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Handbook of HPLC

Danilo Corradini, Terry M. Phillips

Micro-HPLC

Publication details https://www.routledgehandbooks.com/doi/10.1201/EBK1574445541-c3 Heather Kalish, Terry M. Phillips Published online on: 14 Jul 2010

How to cite :- Heather Kalish, Terry M. Phillips. 14 Jul 2010, Micro-HPLC from: Handbook of HPLC CRC Press Accessed on: 02 Oct 2021 https://www.routledgehandbooks.com/doi/10.1201/EBK1574445541-c3

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The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The publisher shall not be liable for an loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 Fused silica is advantageous to glass-lined stainless steel because it is flexible and inert, but it but wall. inert, column inner and the flexible to bonding is chemical itfor allow can because steel stainless glass-lined to advantageous is silica Fused (PEEKSil). PEEK steel, silica–lined stainless fused glass-lined or (polyetheretherketone—PEEK), silica, plastic fused pressure-resistant mainly from manufactured are nano-columns and illary 3.5 3.8 3.7 3.6 3.3 3.2 3.1 Contents umns are manufactured from. col-manufactured the are materials umns the dictate to tends column the of size 3.1. the Additionally, Figure in seen as 0.5 to 0.1 from size in range columns. review of size the on the their based nitions in volumes (as sizes column and shown Tablein 3.1), but Saito coworkersand provided defi-narrower ( sensitivity.Micro-HPLC detection backs includingHPLC, to high standard solventdraw- several consumption, large sample quantity,are and However,decreased there laboratories. analytical commercial and technique academic both separation in used standard a become has (HPLC) chromatography liquid High-performance 3.1 References 3.4 ir-oun rne n ie rm . t 1.0 to 0.5 from size in range Micro-columns

3.8.3 Applications of μ Detectors ElutionGradient Systems 3.5.7 3.5.6 3.5.5 3.5.3 3.5.2 3.5.1 Phases Stationary 3.4.3 3.4.2 Columns Advantages of μ μ Introduction 3.8.2 3.8.1 3.5.4 3.4.1 3 Introdu HPLC SystemsHPLC

...... Other Applications Other Multidimensional Liquid Chromatography Pre-Concentration Method by Sol–Gel the Monoliths Prepared Polymerization by Photo-Initiated Monoliths Prepared by Alkylation Porogen Surface and Monoliths Alteration Prepared Incorporation Nanotube by Carbon Monoliths Prepared by PorogenMonoliths Alteration Prepared Monolithic Micro-Particulate Packed Semi-Packed TubularOpen ...... Heather Kalish and Terry M. Phillips Micro-HPLC ...... c ...... t HPLC SystemsHPLC i ...... on HPLC ......

...... mm i.d., and nano-columns range in size from 0.01 to 0.1 to 0.01 from size in range nano-columns and i.d., mm 1 ...... Micro-columns are made from stainless steel tubing, while cap- while tubing, steel stainless from made are Micro-columns ...... μ . HPLC) is a term that encompasses a broad range of sample of range broad a encompasses that term a is HPLC) ......

mm internal diameter (i.d.); capillary columns columns capillary (i.d.); diameter internal mm 1 ...... 84 1 Commercially available capillary and and capillary available Commercially ...... 84 ...... 83 ...... 84

mm i.d., mm 84 80 92 92 77 88 88 87 77 82 82 82 83 78 91 81 81 81 81 Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 packed with 10–100 with packed and stainless steel columns with i.d. of 0.5–1.0ofi.d. with columns steel stainless and on1.0 materials, and packing supports. Ishii and coworkers continued to work with 0.5 work with coworkers to continued and Ishii supports. packing and construction materials, column size, column the of reduction the in advancements significant made groups research numerous nextdecade, the Overfor LC. weresuperior columns packed the that indicated but investigated columns made ofbut Tefloninvestigated made 30 with packed columns that were slurry licular particles. licular 78 Today nearly all of the major HPLC companies offer a offer companies HPLC major the of all Today nearly 3.2 Technologies(Kyoto, CA),Instruments Scientific(Santa Clara, Shimadzu Japan). and Agilent MA), (Milford, Corporation Waters CA), (Dublin, Technologies Eksigent CA), (Sunnyvale, Packings Dionex/LC CA), (Vista, MicroTechScientific author’slaboratory: the in used following columns the including companies of number a availablefrom are nano-columns FIG to modify a standard instrument to accept micro-bore columns. In our laboratory, we routinely routinely we laboratory, our In columns. use the micro-bore accept to instrument standard a modify to in a series of articles examining the separation of nucleotides.of separation the examining articles of series a in μ 30 long, and (E) A commercial HPLC size exclusion preparative column measuring 7.5 exclusion measuring size column HPLC preparative Acommercial long, (E) and detector interface, and hardware design, which are the subject the of design, review numerous which are hardware and articles. interface, ­detector composition, column of areas the in made have advancements been significant decades, nextthree measuring 75 measuring by 10 HPLC column measuring 100 measuring column HPLC

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cm cm m, m, Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 biomedically important analytes from clinical samples. clinical from analytes important biomedically Micro-HPLC et Sajonz research, forproteomics used are instruments these of majority the 3.4) Although . (Figure Technologies Eksigent from system 2-D the 3.3)and , (Figure Micro-TechScientific from system μ FIG FIG injection port, columns, switching valves, and four of the eight pumps on the Extreme Simple system. Extreme onthe eight pumps of four valves, the and switching columns, port, injection system. Ultimate valves onthe switching and columns, port, (B)ics analysis. injection Aclose-up of the the ultimate “lab-on-a-chip.” Shintani et al. et Shintani “lab-on-a-chip.” ultimate the racemates, these investigators demonstrated rapid analyses, which were comparable to those those to comparable were which analyses, by obtained rapid demonstrated test investigators of panel these a Using ­racemates, separations. “real-time” near providing separations, chiral phase normal Scientific Upchurch Scientific (Oak as such companies from components available commercially using systems tory-built was described was by described Simpson and Brown, with with a laser-induced fluorescence(LIF) detector and usedto measurecoupled was neuropeptidessystem in This 3.5). sub- scientific (Figure Upchurch from obtained being components latter the micro-column; a and port, microinjection a MA), Holliston, Inc., Apparatus, (Harvard pumps described based on dual microdialysis syringe pumps (CMA Microdialysis, been Chelmsford, MA) or has dual syringe system complete A columns. guard from built columns micro-bore accept to samples. (A) HPLC work. The configuration shows the instrument setup as an eight-pump system (B) A close-up of the the of close-up (B) A system eight-pump an as setup instrument the shows configuration The work. HPLC

al. The advent of microfabrication greatly improved greatly microfabrication of advent The n diin o h cmecal aalbe ytm, eea atos ae ecie labora- described have authors several systems, available commercially the to addition In URE URE 24 sd h Esgn Epes eight-channel Express Eksigent the used 3.3 3.2 (A) 26 A further modification of this system was built to perform immunoaffinity isolations of immunoaffinity perform to built system was this of modification further A

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(A) Ultimate The (A) The Extreme Simple 4-D system by Micro-Tech Scientific, used in our laboratory for laboratory our in used Scientific, Micro-Tech by system 4-D Simple Extreme The (A)

Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems HPLC micro-bore laboratory-built reported first WA). Harbor, the of One μ HPLC system by Dionex/LC Packings, used in our laboratory for system HPLC used in proteom- our laboratory Packings, by Dionex/LC 25 which was a simple ofadaptation HPLC systema standard 28 built a multichanneled multichanneled a built μ HPLC system to perform multiparallel, fast fast multiparallel, perform to system HPLC μ 27 (B) HPLC design and will eventually provide eventually will and design HPLC (B) μ HPLC for the separation of separation the for HPLC microliter ­microliter 79 Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 entire entire chip-based systemand colleagues. bychip-based has described Lazar been tip.nanospray similar A integrated an and custom-builtinterface, a spectrometer, mass ion-trap an separation chromatographic on reversed-phase wereparticles, analytes the separated usingdetected to compare well with a conventional HPLC in the fractionation of a protein tryptic digest. tryptic well of fractionation aprotein aconventional compare to with the in HPLC protein analysis. (B) A close-up of the injection port, columns, and switching valves on the NanoLC system. NanoLC valves onthe switching and columns, port, (B) analysis. injection Aclose-up of the protein 80 μ 3.3 al. et Yin Further, optics. fiber on based detector (UV) ultraviolet multichannel a driven by a single HPLC pump and a columns chip-based microinjection monolithic device.of Detection array was employed an systemachieved This with sample. same the within analytes multiple FIG FIG samples in in samples system. The injection valve, gradient system, and detector were controlled via aLabView via were controlled interface. detector and system, valve, injection gradient system. The and mixer.an Upchurch nano-flowgradient (B) A of picture thetwothe gradient that pumps comprise syringe s uh mle o a ailr column. capillary a on smaller much is (dilution) band-broadening volumetric the since ratio, signal-to-noise improved drastically a with sample. original the in contaminants from columns provideanalysisand time all ofprotection analytical small-bore large over-reduce loading the volumes accommodate can dramatically can that pre-columns These HPLC has some significant advantages over traditional HPLC. The delivery of reliably small small reliably of delivery The HPLC. traditional over advantages significant some has HPLC

Biological samples, which are often available in limited amounts, can be separated and detected detected Biologicaland separated be can samples,amounts, often availablewhich are limited in URE URE a μ 3.5 3.4 HPLC system on a microfabricated chip made from laminated polyimide layers. polyimide Following laminated system from chip made HPLC on a microfabricated d v μ

anta (A) HPLC is often obtained using pre-columns, which concentrate large sample volumes.sample large concentrate which pre-columns, using obtained often is HPLC (A) The NanoLC 2-D system by Eksigent Technologies, used in our laboratory for laboratory our in used Technologies, Eksigent by system 2-D NanoLC The (A) (A) A laboratory-built laboratory-built A (A) 1 g (A) es o f

µ H P L μ C HPLC system with dual syringe pumps, an electronic injector port, port, injector electronic an pumps, syringe dual with system HPLC S yste 1 Smaller columns also require less solvent which is an an is which solvent less require also columns Smaller m (B) (B) s 30 The The μ HPLC systemHPLC was reported Handbook of HPLC Handbook 29 developed an an developed μ HPLC HPLC Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 that is bound to the inner wall of the capillary rather than to the particles packed inside. particles to the than rather of capillary wall the inner is to the bound that few reports exist on the use of this column in LC after the early 1990s. early the after LC in column of exist use few onthis the reports expressed in dimensionless magnitudes of reduced plate height, plate reduced of magnitudes dimensionless in expressed often are characteristics kinetic the while properties, thermodynamic the for parameters sentative into glass tubes. The packed tubes are then drawn on a glass drawing machine so that the inner inner the that so 50–200 is machine diameter drawing glass a on drawn then are tubes packed The tubes. glass into flow ­ economical benefit, from thecost the from benefit,solvent ofboth economical the andsolvent disposal. Micro-HPLC o vi mn o te rbes f pn uua clms hl sil eann te datgs of advantages the retaining still while columns tubular open of way feasible problems more the of a offer many avoid columns, to semi-packed than prepare to easier being columns, Packed p 3.4.3 coworkers. and were developed by to packedas referred micro-capillaries, Tsuda Semi-packed originally columns, s 3.4.2 10–20 typically are columns tubular Open o 3.4.1 In 3.4 these columns are often discussed in terms of thermodynamic properties and kinetic character- kinetic and factor, retention properties The istics. thermodynamic of terms in discussed often are columns these size. column even reduced can the due across and neglected fast to transfer be heat reduced are separations with improved results and fewer problems. In narrow bore columns, mismatches thermal to the extensive the to cover review that area. articles each reference is brieflydiscussed making types columns while packed, packed. and Each of three these in broadening band of cause one HPLC. eliminating homogeneous, more becomes structure packing The region. wall packed loosely the by dominated becomes structure support the and ­disappears diameter of diameter 10 havea to needed columns tubular open that calculated Gilbert and effective,Knox be to columns average out any remaining cross-column differences in flow in andretention. differences average cross-column out any remaining their research, many of which are referenced in a reviewal. a et bySaito in referenced are whichof many research, their method this have good mass sensitivity. their Several other research groups used samples, open columns tubular in small require columns the resolving power orequal because exceed conventional HPLC cells columns, and the detectors typically used with single of analysis the for advantageous and Jorgenson and limited quantities. limited tivity of column the more cost effective available are and allows that to use in researchers materials selec- the on effects its and material packing the of structure chemical the between correlation the investigating makes size development reduced the promote The columns. ofnovel umns analytical coworkers establish that semi-packed columns are effective in significantly reducing the plate the reducing significantly in sampleheights, capacities but effective atacost of decreased are columns semi-packed that establish coworkers As the column diameter to particle diameter ratio decreases below 6, the core support region support core the 6, below decreases ratio diameter particle to diameter column the As Three broad categories define the analytical columns used in used columns analytical the define categories broad Three μ μ HPLC systems haveHPLC to exploit liquid chromatographic enabled in also researchers temperature HPLC, there are numerous types of columns used. The comparison and characterization of characterization and comparison The used. columns of types numerous are there HPLC, resistance factor,resistance ϕ Colu 31 Additionally, an analyte can diffuse easily across a column’s cross section, which can can which section, cross column’s a across easily diffuse can analyte an Additionally, d e k c a e n e p m i

