capilary electrophoresis manual

CEC

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okCapillary Electrochromatography -Technology and Applications

Agilent TechnologiesInnovating the HP Way

Edited byGordon A. Ross andGerard P. Rozing

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Table of Contents

Table of Contents

Foreword

1 Introduction

2 Theory

3 Columns

4 Instrumentation

5 Influence of Experimental Parameters on Separation

6 Method Development

7 Analysis of Neutral pesticide and By-products Using On-column Preconcentration

8 Simultaneous Separation of Acidic, Basic, and Neutral Organic Compounds

9 Practical Applications of CEC in the Pharmaceutical Industry

4

Gerard P. Rozing 5

Monika M. Dittmann 10Gerard P. Rozing

Gerard P. Rozing 20

Gordon A. Ross 30

Monika M. Dittmann 39Gerard P. Rozing

Gordon A. Ross 54

Erdmann Rapp, 61Peter Oggenfuss, Aran Paulus and Gerard Bruin

Ira S. Lurie, 69Timothy S. Converand Valerie L. Ford

Melvin R. Euerby 88Christopher M. Johnson

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10 Analysis of Triglycerides

11 Analysis of Fatty Acids and Derivatives

12 Elucidation of cis/trans 18:1 Ratios in Margarine's

13 Analysis of Nucleosides

14 Analysis of Norgestimate and its Potential Degradation Products

15 Analysis of Phenols in Mainstream and Sidestream Tobacco Smoke

16 Practical Aspects of Coupling Capillary Electrochromatography with Mass Spectrometry

17 Summary, Conclusions and Outlook

Table of Contents

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Pat Sandra and Page 95An Dermaux

An Dermaux and 102Pat Sandra

An Dermaux and 109Pat Sandra

Thomas Helboe, 112Jette Tjørnelund and Steen Honoré Hansen

Jian Wang, Daniel E 117Schaufelberger andNorberto Guzmann

M. Saeed, 125M. Depala, D.H. Craston and I.G.M. Anderson

Bob Boughtflower 135and Clare Peterson

Gordon A. Ross 140and Gerard P. Rozing

Table of Content (page 2-3)

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4/143Foreword (page 4)

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ForewordBy J.H. Knox

Capillary electrochromatography, CEC,is the latest addition to the armory ofliquid separation methods. It is a formof miniaturized liquid chromatographywhich uses an electric field to pumpliquid through a packed chromatographycolumn and it is set to play a major rolein separation science over the nextdecade.

The original idea came from the lateVictor Pretorius in 1974, but wasneglected until revived by Jorgensonand Lukacs in 1981. However, at thattime, it was overshadowed by the rapiddevelopment of capillary electrophoresis.In the late 80’s Ian Grant and I undertooka detailed study of the main parametersof CEC, and from that time onwards,CEC has undergone exponentialexpansion, with a doubling period ofabout two years.

While instrument manufacturers quicklyresponded to Jorgenson's original workby developing CE instruments, theyhave been slow to appreciate thebenefits of CEC. Yet, surprisingly, atypical CE instrument requires onlymi-nor modification to handle CEC,namely pressurization of the inlet andoutlet reservoirs to prevent

development of gas bubbles within theCEC column. The HP 3D CE instrumentis the first to achieve this, and it enablesCEC to be carried out routinely.

Packed column electrically drivensystems are likely in the long term toreplace not only conventional drivenHPLC, but also the current open tubularCE. For the latter, monodispersed silicawill be used as the column packing.

This CEC Guidebook is therefore aimedat introducing CEC to those with abasic understanding of HPLC techniqueand terminology. It gives the chromato-grapher a highly readable and informativeaccount of the present state of CEC,without overburdening him or her withtheory. The first six chapters deal withthe basics: history, theory, instrumentation,performance and method development.The remaining ten chapters coverrecent applications. The authorswrite authoritatively, and their unifiedtreatment makes this primer an excellentstarting point for anyone with HPLCbackground who wishes to update toCEC.

The CEC Guidebook will take themystery out of CEC, and will encourageits further application and development.

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Gerard P. Rozing, Agilent TechnologiesGmbH, 76337 Waldbronn, Germany

Capillary Electrochromatography, CEC,is a new technique for liquid phaseanalysis. In CEC, the combined actionsof partitioning chromatography (HPLC)and capillary electrophoresis (CE)achieve separation of the analytes in aliquid sample. In this technique, the bestproperties of HPLC and CE operate insynergy. For HPLC, this is the ability tomanipulate retention and selectivity ofthe separation by simple variation ofmobile phase composition and/or typeof stationary phase. This property ofHPLC, has been the main reason for its growth in popularity over the past 25 years. For CE it is the very highseparation efficiency that can beattained and which was the main reasonfor the appearance ten years ago of thetechnique in the field of analytical sepa-rations. Recently (1-21), the demon-stration of the feasibility of CEC and inparticular of the above abilities, havespurred the interest in the technique.

Since early 1996, the number of reportson CEC in the scientific literature hasgrown rapidly.

In CEC, a stationary phase packed in a fused silica capillary, I.D. 50-200 µm,is used to obtain separation. HPLC typesilica based reversed phase particles, 1-5 µm, have been mainly used as a sta-tionary phase. Mobile phases, typicalfor RP-type separations i.e. organic sol-vent/aqueous buffer mixtures, are used.On application of an electrical fieldalong the axis of the packed capillary,an electro-(endo)-osmotic flow is estab-lished (see chapter 2), which transportsthe solutes and the mobile phasethrough the packed bed. When thesolutes are uncharged, separation isobtained by differential partitioning ofthe solutes between mobile and stationary phase. When the solutes tobe separated are charged, they will have an additional, electro-phoreticvelocity component which will contribute to (or counteract) separation(see chapter 8).

1 Introduction

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The technique is very similar to CZE.Therefore, the equipment used for capil-lary electrophoresis can be used to execute CEC without major modifi-cation. A simple schematic of a systemfor CEC is given in figure 1.1.

History

The possibility of using the electro-(endo)-osmotic flow (EOF) as a meansto transport solvent through a liquidchromatography column was pointed

out by Pretorius et al. in 1974 (1). Theyused 75 to 125 µm particles in a 1-mmI.D. glass tube and were able to showthat band broadening with electro-endo-osmotic flow was considerably smallerthan with pressure driven flow. In 1981,

Jorgenson and Lukacs (2,3) demonstra-ted the feasibility of CEC in 170 µm i.d.pyrex glass capillaries filled with 10 µmPartisil ODS-2 particles. Two years later,Stevens and Cortes published discoura-ging results on measurements of EOF in

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1

2

3

4

5

6

Sample

7

Figure 1.1: Schematic description of a system for CEC. 1, 3 buffer vials, 2 packed fused silica capillary 50-200 µm I.D., 200-500 mm length, 4 electrodes, 5 power supply, 6 point of detection, 7 external pressure 2-12 bar. Further details in chapter 4.

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capillaries filled with particles of 10, 50and 100 µm (4). Their measurementslead to the conclusion that in capillariespacked with particles < 50 µm, EOFvelocities were too low to perform effi-cient chromatography. They concludedthat in these packings, EOF decreasesdue to overlap of the double layers.Knox and Grant, however, demonstra-ted theoretically (5) and practically (6)that no double layer overlap occurswith particles down to 1.5 µm in a 0.01 Melectrolyte. In their landmark paper (6)they showed separations of a mixture ofaromatic hydrocarbons in capillariespacked with 1.5, 3 and 5 µm RP packingmaterials. Reduced plate heights lessthan 1 were achieved.

Concurrently, Tsuda has investigatedfundamental aspects of CEC, whichwere reviewed in 1992 (7). Further stud-ies on the influence of solvent proper-ties on retention and EOF in CEC werepublished more recently (8, 9)

More recently a number of groups havepublished their experimental work inCEC. In capillaries with 50 µm I.D.packed with 3 µm ODS-Hypersil, Erniwas able to demonstrate column effici-encies of 150,000-200,000 plates/meteror reduced plate heights of 1.7-2.2 (10).

In later work this group showed thatthe usage of 0.32 mm I.D. FS-capillariespacked with 5 µm Spherisorb ODSI forCEC led to reduced plate heights of 2-4despite the anticipated efficiency lossesdue to radial thermal gradients in thecolumn (11). Rebscher and Pyellsuccessfully prepared columns by pack-ing FS-capillaries with 3 and 5 µmreversed phase particles (12). They veri-fied the effect of external band spread-ing on the column efficiency obtained.After removing the influence of externalband spreading by calculation, reducedplate heights of 1.3 - 2.7 were found.

Two groups at Glaxo (U.K). have pub-lished their initial results with CEC.Smith (13, 14) demonstrated reducedplate heights 0.9-2 for columns packedwith 3 and 2.2 µm reversed phase parti-cles. This group emphasized the neces-sity of pressurizing the buffer vials withinert gas to 500 p.s.i., to suppress theformation of gas bubbles in the mobilephase at high currents (obtained withbuffer concentrations > 10 mM). Thenecessity to pressurize the whole capil-lary column system and buffer vials hasbeen predicted by Knox and Grant in1991 in their experimental work (6).Boughtflower and Underwood along thesame line obtained comparable column

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efficiencies as Smith (15, 16). Theseauthors also accentuated the im-portance to pressurize the buffer vialson the inlet and outlet side to avoid outgassing and bubble formation. In addi-tion the usage of low conductivitybuffers like TRIS (trihydroxymethyl-aminomethane) and MES (4-morpholino-ethyl-sulfonic acid) was recommendedto reduce the current in the capillary.

In a different approach, two groupshave demonstrated that pressure assis-tance on the inlet side through solventdelivery by an HPLC pump, yieldedcomparable suppression of bubble for-mation in the capillary in CEC and at the same time adds selectivity tuningcapabilities. Unger et al. exploited theinterplay of EOF and hydraulic flow toenhance the separation of solutes whichotherwise co-eluted (17). Behnke andBayer used an HPLC gradient pumpingsystem to assist and enhance separationin CEC by modification of the solventcomposition as in gradient elution(18,19). Zare and coworkers publishedtheir initial results demonstrating theability of CEC to separate polynuclear

aromatic substances with high efficiencyin a short time (20).

Rozing and Dittmann reviewed the state of the art in CEC in a review paper late 1995 (21). Then, since thememorable HPCE'96 meeting where a broad audience was confronted withCEC for the first time, work on CEChas proliferated rapidly. It would bebeyond the scope of this primer toreview this work in detail.

Instead the editors have chosen toselect recent, practical examples ofCEC by practitioners in the field to bepresented in this primer in a conciseway. Complemented with a few chapterson fundamental and practical aspects ofthe technique, this primer should givethe novice user of capillary electro-chromatography an overall impressionof the principles of the technique. Onewill learn about important theoreticaland practical aspects of CEC and find anumber of applications that have solvedpractical problems with CEC.

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References

1. Pretorius, V., Hopkins, B.J. and Schieke, J.D., J. Chrom., 99, 23, 1974.

2. Jorgenson, J.W. and Lukacs, K.D.,Anal. Chem., 53, 1298, 1981.

3. Jorgenson, J.W. and Lukacs, K.D.,J. Chrom., 218, 208, 1981.

4. Stevens, T.S. and Cortes, H.J., Anal. Chem., 55, 1365, 1983.

5. Knox, J.H. and Grant, I.H.,Chromatographia, 24, 135, 1987.

6. Knox, J.H. and Grant, I.H., Chromatographia, 32, 317, 1991.

7. Tsuda, T., LC-GC International, 5, 26, 1992.

8. Kitagawa, S. and Tsuda, T., J. Microcolumn Separations,6, 91, 1994.

9. Kitagawa, S. and Tsuda, T., J. Microcolumn Separations, 7, 59, 1995.

10. Yamamoto, H., Baumann, H. and Erni, F., J. Chrom., 593, 313, 1992.

11. Yan, C., Schaufelberger, D. and Erni, F.,J. Chrom., 670, 15, 1994.

12. Rebscher, H. and Pyell, U.,Chromatographia, 38, 737, 1994.

13. Smith, N.W. and Evans, M.B.,Chromatographia, 38, 649, 1994.

14. Smith, N.W. and Evans, M.B.,Chromatographia, 41, 197, 1995.

15. Boughtflower, R.J. et al.,Chromatographia, 40, 329, 1995.

16. Boughtflower, R.J. et al.,Chromatographia, 41, 398, 1995.

17. Eimer, T. and Unger, K.K., Fresenius J. Anal. Chem., 352, 649, 1995.

18. Behnke, B. and Bayer, E.,J. Chromatography, 680, 93, 1994.

19. Behnke, B., Grom, E. and Bayer, E.,J. Chromatography, 716, 207, 1995.

20. Yan, C., Dadoo, R., Zhao H. and Zare,R.N., Anal. Chem., 67, 2026, 1995.

21. Dittmann, M.M., Wienand, K., Bek, F. and Rozing, G.P., LC-GC, 13, 800, 1995.

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Monika M. Dittmann and Gerard P.Rozing, Agilent Technologies GmbH, 76337 Waldbronn, Germany

2.1 Electro-osmotic flow in apacked capillary

In capillary electrophoresis (CE), thesolvent (electrolyte) is moved throughthe column by electro-motive force. Theorigin of this force stems from the elec-trical double layer existing at the solid/liquid interface between the silica andthe electrolyte in the tube. The occur-rence of the double layer is induced bythe presence of ionizable silanol, -Si-OH,groups forming negative charges at the

interface, depending on the pH of theelectrolyte (Figure 2.1).

