SEPARATION METHODS B Jan Havliš, Ph.D. Masaryk Uni, Fac Sei analytical separation analytical separation methods : SEC, GPC, HCD and FFF :GC /\f : CZE, MEKC, CIEF, ITP, CEC, ACE, NCE and CE-on-chip ®®®® Creative Commons: Attribution-Noncommercial-Share alike 3.0 Unported License Separation methods B - syllabus separation of macromolecules (SM) : definition of macromolecule and its description : separation using molecular sieves (SEC) : separation by field-flow (FFF) gas chromatography : description of GC as continuous extraction : special practical aspects of GC :: injection, detection electromigration methods (EMM) : separation by different migration in electromagnetic field : capillary and slab techniques : combination with chromatography 2 Separation methods - overview separation principle method 1) - two phases method 2) - one phase transport barrier concentration difference volatility distillation solubility zone refining crystalisation distribution constant extraction, distributive chromatography (LL, GL) exchange equilibrium ion exchange and affinity chromatography surface activity adsorption chromatography (LS, GS) foam fractionation geometry of molecules molecular sieve electromigration electrophoresis 3 1556 separation of macromolecules SM history Agricola : separation of gold using gravity in a flow of water 1870 Lord Rayleigh : basic theory on light scattering on small particles 1940 Debye and Zimm; theory on light scattering on large particles 1955 Lindquist and Storgards : gel filtration on starch („molecular sieving") 1959 Porath and Flodin : gel filtration on cross-linked dextrans (Sephadex) (GPC) 1961 Hjertén : use of synthetic gels as stationary phases : Polyacrylamide 4 1962 Pedersen : protein separation on small glass spheres (HDC) 1964 Hjertén : use of natural gels as stationary phases : agarose 1966 Giddings : description of FFF method principles 1969 D i M a rzi o and Guttman : theory of steric exclusion for SEC 1970 first commercial instrument using light scattering for mol. mass characterisation 1974 Small: first HDC experiments on non-porous sorbent 1978 Noel: particle separation in empty capillary (capillary HDC) 5 theoretical base of SM what is that macromolecule? molecule of IVĽ, >10 000 w synthetic polymers monomer, oligomer (10- 100), polymer homopolvmers (PE, PP, PS, PTFE...) : one repeated unit (monomer) nM^[M\ linear branched heteropolymers : more of different units nX+mY ^>XJm n m 6 biological polymers M w 10 000 - 1 000 000 proteins peptidic bond, 21 natural amino acids (Se-Met) complicated complexes of different units, e.g. haem + globin glycana (polysaccharides, oligosaccharides) (starch, glycogen, chitin, cellulose, dextrans, pullulans) nucleic acids (polynucleotides, oligonucleotides) nucleotide = phosphate + nucleoside nucleoside = saccharide + base DNA- saccharide - deoxyribose RNA- saccharide - ribose surface forces (surface charge, ionic strength of surround) primary => secondary, tertiary, ternary structure - native form macroscopic forms random coil size of macromolecule flexible molecule A2 h li A, h \ U r h description of macromolecule rod sphere contour length (L) L = n*l n - number of bonds I - monomer length end-to-end vector length (r,) 8 mean square end-to-end distance (r2) radius of gyration (s2) important quantity for light scattering measurement centre of gravity w2 «£-?#> w1 w3 \ /S4 \ / V__JX w6 ^ w5 w4 s - distance of unit from centre of gravity if monomer units are identical relative molecular mass SM separates mostly according to size = f (molecular mass, cross section, etc) 1 -12 Mr =rn* — m(iZC) 12 SI definition for macromolecules: mix of molecules of different molecular mass, differing in number of units = distribution number average Mr : measured by osmometry weight average Mr : measured by light scattering z-average Mr : measured by sedimentation analysis polydispersity ~ distribution 10 example what will be the number average, weight average molecular mass and polydispersity of polymer sample? Mw _ Z NiM 2< Mw~- 1*12+1*52 1*1+1*5 - = 4.33 average mass average number of units — 1*1+1*5 1 + 1 11 basic modes of macromolecule separation size exclusion chromatography (SEC) : gel filtration chromatography (GFC) : gel permeation chromatography (GPC) : gel filtration (GF) hydrodynamic chromatography (HC) flow-field fractionation (FFF) : sedimentation (SFFF) : thermal (TFFF) : electric (EFFF) : gravity (FFFF) membrane separation : ultrafiltration (hydrostatic pressure) : reversed osmosis (hydrostatic pressure) : dialysis (concentration gradient) : electrodialysis (gradient of electric potentials) separation in force-field : ultracentrifugation (density gradient) : mass spectrometry (electromagnetic field, TOF without field) 12 SEC, size exclusion chromatography gel permeation chromatography (GPC) gel filtration chromatography (GFC) principle: analyte is distributed between MF outside of particles and inside of particles : sieving effect, stehe exclusion : diffusion : pressure of carrier liquid - motion of liquid and its flow profile V»=V +K'„*V- v R v out ~ 1V D v in y =y +y +y tot out in part Vn - retention volume R KD - distribution constant tot- total volume out- MF outside of particles in-MF inside of particles part- volume of particle material v tot part 13 VR=Vout+K'AVHVtot-Vout) where (V -V ) = V. +v V r tnt r nut / r in ' r - m /?arŕ K1^- elution constant • Ct • o* o* •o O pore nalyte molecule time thermodynamic interpretation AG = AH-TAS = -RT]h(k) AH-TAS AS K-- -e RT eR < 1 AH ~ 0 => process is entropically controlled 14 cjn - analyte concentration inside of particles cout - analyte concentration outside of particles VR=kl*\ogMw+k, /cy, k2- numeric constants r max m vR = Vout+ \K'D{R,r)*{r)dr R (p - total pore volume with diameter r to r+dr R - diameter of retained particle separation is given by ratio of diameter of pore and analyte sieve model is in many aspects not exact : flow of liquid out an in pores is different (Fout » Fin) : other interactions: adsorption, L-L distribution, electrostatic repulsion (=> K'D > 1) 15 mechanical separation of A molecules in particles/pores of gel based on their different size not classic LC, no chemical affinity qS -quazi SF, M-MF 16 use of SEC group separation : separation of low and high molecular groups (desalting, extraction agent removal, reaction termination between low molecular mass ligand and biopolymer) fractionation / purification : separation of components with significant Mr difference determination of Mr : comparison with standards (in line increasing Mw) : polymer polydispersity and distribution analysis of ligand-biopolymer binding : emerging complex has higher Mr than components (complex insulin-antibody by diabetics) concentrating samples of biopolymers : dry molecular sieves remove solvent - „dry up" and concentrate sample 17 column filling : pre-filled columns : own filling - SF swelling (uniform, without bubbles) sample introduction : injecting 1 - 5 % of column volume : either on column top or through injection adaptor elution MF not directly influences separation : solvent viscosity and elution MF ratio < 2 : water- uncharged compounds separation, or buffers pH and / keeps ion interactions minimal guarding SF 0.02 % sodium azide 0.05 % trichlorobutanol (Chloreton) 0.005 % ethylmercurythiosalicylate (Mertiolate) 0.002 % Chlorhexidine 18 ideal A . . ,, limited overload matrix influence sample solubility wrong injection calibration set of standards 4-5 defined native proteins with increasing Mw TÖD ™híí.i DG I TGPi y u i.s. {monostearin) y FA log M w AlogM absolute calibration basic parameter defining selectivity - hydrodynamic volume formula for limiting viscosity number of polymer [nj derived from Einstein's equation jn/jn^-l k*Vfí M = lim^----= —f- p^o p M T] *M =k*V R independent on macromolecule structure [j]lA)*M{A) = [TilS)*M{S) = f(yR) Mark-Houwink's equation A - analyte, S - standard CT o vc log([7]*M) = /(VÄ) KAMaAA+x = KSM"S+1 [nl - by viscosimetry 20 selectivity in relation to pore size distribution , i, n increasing pore size distribution AlogMw AlogMw AlogMw i 4 H---------1—1------► '—I-------1------------► '—H-----------1—+ vR vR VR AAA Vc vc vc 21 Separation column : classical tubular