rectly from the source. For example, a values, ideally the solute in SPE has a cannister may be used to collect a gas value of infinity when on the sorbent sample exactly where the scientist desires. and zero when eluted into solution. A river water sample can be obtained Dilute and shoot: A simple sample by lowering a collection vessel directly preparation procedure where one into the water. A thermal desorption merely dilutes the sample with solvent, tube can be used to concentrate volatile mobile phase, or a compatible liquid and semivolatile analytes by passing a and then injects that diluted sample gas stream through the adsorbent con- into a chromatograph without any fur- tained within the tube. This would be ther sample preparation. an example of direct sampling. Dilution: Reducing the concentration Direct thermal sampling: Refers to of a chemical by adding an inert sub- the process of using temperature as a stance; the substance can be a liquid, variable in sample volatile and semi- solid, or gas. volatile substances; static headspace at www.chromatographyonline.com Terminology Guide October 2013 25 a given temperature is an example of of solvent containing extracted ana- thermal sampling; by selecting a cer- lytes (tetrachloroethylene) is removed tain temperature certain sample com- by a microsyringe for direct injection. ponents can be ruled out because they Extraction is almost instantaneous and may have extremely low volatility at a enrichment values are quite high. selected temperature; thermal sampling Dispersive SPE (dSPE): In dSPE, can occur in stages all the way up to loose SPE packing material is added pyrolysis where chemical bonds in the directly to a solution rather than pass- sample are purposely broken to access ing it through the packed material in a the structure of the material. cartridge or tube; dSPE is most often Disk: A number of sample prepara- used as the second step in QuEChERS tion and separation media take the where matrix compounds are removed form of a disk; the most popular disks from the organic solvent salting out ex- are used in filtration and may consist traction of the first step. of any number of porous polymeric Displacement chromatography: materials; the most popular types Chromatographic process where the of SPE disks would have embedded sample is placed onto the head of the particles in a disk made of PTFE or column and then is displaced by a other inert polymeric material or a compound that is more strongly sorbed fiberglass matrix with interdispersed than the compounds of the original sorbent particles; some biological mixture. Sample molecules are then purification media employ the disk displaced by each other and by the format. The stationary phases may more strongly sorbed compound. The contain ion exchange groups or other result is that the eluted sample solute functionality that attracts solutes of zones may be sharpened; displacement interest or impurity that one may techniques have been used mainly in want to get rid of. preparative HPLC applications. Disk cartridge (SPE): See disk. Disposable filter: See syringe filter. Dispersion: See system dispersion. Dissolution: The process of having Dispersive liquid–liquid microextrac- a sample dissolve in an appropriate tion (DLLME): The technique is based solvent. on a three-component solvent system. Distillation: A method of separating The container is usually a centrifuge mixtures based on differences in vola- tube and the appropriate mixture of tility of components in a boiling liquid immiscible organic extraction solvent mixture. Distillation is a unit operation, (usually a few microliters, such as 8 µL or a physical separation process, and of tetrachloroethylene) and a dispersive not a chemical reaction; it can be used solvent (for example, ~1 mL of acetone) to purify organic compounds or to re- is rapidly injected with a syringe into move solvent; fractional distillation is an aqueous solution (~5 mL) contain- used to separate compounds with close ing the analyte of interest. When the boiling points; azeotropic distillation is three solvents are rapidly mixed, a using an azeotrope to remove a solvent cloudy solution is formed consisting of that has a boiling point too close or droplets of extraction solvent; the entire equal to another compound that can- mixture is centrifuged and the droplet not be separated. 26 Terminology Guide October 2013 www.chromatographyonline.com
Distribution constant (coefficient) nal fluid, saliva) as well as other non-
(Kc): The equilibrium concentration biological media have been investigated. of a component in or on the station- Drying: Drying of sample extracts can ary phase divided by the equilibrium be accomplished by heating (evapora- concentration of the component in the tion), vacuum dessication, and other mobile phase; also called the distribution means; water can be removed (dried) coefficient or in partition chromatogra- from organic solvents by using anhy- phy the partition coefficient; in partition drous sodium sulfate. Dwell time: chromatography Kc is used and refers to The time equivalent to the case where the concentration in the dwell volume; determined by the prod- stationary phase is expressed per unit uct of flow rate times the dwell volume. Dwell volume: volume of the phase: VR = VM + KcVS; In LC, refers to the in the case of a solid stationary phase, volume between the point of mixing of
Kg is used and is expressed as per mass solvents (usually in the mixing chamber (weight) of the dry solid phase; in the or at the proportioning valves in the liq- case of adsorption chromatography with uid chromatograph) and the head of the a well characterized adsorbent of known chromatographic column; important in surface area, the concentration in the gradient elution or when changes in sol- stationary phase is expressed as per unit vent composition are made in isocratic surface area. elution so that the column experiences Dividers: A mechanical device used in the composition change in the shortest subdividing solid powder samples into possible time. Low-pressure mixing sys- smaller units; they can be manual or au- tems generally have larger dwell volumes tomated; sample dividers will subdivide than high-pressure mixing systems. material samples into two smaller por- Dynamic coating: The formation of in- tions by a single pass or further subdivi- situ coatings on the packing in HPLC sions can be attained by multiple passes. or on capillary walls in CE by addition The important feature of sample divid- of a substance to the mobile phase that ers is that each subdivision retains the adsorbs (or absorbs) onto the pack- characteristics of the original sample. ing or at the wall surface; the purpose Dried blood spot (DBS) analysis: A of a dynamic coating is to generate a newer method for sampling and trans- new stationary phase or to deactivate porting blood samples; a small (~15 µL) the packing material or capillary wall sample of whole blood is placed on a cel- to prevent unwanted interactions; one lulose or other paper-like material and simple example is the adjustment of is dried for 2 h; the dried blood spot the mobile phase or running buffer to can be extracted to remove analytes of a pH < 3 to protonate silanols and ne- interest for further workup; has poten- gate their effect; another is coating the tial to replace drawing large quantities phase with a hydrophilic polymeric ma- for blood analysis; used in conjunction terial to prevent adsorption of proteins. with LC–MS-MS for high sensitivity In GC, dynamic coating applies a con- and specificity. trolled-thickness coating to the inside Dried media spot (DMS) analysis: In of open-tubular columns as a solution addition to DBS, other biological fluids flows through the column and leaves (for example, plasma, serum, cerebrospi- a thin coating behind, which is then www.chromatographyonline.com Terminology Guide October 2013 27 evaporated to yield a thin coating of any expression is misleading as the suppres- non-volatile material from the solution. sion is chemical. Only the supply of the Dynamic headspace: See purge-and- respective ion is done electrochemically. trap sampling. Electrodialysis: Used to transport salt ions from one solution through ion-ex- E change membranes to another solution Eddy dispersion (diffusion) term (A under the influence of an applied elec- term): The A term in the van Deemter tric potential difference. This is done in equation. It is the contribution to plate a configuration called an electrodialysis height that arises from the heterogeneity cell. The cell consists of a feed (diluate) in axial velocities as a result of the par- compartment and a concentrate (brine) ticle size and geometry of the packing compartment formed by an anion- as well as wall effects: a = 2λdp. Typi- exchange membrane and a cation-ex- cal values of λ for well-packed columns change membrane placed between two are 0.8–1.0. Some theories of chroma- electrodes; can provide good enrichment tography indicate a velocity dependent factors. contribution to the HETP from this Electrolytic suppression: Synonym to process. Also known as eddy diffusion, electrochemical suppression. flow-heterogeneity induced broadening, Electrolytic conductivity detection and the multipath term. The A term is (ELCD): An electrolytic conductivity de- zero in open-tubular GC columns. See tector catalytically reacts halogen-con- van Deemter equation. taining solute with hydrogen (reductive Efficiency: The ability of a column mode) to produce strong acid by-prod- to produce sharp, well-defined peaks. ucts that are dissolved in a working fluid. More efficient columns have more theo- The acids dissociate and the increased retical plates (N) and smaller theoretical electrolytic conductivity is measured. plate heights (H). For asymmetric peaks Other operating modes modify the it is better determined from the peak chemistry for response to nitrogen- or centroid and variance by mathematical sulfur-containing substances. analysis of the peak shape. See Foley- Electron-capture detection (ECD): An Dorsey equation. electron-capture detector ionizes solutes Effluent: The mobile phase leaving the by collision with metastable carrier gas column; the same as eluate. molecules produced by β-emission from Electrochemical detector: Global term a radioactive source such as 63Ni. ECD for all detection modes recording elec- is one of the most sensitive detection trical potential or current (conductivity, methods and responds strongly to halo- amperometric, pulsed amperometric, genated solutes and others with a high coulometric, and potentiometric detec- electron-capture cross-section. tion). More often used for amperometric, Electronic or programmed pressure pulsed amperometric, and coulometric control (EPC or PPC): Any of a number detection only. of pressure and flow control devices that Electrochemical suppression: Con- incorporate electronic pressure or flow tinuously working chemical suppressor sensing and can be programmed from where H+ or OH- are electrochemically the GC microcontroller. Such devices produced by the electrolysis of water. The enable method control of flow, velocity, 28 Terminology Guide October 2013 www.chromatographyonline.com and pressure for GC columns, as well ment chromatography and frontal as providing a convenient means of in- analysis. corporating gas-related parameters into Elution step: This is generally consid- electronic methods. ered to be the fourth step in SPE and Eluate: Combination of mobile phase occurs after the washing (rinsing) step; and solute exiting the column; also in the elution step, analytes are removed called effluent. from the SPE stationary phase by elu- Eluent: The mobile phase used to carry tion with a strong solvent so that the out a separation. analytes are now in a concentrated state; Eluite: The species being eluted: the often, the strong solvent is removed by analyte, or the sample. evaporation and reconstituted in a sol- Eluotropic series: A series of solvents vent more compatible with the chro- (eluents) with an increasing degree matographic system. of solvent strength generally used in Elution volume (VR): Refers to the vol- liquid–solid or adsorption chroma- ume of mobile phase required to elute a tography. In normal-phase chroma- solute from the column. For a symmet- tography, a nonpolar solvent such as ric peak, it is the volume from the point pentane would be at one end of the of injection to the volume at maximum scale, an intermediate solvent such concentration (apex): VR = FtR where F as dichloromethane would be in the is the flow rate and tR is the retention middle of the scale, and a strongly time of the peak of interest. polar solvent such as methanol would Elutriation: A technique used to frac- be near the upper end of the scale. In tionate packing particles by size based on normal-phase chromatography, the the difference in their Stokes terminal reverse order of strength would be velocities. It is most often used for the observed; water would be weak and separation of ion-exchange resins that hexane strong. Thus, when developing need to have a particularly narrow size a method or running a gradient, an range, such as amino acid resins. The eluotropic series is useful for selecting technique involves the upward flow of solvents. water into a large tube. The unsized Elute: To chromatograph by elution beads are added to the moving water chromatography. The term elute is pre- and the particles seek their own level, de- ferred to the term develop used in older pending on their density and particle size. nomenclature. They are then removed at certain levels Elution: The process of passing mobile in the tube. High purity spherical silica phase through the column to transport gels are sometimes sized by elutriation. solutes down the column. Emulsion: A mixture of two or more Elution chromatography: The most liquids that are normally immiscible commonly used chromatographic (nonmixable or unblendable). Emul- method where the sample is applied to sion is usually referred to when both the head of the column as a narrow zone the dispersed and the continuous phase and individual molecules are separated are liquids. In an emulsion, one liquid and eluted at the end of the column (the dispersed phase) is dispersed in the under the influence of a directed flow other (the continuous phase). Emulsions of mobile phase. Compare to displace- are bothersome in LLE because they magenta black cyan yellow innovating routine analysis injectors and detectors and injectors featuring chromatograph © 2013 Thermo Fisher Scientifi c Inc. All rights reserved. system. GC Series 1300 TRACE the by delivered productivity and performance capabilityspecifito applicationsc dailyandworkload. Experience ground-breakingthe instrumenttailorquickly to user theenabling and eliminatingmaintenancedowntime laboratories. 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™ 30 Terminology Guide October 2013 www.chromatographyonline.com sometimes are hard to break so that isolating a compound of interest; sol- the layers can be separated and further vent evaporation is the most often used treated, if necessary. There are numer- sample preparation technique. ous ways to break emulsions. Evaporative light scattering detec- Enantiomeric compound: Chemical tion (ELSD): The effluent from the LC compounds that display chiral activity; column is nebulized and then vapor- such compounds will require a separa- ized in a heated drift tube. This process tion mechanism that can differentiate results in a cloud of analyte particles between the R- or S-enantiomer. Spe- that passes through a beam of light; the cialty columns are available for this particles scatter the light and generate purpose. a signal at a photodiode or photomulti- Endcapping: A technique used to re- plier; it is a universal detection method move silanol groups on silica gel that where molecules are not required to have may remain after reaction with a large a chromophore, be fluorescent, or be silylating agent such as octadecyltrichlo- electrochemically active. rosilane. The column is said to be end- Exchange capacity: See ion-exchange capped when a small silylating reagent capacity. (for example, trimethylchlorosilane Excluded volume: See interstitial vol- and dichlorodimethylsilane) is used to ume. bond residual silanol groups on a silica Exclusion chromatography: See steric- gel-based packing surface. Most often exclusion chromatography and ion ex- used with reversed-phase packings to clusion. minimize undesirable adsorption of Exclusion limit: In SEC, the upper limit basic, ionizable, and ionic compounds. of molecular weight (or size) beyond For polymeric phases with terminal si- which molecules will be eluted at the lanol groups, endcapping reactions are same retention volume, called the exclu- also used to remove them. sion volume. Many SEC packings are Endfitting: The fitting at the end of referred to by their exclusion limit. For the column that permits connection to example, a 105 column of porous silica the injector or detector. Most HPLC gel will exclude any compounds with a endfittings contain a frit to hold the molecular weight over 100,000, based packing and have a low dead volume on a polystryene calibration standard. for minimum band spreading. They Exclusion volume (Vo): The minimum are usually constructed of stainless steel retention volume of a molecule on an but PEEK and other polymeric materi- SEC packing where all molecules larger als are also used. than the size of the largest pore are to- Equilibration: See column equilibra- tally excluded. These molecules are inca- tion. pable of penetrating the pores and elute Equivalent conductivity: The equiva- at the interstitial (interparticle) volume lent conductivity is a function of the of the column. concentration and the limiting conduc- Exponentially modified Gaussian tivity (equivalent conductivity at infinite peak: A theoretical model for peak dilution). asymmetry arising from the column, Evaporation: The process of removing inlet, and detector. The basis for the a volatile compound for the purposes of Foley-Dorsey equations. www.chromatographyonline.com Terminology Guide October 2013 31
External calibration: Calibration mode = 1.5–5 µm). Separation times in the using one or more standard solutions to range of minutes, sometimes seconds, establish the calibration function (cali- are common; sometimes referred to as bration curve). With this function the ultrafast LC. concentration is calculated out of the Fast protein liquid chromatography measured peak area (or height) of the (FPLC): A term coined to cover the spe- sample chromatogram. Linear or qua- cific use of HPLC for the separation of dratic regressions are mainly applied but proteins. Generally, glass columns, mod- other regression modes are possible as erate pressure, and spherical microbeads well (for example, polynomial, point- are used for FPLC. to-point). FFAP: Free fatty-acid phase. Extracolumn effects: The total band- Fiberglass disks: A format where SPE broadening effects of all parts of the particles are embedded in a fiberglass chromatographic system outside of matrix; the disk format is especially the column itself. Extracolumn effects useful for processing large volumes of must be minimized to maintain the ef- sample (for example, water) given that ficiency of the column. Areas of band the larger cross-sectional area allows for broadening can include the injector, a higher flow rate than can be used for injection volume, connecting tubing, a typical cartridge. endfittings, frits, detector cell volume, Filter funnel: A filter funnel is a and internal detector tubing. The vari- laboratory funnel used for separating ances of all of these contributions are solids from liquids via the laboratory additive. process of filtering. To achieve this, Extracolumn volume: The volume be- a disk-shaped piece of filter paper is tween the effective injection point and usually folded into a cone and placed the effective detection point, excluding within the funnel. The suspension the part of the column containing the of solid and liquid is then poured stationary phase. It is composed of the through the funnel. The solid par- volumes of the injector, connecting lines ticles are too large to pass through and frits, and the detector; it determines the filter paper and are left on the the extra column effects. See dead vol- paper, while the much smaller liquid ume. molecules pass through the paper to Extraction: The general term for remov- a vessel positioned below the funnel, ing analytes of interest from a matrix. producing a filtrate. The filter paper is used only once. If only the liquid is of F interest, the paper is discarded; if the FAME: Fatty acid methyl ester. suspension is of interest, both the solid Fast GC: Gas chromatography per- residue on the paper and the filtrate formed on short, narrow-bore open- are kept for further analysis. tubular columns, or on conventional Filter holder: Membrane filters or columns at elevated linear velocities. membrane disks are sometimes fur- Fast LC: The use in HPLC of short nished loose and can be placed in a columns (1.5–7 cm in length) with holder (usually of stainless steel con- conventional internal diameters ( 2–6 struction) for processing samples; after mm) packed with small particles (dp the filter is used the holder is opened 32 Terminology Guide October 2013 www.chromatographyonline.com and the used filter is replaced with a heteroatomic solutes in a hydrogen-air fresh one. flame. The visible-range atomic emis- Filter porosity: Pore size relates to the sion spectrum is filtered through an filter’s ability to filter out particles of interference filter and detected with a a certain size. For example, a 0.5-µm photomultiplier tube. Different inter- membrane will filter out particles with ference filters may be selected for sul- a diameter of 0.5 µm or larger from a fur, tin, or phosphorus emission lines. filtration stream. Filter porosity is typi- FPD is a sensitive and selective detec- cally not related to, nor controlled by, tion method. pore size. These two parameters are es- Flash chromatography: A very fast sentially independent. Porosity is also form of classical liquid chromatography unrelated to thickness. Rather, it is a used by synthetic organic chemists to ef- function of the polymer and casting fect rapid purification. Done primarily process used in the manufacture of the in the normal-phase mode, sometimes filter. with reversed-phase LC. See column Filter vial: A filter vial is a membrane chromatography. filter unit that consists of two pieces. Flexible well plate: See array 96-well One is the filtration plunger, which con- plate. tains a membrane filter suitable for the Flow injection extraction: An on-line solvent being filtered. The second part extraction technique where a sample is of the filter vial is the vial body itself; injected as a plug into an aqueous flow once the sample is loaded and filtered, stream that is divided into small seg- the filter vial can be placed directly in ments by an organic phase; the aqueous the autosampler without transferring the and organic segments are mixed during filtered sample to another vial. their passage down a coil, and eventu- Filtration: The process of passing a liq- ally the phases are separated at the end uid through a paper, membrane, glass, by a special fitting and the amount of or other type of filter for the purposes of extract compound can be measured in removing particulates that could cause the organic phase. problems downstream during a chro- Flow programming: Used to decrease matographic analysis; a chemical filter the retention time of slow-moving com- also removes certain chemical species. pounds. Flow programming is occasion- See chemical filtration. ally used in concert with temperature Fixed-well plate: A 96-well plate with programming in GC. fixed (nonremovable) wells, not an array. Flow rate (F, Fc): The volumetric rate of See 96-well plate and array 96-well flow of mobile phase through a column. plate. For a conventional 4.6-mm i.d. HPLC Flame ionization detection (FID): A column, typical flow rates are 1–2 mL/ flame ionization detector ionizes hydro- min. GC packed column flow rates carbon-containing solutes in a hydro- typically range from 10 to 40 mL/min, gen–air flame. FID is a nearly universal and open-tubular column flows range detection method that responds strongly from less than 1 mL/min up to about to most classes of organic compounds. 10 mL/min. Flame-photometric detection (FPD): Flow resistance parameter (Φ): Φ = 2 A flame-photometric detector burns dp /Bo See permeability. www.chromatographyonline.com Terminology Guide October 2013 33
Fluoro phase: One of a family of Freeze drying: The process of removing aliphatic and aromatic reversed-phase water, mainly from biological samples, materials in which a substantial frac- by using vacuum sublimation. tion of the bonded phase is fluorinated. Frictional heating: Viscous heating of Sometimes called fluorous phases or solvent molecules passing through very perfluoro phases. Typically have dif- small-diameter (micrometer) particles; ferent selectivities than hydrocarbon causes a rise in temperature over the phases. length of the column; reduced by using Fluted filter paper: Filter paper that smaller-diameter columns (smaller heat is folded in a systematic manner to dissipation) and superficially porous allow more air space in the filter fun- particles, which also have improved heat nel; this allows liquid to flow faster dissipation. through the filter paper. Frit: The porous element at either end Foley-Dorsey equation: A correc- of a column that serves to contain the tion of the plate count and retention column or SPE packing. It is placed at time to correct for peak tailing from the very ends of the column tube or in extracolumn sources of broadening. the end fitting. Frits can be stainless (See J.P. Foley and J.G. Dorsey, Anal. steel or another inert metal or plastic Chem. 55, 730–737 [1983].) such as porous PTFE or polypropylene. Forced-flow leaching: Solid mate- The frit porosity must be less than the rial is packed into stainless steel col- smallest particle in the column; other- umn and toluene (or other extraction wise particles will pass through the frit solvent) is pumped into the column and packing will be lost. under pressure and with heating; hot Frontal analysis: A chromatographic solvent leaches (extracts) out extract- technique that involves continuous ad- able compounds which are collected at dition of sample to the column with the exit of the column. the result that only the least sorbed Fraction of analyte extraction (E): compound, which moves at the fast- The fraction of analyte extracted: E est rate, is obtained in a pure state.