The positive counter ions close to theinterface are bound strongly by coulom-bic forces and are therefore immobile(Stern-layer). The positive counter ionsin the electrolyte more remote from theinterface are less strongly bound andtherefore mobile. The positive counterions are present in slight excess in thesolution close to the interface in orderto maintain electro-neutrality over thecross section of the capillary. Uponapplication of a voltage in axial direc-tion, a net movement of cations into thedirection of the negative electrode willoccur. The cations are solvated and

2

Figure 2.1: Depiction of the solid/liquid interface in a fused silica capillary filled with anelectrolyte solution.


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drag along the bulk of the liquid. The ve-locity of this flow, ueo is given by (1):

in which σ is the charge density at thesurface of shear (and proportional tothe zeta-potential), ε is the dielectricconstant, R the gas constant, T the tem-perature, c is the concentration of thebuffer, F is Faraday's constant, η is theviscosity and E is the electric fieldstrength along the axis of the capillary.

The magnitude of the electro-osmoticflow is thus determined by the chargedensity on the surface exposed to themoving liquid, by solvent properties likethe dielectric constant, the viscosity, andthe ion strength and by the operationalparameters temperature and fieldstrength.

It is important to realise that the elec-tro-osmotic flow is generated at

the surface/solvent interface of the cap-illary in contrast to the case where thesolvent is driven through a tube byhydraulic force. In that case, the flow isinhibited at the surface by shear forcesand a parabolic flow velocity distribu-tion is built up over the cross section ofthe capillary. When the solvent is drivenby electrical force, a flat velocity profileis generated over the cross section ofthe capillary.

When the capillary is packed withreversed phase, silica based particlesalso the double layer is formed in andaround the particles. Upon applicationof an axial electric field, the solvent

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starts to move in the direction of thecathode. This is illustrated in figure 2.2.

Inserting reasonable values for the para-meters in equation 1 e.g. 100 mV for the

Stationary phase particles

Electrical double layer

Capillary wall

EOF

Figure 2.2: Depiction of the generation of electro-osmotic flow in a packed capillary. (Takenfrom reference 1, published with permission of the publisher of LC.GC Magazine).

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zeta potential (typical for an unmodifiedsilica surface in an aqueous buffer solution at pH 4), an EOF should beattained with a velocity from 0.8 mm/s

at 100 V/cm to 3.2 mm/s at 400 V/cm. Asexperimental proof, the EOF obtainedwith a CEC column was measured andthe results given in figure 2.3

field strength [V/cm]

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 200 400 600 800 1000

EOF velocity[mm/s]

Figure 2.3: Experimental verification of the magnitude of the EOF in a packed capillary independence of the applied field. Column, CEC-Hypersil C18, 3 µm, 250(335)x0.1 mm; mobilephase, acetonitrile/morpholino-ethylsulfonic acid (MES) 25 mM pH 6 80/20, temperature 20°C.(Taken from reference 7, published with permission of the publisher of J. Chromatography).

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For HPLC separations with columnspacked with 3-5 µm particles, linearvelocities in the range of 0.5 - 3.0 mm/sare a prerequisite. Thus the electro-osmotic effect, in principle, is able togenerate a mobile phase velocity suitedfor liquid chromatography. As an exam-ple, the chromatogram of the separationof a mixture of neutral test solutes isshown in figure 2.4 (column and eluentthe same as in figure 2.3).

The electro-osmotic flow in a packedcapillary, differs from pressure drivenlaminar flow in three important aspects:

a) It has a plug flow velocity profile inthe channels between the particles andthe velocity is independent of the chan-nel width. Therefore, in a packed bedthere is much less cross-sectional flowvelocity difference in electrical drivenflow than in hydraulic flow.

b) Flow velocity is virtually independentof the particle size. No column back-pressure is generated.

c) The presence of an electrical doublelayer on the packing is a prerequisitefor the generation of the EOF. The mag-

1

2

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734

5 6

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1 Thiourea 2 Methlparabene 3 Ethylparabene 4 Propylparabene 5 Butylparabene 6 Pentylparabene 7 Hexylparabene 8 Naphthalene 9 Biphenyl10 Fluorene11 Athrancene12 Fluoranthene

Time [min]0 1 2 3 4 5 6 7

Absorbance[mAU]

0

10

20

30

40

50

60

70

Figure 2.4: Example of a CEC separation. Conditions as in figure 2.3. Voltage is 25 kV. Sampleinjection electro-kinetic. (Taken from reference 7, published with permission of the publisherof J. Chromatography).

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nitude of the flow will therefore dependon the stationary and mobile phaseused.

These factors have a highly favorableeffect on the zone broadening (HETP)that occurs in a CEC column whencompared to an HPLC column.

2.2 Zone broadening in CEC vs. HPLC

The HETP (Height Equivalent to aTheoretical Plate) equation can be expressed as the sum of independentterms, which account for the differentcontributions to solute zone broadeningin chromatography [2].

H=Hdisp.+He, diff.+Hi, diff.+Ht, diff.+Hkin

(1)

Hdisp. is the plate height increment due to axial dispersion of the solute in the interstitial space, He, diff. is theplate height increment caused by filmresistance at the particle boundary, Hi, diff. is the plate height contributionfrom intra-particle diffusion, Ht, diff. isthe contribution from trans-channelmass-transfer and Hkin. the contributionof solute interaction kinetics with thestationary phase.

In packed columns, dispersion in theaxial direction is given by equation 2.

Hdisp. =Ha, diff.+Heddy, diff. (2)

Here Ha, diff. is the plate height contri-bution due to static diffusion in axialdirection and Heddy, diff. is the contribu-tion due to the flow velocity differences(eddy diffusion).

Of all the terms that contribute to zone broadening, only eddy diffusionHeddy, diff. and trans-channel diffusion Ht, diff. depend on the type of flow veloc-ity profile in the column [1]. The trans-channel diffusion term accounts for thefact that a solute has to diffuse throughthe channels between the particles inorder to reach the stationary phase. Dueto the plug like flow-velocity profile thecontribution of trans-channel diffusionis smaller in an EOF driven system thanin a pressure driven system. However, ina packed column, the contribution ofthe trans-channel diffusion term to theoverall HETP value is much smallerthan the eddy dispersion term, becausethe diameter of channels is small (theseaverage 1/6 of the particle diameter). Sothe contribution of this term can beneglected.

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The magnitude of the eddy diffusionterm depends on the differences in flowvelocity in the channels between theparticles (an illustration is given in fig-ure 2.5). The velocity of pressure driven

flow varies with the diameter of thechannels. Solutes may find fast andslow flow paths through the column,resulting in temporal dispersion. In an electrically driven system, the flowvelocity is largely independent on the channel width, such that soluteschanging from one channel to the otherdo not experience a change in flowvelocity. This results in much lowerzone dispersion.

The net result of this is a 30-60 % reduc-tion of the HETP on a capillary HPLC

column driven by electrical force compared to pressure driven mode,depending on the particle size, capacityratio and velocity. This is illustrated bythe chromatogram of figure 2.4 where

the individual peaks have an efficiencyof 60-70,000 plates. In HPLC mode thecolumn will generate 25-30,000 platesmaximally.

The electrical force on the solvated cations is exerted equally over thelength of the column in contrast to thehydraulic force, which is applied to the inlet of the column and dissipateslinearly over the length of the column.The hydraulic pressure needed to drivethe liquid, at a particular velocitythrough the packed capillary, increases

Pressure drive Electro-osmotic drive

flowvelocityprofile

particle

channel flowvelocityprofile

particlechannel

Figure 2.5: Depiction of the flow velocity profile in a packed bed in electro drive and pressuredrive.

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with the inverse of the square of theparticle diameter. The pressure alsoincreases linearly with the length of the

packed bed. As a result, the attempt toincrease the efficiency of an HPLC sepa-ration by reducing the particle sizeand/or increasing the column length ispenalized by a severe increase in pres-sure or decrease in speed of analysis (3). In the electro-driven casethis does not occur. Therefore longercolumns with smaller particles can beused in CEC than feasible in HPLC. Asan example a chromatogram obtainedon a column with 40 cm bed length and

packed with 3 µm particles is given infigure 2.6. In average, all peaks have an efficiency of 100,000 plates. It will

be clear that such a column cannot be used in HPLC mode under the condi-tions specified with the velocity that isachieved in CEC.

For more theoretical details regardingthese considerations see reference 2.The better solvent transport mechanismin the electrodrive case thus leads toless dispersion and the absence of backpressure. As a result, it can be predictedthat in CEC 3-10x better separation

Time [min]2 4 6 8 10 12 14

Absorbance[mAU]

0

2

4

6

8

1 Thiourea2 Nitrobencene3 Naphthalene4 Biphenyl5 Fluorene6 Anthracene7 Fluoranthene1

2

3

4 5

6

7

Figure 2.6: The 100,000 plates column; column, Spherisorb ODS1, 3 µm, 400(485)x0.1 mm; mobile phase, acetonitrile/TRIS 50 mM, pH 8, 80/20; voltage 30 kV, temperature 20°C (Taken from reference 2, published with permission of the publisher of J. Chromatography).

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efficiency than in HPLC can be eventu-ally achieved, leading to columns thatgenerate 250,000-1,000,000 plates permeter in the same time as current HPLCseparations. The upper efficiency limithas not yet been achieved in CEC.Below you find an example from the lit-erature illustrating probably the bestresult obtained sofar.

It must be emphasized that the abovetreatment applies to solutes that areneutral and separate only by partitio-ning between mobile and stationaryphase. If the solutes are charged,

they will undergo an accelaration ordeceleration of their velocity dependingon their actual net charge. However,separation of charged solutes by CEC to date has led to confusing results.Extremely high efficiencies have beenreported but also peak distortion andpoor reproducibility (5,6). Therefore, at this moment, CEC is a techniquewhich can be best used for high effi-ciency separation of neutral or weaklyacidic/basic solutes. Capillary electro-phoresis is the method of choice forstrongly charged solutes like stongacids and bases.

Figure 2.7: Column 238x0.1 mm, 1.5 µm ODS-Chromspher. Solvent ACN/5 mM SDS 1.6 mM sodiumtetraborate 3/2, 30 kV, 25°C, neutral aromatic solutes (Taken from reference 4, published withpermission of the publisher of Chromatographia).

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References

1. Dittmann, M., Wienand, K.,Bek, F. and Rozing, G., LC-GC Magazine, 13, 800, 1995.

2. Horváth, Cs., and Lin, H.-J., J. Chrom., 149, 43, 1978.

3. MacNair, J. E., Lewis, K.C. and Jorgenson, J. W., Anal. Chem., 69, 983, 1997.

4. Seifar, R.M., Kok, W.T., Kraak, J.C., Poppe, H., Chromatographia, 46, 131, 1997.

5. Smith, N.W., Evans, M.B.,Chromatographia, 41, 197-203, 1995.

6. Euerby, M.R., Johnson, C.M., Bartle, K.D., LC-GC-Int., 11, 39, 1998.

7. Dittmann, M.M. and Rozing, G.P.,J. Chromatography A., 744, 63, 1996.


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3 Columns

Gerard P. Rozing, Agilent TechnologiesGmbH, 76337 Waldbronn, Germany

3.1 Preparation, Test, Care & Use,Maintenance

CEC columns are made of poly-imidecoated, fused silica tubing, 0.05 -0.20 mmI.D. and 5 - 50 cm length in which a bed of HPLC type stationary phasesparticles, 1.5 - 5 µm size, is contained.The particles are retained in the tubingby small porous segments, frits, at thebeginning and the end of the packed

bed. The fused silica tubing in mostcases extends beyond the frits in orderto make a hydraulic connection to thebuffer/sample vials. Immediately afterthe outlet frit, the polyimide is removedto allow UV-VIS spectrophotometricdetection over the cross section of thetube. The simple schematic below visu-alizes this description.

