Journal of Pharmaceutical and Biomedical Analysis22 (2000) 957–966

Characterization of bovine collagens using capillaryelectrophoresis — an alternative to slab gel electrophoresis

Paul Chang a,*, Son Kuan a, Gert Eberlein b, David Burke c, Richard Jones a

a Matrix Pharmaceutical, 34700 Campus Dri6e, Fremont, CA 94555, USAb Onyx Pharmaceuticals, 3031 Research Dri6e, Richmond, CA 94806, USA

c Elan Pharmaceuticals, 800 Gateway Boule6ard, South San Francisco, CA 94080, USA

Abstract

A capillary electrophoresis method was developed and characterized for analyzing the spectrum of collagensubspecies in collagen preparations. The Bio-Rad CE-SDS protein kit was used for the dynamic sieving separation ofcollagen subspecies in this CE method (DSCE). The optimized method utilized a 36 cm (or 24 cm)×50 mm uncoatedcapillary, electrophoretic injection at 10 kV for 10 s, a run voltage of 15 kV, a capillary temperature of 20°C, and UVdetection at 220 nm. A preliminary validation of the method was performed. The assay had good repeatability (RSDsfor peaks were 1–5%), and responses were linear for assay solutions with collagen concentrations from 0.125 to 1.25mg/ml. The DSCE electropherogram of bovine skin collagen provided a profile of subspecies similar in number andrelative abundance to that generated by scanning of Coomassie-stained SDS-PAGE gels. © 2000 Elsevier Science B.V.All rights reserved.

Keywords: Capillary electrophoresis; Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Bovine Type I collagen; BovineType III collagen

www.elsevier.com/locate/jpba

1. Introduction

Collagen constitutes the majority of the struc-tural protein in such tissues as bone, skin, tendon,and cartilage, and is a prime component of theextracellular matrix of all tissues. The fibrillarcollagen molecules (e.g. Types I, II, III, and V)are comprised of long triple helices comprisingthree intertwined primary chains. The three chainsin the triple helix molecule, as well as neighboringhelices in collagen fibrils, are held together

through hydrogen bonds. There may also be oneor more interstrand (intramolecular) covalentcrosslinks in individual molecules [1], as well ashelix-to-helix (intermolecular) crosslinks in fibrils.Under denaturing conditions (e.g. in the presenceof sodium dodecyl sulfate, SDS) the hydrogenbonds between the helices or between the threechains of the triple helix break down anddissociate into individual units, as limited by thevarious covalent intra- and intermolecularcrosslinks.

Conventional sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE) has beenused for the identification, purity, and stability* Corresponding author.

0731-7085/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S0 731 -7085 (00 )00246 -6

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966958

testing of collagen materials. On SDS-PAGE gelsthese collagen subspecies bands (a1, a2, b11, b12, g,etc.) typically appear in a characteristic ladder-like array [1]. However, the SDS-PAGE techniqueis time-consuming and involves a multitude ofsteps, including running the gel, staining, destain-ing, and densitometry scanning for quantitation.

The multiplicity of steps in this method oftencreates artifacts and causes variability. The linear-ity range of the band intensity on the gel is alsonarrow [2].

Capillary electrophoresis (CE) has been widelyused in the separation and characterization ofproteins and peptides. Size separation of proteins

Table 1Determination of collagen subspecies by DSCEa and SDS-PAGE

a1 (%) b12 (%) b11 (%) g (%)Method Total \g (%)a2 (%)

DSCE, 24 cm×50 mm capillary (n=6)10.5Mean 37.6 30.3 4.2 8.5 9.00.1 0.3 0.2 0.1 0.1 0.4S.D.

