capillary electrophoresis guidebook

CHAPTER1 Fundamentals of Capillary Electrophoresis Theory Kevin D Altria Introduction This section will describe the fundamental theory, equations, and definitions necessary to comprehend the concepts involved in capillary electrophoresis (CE) This is not an exhaustive treatment, but is considered sufficient to comprehend and appreciate the principles of CE More detailed theoretical background can be obtained from a number of reference books (I-6) Developments in the field of CE are reviewed in detail annually in the journal Analytical Chemistry For example, the 80 papers published in 1992-l 993 were recently reviewed (7) CE can be broadly described as high-efficiency separations of sample ions in a narrow bore (25-100 pm) capillary tube that is filled with an electrolyte solution A typical schematic of an instrument setup is shown in Fig The principal components are a high-voltage power supply, a capillary that passes through the optical center of a detection system connected to a data acquisition device, a sample introduction system, and an autosampler Typically, the CE instrument is controlled by a personal computer The capillary is first filled with the required buffer solution Sample solution (typically l-20 nL) is then introduced at the end of the capillary away from the detector (usually the anode) The capillary ends are then dipped into reservoirs containing high-voltage electrodes and the required buffer solution One electrode is connected to a cable leading to From Methods m Molecular Bology, Vol 52 Capdary Electrophoresrs Ed&d by K Altrla CopyrIght Humana Press Inc , Totowa, NJ Altria n High voltage supply Empty vial Fig Typical instrumental setup the high-voltage output, whereasthe other (situatedat the detector end of the capillary) is connectedto an earthing cable Electrodesare composed of an inert material, such as platinum Application of a voltage (for example, 10-30 kV) across the capillary causes electrophoretic and electroendosmoticmovements(discussedlater in this chapter) resulting in the ionic speciesin the samplemoving along the capillary and passing through the on-line detector A plot of detector response(usually UV absorbance) time is generated, with which is termedan electropherogram 1.1 Electrophoresis This processis the movementof sampleions under the influence of an applied voltage The ion will move toward the appropriateelectrodeand passthrough the detector The migration rate, or mobility, of the solute ion is governed largely by its size and number of ionic charges For instance, a smaller ion will move faster than a larger ion with the same number of charges.Similarly, an ion with two chargeswill move faster than an ion with only one charge and similar size The ionic mobility (pE) is therefore related to the charge/mass ratio (Eq [ 11) (1) Fundamentals of CE Theory Detector response Fig Theoretical separation of a range of catrons where PE = electrophoretic mobility, CJ number of charges, IJ = solu= tion viscosity, and r = radius of the ion Therefore, when we separate a hypothetical mixture of ions havmg different charges and sizes, the smaller, more highly charged ions will be detected first (Fig 2) The actual electrophoretic velocity, or speedof the solute ions, is related to their mobilities and the magnitude of the applied voltage (Eq [2]) v=pEE (2) where v = velocity of the ion and E = applied voltage (volts/cm) 1.2 Electra-Osmotic Flow (EOF) Application of voltage across a capillary filled with electrolyte causes a flow of solution along the capillary This flow effectively pumps solute ions along the capillary toward the detector This flow occurs because of ionization of the acidic silanol groups on the inside of the capillary when m contact with the buffer solution At high pH, these groups are dissociated resulting in a negative charged surface To maintain electroneutrality, cations build up near the surface When a voltage is applied, these cations migrate to the cathode (Fig 3) The water molecules solvating the cations also move, causing a net solution flow along the capillary (Fig 3) This effect could be considered an “electric pump.” The extent of the flow is related (Eq [3]) to the charge on the capillary, the buffer viscosity, and dielectric constant of the buffer: pEOF=(&&/q) (3) where pEOF = “EOF mobility,” IJ = viscosity, and = Zeta potential (charge on capillary surface) Altria Ftg Schematicof electroendosmoticflow The level of EOF is highly dependent on electrolyte pH, since the &, potential is largely governed by the ionization of the acidic silanols Below pH 4, the ionization is small (8), and the EOF flow rate is therefore not significant Above -pH 9, the silanols are fully ionized and EOF is strong The pH dependence of EOF is shown in Fig The level of EOF decreases with increased electrolyte concentration as the potential is reduced The