In most cases, CEC columns are pre-pared by a slurry packing procedurelike used for normal HPLC columns. A suspension of the stationary phaseparticles in a suitable solvent is forced

3

h*

0.05 -0.20 mm

5 - 50 cm

Frit Packed bed

Fused silica tube Poly-imide coating

Frit

Figure 3.1 Schematic representation of a CEC Column.


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under high pressure into the fused silicacapillary. Several publications give de-tails about this procedure (1,2,3,4). Onecommercial supplier uses a proprietary,electrokinetic packing procedure (5).

The critical step in making CEC columns,requiring practical experience, is theformation of the retaining frits. Frits aremandatory to constrain the packingmaterial to the bed zone.

Several attempts have been publishedto make retaining frits (4,6,7,8,9,10,11).Hydrothermal treatment of the packedbed at the location where the frit is to beformed, is currently the most successfulmethod. Figure 3.2 visualizes theprocess.

Homemade CEC columns give flexibilityin the choice of stationary phases usedand reduces purchase cost. But not all,

DC PWS25 V, 4-6 A

600-700 bar,aqueous solvent

Figure 3.2 : Visualization of the frit forming process. Frits are formed by applying power to theheating coil to a temperature of approx. 450°C. The packing solvent, acetonitrile/water ispumped through the bed while heating. Duration 15-45 s.

Columns (page 20-29)

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potential users of CEC will consider investing in the necessary equipmentand time needed to gain experience inpreparing CEC columns. Several HPLCcolumn manufacturers offer columnsfor CEC, which is summarized in 3.2.The following steps described beloware needed in the preparation of CECcolumns for test and usage irrespectivewhether they are prepared in house orpurchased.

First the length of the capillary needs to be adapted to a length matching theCE instrument they are used in. As thefused silica is a glass, cutting the capilla-ry to length is not trivial. The cut needsto be exact and flat (see figure 3.3). Theimportance of a flat end for capillariesused in CE has been emphasized by sev-eral authors (12,13,14, 15). If the capillary end is not flat, the electrical

field at the capillary inlet will becomedistorted. During sample injection, theEOF also will be distorted and zonebroadening will occur.

Cleavage of capillary can be done witha ceramic blade. It is important in thatcase to pull at both sides of the capillaryafter scratching the capillary rather than bending it. In the last case, a sharpedge will remain on the capillary end. In the fiber optic industry, sophisticatedequipment is available to cleave glassor quartz fibers. Such equipment how-ever is prohibitively expensive. Therecently introduced device, SHORTIX™cleaves the capillary like a plumbercleaves water conduits and provides acost-effective alternative (figure 3.4).

Figure 3.3 : Example of a clean cut of a CE(C) capillary.

Figure 3.4 : The Agilent Technologies Shortix™capillary cutter for capillary GC, CE and CEC


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In figure 3.3 it can be seen that at theinlet side, about 5-10 mm of the poly-imide outside coating has been removed.Because in CEC one works with organicsolvents, the polyimide coating may gradually swell and loose consistencyor shape. It may even block the inletside of the capillary.

In case both effects, a poor cut andloose polyimide coating, coincide thequality of the CEC separation may degrade dramatically. In figure 3.5 anexample is given.

Next, the packing or shipping solvent inthe packed capillary has to be replacedby the mobile phase that is used in thechromatography. This is best done byfirst flushing the CEC column with acetonitrile and next with the solventused in the separation. An HPLC pumpequipped with a flow splitter will suffice.

The splitter consists of a standard 1/16''T-piece. One end of the T is connectedto the HPLC pump and a restriction ca-pillary is connected to one of the otherlegs. The restriction capillary should

1 2 3 4 5 6 7 8

Absorbance[mAU]

50

100

150

200

250

Time [min]0 1 2 3 4

20

40

60

80

100

120

0

Absorbance[mAU]

0

Time [min]

Figure 3.5 Improvement of a CEC separation after poly-imide removal and cutting a CEC capillary flat with SHORTIX™. Experimental conditions; Column CEC-Hypersil C18, 3 µm 250x0.1 mm, solvent 80% acetonitrile/20% Tris.HCl, 25 mM, pH 8, Injection electrokinetic,field 25 kV temperature 25°C. Sample thiourea, dimethylphthalate, diethylphthalate, biphenyl,o-terphenyl. Lower trace; capillary inlet damaged; upper trace - after removal of 2-4 mm fusedsilica with SHORTIX™ (with permission of International Laboratory).


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have such dimensions that a pressure of80-100 bar is achieved at 1 ml/min. Thiscan be checked by closing the 3rd leg of the T-piece. After this is calibrated, a short piece of 0.9 mm I.D. 1/16'' SST capillary is connected to the T-piece. To the other side of this capillary a 1/16''internal/external union with 0.75 mmhole (Agilent P/N 5022-2126) is mounted.The capillary connects with the internalside of the union. The SST ferrule isremoved from the external side of theunion and replaced with a special PEEKfused silica adapter (Agilent P/N 5042-1319). The CEC capillary is slipped in atthe outlet side. The capillary is carefullymoved within the connection capillaryand gently sealed in the union. Thepump is switched on and pumping continues till droplets are formed at the capillary inlet side.

The conditioning of the CEC columnwith the solvent used in electrochroma-tography is best done directly in the

CEC system e.g. in the Agilent CE system. For that purpose the capillary is mounted in the cassette and placed in the instrument.

The eluent that is used in our columntest is acetonitrile/25 mM Tris.HCl pH 8,4/1. The buffer is prepared by dissolvingthe appropriate amount of Tris in waterand adjusting the pH to 8 with 30% HCl(high purity grade). The buffer is mixedwith 4 parts acetonitrile. The acetonitrilein the column is displaced by applying12 bar pressure on the inlet side andstepping up the voltage from 5 to 25 kVin 5 kV, 10-15 minutes steps. Next, pressure, 12 bar on both vials and 25 kVis applied for 30 minutes. The currentshould stabilize at about 5 µA for a 25 cm capillary and about 3.5 µA for a40 cm capillary. Wait until the currentand detector baselines are stable.

In practice it is recommended to applystandard conditions to verify the col-


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umn performance. In the example in fig-ure 3.6, such conditions are specified.

The usage of low conductivity and/orzwitter ionic type buffers in CEC isgenerally recommended. These kinds of buffers can be used in higher ionstrength than phosphate or borate buffers while still having a low (< 15 µA)current through the column. Also asone can see in figure 3.6 the voltage isapplied as a ramp in 3-6 seconds. In the Agilent CE system this will minimizeformation of micro-bubbles before thepressure on the vials is fully established.

In practice, the solvent in the inlet andoutlet vial suffices for 5-10 injections

depending on the ion strength and buffering capacity of the solvent. The electrochemical reactions that take place at the electrodes in the vialmay deplete the buffer, change the com-position and the pH of the solvent. MostCE(C) systems provide an automatedfunction for replenishment of the sol-vent.

When the mobile phase needs to bechanged to another composition, this is

1 Thiourea2 Dimethylphthalate3 Diethylphthalate4 Biphenyl5 o-Terphenyl

Time [min]1 2 3 4 5 6 7

Absorbance[mAU]

10

20

30

40

50

60

Currrent

Voltage

12

34

5

Figure 3.6: Standard test for CEC columns. Conditions, Voltage 25 kV; Injection electrokinetic,5s, 5 kV Temp. 20°C; Pressure: 10-12 bar on inlet and outlet vial; Solvent Acetonitrile/Tris.HCl,25 mM pH 8 4/1;


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best done on the CE(C) system directly.Replace the vial and condition with thenew solvent by the application of pres-sure on the inlet vial and stepping up thevoltage as before.

It is recommended that columns arestored in aqueous organic solvent e.g.water/acetonitrile 50/50. The ends of thecapillary are kept in micro-vials filledwith the same solvent. Still one may observe after longer time of storage that parts of the bed have dried out andthat gaps and cracks are formed in the

packed bed. In this case, columns canbe re-wetted by the conditioning proce-dure with an HPLC pump as describedbefore.

Sample preparation for CEC will be described in detail in chapter 6. At thispoint it is recommended to apply cleansamples to a CEC column. Filtration of the sample and removal of proteinsand other high molecular weight bio-molecules is generally recommended. The solvent should be of less or equalelutropic strength as the mobile phase.


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References

1. van den Bosch, S., Heemstra, H.,Kraak, J.C. and Poppe, H.,J. Chrom. A., 755, 165, 1996.

2. Dittmann, M.M., Wienand, K.,Bek, F. and Rozing, G.P.,LC.GC Magazine, 13, 800, 1995.

3. Boughtflower, R.J., Underwood, R.J.and Paterson, C.J., Chromatographia, 40, 329, 1995.

4. Frame, L.A., Robinson, M.L., Lough, W.J.,J. Chrom. A., 798, 243, 1998.

5. Yan, C.,US Patent, 5,453,163, 1995.

6. Cortes, H.J., Pfeiffer, C.D.,Richter, B.E. and Stevens, J.,HRC&CC, 10, 446, 1987.

7. Stegeman, G., Thesis, University of Amsterdam, Amsterdam, The Netherlands, 1994.

8. van den Bosch, S., Thesis, University of Amsterdam,Amsterdam, The Netherlands, 1996.

9. Kennedy, R.T. and Jorgenson, J.W.,Anal. Chem., 61, 1128, 1989.

10. Behnke, B., Grom, E. and Bayer, E.,J. Chrom. A., 716, 207, 1995.

11. Seifar, R.M., Kok, W., Kraak, J.C. and Poppe, H.,Chromatographia, 46, 131, 1997.

12. Cohen, N., Grushka, E., J. Chromatogr-A., 684, 323-328

13. Altria, K. D., Bryant, S. M., Clark, B. J. and Kelly, M. A., LC.GC Int., 10, 157-162, 1997.

14. Guttmann, A. and Schwartz, H.E.,Anal. Chem., 67, 2279, 1995.

15. Rozing, G.P., Amer. Laboratory, 33,December 1998.


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Company

LC Packings

International

Capital

HPLC Ltd.

HYPERSIL

Waters Phase

Separations

Grom Analytik +

HPLC GmbH

Offering

Length 25 cm, I.D. 50, 75, 100 µm.Phase specified by customer.

Customer specific

Length 25, 40 cm,I.D. 100 µm CEC-Hypersil C18, 3 µm

Contact manu-facturer for details

NovoGROM capil-lary columns forCEC are availablewith inner diame-ters of 25, 50, 75and 100 µm. Gromand customer specific stationaryphases.

Address

Baarsjesweg 154,NL 1057 HMAmsterdam, The Netherlands

East MainsIndustrial EstateDunnet Way, Boxburn EH52 5NN,UK

Chadwick Road,Astmoor, Runcorn,Cheshire, WA7 1PRUK

Deeside IndustrialPark, Deeside,Clwyd, CH5 2NUUK

HerrenbergerStrasse 54, D 71083 Herrenberg,Germany

Internet Address

http://www.lcpackings.nl/Prod/ECR01.html

http://www.capital-hplc.co.uk/cprof.htm

http://www.hyper-sil.com/products_f2.html

http://www.phasesep.co.uk/

http://www.grom.de

3.2 Overview of commercial sources for CEC columns


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Company

Unimicro

Technologies

Inc.

Micro-Tech

Scientific Inc

Agilent

Technologies

Supelco

InnovaTech

Offering

Lengths 20, 25, 30, 35 cm, 50, 75,100 µm I.D., C18,C8, Phenyl, Cyano,Amino, Si, 3 µm.

Lengths 15, 25, 45. I.D. 75, 150,320 µm, 3 µm, C18,C8, Si.

Length 25, 40 cm100 µm I.D. CEC-Hypersil C18, C8and Phenyl, 3 µm.

Celect CEC-1820cm X 50µm ID

lengths 15, 25, 35, 45 cm, 50, 75,100 µm I.D., C18,C8, C6, C6/SCX,C18/SCX.Customized touser cassette spec-ification.

