CHROMATOGRAPHY Fundamentals, Methods, Instrumentation, Trends SEPARATION METHOD CATEGORISATION

CHROMATOGRAPHY fundamentals, methods, instrumentation, trends SEPARATION METHOD CATEGORISATION

1. Separations based on distribution of sample components between two Zdeněk Šimek phases

Materials for individual study 2. Separations based on migration rates differences of sample components a) through semi-permeable membrane b) in force field

force field: - electrophoresis membrane separations : - thermal diffusion Masaryk University in Brno - ultrafiltration (hydrostatic pressure) - mass spectrometry - reverse osmosis (hydrostatic pressure) Faculty of Science - ultracentrifugation - dialysis (concentration differences on membrane sites) Research Centre for Toxic Compounds in the Environment - gravitation - electrodialysis (electric potential differences) RECETOX

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Crucial factor of sample component separation is SEPARATIONS BASED ON DISTRIBUTION OF SAMPLE COMPONENTS BETWEEN TWO PHASES DISTRIBUTION CONSTANT

measure of component affinity Types of interfaces separation methods abbreviation (C A ) 1 to contacted phases in K D ( A )  equilibrium (C A ) 2 gas - liquid distillation gas chromatography GLC Equilibrium gas - solid sublimation static and/or dynamic gas chromatography GSC molecular sieve liquid – liquid extraction Phase1 organic adsorbent ion stationary liquid chromatography LLC, exchanger SEC (GPC) solution liquid - solid fractional crystallization Phase 2 water gas solution mobile precipitation liquid chromatography LSC, IEC molecular sieve, inclusion Extraction Adsorption Ion exchange Chromatography

3 4 SEPARATION REQUIREMENT K D( A)  K D(B)  K D(C) CHROMATOGRAPHY

separation factor A,B Definitions separation ratio KD(A) separation coefficient   elution ratio A,B K retention ratio D(B) Method of separation, identification and subsequent quantification of relative volatility - distillation components more or less complex mixtures.

M.S. Cvet (1903-1906) Stationary phase: CaCO3 High easy separation : higher concentration of component A in phase 1 Sample: leaf pigments and component B in phase 2

Two extremes: Physical method of separation in which the components to be separated are

 KD(A)= 6 KD(B) = 2  = 3 components A and B easy penetrate into distributed between two phases, one of which is stationary (stationary phase, phase 1 SP) while the other moves (mobile phase, MP or ‘eluant’) in a definite direction IUPAC (1993)

 KD(A)= 0,6 KD(B) = 0,2  = 3 components A and B remain mainly in phase 2 5 6

Common chromatographic arrangement CHROMATOGRAPHY

Mobile phase Two basic processes occur in the column (eluent) 1. The components are moved apart as Sample a result of their relative affinities for (component A and B) the stationary phase A 2. The spread (dispersion) of the zones (peaks) is constrained so Chromatographic bed that solutes can be eluted (column filling) discretely

Sample mixture Frit injection Peaks (porous disk) separated

Outgoing liquid separation system effluent B column, thin layer,.. 7 8 SEPARATION OF COMPONENTS Chromatographic process IN CHROMATOGRAPHIC SYSTEM

Components are moved through system in the mobile phase direction. stains, strips Chromatography takes for separation of components: Result: chromatographic zones of separated components multiple repeated set up of components equilibrium between two phases Detection: - detector at the end of chromatographic system stationary (SP) and mobile (MP) - physical property of effluent and sample component

Record: chromatogram: chromatographic zones mobile phase flow component transferring profile of analyte peaks waves from the SP to the MP at concentration in the MP the back of the peak response profile

profile of analyte component transferring concentration in the SP from the MP to the SP at stationary phase the front of the peak profile 9 10 time, volume

INTERACTIONS IN CHROMATOGRAPHIC SEPARATION SYSTEMS INTERACTION with MOBILE PHASE physical - chemical interaction Equilibrium is created by various INTERACTION with STATIONARY PHASE (qualitatively and quantitatively) between component and mobile phase component and stationary phase 1. Adsorption - adsorption chromatography - stationary phase = adsorbent  mobile and stationary phase - components are caught on the surface of stationary phase

a) physical adsorption – reversible, energy 0,3 -3 kJ/mol

Types of interaction microscopic view Acting forces: (intermolecular forces) macroscopic view polar (specific) non-polar (non-specific, dispersion) • van der Waals: •adsorption ind. dipole – ind. dipole between permanent dipoles (columbic) between electroneutral molecules •dilution dipole-dipole, dipole - induced dipole permanent dipole vs. induced dipole induced dipoles •chemisorption • electrostatic ion-dipole, ion-ion •precipitation alcohols, carboxylic acids aliphatic hydrocarbons •sieve effect • hydrogen bridge sulfonic acids aromatic hydrocarbons b) chemisorption – irreversible retention of component part energy comparable with energy of covalent or ionic bond 40 - 400 kJ/mol

11 12 characterisation of physical adsorption ADSORPTION ISOTERM INTERACTION with MOBILE PHASE INTERACTION with STATIONARY PHASE continue effect of component concentration in environment on amount of adsorbed the component in equilibrium Attention ! chromatography is dynamic process !!!!!!!!! 2. Partition dissolution in two phases  partition equilibrium creation liquid bonded on the inert surface - film of bonded stationary phase CS = D . Cm D - distribution coefficient  KD invalid for real systems Cs, Cm – concentration in SP, MP 3. Precipitation 1/n precipitant bonded on stationary phase Freundlich: CS = k . Cm k,n - constants separation of component according to solubility product Ksp Langmuir: CS = w . z . Cm / (1+wCm)w- adsorption coefficient z – number of free interaction sites 4. Sieve effect on the surface Inclusion: valid for chromatography ions of similar dimensions and same charge are caught in crystal lattice + K in NH4MgPO4 Occlusion wcm<< 1 isotherm shape Cs wcm>> 1 component, which is not a part of crystal lattice is caught in cavities water in AgNO3 peak shape 13 14 Cm

STATIONARY AND MOBILE PHASE – general information

CHROMATOGRAPHIC METHOD CLASSIFICATION STACIONARY PHASE general: sorbent 1.According to mobile phase -solid particles diameter 1 - 100 ( 200, 300 ) m -thin liquid layer on solid particles Gas - gas chromatography - GC -thin liquid layer on inner wall of capillary Arrangement: thin layer liquid - liquid chromatography - LC tube - column capillary - capillary column Supercritical fluid – supercritical fluid chromatography -SFC supercritical SFC: fluid - supercritical temperature and pressure p liquid diffusion solid MOBILE PHASE density viscosity coefficient CO2, SF6, Xe, NH3 [g.cm-3][g.cm-1.s-1][cm2 . s] CO2 phase diagram Higher values of diffusion coefficients • gas 0,001 0,0001 0,1 in comparison with similar liquids, gas • supercritical fluid 0,1 – 1 0,0001 – 0,001 0,001-0,0001 Lower viscosity T • liquid 1 0,01 <0,00001 15 16 CHROMATOGRAPHIC METHOD CLASSIFICATION

CRITICAL VALUES 2. According to stationary phase:

Solid: Adsorption chromatography Critical temperature - Tc: only one phase exists in a system of Separation is based mainly on differences between the adsorption affinities of the liquid-gas over Tc sample components for the surface of an active solid. substance is in the fluid stage GSC - gas adsorption chromatography Critical pressure-pc: LSC - liquid adsorption chromatography Tlak needed to condensation of fluid substance at critical temperature Liquid: Partition chromatography

How to prepare supercritical fluid Separation is based mainly on differences between the solubility of the sample 1. Substance in liquid form is exposed to temperature and pressure to form components in the stationary phase (gas chromatography), or on differences equilibrium with its vapour : two phase exist between the solubility of the components in the mobile and stationary phases (liquid chromatography). 2. Created system is closed in tube and temperature is increased over Tc to form only one phase disregarding the pressure GLC - gas partition chromatography liquid on support LLC - liquid partition chromatography liquid on support – 17 immiscible with MP 18