-P m T 34 μ 17,36 d e k c a effectively used open tubular columns to analyze single cells. The method was method The cells. single analyze to columns tubular open used effectively m or less ns u b 1 These columns are prepared by packing particles with diameters of 10–100of diameters with particles packing by prepared are columns These r a l u μ m and roughly two to three times the diameter of the particles. the of diameter the times three to two roughly and m . 23 35 and operating columns ofcolumns operating and 10 k , selectivity,, α μ , and the peak asymmetry asymmetry peak the and , m in diameter and characterized by a stationary phase phase stationary bya characterized and diameter in m 37 and significantly analysis significantly longtimes. and μ m or less proved thus to be quite difficult, h , separation impedance, impedance, separation , μ PC Oe tblr semi- tubular, Open HPLC: A 1 However, tubular for open s are believed to be repre- be to believed are 32 1 Finally, col- smaller 37 34 Tsuda and and Tsuda Kennedy Kennedy 38 E , and and , 33 μ 81 m Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 have written an extensivehave an work. related written reviewand techniques of these al. et Lancas tubing. column the to reservoir external an from material packing the transferring in columns. the prepare help to to used used been have liquids, or dioxide, technique carbon supercritical packing gases, including methods, Several the is columns capillary of preparation the in two μ have in most widely the been utilized sintering process, and (4) organic hybrid materials. Of the four techniques mentioned above, a by the first capillary a in material packing particulate (3) porous process, fusing sol–gel a using network 44 Karlsson and Novotny were successful in extremelyobtaining high efficiencieswith acolumn i.d. of columns has focused capillary on the reduction of the i.d., which leads to high separation efficiency. ation, and comparison of packed capillary columns with conventional-size with columns columns, of capillary packed comparison ation,and developed columns monolithic polymeric or phase stationary the as particles silica alkylated with packed ies are found in the literature including the different methods used to pack capillary columns, capillary pack to used methods different the including literature the foundin are micro-columns. 82 Monolithic stationary phases are increasingly considered as a viable alternative for micro- 3.5.2 of reversed-phasethe and phases. preparation characterization stationary extensivelyreviewedhave whoDorsey, and Doyle to according support silica a of surface the to stationary of type bonds siloxaneby common attached species hydrophobic nonpolar, most organic is LC reversed-phase The for phase affinity. or exchange, ion phase, reversed or normal be it whether out, carried being chromatography of type the to specific highly is to and column used the phase pack stationary the by influenced also is separation chromatographic a of outcome The s 3.5 while Hsieh and Jorgenson further reduced the column i.d. to 12–33i.d. to column the reduced Jorgenson Hsiehand further while 3.5.1 columns in HPLC. in columns is easy to pack long columns, lengths of 1 of lengths columns, longpack to easy is values, therefore achieving over 100,000 theoretical plates. theoretical 100,000 over achieving therefore values, h popular popular supports are those that are based on poly(styrene) with cross-linked divinylbenzene, including increased wall smoothness, and good optical characteristics. optical good and smoothness, wall increased including reasons, several for conventionalcolumns in used steel stainless to superior is which silica, fused columns. eliminating the need for retaining frits and enhancing the column’s mechanical stability during during stability column’smechanical the changes. pressure enhancing and frits retaining for need the eliminating sol–gelmethod, monomer, cross-linker, and porogen. and cross-linker, monomer, be can easily adjustedand column permeability surfaces, by selecting functionalize the appropriate organic polymers used to prepare monolithic columns, polymers organic to used prepare common support for normal phase LC is bare silica. bare is LC phase fornormal support common most explored,havethe been Florisil and titania, zirconia, alumina, suchas Jardy,materials while beds glass, azalacetone beads, and hydroxylated and beads, polystyrene media. azalacetone glass, or silica modified are used commonly supports the chromatography, affinity high-performance in

μ Packed capillary columns have been used successfully in in successfully used been have columns capillary Packed m. 21 : (1) polymerization of an organic monomer with additives, (2) formation of a silica-based silica-based a of formation (2)additives, with monomer organic an of (1) polymerization : 39 tat 20–50 to i.d.even column further the reduced Jorgenson successfully and Kennedy M M 18 by in-situ polymerization. Numerous reviews on both packed and monolithic columns columns monolithic and packed both on reviews Numerous polymerization. in-situ by o r c i t i l o n o i onary Phases -P 37 21 Packed columns used in in used columns Packed and a comparison of the efficiency of micro-particulate and monolithic capillary capillary and monolithic efficiencymicro-particulate ofthe of comparison a and 44 e t a l u c i t r a h 18 Furthermore, a large number of readily available chemistries can be applied to to applied be can available chemistries ofreadily number large a Furthermore, c i The monolithic column bed has a uniformly porous integral structure thus thus structure integral porous uniformly a has bed column monolithic The 44 Four approaches have been utilized to prepare continuous prepare to utilized been have approaches Four m or greater, with 5 with greater, or m HPLC monolithic column preparation. monolithic column HPLC μ PC r ete cnetoa fsd iia capillar- silica fused conventional either are HPLC 41 For ion-exchangeFor chromatography,most the 22 silica gel-basedby monolithsthe prepared 37 43 μ μ Additionally, they are made from from made are they Additionally, HPLC for a number of reasons. It reasons. of number a for HPLC m particles and still maintain low maintain still and particles m μ m. 19 37 40 32 Development in packed in Development According to Caude and An additional challenge additional An Handbook of HPLC Handbook 20 the different different the particulate ­particulate 19 42 evalu while μ m, 37 -

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 ing in molecular mass from a few hundred to more than 55,000 surpassed that of conventional of that surpassed 55,000 than columns. ­particle-packed more to hundred few a rang- from proteins mass molecular and in peptides ing of efficiency separation whose columns monolithic reversed-phase produced and process polymerization the in tetrahydrofuran/decanol using by beds column lithic mono- of morphology the altered further al. et Premstaller columns. packed micro-particulate of that surpassed performance whose beds column monolithic in resulted process polymerization the porogens as in bytetrahydrofuran use offormed and The nonporousdecanol bed particles. graphic chromato- a of that resembled morphology its that such tuned be to had monolith the of synthesis columns, so further improvements required. are so further columns, monolithic silica-based modified with compared low remains columns monolithic polymer-based Premstaller and Huber. and Premstaller by researched been has of polymers to form beds polymerization organic monolithic column the in the numerous organic polymers used to prepare monolithic columns. monolithic prepare to used polymers organic numerous the extensive an and detailing beds, monolithic column written reviewto prepare used been has article otides and double-stranded DNA fragments. DNA double-stranded oligodeoxynucle- and otides single-stranded of separation HPLC reversed-phase ion-pair, for beds graphic methacrylates, and dimethacrylates and methacrylates, polymerization of radical to free columns. particle-packed initiated acrylates, Since then, thermally alternative tempting a offered and monomer any almost using prepared be could columns shaped over unmodified poly(styrene-divinylbenzene) over unmodified columns. monolithic peptides of separation LC reversed-phase the improved ultimately alteration surface This groups. C-18 with surface alkyl the alter to ablewere al. solvent,et Huang organic an and halide, alkyl an Micro-HPLC The alteration of monoliths by carbon nanotube (CNT) incorporation (CNT) nanotube carbon by monoliths of alteration The 3.5.4 Porogenic solvent plays a keyof morphology.column role determination in 3.5.3 poly(styrene- ous bed column that incorporates both macroporosity and capacity. and macroporosity both incorporates that column bed ous novel,continu- a of preparation the reported who Frechet, and Svecfollowed by soon was report specific structure, size, and charge of the characteristics of CNT all may play a role. a play may all CNT of characteristics the of charge and size, structure, specific the that proposed is porosity, it column in changes corresponding without enhanced significantly was SWNT of incorporation after retention polymer.analyte Since the into incorporation fortheir may be drawn onto the nanotube surface or channels between nanotubes due to surface tension surface to duetherefore exhibit and effectslonger retentiontimes. capillary and nanotubes between channels or surface nanotube the onto drawn be may nanotubes (SWNT) consist of a graphene sheet rolled into a cylinder, with a typical diameter of diameter 1 typical a with cylinder, a into rolled sheet graphene a of consist (SWNT) nanotubes carbon wall Single surfaces. planar with compared as molecules hydrophobic for stronger affinity show binding to expected are CNT surface, curved their of Because columns. monolithic based polymer-efficiencies separation organic of the tune fine to alternative investigatedanother as been the rapid speed of separations that can be achieved at acceptable back pressures make this col- this make pressures back columns. applications many particle-packed to in superior format acceptable umn at achieved be can that and separations of rates, speed flow rapid high the of tolerance preparation, of ease Their acids. nucleic and oligonucleotides, columns have clearly demonstrated their ability to afford excellent separations of peptides, proteins, divinylbenzene) monolith. the increase resolutionchromatographic of peptides by octadecylating the surface of a poly(styrene- nm. The first monolithic columns reported by Hjerten et al. were based on polyacrylamides. on based were al. et Hjerten by reported columns monolithic first The 51 The challenge is maintaining their unique structure while obtaining a solubility that allows solubilitya that obtaining while structure unique their challenge maintaining is The M M t i l o n o t i l o n o