Address

4713 First Street,Suite 225Pleasanton, Ca 94566 USA

140 S. Wolfe Rd.Sunnyvale, CA94086 USA

Through local salesoffices

Through local salesoffices

The Business andTechnology Centre,Bessemer Drive,Stevenage, Herts,SG1 2DX

Internet Address

http://www.unimicrotech.com

http://www.mtscientific.com

http://www.agilent.com/chem

https://www.sigma-aldrich.com/SAWS.nsf


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30/143Instrumentation (page 30-38)

Inst

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4 Instrumentation

Gordon A. Ross, Agilent TechnologiesGmbH, 76337 Waldbronn, Germany

4.1. Instrumental Requirements4.1.1. Overview

The instrumentation necessary for iso-cratic CEC experiments is broadly simi-lar to that required for conventional

Capillary Electrophoresis. Figure 4.1shows a schematic of the instrumen-tation. This comprises:

High voltage D.C. source (0-30kV)Inert electrodes (e.g. platinum)Sealed inlet and outlet vialsHigh pressure source (e.g. 10 bar) Capillary packed with stationary phaseCapillary thermostating mechanismDetector

4

high voltage

- +

pressure

similar to that for CE withthe option of applyingpressure (ca. 10 bar) toinlet and outlet vials

Figure 4.1: Schematic of instrumentation for CEC.


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CEC instrumentation must support theapplication of an electric field across abuffer filled packed capillary column.Therefore the instrumentation requiresa high voltage D.C. source connected to two electrodes and inlet and outletvials which contain the mobile phase,the capillary ends and the electrodes.By convention and with regard to thedirection of the EOF in silica basedmaterials, the inlet side of the capillaryis usually positive with the outlet atground.

Pressure may be also applied across the capillary by pressurizing the inletand outlet vials up to ca. 8 to 12 bar inorder to eliminate bubble formation.The generation of gas bubbles duringoperation can be a common problem inCEC. Small bubbles appear as spikes inthe electropherogram. The presence ofsmall bubbles leads to a local increaseof the resistance and a consequent rise in the electric field. This leads toincreases in joule heating in the regionof the bubble and an increase in thebubble size. If the bubble becomes largeenough this results in the breakdown ofcurrent, loss of EOF and termination ofthe run. These bubbles are then not eas-ily removed and require flushing withmobile phase on an HPLC pump at high

pressure (100 bar) or overnight flushingat lower pressures (e.g. 12 bar). Bubbleformation can be reduced by a) takingcare when making the mobile phasethat it is extensively degassed and b)reducing the Joule heating by using lowmobility salts and using lower electricfield strengths. However in order toallow the fullest flexibility of conditions to the analyst e.g. highfield strength and the widest range ofbuffer salts and concentrations, it iseasier to pressurize the entire system so that gases remain in solution thusobviating the problem. Further, for long automated runs where the mobilephase may need further degassing, it is essential.

The capillary column is packed withHPLC type packing material, which, in combination with the properties ofthe mobile phase, provides separationselectivity. Columns for CEC are cov-ered in chapter 3.

A thermostating mechanism should alsoincluded in order to control the columntemperature. This serves not only toequilibrate the temperature throughoutthe capillary but also helps to dissipateany Joule heating. As in HPLC, columntemperature is also of some importance

Instrumentation (page 30-38)

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in determining the selectivity of the sep-aration and therefore its control shouldbe facilitated.

Detection is covered in more detail in afollowing section. The entire systemshould be electrically isolated from theoperator with appropriate safety switch-es to interrupt the applied field shouldthe operator attempt to access theinstrument during operation. For repro-ducible analyses it is advisable to useautomated instrumentation.

4.1.2. Injection

Sample injection techniques for CECare similar to those used in capillaryelectrophoresis (CE) and unlike that of LC. In CEC the sample is injecteddirectly onto the capillary and there isno sample loop as in conventional LC.The sample can be electrically injectedusing the EOF or hydrodynamicallyinjected using a pressure. In electro-kinetic injection the sample is placedunder the inlet end of the instrumentand a voltage is applied for a shortduration (5-10s). The EOF will mobilize

an aliquot of sample into the capillary.Hydrodynamic injection is more prob-lematic since adequate pressure is needed to push the sample onto the column against the back pressureexerted by the packed bed. The majorityof reports to date have utilized electro-kinetic injection.

Direct on-column injection eliminatesany extra column dispersion associatedwith injection loops therefore contri-buting to the high efficiency of CEC.

Table 1 shows the reproducibility ofpeak areas for a standard solution con-taining 4 peaks using both electro-kinet-ic and hydrodynamic injection.Although the migration times reproduci-bility are broadly similar the electro-kinetic injection gives more reprodu-cible peak areas than the hydrodynamicinjection mode. This is due to the morereproducible application of the voltageover time than the high pressure whichis primarily designed for vial pressuri-zation and not for injection. Other sam-ple considerations are discussed furtherin chapter 6.


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4.1.3. Gradient Elution in CEC

At the time of writing there are no com-mercially available continuous gradientCEC instruments. However, a numberof workers have built gradient CECinstrumentation. These rely either ondelivery of a solvent flow from an HPLCpump (1,2) or on electrically creating agradient (3).

If using an HPLC pump, the changingmobile phase is played over the inletside of the capillary and usually via a split. This is then introduced into the capillary by the EOF. Given the differences in minimal flow rate generated by the HPLC pump and the much lower flow required by the CEC capillary there is inevitably a waste of mobile phase. There is alsothe possibility of introducing a slightpressure at the inlet end.

An electrically created gradient isachieved by having the inlet capillaryend coupled to a split capillary witheach end in a vial containing mobilephase of differing constitution. Twovoltage supplies are used and by applying voltage to one vial and thenthe other at varying rates, a mix of thetwo mobile phases is achieved using the EOF to mobilize the eluent. Althougha gradient can be generated, its exactcomposition is unknown.

It should also be noted that the flowrate within the capillary is dependentupon the EOF, which is in turn depen-dent upon a variety of other factors.One of these factors is the organic type and proportion (see chapter 5).Therefore the flow rate may changeduring the course of a run as the organic content changes (4).

Injection Electrokinetic 5s @ 20kV Hydrodynamic 15s @ 5 barPeak number 1 2 3 4 1 2 3 4Migration Time %RSD (n=6) 0.37 0.37 0.32 0.26 0.28 0.39 0.40 0.37Peak Area %RSD (n=6) 1.64 1.58 1.40 0.81 2.84 3.10 2.81 3.80


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Table 1.: Comparison of the reproducibility of peak migration time and area for a 4 peak standard solution using electrokinetic and hydrodynamic injection.

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Although not optimum, a step-gradientcan be created by replacing the inletvial at a certain point during the runwith a vial containing a differing mobilephase e.g. higher organic (chapter 9).The results of using such a system areshown in figure 4.2 for the analysis of asteroid mixture. This can be performed

automatically on the Agilent CE instru-ment and any other instrument capableof changing the inlet vial during a run.

4.1.4. Pressure Assisted CEC

In this instrumental set up a HPLC pumpis connected to the capillary inlet end


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4

min5 10 15 20

-20-15-10

-50

a) no gradient (30% ACN)

min5 10 15 20 25 30 min5 10 15 20 25 300

50100150200250

mAU

30% ACN 45% ACN

currentb) 30% to 45% ACN at 18 min

0

50

100

150

200

30% ACN45% ACN

currentc) 30% to 45% ACN at 12 min

Buffer: 25mM TRIS / H2O / ACN(ionic strength constant)

Column: 3µm phenyl 250mm x 0.1mm i.d.Detection : 200,16nmInjection: 5s@ 10kVRun: 30°C, 25kV, 8bar both sides.

Figure 4.2: Effects of a step gradient on the separation of a 10 steroid sample.

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such that a pressure is applied onto thecolumn simultaneously with the appliedvoltage. This provides an increased inthe flow rate and has been combinedwith electrospray MS detection (5)where such an increased flow rate isrequired. This approach can decreaseretention times without compromisingresolution and can also reduce bubbleformation.

4.2. Detection4.2.1. UV-VIS Detection

The majority of detection in CEC has relied on UV-VIS detection throughthe open section of the capillary imme-diately after the second retaining frit. Asin conventional CE, the polyimide isremoved from this section of the capil-lary allowing light to be directedthrough the capillary onto a detector(see figure 4.1). Any absorbing speciesmigrating past the light will register as a peak. This arrangement, however, hassome optical and physical limitations.The detection path length is small sinceit is limited to the internal diameter ofthe capillary (50 – 150µm) and therefore

available sensitivity is limited. Straylight should also be reduced by the useof appropriate slits around the capillaryin order to preserve maximum linearity.The column also becomes very fragileat the detection point due to a combi-nation of the formation of the secondretaining frit and removal of the poly-imide for the creation of the window.This makes the handling of CECcolumns problematic and once installedonto an instrument it is advisable toleave it there. While detection throughthe packed bed is also possible thisresults in an increased noise due toscattered light (6).

One response to such difficulties is touse an off-column detection cell. Onlyone such cell is commercially available,the Agilent high sensitivity detectioncell (7,8). Using this cell the capillarycolumn need only comprise the packedbed and the two retaining frits; muchmore analogous to HPLC. Since there isno fragile polyimide-free zone therobustness of the column is greatlyimproved and its handling is much easi-er. The capillary can then be reversibly


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coupled to the cell. The cell has a pathlength of 1.2mm and therefore providesa much greater sensitivity (figure 4.3).

The data collection rate is also impor-tant in CEC, which is inherently highlyefficient. Peak efficiency values havebeen found to be highly dependent uponthe detector rise time employed. (9).


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min2 4 6 8 10 12

mAU

0

5

10

15

20

25

30

S/N = 315

S/N = 47 Standard CEC column

CEC column with high sensitivity cell

Column: CEC Hypersil C18, 3 µm, 250(350)x0.1 mmEluent: 90% ACN/ 10% Tris-HCl 50 mM, pH 8Voltage: 25 kVInjection 5 kV, 3s (ca. 1mmplug)Pressure: 10 bar both sidesTemp.: 20°C

DAD1 A, Sig=250,80 Ref=off

Figure 4.3: Comparison of detection on-column and using the high sensitivity cell. The path-lenght on column is 100µm while that of the high sensitivity cell is 1.2 mm taking into accountthe asociated rise in noice the overall signal:noise increase is ca. 8-times (with permission ofJ. Capillary Electrophoresis).

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4.2.2. Fluorescence detection

Fluorescence detection can also beused in CEC (10-12) and has been usedfor the analysis of poly-aromatic hydrocarbons. Both on column and on-packing detection has been reported(12) although the technique is not wide-ly applied.

4.2.3. Mass Spectrometric detection

The first demonstration of the couplingof CEC to MS was reported by (13)since which time interest in the combi-nation has grown. CEC has inherentlyappropriate flow rates for interfacingwith atmospheric pressure electrosprayionization (AP-ESI). An electrospraysource typically requires flow rates inthe region of 0.75-500 µl/min while typi-

cal flow rates in CEC are of the order ofµl/min or less. A make up flow is need-ed to stabilise and maintain the flow inthe electrospray source. A FAB probehas also been used for CEC-MS interfac-ing (14). In such examples a degree ofband broadening is observed in theunpacked capillary section between the end of the packed bed and the electrospray source. Practical aspectsof interfacing with mass spectrometryare discussed in chapter 16.

One solution to this problem has beento place the CEC column in the electro-spray probe itself (15), which allows the use of short columns and high fieldstrengths. The CEC column may also be placed in the source itself thus eliminating the need for make up flowsor connecting capillary (16).


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References

1. Benke, B. and Bayer, E., J. Chromatogr. A., 680, 93, 1994.

2. Taylor, M.R., Teale, P., Westwood, S.A. and Perrett, D., Anal. Chem., 69, 2554, 1997.

3. Yan, C., Dadoo, R., Zare, R.N., Rekestraw, D.J. and Anex, D.S., Anal. Chem., 68, 2762, 1996.

4. Ross, G.A., Fundamental studies on

Capillary Electrochromatography.

Poster presented at HPCE ’98,Orlando, Jan 1998.

5. Dekkers, S.E.G., Tjaden, U.R. and Van der Greef, J., J. Chromatogr. A., 712, 201, 1995.

6. Cikalo, M.G., Bartle, K.D., Robson, M.M., Myers, P. and Euerby M.R., Analyst, 123, 87-102, 1998.

7. Ross, G.A., Kaltenbach, P. and Heiger, D., Today’s Chemist

at Work, 6, 31-36, 1997.

8. Dittmann, M., Rozing, G., Ross, G.A.,Adam, T. and Unger, K.K., J. Cap. Elec., 4/5, 201-212, 1997.

9. Euerby, M.R., Johnson, C.M. and Bartle, K.D., LC-GC Int., 11, 39, 1998.


11. Rebscher, H. and Pyell, U. J.,Chromatogr. A., 737, 171, 1996.

12. Yan, C., Dadoo, R., Zhao, H., Zare, R.N. and Rakestraw, D.J., Anal. Chem., 67, 2026, 1995.

13. Veheij, E.R., Tjaden, U.R., Niessen, W.M.A. and Van der Greef, J., J. Chromatogr., 554, 339, 1991.

14. Gordon, D.B., Lord, G.A. and Jones, D.S., Rapid Commun. Mass

Spectrom., 8, 544, 1994.