columns material - mostly soft gels : inert gel matrix (towards analyte and elution solutions) : long-term chemical stability (at different pH and temperature) : mechanical stability (resistance towards high pressure) : small amount of ionised groups : suitable particle size (5 - 250 urn) small particles - high resolution, low rate large particles - fast separation, low resolution fractionation range (FR) Mr range, in which the compounds are separated elimination limit (EL) upper limit of fractionation range column fillings agarose large pores, acidic character elution: polar and non-polar solvents Polyacrylamide FR > 200 000 Sepharose low amount of polar groups; low resolution elution: polar and mild non-polar solvents FR = 1000-3 000 000 Sephacryl, Bio-Gel P mixed SF: agarose-acrylamide chemical very resistant FR = 1000-23 000 000 Bio-Gel A, Ultrogel dextran styrene-DVB strong adsorption effects elution: polar and non-polar solvents FR< 10 000 Sephadex strong hydrophobic interactions elution: non-polar solvents FR = 400-14 000 Bio-Beads, Styragel 23 methacrylate hydroxymethylnnetacrvlate + ethylendimethylmethacrylate elution: polar and non-polar solvents Spheron glvcomethacrylate elution: polar and non-polar solvents detectors : detection of separated compounds : determining molecular mass and polydispersity absorption photometric detector polymers mostly do not contain own chromophores =^> indirect detection refractometric detector : universal fluorimetric (fluorescence) detector : own fluorophores (within proteins Trp, Tyr, Phe), or derivatisation 25 viscosimetric detector Mv e (Mn, MJ, Mv « Mw M=jnf=Bm2Z2!žr Mark-Houwink's equation [r|] - limiting viscosity number [m3/kg] H* - solvent viscosity K, a - Mark-Houwink's constants (for globular macromolecules a = 0) osmometric detector vapour pressure osmometry (VPO) : uses Raoult's law : fast, low sample consumption, temperature interval 25-130 °C : Mr = 40 - 35 000, no volatile compounds T = const., saturated vapours of solvent 1) RT1 and RT2 - droplet of solvent, AT12 = 0, U = 0 2) RT1 - droplet of solvent, RT2 - droplet of sample (solvent + analyte) source adding droplet of sample i solvent vapour tension => condensation of solvent vapours into the droplet => release of condensation heat => t temperature of sample droplet, thus also of thermistor, also of solution tension pressure => Wheatstone bridge unweighing solvent vapour condensation stops when sample vapour pressure is in equilibrium with pure solvent vapour pressure due to higher temperature measured voltage, proportional to the difference of temperatures of both thermistors, is proportional to molar concentration of compound in sample thermal losses => calibration on standard of known Mr value 27 light scattering detector static light scattering scattering of light beam on particles of suspension or colloid solution interaction of light beam electric vector with electron shell => periodic oscillations intensity, polarisation and angular distribution of scattered light depends on size and shape of scattering particles dynamic light scattering studies time fluctuations of scattered light on moving particles : information on diffusion coefficient light scattering on small particles macromolecules particle diameter (d) < A/20 (Rayleigh scattering) a = c(dnl'dc).. *n 0 c - concentration N - number of particles; scattering centres ň^ - refractive index of solvent (dn/dc)M - particle refractive index changes at constant u particles - secondary source of scattered light of the same wavelength 2tt*N L Sn■ *V*a -j— =____________* o %*r N*(l + cos2e) intensity ratio of scattered (is) and original light l0 (non-polarised) V- unit volume A0-wavelength r - distance from particle 9 - angle measured from main light beam number of scattering centres N in case of identical macromolecules (monodisperse sample) NA - Avogadro's number M - molecular mass L 27T2*n0 *(dn/dc)2 *V*c*M I o A40*r2*NA *(l + cos26>) Re- i*r 2 2 /o*V*(l + cosz60 .2 ... 2 K = 2n *n0 *(dnldc) A4 *N /l0 iy A 2 Rayleigh's radius summing constants into one, K in polydisperse sample, M is substituted inter-molecular interactions and non-zero concentrations taken in account (Debye): K*c 1 2 R 6 M + 2A2*c + 3A3*c +... A2, A3... - virial coefficients; mostly A3 and higher are omitted A2 - phys.-chem. measure of thermodynamic solvent quality for given macromolecules good solvent A2 > 0 : macromolecule expands bad solvent A2< 0 : macromolecule shrinks 6-solvent A2= 0 : macromolecule preserves its volume light scattering on large particles macromolecules particle diameter (d) > A/20 (Debye scattering) : large particles => phase shift of light scattering from different parts of molecules : phase difference is dependent on angle 9; for 9 = 0 is the difference 0 : beam interference => angular distribution of scattered light intensity P(9) fi Zimm's equation use of P(9) parameter to express scattering K*c 1 _P(0)_ * 1 „, 1 — + 2A, * c M J if(1-x)-1«(1+x) 32 + 2 A2 * c experimental bases for calculation of gyration radius multiple angle laser light scattering (MALLS) Zimm's graph K*c Mw-double extrapolation to y-axis K*c R o 9 9 = f(ún2- + Ks*c) Ks - arbitrary constant; graphically separates diagram lines 1/M sin2(-«-) + 2000*c different concentrations c of sample laser- A0 source of l0 intensity refractometer (also as concentration detector) - n0 and (dn/dc)M (see constant K) is- scattered light intensity in different angles 9 in known distance r from cuvette 9 -^> 0 (c = const.) blue lines, from blue slope we extract gyration radius (s2) c -» 0, slope ~ A2, interception 1/MW red line 33 low angle laser light scattering (LALLS) at small angles 9 (< 7 °) sin2(9/2) ~ O => P(6) -> 1 then for Mw> 107 or within associated systems this approximation fails instrumentation laser optics adjustable angle advantage: : absolute technique, no calibration needed Mw, A2 for (s2) - standards necessary :fast : connectible with separation technique (GPC, FFF) disadvantages: : dust- demanding high solution purity HC, hydrodynamic chromatography principle: combination of stehe exclusion with surface (colloid) interaction sample-filling, eventually solute retardation behind streamlines of laminar flow with profile (wall effect) non-porous material sample moves with MF flow gravity centre of large macromolecule cannot reach the channel wall (Rp) move in slower flow near to it (wall effect; given by laminar flow profile R0) => heavier (larger) molecules run through channel faster than smaller ones cannot other influences: : electric double-layer : van der Wals interactions © © ©g ©5 © © ■o o < Q) 3 Q), => sample moves in channel hydrodynamically or electrically 35 Separation description t: 1 r, = ŕa„ 1 + BI - CK 'M t - polymer retention factor tj a tM - retention time of polymer and unretained component A - ratio between macromolecule radius and flow channel half-height B and C - constants dependent on channel symmetry, C also on retention model calibration 0.001 0.01 r 0.6 0.7 0.8 0.9 A = f (t) and thus on Mw in case of tubular micro-capillary use and C —> 2.3 porous material pores of filling : 50 - 50 000 nm sample : larger molecules capillary fractionation (CHDF, capillary hydrodynamic fractionation) other influences in account: : colloidal forces : non-linear inertial forces depending of flow-rate gradient and position (lift forces; tubular pinch effect) pump exchangeable resistance capillary waste Zu injection a waste detector HDC capillary FFF, flow-field fractionation principle: physical field inflicts some property of analyte and creates concentration gradient dc/dx => concentration profile c(x) across channel is specific for given analyte , inlet I field flow separation channel outlet i field "N fiowl layer ^ / thirl/nocc ll\ // • \\" v' . > *■•.!