= (CoVo)/(CoVo + CaqVaq) = (KDV )/(1 + The second-least-sorbed compound KDV ) where Vo is the volume of or- is eluted with first eluted compound, ganic phase, Vaq the volume of aque- the third-least-sorbed compound with ous phase, and V is the phase ratio the first and second compound, and so
Vo/Vaq. forth until the original sample passes Fractionation range: In SEC, refers through the column exit. Frontal to the operating range of a gel or pack- analysis is seldom used and is mainly ing. This is the range in which the a preparative technique. packing can separate molecules based Frontal chromatography: The same as on their size. At one end of the range, frontal analysis. molecules that are too large to diffuse Fronting: Peak shape in which the front into the pores are excluded. At the part of the peak (before the apex) in a other end of the range, molecules that chromatogram tapers in advance of the can diffuse into all of the pores totally remainder of the peak (that is, the front permeate the packing are eluted (un- is less steep than the rear). There is an separated) at the permeation volume. asymmetric distribution with a leading 34 Terminology Guide October 2013 www.chromatographyonline.com edge. The asymmetry factor for a front- Gaussian peak: The equation of a ing peak has a value less than 1. The Gaussian distribution can be written in opposite effect is tailing. Fronting can terms of chromatography parameters as 2 2 result at high sample loads because of C = Cmaxexp(–(t – tR ) /2σ ) positive curvature in the isotherm and Gel: The solid packing used in gel from use of poorly packed columns. See chromatography or GPC. An actual gel asymmetry factor. consists of two parts: the dispersed me- Fused-core packing: See superficially dium (solid portion) and the dispersing porous particles (SPPs). medium (the solvent). Also defined as Fused silica: Open-tubular GC col- a colloidal dispersion of a solid and liq- umns and LC nanocolumns are com- uid in which the solid is the continuous monly manufactured from fused-silica phase. tubing that is coated externally with a Gel filtration chromatography protective polymeric material such as (GFC): Also called aqueous size-ex- polyimide. clusion chromatography; carried out Fused-silica open-tubular column with aqueous mobile phases. Gener- (FSOT): Open-tubular GC columns ally refers to molecular size separa- made of fused silica. See open-tubular tions performance on soft gels such as column. polydextrans but highly crosslinked polymers, silica gels, and other po- G rous media may also be used. Most Gas–liquid (phase) chromatography gel filtration separations involve bio- (GLC, GLPC): Solutes partition between polymers and water-soluble polymers a gaseous mobile phase and a liquid such as polyacrylic acid. stationary phase. Selective interactions Gel permeation chromatography between the solutes and the liquid phase (GPC): Size-exclusion chromatography give rise to different retention times in (SEC) carried out with organic mobile the column. phases used for the separation and Gas-phase extraction: See direct ther- characterization of polymers. SEC with mal sampling. aqueous mobile phases is referred to as Gas–solid chromatography (GSC): Sol- gel filtration chromatography. utes partition between a mobile gaseous Ghost peaks: Peaks not present in the phase and a solid stationary phase. Se- original sample. Ghost peaks can be lective interactions between the solutes caused by septum bleed, solute decom- and the solid phase give rise to different position, or carrier-gas contamination. retention times in the column. Gigapores: See perfusion chromatog- Gaussian curve: A standard error curve, raphy. based on a mathematical function, that Golay equation: M.J.E Golay formu- is a symmetrical, bell-shaped band or lated an equation for the theoretical peak. Most chromatographic theory plate height versus the average linear assumes a Gaussian peak. Use of the velocity of open-tubular (capillary) col- peak maximum position as a measure umns. The Golay equation is similar to of retention and equations mentioned the van Deemter equation, except that above for efficiency assume a Gaussian the A term is dropped because there is peak shape. no column packing, and the B and C www.chromatographyonline.com Terminology Guide October 2013 35 terms are modified accordingly as well: heat treatment; this packing material – – h = (B/u) + u (CM + CS) has a strong affinity for polar com- Grab sampling: In gas sampling, an pounds in aqueous samples and water evacuated sample canister can be miscible organic extracts. Commonly opened and sample rapidly collected used in pesticide analysis of food sam- at an uncontrolled rate, usually over ples; also known as graphitized carbon several seconds, until the container black (GCB). Also used as a GC station- reaches equilibrium with atmospheric ary phase. pressure. Generally this qualitative ap- Graphitized carbon packing: A re- proach is used when unknown analytes versed-phase packing material which must be identified, when the air con- consists of pure graphitic carbon; tains high concentrations of analytes possesses interesting sorbent proper- at certain (short) times, or when an ties such as preferential separation of odor is noticed and a sample must be geometric isomers such as o-, m- and obtained quickly. Paired grab samples p-aromatics and cis–trans isomers. (before–after or smell–no smell) often Grinding: Both manual and automated are used to qualitatively diagnose a per- mortar and pestles are available; grind- ceived problem. ing can be performed under wet or dry Gradient: A process to change solvent conditions; by this process particles of strength as a function of time (normally approximately 10 µm can be achieved. solvent strength increases) thereby elut- Guard column: A small column placed ing progressively more highly retained between the injector and the analyti- analytes; typically gradients can be bi- cal column. It protects the analytical nary, ternary, and quaternary solvent column against contamination by sam- mixtures where solvents are blended ple particulates and strongly retained to achieve the proper strength. In GC, species. The guard column is usually an older term that refers to column packed with the same material as is in temperature programming to achieve the analytical column and is often of a similar effect. the same inner diameter. Its length is Gradient delay volume: See dwell much shorter, it costs less, and is usu- volume. ally discarded when it becomes con- Gradient elution: Technique for de- taminated. Integrated guard–analytical creasing separation time by increasing column systems are often preferred to the mobile-phase strength over time minimize extracolumn effects caused during the chromatographic separa- by the use of connecting tubing with tion. Also known as solvent program- separate guard and analytical columns. ming. Gradients can be continuous or For GC, see also retention gap. stepwise. Binary, ternary, and quater- nary solvent gradients have been used H routinely in HPLC. Headspace sampling: Gas-phase sam- Graphitized carbon: Graphitized car- pling technique in which solute is re- bon is a graphitic carbon with more moved from an enclosed space above a or less perfect three-dimensional hex- solid or liquid sample. The headspace agonal crystalline order prepared from refers to the vapors that form above non-graphitic carbon by graphitization liquids and solids; if the sample is in 36 Terminology Guide October 2013 www.chromatographyonline.com thermodynamic equilibrium with the that ionizes solute molecules, which are gas phase in a closed thermostated measured with an electrometer. Meta- vessel, the method is called static stable He may also be harnessed in an headspace sampling; if an inert gas electron-capture mode for halogen- passes over or through the sample and specific response. See also pulsed dis- stripped sample volatiles accumulate charge detection. in an adsorbent or cryogenic trap, the Helium sparging: See degassing. He- method is dynamic headspace or purge- lium has a very low solubility in most and-trap sampling. common liquids. Headspace single-drop microextrac- High performance capillary electro- tion (HS-SDME): A single drop of sol- phoresis (HPCE): A technique where vent (1–2 µL) suspended in the head- small diameter capillaries, buffered space can partition volatile analytes conducting solutions, and high voltage into the solvent; the drop can be with- (up to 30,000 V) are used to separate drawn into the syringe and injected ionic molecules based on their differ- into a GC system. ential electrophoretic mobilities; non- Headspace solid-phase microextrac- ionic (neutral) molecules can be sepa- tion (HS-SPME): Instead of a drop of rated by MEKC. solvent, a polymer-coated fiber can be High performance liquid chromatog- placed in the headspace and once the raphy (HPLC): Modern liquid-phase analytes adsorb on the polymer coat, chromatography technique using small the fiber can be transferred to a GC particles and high pressures. inlet and the sorbed analytes volatilized High-pressure mixing: A configura- by thermal desorption. tion of a gradient HPLC system where Heart cutting: In preparative LC, re- the solvents are mixed on the high- fers to collection of the center of the pressure side of multiple pumps (usu- peak where purity should be maximum. ally two, binary); such a system offers The term is also used for analytical col- a lower gradient delay volume than umn switching for LC or GC in which low pressure mixing systems where the two or more partially resolved peaks solvents are mixed by proportioning eluted from one column are directed valves before a single pump. onto another column of different po- High-abundance protein depletion: larity, or at a different temperature, for By using antibody columns specific improved resolution. for the highest abundance proteins, Helium ionization detection (HID): they can be selectively removed from An ionization detection method for plasma. This process enables investiga- GC. Helium is ionized by a radioac- tion of the lower abundance proteins, tive or other energetic source; the re- which may be biomarkers or other com- sulting He2+ ions interact with solutes pounds of interest. to produce ions that measured with a Hold-up time (tM, t0): The time re- sensitive electrometer. quired for an unretained compound Helium plasma or microwave-in- to be eluted, or the time required for duced detection (HPD): A helium one column volume (VM) of mobile plasma is created in a microwave RF phase to pass through the column. In field. The plasma emits UV radiation reversed-phase LC, uracil is often used www.chromatographyonline.com Terminology Guide October 2013 37 to measure hold-up volume and hold- Homogenization: The process of mak- up times. In GC, methane or another ing a sample more uniform by size re- unretained compound is used to mea- duction and blending; homogenizers sured the column hold-up time. Also with high speed blades are available to called the unretained peak time or dead do the job. time. Hybrid silica: Silica gel consisting of
Hold-up volume (VM or V0): The both organic and inorganic moieties total volume of mobile phase in the with hybrid properties of polymeric column regardless of where it exists. packings and silica packings; synthe- In LC: The hold-up volume consists sized from silanes containing organic of the entire space accessible to a functionality; different selectivity but small molecule able to fully permeate higher high pH stability than bare or all the pores of the packing material. uncoated silica gel. It comprises the interstitial volume Hydrodynamic volume: The molecu- and the unoccupied pore volume. It lar volume defined by the effective di- is denoted as Vo or VM. The system ameter of a molecule in free solution hold-up volume includes the ad- where the hydrodynamic sphere would ditional volume in the tubing used be a sphere defined by the molecule to connect the injector and detector as it revolves around its central axis in to the column. The hold-up volume solution; termed used in size-exclusion is usually approximated by inject- chromatography to define molecular ing a small essentially unretained shape and to explain why molecules species. In reversed-phase LC, ura- with the same molecular weight often cil, acetone, and thiourea are most have totally different elution volumes; commonly used: VM = tMFc. In GC: measured by determining the Stokes The gas-phase volume of the column radius. that corresponds to the hold-up time. Hydrophilic: “Water loving”; refers Measured by injecting an unretained both to stationary phases that are fully species such as methane that fits in compatible with water or to water-sol- all the pores. See hold-up time. uble molecules in general. Many col- 0 Hold-up volume, corrected (VM): The umns used to separate proteins (ion ex- hold-up volume corrected for carrier- change, SEC, affinity) are hydrophilic 0 gas expansion along the column: VM in nature and should not irreversibly = V j sorb or denature protein in the aque- Hollow-fiber liquid-phase microex- ous environment. traction (HF-LPME): A hollow-fiber Hydrophilic interaction liquid chro- (HF) membrane technique where an matography (HILIC): An alternative HF membrane separates two extrac- technique to reversed-phase HPLC tion phases; the membrane serves as a for the separation of highly polar barrier and can be impregnated with analytes that may be only slightly re- solvent to permit liquid–liquid or liq- tained or unretained by reversed-phase uid–liquid–liquid extractions to take LC, HILIC requires a high percent- place; the membrane can be selected to age of a nonpolar mobile phase and allow certain analytes to pass through a polar stationary phase, similar to but not others. the requirements in normal-phase 38 Terminology Guide October 2013 www.chromatographyonline.com chromatography. However, unlike chromatography; usually little or no normal-phase chromatography, which organic solvent is used in the mobile uses nonpolar solvents such as hexane phase in HIC. and methylene chloride and tries to Hydrophobic subtraction model: exclude water from the mobile phase, Developed by Lloyd Snyder and John HILIC requires some water in the Dolan, this model is used to charac- mobile phase to maintain a stagnant terize reversed-phase columns; using enriched water layer on the surface five types of probes, based on their into which analytes may selectively equations, they can predict if a cer- partition. In addition, water-miscible tain column will be close in selectiv- organic solvents are used instead of ity characteristics to another column the water-immiscible solvents used in or totally different (orthogonal); over normal-phase chromatography. With 300 columns have been characterized HILIC, sorbents such as bare silica, using this model. (See L. Snyder and bonded diol, and polyhydroxyethylas- J. Dolan, LCGC 20[11], 1016–1026 partamide are used. Polar analytes are [2002].) well retained and are eluted in order Hydroxyapatite: A porous calcium of increasing hydrophilicity, just the hydroxyphosphate solid that chemi- inverse of reversed-phase LC. cally resembles bone and tooth used Hydrophobic: “Water hating”; re- as a packing material used in bio- fers both to stationary phases that chromatography for nucleic acid con- are not compatible with water or to stituents, monoclonal antibodies, and molecules in general that have little proteins. affinity for water. Hydrophobic Hypercrosslinking: Mainly refers to molecules have few polar functional a new way to synthesize a polymeric groups; most have a high content of monolith; hypercrosslinked monolithic hydrocarbon (aliphatic and aromatic) capillary columns contain an array of functionality. small pores and have very high surface Hydrophobic interaction chroma- areas. tography (HIC): A technique in which Hyphenated techniques: Refers to weakly polar (nonhydrocarboneous) the family of techniques best known packings are used to separate molecules by their abbreviations, including LC– by virtue of the interactions of their hy- MS, LC–FTIR, and MS-MS. drophobic moieties and the hydropho- bic sites on the packing surface. High I concentrations of salt solutions are used Immobilized liquid extraction: Simi- in the mobile phases and separations lar to SPE but a polymeric stationary are affected by changing the salt con- phase is bonded to the inside of a glass centration. The technique is analogous vial; analytes partition into polymeric to “salting out” molecules from solu- phase and loading, washing, and elu- tion. Gradients are run by decreasing tion steps are performed by addition of the salt concentration; often used for various solvents. the separation of proteins that are sen- Immobilized metal affinity chroma- sitive to denaturization by the organic tography (IMAC): See metal affinity solvents used in regular reversed-phase chromatography. www.chromatographyonline.com Terminology Guide October 2013 39
Impinger: Impingers are glass bubble analytes; a UV-absorbing or fluores- tubes designed for the collection of cent compound is added to the mo- airborne hazards into a liquid medium. bile phase that maintains a high back- When using a personal air sampler, a ground signal; when a nonabsorbing known volume of air bubbles is pumped or nonfluorescing analyte is eluted, through the glass tube that contains a the background is diluted and a nega- liquid specified in the method. The liq- tive peak is observed for that analyte; uid is then analyzed to determine air- when an analyte acts to increase the borne concentrations. An impinger may concentration of the indicating species, be mounted on the side of an air sample a positive peak is observed. When a pump or put into a holster and placed negative signal is detected, the detec- near a worker’s breathing zone. tor signals are reversed to the output Imprinted phases: Polymer and silica device. phases generated in the presence of a Infinite diameter column effect “template” or “printing” molecule. Such (IDE): Name given by John Knox to phases have enhanced selectivity for the the following phenomenon: At a cer- templating molecule; also called molec- tain column length, a sample injected ularly imprinted phases (MIPs). into center of a packed bed spreads by In-line filter: A device that prevents radial diffusion but never reaches col- particulate matter from damaging the umn wall, where wall effects can cause column. Modern low-volume, in-line band broadening. Knox showed that filters can be placed between the injec- a sample peak collected in the exact tor and the column without major con- center of the column exit displayed a tributions to band broadening. A filter higher efficiency than a sample peak in this position is used to prevent sample collected near the wall. The infinite particles from entering the packed bed diameter effect depends on column or column inlet frit. length, internal diameter, particle size, In situ derivatization: The act of de- and mobile-phase properties. Very sel- rivatizing a compound of interest in dom applied in HPLC. its native environment; for example, Injection solvent: Solvent used to inject for a tenaciously held analyte on a soil sample into an HPLC column; solvent sample changing its chemical nature should be of equal or lower strength by performing an in situ derivatization, than the mobile phase to prevent pre- the compound may be more easily re- mature movement down the column leased and isolated for further workup due to the presence of a stronger solvent. or analysis; derivatization may also be Inlet: In LC, the initial part of the col- formed in solution and the derivatized umn where the solvent and sample enter. compound extracted by LLE. There is usually an inlet frit that holds Included volume: Also known as totally the packing in place and, in some cases, included volume. The volume at which protects the packed bed. In GC, a de- a small molecule that explores the en- vice between the carrier gas source and tire pore space of a column is eluted. See the column inlet that transfers sample steric-exclusion chromatography. from outside the chromatograph into Indirect detection: Used for non-ul- the column, often by vaporizing the traviolet-absorbing or nonfluorescing sample. See split injection, splitless 40 Terminology Guide October 2013 www.chromatographyonline.com injection, on-column injection, pro- stance being quantitatively determined grammed temperature vaporizer. and should have a retention time fairly Inlet filter: Filtration devices attached close to it. to the inlet lines of the pump that re- Interparticle porosity (ε): The inter- moves particulate matter from the mo- particle volume of a packed column per bile phase before the solvent reaches the unit column volume: ε = Vo/Vc. See also pump; reservoir filters are an inlet filter interstitial porosity. that resides in the solvent bottle. Interstitial porosity (εe): The fraction of Inlet liner: Deactivated glass tube in an the volume in the column located in the inlet system into which liquid sample is interparticle (interstitial) space: εe = Ve/Vc injected. An inlet liner may be open or Interstitial volume (Ve): The volume packed with deactivated glass wool, and between the particles. Does not include it may have various internal structures. the volume in the pores of the particles. The purpose is to vaporize and disperse Also called the excluded volume (see evaporating sample into the carrier gas steric-exclusion chromatography) stream as uniformly as possible while and interparticle volume. Measured by not causing significant sample break- injecting a molecule which does not per- down, adsorption, or discrimination. meate any pores and does not interact Inlet/outlet check valves: The check with the surface of the particles. In SEC valve (or valves) on an LC pump that this volume is denoted Vo. allows mobile phase to flow in one di- Intraparticle porosity (εi): The fraction rection but not in the reverse direction. of the particle volume which lies in the