15. Lane, S.J., Boughtflower, R., Paterson, C. and Morris, M., Rapid Commun. Mass Spectrom., 10, 733, 1996.

16. Schmeer, K., Benke, B. and Bayer, E.,Anal. Chem., 67, 3656, 1995.


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Influ

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5 Influence of ExperimentalParameters on Separation

Monika M. Dittmann and Gerard P. Rozing, Agilent Technologies GmbH,76337 Waldbronn, Germany

5.1 Mobile phase properties

Similar to HPLC, a wide range of mobile phases can be employed in CEC to obtain separation of the samplecompounds. If neutral solutes are to be separated, the same optimizationstrategies (variation of type and concen-tration of organic modifier) as in HPLCcan be used. The major differencebetween CEC and HPLC lies in the factthat in CEC the mobile phase not only

influences retention and selectivity but also the EOF and thus the linearvelocity of the solvent. The impact ofmobile phase parameters on EOF, retention and selectivity in CEC will be discussed in this chapter.

5.1.1 Buffer pH

As has been shown in chapter 2, thevelocity of the electroosmotic flowdepends on the surface charge densityof the surface on which the EOF is gen-erated. In the case of silica based pack-ing particles, the degree of dissociationof the surface silanol groups and there-fore the charge density depends strong-ly on pH of the eluent. It can be expect-

5


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ed, that the EOF-velocity changes withpH. In figure 5.1, the eluent mobility isshown as a function of the pH of the buffer additive for three stationaryphases. In all cases the eluent consistedof 80% acetonitrile and 20 % 25 mM buf-fer tris(hydroxymethyl)aminomethane(Tris) for pH 8, 2-(N-morpholino)ethanesulfonic acid (MES) for pH 6 and

sodiumacetate (NaOAC) for pH 4 andphosphate for pH 2). The pH of thebuffers was adjusted before mixing with acetonitrile. For all stationaryphases the EOF decreases with pH as expected but even at very low pH a considerable eluent velocity can be observed in columns packed with RP-type stationary phases.

Influence of Experimental Parameters on Separation (page 39-53)

Influ

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of E

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Sep

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5

CEC-Hypersil C18

Spherisorb ODS I

ODS Hypersil

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 12pH of buffer additive

Elue

nt m

obili

ty [1

0^-4

cm

^2/V

s]

Figure 5.1: Eluent mobility as a function of pH of buffer additive. Conditions 80% ACN, 20 % buffer, 20°C, voltage 20 kV (with permission of J. Chromatography).

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In figure 5.2 the separation of a testmixture of neutral components isshown at pH 8, 6 and 4. This figuredemonstrates that the selectivity andefficiency of the separation is not influ-enced by the pH of the aqueous portionof the eluent.

5.1.2 Organic modifier

In chapter 2 it was shown that thedielectric constant and the viscosity of the eluent also play an important rolein the generation of EOF. The ratio ofεr/η vs. % organic modifier at 25°C for


Influ

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MES pH 6

Tris-HCl pH 8

min5 10 15

mAU

0

50

100

150

200NaOAc pH 4

12

34 5

6

7

Figure 5.2 : Separation of neutral standard mixture at different pH values. Conditions: CEC Hypersil C18, 3 µm, 250(350)x0.1 mm, 80% ACN, 20% buffer 50mM, 25 kV, 10 bar both sides, 20°C Sample: 1 thiourea, 2 nitrobenzene, 3 naphthalene, 4 biphenyl, 5 fluorene, 6 anthracene, 7 fluoranthene (with permission of J. Microcolumn Separations).

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methanol/water and acetonitrile/wateris given in figure 5.3 (1). From thesedata it is expected that the electro-osmotic velocity will decrease withreduction of acetonitrile or methanolconcentration in the mobile phasethrough a minimum and will increaseagain on low percentage of these organ-ic modifiers.

The EOF velocity at 20°C as a functionof % organic modifier was determinedin a packed capillary (250(335)x0.1 mmpacked with CEC-Hypersil C18) with acetonitrile and methanol asorganic modifiers. The mobile phasewas prepared such that constant ionstrength of the mobile phase mixturewas obtained. This is necessary as a


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methanol

acetonitrile

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 50 100

% organic modifier

ε/η

[1/c

P]

Figure 5.3: Ratio of dielectric constant over viscosity for mixtures of methanol/water and ace-tonitrile water (with permission of J. Microcolumn Separations).

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change in overall ion strength will affectthe zeta-potential and therefore obscurethe observations. The mobility of theeluent is calculated from the elutiontime of thiourea, which is assumed to be non-retained at all compositionstested. By multiplying the eluent mobilitywith the field strength (in V/cm) theelectro-osmotic flow velocity can beobtained directly.

Figure 5.4 shows the eluent mobilitiesfor the acetonitrile/buffer and methanol/buffer mixtures. While the mobilities forthe methanol system shows qualitativelythe behavior expected from the εr/ηdata, the mobilities of the acetonitrilesystem show a steady increase withincreasing acetonitrile content. Otherauthors (2,3) have also observed thisbehavior of the acetonitrile/buffer sys-


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% organic modifier

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 20 40 60 80 100

Elue

nt m

obili

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ACN

MeOH

Figure 5.4: Eluent mobility as a function of organic modifier content (with permission of J. Microcolumn Separations).

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tem. This observation leads to the con-clusion that in addition to the change ofthe ratio of dielectric constant and vis-cosity, also the zeta-potential and thusthe charge density of the particle sur-face in the packed bed is changing withorganic modifier content. (4)

The influence that the concentration oforganic modifier has on the selectivityof the separation is very similar to HPLC. Figure 5.5 shows the separationof a 16 PAH EPA standard on a CECHypersil C8 column with 60%, 70% and80% acetonitrile.

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

min1 2 3 4 5 6

min2 4 6 8 10 12

mAU

0

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1

1.5

2

2.5

mAU

-4

-3

-2

-1

0

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1

2

3

4

1

2

3

45 6

78

9 10

1112

13 14 15

16

80% ACN

70% ACN

60% ACN

Figure 5.5: Separation of the EPA 16 PAH standard with different organic modifier concntrations.Conditions CEC Hypersil C8, 3 µm, 250(335)x0.1 mm, x/10/(100-10-x) ACN/Tris.HCl, 50 mM, pH 8/H2O, 30 kV, 10 bar both sides, 20°C. 1 naphthalene, 2 acenaphthalene, 3 fluorene, 4 acenaphthene, 5 phenanthrene, 6 anthracene, 7 fluoranthene, 8 pyrene, 9 chrysene, 10 benzo(a)anthracene, 11 benzo(b)fluoranthene, 12 benzo(k)fluoranthene, 13 benzo(a)pyrene, 14 dibenzo(a,h)anthracene, 15 indeno(1,2,3-cd)pyrene,16 benzo(g,h,i)perylene (with permission of J. Cap. Electrophoresis).


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The type of organic modifier used alsoaffects the selectivity of a separation.Exchange of acetonitrile for methanolor tetrahydrofuran is common practice

in HPLC. Figure 5.6 shows as an examplethe separation of a test mixture consis-ting of alkyl 4-hydroxybenzoic acidesters (alkyl-parabenes) and polycyclic


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min0 2 4 6 8 10 12

mAU

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20

1

2 36

7

8

4 5

9

min2 4 6

mAU

0

20

40

60

1

2 36

7

8

45 9

min0 2 4 6 8 10 12 14 16 18

mAU

123456 1

23

67

8

45 9

ACN

MeOH

THF

Figure 5.6: Separation of a neutral test mixture with various organic modifiers. Conditions CECHypersil C8, 3 µm, 250(335)x0.1 mm, 80/20 organic modifier/Tris.HCl, 50 mM, pH 8. 30 kV, 10 barboth sides, 20°C. Sample: 1 thiourea, 2 methylparabene, 3 ethylparabene, 4 propylparabene, 5 naphthalene, 6 butylparanvbene 7 fluorene, 8 anthracene, 9 fluoranthene (with permission of J. Microcolumn Separations).

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aromatic hydrocarbons with acetonitrile(top trace), methanol (middle trace)and tetrahydrofurane (lower trace) asthe organic modifier. The column usedwas a 25 cm CEC-Hypersil C18, 3 µm.Mobile phases contained 80% organicmodifier and 20% of 25 mM Tris-bufferadjusted to pH 8 prior to mixing. As hadbeen verified in a separate experiment,the selectivities of the different modifierswere very similar to the same separationin HPLC mode. The major differencehere is the EOF velocity, which underelse identical conditions is about a fac-tor 2 slower for the methanol systemand ca. a factor 3 slower in the tetra-hydrofurane system. The EOF decrease of the methanol system could beexpected from the εr/η data. Values fordielectric constant and viscosity of themixture water/tetrahydrofuran were notavailable. But estimated from the values

of the dielectric constant and viscosityof the pure organic solvents, a decreaseby a factor of 1.5 is expected. The devi-ation is attributed to a substantialchange in accessibility of the surfacesilanol with THF containing solvents.

5.1.3 Ionic strength of eluent

The influence of the buffer ion strengthon the magnitude of EOF in CEC wasinvestigated by variation of the Tris.HClconcentration in the eluent. Bufferscontaining 5-100 mM Tris.HCl at pH 8were mixed with 80% acetonitrile thusthe final ion concentration in the eluentwas between 1 and 20 mM. Thiourea,was used as an non-retained solute tomeasure t0 and to calculate the eluentmobility. The measurements were doneon a 250(335)*0.1 mm capillary columnpacked with CEC-Hypersil C18, 3 µm at


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20, 30 and 40°C. Results are given in fig-ure 5.7.

According to theory, the EOF will in-crease with decrease of buffer concen-tration. With decreasing ion strength thethickness of the diffuse double layerlength increases leading to an increasein zeta-potential. The increase of EOF

with temperature is mainly due to theincrease in the εr/η ratio although thetemperature change will also affect the zeta-potential. Thus to achieve high EOF in packed column CEC, it isrecommended to work at low bufferconcentrations and above ambient tem-perature.

Aqueous buffer concentration [mM]

1.0

1.5

2.0

2.5

3.0

0 20 40 60 80 100 120

Elue

nt m

obili

ty [1

0^-4

cm

^2/V

s]

20 °C

30 °C

40 °C

Figure 5.7: Eluent mobility as a function of buffer ionic strength and temperature (with permis-sion of J. Microcolumn Separations).


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48/143Influence of Experimental Parameters on Separation (page 39-53)

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5.2 Stationary phases

The behavior of various reversed-phasetype stationary phases in CEC wasinvestigated with neutral analytes.While in HPLC the surface properties of a stationary phase influences onlythe selectivity of a separation, in CECalso the flow velocity is determined bythe properties of the packing material.As shown in chapter 2 the EOF velocitydepends on the surface charge densityof the stationary phase particles. Thismeans that only surfaces that possess anet charge can support electro-osmoticflow. In a silica based reversed phasetype packing this surface charge stems

from dissociated silanol groups. It cantherefore be expected that the EOFvelocity depends on the amount of dis-sociated silanol groups per unit area.

The separation of a test mixture of neu-tral solutes on five different silica basedreversed phases with the same mobilephase, temperature and field strength isshown in figure 5.8 (5). The figureshows the behavior of two non-end-capped phases (CEC Hypersil C18 andSpherisorb ODS I) two endcapped phas-es (ODS Hypersil and Spherisorb ODSII) and the base deactivated phase BDS-ODS Hypersil.

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CEC Hypersil C18

Spherisorb ODS I

ODS Hypersil

Spherisorb ODS II

min0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

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10

20

30

40

50

60

70

80 BDS-ODS-Hypersil

1

1

1

1

12

2

2

2

2

3

3

3

3

3

Figure 5.8: Separation of neutral components on different reversed phase type stationary phases. Different samples were used on the columns but all samples contained Thiourea (1),Naphthalene (2) and Fluoranthene (3). Conditions: 3 µm packing particles, 250 (335)*0.1 mm, 80/20 cetonitrile/Tris.HCl 50 mM, pH8, 20 kV, 20°C, 10 bar on both vials (with permission of J. Chromatography).