CHROMATOGRAPHIC METHOD CLASSIFICATION

2a . Special types 3. According to main separation mechanism Exclusion chromatography Separation is based mainly on exclusion effects, such as differences in molecular size and/or shape or in charge. SEC - size exclusion chromatography may also be used when separation is based on  adsorption molecular size.  partition GPC - gel permeation chromatography were used earlier to describe this process  ion exchange when the stationary phase is a swollen gel.  ion paired IEC - ion-exclusion chromatography is specifically used for the separation of  affinity ions in an aqueous phase.  gel permeation adenin ………. aminopurin Ion exchange chromatography, Ion chromatography - IC Separation is based mainly on differences in the ion exchange affinities of the sample components. competition of analyte and mobile phase ions for ionic groups bonded on the stationary phase surface

Affinity chromatography - AFC Particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is utilized for the separation. „affinant“ bonded on chromatographic support specific biological/biochemical interaction of antidote-antigen, enzyme-substrate, hormone-receptor 19 20 5. According to method of component transport through separation system  method of injection

4. According to experimental set-up Elution chromatography

Column Chromatography A procedure in which the mobile phase is continuously passed through or along the A separation technique in which the stationary bed is within a tube. chromatographic bed. The sample is fed into the system as a finite slug. Packed Column: The particles of the solid stationary phase or the support coated with a liquid Sample components are more retained as mobile phase components stationary phase may fill the whole inside volume of the tube or be concentrated on or along the inside tube wall leaving A procedure in which the mobile phase contains a compound (the Displacer) more Open-Tubular Column: strongly retained than the components of the sample under examination. An open, unrestricted path for the mobile phase in the middle part of the tube The sample is fed into the system as a finite slug. Mobile phase is more retained as sample components Planar Chromatography Open-Bed Chromatography. Displacement A separation technique in which the stationary phase is present as or on a plane. Frontal chromatography Paper Chromatography, PC: The plane can be a paper, serving as such or impregnated by a chromatography substance as the stationary bed Thin Layer Chromatography, TLC: Layer of solid particles spread on a support, e.g., a glass A procedure in which the sample (liquid or gas) is fed continuously into the plate chromatographic bed. In frontal chromatography no additional mobile phase is used. Sample solution act as „mobile phase“ Solvent is less retained as sample components 21 22

ELUTION CHROMATOGRAPHY ELUTION: isocratic - composition of the mobile phase remains constant during the Position of zone: elution process. Step 1: Step 2: Step 3: magnitude of interaction stepwise - composition of the mobile phase is changed in steps during a Sample injection Component zone Component identification of component single chromatographic run. into the flow of development in zone elution mobile phase mobile phase flow by mobile phase gradient - composition of the mobile phase is changed continuously or stepwise during the elution process.

small amount 1,2 3 4 1 2 3 4 of component mixture in a V solvent r

Vr’ V mobile phase m

elution of components in separated zones in the order of interaction magnitude with Area/height: lower content of stationary phase quantitative parameter separation of amide C1-C4 components are segregated by clear MP: ethylacetate/heptane ethylacetate in heptane mobile phase zone Eluted components The best separation of sample components dissolved in mobile phase 23 24 DISPLACEMENT CHROMATOGRAPHY DISPLACEMENT CHROMATOGRAPHY

Step 1: Step 2: Step 3: c [mol/l] Result 1: Sample injection Component zone Component zone A, A+B, B, B+C, C, C+D, D development in displacement by mobile phase flow mobile phase

D sample Inserted displacers C D X,Y,Z mobile phase B affinity between A, B, C „displacer“ A volatile A B C displacer

detector response C Sample components are displaced one another Result 2: B according to affinity to stationary phase. Mobile A, A+X, X, X+B, B, B+Y, Y, A phase has greatest affinity. Y+C, C, C+Z, Z, Z+D, D Zone interfaces are diffuse (unequal flow, diffusion, non-homogenous bed, non-homogeneity of displaced interaction zones X,Y,Z evaporate, distil Preparation, sampling of gases and a vapours 25 26

FRONTAL CHROMATOGRAPHY FRONTAL CHROMATOGRAPHY

Step 1: Step 2: Step 3: Step 4: Step 5: A+B+C Continuous feed of Component Saturation of bed Feed of Elution of all component mixture zone A (all components) clear MP components sorption step in MP perforation A+B

A+B+C MP A A+B

A A+B+C desorption step B+C mobile phase MP C

A+B+C in MP C in MP clear only: first component (A with lowest affinity to SP) A+B in MP B+C in MP last component (C with highest affinity to SP) A in MP A+B+C in MP other components in mixtures 27 quantity – area defined by integration curve 28 „Equilibrium“ Generally: Generally: Distribution constant Response of detector Example ofaresultchromatographic analysis c K (component distributionbetween two phases) and THE THEORY OF CHROMATOGRAPHY  D , to THE CHROMATOGRAPHICto THE PROCESS A V n  crucial factorof separation - - ratio of total concentrations of component oftotal concentrations ratio A intwophases - ( ( c SELECTED TERMS RELATEDSELECTED TERMS c A A ) ) 1 2 CHROMATOGRAM  ( ( n n A A ) ) 1 2 . . V V A 2 1 mobile phase  ( ( ( ( n n n n A A A A ) ) ) )  MP SP W O . . V . V V . V A W O MP SP stationary phase extraction chromatography Time ofanalysis 31 29 Meaning of • • • • • k K retention factork A D how muchlonger measure ofthetime equilibrium ratio comparison ofcomponents interactions migration speedofanalyte inchromatographic bed doesn't reflect howcomponentsare shared intwophases!!!! V V  F t Better: u t M R m R M ( ( - retention timeofcompoundretained inthesystem - - - - n - n retention volume u hold-up volume, m retention timeofcompoundnotretained inthesystem flow rate A A A k the volumeofmobilephasepassingthroughcolumninunittime. obile phasevelocity, ) ) often equaltoV - retained compound retained A velocity=L /t - 1 2 dead retentiontime,hold-up compound movesslowlythanmobilephase  ( ( and THE THEORY OF CHROMATOGRAPHY c fmbl hs -[cm of mobilephase- c to travel through column with velocity of mobilephase through columnwithvelocity to travel of component amount of component a samplecomponent isretarded to THE CHROMATOGRAPHICto THE PROCESS A A compound movesasquicklymobilephase ) the samplecomponent resides to thetimeitresides inthemobilephase ) (k 2 1 . . A V V dead volume of retained compound SELECTED TERMS RELATEDSELECTED TERMS m for component A) 1 2 volume ofmobilephaseincolumn,thesampleinjector, – L  the detector, andconnectors.. linear velocity u=Llinear /t K - length ofchromatographic bed D , A = F V V in SP andMP 3 nM n P(K in MP andSP 1 2 / s,ml/min, m  .t M K R,A = F D by stationaryphase thanitwouldtake , A m in the stationary phase relative in thestationary M V .t  V l/ min] R MP [cm /s] SP d R found in literature Mass distributionRatio Capacity Factor Capacity Ratio Partition Ratio - retention distance D , requires V column length continued MP a V 30 32 SP ) ATTENTION!!!!! RF - retardation factor Chromatographic bed, Chromatographic column is dynamic system The fraction of the sample component in the mobile phase at equilibrium; it is related to the retention factor and other fundamental chromatography terms it is impossible to reach equilibrium in particular step of component transportation u A (nA )m n = m / M RF   r u (nA )m  (nA )s TIME can be observed only, (nA)s  0 analyte molecule all the time in MP = no retention  RF  1,0 (nA)m  0 analyte molecule remains in SP  RF  0 time witch molecule spends in both phases during transport through chromatographic system. RF – measure of presence probability of compound A in MP

TIME depends on interaction with mobile and stationary phase Relation between RF a k ????? different interaction  different time spent in column kA = (nA)s / (nA)m  different time spent between inlet and outlet of chromatographic system (n ) / k 1 R  A s A  F ( A) (nA )s / k A  (nA )s 1 k A RETENTION TIME tR 33 34

ADJUSTMENT OF SEPARATION PROPERTIES HOW TO OBTAIN k ???