DSCE, 36 cm×50 mm capillary (n=6)Day 1 (n=4)

29.636.3 11.510.0 8.3Mean 4.4S.D. 0.40.2 0.40.3 0.10.5

Day 2 (n=2)10.5 37.4 29.1Mean 4.5 10.28.3

0.2 0.4 0.1 0.7S.D. 0.3 0.2

SDS-PAGE (n=11)24.2 14.230.810.1Mean 17.33.3

0.7 1.7 1.4 0.4 1.3 2.6S.D.

a Subspecies contents are presented as normalized time-adjusted peak area, which is (time-adjusted peak area/total time-adjustedpeak area)×100%. DSCE conditions: electrophoretic injection (10 kV/10 s), capillary cartridge temperature 20°C, and run voltage15 kV.

Table 2DSCEa system repeatability — electrophoretic injection vs. pressure injection

a1Collagen subspecies b12a2 b11 g Total \g

Electrophoretic injectionb (n=6)Migration time (min)

–10.40 11.12Mean 12.50 12.94 14.410.01 0.010.01S.D. 0.010.01

Normalized peak area (% of total)8.54.230.3 9.037.610.5Mean

0.3 0.2 0.0 0.1S.D. 0.40.1

Pressure injectionc (n=6)Migration time (min)

–13.9512.5812.14Mean 10.8610.170.01 0.01 0.02 0.02 0.02S.D.

Normalized peak area (% of total)37.99.8 8.1Mean 8.04.032.2

S.D. 0.30.10.20.50.40.3

a DSCE conditions: 24 cm×50 mm uncoated capillary, capillary cartridge temperature 20°C, and run voltage 15 kV.b Electrophoretic injection: 10 kV/10 s.c Pressure injection: 60 psi×s.

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966 959

Fig. 1. Plot of peak area of collagen subspecies vs. elec-trophoretic injection time (+ , a2; 2, a1; �, b12; �, b11; × ,g ; and *, \g).

in gel-filled capillaries was first described byHjerten [3] and later by Cohen and Karger [4].The use of non-gel sieving CE, in which capillariesare filled with noncross-linked polymer modifiedsolution, was reported by Chin and Colburn [5].Several workers have published papers on theseparation of collagen using gel-filled CE or non-gel sieving CE [6–9]. However, in those studiesonly qualitative analyses were reported. Thequantitative assessment for the identity, purity,and stability of collagen using DSCE has not beenextensively explored.

In this study the subspecies profile of bovineskin collagen was characterized and quantitatedby non-gel dynamic sieving capillary electrophore-sis (DSCE) and by SDS-PAGE. The possibilitythat DSCE could be used as an alternative toSDS-PAGE was evaluated.

2. Experimental

2.1. Materials

CE-SDS protein sample buffer, CE-SDSprotein run buffer, CE-SDS protein size standard,and CE-SDS internal standard (benzoic acid),acrylamide, SDS, Tris, and dithiothreitol werepurchased from Bio-Rad (Hercules, CA, USA).

Bovine skin collagen solution (pepsinized, con-taining approximately 90% Type I and 10% TypeIII collagen) was produced by Matrix Pharmaceu-tical (Fremont, CA, USA). Types I and III colla-gens were isolated and purified from this bovinecollagen solution by salt fractionation [10]: TypeIII collagen was precipitated from collagen solu-tion using dialysis against 50 mM Tris buffer (pH7.15) containing 1.1 M NaCl, and Type I collagenwas precipitated from the supernatant by the ad-dition of 0.2 M sodium phosphate buffer (pH10.7).

2.2. Instrumentation and methods

2.2.1. CEA Bio-Rad BioFocus 3000 equipped with a

multiple wavelength UV detector was used. Un-coated fused-silica capillaries with length of 24 or

Fig. 2. Electropherograms of bovine skin collagen. DSCEconditions: uncoated 24 cm×50 mm capillary, electrophoreticinjection at 10 kV for 10 s, run voltage at 15 kV, and capillarytemperature at (a) 20°C and (b) 25°C.

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966960

36 cm (19.4 or 31.4 cm to the detector) and ID of25, 50, 75 or 100 mm mounted onto a Bio-Radself-assemble cartridge, were purchased fromJ&W Scientific (Folsom, CA, USA). A polyviny-lalcohol-coated capillary purchased from Hewlett-Packard (Santa Clara, CA, USA) was used forcomparison. Analyte peak areas were normalizedto migration times to compensate for the differentmigrating speeds through the detector.