The inlet check valve allows flow from pores: εi = Vpore/Vparticle the reservoir into the pump, and the Intraparticle volume (V): The volume outlet check valve allows mobile phase inside the pores of the particles. Also to flow to the column from the pump. called the internal volume and included Instrumental bandwidth: The contri- volume. Can be measured by BET or bution of the analytical instrument to mercury intrusion porosimetry. peak broadening; see extracolumn ef- Ion chromatography (IC): An ion- fects for explanation. exchange technique in which low con- Instrumental dispersion: See extracol- centrations of organic and inorganic umn effects. anions or cations are determined using Internal standard (IS): In quantitative ion exchangers of low ion-exchange ca- analysis, precision and accuracy are pacity with dilute buffers. Conductivity greatly improved by use of internal stan- detectors are often used. Ion chroma- dards (IS). The procedure involves the tography is practiced in two forms. In addition of a fixed amount of internal suppressed IC, a second column or a standard to a series of increasing con- membrane separator is used to simulta- centrations of reference sample and to neously remove the buffer counterion to the unknown concentration. The ratio the analyte and replace it with hydrogen of the areas of the reference concentra- or hydroxide ion, which concomitantly tions and the areas of the IS is plotted converts the buffer to an uncharged spe- against the known concentration of the cies, thereby supressing background and reference samples. The internal standard enhancing sensitivity. In nonsuppressed should be chemically similar to the sub- IC, weakly conducting buffers at low www.chromatographyonline.com Terminology Guide October 2013 41 concentration are carefully selected and take place in the exchange process. The the entire effluent is passed through the exchange capacity is expressed in mil- detector — ions are detected above the lieqivalents per gram. A typical styrene- background signal. divinylbenzene strong anion exchange Ion exclusion: The process in which resin may have 3–5 meq/gm capacity. ionized solutes can be separated from Exchangers for IC have very low capac- unionized or partially ionized solutes ity. Capacity of weak anion and cation using ion-exchange resins. Separation exchangers varies dramatically with pH. results from Donnan potential where Ion-exchange chromatography: A ionic solutes exist at a higher concen- mode of chromatography in which ionic tration in solution than in the station- substances are separated on cationic or ary phase whereas nonionic solutes are anionic sites of the packing. The sample evenly distributed between the mobile ion (and usually a counterion) will ex- phase and resin. Therefore, ionic sol- change with ions already on the iono- utes will move faster down the column genic group of the packing. Retention is than non-ionic solutes. Ion exclusion is based on the affinity of different ions for known to take place in reversed-phase the site and a number of other solution LC when anions are separated at pH val- parameters (pH, ionic strength, coun- ues where the silanol groups are ionized. terion type, and so forth). Ion chroma- Ion moderated partitioning chroma- tography is basically an ion-exchange tography: A technique used for the technique. separation of carbohydrates using strong Ion-pair chromatography: Form of cation-exchange packings that are in chromatography in which ions in solu- specific cationic form (for example, cal- tion can be “paired” or neutralized and cium, hydrogen, silver); the separation separated as an ion pair on a reversed- mechanism is complexation rather than phase column. Ion-pairing agents are ion exchange. usually ionic compounds that contain Ion retardation: Refers to the use of a hydrocarbon chain that imparts a cer- amphoteric ion-exchange resins, which tain hydrophobicity so that the ion pair are used to retard ionic molecules and can be retained on a reversed-phase col- allow nonionic molecules or nonelectro- umn. Retention is proportional to the lytes to be eluted preferentially. length of the hydrophobic chain and the Ion suppression: Buffering in an aque- concentration of the ion-pair additive. ous mobile phase at a particular pH to Ion pairing can also occur in normal- suppress solute ionization. For example, phase chromatography when one part weak carboxylic acids can have their ion- of the pair is dynamically loaded onto a ization suppressed by the adjustment of sorbent, but this technique is not as pop- the pH below their pKa. Useful for im- ular as reversed-phase LC. Also known proving peak shape of weak acids and as ion-interaction chromatography or bases in reversed-phase LC. dynamic ion-exchange chromatogra- Ion-trap detector: Mass spectrometric phy, stressing the fact that the precise detector that uses an ion-trap device to mechanistic details of how the additive generate mass spectra. controls retention are not always known. Ion-exchange capacity: The number Ionic strength: Ionic strength is a of ionic sites on the packing that can characteristic of an electrolyte solution. 42 Terminology Guide October 2013 www.chromatographyonline.com
It is typically expressed as the average Isotope-coded affinity tags (ICAT): electrostatic interactions among an Isotope-coded affinity tags (ICATs) electrolyte’s ions. It is related to elec- are a gel-free method for quantitative trolyte concentration, but the main proteomics that relies on chemical la- difference between ionic strength beling reagents. These chemical probes and electrolyte concentration is that consist of three general elements: a reac- the ionic strength is higher if some tive group capable of labeling a defined of the ions are more highly charged. amino acid side chain (for example, The higher the ionic strength of a mo- iodoacetamide to modify cysteine resi- bile phase the more the mobile phase dues), an isotopically coded linker, and competes with the analyte for ionic or a tag (for example, biotin) for the af- adsorptive sites. finity isolation of labeled proteins and Irregular packing: Refers to the shape peptides. For the quantitative compari- of a column packing. These irregular son of two proteomes, one sample is packings are available in micropartic- labeled with the isotopically light (d0) ulate sizes. The packings are obtained probe and the other with the isotopi- from grinding solid materials into cally heavy (d8) version. To minimize small particles and then sizing them error, both samples are then combined, into narrow fractions using classifica- digested with a protease (such as tryp- tion machinery. Spherical packings are sin), and subjected to avidin affinity now used more than irregular pack- chromatography to isolate peptides ings in analytical HPLC but the less- labeled with isotope-coded tagging expensive irregular packings are still reagents. These peptides are then ana- widely used in preparative LC. lyzed by liquid chromatography-mass Irreversible adsorption: When a spectrometry (LC–MS). The ratios of compound that has a very strong af- signal intensities of differentially mass- finity for an adsorbent is injected tagged peptide pairs are quantified to onto a column, it can be adsorbed so determine the relative levels of proteins strongly that it cannot be eluted from in the two samples. The original tags the column. A chemical reaction be- were developed using deuterium, but tween the sample and the surface of later the same group redesigned the tags the adsorbent is an example of irre- using 13C instead to circumvent issues versible adsorption. of peak separation during LC because Isocratic: Use of a time-invariant eluent of the deuterium interacting with the composition in LC. stationary phase of the column. Isolate: Analyte to be isolated from ma- trix background and then analyzed. K Isotherm: See adsorption isotherm. Kieselguhr: A diatomaceous earth used Isothermal chromatography: Use of both in column chromatography and as conditions of constant column tem- a sample cleanup media. Only weakly perature. The vast preponderance of adsorptive, it is also used as a support all LC is done under isothermal condi- in liquid–liquid chromatography and tions, while most GC separations are in supported liquid extraction, see sup- performed with column temperature ported liquid extraction (SLE). Rarely programming. used in HPLC. www.chromatographyonline.com Terminology Guide October 2013 43
Kilopascal (kPa): A unit of pressure. normal volumes of sample in a solvent 100.0 kPa = 1.0 bar = 0.9869 atm. into a capillary GC column; in this 101.325 kPa = 1.0 atm. approach, the bulk of the solvent is Kinetic plot: Kinetic plots are methods evaporated before the sample transfers to characterize the practical limits of to the analytical column; there are two column performance, where theoretical popular LVI techniques: programmed plates (H) and separation impedance (E) temperature vaporization and cool on- are plotted as a function of the pressure- column injection with solvent vapor exit; drop limited plate number (N). The ki- both are approaches to lowering detec- netic plot retains the information shown tion limits. in van Deemter plots but completes it Ligand: In ligand-exchange chromatog- with the information on the bed perme- raphy, refers to the analyte that under- ability. See Poppe plot. goes ligand exchange with the stationary Knox equation: A modification of the phase. In affinity chromatography, re- van Deemter equation developed by fers to the biospecific material (enzyme, John Knox that uses reduced terms, in antigen, or hormone) coupled to the which the A term that represents eddy support (carrier) to form the affinity col- dispersion is replaced with Aν1⁄3 where ν umn. In bonded-phase chromatography is the reduced interstitial eluent velocity. this term denotes the moiety covalently bound to the surface. L Ligand-exchange chromatography: Laminar flow: The smooth time-in- A technique in which chelating li- variant flow that develops when a liq- gands are added to the mobile phase uid is moving under conditions where and undergo sorption onto a pack- viscous forces dominant over inertial ing. These sorbed molecules can act forces. Laminar flow is characterized as chelating agents with certain sol- by a low Reynolds number. For flow utes. An example would be the use of in a cylindrical tube fluid stream-lines copper salt added to the mobile phase near the center move faster than those for the chelatation and separation of at the tube wall which are static result- amino acids. Chelating resins function ing in a radially parabolic distribution in in a similar manner, where chelating axial fluid velocity. This nonuniformity groups are chemically bonded to the of axial velocities in the interstices in a polystyrene backbone. packed bed also leads to substantial peak Limit of detection (LOD): The concen- broadening in packed columns. tration of the analyte at which the re- Langmuir adsorption isotherm: A spe- sulting peak can be distinguished from cific form of an isotherm CS = (N0CM)/ baseline noise. Literature and norms (Kd + CM) where CS and CM are the describe different ways of determining equilibrium stationary and mobile the LOD. Limit of quantitation (LOQ): phase concentrations of the solute, N0 The the total number of surface sites avail- minimum concentration of the analyte able for sorption and Kd the sorption at which the resulting peak can be quan- binding constant. tified with a defined level of certainty. Large-volume injection (LVI): A tech- Typically 3–5 times higher than the nique for introduction of larger than LOD. 44 Terminology Guide October 2013 www.chromatographyonline.com
Linear elution adsorption chromatog- will be found in the organic solvent; by raphy (LEAC): A term coined by Lloyd the use of additives (for example, buf- Snyder; refers to adsorption chromatog- fers or ion-pair reagents), equilibria can raphy carried out in the linear portion be shifted to “force” analytes or matrix of an adsorption isotherm; sometimes compounds into one or other of the two referred to as linear chromatography. layers. Linear range (LR): Also, linear dynamic Liquid-phase microextraction (LPME): range. The range of solute concentration A liquid extraction technique where or amount over which detector response there is a great reduction in the accep- per solute amount is constant within a tor-to-donor phase ratio; a hollow fiber specified percentage. is impregnated with an organic solvent Linearity: In quantitative analysis, it is used to accommodate or protect micro- essential for the detector to yield a linear volumes of acceptor solution. This novel response with respect to solute concen- methodology proved to be an extremely tration; some detectors may show non- simple, low-cost, and virtually solvent- linear performance in certain concentra- free sample-preparation technique that tion ranges, especially on the high end provided a high degree of selectivity and but also on the low end. enrichment by additionally eliminating Liquid chromatography: A separation the possibility of carryover between runs. technique in which the mobile phase Liquid–solid chromatography (LSC): is a liquid; most often carried out in a Same as adsorption chromatography. column. Liquid–solid extraction: The general Liquid phase: In GC, a stationary liq- expression for extraction techniques uid layer coated on the inner column that uses an organic solvent to extract wall (WCOT) or on a support (packed, analytes from a solid material. In its PLOT, SCOT) that selectively interacts simplest form, the “shake flask” extrac- with different solutes to produce differ- tion takes place at room temperature ent retention times. Also refers to the and works well for the case where the stationary phase in LLC. matrix is porous and the analytes are Liquid–liquid chromatography (LLC): easily extractable. Same as partition chromatography. Loadability: The maximum amount of One of the earliest separation modes analyte that can be injected onto a col- of HPLC; it gave way to chemically umn above which it no longer permits bonded phases in the early 1970s. the isolation of product at the desired Liquid–liquid diffusion coefficient level of purity or recovery level; impor-
(DL): See diffusion coefficient. tant in preparative chromatography. Liquid–liquid extraction (LLE): LLE Loading (phase loading versus sam- is an extraction technique for separat- ple loading): The amount of stationary ing interferences from the analytes by phase coated or bonded onto a solid sup- partitioning the analytes between two port. In liquid–liquid chromatography, immiscible liquids (or phases); one phase the mass of liquid phase per gram of is usually aqueous and the second phase packing. In bonded-phase chromatog- an organic solvent; more hydrophilic raphy, the loading may be expressed in compounds prefer the aqueous phase µmol/m2 or %C (w/w); also called cov- while more hydrophobic compounds erage or surface coverage. An alternate www.chromatographyonline.com Terminology Guide October 2013 45
(unrelated) meaning is the amount of the polymer matrix. Bioreactive mol- sample mass that is injected on an ana- ecules can be adsorbed or coupled to lytical or preparative column; prepara- their surface, and used to separate tive columns are often operated in an biological materials such as cells, pro- overloaded condition for throughput teins, or nucleic acids; by the use of reasons. magnets or magnetic fields, the beads Loading step (SPE): The second step can be easily manipulated in test tubes in SPE (after conditioning) where the or 96-well plates. These microbeads sample is loaded onto the SPE phase are used for isolation and handling of (cartridge). specific material or molecules, as well
Log KW: In reversed-phase LC, the ex- as for analyzing sensitive molecules or trapolated intercept of a plot of log k ver- those that are in low abundance (for sus volume fraction of organic modifier example, in miniaturized and auto- in reversed-phase LC. See also solvent mated settings). strength (S). Make-up gas: Extra carrier gas or Longitudinal diffusion: Same as mo- other gas added to the carrier gas as lecular diffusion term; B term in van it flows into or through a detector. Deemter equation; see van Deemter Make-up gas serves to improve peak equation. shapes for open-tubular columns with Low pressure mixing: See high pres- detectors not necessarily designed for sure mixing. them exclusively by reducing the ef- Lyophilization: The process of dehy- fects of detector dead volume. Also, drating a sample, often biological, con- make-up gas may play an active role taining water by using vacuum sublima- in detector operation, as for example tion; also referred to as freeze drying. when hydrogen serves as both make- up and combustion gas in a flame ion- M ization detector. Maceration: The process of breaking Mass spectrometric (MS) detector: down a soft material into smaller parts Chromatography detector that records by tearing, chopping, cutting, and so mass spectra of solutes as they are eluted forth. from the column. Macroporous resin (macroreticular): Mass transfer (inter-phase) (C term): Cross-linked ion-exchange resins that The process of solute movement be- have both micropores of molecular di- tween the moving and stationary zones. mensions but also macropores of sev- The C term of the van Deemter equa- eral hundred angstroms wide. These tion is referred to as the interphase are highly porous resins with large mass transfer term. The faster the internal surface area accessible to large process of mass transfer the better the molecules. efficiency of column. In HPLC, slow Magnetic-bead technology: Micro mass transfer is the most important fac- magnetic beads are uniform polymer tor affecting column efficiency. Its rate particles, typically 0.5–500 µm in di- can be increased by the use of small- ameter, that have iron oxide particles particle packings, thin layers of station- (or other particles that may be at- ary phase, low viscosity mobile phases, tracted to magnets) contained within and high temperatures. 46 Terminology Guide October 2013 www.chromatographyonline.com
Matrix: In sample preparation, the ma- pane, and pyridine are taken together trix normally refers to the substance in to express the overall phase polarity, which the analyst is attempting to mea- or separately to express the station- sure a solute or series of solutes; often the ary-phase behavior toward individual matrix is of no interest, and its concen- compound classes. tration must be reduced or eliminated Mean pore diameter: The average for a separation and measurement to pore diameter of the pore in a porous take place; the matrix can be organic, packing. It is most commonly deter- inorganic, or biological. mined by BET analysis and is reported Matrix adsorption mode (SPE): A lesser as four times the specific pore volume used mode of SPE where the sorbent is divided by the specific surface area chosen to maximize retention of the based on the assumption of uniform matrix and other interferences while the cylindrical pores. The pore diameter is analyte of interest is unretained; the op- important in that it must allow free dif- posite of the normal process (bind–elute) fusion of solute molecules into and out of SPE; there is no concentration of the of the pore so that the solute can inter- analytes. act with the stationary phase. Addition- Matrix solid phase dispersion ally, the pores must be well-connected, (MSPD): Technique uses bonded phase with a minimum of dead ends, so that solid supports as abrasives to produce there are many paths that can allow a disruption of sample architecture and molecule to access any part of the pore as a bound solvent to aid complete space. In SEC, the packings have dif- sample disruption during the sample ferent pore diameters and therefore blending process; the finely divided molecules of different sizes can be sample is gently blended with a mor- separated. For a typical substrate such tar and pestle, transferred to a column, as silica gel, 60- and 100-Å pore diam- and the analytes eluted with appropri- eters are most popular. For packings ate solvents. used for the separation of biomolecules, Matrix-solid phase extraction (MASE): pore diameters > 300 Å are used. See matrix solid phase dispersion. Megapascal (MPa): A unit of pressure; 1 Maximum allowable operating tem- MPa = 10 bar, 10.133 atm, or 145.0 psi. perature (MAOT): Highest continuous Megapores: See perfusion chromatog- column operating temperature that will raphy. not damage a column, if the carrier gas Membrane extraction with sorbent is free of oxygen and other contami- interface (MESI): A version of dy- nants. Slightly higher temperatures may namic headspace where a silicone hol- be permissible for short periods of time low fiber membrane is placed in the during column bake-out. headspace about the sample; an inert McReynolds constants: System for gas is passed through the membrane stationary-phase characterization. and analytes that are permeable to McReyonolds expanded on the ear- the membrane pass from the head- lier Rohrschneider polarity probes. space and are swept to an adsorbent The retention indices of a series of trap; after a period of concentration, test probes such as benzene, 1-buta- the trapped analytes are thermally de- nol, methyl-n-propylketone, nitropro- sorbed to the GC column. www.chromatographyonline.com Terminology Guide October 2013 47
Membrane filtration: Membrane filter; to the desired limits of precision and membrane disk. accuracy in retention, resolution, and Membrane suppressor: Continuous quantitation of the sample components chemical suppression. Ion exchange of interest. through ion-exchange membranes. H+ Micellar chromatography: The addi- or OH- are supplied by the respective tion of micelles to the mobile phase regeneration solution (for example, sul- to effect separations. The micelles furic acid). may act as displacing or partitioning Metal affinity chromatography: A agents and provide another parameter special form of ligand exchange chro- which may be used to change selec- matography used for the separation of tivity. Surfactants above their critical biopolymers with a particular affin- micelle concentration are used in mi- ity for a specific metal cation typically cellar chromatography and in MEKC copper(II), zinc(II), and iron(II). form of CE. Metalophile: A compound that has Micro LC: Refers collectively to tech- high affinity for active (acidic) silanol niques where a column of smaller than groups on silicas surface. Usually a conventional internal diameter is used strongly basic amine. for separation. The term micro LC is Method detection limit (MDL): The most often used for HPLC in <0.5-mm minimum amount of solute that can be i.d. columns; micro LC is used in high analyzed within specified statistical lim- sensitivity analysis when the sample its of precision and accuracy, including amount is limited and with certain ion- sample preparation. ization techniques in LC–MS where the Method development: A process of volume of solvent flowing into the ion- optimizing the separation including ization source must be minimized. the sample pretreatment so as to obtain Microbore: Refers to the use of col- a reproducible and robust separation. umns with smaller-than-usual internal Usually it emphasizes the search for the diameters in HPLC. Columns with stationary phase, eluent, and column internal diameters of 2 mm and below temperature combination that provides are considered to be microbore columns; an adequate separation. columns with internal diameters below Method translation: Several math- 0.5 mm are referred to as micro LC col- ematical techniques for adjusting GC umns. In GC, microbore may signify method parameters for variations in the columns with inner diameters less than carrier gas type or column dimensions, 200 µm. with the objective of maintaining either Microchip devices: Microdevices based the same or ratiometric retention times. on silicon, glass, and other types of mi- Useful when changing from helium to crofabricated chips where experiments hydrogen carrier gas, or when increas- can be miniaturized into single- or mul- ing speed of analysis or resolution by tichannel microfluidic circuits; these adjusting column dimensions. Not use- devices can be used for CE and CEC, ful if changing the stationary phase to a and should be low cost and disposable. chemically different type. Microchip-based GC devices have been Method validation: A process of test- available since approximately 1995. The ing a method to show that it performs use of these devices for separations is 48 Terminology Guide October 2013 www.chromatographyonline.com currently in its infancy, and applications Microporous resin: Same as microre- should expand with time. ticular resin. Microdialysis: Microdialysis is a mini- Microreticular resin: Cross-linked mally invasive sampling technique that synthetic ion-exchange resins that is used for continuous measurement of have pores with openings correspond- free, unbound analyte concentrations ing to molecular sizes. Diffusion into in the extracellular fluid of virtually the narrow pores can be impaired and any tissue. Analytes may include en- low exchange rates can occur, as well as dogenous molecules (for example, neu- poor performance, especially for large rotransmitters, hormones, and glucose) molecules. to assess their biochemical functions in Microwave-assisted extraction the body, or exogenous compounds (for (MAE): The use of microwave energy example, pharmaceuticals) to determine to heat samples in the presence of a their distribution within the body. The solvent allowing for rapid extraction; microdialysis technique requires the in- MAE can be performed in open ves- sertion of a small microdialysis catheter sels. A nonmicrowave-absorbing sol- (also referred to as microdialysis probe) vent is used and the sample contain- into the tissue of interest. After the ing a substance with a high dielectric probe is inserted into the tissue or (body) constant (for example, water) is rapidly fluid of interest, small solutes can cross heated, with the extracted analytes the semipermeable membrane by passive passing into the extraction solvent. A diffusion. The