Quality of separation of sample components u t  t ' ' u  u .R  R, A M t R , A V R , A V R , A  V M V R , A t A F k    1   1    R 1  k A t t M VM VM V M t M Suitable combination of retention times difference L L M  and zone width of separated components t R , A t M (1  k ) V -V = V´ - adjusted retention volume 1 1 R M R  t -t = t´ - adjusted retention time t t (1  k ) R M R R , A M Influence of zone width t  t .k  t M M R , A (transport of analyte through column) kinetic aspect of separation k can be obtained from chromatogram !!!!!!! A Insufficient zone resolution - velocity ratio of analyte and mobile phase - ratio of times spent in chromatographic system for retained and unretained solute

If V = V M m V V V R,A M  K S D Influence of magnitude of interaction of VM VM components with MP and SP V  V  K V R M D S (migration velocity) ' VR  K DVS thermodynamic aspect of separation Retention volume depends on volume of stationary35 phase 36 KINETIC ASPECT OF SEPARATION EFFICIENCY OF CHROMATOGRAPHIC SEPARATION Efficiency of chromatographic column c0 t0 Zone broadening during movement of component through the column Ability of system to separate sample components to independent zones of individual component during elution. Injection of analyte into the middle of column Measure of efficiency – peak Expression – Number of theoretical plate N c1 t1 width 

Plate - part of chromatographic bed or column Zone broadening caused by diffusion makes possible to establish dynamic equilibrium between c2 t2 component fractions in mobile and stationary phase. The faster equilibrium, the higher plate number  standard deviation 2 = 2Dt Einstein From theory of chromatographic plate: 2 – square of distance covered by component molecule   ,  ,  2 2 2 V t L 2 in time t ~ standard deviation standard deviations  t R   V R   L  N          in consistent units peak width in the inflection points= 2   t   V    L 

2 = 2 D. t 4 the segment of the peak base intercepted by tangents drawn Higher  lower number of separated components in time to the inflection points on either side of the peak =4 37 lower separation efficiency 38

HOW TO CALCULATE EFFICIENCY OF CHROMATOGRAPHIC SEPARATION? Effective plate number -Neff

dR, tR, VR A number indicative of column efficiency calculated by using the adjusted Peak width measurement: retention volume (time) instead of total retention volume (time). a) peak-width at base 2 2 ' 2 2 2  t   d  d   t  t   t   d  N 16 R  16 R M   n R M  N 16 R    R  Y 4 eff            Yt  Y  tR   Yt   Y    Y0,607h 2 b) peak-width at half height Yh/2 : sample N is usually calculated for one meter of column Yh/2 2 injection h 10 000 plate / meter  dR  N  5,545  Y  2,355  Yh / 2  Comparison of efficiency of different columns:

c) peak-width at inflection points (at 0,607 height) 0,607h Height Equivalent to One Plate H plate height

2 h/2  d   R  N  4  Y 2 2 2 Y0,607 L LY    H   t  [m] N 16t 2 L d – retention distance R R  N - depends on retention time Yt, Y peculiar to individual component 39 40 HF : the eddy diffusion Why is zone (peak) broadened ????

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1. Theory of chromatographic plate – unable to describe separation correctly

2. Dynamic theory - van Deemter

Four factors causing zone broadening: Different speed of sample particles v in broad and narrow „canals“ High number of streamlines, different local speed 1. eddy diffusion in mobile phase - HF

2. molecular (longitudinal) diffusion in mobile phase - HL Eddy diffusion is proportional to dimension and shape of stationary phase particles

3. resistance to mass transfer in the stationary phase - HS Lower particle: higher bed homogeneity

4. resistance to mass transfer in the mobile phase - HM  lower differences in „canals“ H = HF + HL + HS + HM

41 Flow-round: different for spherical particles and irregular 42

The eddy diffusion is not proportional to linear velocity of mobile phase 2 Zone dispersion  during chromatographic process involving high number of  random steps , H ~ u  parallel line with x-axis Chromatografické metody I F generally 2 = l2.n l = average length of random steps n = number of random steps

speed of moving zone u H molecule remains time te in streamline with speed u* deviation from midpoint of zone te(u*-u) - length of random step difference u*-u is proportional to average speed l = (u*-u) t number of steps n = L/t u e e u*-u = u L/teu is proportional to particle diameter dp n = L/ute = L/dp H = 2d H = 2d F p F p  = constants of proportionality  - geometrical factor 2 2 dp - diameter of sorbent particle L =  dpL = 2dpL u 43 44 HL : molecular diffusion u = L / tM 2 = 2D L / u diffusion in „free“ mobile phase (capillary column) •at both sides of zones Chromatografickém metody I H = 2 / L •transport of molecules from H = 2 D / u higher concentration area to L m H = 2D / u packed column lower concentration area L m  - factor expressed impossibility of „free diffusion in packed columns proportional to quality of bed (canal shape,...) diffusion proceeds: •in the direction of mobile phase flow and in opposite direction •only in longitudinal axis of column H HL ~ u  hyperbola total time of molecule diffusion in Fick‘s law: dn dc mobile phase:  D D : GC ~ 0,1 (1,0 ) cm2 . s -1 dt dx • independent on retention, m • identical with hold-up time LC ~ 10-5 cm2 . s -1  can be vanished

2 - square of distance covered by 2 Einstein's law component molecule in time   2DmtM 45 46 t ~ standard deviation u

HM : mass transfer in mobile phase HS : mass transfer in stationary phase

Molecules penetrate into layer of SP and back. Molecule entering deeper falls behind molecule entering only under surface.

Sample zone in MP runs faster than zone in SP

Different speed near surface or capillary wall

d 2 2 p qd f u k H M  u H S  2 Dm Different speed of sample DS k 1 molecules in MP towards to  - factor dependent on packing type  1,3 SP surface q - configuration factor – packing geometry

df - stationary phase width HM  u  line DS - diffusion coefficient of molecule in SP HS ~ u  line 47 48 TOTAL EFFICIENCY OF CHROMATOGRAPHIC SYSTEM B/u  

H = HF + HL + HS + HM H A 2 2 C.u 2 D m qd f u k  d p u H  2 d p   2  u D S ( k  1) D m B H  A   Cu B/u u A Cu u van Deemter B for GC van Deemter H  A   Cu H u High Dm in gas phase  low HM

1 1 HH H  for LC Giddings H  H  H  Giddings L S 1 1 L S 1 1   H F H M HF HM 50 max efficiency u

 

2 2 2 D m qd f u k d p u H  2 d p   2  PEAK RESOLUTION u D S (k  1) D m Measure of relative separation of two adjacent zones - peaks B GC H  A   Cu u A: open tubular column  eliminated B: significant owing to high diffusion coefficients t  t R j Ri 2Z (high diffusion in gas phase) Rij  Rij  C: thin films  lower 0,5Yi  Y j Yi  Y j H: ~ 0.1 mm tRj dimensionless number LC (HPLC) t t – retention times A: packed columns, high-homogenous particles, high pressure  lower Ri Z R Y – peaks width on base. B: significant owing to relatively high diffusion coefficients in liquid phase (lower than in GC) the higher value R , the better separation C: thin films  lower ij R = 1,0 - zones overlap by 2% H: ~ 0.1 mm ij