• l 0) 3 3 W = t/ = -axn X X X c(x) = c0*e K VÖy dx n - either O or 1 0 - constant flow 1 - depends on position in channel brownian elution mode n = 0 U*t*^2D*t retention ratio is function of A parameters influencing separation: : analyte properties (field-analyte interaction parameter, diffusion coefficient) : strength of applied field field-analyte interaction parameter : effective mass mef m Vpart \Pnart Plia) part k - Boltzmann constant T - absolute temperature F = g*mef; g - gravity acceleration w - height of separation channel 39 stehe elution mode n = 0 U*t»^2D*t particles create a layer near to channel wall concentration of analyte extra muris = 0 rD - particle radius focustion elution mode n = l Ux =-a(x-s) particles create a layer near to channel wall concentration of analyte extra muris = 0 a c(x) = c0*e 2D ■■(x-s) s - position, where resulting force inflicting analyte is = 0; position of zone centre from channel wall importance of hydrodynamic force: its influencing: liquid flow profile channel profile use of FFF : no SF (one-phase chromatography) => no interactions with active surface : MF is carrier liquid, influences separation indirectly only : variables influencing separation may be changed continuously in wide range separation of macromolecules and particles 103 - 1015 Da proceeding FFF instrumentation MF X ::: degas. T pump njection injection pump /—V ÔJ separation^ ^ T channel ľ:::ľ;:::S:™*!Tňí : i : filter o o o ^ control unit II—I B ' ■ i * i * detector TT TJ waste 1 1 ■—. PC =^_^= 0 D o o o 1 1 1 1 ■j™™» — i::m. III pumps : wide range of adjustable flow-rates : no need for high pressure, but for pulseless flow !!! : with constant pressure and flow (reciprocal, peristaltic) injection device similar to LC : septum : multi-way valve : linear injectors (infusion) detectors similar to SEC : refractometer : photometer-absorption, fluorescence, optical rotation, scattering : other-viscosimeter, densitometer, osmometer... SdFFF, Sedimentation flow-field fractionation : the oldest technique : effective force = natural gravity or centrifugal force : rotation 20000 r.p.m. (injection in steady state) A = 6RT/7T*d3p*G*w*Aq G - gravity (g) or centrifugal acceleration Aq - density difference between particles and solvent dp - particle diameter GFFF:> 1 urn SdFFF (G = 105 * g) : 106Daor> 10 nm DNA, proteoglycans, river water colloids, viruses and silicagel SF for HPLC zone 1 ThFFF, thermal flow-field fractionation separation channel - two metallic (cupric) blocks the upper one is electrically heated, the lower one is water cooled => gradient 20 -1000° : distance teflon foil: 50 - 250 urn temperature gradient causes slower flow at colder wall (non-isoviscose liquid) A = w* — * — v T dx j -i DT = thermal diffusion coefficient a - thermal diffusion factor = DT*T / D TFFF: to describe thermal diffusion EFFF, electric flow-field fractionation walls - semipermeable cellulose membranes high voltage gradient; low absolute voltage - low current => low heating A = D/jue*E*w |je - electrophoretic mobility E - electric field intensity EFFF: proteins with different isoelectric point FFFF, flow-field flow fractionation external field - solvent flow orthogonal to flow of basic media tube of semipermeable material => solvent intrusion, not of analyte A = RT*V0/37T*N*r/*Vc*w2*d V0 - channel volume H - viscosity Vc - volumetric orthogonal flow d - effective Stokes diameter FFFF:> 1 nm gas chromatography 1941 GC history Synge and Martin : theoretic base for GC: „...very refined separations of volatile substances should be possible in a column in which permanent gas is made to flow over gel impregnated with a non-volatile solvent......" 1952 James and Martin : practical introduction of GC; separation of volatile fatty a. 1963 GC-MS - first hyphenated technique 1980 capillary columns in GC - distinctive separation improvement 48 theoretical base of GC in principal the same as for LC separation difference: gas is compressible (liquid not) equilibrium on column A (g) + SF (s) <-. > A-SF (s) KD = cs(Ay cM (A)- ¥YS(A) *Ym(A) C s (A) = R*T * p(A) 49 Raoulťs law o p(A) = pu(A)*x(A) x(A) - molar ratio of A in mixture p°(A) - pressure of saturated vapours of A Henry isotherm CD C/) C/) CD CO CO Q. /*** A A* I [ml], ťR>l [min] R,i R,i m V = F *ť y R,i 1 M ĽR,i *R,i *R,i "- m net retention volume VN [min] VR j corrected to carrier gas compressibility specific volume Vh [ml/g] or Vp [ml/m2] VN related to 1 g or 1 m2 SF and to 0 °C VN 1M LR,i J v R,i J il/m2] v _ 273.15 *V„ S*Tk v _ 273.15-V* 55 temperature influence *k > Tboi| A Tinj > Tk A Td > Tk Tinj - injection head temperature Tk - column thermostat temperature Td - detector temperature Ť Tk leads to faster analysis Ť Tk demands Ť MF pressure on column inlet for keeping u through column isothermic analysis: Tk= const analysis with temperature gradient: Tk2 - Tk1 > 0 GC arrangement MF container I Separation I Lb= column -U------- detector injection □ device í j s n zi s íl iJJJUJUJLIJLIJLlJtř ^^5 žä~E output 57 MF delivery gas pressure containers compressor electrolyser 0.5 ml/min -400 ml/min (HP-GC 1200 ml/min) pressure up to 400 kPa (HP-GC 1 MPa) pressure and flow control thermostating carrier gas advanced flow control (AFC) carrier gas advanced pressure control (APC) 1.0 "e £ Q_ I-LU I 0.5 0.0 helium y* 20 40 hydrogen 60 80 u [cm/s] carrier gas N2 (nitrogen) + cheap, safe - low thermal conductivity H2 (hydrogen) + high thermal conductivity, low viscosity - high diffusivity, explosive He (helium) + combines advantages of N2 and H2 - expensive Ar (argon) especially for ECD must be chemically inert - always necessary to remove humidity and 02 purity- pre-set guard column with molecular sieve 59 loading of A onto column : more difficult than by LC injection device tubular columns: 1 -20 |jl capillary columns: ~ 1 nl inject small volume and quickly : slowly and large volume (overload) =^> broad zones and resolution loss sample evaporation necessity to transform liquid and solid samples into gaseous state heated space on the beginning of the column volatility increment chemical derivatisation: silylation (N,0-bis(trimetkylsilyl)acetamide) silanisation (dimethylchlorsilane) and acetylation (acetan hyd ride) 2 ROH + NA-Si -* 2ROI-SÍ.J + O-Si NH2 60 splitless injection proportion valve 1 carrier gas inlet into column sensor pressure | flow regulator with septum I ^^ septum waste ^splitter waste proportion valve 2 to detector with closed valve pressurise using proportion valve 1: flow sensor = 5 ml/min, pressure sensor = 70 kPa septum flow set to 2 ml/min => slow flow of 3 ml/min onto column sample introduced into injector and is carried onto column after certain time without splitting (10 - 40 s /optimum 20 s/, splitless time), which happens after injection, the valve is open and rest of the sample is washed out 61 it demands sample reconcentration : prevents zone broadening cold trapping : first few centimetres of column has negative temperature gradient (~ 250 °C /injection/» 40 °C capture region; ca < 150 °C than TboN) => mobility of components with high TboN is zero => their reconcentration solvent effect : first few centimetres of column has negative temperature gradient (~ 250 °C /injection/» capture region is ca 20 °C bellow solvent TboN) => sample components with low TboN condensate with solvent from the created thin film, the solvent is slowly evaporating => reconcentration of components with low TboN split injection splitter allows: easy injection of small volume : is related to sharp zone entering onto column and column capacity S - degree of sample splitting, FM - column flow rate, Fs - splitter flow rate (proportion valve 2) disadvantages: : unsuitable for trace analysis : depends of splitter geometry today the most used way of injection 63 proportion valve 1 carrier gas septum waste splitter waste proportion valve 2 to detector pressurise using proportion valve 1: flow sensor = 103 ml/min, pressure sensor = 70 kPa septum flow set to 2 ml/min => slow flow of 3 ml/min onto column pressure sensor sets proportion valve 2 to 100 ml/min => onto column 1 ml/min => through inlet MF flow quickly, 101 ml/min sample introduced into injector and according to split equation, part goes onto column, part out to waste 64 on-column injection : injects precise amount : suitable for analytes with high Tboi| - no evaporation during injection instrumentally demanding - restrict pressure losses within injection overloads column with liquid (1 pi for 50 cm of column) =^> peak broadening : solution as within splitless injection : gas entrance to column is sealed : with closed valve pressurise using proportion valve 1: flow sensor = 7 ml/min, pressure sensor = 70 kPa, : septum flow set to 2 ml/min : sample introduced into injector and carried onto column by flow rate 5 ml/min : after certain time without splitting (splitless time), which happens after injection, the valve is open and rest of the sample is washed out 62 Separation column tubular : analytical : preparative capillary : open : filled length: 0.5- 10.0 m diameter: 1 - 6 mm length: 2-6 m diameter: > 6 mm length: 10- 100 m diameter: 0.1 - 0.5 mm length: 0.5-50.0 m diameter: 0.3 - 1.0 mm Separation efficiency comparison of different column types 200 220v GC separation of calamus oil components A - 50 m capillary column B - 4 m tubular column 67 column filling tubular columns cover: glass, steel, copper, polymers carriers modified infusorial earth active centres (silanols and siloxanes) =^> tailing of more polar components suppression - silylation adsorbents : unspecific (active carbon) : specific (silicagel, alumina, molecular sieves etc.) carrier- fine, solid and inert material (spherical silicagel) serves directly as SF (GSC), or is covered by thin film of liquid phase (GLC) solid SF non-polar : methylated polysiloxane, squalene mildly polar : phenylated polysiloxane -HO H H c — —o L H H H strongly polar : polysiloxane with CH2-CH2-CN, -CH=CH-CN, Carbowax 20M (based on PEG) capillary columns silica surface enlargement by etching polyimide cover => increase of mechanical stability SF universal non-polar silicon phases or immobilised Carbowax i.d. 100-530 um wall-coated open tubular columns (WCOT) liquid SF directly on the capillary wall fused silica open tubular (FSOT) thin wall with outer polyimide cover (mechanical stability) film thickness 0.1 -8 jjm i.d. 320 - 530 um film layer thickness 6-60jjm support-coated open tubular columns (SCOT) carrier is on capillary wall, SF is on it i.d. 320-530 [jiti porous-layer open tubular columns (PLOT) layer of solid active sorbent on an inner capillary wall layer thickness 5-50 jjm 70 column thermostat importance of temperature of GC : evaporation of liquid or solid sample : kinetic aspects of separation kept with precision of 0.1 °C; thermostat range (T,ab + 4 °C) - 450 °C optimal loading temperatures - TboN of component with highest value + 30-50 °C optimal column temperature ~ TboN of analyte column temperature > TboN => tR = 2 - 30 min minimal temperature => better resolution, but higher tR wide range of TboN of separated components => => temperature programme /column gradient (A temperature during experiment) temperature may be increased gradually or in steps 71 detectors detected compound is volatile, in gaseous state concentration dependent detector (CDD) : non-destructive, dilution with carrier gas decreases sensitivity mass dependent detector (MDD) : destructive, carrier gas interferes not, depends on introduction rate into detector ignition spirále flame ionisation detector collection electrode +300 V FID MDD signal: current created by pyrolysis of carbon sample hydrogene column noise 10"13 dyn. range 107 sensitivity 1010 g/ml 72 \A/^—i thermal conductivity detector TCD catharometer amplifier : noise 105 : dyn. range 106 : sensitivity 109 g/ml CDD signal: sample molecules change (decrease) thermal conductivity of carrier gas : carrier gas must have high thermal conductivity (He, H2...) : temperature dependent, universal electron capture detector ECD noise 10"12 dyn. range 105 sensitivity 1014 g/ml 63Nj -(•) waste current measuring CDD signal: analyte molecules decrease current generated by ß-emitter : halides, nitrites, cyano-compounds, peroxides, anhydrides, organometals from column 73 nitrogen phosphorus detector NPD - thermoionisation detector : noise 1012 : dyn. range 106 : sensitivity 1011 g/ml MDD connections- of heating heating spirále hydrogen V anode rubidium or caesium glass flame air ^=1- carrier gas (nitrogen) signal: Rb/Ce glass thermoionisation electron emission enhanced by N or P presence inlet of fluorine pump outlet chemoluminiscence detector data column : noise 1013 : dyn. range 104 : sensitivity 1012 g/ml irradiation CDD signal: reaction of F (strong oxidant) with analyte 74 flame photometric detector FPD region of chemoluminiscence waste thermostat wall source : noise 1012 : dyn. range 107 : sensitivity 1011 g/ml into amplifier MDD signal: chemoluminiscence : selective S (394 nm), P (526 nm) electrolytic conductivity detector ELCD : noise 1013 : dyn. range 106 : sensitivity 1012 g/ml rf -^ solvent recyclat. conductom. cell meas. O o «J o 1_ inli ?t of MDD signal: appearance of special products their conductivity measurement after mixing with solvent * reactive gas column 75 photoionisation detector PID : noise 1013 : dyn. range 107 : sensitivity 1012 g/ml CDD signal: UV-irradiation ionisation cooler mirror atomic emission diode array data microwave „ignition" microwave ^ reactor \ column grid thermostat wall column lightproof cover heated waste ionisation cell or secondary detector UV-transparent window atomic emission detector AED : noise 1014 : dyn. range 104 : sensitivity 1012 g/ml MDD signal: microwave induced plasma : selective according to chosen emission wavelength : very expensive 76 500 V ^__ discharge electrodes ionisation chamber auxiliary gas discharge chamber collection tables column waste ■*== meter 150 V gas density balance helium ionisation detector HID : noise 1014 : dyn. range 106 : sensitivity 1013 g/ml MDD signal: auxiliary gas is ionised first (He, Ar), its ions then secondary ionise sample molecules vapour bar catharometer ■ -r -r GDB : noise 108 : dyn. range 103 : sensitivity 107 g/ml waste MDD signal: pressure difference between upper and lower passage of gas in presence of eluent vapours column outlet catharometer 77 infrared detector IRD waste column o o ■a QĹ o gilt glass tube ■'yyyykXV^^^< KBr window o c o (D : noise 1012 : dyn. range 105 : sensitivity 1011 g/ml CDD signal: IR absorbance mass spectrometric detector MS noise 1014 dyn. range 103 sensitivity 1016 g/ml CDD signal: ion count universal ionisation: : electron impact (El) : chemical i. (CI) analysers: : quadrupole (Q, Qq) : ion trap (IT) : magnetic sector : time-of-flight (TOF) definition of chromatographic system in GC carrier gas type flow/ pressure (ml.min-1 / kPa) injection (X pi) injection type (event, splitting rate) stationary phase type length, inner diameter, manufacturer, SF type, film thickness 25m x 0.32 ID J&W DB-5 DF - 1.0 temperature gradient profile initial temperature and its period, temperature increase; inlet temperature (e.g. 130 X 1 min, 130 - 250 X at 5 X/min, 250 X 5 min; 250 X) detector basic characteristic according to type 79 analytical information in chromatogram qualitative information retention time « retention factor, distribution constant : compound identification (standard method) spectroscopic detectors: UV-Vis spectra MS spectra (ESI / APCI; Qq / IT / o-TOF) NMR spectra (1H,13C) retention time formulation specific retention volume (Vp) = 273.15*7, P S* T, relative retention time (rA B) : comparison with internal standard rA,B ť Ab) Kovats retention indices (rA B) : linear dependence pf retention time logarithm of homologues on carbon number 80 quantitative information peak area « amount, concentration of compound internal normalisation method : all components are eluted (solvent does not count) : all they have same/similar response factor c% A%j 100 *Aj A tot external standard method (absolute calibration; calibration curve) : always same measurement conditions, same injection volumes : indispensable matrix influence A c unknown unknown A *c known known c unknown internal standard method : need not to know injection volume : standard must be chemically similar to analyte A A IS l unknown Á Á ^IS2 ^known *c known standard addition method : presumes calibration curve linearity A,, - analyte peak area, unknown concentration c, A2 - analyte peak area of unknown concentration c, after addition of standard of known concentration c, V,, - sample volume, Vs - standard solution volume electromigration methods driving force - electric field : charged particle motion in electric field : extraction L-S electrolyte (liquid able to conduct current) separation channel wall (carries charge) stationary phase (SF, solid matter, micelles) mobility