microdialysis probe is de- variation of this technique involves the signed to mimic a blood capillary and addition of an inert microwave-ab- consists of a shaft with a semipermeable sorbing solid substance that transfers hollow fiber membrane at its tip, which the heated energy to the surrounding is connected to inlet and outlet tubing. solvent. MAE can also take place in Microextraction: The general process closed vessels that are non-microwave- of liquid extraction using small amounts absorbing containers. of organic solvent where the phase ratio Migration time (tM): The time it takes Vo/Vaq is quite low; other techniques for a charged molecule to move from the using hollow microfibers as a barrier point of injection to the point of detec- are also referred to as microextraction. tion in a CE capillary. Microparticulate: Refers to the small Milling: Devices for reducing the par- particles used in HPLC. Generally ticle sizes of solid materials. Disk mills packings with a particle diameter of less pulverize <20-mm diameter hard sam- than 10 µm and that are totally porous ples by feeding between stationary and are considered microparticle packings. rotating disks with adjustable gap set- Micropipette tip: A form of SPE in tings; samples are generally reduced to which the packing material is embed- a 100-µm diameter; rotor speed mills ded or adsorbed on the inner walls combine impact and shearing processes of a pipette tip; useful for the SPE of to grind soft-to-medium hard and fi- very small amounts of liquid sample; brous materials to 80 µm; ball mills often used with xyz liquid handling grind material to submicrometer size systems for automation purposes. See by developing high-grinding energy via pipette tip. centrifugal or planetary actions using www.chromatographyonline.com Terminology Guide October 2013 49 agate, tungsten carbide, or PTFE- Mobile-phase modifier: Modifiers are coated stainless steel balls. materials (usually organic or inorganic Mincing: The process of breaking down compounds ) added to the mobile phase a meat or vegetable product into smaller to alter its elution properties. parts by tearing, chopping, cutting, dic- Mobile-phase strength: See solvent ing, and so forth. strength. Minimum detectable quantity (MDQ): Modifier: An additive that changes The amount of solute that produces a the character of the mobile phase. In signal twice the noise level. reversed-phase LC, for example, water Mixed-bed column: Combination of is the weak solvent and methanol, the two or more stationary phases in the strong solvent, is sometimes called the same column, used most often for ex- modifier; sometimes other additives change separations (IEC mixed anion such as competing bases like triethyl- and cation resins) and SEC (mixture of amine or ion pair reagents are referred to different pore size packings). The ad- as modifiers but they should more cor- vantage in IEC is the total removal of rectly be called additives. See additive. both cationic and anionic compounds; Molecular diffusion term (B term): the technique is useful in SEC because Refers to the B term (second term) of a wider molecular weight range can be the van Deemter and Golay equations. accommodated by the same column. Also called longitudinal or axial diffu- Mixed-mode separation: A separation sion term. It dominates band broaden- that takes place in a single column as ing only at very low flow rates below the a result of retention and selectivity pro- minimum plate height where the diffu- vided by a dual retention mechanism. sion of individual solutes can occur in a For example, at intermediate-to-high longitudinal (lengthwise) direction on pH values, a reversed-phase column the column. See van Deemter equation, with residual silanols can separate by Golay equation. hydrophobic interactions as well as Molecular sieve: GC column packing ionic interactions by virtue of the ion- that retains solute by combined mo- ized silanols; sometimes mixed-mode lecular size and adsorptive interactions. separations can be quite beneficial to Molecular sieves can separate light gases the selectivity (band spacing) but can and hydrocarbons. cause peak asymmetry; the precise bal- Molecular weight distribution: The ance of interactions may be difficult to distribution of the molecular weight of reproduce with subsequent batches of molecules in a polymer sample. Distri- packing. bution can be defined as weight average Mobile phase: The fluid that moves and number average. solutes through the column. In LC, the Molecularly imprinted phases (MIPs): mobile phase interacts with both the sol- See imprinted phases. ute and the stationary phase and there- Monolith: A monolithic HPLC col- fore can have a powerful influence on umn is a special type of column used the separation. In GC, the mobile phase, in HPLC with porous channels rather as an inert gas, has little interaction with than beads; monoliths, in chromato- stationary phase and analytes and serves graphic terms, are porous rod struc- to move the sample through the column. tures characterized by mesopores 50 Terminology Guide October 2013 www.chromatographyonline.com and macropores. These pores provide tryptic peptides to analyze an entire monoliths with high permeability, a proteome of a cell or tissue type pro- large number of channels, and a high tein extract. The approach uses a dual surface area available for reactivity. enzymatic digestion (Lys-C followed The backbone of a monolithic column by trypsin) to increase the number is composed of either an organic (poly- of peptides observed. The peptides meric) or inorganic (silica) substrate, are separated using strong cation ex- and the column can be chemically al- change and are identified by MS-MS tered for specific applications. detection. Monomeric phase: Refers to a bonded Multimodal SPE: The practice of SPE phase where single molecules are bonded where two different phases or modes are to a support. For silica gel, monomeric used to clean up a sample; the process phases are prepared by the reaction of an can consist of two separate cartridges alkyl- or arylmonochloro- or alkoxysi- placed in series with the analytes sepa- lane. Polymeric phases generally are pre- rated on the two different cartridges; a pared from a di- or trichlorosilane or an second process is where two different alkoxysilane reactant. phases are present in the same cartridge Moving zone: The moving zone is that or even on the same packing; sometimes fraction of the mobile phase in the col- referred to as mixed-mode SPE. umn that occupies the interstitial spaces. Multidimensional chromatography: N The use of two or more columns or Nano LC: LC practiced with columns chromatographic techniques to ef- that have internal diameters less than fect a better separation. It is useful for 100 µm; usually requires specialized in- sample cleanup, increased resolution, strumentation; often used in proteomic and increased throughput. Separa- studies where sample is limited and sen- tion is carried out with two or more sitivity is required. columns in which peaks are selectively Narrow-bore column: Columns of less directed onto or removed from at least than 2 mm i.d. used in HPLC, and less one of the columns by use of a timed than 320 µm i.d. in GC; also referred to valve system. In GC, a Deans fluidic as microbore. switch is often used. It also can be Nitrogen–phosphorus detection used off-line by collecting fractions (NPD): The nitrogen–phosphorus detec- and reinjecting onto a second column. tor catalytically ionizes N- or P-contain- Also called coupled column chromatog- ing solutes on a heated rubidium or ce- raphy, column switching, multicolumn sium surface in a reductive atmosphere. chromatography, and boxcar chromatog- NPD is highly selective with sensitivity raphy. See backflushing, heart cut- somewhat better than FID. ting, precut. Noise: See baseline noise. Multidimensional protein iden- Non-aqueous reversed phase chroma- tification technology (MudPIT): tography (NARP): Refers to reversed- Multidimensional protein identifi- phase chromatography performed with- cation technology combines both a out water as a component of the eluent cation-exchange prefractionation and on a reversed-phase packing. Used for reversed-phase HPLC separation of compounds that are very nonpolar that www.chromatographyonline.com Terminology Guide October 2013 51 either cannot be eluted or are poorly Nonporous packing (NPR, NPS, NPZ): eluted from a reversed-phase column Particles similar to porous-layer bead with 100% methanol or acetonitrile; in but with particle diameters in the sub- these cases, solvent A would be aceto- 5-µm range, often particles are in the nitrile and solvent B would be a stron- sub-2-µm range; used for high-speed ger solvent such as tetrahydrofuran; for separations in short columns; NPS refers NARP, reversed-phase rules apply (that to nonporous silica, NPR to nonporous is, the more nonpolar the analyte, the resins, and NPZ to nonporous zirconia. greater the retention). Normal-phase chromatography: A Nonpolar: A nonpolar molecule is one mode of chromatography in which the in which the electrons are distributed stationary phase is more polar than the more symmetrically and thus does not mobile phase. Adsorption chromatogra- have an abundance of charges at the phy on silica gel or alumina using mix- opposite sides. The charges all cancel tures of less polar eluents (for example, out each other. Nonpolar compounds, hexane–diethethyl ether) as a mobile solvents, or bonded phases readily dis- phase would be a typical normal-phase solve in organic solvents, such as hexane, system. Also refers to the use of polar or prefer such solvents in place of water. bonded phases, such as -CN or NH2. Nonpolar substances do not readily dis- Sometimes referred to as straight phase solve in water. chromatography. Nonporous particle: Refers to a solid particle used as a support for a porous O coated or bonded phase; pellicular par- Octadecylsilane (ODS, C18): The most ticles are nonporous particles with large popular reversed phase in HPLC. Oc- particle diameters (approximately 40 µm) tadecylsilane phases are bonded to silica and nonporous silicas and resins with or polymeric packings. Both monomeric small particle diameters of less than 3 µm and polymeric phases are available. usually consist of a microbead with a thin Octylsilane (C8): A popular stationary porous outer coating of silica gel, bonded phase in reversed-phase chromatogra- silica gel, or polymeric phase. phy; usually has slightly less retention Nonsuppressed ion chromatography: than the more popular C18; both mo- Direct ion chromatography. After the nomeric and polymeric phases available separation column the eluent is directly Off-line SPE: The normal practice of fed into the conductivity detection with- SPE where SPE cartridges, disks, pipette out prior chemical suppression. The tips, and so forth are handled using conductivity measurement takes place manual processes (for example, vacuum on the high background conductivity. manifolds or pipette transfer); opposite This requires a very high quality detec- to on-line SPE. tor with perfectly stable temperature. On-column detection: The column it- The nonsuppressed approach allows de- self serves as the flow cell in HPLC, CE, tection of weak acids or bases that are or CEC. Generally the term used when undissociated after suppression — for fused-silica capillaries are employed; the example, silicate or borate. For cation outer polyimide layer is removed and op- analysis the peaks for the components of tical beam is directed through the capil- interest are larger than with suppression. lary; a measuring device (for example, a 52 Terminology Guide October 2013 www.chromatographyonline.com photomultiplier tube) is located on the 750 µm. In HPLC, SFC, and capillary opposite side of the capillary. electrophoresis, small internal diameter On-column injection (OCI): In GC, re- (less than 100 µm) columns are used. fers to the process of injecting the entire The most frequently used column mate- liquid sample directly onto the head of rial is fused-silica tubing. Used very little the column using a fine needle that will in routine HPLC or SFC but routinely fit inside the capillary. Usually carried in CE and GC. Also termed capillary out at an initial column temperature column. less than the solvent boiling point, also Open-tubular column, packed: A cap- termed cold on-column injection. illary-dimension column that is packed On-line column switching: See multi- with stationary phase particles. Also dimensional chromatography, on-line called micropacked columns, particularly SPE. in GC. On-line preconcentration: A precol- Optically active resin: Incorporation umn is placed in front of the separation of optically active groups into an ion- column to concentrate analytes before exchange resin to allow separation of their separation; different mechanisms optically active isomers. Not many are may be used (for example, hydrophobic commercially available in HPLC. interaction, adsorption, or enzymatic re- Organic modifier: Water-miscible action) to retain analytes as a function of organic solvent added to an aqueous time and then by a displacement process mobile phase to effect separations in re- (such as solvent elution or pH change) versed-phase HPLC. Common organic concentrated analytes are transferred to modifiers are acetonitrile, methanol, iso- the separation column. propanol, and tetrahydrofuran. On-line SPE: Refers to the use of small Orthogonality: Refers to two separation stainless steel cartridges packed with dimensions for which the elution times SPE packings placed across two ports in the two dimensions can be treated of a 6- or 10-port injection or column- as statistically independent; ideally, the switching valve. The SPE trap is loaded two dimensions should have totally dif- with sample by an external pump or ferent retention mechanisms (for exam- syringe transfer and then the valve is ple, reversed phase and normal phase; switched so that the SPE trap becomes ion exchange and reversed phase; polar part of the HPLC flow stream and ana- and nonpolar) lytes can be swept into the column based Overload: In preparative chromatog- on the solvent being used for displace- raphy, the overload is defined as the ment. On-line SPE columns are usually sample mass injected onto the column used multiple times whereas off-line SPE where efficiency and resolution begins cartridges are generally used once. to be affected if the sample size is further Open-tubular column: Also termed increased. See sample capacity. capillary columns, open-tubular col- umns for GC have the stationary phase P coated or chemically bonded on the Packing: The adsorbent, gel, or solid sup- inner walls or have support particles port used in the chromatography column. deposited on the inner walls. Internal Most modern analytical HPLC packings diameters range from ~100 µm up to are less than 10 µm in average diameter www.chromatographyonline.com Terminology Guide October 2013 53 with 5 µm currently the favorite. GC col- escape from the column exit may cause umn packings range in size from 60–80 detector noise. mesh down to 100–120 mesh. Partition chromatography: Separa- Paired ion chromatography: The same tion process where one of two phases is as ion-pair chromatography. held stationary on a solid support or the Paper filtration: Using porous filter column inner wall (stationary phase or paper (mainly cellulose) to remove liquid phase) while the other is allowed particulates from liquid samples; pa- to flow freely down the column (mo- pers with different porosities are avail- bile phase or carrier gas). Solutes parti- able. Low porosity filters will remove tion themselves between the two phases very fine particulates but may have a based on their individual partition co- lower flow rate while high porosity efficients. LLC is an example; modern filters filter out larger particulates at bonded-phase LC can be considered to a higher flow rate; paper filtration is be a form of partition chromatography often used in wet chemistry to filter, where one of the liquid phases is actu- combust, and then weigh insoluble ally bonded to the solid support. Mech- materials; ashless filter paper is used anistically, partition chromatography for this purpose. implies that the solute becomes at least
Particle diameter (dp): Average diam- partially embedded within the station- eter of the column packing particles. ary phase, which is impregnated, coated, Particle size distribution: A measure of or bonded to the substrate, in contrast the distribution of the particles used to to an adsorption process in which the pack the LC column. In HPLC, a nar- solute does not penetrate into the reten- row particle size distribution is desirable. tive surface or interphase. Partition coefficient (K): A particle size distribution of dp ± 10% The ratio of would mean that 90% of the particles the equilibrium concentration of solute fall between 9 and 11 µm for a 10-µm in the stationary phase relative to the average dp packing. equilibrium concentration of solute in Particle size reduction: The general the mobile phase. In GC, the relative process of reducing larger particles down concentration of solute in the mobile to a size that can be more conveniently and stationary phases is a function of extracted; the smaller the particle the k and β: K = βk. Also called distribu- more quickly it will dissolve or if in- tion coefficient, KD, and distribution soluble the more quickly analytes can constant, Kc. be extracted for further sample cleanup. Passive sampling: In passive gas sam- Typical methods for reducing particle pling, an air sample is pulled through a size include pulverizing, milling, ho- flow controller into an evacuated canis- mogenizing, chopping, blending, and ter over a chosen period of time, rang- so on. ing from 5 min to 24 h. The sampling Particulates: Generally refers to a period and the flow rate determine the small particles found in the mobile canister volume required. phase that can cause back pressure Peak: The profile of an analyte com- problems by lodging in frits; it can pound as it is eluted from a column also refer to the small particles packed through a detector; usually depicted on into HPLC columns. Particulates that a visual output on a recorder or printer 54 Terminology Guide October 2013 www.chromatographyonline.com based on the detector’s electrical re- other column degradation or drift in the sponse. chromatographic conditions. 2 Peak area (Ap): The area measured Peak variance (σ ): The second central under a chromatographic peak; usu- moment of the peak about the retention ally measured by an integrator or data time. For a Gaussian peak the variance system; the peak area is related to the is the fundamental parameter control- amount of substance eluted in a peak. ling peak width. See Gaussian peak.
Peak capacity (n): The number of Peak volume (Vp): The volume occu- equally well resolved peaks that can be fit pied by a chromatographic peak from in a chromatogram between the hold-up starting basepoint to ending basepoint volume and some upper limit in retention as it passes through the detector: Vp = k . For R = 1, n can be expressed by the F w n — c b approximation n = 1 + √N /4 · ln(1 + kn) Peak width at base (wb): The width where N is the plate number and kn is the of the chromatographic peak at the retention factor of peak n. baseline as eluted from the column. It Peak dispersion: See band broadening. is measured at the baseline by drawing Peak doublet: A split peak generally tangents to the inflection points on the caused by column void, poor injection sides of the Gaussian curve representing technique, or solvent flooding in GC. the peak. Smaller peak widths usually Split peaks also could be closely eluted represent efficient separations; also re- compounds. ferred to as band width. It is sometimes
Peak height (hp): The maximum height convenient to estimate the peak width of a chromatographic peak as measured at base from the peak area and height: from the baseline to the peak apex; the wb = 1.596 Ap/hp (see Figure 2). peak height is related to the amount of Peak width at half-height (wh): The substance eluted in a peak. width of the chromatographic peak at Peak overload: When too much of any half of the peak height above the base- one solute is injected its peak may be line. Smaller peak widths usually repre- distorted into a triangular shape. sent efficient separations; also referred Peak shape: Describes the profile of a to as band width. It is sometimes conve- chromatography peak. Theory assumes nient to estimate the peak width at half- a Gaussian peak shape (perfectly sym- height from the peak area and height: wb metrical); peak asymmetry factor de- = 0.94 Ap/hp (see Figure 2). scribes shape as a ratio. See asymmetry. PEEK: Polyether ether ketone (PEEK) is Peak tracking: A method of matching a colorless organic polymer. It is used as of peaks that contain the same com- a material for inert capillaries and fit- pound between different experimental tings in HPLC and IC systems runs during method development; relies Pellicular: See porous layer bead. upon detection parameters of each pure Percent B (%B): Refers to the stronger analyte; diode-array detectors and mass solvent in a binary solvent mixture; %A spectrometers are among the best detec- would be the weaker solvent analog. tors for peak tracking because of their Perfusion chromatography: Re- specificity. Also refers to data-system fers to chromatography performed tracking of gradual changes in retention using particles with very large times caused by stationary-phase loss or pores (for example, 4000–8000 Å) www.chromatographyonline.com Terminology Guide October 2013 55 called throughpores (megapores or Phenol extraction: A sample prepara- gigapores). Eluent flows through tion technique used for the isolation of the particle as well as smaller in- DNA from biological samples. terconnecting pores (for example, Phenyl phase: A popular nonpolar 300–1000 Å) called diffusive pores bonded phase prepared by the reac- between the large pores. Best suited tion of dimethylphenylchlorosilane or for the preparative separation of mac- alkoxysilane with silica gel for LC, or romolecules. as the components of a cross-linked
Permeability (Bo): Also referred to as or bonded phase for GC. Claimed to column permeability and specific perme- have affinity for aromatic-containing ability; a term expressing the resistance compounds and does impart a different of the packed column to the flow of selectivity compared to alkyl bonded mobile phase. For a packed column: Bo phases. 2 3 2 2 Photoionization detection (PID): = (dp /180) · ε /(1 - ε) ≈ (dp )/1012.For The 2 an open-tubular column: Bo = dc /32. A photoionization detector ionizes solute column with high permeability gives a molecules with photons in the UV en- low pressure drop. ergy range. PID is a selective detection Permeation: In SEC, refers to the pro- method that responds to aromatics and cess where a solute can enter a mobile olefins when operated in the 10.2-eV phase filled pore of the packing. photon range. It can respond to other Phase collapse: See phase dewetting. materials with a more energetic light Phase dewetting: A term used in source. reversed-phase LC where very dense PIONA: Refers to the analysis of paraf- bonded-phase coverage and a high per- fins, isoparaffins, olefins, naphthenes, centage of aqueous content in mobile and aromatics. phase can lead to expulsion of water Pipette tip: Replaceable tips used in from the pores, which prevents the automation of liquid handling chores; normal partitioning process from tak- used once and discarded to avoid con- ing place. Phase dewetting may occur tamination. with as high as 10% organic content Pirkle column: Chiral “brush type” sta- in the mobile phase but can occur at tionary phases based on 3,5-dinitroben- lower %B values; results in earlier than zoyl-phenylglycine silica that are used in normal elution of analytes, poor peak the separation of a wide variety of en- shape, and nonreproducible retention antiomers. Named after the developer, times. Dr. William Pirkle, University of Illnois.