co -divinylbenzene) prepared in nanospray needle, nanospray in prepared -divinylbenzene) h h s s P P d e r a p e r d e r a p e r 49,50 50 52 Premstaller et al. first experimented with using monolithic chromato- monolithic using with experimented first al. et Premstaller Then by treating it with a solution containing a Friedel-Crafts catalyst, it by with a a solutionFriedel-Crafts Then treating containing

b b y y C P 46 n e g o r o r a and ring-opening metathesis polymerization of norbornane, polymerization metathesis ring-opening and b n o N A 49 u t o n a In order to accomplish successful separations, the the separations, successful accomplish to order In n o i t a r e t l 22 b e I n o i t a r o p r o c n 48 51 and other monomers have been been have monomers other and Huang and coworkers and Huang sought to 46 22 51 22 They noted that these rod- these that noted They or surface alkylation surface or Polymer-based monolithic Polymer-based However, efficiency the of 44 Altering the porogensthe Altering 51 Analytes Analytes 45 52 This This has has 83 47

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 20 sought ers improvetoefficiencies separation a the monolithic of columns silica-based preparing by switching valves, and high-temperature programming. switching valves, high-temperature and multiport and loops high- gradient preformed formation, of gradient exponential pumps, gradient miniaturization pressure splitting, flow including gradients reproducible and accurate deliver to is achieved by inserting a variety of devices between the pumps and the injector. While Chervet Chervet While injector. the and pumps the between devices of variety a inserting by achieved is gel method of preparation in great detail and provides an extensive list of references. of list extensive an provides and detail great in preparation of method gel C with packed columns conventional than rates flow high at performance better muchshowed rods ene glycol dimethacrylate) monolithic column was used by poly(glycidylLi coworkersand alkylated a surface to manufacture ethyl- methacrylate reaction polymerization the in porogens the of alteration and alkylation surface of combination A 3.5.5 84 The delivery of accurate and reproducible gradients in in reproducible gradients and deliveryof accurate The 3.6 technology. column in change majortechnological a as regarded be can columns inventionsilica-based monolithic ofThe 3.5.7 poly(butyl of preparation the for methylacrylate- polymerization photo-initiated a on report coworkers and Lee 3.5.6 and show potential in analyzing small amounts of proteomic samples. Continuing to modify the the may lines provide of preparation columns along these i.d. smaller similar modify well. columns as to Continuing samples. proteomic of amounts small analyzing in potential show and and concentrations and final ofthe more efficient component B. initial the and ofgradient, the 3.6:slope curvature Figure the and in seen ofgradient, shape the the as respects, three in other each from differ solve. to gradients Simple trying are researchers many does not deteriorate with time or number of injections and their reproducibility is excellent. reproducibility of their or number injections and time with notdoes deteriorate ability separation their since robust are columns resulting The formed. column monolithic the of control over greater to leads far size and length This the exposed to irradiation. are that areas those polymerization is well suited to monolith formation in spaces restricted since polymer forms only in tetramethoxysilane accompanied by phase separation in the presence ofpresence the by in water-soluble separation phase accompanied tetramethoxysilane polyorganic mers. methacrylate ethylenemethacrylate glycol monolithic columns. dimethacrylate) poly(glycidyl nonfunctionalized over proteins forresolution separation the in improvementciable or perature, the concentration of initiator. Alkylation with octadecyl linear groups showed an appre- numerous factors including using the appropriate monomer, by cross-linker, altered porogens, be the could monolith reaction polymeric tem- the of porosity The functions. retentive used widelymost the are groups alkyl since alkylation by functionalized further and solventsporogenic as toluene irpru slc skeletons. silica microporous or meso- and through-pores micrometer-sizeof consists that structure biporous a with rod silica a by Nakanishi and Soga. and Nakanishi by organic organic polymer-based monoliths, to improve and coworktheir separation Minakuchi performance. preparation of silica-based monoliths, research has been done on them, waysin modifying similar to ers derivatized ers silica-based derivatized monoliths C to incorporate 18

μ Splitting Splitting the solvent flow,delivered bythe pumps, downto the flowrequired rate fornano-HPLC particles; however, the separation efficiencies obtained were not impressive. not were however,efficiencies obtained particles; separation the m i.d. monolithic column. monolithic i.d. m 56 Grad The morphology, by phase determined separation, is solidified by gel formation,resulting in M M M t i l o n o t i l o n o t i l o n o i ent E co ehln dmtyarlt) ihn ue slc capillaries. silica fused within dimethylacrylate) -ethylene h h h s s s P P P lut 21 d e r a p e r d e r a p e r d e r a p e r The preparation of a porous silica rod by the sol–gel method was reported reported wassol–gel method bythe rod silica porous a of preparation The 54,55 i The porous silica rods were prepared by hydrolytic polymerization of hydrolyticpolymerization by prepared were rods silica porous The on S 56 57 Siouffi has published an extensive review that describes the sol– the describes that review extensive an published has Siouffi

These columns provided a high separation efficiencyand sensitivity b b b y y y P

P ystem t h h n e g o r o e o t o S l o -I s –G d e t a i t i n A 44 l e n o i t a r e t l that was copolymerized in situin was that and with dodecanol copolymerized M t e P h 18 y l o μ d o alkyl groups on the surface. HPLC systems is one of the problems that problems that of systemsonethe is HPLC