of ions is influenced by charge, molecule size and surrounding ions basic electromigration techniques : column arrangement (in tube, in capillary) : slab arrangement (in gel) 83 EMM history 1808-93 I first experiments in U-tubes - F. von Reuss (1808), G. Wiedeman (1856), H. Buff (1858), O. Lodge (1886), W. Whetham (1893) 1897 Kohlrausch - basic equation for ion migration in electrolyte solution 30. léta Tiselius - gel elfo with glucose as medium 1937 Tiselius - first fully functional electrophoresis instrument, 1948 Nobel price 1955 Smithies - use of starch gels for elfo 1958 Hjertén - ZE in rotating tubes 1-3 mm 1959 Raymond and Winstraub - acrylamide gels, setting up gel porosity & stability 1965 Tiselius - ZE in 3 mm tubes 1967 Hjertén - elfo in tube, i.d. 1 - 3 mm, with inner coating against EOF 1969 Vesterberg and Svensson - IEF of proteins in ampholytes 1970 Laemmli - denaturing separation in gel, SDS and concentration gel use Everaerts - ITP on own instrument 1974 Pretorius - EOF as a MF driving force through sorbent 1974-79 Virtanen, and Mikkers et al. - glass and teflon capillaries, i.d. 200 |jm 85 1975 O'Farrell - 2D GE, presetting IEF in gel to SDS elfo 1981 Jorgenson and Lucas - borosilicate glass capillary, i.d. 75 |jm 1983 Hjertén - CGE for biological samples 1984 Terabe - micellar electrokinetic chromatography 1985 Hjertén - CIEF for biological sample 1987 Karger and Cohen - high efficiency CGE for NA Knox and Grant - CEC in 50 |jm capillaries with ODS 1988 Beckmann Instruments - first commercial instrument 86 theoretical base of EMM motion of free charged particle in electric field : charge and field orientation decided on direction and velocity v = jU*E = ju = u * u_ i v- ion motion velocity p - electrophoretic mobility [m2 V-1 s_1] E - electric field intensity U - voltage I - length of voltage gradient influencing the motion by ionic atmosphere => => decrease of velocity with increase of electrolyte concentration |j0 ionic (net) mobility - u at zero ionic strength 10"9 m2V"1 s-1 = 1 tiselius (Ti), sign implies ion polarity (anion has negative p) temperature influence: ŤT => Ťu0; with 1 °C about 2 % ^ = //*[i+o.Q2*(r-r0)] T - working temperature T0 - standard, tabulated temperature 87 ion mobility estimation in a case, when value is not known (tabulated) Stokes mobility; a - acceleration of spherical charged particle motion a = 0 FE=FF FE FF q*E 4 Ô7T* ľ] *r *v 6n*r]*r* ju q - charge H - solution viscosity r- ion radius v- ion motion velocity relation of ion mobility and diffusion coefficient z - relative charge F - Faraday constant R - gas constant T - temperature D - diffusion coefficient ion mobility estimation for small molecules Jokl equation M - molecular mass a, b - empiric constants a~485x10-9rrr2V-1 s1 b~9.6x10-9rrr2V-1 s1 estimation error is ca 10 % actual ion mobility Onsager equation z+, z_ - relative ion and counter-ion charge I - ionic strength effective mobility mobility of weak bases, acids or zwitterions resulting mobility of all ion forms M n ^=z# I I i=l Uj - mobility of one ion form X: - its molar ratio free mobility mobility extrapolated to zero gel concentration migration time entry useful for mobility calculation / */ 11 tot leff ±/ * J- x U ŕ_ t0 m 'tot - separation channel total length leff - separation channel effective length tm- migration time t0- migration of neutral particle (EOF) Mtot Meff + M EOF I eff t *E m Kff^tot t *u m fully charged X^a = 1 pKa1 J J pKa2 a = 0.5 a = o// uncharged pH 12 90 wall is charged negatively - until said others electroosmotic flow (EOF) Si o t undisociated silanol SiOH capillary = endo-osmotic pump capillary made of fused silica with exposed hydroxyl groups dissociation of hydroxylgroups leaves a negative charge on the inner wall 2. -h (-) (-) (-) t-) (-) c-) c-) (-) (-) (V> (-) O g 3 o Ö" switching voltage on, liquid starts to move to cathode - it is mobilised by endoosmotic flow ! 91 eeefeQD O o + c (Š 9 9 <9® 9 ^e«e o8<*e e©e cations migrate towards cathode and carry solvent molecules in the same direction electroosmotic flow neutral molecules are moving in the same direction as electroosmotic flow with negligible mutual separation anions are slowed on their way towards anode, electroosmotic flow is stronger than their electrophoretic mobility => they proceed towards cathode too EOF = 0 => no mass flow, only ion exchange 92 r v EOF s*E, \ K V J * E e - dielectric constant 5 - zeta potential (electrostatic), appears as a consequence of charge on capillary wall H - viscosity . EOF positively influences peak shape EOF flow íl 93 influencing the EOF high EOF - electrolyte carries cationic analytes out before reaching separation low EOF - adsorption of cationic analytes some EMM modes demand EOF suppression (IEF, ITF, GE) what influences EOF? : surface wall charge : electrolyte viscosity : electric field intensity influence of voltage : change of EOF is directly proportional : low voltage => low efficiency of separation and resolution : high voltage => high Joule heat influence of ionic strength or background electrolyte concentration increasing value lowers ^-potential and thus EOF high values increase current and thus Joule heat high values may cause analyte salting-out and adsorption to wall low values supports adsorption to wall and limits sample concentration changes peak shape, if electrolyte conductivity differs much from analyte influence of organic solvent addition : decreases ^-potential and viscosity :: may change selectivity, gathered only empirically Meof 2.0 2.5 3.0 3.5 4.0 4.5 5.0 In c influence of tensides : changes ^-potential, may change wall polarity; anionic tenside increases EOF, cationic decreases (if wall if negatively charged) 95 influence of background electrolyte pH : directly proportional EOF change; low pH => low EOF, high pH => high EOF :: may change charge or structure of analyte Meof 4- influence of temperature 3_ : changes viscosity, higher temperature => higher EOF :: thermolability of some samples i- influence of covalent wall surface modification : changes ^-potential and wall charge polarity "ph pH influence on EOF influence of neutral hydrophilic polymers : changes ^-potential (decrease) and viscosity (increase), decrease EOF by charge shielding EOF measuring B.A. Williams, G. Vigh, Anal. Chem., 68, (1996) 1174-1180 outlet detector i N1 | inlet N1 N1 N2 II first EOF marker injection shifting the marker zone to detector by pressure second EOF marker injection shifting both marker zones to detector voltage application - electrophoretic mobilisation third EOF marker injection and consequent application of pressure - shifting all marker zones to detector + N1 N2 H N1 N2 □ N1 N2 N3 i 97 / =(t —2*t +t)* lEOF V 3 ^ L2 ^ l\) l eff u + ř,, / 2 3 inj M = l *l ''EOF Ltot U*(t -t l2-tJ2) \ m. ru rd / rw rd lE0F - length, which marker travels during electrophoresis t,, t2, t3 - migration times of zone N1, N2, N3 tinj- time period of marker injection by pressure leff - effective capillary length ltot - total capillary length U - applied voltage tm - time period of electrophoretic shifting tru and trd - time periods, for which the voltage (inc-/dec-)reases linearly to given value common EOF calculation Mtot Meff + M EOF I eff t * m ^ejf^tot t *u m 98 description of separation maximum function lsign = f (t) electrophoretic peak (Gaussian peak) width of zone A in separation channel a) peak width at baseline w = Ac b) peak width in half of peak height c) peak width between inflex points a2 - dispersion; defines zones broadening peak width is given in temporal units peak area w1/9 = 2,354cr W: = 2 better peak depicture 102 sorption influence sorption causes peak tailing 2 _ * VEOF hjf % ads ( ,\2 'r2*k (l + f) \ V AD K d J j ' __ m,ret m,unret t m,unret k' - capacity factor Wet- stained analyte migration time Kd - first order dissociation constant tm unret - unretained analyte migration time sorption could be prevented by capillary inner coating : serves to change also other system properties (reverts EOF...) injection length influence : injection length must be shorter than diffusion controlled zone width : low sensitivity demands often longer injections ť 2 inj inj ~ -j ry tini - injection pulse length Joule heat influence leads to temperature gradient and laminar flow Ar,= Q*r{ 1 í Ksil *ln A o.d.sil V i.d.sil J + 1 í *ln K polyim \ o.d. poly im + 1 V o.d.sil J o.d. poly im /Z Q - output r- radius k - thermal conductivity h - heat transfer rate off capillary c o 1-1- 3 O max. separation voltage 390 375 375 390 voltage d [(j m] decreasing voltage : decreasing generated heat, low sensitivity and resolution lowering capillary i. d. : current decrease with i. d. square, low sensitivity, adsorption! decreasing BGE concentration : decreasing current, increasing adsorption thermostating : draining heat 104 electromigration dispersion influence influences peak shape difference between conductivity of sample and electrolyte leads to 1) peak tailing 2) focusation (low sample conductivity), broadening (high sample conductivity) 3) ITF effect (peak fronting) because of certain ion surplus (e.g. Ch) + — 1 ©© © © © © © © © © © time Ms > Mbge => front 9ets broad and tail focuses + — ©© 1 © © © © © © © © time Ms < Mbge => front focuses and tail gets broad time Ms = Mbge => sharp zone 1 resolution R„, = 2-(t -t .) 