CE A: open tubular column  eliminated B: significant owing to relatively high diffusion coefficients in liquid phase Yi Yj C: only one „phase“ , no equation between phases  eliminated H: ~ 0.001 mm 52 Effect of chromatographic parameters on resolution PEAK RESOLUTION Measure of relative separation of two adjacent zones - peaks - difference of elution times ~ thermodynamics of separation - zones (peaks) width ~ kinetics of separation

full separation k - retention factor (capacity ratio) of t R  t R i R  j i component i ij 0 ,5 Y  Y i j  -ki /kj - separation factor t  t (1  k ) R M n (k j  k i ) R1,2 = 1.0 R1,2 = 1.5 R  2 ij 4 (1  k )  t R  i n  16    Y t  overlap 2% overlap 0,1% n k 4 i Rij   1 Y t  t R 4 1  k n i  tM (k j  ki ) n  11 k j Rij  n R    If Y =Y 4t ij 2 1 Ri 4    k j

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G A S CH R O M A T O G R A P H Y THREE INDEPENDENT TERMS AFFECTING RESOLUTION Mobile phase: gas – inert – transport of components – carrier gas

hydrogen, nitrogen, helium, argon, CO2 according to type of detection, influence on separation efficiency

o o o n k i Carrier gas DG (30 C)  (50 C)  (150 C) R ij    1 H 0.277 94 112 4 1  k i 2 He 0.248 208 249

N 0.073 188 227 KINETIC TERM THERMODYNAMIC 2 CAPACITIVE TERM TERM Ar 0.059 242 296 can be changed by: can be changed by: CO 0.059 162 206 can be changed by: 2 Diffusion coefficients D (cm2s-1) of n-octane •amount SP in the column G •mobile phase velocity •change of MP and/or SP and viscosity  (P) of most used carrier gases •temperature •column length •change of MP and/or SP H •sorbent particle diameter 2 disadvantage: explosive in mixture of air - Attention •diffusion coefficients advantage: fast separation (low analysis time),

55 separation of strong retained components 56 Sample components to be separated, main application: • gas mixtures I N S T R U M E N T A T I O N • volatile organic compounds b.p. < 400°C in Gas Chromatography (requirement for sample conversion into gas phase)

Gas Chromatograph Components Interaction with stationary phase: • carrier gas source • sample introduction (injector) • Adsorption gas chromatography GSC gas-solid phase • chromatographic column gases, liquids (low M ) r • thermostated compartment • detector • Partition gas chromatography GLC film of non-volatile liquid • data station on the surface of solid carrier

Elution:

• isocratic constant temperature • gradient variable temperature

57 58

Isocratic GC - Isotherm TEMPERATURE MODES

I N S T R U M E N T A T I O N Two basic characteristics: in Gas Chromatography Chromatografické metody I retention data  number of methylated groups (-CH2-) (vapour pressure, boiling point) Sources of carrier gas: Tc – column temperature gas cylinders  1/Tc (log tR = f (1/Tc) generators (molecular sieves) linear dependence of homologous series (n-alkanes, n-alkylbenzenes, …) couplings, connections : disadvantages : metal capillary, ideal gas tightness  it is not possible to find temperature suitable for separation of components with b.p. difference than 100 °C gas purifying :  poor separation of early eluted peaks moisture, low molecular hydrocarbons and oxygen removing  poor delectability of later eluted peaks (broadened peaks)

flow regulation : Gradient GC – Temperature programming a) mechanical regulators • analytes retention times decreasing in samples with broad range of boiling points b) electronic regulators • improving of delectability • large volumes of injected samples gas flow : 1 - 100 ml/min.,  1 - 2 % accuracy • optimum: temperature slightly over average b.p. of sample components  0.2 % repeatability of set value temperature stability: ± 0,1°C, change: about 1°C, temperature range: up to 450°C 59 60 INJECTORS for GC

Function: Methods of sample injection  Sample injection on the column beginning as a narrow zone  Conversion of sample into the gas phase  Mixing of sample and carrier gas ahead of column entry  over the column inlet – packed column  on-column – capillary columns Requirements (ideal) :  injection without decreasing of separation efficiency  injection without temperature degradation and sample adsorption

 injection without discrimination according to b. p, polarity or Mr  injection with total recovery of all sample components

Injection devices depend on sample state gases - gastight syringes, injection valves (volume up to 1 ml) liquids - syringes, autosamplers (volume 0.5 - 5 µl)

61 62

Syringe injection methods Syringe injection methods

solvent flush air flush cold needle hot needle

Small amount of solvent is full field into the Syringe is full field more than required syringe, following by air, sample and again volume and tip into required volume. Fast injection with Needle remains in hot injector space air. minimum delay in injector space approx. 5 sec before sample injection. Liquid sample is retracted in to the syringe. following fast extraction from injector. Polar compounds absorbable on glass Samples with high boiling point of and/or needle Sample is fast injected in to the GC and Liquid remains in needle. components. piston is released Hot needle increases evaporation speed. Syringe is fast taken out the injector. Injection of sample in needle and syringe Complete sample injection, low loss of cylinder. volatile compounds, excellent repeatability 63 64 SPLIT LESS – without flow splitter Methods of sample injection into capillary columns  diluted samples  approx. 80% of sample Splitter closed 1 - 2 minutes, sample enters column any longer SPLIT – flow splitter Use :  high number of components • qualitative analysis at high injection ways :  < 10% of sample chromatographic resolution • into hot column • less for quantitative analysis • into cold column (temperature 20 - 30 C lower, than Disadvantages : b.p. of solvent used) • discrimination of compounds with higher b.p. • loss of injected sample according to advantages : split opening • analysis of diluted samples without preconcentration (the whole sample is injected into a column) • analysis of impure samples (change of injection liner and retention gap) 65 66

ON COLUMN INJECTOR - direct into capillary column Retention gap under b.p. of solvent, thermolabile components reduction of the length of submerged zone in capillary column injection ways (deactivated fused silica capillary, 1 - 10 m)

• direct on column (micro syringe with thin needle, • injection into short column with i.d. 0.53 mm)

temperature 20 - 30 C lower, than b.p. of solvent used

difficulties:

• Sample could be broken into separated parts. • Bubbles generated et the inlet of column separate sample into different pars of column depending on way of solvent evaporation • Zone with different concentration are formed, each behaves as individual injection. • Chromatogram with more peaks of the same component. 67 68 GAS CHROMATOGRAPH

PTV INJECTOR (programmed temperature vaporization) injection port detector port capillary column fan (FID) wound around holder Control of temperature of injector body with liner packed with chromatography bed. advantage combination of split, split less and on-column injectors. control panel advantages • minimum discrimination caused by injection from needle of microinjector • minimum discrimination according to b.p. of analytes • not necessary to use special needle as for on column injection • large volume injection oven • elimination of solvent and low molecular compounds before analysis • retention of non-volatile compounds in injection liner • high repeatability of retention times and peaks areas.

69 70

COLUMN FOR GC / STATIONARY PHASES

GAS CHROMATOGRAPH PACKED COLUMNS:

N2 inlet tubes: Al, Cu, Ni, stainless steel, glass, Air inlet 2-6 mm, 1-5 m (detector) H inlet 2 particles: 0,13 až 0,40 mm (detector) adsorbents (GSC): silica gel, alumina, active carbon, molecular sieve, porapak (styren-divinylbenzen copolymer)

bonded phases (GLC): non-volatile, chemically inert liquids siloxane, polyethylenglykole, esters, hydrocarbons heated fan (squalan, C30H62),silicone He inlet (carrier gas) inert carriers: siliceous earth, modified siliceous earth

Properties of packed columns: worse resolution higher volume of stationary phase, preparative purposes 71 low-boiling components, less retained gases 72 Chromatographic carriers for liquid stationary phases Column packing device Sorbents Graphitized active carbon (heated to 3000 K) non-porous, non-specific, high inert material adsorbent and solid carrier Carbopack , Carbotrap, Spherosil, Spherocarb