2.2.2. SDS-PAGECollagen samples were denatured in SDS, then

run on 4–15% gradient PAGE gels and stainedwith Coomassie Blue or run on 4–20% gradientPAGE gels and stained with silver. The relativeamounts of the collagen subspecies in theCoomassie-stained gels were quantitated with adensitometer (personal densitometer SI, Molecu-lar Dynamics, Sunnyvale, CA, USA). A gel eluter

(SixPac, Hoefer Scientific Instrument, USA) wasused for isolating the collagen subspecies fromunstained gels.

2.3. Method de6elopment: optimization for DSCEconditions

Using a generic method developed for proteinanalysis by Bio-Rad Laboratories [11] as the pointof departure, experiments were performed to opti-mize both sample preparation and DSCE operat-ing parameters for the analysis of collagensamples. These included the evaluation of differ-ent capillaries, injection modes, injection times,capillary temperatures, and run voltages.

2.3.1. CapillaryAlthough the resolution for the CE separation

can often be improved by increasing the capillarylength and decreasing the capillary internal di-ameter, there are trade-offs in the runtime, runvoltage, and sensitivity. In this study uncoatedfused-silica capillaries with 25, 50, 75 or 100 mmID, and 24 or 36 cm length were evaluated. Both24 and 36 cm×50 mm capillaries provided rea-sonable resolutions, sensitivities, and runtimes forthe characterization of collagen subspecies. The36 cm×50 mm capillary provided better resolu-tion but took a longer runtime (40 min) comparedto the 24 cm×50 mm capillary (20 min). The24-cm and 36-cm capillaries provided equivalentresults (Table 1). A polyvinylalcohol-coated capil-lary was also examined. The migration patternsand migration times of bovine skin collagen incoated and uncoated capillaries were very similar(electropherograms not shown). Since the pH ofthe electrophoresis buffer is high (ca. 8.9) and thedenatured collagen/SDS complex forms a chargedmicelle-like species, electroosmosis and proteinadsorption effects are not significant in this sys-tem [12], and a coated capillary is not needed.

2.3.2. Injection modeBoth electrophoretic and pressure injections

were evaluated. In theory, during electrophoreticinjection sample ions migrate into the capillary inproportion to their electrophoretic mobilities.Consequently, electrophoretic injection may be

Fig. 3. (a) Coomassie blue-stained SDS-PAGE gel, (b) opticaldensity trace of SDS-PAGE and (c) DSCE of bovine skincollagen.

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Fig. 4. DSCE electropherograms of a and b fractions collected from SDS-PAGE gel. (a) Collagen; (b) a bands from SDS-PAGEgel; and (c) b bands from SDS-PAGE gel.

component selective and may introduce a samplebias. Pressure injection produces a sample zonewith analytes at the same concentrations as in thesample solution.

For bovine skin collagen material, the electro-pherograms and the resultant contents of collagensubspecies obtained from electrophoretic injection(10 kV for 10 s) and pressure injection (60 psi×s)were compared (Table 2). The relative peak areasseen after electrophoretic injection were essentiallyequivalent to those obtained after pressure injec-tion, showing that component selectivity was notsignificant during electrophoretic injection. How-ever, the system precision obtained from elec-trophoretic injection was slightly better (i.e. withlower relative standard deviation, R.S.D.) thanthat from pressure injection (Table 2). In addi-tion, electrophoretic injection produced a betterbaseline and a higher sensitivity (signal-to-noiseratio) than pressure injection (data not shown).

2.3.3. Injection timeElectrophoretic injection with various injection

times between 5 and 25 s was evaluated. A colla-

gen concentration of 0.75 mg/ml and an injectionvoltage of 10 kV were used throughout. The peakareas showed good linearity for a2, a1, b12, andb11 peaks for the injection range from 5 to 20 s,whereas the peak areas for g and \g peaks werelinear only up to 15 s. Above 15 s there was poorresolution between g and \g peaks. Based on thisstudy, an optimal electrophoretic injection time of10 s was selected (Fig. 1).