d n a m 58 n o i t a z i r e Several havemethods developedbeen S e c a f r u A n o i t a l y k l 53 Handbook of HPLC Handbook UV light-initiated light-initiated UV 57 Luo and cowork 56 The C The 21 Since the the Since 53 18 silica silica - - - Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 consisting of solventthree reservoirs and channels etched in glass. device microfabricated a constructing bysystem pumping the miniaturized further Aebersold and of flexibility, and the inability to reequilibrate columns for future analysis without reversing the reversing without solvents reservoirs. and pumps the in analysis future for columns reequilibrate to inability the and flexibility, of separating proteins, but exponential have gradients successfully several drawbacksof including deviations capable from linearity, lack gradients exponential delivered methods three All both reservoirs. in compositionssolvent initial the altering and A, the reservoir in solvent altering the of reservoirs, height two starting the between tubing connecting different using by modulated be can produced are that gradients The A. reservoir to B reservoir from flow gravity-induced a generates FIG Micro-HPLC and coworkers used a micro-flow processor from LC Packings LC from processor micro-flow a used coworkers and ­composition may delayed be variation flow due reduced to rates. gradient distortions may occur due to column back pressure altering the split ratio, and used, mobile commonly phase those than higher rates flow requires delivery gradient flow However,split since grams, the gradient ends with solvent A being restored as the major component. the as solvent with ends restored Abeing gradient the grams, by Simple Xtreme the in Microtech, which on solvent B gradient is quickly and introduced steep used for a the majority ofof the example elution. In An both chromato- (B) period. long a over gradually introduced is B μ previously, by ultrahigh pressure LC and by a gradient elution system known as as known systemelution gradient a by and LC pressure previously,ultrahigh by dients has bybeen attained the micro-fluidicsdevice developed by Figeysand Aebersold, mentioned advantageous they because provide of a solvents.thorough mixing deliveryThe of exponential gra- exponential gradients for protein analysis. protein for gradients exponential deliver to ability its demonstrated and system LC ultrahigh-pressure an developedcoworkers and ble of generating highly accurate and reproducible micro-flows. pumping systempumping achieved but elutions also gradient by exponentialoverall producing gradients. the of miniaturization the in success a only not was module microfluidic microfabricated newly The capillary. transfer of constructed the chemistry surface the and reservoirs the to applied and byvoltages controlled be could gradient ofthe aqueous direction slope,solvents shape, and organic the and of pumping electroosmotic differential computer-controlled the by generated were “ Bihan Bihan and coworkers. Alexander and coworkers used a modified stainless steel fused silica makeup adapter manufac- Valco,adapter by makeup tured silica fused steel stainless modified a used coworkers and Alexander and provide conditions-independent flow rate accuracy and highly reproducible gradient. reproducible highly and accuracy rate flow conditions-independent provide and could overcome mechanical limitations and imperfections of conventional piston and syringe pumps thethat new micro-flow its piston results deliveryconfirmed performs system stroke. Experimental pre-compress the solvent in the piston chamber up to the pressure at the pump outlet, while the other flow. to constant used piston and One was continuous a generate systems to drive independent two (A) η HPLC without flow andcoworkers HPLC Zhou splitting. developed micro-flowpumping a system capa-

grad,” for the direct and reproducible delivery of exponential gradients was completed by Le Le by completed was gradients exponential of delivery reproducible and direct the for grad,” Percent (%) Exponential gradients deviate from linearity, either with concave or convex slopes and are are and slopes convex or concave with either linearity, from deviate gradients Exponential for rates flow optimal at solventgradients generates system pumping overall the Miniaturizing URE 0 369 3.6 12

51 124 21 18 15 (A) An example of a gradual gradient on the Xtreme Simple by Microtech, in which solvent which in Microtech, by Simple Xtreme the on gradient gradual a of example (A)An 61 Composite percent many groups simply use a stainless steel tee to split the flow pre-column. flow the split to tee steel stainless a use simply groups many Time (min) 70 73 33 94 54 15 760 57 54 51 48 45 42 39 36 33 30 27 The device is composed of two reservoirs whose difference in solvent height 68,69 The development of the simple and low-cost device,and developmentsimple The the of 100 10 20 30 40 50 60 70 80 90 (B) Percent (%) 036 91 215182 66 65 The system uses dual pistons, with 59,60 67 Composite percent The solvent gradients and flows to generate the split flow and flow split the generate to Time (min) 12 42 7 03 63 24 815 760 57 54 4851 45 42 39 36 33 30 η grad. MacNair MacNair grad. 67 66 Figeys 62–64 100 85 10 20 30 40 50 60 70 80 90

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 86 FIG part consists of a conventional semi-micro-flow gradient pump, an isocratic nanoflow pump, a 10-port a 10-port pump, nanoflow isocratic an pump, gradient 1.0two (10-PV)valvewith semi-micro-flow conventional a of consists part valve. switching 10-position a and manifold 10-port a between connected reservoirs, solvent 10-channel rates flow suitableand for columns.packed Natsume capillary conditions, et al. expanded ideathe of gradient loopsinitial to include to recycling loading, sample rapid for control flow programmed flows, pressure split of use the without operation gradient includedsystem this of advantages The 2 injection loops installed, a backpressure column or coil after the 10-port switching valve, and a a and valve, switching 10-port the after coil or column backpressure a installed, loops injection 2 capableswitching deliveringsolvent valveofwith pumpone isocratica nano-flow 10-port rates, at low-pressurewith pump gradient micro-flow atcapability rates gradient (micro-flowpump), GR an length of narrow bore tubing (gradient loop) mounted on a standard high-pressure switching valve. switching high-pressure standard a on loop) mounted (gradient tubing bore narrow of length a 1995,in coworkers solventIn stored a Davisand designed gradients ofsystemdeliveryconsisting deliveryfor of solventthe quentlyvalves,ports, switching numerous more and more with gradients. a unit consisting of an isocratic nanoflow pump and valves indicated by the dotted line. (From Ito, S. et al., et J. S. Ito, (From line. dotted the by indicated valves and pump nanoflow isocratic an of consisting unit a and filter, a and mixer a including pump gradient semi-micro-flow a of consists generator AT10PV nanoGR The auto-sampler). an and pump gradient semi-micro-flow conventional a (i.e., unit injection sample a and for generationthe ofwas smooth developedgradients by Cappiello coworkers.and dient by diffusion of the solvent boundaries during transfer to the column. A 14-port switching valve valve is equipped with 14 ports: 1 inlet, 1 outlet, and 12 ports, which support six loops, each contain each loops, six support which ports, 12 and outlet, 1 inlet, 1 14 ports: with equipped is valve Technologies), as seen in Figure 3.7,coworkers Figure Technologies), and in Itodeveloped by seen as High- Hitachi by produced system, gradient exchange dual the as known (also generator gradient) valve multiport the 10-port-valve desired between (AT10PV)any asymptotic-trace (GR being injector.Finally,the nanoGR and chamber mixing a placing by overcome is This be stepwise. can and compositions between sharp transitions loops, of number limited the to due but time, switching eluentvariables,compositiontwo of and manipulation the bygenerated be can shape gradient any with the strongest, and an electric switch allows selection of filled a loop last the given eluent, weakest the loopwith filled atis loop anyfirst eluents.ofThe given mixture time. selected a Basically, ing

Chromatogr. A Chromatogr.

Recently,have groups several employedresearch loops conse together with gradient pre-formed URE 72 Their device, called the revolving (ReNCon)nano-connection system, generated a gra linear 3.7 Nanoflow pumpandvalveunit Semi-microflow gradientpump

Schematic diagram of NanoFrontier (capillary HPLC/ESI-IT-TOF MS) system. The HPLC HPLC The system. MS) HPLC/ESI-IT-TOF (capillary NanoFrontier of diagram Schematic S D , 1090, 179, permission.) With 2005. Elsevier Copyright 2005. P1 CV μ L injection loops, two 6-port values for drain (DV) and sample trapping (TV), (TV), trapping sample and (DV) drain for values 6-port two loops, injection L P2 Sample injectionunit DV Drain Filter Mixer 10-PV Drain TV Column and column Trap ESI tip 73–75 consists of a conventionala of consists Handbook of HPLC Handbook 65 Their multiportTheir TOF MS IT- 71 - - -