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The endcapping and in particular thebase deactivation results in a reducednumber of surface silanols which isreflected in a reduced EOF under else

identical conditions. Other highly basedeactivated phases (Zorbax EclipseXDB-C18, Inertpak C18) do not exhibitany EOF (6). Figure 5.9 shows a com-


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-4

0

4

8

12

mAU

0

20

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60

min2 4 6 8

min2 4 6 8

min2 4 6 8

mAU

0

20

40

60

1

2 3 5 6 7

8

910

11

12

1 2 3 4 5 6 7

910

11

12

1 2 3 4 5 6 7

9

10

11

12

APEX-1 ODS n-ec

APEX-1 ODS ec

CEC-Hypersil C18 n-ec

8

Figure 5.9: Separation of polycyclic aromatic hydrocarbons and parabenes on endcapped anda non-endcapped Apex-1 ODS 1 compared to CEC-Hypersil C18. Conditions: Particle size 3 µm,250(350)x0.1 mm, 80/20 ACN/Tris.HCl, 50 mM, pH 8, 25 kV, 20°C, 10 bar both sides. Sample: 1 Thiourea, 2 Ethylparaben, 3 Propylparaben, 4 Butylparaben, 5 Pentylparaben, 6 Hexylparaben, 7 Heptylparaben, 8 Naphthalene, 9 Fluorene, 10 Phenantrene, 11 Anthracene,12 Fluoranthene.

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parison of CEC Hypersil C18 with Apex-1 ODS endcapped and Apex-1 ODS non-endcapped materials. Here again theEOF velocity of the non-endcappedphases differs considerably from that ofthe endcapped phase (see also table 5.1).

Figure 5.10 shows the separation of a standard mix on stationary phasesthat consist of the same base silicamodified with different chemistries(CEC Hypersil C18, CEC Hypersil C8 andCEC Hypersil Phenyl). These phases


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min2 4 6 8

mAU

0

10

20

30

40

50

60

CEC-HYPERSIL Phenyl

CEC-HYPERSIL C18

CEC-HYPERSIL C8

1

2

34

5

1

23

4

1 23

4

5

5

6

Figure 5.10: Separation of a neutral standard mix on CEC Hypersil C18, CEC Hypersil C8 andCEC Hypersil Phenyl. Conditions: Particle size 3 µm, 250(350)x0.1 mm, 80/20 ACN/Tris.HCl, 50mM, pH 8, 25 kV, 20°C, 10 bar both sides. Sample: 1 Thiourea, 2 Dimethylphthalate, 3 Diethylphthalate, 4 Biphenyl,5 o-Terphenyl, 6 isomeric esters of Dioctylphthalate.

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show large difference in selectivity but exhibit very similar EOF velocities.By using the same columns in pressuredriven mode it was verified for the threeHypersil phases that the selectivities (k’ values) were very similar to those inCEC mode. One can therefore concludethat except for the changes in flowvelocity reversed phase type stationaryphases behave similar in HPLC andCEC.

Table 5.1 gives the EOF velocities andthe k’ values for o-terphenyl or fluoran-thene at a field strength of 715 V/cm at

20 °C in a 80/20 acetonitrile/Tris 50 mMpH 8 buffer for a variety of different RP-type stationary phases. Most phaseshave an EOF velocity of ca. 1.5 mm/sunder these conditions. Only ODSHypersil and BDS-ODS Hypersil have a considerably lower EOF. The k’ valueof o-terphenyl and fluoranthene (both1.40 on CEC Hypersil C18) vary over alarge range from 0.5 on CEC HypersilPhenyl to 2.85 on Supersphere C18 butthis is due to the different coatingchemistries and carbon loading of thedifferent phases.


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References

1. Schwer, Ch. and Kenndler, E., Anal. Chem., 64, 1801, 1991.


3. Adam, T. and Unger, K.,Poster at Analytica Conference

Munich, April 23-25.

4. Dittmann, M.M. and Rozing, G.P., J. Microcol. Sep., 5, 399-408, 1997.

5. Dittmann, M.M. and Rozing, G.P., J. Chrom. A., 744, 63-74, 1996.

6. Dittmann, M.M. and Rozing, G.P.,unpublished results


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EOF (25 kV) k' o-terphenyl k' fluoranthene[mm/s]

CEC Hypersil C18 1.60 1.40 1.40CEC Hypersil C8 1.45 1.00 .-CEC Hypersil Phenyl 1.50 0.50 .-Hypersil ODS 1.00 - 1.50Hypersil BDS-ODS 0.70 - 1.65Spherisorb ODS I 1.60 - 1.3Spherisorb ODS II 1.45 - 3.2Zorbax SB C8 1.45 1.30 .-Zorbax SB C18 1.35 2.55 .-APEX ODS-1 ec 1.40 - 1.60APEX ODS-1 n-ec 1.50 - 1.40Super-Sphere C18 1.40 2.85 .-Nucleosil C18 1.30 2.35 .-

Table 5.1: EOF values and capacity ratio's on different stationary phases under equal conditions

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54/143Method Development (page 54-60)

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6 Method Development

Gordon A. Ross, Agilent TechnologiesGmbH, 76337 Waldbronn, Germany

Introduction

The effects of mobile phase and station-ary phase properties on CEC separa-tions have been discussed in Chapter 5.From this the effects of changing theseparameters during method developmentcan be estimated. Therefore to summa-rize, the flow rate via the EOF is depen-dent upon a range of chemical parame-ters and when changing either station-ary or mobile phase parameters itshould be expected that the flow ratecan and probably will change.However, in the separation at least ofneutral species the established theoriesused in HPLC method development aredirectly applicable to CEC (1).

In chapter 8 the analysis of charged andneutral species is discussed and thishighlights that the selectivity of chargedspecies is quite different to that foundin LC. There is still a great deal of workto be done in order to characterize a CEC method development route forsuch analytes. In the other chaptershighlighting applications of CEC in thisprimer, there is much detail, whichshould help the analyst to develop CECmethods.

6.1. Sample Considerations

While electrokinetic injection is usefulfor neutral species it should be notedthat with charged compounds theinjected aliquot would possess a biastowards analytes with a higher mobility.Given the increased interest in simul-taneous analysis of charged and neutralspecies (Chapter 8) this is one aspect,


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which should not be overlooked by theanalyst especially if a quantitative analy-sis is envisioned.

The injected amount is, however, alsodependent upon the sample matrix (2).Figure 6.1 shows a separation of a stan-dard solution under similar conditionswhere the sample matrix was varied.The amount injected for the same elec-trokinetic injection (10s @ 5kV) varies

such that less is injected for the 50mM TRIS / acetonitrile (1:4) than for the other sample matrices. This is most probably due to the smallerelectric field over the sample duringinjection, which will affect the amountinjected. In this case the ionic strengthof the sample is greater than that of themobile phase and therefore the fieldstrength during the sampling period isreduced.

Figure 6.1: Comparison of the effects of sample matrix on anlysis of a 4 component standardmixture (with permission of J. Capillary Electrophoresis)

Method Development (page 54-60)

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If peak one is chosen as an internalstandard and the peak areas examinedas proportional to this peak area, thenthis illustrates another effect of samplematrix on the injected amount (figure6.2). The relative amounts injected are the same for all peaks in all matricesexcept for water. With water as samplematrix the amounts injected are dis-proportionate with peak 2 having anincreased relative amount and peaks 3and 4 injecting less. Since peak 1 isdimethylphthalate and peak 2 is diethyl-phthalate it is possible that in water,

peak 1 is injected to a lesser degreesince it will have a greater negativemobility than peak 2. However this does not explain the reduced relativeamounts of peaks 3 and 4 (o-terphenyland biphenyl). In this case the differenceis more probably due to decreased solu-bility of the neutral peaks 3 and 4 in water. In any case the exampledemonstrates that more than one mech-anism can affect the amount injecteddepending upon the nature of the ana-lytes and the sample matrix.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

2 3 4

ACNH2O50% ACN10% ACN

Relative peak area(pkx/Pk1)

Peak

Figure 6.2: Effect of sample matrix on relative peak areas from figure 5.1.


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While the majority of applications ofCEC have involved the analysis of com-pounds in ideal, completely definedsample matrices, there are exceptions,which highlight the efforts necessary to ensure that the sample is suitable forCEC analysis. One excellent example isthat provided by Taylor et al (3) in theanalysis of steroids in equine urine andplasma. In this example a C8 clean upof the urine followed by SAX extractionwas necessary to ensure long term func-tion of the column. The column wasstill functioning well after over 200 runs (it is this authors experiencethat direct injection of a urine sampleonto a CEC column is sufficient to killthe column within a couple of runs).Patterson et al (4) reported the C2 solidphase extraction of a potential drugcandidate and 13 related compoundsfrom plasma followed by unequivocaldetermination by CEC-MS. In theseexamples the potential hazard of injec-ting high salt or high protein/organiccontaining matrices was circumventedby sample extraction. Constructing thesample in a matrix which has a lowerelution strength than the mobile phaseis also useful for lowering detection lim-its (4, also see Chapter 7).

6.2 Method Transfer from HPLC to CEC

The main point of difference betweenLC and CEC is in the motive force,which propels the mobile phase throughthe stationary phase. In LC this isachieved through hydraulic pressure,whereas in CEC this is achieved via the EOF. Therefore, in the first instancean EOF must be established in the CECanalysis and when changing parameters,in order to achieve an optimal separa-tion, their possible effect on the EOFshould be appreciated.

Figure 5.6 showed the effects of differ-ent organic modifiers on the CEC separation of a standard mixture.Compared to acetonitrile the flowobserved with methanol is much slow-er. This has the additional effect thatwhen injecting electrokinetically, muchless sample is loaded with methanolthan with acetonitrile due to the lower EOF. THF has a lower flow again although the amount injectedis not so seriously compromised.Therefore although selectivity changesare observed with both MeOH and withTHF, they have deleterious effects on


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the EOF. (This is not true, the efficien-cies are similar for all three solvents,the peaks are broader because thevelocity is lower)

Figure 6.3 shows the separation of twosteroid isomers on LC and CEC. In both cases the method was not opti-mized. On transferring to CEC initialconditions were chosen to provide ade-

quate linear velocity. Therefore a highpH buffer (Tris pH 9.0) was used with avoltage of 25kV. However, using thesame concentration of acetonitrile as inthe LC separation gave no peaks. Thiswas then increased to 95% acetonitrileat which point both isomers could beobserved with better resolution thanwas obtained with LC.

min2 4 6 8 10 12 14

mAU (214 nm)

HPLC CECInstrument - HP 1090 seriesMobile phase - NH4 Ac pH 7.0 /acetonitrile 50%; Column - 3µm ODS125mm x 2mm; Flow - 0.26ml/min; Temp - 38°C.

min5 10 15 20 25 30 35 40

0

10

20

mAU (192 nm)

Instrument - Agilent CEMobile phase - 25mM TRIS pH 9.0 / ACN 95:5;Column - 3µm CEC ODS 1 250mm x 0.1mm; Voltage - 25kV; Temp - 30°C.

-30

-20

-10

0

10

20

Figure 6.3: Analysis of steroid isomers by HPLC and CEC.


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Figure 6.4 shows the transfer of amethod for explosive analysis from gra-dient -LC to CEC. In this case the trans-fer was rather more complex. Firstlythe LC method used a BDS phase whichhas shown to have very low associatedelectro-osmotic flow. Secondly theorganic modifier used in the LC methodwas a methanol gradient which again

gives a low EOF. Therefore primarilythe separation was performed using iso-cratic CEC with acetonitrile as modifier.Despite variation of a range of parameters e.g. organic modifier,temperature, the separation shown in figure 6.4a was the best obtained, and this at a temperature of 50°C. Upon changing to a phenyl column

Instrument: Agilent CEMobile phase: 25mM TRIS pH 9.0 / H2O / ACN

( 20: 35: 45)Column: 3µm phenyl 250mm x 0.1mmVoltage: 25kVTemp: 30°C

min2 4 6 8 10 12 14 16 18

mAU (200nm)

0

10

20

30

40

50

60

70

80

min5 10 15 20 25 30

0

50

100

150

200

250

300

350

Instrument: Agilent CEMobile phase: 25mM TRIS pH 8.0 / ACN

( 50:50)Column: 3µm CEC ODS 250mm x 0.1mmVoltage: 25kVTemp: 50°C

mAU (200nm)

Figure 6.4: Effects of changing stationary phase for the separation of explosives.


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the resolution was significantlyenhanced and the separation markedlyimproved although still being performedisocratically.

In transferring methods from LC to CEC,gradient LC methods can be replaced by isocratic CEC methods. If possibleacetonitrile should be used as theorganic modifier since it gives a goodlinear flow velocity.