2-Ař V m,i m,j ' ^ L-^1 m w.+w. w.+w. 1 J l J Am - difference, (m2 - Mi) M - median, (m2+ MiW2 EMM arrangement capillary rciA/ kV voltage source output 107 instrumentation hydrostatic siphon effect t rozdíl hladin injection device typical volumes: 10- 100 nl (capillary ~ 1 -2 ul) normal - longer part before detector reverse (short-end) - the other end hydrodynamic AP*d4*7r*t. V — - inj 128* tj* ltot injected volume Vinj AP - pressure difference d -capillary i. d. tinj - time length of injection ltot - total capillary length H - background electrolyte viscosity pressure ľ LÜ pressure —>■ 1 108 electrokinetic for CGE the only possible : non-quantitative - more mobile ions go easier stacking effect sample conductivity < electrolyte conductivity => sample ions carry the current => stacking/concentration on inter-phase sample-electrolyte ©"e n electrolyte weak field high conductivity strong field low conductivity sample V- (+) tT electrolyte t *u V. .=7T*r2*K*^-----^~ mJ eff %jj lEOF U sep +) weak field |i)high conductivity injected volume V, inj Uinj - injection voltage Usep - separation voltage r-capillary i. d. Ieff - capillary effective length tinj - injection time length tE0F - EOF marker migration time 109 voltage source typical range: 0-30 kV; recommended gradient 400 V/cm 0 - 300 mA too high voltage decreases analysis time, lead to discharges (ca 20 - 25 kV) ZE - constant voltage, ITF - constant current one electrode always grounded - that one closer to detector separation channel anode cathode electrolyte nter-phase sinks the oldest (proposed 1892, done 1930) glass U-tube electrophoresis in free solution : separation detection by moving inter-phase observation : coloured solution and clean electrolyte solution sample solution 110 capillary fused silica i. d. 10-200 Mm o. d. 350 - 400 Mm length 10 (CGE) - 100 cm; 50 - 75 cm most common outer coating - polyimide (mechanical properties) conditioning: establishing the properties of capillary inner surface surface cleaning: 1 M NaOH, 0.1 M HCl, BGE other: strong acids, organics (DMSO), detergents teflon reproducible EOF worse heat conductivity other materials based on Si00 - glass (Pyrex) covalent coating inner coating suppressing EOF, in range pH 4 - 5 relatively low (~ 0), pH 6 - 7 slowly increases at high pH is almost about 4/5 lower than in un-coated silica capillary Si-O-Si-R Polyacrylamide-, arylpentafluoro-, 3-glycidoxypropyltrimethoxy-siloxan protein or amino acid, sulphonic acids, maltose, PEG, polyvinylpyrrolidon : relatively easy preparation : limited long-term stability Si-C Polyacrylamide using Grignard reaction : stabile between pH 2 - 10 : difficult to prepare SF from GC and LC C2-18, PEG, phenylmethylsilicon : easy to hydrolyse : increased adsorption 112 adsorbates cellulose, polyethylene glycol, polyvinyl alcohol, polyethylene imine : only short-term stability in acidic range pH 2 - 4 (PEG, PVA) : stabile in neutral pH (PEI) : relatively hydrophobic : reverts EOF (PEI) dynamic coating part of BGE, stems in the praxis of adsorbates use pH extremes reduction of coulombic interactions : pH range 2-12 : EOF elimination at low pH, EOF high at high pH : unsuitable for proteins - denaturation : decreasing the charge differences decreases separation efficiency high BGE concentration (ionic strength) reduction of coulombic interactions : decrease of EOF often limited by Joule heat hvdrophilic polymers alkylcellulose, polyvinyl alcohol, dextrans, Polyacrylamide shield wall charge of capillary and decreases EOF : increases viscosity : in high concentration = entangled gel electrophoresis (CEGE) tensides anionic: sodium dodecylsulphate (SDS), cationic: cetyltrimethylammonium bromide (CTAB) non-ionic: Brij-35, BRIS zwitterionic: 3-[(-cholamidopropyl)dimethylammonio]-1 -propansulphate (CHAPS) deactivate capillary surface by hydrophobic or ionic interactions : wide possibility of compounds, easy use : decrease or revert EOF : may irreversibly denaturise protein : suitable in combination with RP-LC surfaces quaternary amines decrease or revert EOF : work also as ion pairing agents (MEKC) 114 paper / membrane 100% cotton/cellulose 0.17-0.30 mm thick pore size 2.5 |jm electrolyte source two glasses zones electrophoretic paper acetate cellulose pore size 0.2 |jm nitrocellulose pore size 0.2 um visualisation nafion (PTFE, sulphonated tetrafluoroethylene) 1 -2 nm and 5-6 nm m hbN bromophenol blue dimethylthionine (azure A) toluidine blue alcian blue sudan black naphthalene black ci ChU CI R=CH2-S^N.CH3 H 3C C H 3 116 agarose gel non-toxic, cheap, no additional components for polymerisation fragile 0.8% large molecules 1 - 2% common separation 4% small molecules % w/v agarose solution resulting gel structure D-galactose 3,6-anhydro-L-galactose 117 Polyacrylamide gel toxic (bis-acrylamide), inert fragile, reinforcement by RhinoHide™ or DurAcryl™ N H, acrylamide 0 H H ° -N, .N O \ O bis-acrylamide ch, -«- brjdge methylene-bis-acrylamide acrylamide H2C=CH/CONH S20|"> SOi + persulphate nn persulphate /ammonium/ - initiator tetramethylene ethylenediamine (TEMED) - catalyser cor H2O^H-OONH2 + „^.„jhJ^ CONH2 \ CONH2 H 2C-CH-CH 2-CH-CH2-CH- CONH. CONH. gel density (cross-linking percentage; acrylamide and bis-acrylamide ratio) i % cross-linking => easier motion of very large molecules 12% - common for 15 kDa - 60 kDa 8% - molecules 30 kDa - 120 kDa 25%-< 15 kDa; special protocol according to Schägger-von Jagow 12%-gel viscosity cavity diameter (12%) -100 m2s-1 -4.4 nm isocratic (continuous) (8-15 %) discontinuous gel (4% concentration and 12 % separation) gradient gel (Schäger-von Jagow) 119 Pyoo^r HjN \=S 0.3 M CuCI2 0.2 M ZnS04 0.2 M imidazole 0.02% Na2S203 0.1%AgNO3 37% HCOH 1%CH3COOH 120 (CE-on-chip) simpler arrangement than LC-on-chip : easy application of driving force : simple separation channel : suitable detection ZE, ITF, IEF... (má electrochemical detection injection channel access point separation channel lab-on-chip LC + CE absorption photometric detector diode array detector flow window in polyimine coating light source capillary i.d. 75 urn detectors problems : beam focusation : optical path length focusing optics - two spherical lenses absorbance : sensitivity 107 g/ml indirect detection : sensitivity 105 g/ml 122 prolongation of optical path bubble cell Z-cell optical path optical path flow reflexive inner coating capillary 4 ~ photomultiplier 123 radioactive (scintillation) detector ß-particle light photomultiplier tube Nal crystal . photocathode optical window anode fluorescence detector laser induced fluorescence (LIF) fluorescence : sensitivity 109 g/ml LIF : sensitivity 1011 g/ml scintillation : sensitivity 1011 g/ml photo-multipl. laser — — aperture A lenses V \ mirror i 4 capillary laser lens ^7 CCD camera capillary 124 amperometric detector < -s D) ground < o c 3 2> 0> ^sräszzzaj 2Z223IZZ7ZZZZ1 electrodes ground i ■ čd 2, = ňT >< 1 Q) O I 3 3 (D electrodes working amperometry : sensitivity 108 g/ml conductivity detector conductivity : sensitivity 106 g/ml electrodes actuator pick-up HI m ll flow : two metallic electrodes around capillary : when applying AC voltage on an actuator, the current flows