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 between micro-bore HPLC transform (FTIR) and spectroscopic infrared Raman/Fourier detectors. interface solvent a as elimination used havedevelopworked micro-dispenser ers to flow-through a found Table in 3.2. this area. this in limited is research the impressiveand not are gradients the of reproducibility and accuracy the gradients, which reduced temperature contribute to band broadening, the within columns. However, dimensions of packed columns capillary enable a faster response to changes temperature and exhibit is made difficult by the low concentration sensitivity of this technique. this of sensitivity concentration low the by difficult made is with with more sensitive detection schemes. controller to control the pumps and switching valve. Micro-flow GR pumps create an original gradi original an create pumps GR Micro-flow valve. switching and pumps the control to controller Micro-HPLC μ d 3.7 sample sizes that are collected at the end of a separation. While some of these detectors will be be will detectors these of briefly,discussed somean extensive published review been al. by has use et Vissers their of While separation. a of end the at collected are that sizes sample small the suitablemore to someare and disadvantages, offer and advantages detectors ofthese All (ICP-MS). spectrometry mass plasma coupled inductively and (ESI), ionization electrospray and light-scattering evaporative spectroscopy, resonance magnetic nuclear electrochemical, rescence, fluo- absorbance, (IR) infrared includeand UV, detectors These Raman, column. separation the to A dispensingA frequencyof 10 provide. (e.g., 100 ent profile bymixing reservoir solvents Aand B.The well-mixed solventsare delivered at a flow rate more reproducible allows measurements,for Raman detection of ofthe amounts sample. minute spectroscopy provides FTIR more of average suring While an beam. ofRaman the droplet the than mea- size beam IR larger a signal to due spectroscopy Improved sensitivity.Raman than IR and with obtained was reproducibility reproducibility higher assured which beam, IR the of diameter period of switching period 10-port the valve. systemsthe All mentioned above were able to effectively con theswitching simply nano-flow-gradient beany generated can and byprofile shortening samplerun the throughout switch B and A roles of The nano-flowrate. a at column capillary the to loopits in trol the shapes of the gradients, producing accurate and reproducible and gradients. accurate producing gradients, of shapes the the trol with a temperature gradient elution to study organo-tin and organo-lead detection limits. detection organo-lead and organo-tin study elution to gradient temperature a with elution gradient liquid a replaced and ICP-MS with HPLC capillary coupledhave coworkers and applications. Trones most in unusable therefore and unstable technique the makes rates flow high height and area weregood. area heightand of limits detection were much lower those than of conventional HPLC, and of the repeatability peak systems detection nosuch to other had effect ICP-MS,UV, as on the ramping trast the temperature HPLC systemsHPLC have been designed with numerous which detectors, connected ideally are directly μ and ofmid-IR detectors are Raman interestspecial due to the molecularthey specific fingerprint High-temperature generated gradients have been explored in exploredin have been gradients generated High-temperature HPLC has been successfully coupled to ICP-MS to coupled successfully been has HPLC T E TABLE 3.2 PCIPM Selenomethionine,carboxymethylated selenocysteine HPLC-ICP-MS echnology Used 78 xamples of A xamples ete μ In addition, their nondestructive character allows their use in sequence as well as coupling L/min) into injection loop A or B. A While is being loaded, B is delivering solvent loaded

c tors nalytes Investigated bynalytes μ 77 calmodulin selenomethionine γ-gluyamyl-Se-methylselenocysteine, selenosugar, trimethylselenonium, Additional examples of analytes investigatedby examplesanalytes ofAdditional

Hz was chosenHz deposits assure tothe to closer with diameter and size 85 ; andtrypticdigest ofselenomethionyl calmodulin 78 However, application direct ofand IR spectroscopy Raman 83 ; Asp-Tyr-SeMet-Gly-Ala-Ala-Lys peptide H P L 79–81 C-ICP-M Analyte(s) ; however, the use of gradient elution however,and ; gradient of use the S μ HPLC by Trones et al. Tronesbyet HPLC 82 ; Se-methylselenocysteine, 78 Surowiec and co work 84 86 ; selenomethionyl μ HPLC-ICP-MS are HPLC-ICP-MSare 23 33,76,77 77 In con- In The The 78 87 78 - - -

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 , it is ESI ESI is it 3.3)proteins. , Table in seen (as samples copious has analyze that to used and explored been have LC current in-gel digestion methods and and in-geldigestionmethods current which may clog μ remove impurities, and any analyte the volume,concentrating sample thus employedthe reduce to nozzle, set in an arrayed pattern on a silicon wafer. The advantage of this interface is that each each that is interface this contamination. cross of eliminating sample anew is sprayed nozzle,advantage virtually through thus The wafer. silicon a on pattern arrayed an in set microfabricated nozzle, a with tip pipette specialized a aligns instrument This NY). (Ithaca, Biosystems references havetechnique. and that additional this used tions of chapter this discuss the use of LC-ESI MS separation products. to characterize 88 peak broadening, and poor separation performance. separation poor and broadening, peak However, sample is not of possible injection diluted of the amounts large without overload, column μ 3.8.1 3.8 coworkers. and Ito from chromatogram sample a and rates. flow overmicroliter magnitude 1–2orders tides 15 to 150 from diameters Column principle, detection concentration-sensitive a as proteins. and peptides of analysis the for nique al. et many surfaces, which may contain a minimal number of active sites where losses can occur. ofactive siteslossesnumber can where minimal a maycontain which surfaces, many capillary LC. capillary inside of diameter the the inner nebulizer, the capillary ELSD is relatively simple to adapt to micro/ the response with ELSD. Despite the predictable effect on droplet size in relation to the reduction of of linearity the increased homogeneityhas better distribution, droplet in flow of resulting size rate, centrations, such as the femtomole such the centrations, as (fm lowercon- at losespeptide but samples, negligibleforconcentrated be may loss sample of amount components in the LC mobile phase is proportional to the square of the column internal componentsdiameter. LC in the mobile of to square column the phase internal the is proportional HPLC coupled to MS is fast becoming the most widely used technique in proteomic research. research. proteomic in technique used widely most the becoming fast is MS to coupled HPLC

Pre-concentration by vacuum centrifugation is a common technique used in many versions of versions many in used technique common a is centrifugation vacuum by Pre-concentration A chip-based A nanospray chip-based between interface an HPLC and the MS has bybeen introduced Advion Miniaturizing the column i.d. is of great benefit to the sensitivity of ESI-MS, which behaves which ESI-MS, of sensitivity the to benefit great of is i.d. column the Miniaturizing - tech powerful a is (LC–MS) spectrometry mass tandem to coupled chromatography Liquid device evaporative(ELSD) to the Adapting scattering light aPP 87 p Quantitative analysis by ELSD is often hindered by nonlinearity; however, the nonlinearity; ofby reduction hindered often is ELSD byanalysis Quantitative transformed LC–MS into a routine procedure sensitive enough to analyze peptides and and peptides analyze to enough sensitive procedure routine a into LC–MS ­transformed 88 PCMLIMonosaccharideanhydride levoglucosan, galactosan, andmannosaninthe Proteinsexpressing differences amongisolatesofMeloidogyne spp. HPLC-MALDI N-linked glycosylationstructuresinhumanplasma UrinarymetabolicprofilesofsamplesfrommaleandfemaleZucker rats HPLC-Ion Trap MS HPLC-LTQ/FTMS HPLC-TOF-MS T E TA e r echnique xamples of xamples l B - ic C 87 LE n o i t a r t n e c n o at 3.3 HPLC analytical columns. analytical HPLC i ons o A nalytes Investigated bynalytes μ f μ (MHC)-associated peptides PM10 fractionofambientaerosols H P

μ L m with flow rates 20–200 rates flow with m C μ

= HPLC.