Fig. 5. Silver-stained SDS-PAGE gel of a, b and g fractionscollected from DSCE. (lane 1, protein standards; lane 2 andlane 6, bovine skin collagen)

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966962

Fig. 6. A plot of log MW vs. migration time from DSCEelectropherograms of protein standards and collagen subspe-cies ( , protein standards and 2, collagen subspecies).

Fig. 7. Linearity of collagen subspecies separated by DSCE.(+ , a2; 2, a1; �, b12; �, b11; × , g ; and *, \g).

Fig. 8. A plot of percent normalized peak area of collagensubspecies vs. collagen concentration ( , a2+a1; 2, b12+b11; �, \g ; and �, g).

2.3.4. Capillary temperature/run 6oltageRaising the capillary temperature or run

voltage should decrease the viscosity of the runbuffer and increase the electrophoretic mobility ofthe analyte. It may result in shorter migrationtimes but lower resolutions for the analytes [13].Bovine skin collagen material was electrophoresedat various capillary temperatures (i.e. 15, 20 and25°C) and run voltages (i.e. 15 and 20 kV). Somehigher molecular weight peaks (g and \g) werelost when the samples were electrophoresed at15°C, probably because of higher viscosity of therun buffer. Satisfactory results were obtainedwhen the samples were electrophoresed at 20 or25°C (Fig. 2). The electropherogram run with 15kV at 20°C provided the best resolution andbaseline with a reasonable runtime.

The proposed DSCE operating conditions forthe characterization of bovine collagen are there-fore as follows: capillary, 24 cm (or 36 cm)×50mm ID uncoated fused-silica capillary; polarity,negative to positive; buffer, CE SDS-protein run

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966 963

Table 3Comparison of DSCE to SDS-PAGE in the characterization of collagen

SDS-PAGEDSCE

Total analysis 0.5–1.5 days 2–3 daystime

Preparation Pour gel, prepare SDS-protein samples, prepare runAssemble capillary cartridge, prepare SDS-proteinsamples, load samples and reagents into carousels, buffer, load samplesprogram the software

Run gel electrophoresis, fixation, Coomassie blueRun capillary electrophoresisProcedurestaining, destaining

Analysis Scan gel by densitometer, draw grids manually, per-Analyze electronic dataform integration

Run buffer 0.5–2 ml 5–10 lneeded

2–10 mlWaste gener- 5–15 lated

12 samples per gelMaximum 22 samples per carouselnumbers ofsamples

NoAutomation YesDirect UV detection NoOn-line detec-

tion

buffer; electrophoretic injection, 10 kV for 10 s;run voltage, 15 kV; detection, 220 nm; cartridgetemperature, 20°C; and the runtime, about20 min (ca. 40 min if using a 36 cm×50 mmcapillary).

2.4. DSCE sample preparation

A collagen solution (200 ml) in 10 mM HCl wasmixed with 200 ml Bio-Rad CE-SDS samplebuffer and 10 ml internal standard. The mixturesolution was incubated in a 90–100°C water bathfor 10 min. The sample solution was cooled toroom temperature and centrifuged at 6000 rpmfor 2 min prior to injection. For reduced samplepreparations, dithiothreitol solution was added toa final concentration of 2.5%.

3. Results and discussion

3.1. Characterization of bo6ine skin collagen

Bovine skin collagen samples were preparedand electrophoresed by SDS-PAGE and DSCEmethods. The SDS-PAGE gel banding pattern in

the sequence of a2, a1, b12, b11, g, \g (Fig. 3),was similar to that in the literature [1]. The opticaldensity trace of the collagen subspecies from thedensitometer scan of Coomassie-stained SDS-PAGE gel and the electropherogram of the samelot of collagen from DSCE were similar in termsof both peak spacing and relative peak areas. It isof note that due to the different separation mech-anisms the migration directions of the collagensubspecies on the SDS-PAGE gel and DSCE elec-tropherogram are reversed.