Chromatographic carriers Silica gels for stationary phases particles based on silicon dioxide activity depends on amount of adsorbed water Porasil, Chromosil, Chromosorb T, Chromosorb G, Supelcoport Porous/nonporous particles of suitable chromatographic properties Activated alumina particles based on aluminium oxide Particles: - 0.5 - 0.1 mm Siliceous earth - narrow distribution of particle diameter Siliceous earth micro amorphous silicate rock (formation) treated by calcinations at - low specific surface (1 - 7 m2.g-1) Molecular sieves 1000 C, sintered particles separated suppression of adsorption aluminosilicate, zeolite - sufficient porosity (0.1 - 1 ml.g-1) according to size good wetting by liquid SP Graphitized molecular sieves separation of permanent gases and low molecular organic compound Carbosieve, Carboxen, Supelcarb Properties: -inert Polymers chemical reactivity – peak deformation STY-DVB, Acryl ester, Polystyrene, EVB-DVB, ACN-DVB or disappearance Chromosorb (polar copolymer), Durapak, Porapak, Tenax specific adsorption – peak tailing 73 Teflon PTFE 74

Liquid stationary phases for GC

Liquid stationary phases for GC Product Tempe Use Properties Type name rature °C requirements: High molecular Non-polar Squalan  300 Hydrocarbon easy oxidizable hydrocarbons Apiezon s Non-polar - low volatility (vapor pressure 1 - 10 Pa) compounds

- suitable chemical composition (from non-polar to polar phase) Perfluorinated Polar Fomblin  250 Halogens High reactivity - good solubility of separated components in the liquid phase alkanes Fluorinated Fluorad Amines Low temperature alkylesters Phenols stability - different solubility of separated components Carboxylic acids • High molecular hydrocarbons - no chemical reaction with analytes Polysiloxanes - R SiO – OV 350 Non polar- High temperature • Perfluorinated alkanes 2  Variable polar stability • Polysiloxanes SE polarity • Polyethylenglykoles easy oxidizable • Polyfenylethers, Phtalates, Polyethylenglykoles Polar Carbowax 220 oxygen Low temperature • Liquid crystals Superox  300 compounds stability Alcohols easy degradable 75 HO(CH2CH2O)n CH2CH2OH 76 Selected commercially available polysiloxane phases Liquid crystals

Mark Type Structure  polar liquids  strong columbic interaction of ions with analyte  positional isomers OV-1 Dimethylsiloxane CH3 OV101 Dimethylsiloxane CH 3 Cation Anion

OV-7 Phenylmethyldimethylsiloxane C6H5 (20%) Tetrabutylamonium Perfluorooktansulfonate 4-toluensulfonate OV-17 Phenylmethylsiloxane C H (50%) 6 5 Tetrafluoroborate OV-25 Phenylmethyldiphenylsiloxane C6H5 (75%) Picrate

OV-210 Trifluoropropylmethylsiloxane CH2CH2CF3 (50%) Tetrapentylamonium 4-toluensulfonate

OV-225 Cyanopropylmethylsiloxane C6H5(25%) Tributylbenzylphosphonium Chloride OV-275 Dicyanoalkylsiloxane C H CN(20%) 3 6 Ethypyridinium Bromide

77 78

Bonded liquid phases CAPILLARY COLUMNS

carrier stacionary phase polarity temperature use (w/w) limit (C)  capillaries with low inner diameter wetted by thin layer of liquid Porasil C 3-hydroxypropionitrile middle 135 hydrocarbons,  efficiency 100 times higher than packed columns. (3%) aromates Marcel Golay 1957 Porasil C Carbowax 400 non-polar 175 alcohols (7.86%) • glass, fused silica, organic polymers (PAD, PES, PTFE, FEP) Porasil C n-Octanol polar 175 alcohols, • metals (stainless steel, Ni, Al, Cu) hydrocarbons • i. d. 100 – 530 m (700) Porasil S Carbowax400 non-polar 230 hydrocarbons • length 15 – 100 m (16.75%) • layer 0.1 - 10 m Porasil S Carbowax 4000 polar 230 aromates, chloraromtes Porasil F Carbowax 400 non-polar 230 waxes,steroids, PAHs (1.41%) According to stationary phase immobilization • Porasil C (100 m2/g, pores 30 nm) • classical capillary columns • Porasil S (300 m2/g) disperse forces-bonded stationary phase, • Porasil F (10 m2/g, pores 300 nm) permanent dipoles, induced dipoles, hydrogen bonds, ….

• capillary column with bonded phase 79 SP chemically bonded to inner wall of capillary column. 80 CAPILLARY COLUMNS with NON- FILLED FREE SPACE 100% dimethylpolysiloxane PLOT (Porous Layer Open Tubular column) thin layer of solid sorbent (10 µm) on inner wall

trifluoropropyl-methylpolysiloxane (trifluoropropyl) WCOT (Wall Coated Open Tubular column) thin film (0.01 - 1 µm) of SP created directly on inner surface of column, phenyl –dimethylpolysiloxane small inner diameter (narrow bore) - pod 0,1 mm I.D. conventional diameter - 0.32 - 0.15 mm I.D. big inner diameter (wide bore) - 0.53 mm I.D.

diphenyl –dimethylpolysiloxane FSOT (Fused Silica Open Tubular column) - type of WCOT, thinner film, smaller diameter phenylmethylpolysiloxane - rigidity increased using polyimide laser, flexible - low reactivity

SCOT (Support Coated Open Tubular column) thin layer of carrier (1 - 5 µm) wetted by SP on inner wall of capillary

cyanopropyl-phenyl –dimethylpolysiloxane

polyethylene glycol 81 82

DEACTIVATION

elimination of negative effect of silanol groups from inner CAPILLARY COLUMNS FILLED in WHOLE VOLUME surface of capillary

Advantage: silylation column capacity improving aliphatic or cyclic silylation dyes

polycondensation packed column with floating and irregular packing reaction of silanol groups with deposited thin layer film of Carbowax or silicone stationary phase at high temperature capillary tube drawn from tube field by bulk chromatography carrier followed by wetting of stationary phase esterification reaction of silanol groups with aliphatic alcohols C4 - C10 and tetraethylglycole at high temperature packed column with regularly loaded bad polyimide layer layer of polyimide (1 - 5 µm) on the inner surface of capillary capillary packed in ultrasonic bath using inert gas

83 84 Retention time in gas chromatography  B  p 2  p 2 up p up   o  i o  o o  up j  Gas compressibility - change of mobile phase volume   L  2 p p o  Decry's law for liquid flow trough nonporous bed  o 

p - middle pressure in column 3 3 Bo - specific permeability constant B o dp 2 pi  po u  o - antiparticle porosity p  2 2 u(p) - velocity at middle pressure o dz  - liquid viscosity 3 pi  po dp/dz – fall in pressure in the flow direction j - James-Martin‘s compressibility factor  2   p i  pi a po - absolute pressure at inlet and outlet 3     1  Bo    u   pp L - column length   p o   io j     o L 3   p   2   i   1  p    o   pro GC  B  p 2  p 2 u p   o  i o o    L  2 p L(1  k ) L(1  k )  o  o t   R u u( p ) j 85 0 86 u(po) – velocity at pressure po,

Hold-up volume Detectors - classification Dead volume according to the basis of response: retention volume - corrected to 0°C concentration-sensitive - response depends on change of sample concentration in - correlated to mass unit (ms) of stationary phase Vg detector (g/ml) TCD, ECD PID surface unit (S) of adsorbent VS mass-flow-sensitive - response depends on mass component in detector (g/s) FID, TID, FPD, j (t R  t M ) F m .273  1 V g  ml .g according to detector selectivity m s .T c universal - response to every component in the effluent except the mobile phase selective - response to a related group of sample component in the effluent j(t R  t M ) Fm .273  2 V S  ml .g .m specific - response to a single component or to a limited number of components having S .Tc similar chemical characteristics

according to detection principle Values of specific retention volumes (times) affected by experimental errors: ionization of molecules (FID, TID, PID, ECD, HID) bulk physical properties of molecules (TCD) Vg – dependent on accurate mass determination of used SP loss of liquid SP as o result of volatility optic properties of molecules (FPD) electrochemical properties of molecules (HECD)