10 −15

M) levelM) (see Table 3.1) markedly., increase 125 Throughout the process, the sample is exposed to to exposed is sample the process, the Throughout 1 88 because the concentration of equally abundant abundant equally of concentration the because While numerous methods for coupling MS to to MS coupling for methods numerous While H 94 92 P ; proteolyticdigestofglycoproteins 1 Pre-concentration techniques are currently currently are techniques Pre-concentration 75 Several references referred to in other sec- other in to referred referencesSeveral L C–M is seen in Figure 3.8. Table 3.4 provides 3.4Table 3.8. Figure in seen is A nalyte 91 ; majorhistocompatibilitycomplex S

nL improve detection limits of pep- of limits detection improve nL μ ( HPLC was investigated HPLC by Gaudin O ther ther T han han ES Handbook of HPLC Handbook I) 93 90 49,50,64,67,70,73,95 89 95,125 95 The The 94

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 a flowrate of 50 tonitrile. The composition of10%The from solvent tonitrile. at 0.0 increased B was linearly silica-ODS column (30silica-ODS 1 switching: valve 10-port of 5 2005. Copyright Elsevier 2005. With permission.) With 2005. Elsevier Copyright 2005. umn umn by injecting 2 is placed micro-cartridge in-line with a column capillary and the peptides are to transferred this col- The exchangecation strong material. a containing micro-cartridge, a on trapped geland the ofout decreasing sample size. decreasing gave modest improvements in sensitivity over extraction the standard method, which with increased Micro-HPLC FIG because, at low flow rates, the void volume of trapping columns is often too large to allow rapidanalysis. allow to large too often is columns trapping of volume void the rates, flow low at because, Aebersold. of 1.0of pressure of the membrane unit is much lower, samples can be loaded at 50 times higher flow rate, rate, flow higher times 50 at loaded be lower, muchcan is samples unit membrane the of pressure back they membrane, have because the capacity isand ume 10 trapping than same higher the times column trapping vol- typical the While groups. octadecyl with modified support silica-based a and polytetrafluoroethylene offibers made membrane a and unit membrane forthe frit stainless a with μ m, m, 150 Murata and coworkers explored replacing the traditional trapping column with a membrane a with column trapping traditional the replacing exploredcoworkers and Murata Electroextraction has been developed as a pre-concentration technique by Timperman and and Timperman by technique pre-concentration a as developed been has Electroextraction 3.8 URE L/min; and the scan mass range ( range mass scan the and L/min; Intensity Intensity Intensity 100 100 100 127 50 50 50 μ 0 0 0 m i.d., 10 95 The membrane unit consists of an in-line filtermade of steel stainless usedas the holder, 0 03 0 Following in-gel digestion of the proteins, the resultant peptides are electrophoresed electrophoresed are peptides resultant the proteins, the of digestion in-gelFollowing

Total ion-intensity chromatograms (TIC) of BSAof (100 (TIC) chromatograms Total ion-intensity nL/min. Solvents A: nL/min. 0.1% B:and acid in 2%formic 0.1% acetonitrile acid in 98%formic ace- μ mm in length).in mm ESI voltageThe was 1.6 1 L of elution buffer with onto a the pressure micro-cartridge vessel. This method Retention time(min) Retention time(min) Retention time(min) μ m i.d., 150 min. BSA peptide sample 1.0 sample peptide BSA min. 2 30 30 0 3 mm mm in length) a size column (particle through packed trapping silica-ODS 4 m / z ) was 300–2000. (From Ito, S. et al., al., et S.Ito, (From 300–2000. was ) 00 60 60 60

Intensity Intensity Intensity 100 100 100 μ 50 50 50 kV; (nitrogen) gas was at used a curtain flow rate L (i.e.,100 L 0 0 0 0 0 fmol) peptides of six sequential runs at at runs sequential six of peptides fmol) fmol) was injected into a monolithic monolithic a into injected was fmol) Retention time(min) Retention time(min) Retention time(min) min to at 40% 70.0min J. Chromatogr. A Chromatogr. J. 30 30 30 min. Period Period min. , 1090, 181,1090, , 60 60 60 89 126

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 E TABLE 3.4 PCEIQTFM CereulidefromBacilluscereus HPLC-ESI-Q-TOF-MS Integral plasmamembraneproteinsfromahumanlungcancercellline(62prenylated proteins HPLC–MS-MS (ESI) T HPLC-ESI changes to the unit structure. unit changes the to was membrane durability that was after 40 impaired injections, but this could be improved by minor scale LC–MS. scale teins in small samples of human serum. Likewise, Starkey et al. et Starkey Likewise, serum. human of samples small in teins the ligandthe affinity ofinterest of ranges pre-columns fromaffinity sol–proteinmaking gel-derived monolithic columns containing 90 with as with muchas 50as pre- whose volumes can be several times that greater than of the negating pre-column, any sample initial voidvolumes,by plagued be can concentration sample for pre-columns However,of use umn. the powers, elution low col- with separation analytical the on injection phases to clean-up, prior and concentration forsample allowing mobile using pre-columns onto Large elutedconditions. be experimental by can volumesdictated is sample used pre-column of type The section. this to tion introduc- the in mentioned problems the avoidhelp techniques switching column with combined pre-analytical concentration ofpre-analytical samples has become popular in sciences.the analytical for ligands immunoaffinity and affinity of addition The pre-column. ligand affinity an of poration et al. et created a 2 a created methods and equipment. and methods levels. detection current subfemtomole in changes low-to minimal requires technique retaining pre-concentration this Additionally, implementing while volumes peak elution (nL) nanoliter 50 echnique xamples of A xamples