In order to identify the collagen peaks in theDSCE, the collagen subspecies separated on SDS-PAGE were eluted from the gel and elec-trophoresed on DSCE. The migration times of thefirst four bands (putatively identified as a2, a1, b12

and b11) well matched those of the first four peaksof collagen on the electropherogram. The relativepeak areas were also consistent with the peakidentities (Fig. 4).

Fractions from the DSCE separation of colla-gen were also collected and verified by SDS-PAGE. Fraction 1 contained the first two peaks,fraction 2 contained the third and fourth peaks,and fraction 3 contained all the other peaks.These fractions were run on SDS-PAGE and the

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966964

protein bands were visualized by silver staining.Using the collagen reference material, the frac-tions from DSCE were positively identified as thea bands, b bands and g bands (Fig. 5).

3.2. Anomalous migration times for bo6ine skincollagen subspecies

A plot of log MW versus migration time from atypical electropherogram of CE-SDS protein stan-dards (Bio-Rad) is shown in Fig. 6. This standardsolution contains lysozyme (14.4 kDa), trypsininhibitor (21.5 kDa), carbonic anhydrase (31kDa), ovalbumin (45 kDa), serum albumin (66.2kDa), phosphorylase B (97 kDa), b-galactosidase(116 kDa), and myosin (200 kDa). The molecularweights of a2, a1, b12, b11 and g collagen subspe-cies based on the primary sequence are 93, 93,186, 186 and 279 kDa [1]. The log MW versusmigration time plot of the collagen subspecies didnot fall onto the standard curve (Fig. 6). Theapparent discrepancy in the migration behavior ofthe collagen subspecies may be due to differencesin SDS binding stoichiometry between collagenand the CE-SDS protein size standards and/ordifferences in shape of the denatured mass. Colla-gen differs significantly from other proteins in itsamino acid composition and amino acid sequence[1]. Collagen is high in glycine and proline, andcontains the rare amino acids 4-hydroxyprolineand 5-hydroxylysine. In addition, the proline con-tent may make the collagen–SDS complex morerigid than other protein–SDS complexes, thusincreasing the hydrodynamic radius of the colla-gen–SDS complex. It is of note in this regard thatthe collagen subspecies showed higher apparentmolecular weights by SDS-PAGE.

3.3. System performance

3.3.1. PrecisionExperiments were performed using three sample

vials. Two electrophoretic injections (10 kV for 10s) were taken from each sample vial and injectedonto a 24 cm×50 mm uncoated capillary. TheRSDs for the migration times of each subspecieswere only in the range 0.1–0.2%. The RSDs forthe time-adjusted peak area percents (Table 2)were less than 5%.

3.3.2. Linearity and rangeLinearity was examined using Matrix bovine

collagen at concentrations of 0.125–1.25 mg/ml.

Fig. 9. DSCE electropherograms of (a) a nonreduced pureType I collagen, (b) a nonreduced Type III collagen, (c) areduced bovine skin collagen, and (d) a nonreduced bovineskin collagen. DSCE conditions: uncoated 36 cm×50 mmcapillary, electrophoretic injection at 10 kV for 40 s, runvoltage at 15 kV, and capillary temperature at 20°C.

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966 965

Each sample was injected in duplicate. Good lin-earity was demonstrated for a2, a1, b12, b11, g, and\g peak areas for the whole concentration rangestudied (Fig. 7). The r2 values for a2, a1, b12, b11,g, and \g peak areas were 0.996, 0.987, 0.988,0.914, 0.998 and 0.985, respectively. The aboveexperiments show that the peak responses arelinear with respect to collagen concentration overa 10-fold range from 0.125 to 1.25 mg/ml.