through wall, in-between electrodes towards the pick-up electrode signal is then amplified 125 mass spectrometry matrix assisted laser desorption / ionisation MALDI discrete points (fractions) mixing with matrix : before outlet : after outlet liquid junction spotting probe vacuum chamber target capillary BGE reservoir o j---------------------------------V. s_____________t target continuous trace mixing with matrix : in liquid junction : pre-spotted matrix trace ion count : sensitivity 108 g/ml 1 sheath gas nebulising gas sheath capillary capillary t-connection spraying needle nuclear magnetic resonance may use bubble cell 1Hand13C-NMR 127 NMR : sensitivity 106 g/ml electrosprav ionisation ESI key point liquid junction separation capillary PEEK-seal spraying capillary block spraying buffer vial with BGE detection coil (5 nl) main magnetic field f current induced magnetic field output / capillary vial with BGE preparation small volumes (nl) => elution into collection vials (10 - 15 ul) peak detection => volume calculation / distance from capillary end pressure elution: (CZE, ITP; MEKC, IEF; CGE - no) : pressure application (5 kPa) during pre-calculated time period electrokinetic elution: (CZE, ITP, CGE, MEKC; IEF -no) : voltage application during pre-calculated time period : collection vial must contain BGE or other electrolyte elution in IEF mode: : it is necessary to consider that u = 0 collection electrolytes: CZE 2% acetic acid ITP 2% acetic acid CGE BGE MEKC BGE IEF ampholyte fJS electrolyte detector pressure or voltage ■■ j y electrolyte collection vial 128 definition of electrophoretic system composition: buffer concentration, pH, additives injection: type, its characteristics (time, pressure, voltage) separation channel type capillary length, i. d., material, manufacturer 30 cm x 50 |jm i. d., fused silica, J&W Scientific conditioning - coating, rinsing slab size (height x length x thickness), material 6.5 x 10 cm x 1 mm, Polyacrylamide continuous, discontinuous, gradient; leading colour applied voltage, current or output application time period detector basic characteristic according to type 129 analytical information from electrophoretogram electropherogram, electrophoregram, electrophoreogram migration time normalisation: wrong reproducibility; adsorption or EOF changes : on one marker (either EOF or very fasf) : on two markers inclosing separated components first: carries no charge, moves with EOF second: highest mobility peak area normalisation: peak area is function of migration velocity (migration time) only within EOF changes; within ionic strength or injection length changes - no correction effect correction of injection length change within pressure injection IS - internal standard; might be a peak in mixture 130 A_ = A, IA W2 TV lN,IS basic modes of electromigration methods electrophoresis (ZE) isoelectric focusation (IEF) isotachophoresis (ITF) electrochromatography (EC) micellar electrokinetic chromatography (MEKC) affinity electrophoresis (ACE) non-aqueous electrophoresis (NCE) 131 CZE, capillary zone electrophoresis electrophoresis - greek rjAcKTpov (amber) and cpopéco (I carry) one background electrolyte (BGE) => constant electric field intensity in whole separation channel ä r A A A t = 0 t>0 a = Ma- Mb M B selectivity of separation, analytes A and B 132 choice of background electrolyte : sufficient buffering capacity in chosen pH range : low background signal in detector : low mobility (large, low charged molecules) => low Joule heat additives tensides all types changes EOF; give charge to non-polar molecules changes CZE into MEKC (if the critical micellar concentration is exceeded) zwitterions CHAPS (3-[(-cholamidopropyl)dimethylammonio]-1 -propansulphate) increases ionic strength without increase in conductivity (heat) influences selectivity chiral selectors cyclodextrins, crown-ethers ... similar to chiral additives in MF within LC metal ions K+, Na+, Cu2+, Li+... influence selectivity in MEKC and GE chaotropic agents urea ... solubilise NAand proteins; influence selectivity in MEKC linear hydrophilic polymers methylcellulose, Polyacrylamide, polyethylene glycol, polyvinyl alcohol... decrease EOF; decrease analyte adsorption in low concentrations, ZE => GE organic agents methanol, acetonitrile ... generally decrease EOF; influence selectivity in MEKC and chiral separations complexing buffers borate ... allow separation of saccharides and catechols 135 CGE, capillary gel electrophoresis j^'jg^sžgyígď^as^'- ^t^:'.Rafl-:ŕ*2á-.--s>r gel matrix t>0 classical - cross-linked gel in capillary relatively fast, reproducible and quantitative compared to slab gel electrophoresis : on-line detection in UV-VIS without visualisation disadvantages: capillary filling (homogeneous polymerisation, bubbles...) commercially filled capillaries - high price chemical gels: Polyacrylamides - porous structure with strong covalent bonds physical gels: agarose - weak intermolecular bonds of different molecule parts 136 entangled gel - linear gel as part of BGE entangling medium (e.g. polymerous net) is present in background electrolyte similar to physical gels - characteristic intermolecular interactions rapid increase in viscosity ( = f(lvlw)) at liminal concentration values mostly used polymers : linear Polyacrylamide N-substituted acrylamides N-acryloyl aminopropanol (AAP) N-acryloyl aminobutanol (AAB) N-acryloyl aminoethoxyetanol (AAEE) polyethylene glycol (PEG) cellulose derivatives methylcellulose (MC) hydroxyethylcellulose (HEC) hydroxypropylcellulose (HPC) : polyethylene oxide (PEO) hydroxypropylmethylcellulose (HPMC) polyethylene alcohol (PEA) : galactomannan (GalMan) : polyvinyl alcohol (PVA) : glucomannan (GluMan) ^ capillary filling bubbles : monomer solution looses volume when polymerising => isotachophoretic polymerisation capillary and anodic space: acrylamide, bisacrylamide, triethanol amine (catalyser) cathodic space: ammonium persulphate (initiator) when the source is switched on, the initiator enters the system ITF interface chloride / persulphate keeps initiator zone sharp => supervised polymerisation such a voltage that initiator flow ~ rate of polymerisation (ca 2 - 4 V/m) 138 GE, slab-gel electrophoresis ■w denaturing (SDS, Lämmii) - separation according to Mv non-denaturing (native) - separation according to pi, shape and Mw one dimensional gel electrophoresis (1D-GE) slab gel polymerises between glass plates, separated by spacers loading jars are created by special spacer - comb nskvvvmnrifuuxn (4%) concentration gel (12%) separation gel basic procedure 1. sampling buffer is added to sample 2. sample is loaded into jars 3. gel is put in-between buffers and voltage is applied 4. gel is washed and stained 140 two dimensional gel electrophoresis (2D-GE) two dimensions: 1.IEF 2. SDS-GE 1. isoelectric focusation (IEF) immobilised pH-gradient in gel strip 2. denaturing gel elfo (SDS-GE) SDS is not in gel since polymerisation (as with 1D) - micelles would be created sample decreasing pi ' m m *ŕ ■ m 1st dimension IEF o o 2 H -N N- -C=CH, H 2 IEF strip on SDS gel i « ľT i ! i | + =ŕ 2nd dimension SDS-GE decreasing pi necessary to cool more than 1D(5-12 °C) as cross-linking agent piperazine diacrylyl (PDA), diallyltartarate diamide (DATD), bisacrylyl cystamine (BAC) H,C=C-C-NH-2 H H, OHOH HN-C-C=CH, H2 H 2 H9C=C 2 H O -SH-SH' J- C=CH9 H 2 sodium thiosulphate in gel - low background with Ag-staining 141 in 2D density gradient (9-16 %) is used in connected containers are mixed A) solution without cross-linker B) solution with max cross-linker concentration : at outflow, increasing cross-linker gradient is formed gradient profile is given by the shape of containers new- non-linear pH gradients in IEF after staining : densitometry :: UV-Vis :: fluorimetry ( T* L CCD camera focusing optics passed light stained gel light filter : prior to analysis, sample is denatured (+ EtSH, 95 °C, 5 min) :: breaking of di-sulphidic bonds :: turn into random coil conformation : leading colour: bromphenole blue non-denaturing (native) GE separation of acidic and basic proteins - separately: : leading colour: bromphenole blue for acidic methylene blue for basic H3C. denaturing GE ^^7 merkaptoethanol 'dismisses S-S bridges t#-\U AAR