87 88 Signal noise - changes of output signal (base line) not caused by eluted sample component Detectors - requirements a) short-time - frequency higher than eluted peak frequency b) long-time - frequency similar to eluted peak frequency • high sensitivity •low limit of detection • good stability and repeatability of signal •low noise and drift of signal •fast response independent on flow of MP A – peak area • linear response over several orders F – flow through detector w – sample amount sensitivity – slope of calibration curve S = A . F/ w ( detector with concentration response) detector signal S = A / w ( detector with mass response)

Limit of detection - the lowest concentration or amount of detected time compound which caused signal magnitude three times higher than magnitude of detector noise Signal drift – frequency lower than frequency of eluted peaks 89 90

Flame ionization detector - FID

Linear dynamic response range of detector working principle

 hydrogen-air flame between two electrodes Range of concentration or mass flow of analyte with linear response  difference in potentials  500 V  burning of organic compounds – ion generation  ions increase flame conductivity  generated current equal to concentration of organic compound in carrier gas signal •organic compounds detection Upper limit • limit of detection (up to 10-11 mol) • linear dynamic range (up to 107). five percent deviation of signal • low sensitivity for : from linearity inert gases, hydrogen, nitrogen, oxygen, Lower limit chlorine, ammonia, peak height equal to double noise sulphane, water, CO S/N=2 2 height of signal/noise ratio • small hold-up volume concentration

91 92 Termionic ionization detector -TID Alkali-flame ionisation detector -AFID (FID with alkali metal) Photoionization detector - PID Nitrogen phosphorus detector – NPD

• selective for N, P, S, B and halogenated compounds • pesticide, drugs • selected metals (Sb, As, Sn, Pb) • limit of detection (10-13 -10-14 mol) • linear dynamic range (105 )

High noise Low signal stability

working principle • compounds absorbed UV radiation  similar to FID detector • limit of detection (10-14 mol)  carrier gases N2, O2, air, air + H2 • linear dynamic range (107 ).  alkali metal salt (Rb2SO4) working principle in bead on platinum wire or ceramic ring  absorption of radiation emitted from UV lamp  difference i potentials  180 V  ion current detection in ionization chamber  plasma production in bead  800C  high amount of ions, high current  non-destructive detector 93 94

Electron-capture detector - ECD Thermal conductivity detector -TCD (catarometer) • detection of compounds generated stabile ions: alkylhalogenide, carbonyle compounds, nitrile, • organic and inorganic compounds organometalic compounds, water vapour • non-hydrocarbons gases • low-molecular hydrocarbons compounds with P and S, NO2,, oxygen, ozone • halogenated compounds • limit of detection (10-8 mol) • limit of detection (10-15 mol) • linear dynamic range (104) • linear dynamic range (105)

Electrodes (anode, cathode) working principle -emitter  carrier gases N2, H2, He  proportional counter for measurement of intensity of radiation  beta-source (63Ni, 3HonPtorTilayer). working principle  ionization of carrier gas caused by electron from emitter  thermal conductivity changes of carrier gas outgoing from column  strong production of „thermal electrons“.  heated Pt, W, Au fibre  absorption of thermal electrons during collision with positive charged particles  measurement of temperature changes of sensor (thermistor, metal fibre)  decreasing of number of negative charged particles  current decrease 95 96 Atomic emission detector - AED Flame photometric detector -FPD

• selective for compounds with S or P

• particles S2 or POH • chemiluminiscence blue (394nm) Selective detection of green (526nm). elements in organic molecules compounds N, halogens, B, Se, Ge (analysis of elements in -13 -12 working principle • limit of detection (10 g P/s, 10 g S/s) solute). 4  similar construction as FID, • linear dynamic range (10 ).  separated optical system and photomultiplier working principle  excitation of molecules in flame plasma source – excitation of atoms (C, H, D, O, N, S, P and halogens) detection of emitted irradiation  emission of specific irradiation 97 98

Comparison of most used GC detectors

Chemiluminiscence detector – CLD Limit of detection Detector Signal generation Signal production Advantages Disadvantages (g analyte /ml type carrier gas)

TCD thermal change of universal, simple, wide low 10-8 conductivity of electrical dynamic range, non- sensitivity (10-100 ppm) gaseous analyte resistance of destructive number of filament in stream compounds of analyte FID ionization of current of ions high sensitivity, wide destructive 10-13 analyte in H2/air dynamic range, broad flame applicability

TID ionization of current of ions selective for org. P or N 10-13 analyte in H2/air Not for N a P flame

ECD decreasing of current of ions selective for org. narrow 10-15 ionisation of carrier decreased in the compound with dynamic gas caused by presence of electronegative function range radioactive source organic molecule groups, non-destructive, working principle high sensitivity chemically produced vibration- or electron- excited particles MS ionization of ions of analyte, universal. complex price 10-12 analyte separation mixture of organic excited particles emit photons according to compound, speed, high mass/charge ratio sensitivity, identification of compounds 99 100 Detectors for GC range of applicability L I Q U I D C H R O M A T O G R A P H Y

Differences in comparison with GC:

• compressibility of mobile phase is not considered • lower influence of temperature on retention characteristics • active role of mobile phase • 85 % compounds are non-volatile , bad volatile, unstable

„Classical“ LC: Open system, large-size particles > 100 m, MP flow controlled by gravitation time consumed separation – a number of hours fractions analyzed separately Cl-, Br-,J- low efficiency injection amount

101 102

GC theory  LC theory  technique for faster LC separation  HPLC HIGH PERFORMANCE LC CHOMATOGRAPHIC SYSTEMS IN LIQUID CHROMATOGRAPHY Result: low particles 3-10m 30-90 thousand plates per meter • homogenous filling – narrow particle distribution • homogenous film of stationary phase according to dominant mechanisms of interaction of sample   components with SP and MP H = HF + HL + HS + HM LSC - liquid adsorption chromatography 2 D qd 2u k d 2u B m f p LLC - liquid partition chromatography H  2 d p   2  H  A   Cu u DS (k 1) Dm u SEC - size exclusion chromatography

dp : lower eddy diffusion:  A GPC - gel permeation chromatography Dm: lower molecular diffusion in the liquid:  B  u, d , D : faster mass transport between phases :  C f s 1 IC - ion chromatography HH H  L S 1 1  better for HPLC - Giddings H H F M 104 LSC - Liquid adsorption chromatography Alumina (aluminium oxide): Stationary phases for LSC - solid particles ◘ surface - hydroxyl groups and electron-acceptor centres particle character irregular spherical spherical - strong electrostatic field, creation of induced dipoles ◘ pH = 8 - 11 - separation of weak acidic compounds from neutral particle property fully porous fully porous surface porous - strong acids – chemisorption (no requested phenomenon) specific surface 100-500 m2/g 100-500 m2/g 5-15 m2/g ◘ activation - 400 °C 6 - 16 hod, activity control with water addition capacity high high low ◘ capacity - lower than silica gel permeability lower high high efficiency lower high high Silica gel: ◘ surface - hydroxyl (silanol) groups area of applicability preparative analytical analytical - creation of H-bridges price low high high ◘ pH  8 - chemically labile ◘ pH 3 – 5 - strong retention of basic compounds Basic material for SP: - polar adsorbents - silica gel (protonated basic compounds + dissociated silanols) - aluminium oxide ◘ activation - 180°C 3 hour Two forms: wide pores > 10 nm surface area: 100 - 500 m2/g narrow pores < 10 nm > 500 m2/g Adsorption of analytes increases with increasing surface activity and area Polymeric packing decreases with increasing mobile phase polarity - stabile in broad range of pH: 2 - 12 depends on geometrical distribution of functional groups -HEMA 105 106

MAGNITUDE of ANALYTES POLAR INTERACTIONS and ADSORBENT depends on analytes polarity and MP polarity