presence of EOF allows the separation and detection of both cations and anions within a single analysis, smce EOF is sufficiently strong at pH 7, and above, to sweep anions to the cathode regardless of their charge Analysis of a mixture of cations, neutral compounds, and anions would result in the electropherogram shown in Fig The migration times correspond to the time the individual peaks pass through the detector The smaller anions fight more strongly against the EOF and are therefore detected later than anions with a lower mobility Multiply charged anions will migrate more strongly against the EOF and will be detected later Therefore, pH is clearly identified as the major operating parameter affecting the separation of ionic species, smce it governs both the solute charged state and the level of EOF The overall migration time of a solute is therefore related to both the mobility of the solute and EOF The term apparent mobility @A) is measured from the migration time, and is a sum of both yE and pEOF: PA = pE + JJEOF= (ZL/ tV) (4) where I= length along the capillary (cm) to detector, V = Voltage, and L = total length (cm) of the capillary Fundamentals of CE Theory 15 05 I PH Fig Varlatron of EOF with pH Mobility values can be calculated from migration times when both ionic and neutral components are measured For instance, in the separation of a five-component mixture shown in Fig 5, the mobility values for the peaks are calculated and given in Table Example peak = IA = (1L/ Vt) = (50 x 57 / 30,000 x 500) = 1.9 x lOA vEOF (from peak 3) = (IL / Vt) = (50 x 57 / 30,000 x 600) = 1.58 x 10q j~E=pA-pEOF=0.32x IO4 The negative values of PE for peaks and indicate that they are anions The separation of ions is the simplest form of CE and is often termed Free Solution Capillary Electrophoresrs (FSCE) The separations rely Altria Frg Theoretical separation Calculated Mobility of a range of ionic and neutral solutes Table Values for the Peaks m Fig Peak no Mlgratlon time, s PA cm2/Vs 400 500 600 750 900 2.38 x lo” 190x1@ 1.58 x 1W’ 1.27 x lo” 1.06 x lti PE 080x lOA 32 x lOA -031 x lOA -052 x 10“ I = 50 cm, L = 57 cm, and V = 30,000 V principally on the pH-controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute In FSCE, all neutral compounds are swept, unresolved, through the detector together (Fig 5) Separation of neutrals is generally achieved by incorporation of anionic surfactant, at sufficient concentration to form micelles These anionic micelles migrate against the EOF and can chromatographically interact with neutral solutes Solutes having a large interaction will migrate later than those having little or no interaction Use of micellar solutions is known as micellar electrokinetic capillary chromatography (also called micellar electrokinetic chromatography) and is covered in depth in Chapter 12, which is coauthored by the originator of the technique When dealing with large biomolecules, such as nucleic acids, their electrophoretic mobilities may be very similar, and FSCE is often insufficient for adequate resolution In this case, separations are performed in Fundamentals of CE Theory capillaries filled with gel solutions In Capillary Gel Electrophoresis (CGE), a sieving effect occurs as solutes of various sizes migrate through the gel filled capillary toward the detector Chapter 13 describes the exceptional, efficient separations that can be obtained in gel filled capillaries The separation and quantitation of chiral samples are an important area in many industries Highly efficient chiral CE separations (Chapter 14) can be obtained by the addition of chirally selective substances, such as cyclodextrins, into the electrolyte Capillary electrochromatography (CEC), which is a hybrid between CE and HPLC, has been developed In this technique, CE equipment is used to generate HPLC-type separations Capillaries are filled with HPLC packing material, and the application of a voltage results in the EOF pumping the mobile phase through the capillary The full details of this technology and some applications are given in Chapter 15, which is written by one of the initial developers of the technique 1.3 Sample Introduction Sample can be introduced into the capillary by three techniques, all of which involve immersing the capillary end into the sample solution and exerting a force to inject sample into the capillary The three mechanisms for introduction of sample solution into the capillary are hydrodynamic, gravity, and electrokinetic All these methods are quantitative, and equations describing the volumes injected have been derived Figure shows the principles of operation for the three methods 1.3.1 Pressure Differential In this method, the sampling end of the capillary is immersed in the sample solution and a pressure difference applied (positive pressure or vacuum) The volume of sample solution injected onto the column can be calculated: Volume = AP d411t / 128 q L (5) where AP = pressure difference (mbar), q = buffer viscosity, L = total capillary length, and d = capillary diameter (pm) Table gives injection volumes (9) for l-s injections using 65-cm capillaries of varying bore, TJ= 1, and various AP values These volumes generally correspond to sample plug lengths of 122 f 2.2 Soft capsule Bl (10 mg) PP (30 mg) 111 + 1.8 112 I +3 B2 (7 mg) 1086k 1.8 B6 (5 mg> Repnnted wtth pernnsslon from ref 75 n/a = not analyzed Tablet MECC HPLC 1236_+26 108.0 X!I1 994+2 113 7* 17 112.4 3~ 1094+09 119.3 + 2.9 106 2-1: 3.1 126.6 21 1086k 17 1149k 16 108 Z!I 1.6 1238+36 108752 1039+07 1124+3 111.6+ 16 1115+34 117.2 + 2 1132+39 n/a n/a n/a n/a solid-phase extraction (84) However, the rugged nature of the CE capillary format can also allow direct mjection of biofluids, such as serum (90) and urine (86), with obvious savings in analysis time and cost of consumables The majority of direct injection analyses are performed by MECC, since the SDS micelles strongly interact with the sample proteins causing the proteins to be eluted later, and the small solutes of interest are then visible free from protein interfaces Figure 11 shows that antiepileptic drugs can be directly monitored in patient serum as the proteins migrate after the peaks of interest (90) The performance of CE methods in clinical assays has been assessed by many workers and general comments indicate that CE is not as sensitive as HPLC, but has benefits in terms of simplicity and possible sample pretreatment reductions Validation parameters, such as linearity, recoveries, and precision, show acceptable performance (81,83,86) Crossvalidation of CE results with other techniques, such as HPLC (86) and immunoassays (90,91), show that CE is capable of generating accurate results Table compares levels of creatinine and uric acid as determined by both an enzymatic method and by MECC (92) Despite the interest and research focus in this area, increased routine application may require Additional Application Areas 331 Proteins Phenobarbital 1, , 10 15 2( Fig 11 Separation of antieplleptic drugs by direct qectlon of patient serum ReprInted with permission from ref 90 Separation conditions: 75 n-&I SDS, mA4 borax, 10 mA4 phosphate, 220 nm, 35°C Comparison Table of Levels as Determmed by MECC and Enzymatic Methods Creatmine, yglmL Plasmasample I Uric acrd, pg/mL Enzymatic MECC 7 7 N/R Enzymatic 28 50 38 27 66 12 56 60 MECC 32 51 N/R 32 66 11 58 60 Reprinted with permIssIon from ref 92 N/R = no result obtamed further advances in sensitivity by instrument improvements sample introduction procedures or by better Alternative Detection Systems (Including CE-MS) Currently, all commercial CE systems incorporate a UV-absorbance detector Some systems are modular, or partially modular, and alternative detection systems can be employed The principal detection alternatives to UV-absorbance detectlon that are commercially available are fluorescence and mass spectrometry Other less-developed detector Altria 332 options include electrochemical (93,94) and conductivity detection (9.5) An increasing number of commercial instruments are also available with UV-absorbance photo-diode array facilities Diode array detectors have been shown to be of use in peak identification and peak purity assessments (96,9 7) 7.1 CE-Mass Spectrometry There has been a great deal of activity in the area of interfacing CE to mass spectrometers, and the advances to date have recently been reviewed (98) A number of interfaces have been successfully demonstrated, include continuous-flow fast atom bombardment (99), electrospray (100, 10 I), and ionspray (102) Generally, reports have indicated that CE-MS is less sensitive than UV-absorbance detection, which may have limited a more substantial exploitation of this detection mode However, the significant potential sensitivity advances offered by ion-trap MS (103) or the use of on-line stacking in CE (104) may lead to an alleviation of this issue Amino Acid Analysis Much of the early development work in CE was performed using derivatized amino acids as test solutes A range of derivatization agents are avaliable for this purpose (Table 8) The vast majority of separations have been achieved employing SDS-based MECC conditions with the addition of organic solvent (105) Figure 12 shows the highly efficient resolution of 23 dansylated amino acids using 102 mMSDS, pH 9.2, electrolyte and a temperature of 10°C (105) Underivatized amino acids have not been widely analyzed, since they generally possess very limited chromophores However, native amino acids have been separated and monitored by indirect detection, employlng mM sodium salicylate at pH 9.7 (11.5) Particulates, Bacteria, and Dyes 9.1 Analysis of Particulates CE has been successfully applied to several separations of large polymeric species, such as polysterene latex particles (I I6) and Jeffamine polymers having molecular weights up to 2100 (II 7) Separations are performed in free solution with resolutions owing to size differences Silica gel sols, which are used in the preparation of HPLC packing mate- Additional Application Areas 333 Table Derwatlzatlon Reagents Employed m Amino Acid Analysis Derwatwe Reference no PTH FITC CBQCA TBQCA Dns OPA NDA 106 107,108 109,rro Ill 112,105 113 113,114 PTH (phenythlohydantom), FITC (fluorescem lsothlocyanate), CBQCA (3-[4-carboxybenzoyll-2-qumolmecarboxaldehyde), Dns (Dansyl), OPA (o-phthaladehyde), TCQCA (3-[4-tetrazolebenzoyll-2-qumolmecarboxaldehyde), NDA (napthalene dlaldehyde) 10 12 14 16 7.0 22 mln Fig 12 Resolution of 23 dansylated amino acids usmg MECC at 10°C Reprmted with permlssion from ref 105 Separation conditions: 20 mA4borax, 100 mMSDS, 10°C, 214 run rial, have also been characterized by CE (118) using pH 9.0 buffers and detection at 190 nm Silica sol colloids up to 500 nm diameter were separated (118) of Bacteria by CE Mixtures of various viable bacteria, such as Enterococcus, Streptococcus, and Staphylococcus strains, were resolved by CE (129) using Tris/Borate/EDTA buffer The pH of this buffer is high, and the bacteria 9.2 Separation and Isolation were resolved as anions UV detection at 190 or 200 nm, together with use of a loo-pm capillary, allowed sufficient sensitivity Preparative CE was performed to collect specific bacteria from mixtures Positive iden- Al tria 334 tification of collected fractions was achieved by several techniques, including metabolic fermentation Purities of recovered fractions were >99% The authors concluded that CE could afford the microbiologist a new tool for studying the composition and distribution of microorganisms in mixed populations It 1s noted that these separations were conducted on homemade equipment and that sophisticated commercial equipment may offer significant advantages in terms of improved performance and sensitivity 9.3 Determination of Dyes by CE Currently HPLC IS predominantly employed in the separation and determination of levels of cationic, anionic, and neutral dyes, and dye intermediates CE has been shown to be of use in this area (120,121) Many dyes have two or three membered ring structures and are water soluble, making them very suitable for analysis by CE Notes Added in Proof Analysis of small ions by capillary electrophoresls: An optrmized separatlon has been reported (122) that allows simultaneous determmatlon of ammonium, alkali, alkalme-earth, and various transition metals using an electrolyte containing lmidazole, crown ether, methanol, and HIBA Low ppb detection levels were possible with electrokinetic mJectlon Laserinduced indirect fluorlmetrlc detection of cations has been reported (123); the electrolyte employed contained fluorescem sodium and EDTA, and low-mid ppb levels of metal ions could be detected using pressure mJectlon Recent advances in the determination of anions has centered on the optlmlzation of electrolyte systems, For example, the use of p-aminobenzoate as an electrolyte additive has been shown (124) to be useful for the detection of orgamc acids 2,6-napthalene dicarboxylic acid has been employed as a UV absorber and has been shown (125) to be a considerable improvement over pthalate Migration time drifts can occur using the standard electrolyte containing chromate because of electrolyte depletion This problem can be overcome (126) by the addition of r&k! 5,Sdiethylbarblturate to the electrolyte Experimental design: Multivariate regression analysis has been employed to study the effect that various ratios of EDTA and borate concentrations have on the migration time of metal complexes (127) A central composite design has been utilized for optimization of electrolyte composition (122) A full factorial design was then used to measure the mam effects of several parameters on the EOF velocity Additional Application Areas 335 Carbohydrate analysis: A recent survey (128) has been published concernmg the application of CE to carbohydrate analysis It comprehensively reviews the derivative types available and a range of appltcations Another recent report (129) dlscussed the use of tags, such as ANTS Underlvatized carbohydrates have been separated using NaOH electrolytes with electrochemical detection (130) Indirect UV detection using a pH 12.3 electrolyte containing sorbic acid has been used (131) to assay the carbohydrate content m fruit Juices and good agreement with HPLC data was obtained Vitamin analysis: Surpnsmgly, there contmues to be relatively few reports of analysis of vitamins by CE One notable exception is the work concernmg the determmatlon of vitamin A in dried blood spots Laser-based fluorescence measurements allowed (232) a detection limit of pg/L to be obtained for retinol The analysis could be conducted from one or two drops of blood Biomedical