SDS 1 unit charge i GH3 Cr CH3 : separation of acidic and basic proteins - together: giving them a unit charge without denaturation blue native PAGE (BN-PAGE) - CBB R-250 (~ 1 g to 1 g of protein) clean native PAGE (CN-PAGE) - n-dodecyl-ß-maltoside and digitonin 143 polyacrylamidove gel electrophoresis - PAGE : for separation of proteins in native and denaturing mode; 1D and 2D agarose gel electrophoresis - AGE : for nucleic acids separation 0.8% 50 - x1000 kbp only one mode (1D) 1 - 2% 20 - 50 kbp ^^^^H NAs already have unit charge 4% < 20 kbp leading colours: xylene and bromophenol blue, cresol red, orange G _*—■ separation conditions: TRIS-acetate EDTA (TAE) : low voltage, large molecules (50 - xOOO kbp) TRIS-borate EDTA (TBE) : 20 - 50 kbp sodium borate (SB): high voltage (35 V/cm), small molecules < 5 kbp column continuative elution gel electrophoresis (CEGE) : new technique similar to slab GE - primarily preparative :: mostly SDS-PAGE :: native isoelectrofocusation QPNC-PAGE (quantitative preparative native continuous) : suitable for on-line connection with detection techniques (MS) separation buffer lution buffer ^- cooling core collection channel gel column separation zones - elution chamber V fraction collector eparation buffer 145 CIEF, capillary isoelectrofocusation isoelectrofocusation - greek \ooq (same), rjAcKTpov (amber) and latin focus solution contains ampholytes during separation, the pH gradient is established pH = pi, analyte is not moving mixture of ampholytes and sample C t D F G B . EA C 9 F H • G A B • G n# H ■ i A E A • A • Ac ■ g E A FA D A F * A W F B H A • A A D * • B • H E • u G A A low pH <---------- pH gradient -----------> high pH A A B B A A B B A A A A CC DD E E CC DD E E ■ _ ■ F F F F • • • • G G H H G G H H 146 zones are sharp, self-focusation effect wA - zone width x - length coordinate resolution in IEF E - electric field intensity [V/cm] dpH / dx - pH gradient du / dpH - mobility slope at given pi CITF, capillary isotachoforesis isotachophoresis - greek íooq (same), Taxúq (speed) and cpopéco (I carry) two electrolytes : leading - leading ion has absolutely highest mobility in system : terminal (trailing) - terminal ion has absolutely lowest mobility in system => electric field intensity increases from leading to terminal ion t = 0 terminal electrolyte (T) Á A Á Á • * t>0 component concentration in zone is according to Kohlrausch co-function analytical concentration of compound A, cA: c = c * M, Ma Mci * Ml Mci M L for strong univalent electrolytes CI - analyte counter-ion 148 self-focusing effect zones are sharp and do not broaden => concentrating minor components in few orders if ion L because of diffusion goes to zone X, because of t E also increases its migration velocity and it goes back to zone L if ion X because of diffusion goes to zone L, because of i E also decreases its migration velocity and it goes back to zone X 333 379 350 370 390 410 time [min] isotachophoretogram typical detection - resistance; others methods - conductivity, thermometry, UV-Vis 14g MEKC, micellar electrokinetic chromatography one electrolyte containing ionogenic tenside over critical micellar concentration => micelles are created analyte is separated between micelles and electrolyte ace. distribution coefficient (K) MEKC may be seen as ZE of two entities - analyte and micelles with it analyte does not enters micelles => K = 0, analyte enters completely =^ K = oc tenside / micelles t = 0 t>0 V = ^m ^M tM^-(tm/tmc)) = K*(VSF/VMF) k' - capacity factor tM- void retention time tm- retention time tmc- retention time of micelles 150 commonly used tensides anionogenic : sodium dodecylsulphate ... cationogenic : cetyltrimethylammonium bromide, septonex to decrease migration velocity of micelles non-ionogenic tenside (Triton X-100) is added micelles may be substituted with microemulsion or polyions addition of organic phase: solvatation changes, micellar structures, smoother setting - mixture of tensides resolution in MEKC efficiency selectivity retardation a - selectivity N - number of theor. plates disadvantage: difficult reproducibility 151 TLE, thin layer electrochromatography paper electrophoresis, slab electrochromatography charged (mostly negative) SF; often silicagel, cellulose and its derivatives analyte is separated between SF and electrolyte ace. distribution coefficient (K) charged surface groups O- 0 0 0 0" 0" compact CjO Ci) © © layer ------------------------------------------ diffusion S~\ s~\ /""n EOF _____ solvent _____J---- fast: applied voltage is driving force; comparing to TLC where it is capillary elevation : fast also comparing to capillary variant (up to three orders of magnitude) : voltage 160 V/cm => migration velocity 100 um.s-1 152 CEC, capillary electrochromatography charged (mostly negative) SF; porous particles of o.d. 1.5- 5.0 urn column: either broader (320 urn) or narrower capi Nary (50, 75 or 100 urn) analyte is separated between SF and electrolyte ace. distribution coefficient (K) : applied voltage is separation driving force => flow of the liquid is not laminar : EOF is created on the surface of SF rather than on a wall of separation channel low currents: max 10 uA Joule heat 0.1 W.cnrr2 (1500x more heat than within pressure heating by H PLC) o o o o o o o o o o o o o o o o ° o o o o t o0o°o Q °oo o Q °o o o Q °o o o o0 153 SF C18 bound on silicagel (reverse CEC) ß-CD bound on silicagel (chiral CEC) SCX cation exchanger (-CH2CH2CH2S03H) testing mixture F F H thiourea GR 57888X, GR 57994X fluticason proprionate, des-6-a-fluoro-fluticason proprionate O =>^ H thiourea indicates EOF components 2 and 3 determine hydrophobicity components 4 and 5 determine resolution COSCH2F 154 electric field advantages : higher efficiency than HPLC up to 300 000 plates / m (i.e. 3 - 4x) frit column frit : may use very small particles no high back pressure : separation of neutral, lipophilic and water-insoluble analytes : low sample and MF consumption : isocratic and gradient elution : may use MS detection : same instrumentation as for CZE, CEC or CLC 320 urn i.d. inlet frit window disadvantages column outlet frit : column filled capillaries with frits; fragility : bubbles (EOF differences, Joule heat) : electrokinetic injection (internal standard) : lower sensitivity 75 urn i.d. 280 urn o.d. window AE, affinity electrophoresis uses combination of separation in filed and affinity separation affinity separation - specific interaction of analyte and ligand enzyme nucleic acid antigen receptor coenzyme, substrate, inhibitor complementary chain, histone antibody signal molecule afinant carrier analyte A analyte B MZS EOF aa separation in capillary and in gel : separation highly selective : purification shot-gun : interaction study compatibility association constants 156 blotting blotting paper analyte zones absorbent support Southern blot - DNA Northern blot - RNA Western blot - proteins blotting solution immunoelectrophoresis interaction antigen (Ag) + antibody (Ab) membrane membrane with blotted analytes 1D gel immunophoresis 1st dimen. - Ag separation 2D gelová immunophoresis 1 NAE, non-aqueous electrophoresis separation in non-aqueous solvents 1978 - non-aqueous TLE 1984 - non-aqueous CE (NACE) advantages: : elimination of levelling effect of solvent => higher selectivity of separation : low current : separation of hydrophobic (water-insoluble) analytes solvent choice: : volatility : ability to solve BGE and analyte : viscosity : dielectric constant : transparency in UV solvents: water content max 1 % amphiprotic : neutral : protogenic : protophilic : dipol. protophilic aprotic : dipol. protophilic : inert (+ (+ (-(- +) -) +) +) MeOH, glycerol, phenol, te/f-butylalcohol sulphonic a., formic a., acetic a. liquid ammonium, formamide, N-methylformamide DMSO, dimethylformamide, THF, 1,4-dioxan, pyridine (-;-) : AcN, acetone, nitrobenzene, sulpholane, PC (-;-) : alif. hydrocarb., benzene, 1,2-dichloret., tetrachlorom. relatively basic or acidic (*;*) background electrolytes: : ammonium acetate, sometimes with addition of acetic a. or sodium acetate : quaternary ammonium salts : Tris, magnesium acetate, citric a., formic a., trifluoroacetic a. ... additives: polyalcohols and surfactants => decreasing EOF 159