Retention order of compounds Mobile phase for LSC LLC - Liquid partition chromatography on polar adsorbents Organic solvent and/or water  different polarity Aliphatic hydrocarbons MP polarity must be lower than SP polarity - separation of analytes between two immiscible phases (liquids) Aromatic hydrocarbons analytes penetration through phase interface into whole space of SP – absorption depends ontypeofsubstituent Halogenated compounds NORMAL PHASE CHROMATOGRAPHY - similar as extraction Advantage over LSC: Ethers SYSTEMS - retention depends on stationary phase amount Tertiary amines NPLC, NP-HPLC Nitryl k = KD VS/Vm Liquid stationary phases: - immobilization of different amount of SP Nitro compounds Polarity of MP for LSC Esters of carboxylic acids • mechanically immobilized on suitable carrier (silica gel) – physically bonded Heptane Ketones low solubility in mobile phase  saturation column Pentane Aldehydes Cyclohexane Primary amines • chemically bonded on reactive carrier Benzene Amides of carboxylic acids Ethyl ether - insoluble in mobile phase Alcohols Dichloromethane - possible use of gradient elution Phenols Acetone - possible temperature changes Carboxylic acids 2-propanole Sulfonic acids water 107 108 Chemically bonded stationary phases Bonds with surface silanols 1. No polar, hydrophobic 2. Polar hydro carbonic

C18

C18 ec  Si -O -C  Si -O -Si -C C8ec  Si - C C8  Si - N - C

C4 SA

SB 109 C2 110

REVERSED PHASE CHROMATOGRAPHY SYSTEMS „ENDCAPPING“ SP – non-polar MP viscosity changes Surface of silica gel covered with SP - long hydrocarbon chain as a function of organic/water ratio

with free silanol groups usually C18, C8, C4 backpressure

MP- polar - acetonitrile Polar protective group - tetrahydrofuran in carbon chain - dioxane strongly interact with - diethylether silanol groups - methanol - propanol

Inserted polar group mixture with water: decreasing of high elution strength of organic solvents Surface coverage with high density Modification of mobile phase properties: of alkyl groups change of pH: increasing/decreasing of solute ionisation and a polar groups at the surface of stationary phase increasing pH: - higher retention of basic sample components - lower retention of acid sample components cross-linked carbon skeleton 111 112 Reversed phase chromatography systems Effect of higher water content > 95% in MP

Effect of SP surface modification on the • plainly visible deterioration of retention of aromatic hydrocarbons column efficiency Analytes separated in reversed phase systems • non-polar alkyl chains lose increase ofretention their n - alkanes “brush-type-structure“ aromatics halogenated hydrocarbons • drastically decrease of ethers retention times and nitro compounds resolution esters amines amides acids sulfonic acids

113 114

SEC - Size exclusion chromatography

- separation according to size of molecules - mechanical separation according to particle hydrodynamic diameter

SP: particles with high number of pores - defined pore size - pores filed by MP - no interaction with MP and analyte MP: good dissolution of analyte

Small molecules - diffusion into the SP - highest retention

Middle and large - only widest pores Exclusion limit: - pores accessible for molecules from certain size KD = 0 Larger than pore diameter - no retention Total exclusion: - size area over exclusion limit - Mr, min - exclusion limit ´ VR = A - B log Mr,A Total penetration: - pores accessible for all molecules KD = 1

115 116 Materials for GPC (SEC) Materials for GPC (SEC)

Gels: hard - aero gels, inorganic materials, non-swelled porous glass Sephadex semi-hard, soft - xero gels, organic materials, swelled Sephadex Sepharose hybrid - MP change causes only low volume changes Spheron

CONH2 Hydrophilic: Hydrophobic: Sephacryl CONH CONH cross-linked dextran PS/DVB Nucleogel, Supelcogel 2 2 (epichlorhydrin) Sephadex acrylate, polyvinylacetate CONH CONH2 • stabile polymers with high pressure (N,N -methylbisacrylamid) Sephacryl stability CONH Sepharose • minimum changes in volume caused CONH2 glycolmethacrylate Spheron • by MP polarity change polyamides CONH • suitable for different temperatures 2

117 118

IC – Ion chromatography GPC application molecule mass determination separation of ions and charged particles biochemical analysis of macromolecules SP: Ion exchangers: chemically bonded ion groups on the surface of oligomeric compounds Chromatogram of styrene oligomers n =1-14 inorganic carrier (silica gel) or organic carrier (PS/DVB) MP: Excluded molecules aqueous solution of salt of different pH and ionic strength elution volume  volume IC modes: of MP in interparticle Styrene monomer space V z size of small molecule 1. common ion exchange: equal to molecule MP Sample and MP ions competition for ionic elution volume  hold-up sites on the SP surface volume VM + + SP pore volume Na + ~~ SO3H  ~~ SO3Na + H

V -V = V V M z p Vz M MP: acid (diluted HCl) tR = f(pH, I, cH+ ) exclusion volume hold-up volume

119 120 Ion chromatography interaction scheme IC modes (continue)

2. Acid-base reaction separation of weak acids + - + - ~~ CH2N (CH3)3OH + CH3COOH  ~~ CH2N (CH3)3CH3COO + H2O

3. Ligand exchange Competition of sample and MP components for metal on SP surface retention SP ion exchanger with bonded metal element MP ligand created complex compound with bonded metal Sample creates similar complex as MP component

Solutes retentions ratio of complexes constants of bonded metal with MP and sample ligand elution Separation of amino acids - bonded metals: Cu, Ni

- mobile phase ligand: NH3 - sample ligand: NH2 – groups of amino acids

121 122

Effect of pH mobile phase change on basic, neutral and acidic compounds retention in reversed phase system The choice of suitable bonded phase

C18 Rules CN Retention times are changed with the chains length of bonded phase.

Retention order of main compounds on „aliphatic“ SP (C18, C8, C4) is the Phenyl C8 same.

The change of elution order or resolution of zones can be better realised using phenyl or CN phase C4 Column: than mobile phase change. ACE 250 x 4.6 mm i.d., 5 m

Higher resolution can be achieved on Mobile phase: Resolution changes 20% 25 mM KH PO , pH6 SP with low particle diameter. 2 4 R = 3,0  1,4 80% MeOH Flow rate: 1.0 mL/min caused by 0,1 pH change 1. Norephedrine, 2. Norteiptiline, 3. Toluene, 4. Imipramine, 5. Amitriptyline Temperature: 22°C

123 124 INSTRUMENTATION in COLUMN LIQUID CHROMATOGRAPHY Instrumentation in column liquid chromatography (continue)

Mobile phases reservoir with degasser Scheme of liquid chromatograph

Detectors

Injector

Pump

Thermostated oven with column holder

Control and data station

125 126

Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

1. Mobile phase pumps common analytical flow range: 0,1 - 10 ml/min flow precision < 2% Piston pumps types: pulsed - piston - membrane non-pulsed - linear „syringe“

pump accessories pulse dampener - capillary resistor - two (or more) pump coupling - programmed change of piston speed

gradient - two linear syringes programmer - continual mixing of two or more component of MP

127 128 Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

Syringe pump Membrane pump Flow/pressure profile

Pulse dampener

129 130

Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

2. Sample injection High pressure gradient direct injection - septum, onto the MP flow - stop flow Low pressure - poor reproducibility gradient injection valve automatic injection

131 132 Instrumentation in column liquid chromatography (continue) Instrumentation in column liquid chromatography (continue)

3. Columns Type, proportions ( according to application) Construction details of selected column for analytical LC metals stainless steel diameter [mm] length [mm] glass Preparative > 50 500 - 2000 Stainless steel PEEK 10 - 50 250 - 1000 Semi preparative 8 - 10 150 - 500 „Classical“ (SEC, GPC, IC) 6 - 10 150 - 1000 Analytical 2 - 6(4,6) 50 - 300 with guard column Micro columns 0,5 - 2 50 - 1000 PEEK Capillary columns  0,3 100 - 1000 packed open tubular with SP chemically bonded on the capillary wall