The time-adjusted peak area percents for the a,b, g and \g peaks (Fig. 8) were most consistentover the collagen concentration range from 0.25to 1.0 mg/ml, with RSDs of 3.3, 3.1, 5.9 and9.7%, respectively, for the four peaks.

3.4. Comparison of DSCE to SDS-PAGEanalysis for bo6ine skin collagen

Quantitative results from the analysis of bovineskin collagen by DSCE and SDS-PAGE are sum-marized in Table 1. The DSCE data were takenfrom the system precision experiment and theSDS-PAGE data were taken from multiple gels ofa collagen material. Compared to the SDS-PAGEdata, DSCE peak area percents were slightlyhigher for a and b peaks and slightly lower for g

and \g peaks. The differences between the SDS-PAGE and DSCE data may reflect differences inCoomassie blue staining among the different col-lagen subspecies in SDS-PAGE or differences inUV absorptivities of the various subspecies inDSCE. The repeatability of the DSCE assay wasbetter than that of the SDS-PAGE assay. Ingeneral the DSCE method provided better resolu-tions between a2 and a1, and b12 and b11 peaksthan did SDS-PAGE gels; on the other hand, theresolution between the g and \g peaks withDSCE was not as good as on the SDS-PAGE gels(Fig. 3).

The advantages and disadvantages of DSCEand SDS-PAGE methods in bovine skin collagenanalysis are compared in Table 3. Compared toSDS-PAGE, DSCE has several advantages. Theseinclude a shorter runtime, smaller volumes of runbuffers needed, in-line UV detection, automationand reduction in waste. In the collagen assayDSCE provides better resolutions between a2 anda1 and between b12 and b11 peaks. The disadvan-

tages of this DSCE method are, besides the highercost of the equipment, the lower resolution be-tween g and \g peaks and the lower sensitivity(signal/noise). The latter, however, can be im-proved using a bubble-cell capillary, a Z-shapedcapillary, or other techniques [14].

3.5. Bo6ine Types I and III collagens

The bovine Type III collagen triple helix isdisulfide-bridged. Under nonreduced conditionsthe inter-chain disulfide bonds are intact. It isexpected, then, that the [a1(III)]3 chains shouldhave mobility on SDS-PAGE similar to that ofthe g(I) and the \g(I) bands. Under reducingconditions, in contrast, the disulfide bonds arecleaved and the a1(III) monomers should migratesimilarly to the a1(I) chains.

Pure Type I and Type III bovine collagensisolated from bovine skin collagen were analyzedby DSCE in nonreduced form (Fig. 9a and b).Bovine skin collagen samples (containing about90% Type I and 10% Type III collagen) in re-duced and nonreduced sample buffers were alsoprepared and analyzed by DSCE (Fig. 9c and d).By comparing the electropherogram of nonre-duced collagen (Fig. 9d) to that of the nonreducedType III collagen (Fig. 9b) it is clear that the \g

peaks in the nonreduced collagen sample containType III collagen. This conclusion was furtherconfirmed using the reduced collagen samples.Compared to the nonreduced sample the reducedsample (Fig. 9c) showed a decrease in the \g

peaks and the appearance of two new peaks, oneimmediately following the a1(I) peak and oneafter the b11(I) peak. This experiment suggeststhat it would be possible also to quantitate TypeIII bovine collagen in collagen samples by DSCE.

4. Conclusion

This study shows that the DSCE electrophero-gram of bovine skin collagen was very similar innumber and relative abundance of subspecies tothe profile generated by SDS-PAGE. The identityof the banding pattern was established. SDS-PAGE is selective but is labor-intensive and oper-

P. Chang et al. / J. Pharm. Biomed. Anal. 22 (2000) 957–966966

ator-dependent. The DSCE method is more re-producible, simple, rapid, and automated and canseparate collagen subspecies to the same bandingpattern as SDS-PAGE. Therefore, the DSCEmethod may provide an attractive alternative toSDS-PAGE as a routine method for quality con-trol and stability testing of relative subspeciesdistribution for bovine collagen materials.

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