apphcations: The number of biomedical apphcatlons of CE contmues to expand rapidly Some particular examples are discussed covering both applications and methodology approaches Theophylline and metabolites have been determined in urine (133) using solld-phase extraction pretreatment A variety of ephedrine alkaloids were determined m urine with direct sample injection (234), and levels of free and total 7-hydroxy-coumarin were determined (23.5) in both urine and serum samples The use of SDS solution as a rinse solution between analyses of blosamples has been shown to be more effective (136) than conventional rinsing regimens The different approaches to quantifying drug m human serum followmg direct sample Injection have been compared (23 7) CE-MS: The apphcatron of CE-MS combinations as separation-detectlon systems continues to grow For example, peptides (138) and DNA fragments (139) have been detected by MS followmg their separation by CE References Beck,W andEngelhardt,H (1992) Capillary electrophoresls orgamcand morof ganlc cationswith mdlrect UV detection.Chromatographza 33,3 13-3 16 Chen, M andCassldy,R M (1993) Separationof metal ions by capillary electrophoresis.J Chromatogr 640,425-43 Weston,A., Brown, P R., Heckenberg,A , Jandlk, P., and Jones,W R (1992) Effect of electrolyte composltionon the separationof morgamc metal catlonsby capillary ion electrophoresls Chromatogr 602, 249-256 J Shi,Y andFritz, J S.(1993) Separation metal ionsby capillary electrophoresls of with a complexlng electrolyte.J Chromatogr 640,473-479 Weston,A., Brown, P R., Jandlk, P , Jones,W R., andHeckenberg,A L (1992) Factors affecting the separationof inorganic metal cations by capillary electrophoresis.J Chromatogr 593,289-295 Altria Jackson, P E and Haddad, P (1993) Capillary electrophoresis of inorganic ions and low-molecular-mass iomc solutes TRAC 12,231-238 Quang, C and Khaledi, M G (1994) Prediction and optimisatton of the separation of metal cations by capillary electrophoresis with indirect UV detection J Chromatogr 659,459-466 Shi, Y and Fritz, J S (1994) New electrolyte systems for the determmation of metal cations by capillary zone electrophorests J Chromatogr 671,42%-435 Backmann, K , Boden, J., and Haumann, I (1992) Indirect fluorimetric detection of alkah and alkaline earth metal ions m capillary zone electrophoresis with cermm (III) as carrier electrolyte J Chromatogr 626,259-265 10 Altrta, K D., Goodall, D M , and Rogan, M M (1994) Quantitative determmatton of drug counter-ton stoichiometry by capillary electrophoresis Chromatographla 38,637-642 11 Swartz, M E (1993) Capillary electrophoretic determmation of morgamc tons in prenatal vitamin formulation J Chromatogr 640,44 l-444 12 Koberda, M., Konkowskt, M , Youngberg, P., Jones, W R , and Weston, A (1992) Capillary electrophoretic determination of alkali and alkaline-earth cations in various multiple electrolyte solutions for parenteral use J Chromatogr 602,235-240 13 Morawski, J , Alden, P , and Sims, A (1993) Analysis of cationic nutrients from foods by ion chromatography J Chromatogr 640,359-364 14 KaJiwara, H , Sato, A , and Kaneko, S (1993) Analysis of calcmm and magnesium ions in wheat flour by capillary zone electrophoresis BIOSCL Blotech, Bzochem 57, lOlO,lOll 15 Motomizu, S , Oshima, M., Matsuda, S -Y., Obata, Y., and Tanaka, H (1992) Separation and determmatton of alkaline-earth metal ions as UV absorbing chelates with EDTA by capillary electrophoresis Determmation of calcium and magnesium m water and serum samples Anal Scz 8,619-624 16 Buckberger, W , Semenova, P., and Timerbaev, A R (1993) Metal ton captllary zone electrophoresis with direct UV detection separation of metal cyanide complexes JHRCC 16, 153-156 17 Aguilar, M., Farran, A , and Martinez, M (1993) Determmatton of gold (1) and silver (1) cyanides m ores by capillary zone electrophoresis J Chromatogr 635, 127-13 18 Swaile, D F and Sepamak, M J (199 1) Determmatton of metal ions by capillary zone electrophoresis with on-column chelation usmg 8-hydroxyquinoline-%sulfomc acid Anal Chem 63, 179-184 19 Timerbaev, A R , Buchberger, W , Semenova, P., and Bonn, G K (1993) Metal ion capillary zone electrophorests with direct UV 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modlficatlons m DNA Rapzd Comm Mass Spectrom 8,1035-1040 ... Application of voltage across a capillary filled with electrolyte causes a flow of solution along the capillary This flow effectively pumps solute ions along the capillary toward the detector This... capillaries filled with gel solutions In Capillary Gel Electrophoresis (CGE), a sieving effect occurs as solutes of various sizes migrate through the gel filled capillary toward the detector Chapter... introduced into the capillary by three techniques, all of which involve immersing the capillary end into the sample solution and exerting a force to inject sample into the capillary The three