Phase ratio (β): The relative amount of pKa: An acid dissociation constant, stationary to mobile phase in the col- Ka, (also known as acidity constant, or umn. In partition chromatography: β = acid-ionization constant) is a quantita-
VS/VM where VS and VM are the volume tive measure of the strength of an acid of stationary and mobile phase in the in solution. It is the equilibrium con- column respectively. For open-tubular stant for a chemical reaction known columns: β ≈ rc/2df. Thicker station- as dissociation in the context of acid- ary phase films or higher phase loading base reactions. The equilibrium can be gives longer retention and higher peak written symbolically as: HA ←→ H+ + capacity. A- where HA is a generic acid that 56 Terminology Guide October 2013 www.chromatographyonline.com dissociates by splitting into A−, known solvent and is useful for identifying suit- as the conjugate base of the acid, and able mobile phase solvents or extraction the hydrogen ion or proton, H+, which, solvents. The polarity index increases in the case of aqueous solutions, ex- with polarity; examples: hexane, P′ = ists as the hydronium ion — in other 0.0; isopropanol, P′ = 3.9; tetrahydrofu- words, a solvated proton. The disso- ran, P′ = 4.0; methanol, P′ = 5.1; aceto- ciation constant is usually written as a nitrile, P′ = 5.8; water, P′ = 9.0 quotient of the equilibrium concentra- Polyacrylamide gel: Neutral hydro- tions (in mol/L), denoted by [HA], [A−], philic polymeric packings used in + + − and [H ]: Ka = ([H ] [A ])/[HA]; due to aqueous SEC. They are prepared by the the many orders of magnitude spanned copolymerization of acrylamide with by Ka values, a logarithmic measure of N,N′-methylene-bis-acrylamide. the acid dissociation constant is more Polyaromatic hydrocarbon (PAH): commonly used in practice. The loga- Members of a class of hydrocarbon rithmic constant, pKa, which is equal to molecules characterized by one or more −log10 Ka, is sometimes also (but incor- fused aromatic rings. rectly) referred to as an acid dissociation Polychlorinated biphenyl (PCB): Bi- constant. phenyl molecule with two or more Planar chromatography: A separation chlorine substitutions. technique in which the stationary phase Polyethylene glycol (PEG): Polymeric is present as or on a plane (IUPAC). hydrocarbon used as a GC stationary Typical forms are paper and thin layer phase; possesses moderately polar reten- chromatography. tion characteristics. Plate height (H): See theoretical plate Polyethyleneimine (PEI): Polyethyl- height. eneimine, an anionic polymeric phase Plate height, effective (Heff): The used to coat or bond onto silica or a column length divided by the number polymeric packing. Most often used for of effective theoretical plates: Heff = L/ the separation of proteins and peptides. Polymeric packings: Neff Packings based Plate number (N): See theoretical on polymeric materials, usually in the plate number. form of spherical beads. Typical poly- Polar: A polar molecule may be polar mers used in LC as well as GC are as a result of polar bonds or as a result polystyrene–divinylbenzene (PS-DVB), of an asymmetric arrangement of non- polydivinybenzene, polyacrylamide, polar bonds and nonbonding pairs of polymethylacrylate, polyethyleneoxide, electrons; polar molecules are generally polydextran, or polysaccharide. Polymeric phase: able to dissolve in water (H2O) because Refers to chemically of the polar nature of water; polar mol- bonded phase where a polymer species ecules do not prefer nonpolar organic is bonded to silica-based particles or to solvents such as hexane. Polar molecules the wall of an open-tubular column. have slightly positive and slightly nega- Polymeric SPE: The use of a polymeric tively charged ends; we often refer to a base material (for example, PS-DVB or compound’s polarity. methacrylate) rather than an inorganic Polarity index (P’): The polarity index material (for example, silica or alu- is a measure of the relative polarity of a mina); polymers generally have a wider www.chromatographyonline.com Terminology Guide October 2013 57 pH range and higher sample capacity a submicrometer-thick shell; wide pore than some of the inorganic materials. versions (>300 Å) allow rapid diffusion Polystyrene–divinylbenzene (PS- of macromolecules and smaller pore DVB) resin: The most common base versions (90–120 Å) are for small mol- polymer for ion-exchange chromatog- ecules; give similar efficiency to sub- raphy. Ionic groups are incorporated 2-µm particles but at much lower pres- by various chemical reactions. Neutral sure because of their larger particle size. PS-DVB beads are used in reversed- Porosity: For a porous substrate, the phase LC. Porosity and mechanical ratio of the volume of the pores in a stability can be altered by variation of particle to volume occupied by the par- the crosslinking through the variation ticle. The pore volume is a measure of of the DVB content. In GC, a number the porosity and is expressed in mL/g. of porous polymer stationary phases are Porous layer bead: A small glass bead available for gas and light-compound coated with a thin layer of stationary separations. phase. The thin layer can be an ad- PONA: Refers to the analysis of paraf- sorbent, resin, or a phase chemically fins, olefins, naphthenes, and aromatics. bonded onto the adsorbent. These pack- Poppe plot: A kinetic plot named after ings were among the first to be used in Professor Hans Poppe (J. Chromatogr. HPLC. They were of larger particle size A 778, 3 [1997]), University of Amster- (20–40 µm) than the microparticulate dam, the Netherlands, where the plate packings of today but were easy to pack time log (t0/N) is depicted as a func- and gave adequate efficiency. Also re- tion of the number of theoretical plates ferred to as controlled surface porosity to assess the limits of column perfor- supports and pellicular materials. mances as a function of particle size, Porous particle: Refers to column column pressure drop, and so forth. packing particles possessing intercon- Pore diameter: Same as mean pore necting pores of specified diameter and diameter. pore volume; generally in HPLC porous Pore size: The average size of a pore in particles with diameters below 10 µm a porous packing. Its value is expressed are the most popular, and in prepara- in angstroms or nanometers. The pore tive chromatography larger particles size determines whether a molecule can are used because of their lower cost and diffuse into and out of the packing. See higher column permeability. mean pore diameter. Porous polymer: A packing material,
Pore volume (Vi): The total volume of generally spherical, based on organic the pores in a porous packing, usually polymers or copolymers; popular ex- expressed in mL/g. Better termed the amples would be polystyrene–divinyl- specific pore volume. It is measured by benzene, polyacrylates, polydextrans, the BET method of nitrogen adsorp- polyacrylamides, and polybutadienes. tion or by mercury intrusion where Hg Retains solutes by selective adsorption is pumped into the pores under high or molecular size interaction. pressure. Porous-layer open-tubular (PLOT) col- Poroshell: Similar to nonporous par- umn: An open-tubular column used in ticles and porous-layer beads; particles GC or HPLC that has particles coated are generally in the 2–5 µm range with or uncoated with stationary phase 58 Terminology Guide October 2013 www.chromatographyonline.com attached to the inner walls, which al- Precolumn reaction: Analytes are con- lows more rapid mass transfer. In GC, verted into components with better small porous particles such as polymer, detectability (for example, UV–vis ab- alumina, silica, and so forth are attached sorbance) by a chemical reaction before to the walls or the wall may be modified injection. The analytes are separated and by etching or other treatment to increase detected by UV–vis detection. Complex- the inner surface area and provide gas– ing agents such as EDTA, NTA, and so solid chromatographic retention behav- forth are used as their Fe(III) complexes. ior. In LC, porous polymers have been Precolumn: A short section of similar used occasionally. In GC, the technol- column placed before the analytical col- ogy is more developed. umn; used to physically retain undesired Postcolumn derivatization: See postcol- compounds or to saturate the mobile umn reaction. phase with stationary phase that may be Postcolumn reaction: In LC and IC, packed into the precolumn (for example, after the analytical column a UV-trans- a silica precolumn saturates the mobile parent ion or molecule is converted into phase with dissolved silica and prevents a component with better detectability mobile phase from dissolving silica in the (that is, UV–vis absorbance, fluores- analytical column). cence) by adding a specific reagent. This Precolumn filter: A filter used between product is then detected with UV–vis the injector and the column (or guard or fluorescence detection. The reaction column) to keep unwanted sample of the analyte and the reagent is usually components from reaching the column; very selective and yields often in a col- sometimes called in-line filter, occasion- ored product (visible detection), that is, ally inlet filter. chromate + diphenylcarbazide complex Preconcentration: See also trace enrich- (540 nm); bromate + iodide → triiodide ment. (352 nm). Parameters that will influence Precut: Peaks in the beginning of a chro- the sensitivity are the reaction time (flow matogram are removed to vent or are di- rate/length of reaction coil), the reaction rected onto another column of different temperature, pH, concentration of cata- polarity, or at a different temperature, for lysts. In GC, postcolumn methanization improved resolution. See heart cutting, may be used to convert CO and CO2 to multidimensional chromatography. Prefilter (SPE): CH4 with hydrogen and a heated nickel In cases where samples catalyst to achieve flame ionization de- contain a large amount of particulates, tection, more sensitive than thermal regular SPE cartridges and disks may conductivity detection. become clogged and flow is reduced. Potentiometric detection: Ion selec- Prefilters are filter devices that have tive electrodes in a flow-through cell higher porosity that will filter out large are used to detect the analyte ions. Not particles and allow the SPE bed to oper- a very common type of detection. ate more efficiently. Some SPE devices Pounds per square inch (psi): A unit of have prefilters built in; in others one can pressure: 14.6959 psi = 1 atm = 101.325 add a prefilter. In some cases, the use kPa = 1.013 bar. of an inert packing such as glass beads Precolumn derivatization: See precol- serves the same purpose as an actual umn reaction. filter. www.chromatographyonline.com Terminology Guide October 2013 59
– 2 Preparative chromatography: Refers proximated as Δp ≈ (1012L η u)/(dp ), where to the process of using chromatogra- u– is the average linear velocity. In open-tu- phy as a technique for the isolation of a bular column GC, the pressure drop is Δp – 2 sufficient amount of material for other ≈ (8 L η u)/(rc ). The equations for GC will experimental or functional purposes. overestimate the required pressure drops by For pharmaceutical or biotechnological more than 10% at inlet pressures above 400 purifications, large columns of several kPa (58 psig) because of gas compressibility feet in diameter can be used for puri- effects. Pressure drop can be expressed in fying multiple grams of material. For pressure units of psig, bar, atm, kPa, or MPa. isolating a few micrograms of valuable The above equations will yield pressures in natural product, a 4.6-mm i.d. analytical pascals if the dimensions are expressed in column can be used. Both of these sepa- centimeters, times in seconds, and viscosi- rations can be considered preparative ties in pascal-seconds. chromatographic approaches. Prepara- Pressure, relative (P): Relative pressure tive LC is often employed; preparative across the column: P = pi /po GC is seldom used. Pressurized-fluid extraction (PFE): Pressing: The general process of squeez- Pressured fluid extraction is a liq- ing liquid from a semisolid material uid–solid extraction process where (such as plants, fruit, or meat). the sample and solvent are placed in a Pressure drop (Δp): The pressure drop closed container and heated well above across a column: Δp = pi – po, where pi the solvent’s normal boiling point. The and po are pressure at the column inlet combination of increased temperature and outlet, respectively. and resultant pressure extracts analytes Pressure injection (CE): Pressure-in- and matrix compounds into the super- duced injection; the use of pressure or heated fluid, often in a few minutes. vacuum to inject small (nanoliter) vol- Because the technique extracts a wide umes of sample into a capillary column; variety of soluble compounds, addi- best for narrow-bore capillaries (<10 µm tional cleanup steps may be required i.d.); a version of hydrostatic injection. after PFE is completed; method de-
Pressure, absolute inlet (pi): The col- velopment involves selecting the best umn inlet pressure expressed relative to solvent for analytes and the poorest a vacuum. solvent for the matrix and other in-
Pressure, absolute outlet (po): Pressure terferences that may be present. The at the column outlet, relative to vacuum. technique has been approved for vari- Pressure, back: Same as head pressure, ous environmental samples by the U.S. column pressure. EPA under the generic name of PFE or Pressure, head (Δp): The pressure dif- pressurized-solvent extraction. See ference between the inlet and outlet of accelerated solvent extraction. the column. In LC, governed by the Pressurized-solvent extraction (PSE): following approximate equation for a See accelerated solvent extraction, column packed with spherical particles: pressurized-fluid extraction. 2 Δp ≈ (3000Lη)/(t0dp ) where η is the mobile Primary sampling: The collection of phase viscosity, t0 the column holdup time, one or more increments or units ini- and dp is the particle diameter. In packed- tially taken from a population; the column GC the pressure drop can be ap- primary sample is that taken from the 60 Terminology Guide October 2013 www.chromatographyonline.com primary source; proper statistical sam- the temperature of the inlet is heated pling protocols are recommended. very rapidly to transfer the sample into Process-scale chromatography: Re- the column; using PTV there is the fers to the use of liquid chromatography potential for less sample discrimination at the industrial scale level outside the and less thermal degradation of sensi- laboratory; generally requires specially tive compounds compared to hot inlet designed columns (usually with di- injections. ameters > 5 cm), recoverable solvents, Programmed temperature vaporizer lower-cost packings (with larger and ir- (PTV): An inlet system designed to per- regular-shaped particles), and overloaded form programmed-temperature injection. operating conditions compared to those Protein crashing: The term used in of laboratory-scale HPLC. removing or reducing the protein con- Programmed elution: A procedure in centration in a biological fluid such as which the conditions of separation are plasma. After slight dilution, an organic changed in a programmed manner. Un- solvent such as acetonitrile is added to like LC, in GC and SFC both tempera- the plasma and the proteins, which are ture and pressure can be programmed, insoluble, precipitate (crash). Centrifuga- separately or simultaneously. tion or filtration is used to remove the Programmed temperature chroma- protein, and the supernatant liquid is in- tography: Use of conditions in which jected into an HPLC system or worked the temperature is varied during the run up further. in a controlled manner. Widely used in Protein precipitation: See protein GC; seldom seen in LC. crashing. Programmed temperature injection Pulsating flow: Flow originating from (PTI): A cold injection technique in a reciprocating pump. Normally the which the inlet temperature is specifi- pulses are dampened out by a pulse cally programmed from the GC. damper, an electronic pressure feedback Programmed temperature rate: The circuit, or an active damper pump head. rate, in °C/min, at which the GC oven Some detectors (for example, electro- temperature is increased during a con- chemical, refractive index) are greatly trolled temperature program ramp. The affected by flow pulsations. program may consist of multiple ramps Pulsed amperometric detection: with variable hold times before and after Electrochemical detection applying dif- each. Typical GC programming rates ferent potentials (pulses) to the working range from <0.5 °C/min up to 40 °C/ electrode. Components that can be an- min. Programming rates up to 200 °C/ alyzed include those that are oxidized min and higher have been applied to or reduced at the electrode and those high-speed gas chromatography. that react with the electrode surface or Programmed temperature vaporiza- cover it. To remove reaction products tion (PTV): In PTV, the sample is intro- that could foul the electrode, highly duced into the inlet liner at a tempera- oxidative and reductive potentials are ture slightly below the boiling point of applied to the working electrode after the solvent; the solvent is continually the measuring potential. This removes evaporated and vented through the the reaction products from the previ- inlet split line; after the solvent is gone, ous measuring cycle and renews the www.chromatographyonline.com Terminology Guide October 2013 61 electrode surface. Typical applications lyzed by GC. Often applied to polymer are carbohydrates and amino acids. characterization. Pulsed discharge detection (PDD): Several ionization detectors use a pulsed- Q discharge ion source to improve detec- Quaternary methyl amine (QMA): A tivity compared to constant-discharge strong anion-exchange functionality detectors. popular in resin-based packings; usually Pulsed-splitless injection: A form of supplied in chloride form. GC injection recommended for large Quaternary mobile phase: See quater- volumes (up to 5 µL) of sample where nary-solvent mobile phase. a short-term high pressure pulse is im- Quaternary-solvent mobile phase: A posed on the inlet such that there is not mobile phase consisting of four separate a large volume of solvent vapor gener- solvents that allow the mobile-phase ated and most or all of the sample is di- composition to be fine-tuned; most rected to the column; after the sample often this mobile phase is delivered by a is transferred, then normal pressure is low-pressure quaternary pump. resumed. Using this technique, highly QuEChERS: A technique initially used volatile compounds are less likely to be for the extraction of pesticides from lost through the split vent line and ther- fruits and vegetables. It consists of mally unstable compounds spend less two steps: salting out extraction using time in the hot injection port so there is buffered or unbuffered solvent (usually less degradation. acetonitrile), and dispersive SPE where Pulverizing: Electromechanically a solid adsorbent is used to treat an driven rod or vibrating base devised to aliquot from the first step to remove reduce the particle size of solid samples. interferences and matrix compounds. A freezer mill can be used with liquid QuEChERS (which stands for quick, nitrogen to treat malleable polymers or easy, cheap, effective, rugged, and safe) those with low glass transition tempera- is mostly used with GC–MS and LC– tures. MS (or MS-MS) to more selectively an- Purge-and-trap sampling: Dynamic alyze pesticide extracts. More recently, headspace technique where the head- QuEChERS has expanded to matrices space vapors over a liquid or solid such as cooking oil, meat, fish, and sample are continuously removed by a biological fluids, and to other analytes, flow of gas over the sample (purging) or such as pharmaceuticals, antioxidants, through the sample (sparging); volatil- and toxins. ized analytes are usually concentrated by trapping on an adsorbent or by cryogenic R means. The trap is then heated to desorb Radial compression: The use of radial trapped components into a GC column. pressure applied to a flexible wall col- Most often used for volatile trace ana- umn to cut down on wall effects. lytes where concentration is needed. Radial diffusion or dispersion: Diffu- Pyrolysis gas chromatography: The sion or dispersion across the column in a process of heating a sample enough to radial direction. If the sample is injected break its chemical bonds, thereby form- into the exact center of a column, it will ing smaller molecules that can be ana- spread not only in a longitudinal 62 Terminology Guide October 2013 www.chromatographyonline.com direction as it moves down the column Refractive index peak: A pseudo peak but radially as well, allowing the solute normally found near the hold-up volume to reach the wall region where the eluent resulting from the refractive index sensi- velocity is different than in the center of tivity of absorbance and other detectors. the column. Regeneration: Regeneration of the Recovery: The amount of solute (sam- packing in the column to its initial state ple) that is eluted from a column rela- after a gradient elution. Mobile phase tive to the amount injected. Excellent is passed through the column stepwise recovery is important for good quanti- or in a gradient. The stationary phase is tation, for preparative separations, es- restored (solvated) to its initial condition. pecially for biomolecules, and for good In ion exchange, regeneration involves peak shape and resolution. Reasons for replacing ions taken up in the exchange inadequate recovery can be solute inter- process with the original ions which action with active sites on the packing, occupied the exchange sites. Regenera- with column frits, and with column tion can also refer to bringing back any tubing. Compound decomposition column to its original state (for example, during the separation process can also the removal of impurities with a strong effect recovery. solvent). Recycling chromatography: A tech- Relative retention (r): Retention rela- nique where the column effluent is re- tive to a standard peak: r = t′R/t′R(st) where circulated onto the head of the column t′R is the retention time of the compo- in an attempt to gain the advantage of nent of interest and t′R(st) is the retention extended column length. Can be car- time of the standard peak. Also: r = ki/ ried out on a single column by passing kst where ki and kst are the correspond- the effluent back through the pump. An ing retention factors. For two adjacent alternative technique uses two columns peaks, the separation factor, α, expresses connected by a switching valve where the relative retention. See separation the effluent of one column is directed factor, resolution. onto the head of the other column. Very Relative standard deviation (RSD, seldom used in HPLC, and then only in %RSD): In probability theory and exclusion chromatography. statistics, the relative standard devia- Reduced plate height (h): The plate tion (RSD or %RSD) is the absolute height expressed in terms of the average value of the coefficient of variation. It particle diameter for packed columns: h is often expressed as a percentage. A