References

1. Euerby, M.R., Johnson, C.M., Roulin, S.C.P., Myers, P. and Bartle, K.D., Anal. Commun., 33, 403, 1996.

2. Dittmann, M.M., Rozing, G.P., Ross, G.A., Adam, T. and Unger, K.K., J. Cap. Elec., 4, 201-212, 1997.

3. Taylor, M.R., Teale, P., Westwood, S.A. and Perrett, D., Anal. Chem., 69, 2554, 1997.

4. Paterson, C.J., Boughtflower, R.J., Higton, D. and Palmer, E.,Chromatographia, 46, 599, 1997.


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61/143Analysis of Neutral Pesticide and By-products Using On-column Preconcentration (page 61-68)

and

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n7 Analysis of Neutral Pesticide and By-products Using On-column Preconcentration

Erdmann Rapp1*, Peter Oggenfuss2,Aran Paulus1**, Gerard J. M. Bruin1 ,

1 Novartis Pharma AG , CH-4002 Basel,Switzerland 2 Novartis Crop Protection MünchwilenAG, CH-4333 Münchwilen AG ,Switzerland* Present address: Institut fürOrganische Chemie, UniversitätTübingen, D-72076 Tübingen, Germany** Present address: ACLARABiosystems, Hayward (CA), USA

Introduction

CEC has emerged as a promising, highlyefficient new tool for the separation ofneutral and charged compounds (1,2).However, it is not yet established forroutine applications in an industrialenvironment, although it can be claimedthat it has several advantages comparedto standard HPLC methods.

Since the electroosmotic flow (EOF) isgenerated by an electric field it has aplug-like velocity profile. Besides that aCEC column exhibits no pressure dropwhich enables separations with higher

efficiency and in addition facilitates theuse of capillaries packed with particlesof 3 µm or smaller (3). Others and wehave shown that plate numbers up to700,000 per meter could be generatedusing 1.5 µm particles (4-8).

Most of the separations in the publishedliterature have been performed inpacked capillaries with inner diameters(I.D.) between 50 and 100 µm. As a consequence, consumption of sample,packing material and mobile phase withflowrates between 50 and 200 nl/minute,depending on the I.D., field strength andelectroosmotic mobility, are minimal.However, the concentration sensitivityseriously limits the usefulness of CECfor a number of interesting applications.

To overcome the limitation of low sensi-tivity with UV detection, two differentapproaches have been described. Eitherdetection cells with longer light path-lengths, such as U-, Z-, or bubble-cellscan be used (9,10) and/or the implemen-tation of off-line (11) or on-line (12) preconcentration methods to create ahigher sample concentration within thedetection cell.

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In this work, an on-line preconcentra-tion method, routinely applied in HPLC (13, 14), was applied under CECconditions. In this technique, named on-column focusing, the low sensitivityof UV-detection in CEC is compensatedby the electrokinetic injection of samplecompounds from a non-eluting samplesolution (15-16). The on-column focusingtechnique permits the injection of largesample volumes without compromisingthe separation performance. Using injection times up to 90 seconds com-pared to only a few seconds in mostother CE methods, an injection from a hydrophilic solution allows a 30-foldpreconcentration with a linear depen-dence of peak areas on injection time.This preconcentration technique isdemonstrated with the separation of a commercial pesticide, Cinosulfuron,from some of its by-products which are present at concentrations of lessthan 0.3 % of the main component. Asensitivity increase without sacrificingseparation performance is clearlydemonstrated.

Experimental

Materials. Acetonitrile (HPLC grade)and buffer salts were obtained fromFluka (Buchs, Switzerland). The pesti-

cide Cinosulfuron was synthesized at Novartis Crop Protection SA. Water was purified using a Milli-Q system(Millipore, Watford, UK.) The mobilephase was filtered and thoroughlydegassed by helium directly before use.

Instrumentation. CEC was performedwith a Agilent CE instrument (AgilentTechnologies, Waldbronn, Germany). A commercially available packed fused silica capillary, ElektropakTM

EP-100-25-C8, with 25.0 cm packedlength and 33.5 cm total length and 375 µm OD and 100 µm ID was pur-chased from Unimicro Technologies(Pleasanton, CA, USA). This CEC capil-lary has been electrokinetically packedwith 3 µm SyncroPak C8 (non-endcapped) particle (17).

The sample containing Cinosulfuron(0.73 mM) as the main component andits by-products was dissolved in 20 mMsodium dihydrogenphosphate (pH 4.0) /acetonitrile (90/10). Injections weremade electrokinetically at a constantvoltage of 5 kV with injection timesvarying from 3 to 90 s. The thoroughlydegassed mobile phase consisted of amixture of 20 mM sodium dihydrogen-phosphate buffer (pH = 4.0) /acetonitrile(40/60). Separations were performed

Analysis of Neutral Pesticide and By-products Using On-column Preconcentration (page 61-68)7

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under isocratic conditions at 15 kV at30.0°C with an applied pressure of 8 barat both inlet and outlet vial.

Preconditioning of the capillary wasdone by ramping up the voltage from 0 to 15 kV over 30 minutes followed bya 30 minute application of 15 kV.

Results

On-column focussing is achieved by dis-solving the sample in a solvent mixturewith low elution strength which causesthe sample constituents to adsorb ontothe packing material at the top of thecapillary in a very narrow sample band

without inducing volume overload ofthe capillary. Utilising this compressionof the injected volume makes the injec-tion of large sample volumes possible.

In most of the reversed phase applica-tions dealing with on-column focusing,the sample is dissolved in an aqueoussolution. In our case, the Cinosulfuronsample was dissolved in aqueous solu-tion containing only 10 % acetonitrileand injected electrokinetically with con-stant voltage. Injection time rangingfrom 3 to 90 seconds was used with an applied voltage of 5 kV. A CEC sepa-ration of Cinosulfuron and its by-prod-ucts after a "normal” electrokinetic


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injection is depicted in Figure 7.1. Baseline resolution of the main com-pound and some closely related struc-

tures was obtained with theoretical platenumbers between 30,000 and 40,000.

N

UV-absorbance at209 nm [mAU]

Time [m]5 6 7 8 9 10 11 12

0

20

40

60

80

100

120

5 6 7 8 9 10 11 12-1

0

1

2

3

Cinosulfuron

631 Methanol

2 Thiourea

4 5

0,2% 0,2% 0,8% 0,7%

SO2 NH CO NHN OCH3

NOCH3

NOCH2CH2OCH3

1

1 2 3 4 5 6

23 4

5

6

H3CO

H3CO

NN

NNH2

OCH2CH2CCH3

SO2 NH CO NHN

N

NH2

OCH3SO2 NH2

OCH2CH2CCH3

OCH2CH2CCH3

SO2 NH CO NHN

NN

CI

OCH3

Figure 7.1: CEC separation of Cinosulfuron and by-products. Capillary: 25.0/33.5 cm, 100 µm ID from Unimicro Technologies. Stationary phase: C8, non-endcapped (Synchropak) 3 µm; mobile phase: ACN/20 mM sodiumdihydrogenphosphate, pH 4.0 (60/40). Separation voltage: 15 kV. Injection: 5 kV, 3 s. Applied pressure: 8 bar at both sides. Sample: 2.4 mM Cinosulfuron, dissolved in mobile phase.


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Using injection times up to 90 s resultedin a linear dependence of peak area oninjection time, as can be seen in Figures7.2 and 7.3A.

Compared with the peak area after aninjection of 3 s, a roughly 30-fold higherpeak area after a 90-s injection was

obtained. A good linear relationshipbetween injection time and peak areasof Cinosulfuron and some of the by-products was found. Separations withinjection times longer than 90 s showeda decreased separation performanceand loss of linearity of the injectiontime vs peak area curve. Up to 90 s

5 10 15 20 25 30 35 40 45

0

200

400

600

800

900

3s

30s40s

50s60s

70s80s

90s

Time [min]

UV-absorbanceat 209 nm [mAU]

Figure 7.2. Preconcentration of Cinosulfuron and by-products by on-column focusing.Electrokinetic injection at 5 kV with injection times between 3 and 90 s. Sample: 0.73 mM Cinosulfuron, dissolved in 90 % 20 mM sodiumdihydrogenphosphate, pH 4.0,10 % ACN. Rest of experimental conditions as in Fig. 7.1.


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injection time, no loss in separation per-formance was observed (see Figure 7.3B). Surprisingly, even a slight

increase in plate numbers was found forthe peak of by-product 3.

Peakarea[mAU*s] Peakarea

[mAU*s]

Plate number [N]

0 10 20 30 40 50 60 70 80 900

1000

2000

3000

4000

5000

6000

7000

8000

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0 20 40 60 800

100200300400500

Time [s]

By-product 3By-product 5

A

30 40 50 60 70 80 900

10000

20000

30000

40000

50000

Time [s]

By-product 3By-product 5Cinosulfuron

0 10 20

60000 B

Figure 7.3: A) Peak area of Cinosulfuron and by-products 3 and 5 vs injection time. B)Separation performance vs injection time. Experimental conditions as in Fig. 2.


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With on-column focusing, impuritieswith concentrations of less than 0.3 %of the main compound could be quanti-tated.

Conclusions

Capillary electrochromatography can be applied for the separation of complexindustrial samples. Compared withHPLC, CEC is faster, more efficient andalso reliable. The inherent low sensitivityof this miniaturised separation technique

can be partly compensated by an on-column preconcentration method. Theonly prerequisite is that the compoundsto be separated must be soluble in anaqueous medium. CEC combined withon-column focusing and UV-detectionopens up the possibility to analyse sam-ples with concentrations at the submi-cromolar level. Also with other detec-tion methods, such as mass spectro-metry [18] and NMR [19], on-columnfocusing will be an efficient and easy-to-implement tool to improve sensitivity.


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References

1. Knox, J.H., Grant, I.H.,Chromatographia, 24, 135, 1987.

2. Dittmann, M., Wienand, K., Bek, F.,Rozing, G.P., LCGC, 13, 800, 1995.

3. Knox, J.H., Grant, I.H.,Chromatographia, 32, 317, 1991.

4. Mayer, M., Rapp, E., Marck, C.,Bruin, G.J.M., Electrophoresis, 20, 43-49, 1999.

5. Seifar, R.M., Kok, W.Th.,Kraak, J.C., Poppe, H.,Chromatographia, 46, 131, 1997.

6. Lüdtke, S., Adam, T., Unger, K.K.,J. Chromatogr., 786, 229, 1997.

7. Behnke, B., Grom, E., Bayer, E.,J. Chromatogr., A 716, 207, 1995.

8. Dadoo, R., Zare, R.N., Yan, C., Anex, D.S.,Anal. Chem., 70, 4787, 1998.

9. Chervet, J.P., Ursem, M.,Salzmann, J.P., Anal. Chem., 68, 1507, 1996.

10. Heiger, D.N., Herold, M., Grimm, R.,Applications of the Agilent

Capillary Electrophoresis System,Volume1, Agilent Technologies, Pub. No. 5962-6957E

11. Maier, M., Fritz, H., Gerster, M.,Schewitz, J., Bayer, E.,Anal. Chem., 70, 2197, 1998.

12. Barmé, I., Bruin, G.J.M.,Paulus, A., Ehrat, M.,Electrophoresis, 19, 1445, 1998.

13. Mills, M.J., Maltas, J., Lough, W. J., J. Chromatogr. A., 759, 1, 1997.

14. Naish, P.J., Goulder, D.P.,Perkins, C.V., Chomatographia, 20, 335, 1985.

15. Ding, J., Vouros, P.,Anal. Chem., 69, 379, 1997.

16. Stead, D.A., Reid, R.G., Taylor, R.B.,J. Chromatogr. A., 798, 259, 1998.

17. Yan, C., US Patent 5,453,163, 1995.

18. Lane, S.J., Boughtflower, R.,Paterson, C., Morris, M.,Rapid Commun. Mass Spectrom., 10, 733, 1996.

19. Gfrörer, P., Schewitz, J., Pusecker, K.,Tseng, L.-H., Albert, K., Bayer, E.,Electrophoresis, 20, 3-8, 1999.


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8 69/143Simultaneous Separation of Acidic, Basic, and Neutral Organic Compounds (page 69-87)* Originally published in Anal. Chem., 1998, 70, 4563

8 Simultaneous Separation of Acidic, Basic, and Neutral Organic Compounds*

Ira S. Lurie, Timothy S. Conver, and Valerie L. Ford. Special Testing and Research Laboratory, U.S. DrugEnforcement Administration, 7704 Old Springhouse Road, McLean,Virginia 22102-3494

Introduction

The separation of strongly basic, mo-derately basic, weakly basic, stronglyacidic, moderately acidic, weakly acidic,and neutral compounds in a single runusing capillary electrochromatography(CEC) is presented. This is accom-plished using a 3 µm, CEC Hypersil C8 capillary with high organic contentacetonitrile/phosphate (pH 2.5) mobilephases containing hexylamine. Fifteenbasic, acidic, and neutral drugs of foren-sic interest are resolved using a step gradient. Strong and moderatelybasic drugs separate before to, appa-rently by a combination of free zoneelectrophoresis (CZE) and chromato-graphic phenomena. Weak bases sepa-rate after to, also by a combination ofCZE and chromatographic processes.Due to large selectivity differencesbetween CEC and CZE for bases, thereis evidence that the stationary phase is

playing a significant role in the separa-tion of these solutes. The CEC approachpresented offers unique selectivity,expanded peak capacity, and the abilityto solubilize both hydrophilic andhydrophobic solutes in an injection sol-vent that is compatible with the chro-matographic system.