133 134

Instrumentation in column liquid chromatography (continue) DETECTORS for HPLC

Photometric detector 4. Detectors 1. Optical - photometric - fluorescence - universal Types - selective - refractometric UV - VIS detector: 2. Electrochemical - voltammetric - fixed wave length: 254 nm - conductimetric 3. Mass - filter detector : 254, 280, 313, 340, 365, 405, 436, 546 nm - continuously changed wave length : 190-400 (600) nm Requirements - fast, linear concentration response - high sensitivity - fast record of optical spectrum: DAD - Diode Array Detector - low noise photodiodes - minimal effect of change pressure, MP flow and temperature simultaneous detection and quantification at different  - minimal contribution to peak broadening simultaneously obtained chromatogram and optical spectrum - gradient elution

135 136 DETECTORS for HPLC (continue)

DETECTORS for HPLC (continue) Absorbance maximum wave length for compounds with selected groups

chromophor wave length [nm] absorption coefficient Scheme of UV-VIS detector acetylide -C=C 175-180 6,000 aldehyde -CHO 210 1,500 Scheme of diode array detector - DAD aminee -NH2 195 2,800 azo group -N=N- 285-400 3-25 bromidee -Br 208 300 carboxyle -COOH 200-210 50 - 70 disulphide -S-S- 194 5,500 ester -COOR 205 50 ether -O- 185 1,000 ketone >C=O 195 1,000

nitrate -ONO2 270 12 nitrile -C=N 160 - nitrite -ONO 220 - 230 1000-2000

nitro group -NO2 210 high 137 138

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Scheme of fluorescence detector Voltammetric (amperometric) detector: - oxidizable / reducible compounds - current is recorded according to impressed voltage between working polarizable and auxiliary electrode - property: good MP conductivity Phenoles, thioles, peroxides, aromatic amines, ketones aldehydes, nitrocompounds, nitriles, esters

Conductivity detector - ionic compounds - dominant in IC - two electrodes connected to alternating current

139 140 DETECTORS for HPLC (continue) Comparison of selected detectors for HPLC

Electrode lay-out in electrochemical Detector limit of detection dynamic range gradient temperature detectors g/ml application effect photometric 10-9 104 yes low

fluorescence 10-10 103 yes low

refractometric 10-6 104 no high

voltammetric 10-10 104 no low

conductivity 10-8 104 no low

141 142

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Evaporative Light Scattering Detector (ELSD) Transport detector working principle

effluent from column is dispersed by spray into small droplet droplets are evaporated and leave an analyte into the form of fine particles suspended in a gas suspended particles absorb radiation of primary source absorbed radiation is proportional to component amount

143 144 DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Low Angle Laser Light Scattering (LALLS) Detector Multiple Angle Laser Light Scattering (MALLS) Detector working principle working principle • Laser beam goes through set of optical •dispersed irradiation is collected elements and is focused on inlet cell under more angels than LALLS (till window of detector 16, not in the direction of primary •Ring form mask situated between outlet cell window of detector and transferring beam) lens selects beams dispersed on eluate •significant decrease of effect of particles under small angle and prevents beam dispersed by effluent primary laser beams to enter photomultiplier impurities • Dispersed beams • recording of relation of mean are focused to quadratic value of molecule diameter photomultiplier and molecular ass

145 146

DETECTORS for HPLC (continue) DETECTORS for HPLC (continue)

Multi–Electrode Array Detector Multi–Electrode Array Detector working principle Porous large surface, permeable for mobile phase Record of coulometric response of electrochemical reaction on electrode

 Limits of detection – femtomoles  3D resolution of co-eluted peaks  gradient elution

147 148 efficiency Trends in preparation of packing of HPLC column separation velocity Trends in preparation of packing of HPLC column - continue chemical/pressure stability Separation of peptides and proteins miniaturization „Perfusion“ packing Lower particles - dp on surface porous particles C 18 75 x 2.1 mm i.d. . 10 m  5 m  3 m  2 m  < 2 m diffusion pores . decreased time of transport of analyte into and from pores through-hole pores . efficiency increasing . increasing of permeability of chromatographic bed . flow of MP through particles . increasing of pressure  special pump construction and . mass transport speed increasing injection requirements (max 450 bar  5000 bar) . narrower zones, higher efficiency . decreasing of analysis time . UPLC ultra-high pressure liquid chromatography . UHPLC ultra-high performance liquid chromatography

5m

Non-porous particles Surface porous particles column length 25 cm non-porous silica gel (NPS) viscosity MF 1,0 cP – thin layer 3m non-porous resin (NPR) dp 5 m dp 1,5 – 2,5 m m • separation of bio molecules • high mass transport speed and separation • high mass transport speed 1000psi = 70 bar • low capacity of column • acceptable column capacity 149 150 • good recovery

Monolithic packing Polymeric monolithic columns Column packing created as „uninterrupted“ homogenous porous phase •„uninterrupted“ cross-linked porous polymer • polymethylacrylte, metylacrylat copolymer, PS-DVB Types: • agglomeration of polyacrylamide particles • polyacrylamide block • agglomeration of micro particulate silica gel bed • PS-DVB block • silica gel rod Separation of oligonucleotide on • membrane of different types polymeric monolithic column CIM DEAE a) disk monolithic column b) cylindrical monolithic columns in glass and ion exchanger stainless steel shell poly(glycidylmethacrylate-ethylenglycol) dimethacrylate Silica gel rods: disk: ø16 mm, thickness 3mm • through-hole pores, diffusion pores, to be modified (C18,...) • efficiency equal to columns with particles 3-5 m monolithic cylinders Fm = 6 ml/min • pressure drop 30-40% in comparison with 5 m particle packing  • column coupling for higher efficiency 151 152 Non-silica gel stationary phases Silica gel packing with very low content of trace metals •PS-DVB cross-linked copolymers Future trends in LC lower efficiency than silica gel, •decreasing of interaction of metal with separated compounds high resistance against pH •effect on acidity of residual silanols

Hydroxyapatite • Lower particles of silica gel packing (max 3 mm) in short columns interaction with sensitive bio molecules (7,5-15 cm) (LC/MS).

•Zirkonia (ZrO2) • Columns with low dimensions with inner diameter lower than 100 m high stability against high pressure and temperature, with low particles (proteomics, LC/MS). resistant against high pH (to pH 14) surface without silanols, contain centres Lewis acidic centres strong affinity to Lewis base (hydroxyl, phosphate, fluoride,..) • Monoliths in small diameter columns.

•Graphitised carbon • Monoliths in 100 mm column diameter created in situ

hydroxyapatite particle Hybrid inorganic-organic stationary phase • TiO2 particles

• polymerization of tetrachloro- or tetraethoxysilane • Chip column „lab-on-a-chip“, creation of silica gel polymer with silanols on the surface column „packing“ created directly on inner capillary column wall electroosmotically generated liquid flow - see CEC capillary electrochromatography •triethoxysilane connected with ethylene

(RO)3SiCH2CH2Si(OR)3 high pH stability 153 154

Column format trends in HPLC Efficiency of capillary columns Scheme of LC column on chip and part HETP corresponds to particle of packed capillary size 150 x 0,5 mm 3,7 m

Analysis of BSA digest on „ChipLC column“

Column dimensions: 40 x 0,075 x 0,050 mm 5m particles

Separation on capillary column packed with micron particles 370 x 0,030 mm i.d. 1m

detection inner part of injection valve

155 156 Other separation examples Selected examples of chromatographic separations

Separation of mixture of BSA digest, Separation of peptides on nanoflow myoglobine a alfa-lactalbumine HPLC column on monolithic capillary column C 18 150 x 0,075 x 0,050 3,5 m injection 100 fmol 1 l PS/DVB monolith 50 x 0,2 mm

Fm = 2,5 l/min detector cell volume 3nL

157 Optima 17 - Phenylmethylpolysiloxane Optima 5 – 5% diphenyl-95% dimethylpolysiloxane158