= H/dp where dp is the particle diameter, similar term that is sometimes used is or in terms of the column inner diam- the relative variance which is the square eter for open-tubular columns: h = H/dc of the coefficient of variation. Also, the where dc is the column inner diameter. relative standard error is a measure of a Refractive index detection (RI detec- statistical estimate’s reliability obtained tion): Based on differential refractive by dividing the standard error by the index between the mobile-phase sol- estimate; then multiplied by 100 to vent and the eluted analyte in mobile- be expressed as a percentage. The rela- phase background; not useful in gradi- tive standard deviation is widely used ent work; often used in size-exclusion in analytical chemistry to express the chromatography. precision and repeatability of an assay. www.chromatographyonline.com Terminology Guide October 2013 63
Removable well plates: See array 96- They contain an outer hydrophilic sur- well plate. face that provides minimal interaction Representative sample: A sample re- with proteins and when combined with sulting from a statistically worked out small pores on the sorbent exclude the sampling plan; it can be expected to ad- proteins. The inner surface is hydropho- equately reflect the properties of interest bic, and when small molecules diffuse of the parent population. into the pores they interact by reversed- Residual silanols: The silanol (-Si–OH) phase mechanisms and are retained. The groups that remain on the surface of a small molecules such as drugs and their packing after chemically bonding a metabolites can be removed by rinsing phase onto its surface. These silanol with an organic solvent. RAMs are most groups, that may be present in very successfully used in a column switching small pores, may not be accessible to setup where the secondary column is the reacting bulky organosilane (such as used to resolve the small molecules and octadecyldimethylchlorosilane) but may the proteins are directed to waste so as be accessible to small polar compounds. not to foul the secondary column. Often they are removed by endcapping Retention factor (k): The measure with a small organosilane such as tri- of time the sample component resides methylchlorosilane. See endcapping. in the stationary phase relative to the Resin: A solid polymeric packing used time it resides in the mobile phase: k in ion exchange separations. The most = (tR – tM)/tM. Formerly, kʹ was used popular resins are polystyrene–divinyl- and it was called the capacity factor or benzene copolymers of small particle size capacity ratio. (<10 µm). Ionic functionality is incorpo- Retention gap: A short piece of deac- rated into the resin. tivated but uncoated column placed
Resolution (Rs): Peak resolution; incor- between the inlet and the analytical porates both efficiency and separation. A column. A retention gap often helps resolution of 1.5 is said to be “baseline” relieve solvent flooding. It also entrains resolution, and a minimum resolution of nonvolatile sample contaminants from 1.7–2.0 is considered essential for robust- on-column injection. ness. For two closely eluted peaks: R = Retention index (I): A uniform system
(tR,2– tR,1)/wb where the subscripts 1 and of retention classification according to a 2 refer to the first and second— peaks. solute’s relative location between a pair α From N, k2 and : Rs= (√N /4)((α – 1)/α) of homologous reference compounds on (k2/(k2 + 1)) (k2 is the retention factor of a specific column under specific condi- the second peak). tions. A series of normal straight-chain Resolution equation: See resolution. hydrocarbons, fatty acid esters, or mul- Response factor (RF): Defines the rela- tiring polycyclic aromatic hydrocarbons tionship between the measured peak area have been used for the reference com- or height and the quantity of substance pounds. For a solute i that is eluted at tʹRi represented by a peak. between two hydrocarbons with chain Restricted access media (RAM): RAM length z and z + 1: I = 100[z + (logt′Ri – are sorbents are used for the direct injec- logt′Rz)/(logt′Rz1 – logt′Rz)]. tion of biological fluids such as plasma Retention time (tR): The time between or serum into an HPLC flow stream. injection and the appearance of the peak 64 Terminology Guide October 2013 www.chromatographyonline.com maximum. It is usually measured from lipophilicity) of the solutes; the more the point of injection to the apex of the hydrophobic, the stronger the retention. peak. For asymmetric peaks it should be The greater the water solubility of the measured to the center of the mass of the analyte, the less it is retained. There are peak. Also called the total retention time many variations of reversed-phase LC (IUPAC). See retention volume; reten- where various mobile phase additives tion time, adjusted. are used to impart a different selectivity.
Retention time, adjusted (t’R): A mea- For example, for the reversed-phase LC sure of the retention time adjusted for of anions, the addition of a buffer and the void time or unretained peak time: a tetraalkylammonium salt would allow t′R = tR – tM where tR is the retention time ion pairing to occur and effect separa- and tM (or t0) is the hold-up time, void tions that rival ion-exchange chromatog- time, or unretained peak time (that is, raphy. More than 90% of HPLC users the time it takes for a small, unretained employ reversed-phase LC. compound that completely permeates Reynolds number (Re): For flow in a the pores to be eluted from the chro- smooth unpacked pipe where u– is the matographic column). average velocity (cm/s), dc is the pipe Retention volume (VR): The volume diameter, η is the viscosity (Pa·s) and 3 – of mobile phase required to elute a sub- ρ is the density (g/cm ): Re = (udcρ)/η. stance from the column: VR = FctR or The Reynolds number is the ratio of VR = VM + KDVS where VM is the void viscous to inertial energy of the mov- volume, KD the distribution coefficient, ing fluid. At low Re viscous friction and VS the stationary phase volume. Also dominates and controls fluid motion, termed the total retention volume. See re- making it slow and steady. In an un- tention time. packed tube flow becomes fully turbu- Retention volume, adjusted (V′ ): R Ad- lent when Re exceeds 4200. In a packed justs the retention volume for the holdup bed u– is replaced with the average inter- volume (or V0) where VR is the retention stitial velocity and dc with the average volume of the peak of interest and VM is particle diameter. Flow becomes turbu- the hold-up or void volume, the volume lent in a packed bed at Re above about corresponding to the holdup time: V′R 10 but is not fully turbulent until Re = VR – VM exceeds 100–200. 0 Retention volume, corrected (VR ): Riffler: A mechanical device used in Corrects the retention volume for the subdividing solid powder samples into effect of carrier-gas expansion along the smaller units. Rifflers can be manual or 0 column: VR = VR j automated. Rifflers will subdivide mate- Reversed-phase chromatography: The rial samples into two smaller portions by most frequently used mode in HPLC. It a single pass or further subdivisions can uses low polarity packings such as octa- be attained by multiple passes. decylsilane or octylsilane phases bonded Rinse step: In SPE, the rinse (wash) to silica or neutral polymeric beads. The step is the third step in the process. mobile phase is usually water or water- After the sample is loaded, the rinse step miscible organic solvents such as metha- is designed to eliminate interferences nol or acetonitrile. Elution usually occurs including various matrix compounds. based on the relative hydrophobicity (or A solvent (or solvents) or buffer is selected www.chromatographyonline.com Terminology Guide October 2013 65 to remove interferences but not the ana- onto a column without overload and lytes of interest. loss of column efficiency. Often ex-
Room temperature (To): The room or pressed as grams of sample per gram laboratory temperature can be used as a of packing. Overload is defined as the reference temperature for gas measure- sample mass injected that will cause ments, for example 20 °C or 25 °C. the column efficiency to decrease by Round-well plates : 96-well plates that 10% from its normal value. Sometimes have round-shaped wells resembling 96 referred to as sample loading. small test tubes. See 96-well plate. Sample discrimination: The charac- teristic of systematic change in sample S composition according to a specific Salting-out effect: The use of a high sample property. For example, a GC concentration of salt buffer in the mo- inlet may exhibit mass discrimination bile phase to cause a low polarity ana- and admit relatively higher amounts of lyte to have a decreased solubility in low-boiling sample components than water and therefore precipitate or come high-boiling components in the same out of solution; most often used for the sample or injection. hydrophobic interaction chromatog- Sample division: The process of sam- raphy of proteins where proteins are ple reduction to divide the sample into first precipitated at high salt con- smaller portions while retaining repre- centrations then eluted by gradual sentative characteristics of the primary dilution using reverse gradient elu- sample. See sample size reduction. tion. Salting-out is also used in Sample loop: Part of an injection headspace sampling to increase valve that delivers an accurate volume the ionic strength of the sample of liquid or gas to the column, giv- solution and thereby decrease the ing a “slug” injection; loops come in solubility of dissolved analytes and in- different volumes depending on the crease their headspace concentrations. needs of the analysis and the size of Can also be used in liquid–liquid the column. extraction; see salting-out extraction. Sample pretreatment: Often syn- Salting-out extraction: In this extrac- onymous with sample preparation; the tion approach, high concentrations of process of manipulating the sample to salt in the aqueous phase will cause make it easier to analyze. certain compounds to migrate into an Sample size reduction: The process of organic phase or perhaps vice-versa; sample reduction to divide the sample high concentrations of salt also will into smaller portions while retaining force normally miscible solvents (such representative characteristics of the as water and acetonitrile) to become primary sample. See sample division. immiscible and be used for further Sample tracking: The process of track- partitioning more polar analytes than ing primary, secondary, laboratory, and could be achieved by an extraction further samples through the analyti- using a non-polar organic solvent. See cal cycle; it is important for chain of QuEChERS or salting-out effect. custody reasons to be able to ensure Sample capacity: Refers to the that the sample analyzed in the instru- amount of sample that can be injected ment was the original sample collected 66 Terminology Guide October 2013 www.chromatographyonline.com at the source; sample tracking can be as reproducibility of results on columns of simple as writing a sample number on different internal diameters when using a container but can be more complex, the same particle size and bonded phase; such as using bar-coded vials or radio normally a larger diameter column is frequency identification (RFID) tags to used to increase capacity; a linear scale- automatically keep track of sample flow. up process minimizes time required to Sampling: The process of collecting optimize preparative separations. a representative sample at the source. Scavenger: Special type of solid-phase Sampling can also refer to further sam- particle that uses chemical reactions (un- ple division as it more closely approaches like SPE, which uses molecular interac- the laboratory analysis; it is important tions) to remove undesired species such to make sure that the final sample ana- as undesired reaction products or excess lyzed represents a subsample of the origi- starting material from an organic syn- nal sample without any imposed bias or thesis. Scavengers mostly operate on the discrimination. basis of covalent bonding. Packing ma- Sampling error: In statistics, sampling terials contain reactive groups that can error is incurred when the statistical be used for organic or inorganic species characteristics of a population are esti- such as catalysts. mated from a subset, or sample, of that Secondary sampling: Refers to the pro- population. Because the sample does not cess of taking a representative portion of include all members of the population, the primary sample to further reduce its statistics on the sample, such as means particle size or to prepare a laboratory and quantiles, generally differ from sample for eventual analysis. parameters on the entire population. Sedimentation: A technique used for Because sampling typically is done to the sizing of resins for ion-exchange determine the characteristics of a whole chromatography; a broad distribution of population, the difference between the beads is placed in a solvent, often water, sample and population values is consid- in a container that is affixed to a station- ered a sampling error. Exact measure- ary surface. Based on particle size and ment of sampling error generally is not particle density the beads will settle at feasible because the true population different velocities into a gradient of sizes values are unknown; however, sampling and the fraction of interest is removed. error can often be estimated by probabi- Very narrow cuts of particle size can be listic modeling of the sample. obtained by sedimentation. Sampling rate: See data acquisition Selectivity: The fundamental ability rate. of a stationary phase to selectively re- Sandwich technique: Injection tech- tain substances based on their chemical nique in which a sample plug is placed characteristics, including vapor pres- between two solvent plugs in the syringe sure and polarity. In LC, selectivity so as to wash the syringe needle with sol- is strongly influenced by the mobile- vent and obtain better sample transfer phase composition. In GC, carrier gas into the inlet. has less, if any, impact on chromato- Saturator column: See precolumn. graphic selectivity. Scaleability: In going from analytical to Selectivity (α): Term replaced by the preparative chromatography, refers to the separation factor. See separation factor. www.chromatographyonline.com Terminology Guide October 2013 67
Selectivity coefficient (kA/B): In ion-ex- (LOD). For a concentration-sensitive change chromatography, the equilibrium detector such as a thermal conductivity coefficient obtained by application of the detector or UV–vis detector: S = mmax/c law of mass action to an ion exchanger where mmax is the peak height and c is and characterizing the ability of an ion the solute concentration in the detec- exchanger to select one of two ions pres- tor; units of sensitivity for a concentra- ent in the same solution. For example, tion-sensitive detector that responds in + + the exchange of Na for H in: KNa/H = millivolts are mV·mL/g. For mass-flow [Na]S*[H]S/[Na]M[H]M sensitive detectors such as the flame- Selectivity triangle: An approach to ionization detector: S = mmax/Wt where classify the properties of stationary Wt is the mass of solute passing through phases in reversed-phase LC. Results the detector per unit time; the units of can be represented in a “selectivity S are then expressed as A•s/g or C/g. triangle” in which the apices of the Separation: The degree of separation of triangle represent the relative con- two peaks in time. See separation fac- tributions of steric hindrance (χS), tor (α), relative retention, resolution. hydrogen bonding basicity (χB) and Separation factor (α): The separa- cation exchange capacity (χC) to se- tion factor α expresses the relative lectivity. A graphical visualization of retention of two adjacent peaks: α = the column selectivity allows three- t′R2/t′R1 = k2/k1 where t′R2 is the reten- dimensional data to be presented in tion time of the second peak and t′R1 a two-dimensional space. Provides an is the retention time of the first peak; informative yet universal approach for k2 and k1 are the corresponding reten- phase classification compared to other tion factors. models. With this model, selection of Separation impedance (E): A figure columns of either equivalent or dif- of merit developed by John Knox to ferent selectivity is readily achievable, compare the efficiency of two chro- which should further facilitate the ap- matographic systems by normalizing for plication of reversed-phase LC. both analysis time and pressure drop: E Selectivity tuning: 2 Several techniques = tRΔp/N ν(1 + k) where tR is the re- for adjusting the selectivity of separa- tention time, Δp is the pressure drop, N tions involving more than one column or is the theoretical plate number, ν is the stationary-phase type. Serially coupled reduced velocity, and k the retention fac- columns and mixed-phase columns can tor. The lower the value of E, the better be selectivity-tuned. the system. Semipreparative chromatography: Re- Separation number (SN): Separation fers to preparative LC carried out on an number, or Trennzahl (TZ). A mea- analytical size (4–5 mm i.d.) or a slightly sure of the number of peaks that could larger (6–10 mm i.d.) column. Normal be placed with baseline resolution be- injection size would be in milligram to tween two sequential peaks, z and z+1, low gram amounts. in a homologous series, such as two Sensitivity (S): Degree of detector hydrocarbons: SN = (tR(z+1) – tR(z))/ response to a specified solute amount (wh(z+1) + wh(z)) per unit time or per unit volume, often Septum: Silicone or other elastiomeric defined by lower limit of detection material that isolates inlet carrier flow 68 Terminology Guide October 2013 www.chromatographyonline.com from the atmosphere and permits sy- each other and based on the metal con- ringe penetration for injection. tent of the silica. The strongest silanols Septum purge: Carrier gas is swept are acidic and often lead to undesirable across the septum face and out to a interactions with basic compounds dur- separate vent so that material emitted ing chromatography. from the septum does not enter the Silanophile: A compound that has high column. affinity for active (acidic) silanol groups Sequential suppression: Combina- on the silica surface. Usually a strongly tion of chemical suppression (MSM) basic amine. Silica gel: and CO2 suppression (MCS). The The most widely used HPLC background conductivity of carbonate packing. It has an amorphous structure, and hydrogencarbonate eluents after is porous, and consists of siloxane and suppression is approximately 10–20 silanol groups. It is used in all modes of µS/cm. This is a result of the dissolved LC as a bare packing for adsorption, as carbonic acid that partially dissociates. the support for LLC or for chemically
The MCS removes the CO2 from the bonded phases, and, with various pore suppressed eluent and therefore reduces sizes, as an SEC packing. Microparticu- the background even further (typically late silicas of 3-, 5-, and 10-µm average >1 µS/cm). particle diameter are used in HPLC. Shell particle: See superficially porous Compared to irregular silicas, in mod- particles. ern analytical HPLC columns, spheri- Sieving: Process of passing a sample of cal silicas are preferred because of their solid particles through a metal or plastic packing reproducibility and because they mesh of a uniform cross-sectional area have lower pressure drops; sometimes re- (square opening from 3 µm to 123 mm) ferred to as silica. Also used as a gas-solid to separate particles into uniform sizes; adsorbent in GC. can be performed under wet and dry Siloxane: The Si-O-Si bond. A principal conditions. bond found in silica gel or for a silylated Signal-to-noise ratio (S/N): The ratio compound or bonded phase. Stable ex- of the peak height to the noise level. A cept at high pH values; has little effect detector gives a valid signal if there is on the HPLC separation. some measurable response above the Silylation: The process of reaction of normal background noise; both detec- an organochlorosilane or organoalk- tor sensitivity and limit of detection are oxysilane with a compound containing dependent on the level at which the sig- an reactive group. In LC it refers to the nal can be distinguished. A minimum process of derivatizing the solute before S/N is equivalent to 2 but for quantita- chromatography to make the solute de- tive methods sometimes a higher value is tectable or to prevent unwanted station- chosen (such as S/N = 6), meaning that ary phase interactions. It can also refer the signal is 6 times that of the baseline to the process of adding a chemically noise. bonded phase to a solid support or to Silanol: The Si-OH group found on deactivating the packing to cut down the surface of silica gel. There are dif- on surface activity. ferent strengths of silanols depending Simulated distillation (SIMDIS): on their location and relationship to Boiling-point separation technique www.chromatographyonline.com Terminology Guide October 2013 69 that simulates physical distillation of Solid-phase extraction (SPE): A tech- petroleum products. nique for sample preparation using Simulated moving bed : A chromato- a solid phase packing (dp of 20–40 graphic system involving a series of µm) contained in a small plastic car- columns and valves set up to simulate tridge or disk or in a well of a 96-well the countercurrent movement of the flow-through plate. The solid station- mobile and stationary phases to allow ary phases used are no different than for the continuous removal of product HPLC packings. However, although and reapplication of sample. A complex related to chromatography, the prin- form of recycle chromatography used in ciple is different and is sometimes preparative-scale chromatography. referred to as digital chromatography. Single drop microextraction (SDME): The process as most often practiced A single drop of solvent (1–2 µL) sus- requires four steps: conditioning the pended in the headspace can partition sorbent; adding the sample; washing volatile analytes into the solvent; the away the impurities; and eluting the drop can be withdrawn into the syringe sample in as small a volume as possible and injected into a GC instrument. with a strong solvent. SPE can be per-