Licklider and co workers experimented with automating the sample introduction step in nano- in step introduction sample the automating with experimented workers co and Licklider One type of pre-analytical concentration that is particularly well suited to to suited well particularly is that concentration pre-analytical of type One The most common form of pre-concentration involves the use of pre-columns. of use involvesthe pre-concentration of form common most The concentration and leading to poor separation performance. separation poor to leading and ­concentration 131 employed immobilized lectin micro-columns prior to reversed-phase analysis of glycopro-of analysis reversed-phase to prior micro-columns lectin employed immobilized cm vent after the head of the analytical column. Experimental results demonstrated demonstrated results Experimental column. analytical the of head the after vent cm 62 nalytes Investigated bynalytes μ In order to achieve pre-concentration and desalting prior to sample analysis, they they analysis, sample to prior desalting and pre-concentration achieve to order In μ g sample, spitein volume. of small the drawbackOne observed by researchers Flavonoids oflemon,grapefruit,bergamot, orange,andmandarin proteins inwine from isolatedlocustcorporacardiaca proteins AIDS patients inhibitors andnonnucleosidereverse transcriptaseinhibitorsindriedbloodspotsfromHIV/ minor populatedisoformsofantithrombin (Mandolina) andanabioticstress-susceptible(Jubilant)barley cultivar acids nitrobenzoxadiazole (NBD-F)and1-fluoro-2,4,dinitrobenzene(DNB-F)taggedamino samples derived componentsofredwines wines phosphothioate oligonucleotidesG3139andmetabolitesinplasma peroxidase glycosylation antennae betweenappressedandnonappressedthylakoid membranes Ser(150), Ser(418),andSer(476)ofhumanGrb10zeta acid samples and 45Rasfamily proteins) 112 130 115 100 ; sulphametoxazole,bezafibrate,metoprolol,carbamazepine,andbisoprololin water ; sixquinicacidderivatives isolatedfromBaccharis usteriiHeering to the employment of ligandstrue bioaffinity and antibodies. Madera 113 111 103 ; ochratoxin A ingrapes ; 16mycotoxinspossiblyrelatedto“SickBuildingSyndrome” ; humanmilkoligosaccharidesderivatized withvarious estersofaminobenzoic ; antiphosphotyrosineantibodies 117 121 ; urinary8-hydroxy-2′-deoxyguanosine H 119 P LC- ; “trypsinosome”fromspecificpeptidecharacteristics 106 122 ; urinaryproteins ES ; humanplasmaproteome 101 98 ; light-inducedlateralmigrationofphotosystemII I ; α-bisabololinhumanblood 124 Analyte(s) 109 104 ; regulatory lipidsinbreathcondensate; ; N-acylhomoserine lactones 107 ; AGP-derived glycoproteins 132 used a combination of phenyl of combination a used 110 118 ; dissolved proteinsinseawater 103 ; site-specifichorseradish ; abioticstress-tolerant 99 96 ; anthocyanins inSicilian ; Bcl-2antisense 97 μ ; anthocyanins and Handbook of HPLC Handbook 102 HPLC is the incor- the is HPLC 123 ; intactyeast ; neuropeptides 114 105 94 116 ; 7-fluoro-4- Pre-columns Pre-columns 128,129 ; protease 108 ; majorand 120 The use ; soluble Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 Mentioned in S Mentioned T E TABLE 3.5 HPLC HPLC-NMR HPLC-diode array Changes incatecholaminesand3-O-methyl metaboliteconcentrationsinhumanplasma HPLC-fluorescence HPLC-chemiluminescence HPLC-electrochemical HPLC-UV-NMR HPLC-UV-MS HPLC-UV-electrochemical HPLC-UV section, section, see Chapter 4 in handbook on this comprehensivetwo-dimensional liquid chromatography. LC) to achieve to LC) efficient sampleseparation. in umns 1995 and since then, there have been numerous ofreports combining two LC columns (2D- Micro-HPLC Holland Holland and Jorgenson 3.8.2 and chromatography micro-column by followed pre-concentration affinity borate ot plctos ae mlyd h mcoclms s muofiiy hoaorpy with chromatography immunoaffinity measurement of as direct Hodgsonanalyte. the captured and colleagues micro-columns the employed have applications Most animal brain tissue. brain animal experimental in catecholamines major the and salsolinal measure to MS photoionization ­pressure chromatography (see Chapter 13) but are only just becoming introduced into into introduced becoming just only are but 13) Chapter (see chromatography immunoaffinity pre-analytical concentrators have been described in capillary electrophoresis. capillary in have described been concentrators pre-analytical immunoaffinity concentrator. tion. However, as the authors rightly point out, such columns could easily be used as a pre-analytical detec- by LIF measured being analyte released the with was concentrator immunoextraction an as used column the Here chromatography. immunoaffinity nanoflow perform to column monolithic echnique detection detection xamples of A xamples Antibody-based immunoaffinity pre-analytical concentrators have been widely used in routine routine in used widely been have concentrators pre-analytical immunoaffinity Antibody-based M i d i t l u

nalytes Investigated bynalytes A ection 3.7 ection m l a n o i s n e 136 reported separating amines using anion amines exchangeseparating reported and reversed-phase col- Clenbuterol in pork, beef, and hog liver Shape constrainednaturalcompounds(tocopherolhomologues,vitaminE) Azoxystrobin, kresoxim-methyl, andtrifloxystrobinfungicides Erythropoietin inpharmaceuticalproducts Baicalin andbaicaleininratplasma Bioactive compoundsfromBlumeagariepina Bioactive compoundsfromBlumeagariepina Disodium-2,2′-dithio-bis-ethane sulfonate(BNP7787)intracellularconversion products Mebeverine hydrochloride inraw materials,bulk drugsandformulation honokiol andmagnololinfreshMagnolia obovata aromatic aminesinwater glycyrrhetic acidinlicoricerootsandcandies platinum inbloodandurinesamplesafteradministrationofcisplatindrug natural andunnaturalsulfurcontainingaminoacids kinase ZAP-70trypticfragmentcontainingaminoacids485–496 a novel glucocorticoidprescribedforinflammatory bowel disease spinach sample USEPA classified11prioritypollutantphenols dipeptides andtripeptides Magnolia obovata 3,4-methylenedioxyamphetamine, amphetamine,andmethamphetamineinraturine or 5-methyl-4-(2-thiazolylazo)resorcinol (5MTAR) pharmaceutical formulationsandbiologicalsamples ketorolac, piroxicam,tolmetin,naproxen, flurbiprofin,diclofenac,and ibuprofen in products L d i u q i 159 ; resveratrol andresveratrol-glucosides inSicilianwines C h 173 o r ; isoflavines inRadixastragali 162 dditional D dditional 137–143 m ; quercetininhumanplasma p a r g o t a 154 157 In addition to the few addition the to In references this mentioned in ; cetirizineinhumanplasma ; gossypolincotton h 165 etection Methods T Methods etection y ; honokiolandmagnololinbranchesleaves of 145 Analyte(s) ; Co(II) ion as 4-(2-thiazolylazo)resorcinol (TAR) 168 ; 3,4-methylenedioxymethamphetamine, 163 163 160 147 ; salicin,salicylic acid,tenoxicam, 158 174 166 ; polyprenolanddolichol ; West NileandSindbisvirusPCR 162 146 156 135 164 ; totalphenolsafternitrosationof ; glycyl- anddiastereomeric used an antibody-entrapped 155 ; Fmocandz-derivatives of 170 han T han 172 152 100 ; cartenoidsfroma μ ; amikacin HPLC, although although HPLC, ; glycyrrhizin and hose hose 149 ; meloxicam 171 151 148 atmospheric ­atmospheric ; protein ; budesonide, 153 ; 169 133,134 150 161 167 91 ; ;

Downloaded By: 10.3.98.104 At: 03:24 02 Oct 2021; For: 9781420016949, chapter3, 10.1201/EBK1574445541-c3 increases increases by combining two orthogonal separation techniques. systemdramatically LC the of capacity peak the gels,and 2D with overcomeassociated problems loading pump, and the sample loading time. sample loading the pump,loading and that the success of 2-D by HPLC is the ofdetermined nature the loading solvent, the flowrate ofthe to according hydrophobicity. proteins systemThe they used is separate automated, samplefully loss is to low, second and they demonstrated used was column reverse-phase a whereas distribution, and charge charge state their electric on peptides exchangebased to separate was first column used directed the 2D-LC field toward capillary columns. fieldhas toward capillary 2D-LC the proteomics directed of field the columns, LC conventional using accomplished be can 2D-LC While 92 Ref Table etc. contaminants, environmental proteins, reagents, of analysis specific for techniques additional tech- the validate to use references numerous that analytes are references, there these to addition In researching. specific are they niques use chapter this throughout cited references the of Many o 3.8.3 with these methods of detection to study the specific analytes listed. analytes specific of study to the detection methods these with tein digests using a 2D-LC system digests usingtein was a2D-LC investigated by Mituloviccoworkers. and

10. 11. 12. 13.

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