Single-ion conductivity (Ki): The formed in a variety of formats, such as single-ion conductivity is proportional cartridges, disks, pipette tips, and 96- to the concentration and the equivalent well plates, and in a variety of modes conductivity of the respective ion. such as reversed phase, ion exchange, Size-exclusion chromatography (SEC): and normal phase. It is a widely used Same as steric exclusion chromatogra- sample preparation technique. phy. Solid support: The same as support. Slurry packing: The technique most Solid-core packing: See superficially often used to pack HPLC columns porous particles. with microparticles. The packing is Solid-phase microextraction (SPME): suspended in a slurry (~10% w/v) and A technique in which a small polymer- rapidly pumped into the empty column. coated solid fiber is placed into a solu- Special high pressure pumps are used. tion or above the headspace of a solid or Snyder solvent strength parameter liquid sample; analytes will diffuse into
(E0): Solvent strength parameter in ad- the coating until equilibrium is estab- sorption chromatography; the energy of lished; for GC, the fiber containing the solvent adsorption per unit surface area sorbed sample is transferred to the GC occupied by the solvent. and the trapped analytes are thermally Soap chromatography: The earlier desorbed into the column. In HPLC, name for ion-pair chromatography. Long- solvent is used to rinse the sorbed ana- chain soaps or detergents were used as lytes for eventual injection into the LC the mobile phase additives. column; less popularly used in LC than Sol gel: Silica gel formed by the aggrega- in GC. tion of silica sol; results in type B silica Solid-phase trapping: The use of an gel with lower surface acidity, lower trace SPE cartridge or packed column to trap metal, lower surface area and porosity, specific analytes that flow through the and higher high pH stability than older device; the packing material is chosen to type A silica gels. selectively retain the analytes of interest 70 Terminology Guide October 2013 www.chromatographyonline.com and let other compounds pass through tile sample residue and partially restore unretained. column performance. Some stationary Solute: The dissolved component of a phases may be damaged by solvent rins- mixture that is to be separated in the ing or flushing. chromatographic column. May be re- Solvent selectivity: Ability of a solvent ferred to as the analyte. to influence selectivity; there is solvent Solvent: The liquid used to dissolve a strength selectivity where a change in sample for injection into a chromatogra- solvent strength (say from 5% B to phy column or CE capillary; sometimes 10% B) will change band spacing or refers to the mobile phase used in LC. solvent-type selectivity where change Solvent demixing: Occurs when two from methanol to acetonitrile as a solvents with very different strengths reversed-phase organic modifier will (A = weak solvent and B = strong sol- change band spacing. vent) are used with unmodified silica Solvent selectivity triangle: A useful or alumina; the strong solvent (B) will guide for choosing among different sol- be preferentially adsorbed by the active vents for the purposes of changing band surface of the stationary phase until it spacing; solvent selectivity is dependent is saturated; until this occurs, the weak on dipole moment, acidity, and basicity solvent (A) will be enriched (demixed) as of the solvent molecule. For details, see it travels down the column; eventually L.R. Snyder, P.W. Carr, and S.C. Rutan, when the entire column is saturated with J. Chromatogr. A 656, 537–547 (1993). B, this solvent will elute mixed with A at Solvent strength (S): Refers to the abil- the initial strength and sample compo- ity of a solvent to elute a particular solute nents are eluted with the sudden change or compound from a column. Described in solvent strength. for HPLC by Lloyd Snyder for linear Solvent effect: A solute-profile sharp- elution adsorption chromatography on ening technique used with splitless and alumina, solvents were quantitatively on-column injection. Condensed solvent rated in an eluotropic series; S varies in the column during and shortly after with modifier type, stationary phase, injection traps volatile solutes into a nar- and temperature. Less extensive data are row band. See also retention gap. available for silica and carbon adsorbents. Solvent exchange: The process of ex- Sonication: The use of ultrasound to changing one solvent that may not be create vigorous agitation at the surface of compatible with the analysis method a finely divided solid material. The direct for a solvent that is more compatible. In method uses a specially designed inert some cases, evaporation is used to re- acoustical tool (a horn or probe, called a move a volatile solvent and the sample is sonotrode) placed in sample–solvent mix- reconstituted in a different solvent. ture. In the indirect method, a sample Solvent flooding: A source of peak- container is immersed in an ultrasonic shape distortion caused by excessive bath with solvent and subjected to ul- solvent condensation inside the column trasonic radiation. Dissolution is aided during and after splitless or on-column by the ultrasonic process. Heat can be injection. added to increase the rate of extraction. Solvent flushing: A column rinsing The method is safe and rapid and is best technique that may remove nonvola- for coarse, granular material. With the www.chromatographyonline.com Terminology Guide October 2013 71 indirect method, multiple samples can Spin filter: Similar to a spin column but be done simultaneously. instead of the column packing a mem- Sorb: The process of being retained by brane filter is used; the purpose of the a stationary phase when the retention filter is to remove particulates. mechanism — adsorption, absorption. Spin tube: See spin column. partitioning — is not clear. Split injection: An injection technique Sorbent: Refers to a packing used in ad- for GC where only a portion of the sorptive chromatography LC. Common sample is directed to the column. The sorbents are polymers, silica gel, alumina, ratio of the vented volume to the injected titania, and zirconia and chemically volume is called the split ratio, which has modified materials. typical values of 100:1, 50:1, and so on. Soxhlet extraction: A well accepted Split injection tries to avoid overloading technique for the extraction of com- the column; it ensures a representative pounds from a solid sample; the sample sample reaches the column. The tech- is placed in a disposable porous container nique is simple and rugged and protects (thimble); constantly refluxing fresh the column. However, sample discrimi- condensed solvent flows through the nation is possible; splitless injections are thimble and dissolves analytes that are usually performed automatically. continuously collected in a boiling flask; Split ratio (s): The ratio of the sample special glassware called a Soxhlet extrac- amount vented to the sample amount tor is designed to perform the extraction entering the column in split injection. unattended. Higher split ratios place less sample on Specific surface area: The surface area the column. Usually measured as the of an LC packing based on measure- ratio of total inlet flow to column flow: ment by an accepted technique such s = (Fs + Fc)/Fc as the BET method using nitrogen Split vent flow rate (Fs): Carrier gas adsorption. flow rate from the split vent, measured Spherical packing: Refers to spherical at room temperature and pressure. solid packing materials. In analytical Splitless injection: Derivative of split in- HPLC spherical packings generally are jection. During the first 0.5 to 4 min of preferred over irregular particles but in sampling the sample is not split, and en- preparative work irregular particles are ters only the column. Splitting is restored often used because of their lower cost. afterwards to purge sample remaining in Spin column: A small column that the inlet. Up to 99% of sample enters the usually contains a packing material for column. Ensures higher sensitivity than sample cleanup or isolation; the sample split injections but flashback can occur is added to the column, which has a se- and a higher possibility of sample deg- lective packing material; the column, in radation is possible as a result of longer turn, fits into a small collector tube that residence time in the hot injection port. is placed in a centrifuge, and the liquids Square-well plates: 96-well plates that in the tube are separated by centrifu- have square-shaped wells instead of the gation. Spin tubes are very popular in normal round-bottom wells. handling biological samples for isolating Stagnant mobile phase: The fraction DNA, RNA, and other biocompounds of the mobile phase contained with the of interest. pores of the particle. 72 Terminology Guide October 2013 www.chromatographyonline.com
Standard addition: Process used to im- evaporates, a thin uniform film of sta- prove quantitation; necessary to have a tionary phase is left behind. pure standard of known concentration. Stationary phase: The immobile phase An unknown concentration of sample is involved in the chromatographic process. first injected to give a peak area; then to The stationary phase in LC can be a the unknown concentration a measured solid, a bonded, immobilized, or coated amount of pure compound is added. As phase on a solid support, or a wall-coated a result of the new peak area, one can phase. The stationary phase used often determine the original concentration. An characterizes the separation LC mode. alternative procedure is to add a constant For example, in LC, silica gel is used in amount of unknown concentration to a adsorption chromatography, whereas an series of standards of pure substances octadecylsilane bonded phase is used in and to plot the peak areas obtained reversed-phase chromatography. In GC, against the known concentrations of the liquid or polymeric stationary phases original standards. The slope of the line are used for liquid–liquid partitioning obtained gives the concentration of the separations, and porous-polymer, silica, unknown. alumina, or molecular sieve packings are Standards: A sample that contains used for adsorption and molecular size- known quantities of the compounds based separations. of interest. Standards are used to help Stationary phase film thickness (df): identify sample peaks by comparing The average thickness of the stationary- the time in which they are eluted to the phase film coated on the walls of an retention times obtained through the open-tubular (capillary) GC column. injection of the sample under the same Most open-tubular GC columns have conditions. For quantitation, external film thicknesses of 0.1–5 µm. standards are compounds that are used Stationary zone: To be distinguished to construct calibration curves of detec- from the stationary phase. The station- tor output (peak area or peak height) ary zone includes the stagnant mobile versus concentration; the concentration phase and the chromatographically ac- of unknowns is determined by fitting tive stationary phase. the detector output to the calibration Step gradient: See stepwise elution. curve. Internal standards are com- Stepwise elution: Use of eluents of pounds of known concentration with different compositions during the different retention times that are added chromatographic run. These eluents to the sample and relative detector re- are added in the stepwise manner with sponses between the internal standard a pump or by a selector valve. Gradi- and the unknown are compared in ent elution is the continuous version of order to quantitatively measure un- changing of solvent composition. known compounds. Steric exclusion chromatography Static coating: A technique for station- (SEC): A major mode of LC in which ary-phase deposition in open-tubular samples are separated by virtue of their columns. The column is filled with a size in solution. Also known as size exclu- solution of stationary phase and one sion chromatography, gel permeation chro- end sealed. A vacuum, heat, or both are matography, gel filtration chromatography, applied to the open end. As the solvent or gel chromatography. SEC is most often www.chromatographyonline.com Terminology Guide October 2013 73 used for polymer separation and charac- average particle diameter; current prod- terization, the separation of proteins, and ucts vary from 1.5 to 2.0 µm. the desalting of biological samples. Sulfonyl cation exchanger: A strong Sterically protected bonded phase: cation-exchange functionality found Bonded phase that has sterically pro- in resin-based packings; usually propyl- tecting bulky functional groups such SO3H; may come in other cationic forms as isopropyl and isobutyl surrounding such as sodium, ammonium, silver, and siloxane covalent surface bond; prevents calcium. attack on siloxane bond and prevents Sulfur chemiluminescence detection catalyzed hydrolysis and loss of bonded (SCD): Detection method that responds phase at low pH (< 3). to sulfur-containing compounds by gen- Stir-bar sorbent extraction (SBSE): erating and measuring light from chemi- Principle similar to solid-phase mi- luminescence. croextraction (SPME) but instead of a Supercritical fluid: The defined state coated fiber a polymer-coated stir bar of a compound, mixture, or element is used, greatly increasing the surface above its critical pressure and critical area, thus providing higher capacity temperature. and greater mass sensitivity. Similar Supercritical fluid chromatography to SPME, equilibration requires tens (SFC): A technique that uses a supercriti- of minutes. For GC, a special thermal cal fluid as the mobile phase. The tech- desorption unit is needed to handle the nique has been applied to the separation stir bar; in LC, the stir bar is usually of substances which cannot be handled rinsed off-line. effectively by LC (because of detection Straight phase chromatography: Same problems) or GC (because of the lack as normal-phase chromatography. of volatility). Examples are separations Strong anion exchanger: Anion- of triglycerides, hydrocarbons, and fatty exchange packing with strongly basic acids. GC detectors and HPLC pumps ionogenic groups (for example, tetraal- have been used together in SFC. kylammonium). Supercritical fluid extraction (SFE): Strong cation exchanger: Cation- Uses supercritical fluid, most often exchange packing with strongly acidic carbon dioxide alone or containing a ionogenic groups (for example, sulfonic). small percent of organic modifier for Strong solvent: In general, refers to more polar analytes, to extract ana- a solvent which is a good solvent for a lytes from solid materials; supercritical chemical compound; in chromatography, fluid has the diffusivity of a gas and refers to the mobile phase constituent the solvent power of a liquid; requires a that provides a higher solvent strength special SFE unit where the pressure and that causes an analyte to elute more temperature can be precisely controlled; quickly from the column; in a water- analytes are collected in a cold trap, on acetonitrile binary solvent system for an adsorbent or in a liquid; a “green” reversed-phase liquid chromatograhy, extraction technique. acetonitrile would be considered to be Superficially porous particles (SPPs): the strong solvent. Same as porous-layer bead. Recently Sub-2-µm packing: A term that refers to there has been a revival of superficially the use of porous packings below 2 µm porous particles based on smaller 74 Terminology Guide October 2013 www.chromatographyonline.com particles (1.3–5.0 µm) with thicker po- Suppression: Method to reduce the rous shells (0.3–0.6 µm); such particles background signal before detection (see give similar or better performance than electrochemical suppression, chemi- sub-2-µm particles. cal suppression, and sequential sup- Superheated water extraction: Water pression). Typically used together with is heated well above its boiling point conductivity detection. in a closed pressurized system; heating Suppressor column: In ion chromatog- changes dielectric constant and increases raphy, refers to the column placed after the solvating power such that it becomes the ion-exchange column. Its purpose is “organic-like.” It is a “green” method for to remove or suppress the ionization of extracting organic analytes from solid buffer ions so that sample ions can be matrices. observed in a weakly conducting back- Support: Refers to solid particles. Sup- ground with a conductivity detector; port can be naked, coated, or have a sometimes rather than a column, mem- chemically bonded phase. The solid brane suppressors are used. support doesn’t contribute to the liquid– Surface area: In an adsorbent, refers to liquid chromatographic process but is the total area of the solid surface as de- active for adsorptive processes. termined by an accepted measurement Support-coated open-tubular column technique such as the BET method, (SCOT): A capillary column in which which uses nitrogen adsorption. The stationary phase is coated onto a sup- surface area of a typical porous adsor- port material that is distributed over the bent such as silica gel can vary from 100 column inner wall. A SCOT column to 600 m2/g. generally has a higher peak capacity Surface coverage: Usually refers to the than a wall-coated open tubular col- mass of stationary phase per unit area umn (WCOT) with the same average bonded to an LC support. Often ex- film thickness. See wall-coated open pressed in micromoles per square meter tubular column WCOT. of surface. Sometimes %C is given as an Supported liquid extraction (SLE): A indicator of surface coverage. technique based on the principles of liq- Surrogate samples: A pure analyte that uid–liquid extraction where the aqueous is extremely unlikely to be found in any phase is supported on a bed of highly sample, and which is added to a sample purified, high surface area diatomaceous aliquot in known amounts before ex- earth (in a tube, cartridge, or 96-well traction and is measured with the same format); this aqueous phase may be procedures used to measure other sample buffered and may contain the sample components. A surrogate behaves simi- to be partitioned; the organic phase is larly to the target analyte and is most then percolated through the packed bed often used with organic analytical pro- allowing for intimate contact with the cedures. The purpose of a surrogate ana- dispersed aqueous phase. The effluent lyte is to monitor method performance collected at the exit of the column con- with each sample. tains the extracted analytes; compared Swelling or shrinking: Process where to LLE, the SLE technique is miniatur- resins and gels increase (or decrease) ized, easily automated, and provides ex- their volume because of their solvent en- cellent extraction efficiency. vironment. Swelling is dependent on the www.chromatographyonline.com Terminology Guide October 2013 75 degree of crosslinking; low cross-linking A typical example of a tailing phenom- resins will swell and shrink more than enon would be the strong adsorption of highly cross-linked resins. If swelling amines on the residual silanol groups of occurs in a packed column blockage or a low coverage reversed-phase packing at increased back pressure can occur. In ad- intermediate pH values. Tailing can also dition, column efficiency can be affected. result from injecting an excessive mass Syringe filter: A small plastic holder or sample, from badly packed columns, containing a membrane filter that has from excessive extracolumn volume, poor Luer-lock fittings at both the top and the fittings, and excessive detector volume, bottom so that it can be affixed to a sy- or slow detector response. Tailing peaks ringe (which also has a Luer-lock fitting) show an asymmetry factor greater than to pass a sample through the filtration 1.0; see asymmetry factor. media. Syringe filter diameters can range Tailing factor: U.S. Pharmacopeia mea- up to 90 mm. sure of peak asymmetry defined as the System dispersion: The contribu- ratio of the peak width at 5% of the apex tion to band broadening outside of the to two times the distance from the apex column itself; it generally refers to the to the 5% height on the short time side instrumental contributions as well as of the peak. Greater than unity for tailed other extra column contributions. With peaks. See Figure 1 and asymmetry fac- newer high efficiency columns, decreas- tor. ing the system dispersion contributions TCDD: Tetrachlorodibenzo-p-dioxin. will result in better chromatographic TCEP: Stationary phase for GC: tris-cya- performance. noethoxypropane. System peak: The system peak is the Tedlar bags: Used for grab sampling peak of the eluent ion. There is no pos- of air or other gases; Tedlar (Dupont) sibility of quantifying that peak. It is an sampling bags are a whole-air sampling unwanted peak in the chromatogram. A device for high-level volatile organic com- possible explanation of the system peak: pounds (VOCs) and permanent gases. Because of the sample injection, the ion Several EPA, NIOSH, and OSHA meth- exchange equilibrium of the eluent ions ods exist for bag sampling for a variety of get disturbed. The re-equilibration pro- applications: stationary sources emissions, cess yields in this additional peak. It ap- workplace atmospheres, ambient, indoor pears in suppressed and nonsuppressed air quality, and breath analysis. The IC, but it is pretty small with suppression. unique design of these sample bags in- Using sequential suppression minimizes corporates the sampling septum directly the system peak. in the valve (polypropylene or stainless steel construction), providing easier use T and lighter weight than other styles. Tailing: The phenomenon where the Ternary mobile phase: Mobile phase normal Gaussian peak has an asymme- consisting of a mixture of three individ- try factor greater than one. The peak will ual solvents or buffers or both. have an extended trailing edge. Tailing is Theoretical plate: A hypothetical entity caused by sites on the packing that have inside a column that exists by analogy to both a stronger-than-normal retention for a multiplate distillation column. As sol- the solute and slower desorption kinetics. utes migrate through the column they 76 Terminology Guide October 2013 www.chromatographyonline.com partition between the stationary phase inner column diameter and, ignoring and the carrier gas. Although this process stationary-phase contributions to band 2 is continuous, a stepwise model is often broadening: Hmin = (dc/2)((1 + 6k + 11k )/ visualized. One step roughly corresponds (3(1 + k)2 ))1/2 to a theoretical plate. Theoretical plate height, reduced (h): Theoretical plate height (H): The dis- Used to compare efficiencies of different tance along a chromatographic column columns. A reduced plate-height value that corresponds to a single theoretical of 2 or less at the optimum velocity is plate. H = L ⁄N where L is column length considered to be a well-packed column. and N is the number of theoretical plates. For packed columns: h = H/dp. For open- A carryover from distillation theory; a tubular columns: h = H/dc measure of efficiency of a column. For Theoretical plate number (N): The a typical well packed HPLC column, H number of theoretical plates measured in should be about 2–3 dp for 5-µm par- a column. A concept described by Mar- ticles, usually in the range of 0.01–0.03 tin and Synge. Relates chromatographic mm; modern superficially porous and separation to the theory of distillation. sub-2-µm particles sometimes show plate The length of column that corresponds heights of less than 2 dp. In open-tubular to a single theoretical plate relating to column GC, H should be between 0.5–2 this concept is called the plate height or times the column inner diameter. HETP height equivalent to a theoretical plate. is a deprecated term for the plate height. The larger the plate number, the more The combined van Deemter–Golay equa- theoretical plates the column possesses. tion gives the theoretical plate height for a A typical well-packed HPLC column chromatography column: H = A + B/u– +u– with a 5-µm porous packing in a 15-cm
(CM + CS) where A is the contribution due column of 4.6-mm i.d. should show to eddy diffusion and multipath flow and 10,000–12,000 plates, which is the same B is the contribution from longitudinal number of plates for a 5-cm column of solute diffusion in the mobile phase. The the same internal diameter packed with C terms are related to the effects of diffu- sub-2-µm particles or superficially po- sion on mass transfer; CM in the mobile rous particles. A typical 25-m, 0.25-mm phase and CS in the stationary phase. See i.d. open-tubular GC column with a thin A term, B term, C term, Golay equa- stationary-phase film of 0.25 µm or less tion, and van Deemter equation. should exhibit 50,000 theoretical plates Theoretical plate height, minimum or more. The theoretical plate number is
(Hmin): The minimum of the van Deem- calculated from a chromatogram as fol- 2 2 ter curve that results from a plot of H lows: N = 16(tR/wb) = 5.54(tR/wh) where – versus u (LC) or H versus u (GC). This wb is the width at the peak base and wh is value represents the most theoretical the peak width at half-height. See theo- plates that can be obtained for a certain retical plate height. column and mobile phase system. Usu- Theoretical plates, effective (Neff): The ally occurs at excessively slow flow rates. true number of theoretical plates in a col- Also known as the optimum plate height. umn. The number of effective theoretical For well-packed columns it is typically plates corrects theoretical plates (N) for 2 2–3 times the particle diameter; for hold-up volume: Neff = 16(t′R/wb) where open-tubular columns 0.5–2 times the t′R is the adjusted retention time and wb www.chromatographyonline.com Terminology Guide October 2013 77 is the peak width at base. It is a better Time-integrated sampling: In gas figure of merit than simple plate number sampling, to obtain a more representa- when comparing devices of very different tive sample requires time-integrated geometries and phase ratios; sometimes sampling. A flow restrictor is used to referred to as effective plate number. spread the sample collection flow over a
Theoretical plates, required (Nreq): specific time period to ensure an “aver- Number of theoretical plates required age” composited or time-weighted aver- to yield a particular resolution (R) at a age (TWA) sample. A TWA sample will specific peak separation (α) and reten- accurately reflect the mean conditions of 2 2 tion factor (k): Nreq = 16R (α/(α – 1)) the ambient air in the environment and ((k + 1)/k)2 is preferred when, for regulatory or health Thermal desorption: The use of heat to reasons, a typical exposure concentration desorb analytes from SPME fibers, an is required for a situation that may have SBSE bar, or solid matrices placed in a high variability, as in an occupational thermal desorption tube. setting. Thermal extraction: Titania: Uses high temper- TiO2, is an uncommon adsor- atures (below pyrolysis temperatures) to bent used in adsorption chromatography; extract stable analytes from porous solid also used as an SPE sorbent primarily for matrices; samples are placed in thermal removal of phosphorous-containing com- desorption tubes just as in thermal de- pounds such as phospholipids. sorption. TMS: Trimethylsilyl (a chemical deriva- Thermal-conductivity detection (TCD): tive). In LC, the TMS group is frequently A thermal-conductivity detector mea- found on endcapped silica gel-based col- sures the differential thermal conductiv- umns. ity of carrier gas and reference gas flows. Tortuosity (tortuousity factor) (ω): A Solutes emerging from a column change property of a packed column that con- the carrier-gas thermal conductivity and trols the inhibition of longitudinal dif- produce a response. TCD is a universal fusion of the solute as it diffuses along detection method with moderate sensi- the column axis. The B term in the van tivity. Deemter equation is proportional to the Thermally tuned tandem column chro- tortuousity. matography (T3C): A form of LC in Total mobile-phase volume (Vt): In which two columns with distinctly dif- SEC the total volume of mobile phase ferent selectivities are placed in tandem in the column. The same as VM. Also and operated at two different tempera- known as the totally included volume. tures so as to optimize the resolution and/ Total permeation volume (Vp): The or speed of analysis. A common eluent retention volume on an SEC packing is used in both columns and the entire where all molecules smaller than the sample passes through both columns smallest pore will elute. In other words, and is detected with a single detector. It at Vp all molecules totally permeate all of is not a two-dimensional technique in the pores and are eluted as a single peak. that each sample component gives only Total porosity (εT ): The ratio of the total a single peak. volume of mobile phase in the column to Thermionic specific detection (TSD): the total column volume: εT = VM/Vc = See nitrogen–phosphorus detection. εe + εi(1 – εe) 78 Terminology Guide October 2013 www.chromatographyonline.com
Totally porous packing: A stationary characterized by chaotic and stochastic phase that is a porous matrix. Solutes property changes. Flow in which the ki- penetrate the porous to interact with the netic energy dies out as a result of the ac- stationary phase. tion of fluid molecular viscosity is called Trace enrichment: Technique where laminar flow. Although there is no theo- trace amounts of compounds are re- rem relating the nondimensional Reyn- tained on an HPLC or precolumn olds number (Re) to turbulence, flows at packing out of a weak mobile phase Reynolds numbers larger than 4200 are or solution and then are eluted by the typically (but not necessarily) turbulent, addition of a stronger mobile phase in whereas those at low Reynolds numbers a concentrated form. The technique usually remain laminar. has been most successfully applied in Turbulent flow: A form of fluid motion the concentration of trace amounts of in which the flow ceases to be smooth hydrophobic compounds (for example, and steady, and becomes chaotic and polynuclear aromatic hydrocarbons) out fluctuates with time. It is characterized of water using a reversed-phase packing. by a pressure drop significantly higher A strong solvent such as acetonitrile than that which would be extrapolated serves to elute the enriched compounds. from the laminar region to achieve the Trapping: Process of using a solid mate- same volumetric flow rate. rial (such as silica gel, polymer, or in- Turbulent flow chromatography: organic sorbent) or liquid solution to Chromatography performed at very physically or chemically retain solutes of high linear velocities with large particles, interest from a diluted stream of liquid if present, under conditions using high or gas. Frequently used to concentrate Reynolds numbers. At these conditions analytes for more sensitive analysis. the H versus u curves show a decrease Trennzahl (TZ): See separation num- in H with increase in u. Turbulent flow ber. chromatography can be used for separa- Triethyl amine: A very common addi- tion or sample preparation. tive used to block silanol groups in re- Two-dimensional chromatography: A versed-phase LC when separating basic procedure in which part or all of the sep- analytes. arated sample components are subjected Trifluoroacetic acid: A very common to additional separation steps. This can mobile phase additive in reversed-phase be done by conducting a particular frac- LC for peptides and proteins. Also a tion eluted from the first column into derivatization reagent for amines and a second column or system having a carboxylic acids. different separation characteristic. It Tryptic digestion: A method of selec- includes techniques such as two-dimen- tively and reproducibly dissecting pep- sional thin-layer chromatography using tide chains of proteins to yield a char- two eluent systems, where the second acteristic pattern of smaller units that eluent is applied after rotating the plate allows analysis of the parent protein by through 90°. This also includes LC or gradient elution reversed-phase liquid GC followed by GC, or one LC mode chromatography. followed by a different mode — for ex- Turbulence: In fluid dynamics, turbu- ample, reversed-phase LC followed by lence or turbulent flow is a flow regime SEC. www.chromatographyonline.com Terminology Guide October 2013 79
Two-dimensional electrophoresis: cess is used for purifying and concen- Two-dimensional gel electrophoresis, trating macromolecular (103–106 Da) abbreviated as 2DE or 2D electropho- solutions, especially protein solutions. resis, is a form of gel electrophoresis Ultrafiltration is not fundamentally commonly used to analyze proteins. different from microfiltration, nano- Mixtures of proteins are separated filtration or gas separation, except by two properties in two dimensions in terms of the size of the molecules on 2D gels. 2D electrophoresis be- it retains. Ultrafiltration is applied gins with 1D electrophoresis but then in cross-flow or dead-end mode and separates the molecules by a second separation in ultrafiltration undergoes property in a direction 90° from the concentration polarization. first. In 1D electrophoresis, proteins Ultrasonic sieving: Used for the accel- (or other molecules) are separated in eration of sieving processes alternatively one dimension, so that all the proteins or complementary to the classical low will lie along a lane; the molecules are frequency vibrators. Especially useful for spread out across a 2D gel. Because very fine powders where ultrasound is it is unlikely that two molecules will often the only possibility to enable the be similar in two distinct properties, sieving process at all. molecules are more effectively sepa- Ultrasonication: The irradiation rated in 2D electrophoresis than in of a liquid sample with ultrasonic 1D electrophoresis. (>20 kHz) waves resulting in agita- Type A silica: Silica gel formed by gell- tion. Sound waves propagate into ing soluble silicates; generally higher the liquid media result in alternating acidity, higher surface area and poros- high-pressure (compression) and low- ity, more trace metals, poorer high-pH pressure (rarefaction) cycles. Dur- stability than Type B silicas. ing rarefaction, high-intensity sonic Type B silica: See sol gel. waves create small vacuum bubbles or voids in the liquid, which then U collapse violently (cavitation) dur- Ultrahigh-pressure liquid chroma- ing compression, creating very high tography (UHPLC): Ultrahigh-pressure local temperatures; several regulatory liquid chromatography is often used methods for environmental samples loosely for any separation performed (for example, soils or solid waste) at pressures greater than provided by specify ultrasonication. conventional pumps (400 bar); origi- USP categories for chromatographic nal meaning was for separations in the columns: United States Pharmacopeia 20,000 psi+ range. characterizes columns for use in their Ultrafiltration: Variety of membrane HPLC methods by an “L” designation: filtration in which hydrostatic pres- L1 = octadecylsilane, L7 = octylsilane, sure forces a liquid against a semiper- L8 = aminopropyl, and so on. For GC meable membrane. Suspended solids columns, a “G” designation is used: and high-molecular-weight solutes G1 and G2 are dimethylpolysiloxane are retained, and water and low-mo- columns, G3 is a 50% phenylmethyl– lecular-weight solutes pass through polysiloxane column, G16 is a polyeth- the membrane. This separation pro- ylene glycol column, and so on. 80 Terminology Guide October 2013 www.chromatographyonline.com
UV–vis detection: The absorbance of van Deemter equation: An equation light is the signal for measuring the used to explain the band broadening chromatogram. There are four differ- in chromatography. The equation rep- ent ways of applying UV–vis detection resents the height of a theoretical plate in IC: direct UV–vis, indirect UV–vis, (H) and has three terms. The A term is UV–vis after postcolumn reaction, and used to describe eddy dispersion (dif- UV–vis after precolumn reaction. fusion) that results from axial velocity heterogeneity. The B term is for the V contribution from molecular diffusion Vacancy chromatography: Technique or longitudinal diffusion for the solute where a mobile-phase additive causes a while passing through the column. The positive detector signal output. When C term is the contribution from inter- a solute is eluted from the column, it phase mass transfer and allows for the dilutes the signal and yields a negative finite rate of transfer of the solute be- peak (“a vacancy”). The technique has tween the stationary phase and mobile been mostly been applied to single col- phase. In its simplest representation it umn ion chromatography where mo- is expressed as follows: H = A + B/u + bile phases such as citrate and phthal- C u. The van Deemter equation applies ate buffers absorb in the UV. When a to packed columns both for LC and for nonabsorbing anion is eluted it dilutes GC. The related Golay equation ap- the UV-absorbing background and plies to open-tubular or capillary GC causes a negative peak; the detector columns. See Golay equation. output leads are usually reversed so that Velocity (u): The same as velocity, lin- the chromatogram looks normal. The ear. technique has also been used in CE for Velocity, average linear (u–): The aver- detection. age speed at which a molecule of GC Vacuum compensation: Method of carrier gas or LC liquid mobile phase – carrier-gas pressure control in GC with passes through a column: u = L/tM the column exit at mass-selective de- where L is the column length and tM tector vacuum levels. Enabling vacuum is the hold-up or unretained peak time. compensation adjusts the column inlet Velocity, column outlet (uo): In GC, pressure to maintain a set flow or ve- the carrier gas velocity at the column locity when the column exit is not at outlet. Equal to the average carrier gas room pressure. velocity divided by the compressibility Vacuum filtration: – Using a vacuum to correction factor: uo = u/j. The carrier help pull liquids through a filter; espe- gas expands as it pass through the col- cially useful for viscous liquids or very umn from the inlet to the outlet pres- fine, low porosity filters. sure, which causes the local carrier-gas Vacuum manifold: A manifold de- velocity to increase along the column. signed for SPE cartridges and SPE The outlet velocity is always greater disks that uses a vacuum to pull liquids than the average velocity. See com- through the packed beds; pressurized pressibility correction factor. manifolds are also available. Vacuum Velocity, interstitial (ue): The ac- manifolds can process multiple samples tual velocity of the eluent as it moves from ranging from 8 to 24 at a time. through the column flowing around www.chromatographyonline.com Terminology Guide October 2013 81
the particles: ue = F/(Ac εc). The inter- operated unpacked but with the same stitial velocity is the basis for computa- flow rate: us = F/Ac tion of the reduced velocity. Velocity, zone (uz): The velocity of Velocity, linear (u): The velocity of travel of the solute zone: uz = u/(1 + k) the mobile phase moving through the = L/tR column. Expressed in cm/s. In LC it is Viscosity (η): Also referred to as mo- directly related to column flow rate by bile phase viscosity. The viscosity of the cross-sectional area of the column the mobile phase varies with the tem- and is determined by dividing the col- perature of the column. Column back umn length (L) by the retention time pressure is directly proportional to of an unretained compound: u = L/tM. solvent viscosity. Low-viscosity mobile In GC, the speed at which carrier gas phases generally give better efficiency moves through the column usually is than less viscous ones because diffu- expressed as the average linear velocity sion coefficients are inversely related to account for carrier-gas compressibil- to solvent viscosity. For example, in ity. See hold-up time; velocity, aver- reversed-phase LC, column efficiency age linear; velocity, column outlet. is higher with acetonitrile as an organic
Velocity, mobile phase (uM): The ve- modifier than with isopropanol which locity at which the liquid mobile phase is more viscous. In GC, the viscosity percolates through the bed of particles of the gaseous mobile phase increases in an LC column: uM = L/tM. See veloc- with temperature, which causes the ity, linear; velocity, average linear. carrier-gas flow rate and linear veloc-
Velocity, optimum linear (Uopt): The ity to decrease during temperature pro- mobile-phase velocity corresponding to gramming if the inlet pressure is held the minimum theoretical plate height, constant. Different GC carrier gases ignoring stationary-phase contributions such as nitrogen, helium, or hydrogen to band broadening. In open-tubular have different viscosities. 2 Void: GC: uopt = 8 (DG/dc) ((3(1 + k) )/(1 + The formation of a space or gap, 6K + 11k2))1/2 usually at the head of the column, Velocity, reduced (ν): Along with the caused by a settling or dissolution of reduced plate height, the reduced veloc- the column packing. A void in the col- ity is used to compare different chro- umn leads to decreased efficiency and matographic columns. It relates the loss of resolution. Even a small void solute diffusion coefficient (DM) in can be disastrous for small particle the mobile phase to the particle size of microparticulate columns. The void the column packing (dp): ν = u dp/Dm can be removed sometimes by filling where u is the interstitial mobile-phase it with glass beads or the same porous linear velocity in packed columns, or packing used in the remainder of the the average carrier-gas linear velocity column. in GC. For open-tubular columns, the Void time: See hold-up time. column internal diameter is used in- Void volume: See hold-up volume. stead of the particle diameter. Volume, liquid phase: See volume,
Velocity, superficial (us): The hy- stationary phase. pothetical velocity the mobile phase Volume, mobile phase (VG or VM): would have if the same column were For wall-coated open-tubular columns 82 Terminology Guide October 2013 www.chromatographyonline.com
(WCOT), ignoring the stationary phase Weak solvent: In general, refers to a 2 film thickness (df = 0): VG ≈ L(πdc )/4 solvent that is a poor solvent for a par- Volume, stationary phase (VL or VS): ticular chemical compound; in chro- Volume of the liquid stationary phase matography, refers to the mobile phase contained in the column. The ratio of constituent that provides a low solvent the mobile-phase volume to the station- strength and causes an analyte to be ary liquid-phase volume is the phase eluted more slowly from the column. In ratio of a GC column. See phase ratio. a water–acetonitrile binary solvent sys- tem for reversed-phase LC, water would W be considered the weak solvent; in a bi- Wall effect: The consequence of a looser nary solvent eluent would normally be packing density near the walls of a rigid the “A” solvent. HPLC column. The mobile phase has Wide-bore open-tubular column a tendency to flow slightly faster near (WBOT): Open-tubular (capillary) GC the wall because of the increased local column with a nominal inner diameter permeability. The solute molecules that dc of ≥530 μm. happen to be near the wall are carried Wilke-Chang equation: A semiempiri- along faster than the average of the cal equation used to estimate diffusion solute band and, consequently, band coefficients in liquids as a function of spreading results and there is a loss of molecular size of solute and solvent column efficiency. viscosity. Wall-coated open-tubular column (WCOT): An open-tubular (capillary) GC XYZ column in which a uniform stationary Xerogels: Gels used in SEC that will phase film is coated directly onto the col- swell and shrink in different solvents; umn wall. See also porous-layer open tu- also refers to silica-based packings that bular (PLOT) column, support-coated are prepared from acidification of solu- open-tubular column (SCOT). ble silicates to give a amorphous, high Wash step: See rinse step. surface, high porosity, rigid particle. Water dip: Indicates the hold-up time in Zero dead volume: Any fitting or com- suppressed IC. Usually a negative peak ponent in which all of the volume is that corresponds to the volume of sample swept by the eluent. See dead volume. water. The area depends on the difference Zirconia: Porous zirconium oxide; used in concentration of the eluent anions be- as a chromatographic sorbent usually tween eluent and sample. The water dip coated or bonded with polymeric or- is large if the sample is almost ultrapure ganic phase. water. If the sample is diluted with eluent Zone: See band. there will be almost no water dip. Zwitterionic packing: A packing ma- Weak anion exchanger: Anion- terial for HPLC that carries both posi- exchange packing with weakly basic tive and negative charges on its surface; ionogenic groups (for example, amino zwitterionic packings are useful in the or diethylaminoethyl). HILIC mode. Weak cation exchanger: Cation-ex- Zwitterions: Compounds that carry change packing with weakly acidic iono- both positive and negative charges in genic groups (for example, carboxyl). solution. 36613100325_2536500.pgs 09.14.2013 03:39ADVANSTAR_PDF/X-1a magenta black cyan yellow for biologics development, samples— complex for sensitivity and capacity peak highest the ensures with Scientific path Thermo flow low-dispersion biocompatible The information. structural increased Bio for columns resolution UHPLC high new Scientific Thermo UltiMate new The separations. fast and results, accurate data, extensive requiring demanding, be can laboratories biopharmaceutical in workflows development Drug HPLC and UHPLC © 2013 Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are identification, and QC the property of Thermo Fisher Scientifi c Inc. and its subsidiaries. ™ 3000 BioRS system is specifically designed to power the proteins, peptides, and biotherapeutics. and peptides, proteins, now in one system ™ ™ Dionex • Streamline yourbiopharmaceuticalanalyticsworkfl ow: ™ Viper ™ Fingertight fitting technology technology fitting Fingertight thermoscientific.com/BioRS ™ ™ Dionex ™
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