Capillary electrochromatography (CEC),which combines the best features of CE (i.e., separation efficiency) with the best features of HPLC (i.e., well-characterized retention and selectivitymechanisms, ability to handle thermallylabile solutes and highly polar com-pounds, and increased sample capacity)has recently generated much interest (1-3). However, a recent survey of lead-ing practitioners in CEC indicated thatone of the major drawbacks of the technique is its limited ability toseparate strong bases and its inability toseparate strongly basic, strongly acidic,and neutral compounds in a single run(4). In order for CEC to fully realize itspotential for the separation of smallmolecules, it must be applicable to awide range of solutes, including weak,moderate, and strong bases, neutrals,and weak, moderate, and strong acids.

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This requirement is particularly signifi-cant in the pharmaceutical, clinical, andforensic fields, where all these differenttypes of components can be present ina single sample.

Some preliminary work with strong and moderate bases has been reported.Smith separated tricyclic antidepres-sants via CEC using a strong cation-exchange stationary phase (5). However,although extremely narrow peaks wereobtained for these compounds in certainruns (presumably due to some kind of focusing effect), broad, as well asmultiple peaks have also been observedfor these solutes (6). Very recently, the use of bare silica with buffered ace-tonitrile/water mobile phases has beenreported for the CEC separation of basic drugs (7). The separation mecha-nism in this latter study is presumably amixture of cation exchange and normalphase chromatography.

This article describes the simultaneousCEC separation of strongly basic, mode-rately basic, weakly basic, neutral,weakly acidic, moderately acidic, andstrongly acidic compounds in a singlerun. To our knowledge, this is the firstreport of the successful CEC separationof this range of solutes. In addition, the

CEC separation of strong and mode-rately basic drugs using a C8 bondedphase column at low pH is described.

Experimental Section

Instrumentation: An AgilentTechnologies Model Agilent CE capillaryelectrophoresis system (Waldbronn,Germany) was used for all CEC andCZE experiments. A Beckman Pace5500 capillary electrophoresis system(Fullerton, CA) was used for all micel-lar electrokinetic capillary chromatogra-phy (MECC) experiments. Finally, anAgilent Technologies model 1100 wasemployed for all HPLC experiments.

For CEC, a 100 µm I.D./350 mm O.D.column with a packed bed length of 25 cm (CEC Hypersil C8, 3 µm) wasobtained from Agilent Technologies. Forall separations, the total column lengthwas the packed bed length plus 8.5 cmof polyimide-coated fused-silica tubing.The column was conditioned withmobile phase by first pressurizing theinlet at 10 bar and ramping the voltageto 25 kV over a 30-min period. Both theinlet and outlet were pressurized at 10 bar and the voltage was maintainedat 25 kV for another 30 min. Changingmobile phases was also accomplished

Simultaneous Separation of Acidic, Basic, and Neutral Organic Compounds (page 69-87)

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electroosmotically with pressurizationof the inlet and outlet to 10 bar.

For CZE, a 33.5 cm (25 cm to detectionwindow) 50 µm I.D./350 µm O.D.uncoated fused-silica capillary wasobtained from Polymicro Technologies(Phoenix, AZ). The capillary was con-ditioned with 1 N sodium hydroxide for10 min, followed by water for 10 min,and finally run buffer for 30 min.

For MECC, a 57 cm (50 cm to detectionwindow) 50 µm I.D./350 µm O.D.uncoated fused-silica capillary (Polymi-cro Technologies) was used. The capil-lary was conditioned as above for CZE.

For HPLC, a 12.5 cm x 4.0 mm I.D.HPLC column (ODS Hypersil, 5 µm)was obtained from Agilent Technologies.The column was conditioned with start-ing mobile phase for 30 minutes.

Reagents: Sodium dodecyl sulfate (SDS)obtained from Mallinckrodt (Paris, KY)was used as received. Sodium phosphate(monobasic), sodium phosphate (diba-sic), phosphoric acid, hexylamine, andsodium hydroxide were reagent grade.Deionized water was obtained from aMillipore Milli-Q water system(Bedford, MA). LC grade acetonitrile

was used. All drug standards except for cannabinol (CBN) and 9-tetrahydro-cannabinolic acid (9-THCA-A) wereobtained from the reference collectionof the Special Testing and ResearchLaboratory (McLean, VA). CBN wasacquired from RTI (Research Triangle,NC), while 9-THCA-A was obtainedfrom the Research Institute of Pharma-ceutical Sciences, School of Pharmacy,The University of Mississippi (Univer-sity, MS).

The solutions used for both CEC mobilephases and CZE run buffers were pre-pared by combining 25 mM monobasicphosphate and hexylamine so that thefinal amine concentration after the addi-tion of acetonitrile was either 1 or 2 µL/mL. The phosphate/hexylaminebuffer was then adjusted to pH 2.5-2.6using phosphoric acid.

The MECC run buffer was prepared by combining 85 parts of 50 mM SDS/20 mM dibasic phosphate buffer pH 8.5with 15 parts acetonitrile.

The HPLC mobile phases were mixedinternally from solvent reservoirs con-taining acetonitrile and phosphatebuffer with 3.4 mL/L hexylamine at pH 2.0. The phosphate buffer consisted


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of a mixture of 3480 mL of water, 120 mL of 2 M sodium hydroxide, and40 mL of phosphoric acid.

Procedures: For CEC and CZE, standard compounds were dissolved in either mobile phase or run buffer at a concentration between 0.3 and 0.5 mg/mL prior to 3-s electrokineticinjections at 5.0 kV.

For MECC, standard compounds weredissolved in methanol at a concentrationbetween 0.3 and 1.0 mg/mL prior to 1-spressure injections at 0.5 psi.

For HPLC, standard solutes were dis-solved in 1 part mobile phase buffer and 1 part acetonitrile at a concentration

between 0.3 and 0.5 mg/mL prior to 5 µLinjections.

Results and Discussion

Effect of Hexylamine on CEC of

Strong and Moderate Organic Bases:

Strongly and weakly acidic organiccompounds (e.g., cannabinoids) havebeen previously separated using a C8column with a mobile phase containingacetonitrile and phosphate buffer at pH 2.5 (8). This system is also viable for weakly basic and neutral organicsolutes, which are un-ionized at this pH.These CEC conditions were thereforeinvestigated for strongly and moderatelybasic organic solutes. However, heroin(a moderately basic solute, pKa 7.6 (9))


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exhibited very poor chromatographicperformance, with multiple peaks beingobtained (see Figure 8.1A). Adsorptionof heroin onto the unbonded silanolgroups is presumably contributing to

this phenomenon. Hexylamine has beenpreviously used in HPLC as a modifierto minimize tailing of basic solutes(10,11). This additive minimizes silano-philic interactions by competing with

Figure 8.1: Effect of hexylamine on CEC of basic compounds. Conditions: acetonitrile/25 mM phosphate buffer pH 2.5 (75:25) with voltage 25 kV and temperature 20°C. A CEC Hypersil C8, 3 m (100 µm x 34 cm) (25 cm length to detector) column is used. Other conditions described in Experimental Section. The hexylamine concentrations are (A) 0 (only heroin injected), (B) 1, and (C) 2 µL/mL, respectively. Compound key: (a) amphetamine; (b) methamphetamine; (c) procaine; (d) cocaine; (e) heroin;(f) quinine; (g) noscapine; (h) thiourea.


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the solutes for the unbonded silanolgroups. A major concern for its use inCEC would be the minimization and/orreversal of osmotic flow. However, as shown in Figures 8.1B and 8.1C,appreciable osmotic flow still existsafter the addition of hexylamine, as evi-denced by the time of the neutral mark-er (to); the retention time of thioureaonly increased from 5.2 to 7.7 min after the addition of 2 µL/mLhexylamine. In addition, as shown inFigure 8.1C, improved chromatographicperformance was also obtained for theindividual moderate and strong basessuch as amphetamine (pKa 9.9) (9)methamphetamine (pKa 10.1) (9) pro-caine (pKa 9.0) (9) cocaine (pKa 8.6) (9)heroin (pKa 7.6) (9) quinine (pKa 4.1,

pKa 8.5) (9) and noscapine (pKa 6.2) (9).These solutes all migrated before to,indicating that electrophoresis is play-ing a major role in the separationprocess. It has been postulated that theCEC separation process for chargedsolutes would include CZE (3). Theseresults confirm that postulation. Asshown in Figure 8.1B, lowering thehexylamine concentration from 2 to 1 µL/mL resulted in peak splitting forcertain solutes (cf. amphetamine, pro-caine, and cocaine). However, higherconcentrations of hexylamine wouldresult in lower osmotic flow, higher cur-rent and increased risk of bubble forma-tion, and possible band spreading dueto Joule heating. Therefore, 2 µL/mLappears to be the optimal concentration.


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Effect of Acetonitrile Concentrationon CEC of Strong and Moderate OrganicBases: The effect of acetonitrile con-centration on the CEC of strong andmoderate bases is shown in Figure 8.2.Lower separation voltages were used at lower acetonitrile concentrations in order to operate at smaller currents.In general, lowering the amount of ace-tonitrile in the mobile phase increasesresolution and alters selectivity, especial-

ly at 30% acetonitrile. Except for qui-nine, the elution order remains thesame with changes in acetonitrile con-centration. As the acetonitrile concen-tration is increased, the relative hydro-nium ion concentration is lowered, lead-ing to an increase in apparent pH. Inaddition, the pKa of bases is lowered with increasing acetonitrileconcentration.

8

Figure 8.2: Effect of percent acetonitrile on CEC of basic compounds. Conditions identical toFigure 8.1C, except for percent acetonitrile (ACN) and voltage (V). Compound key: as per Figure 8.1.


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The behaviour of quinine can beexplained by considering that the appar-ent pH of the mobile phase is near its first pKa. It is less likely thatselectivity effects for the other basicdrugs can be explained by pH and pKaconsiderations. These solutes all havepKa values greater than 6.2, and in addi-tion, acetonitrile (a relatively nonbasicsolvent) would not be expected to havesuch a large effect on lowering the pKa.At all acetonitrile concentrations, thesolutes eluted before to. The time of the neutral marker (thiourea) signifi-cantly increased with a decrease in ace-tonitrile concentration (this effect willbe discussed in more detail later in thepaper). As shown in Figure 8.2, the bestseparation of the bases in terms of reso-lution and speed of analysis wasobtained at 60% acetonitrile. Good platecounts of 82000, 85000, 101000, 110100,and 130 000 plates/m were obtained forheroin, cocaine, procaine, noscapine,and amphetamine, respectively, underthese separation conditions. Metham-phetamine and quinine (which bothexhibit significant tailing) have platecounts of 34 000 and 42 000, respectively.

These results compare quite favorablywith the 14 000 plates/m obtained forheroin via HPLC using a 3 µm C18 col-umn with a similar mobile phase (11) Itis noted that this stationary phase mightbe expected to have less unbondedsilanol groups than the one used for CEC, since it is end capped(12)(unlike the CEC phase (1)). For CEC,no general trends were observed for the effect of acetonitrile concentrationon plate height, except that the highestefficiencies were obtained at 60% ace-tonitrile.

The effects shown in Figure 8.2 couldrepresent a combination of electropho-retic and chromatographic phenomena.Electrophoresis would involve a CZEmechanism while chromatography con-siderations include hydrophobic andsilanophilic interactions. Hydrophobicinteractions involve the mobile phaseand the C8 ligands, while the silano-philic interactions include the mobilephase and unbonded silanol groups.The peak tailing observed for the basicsolutes indicates the latter interaction is occurring.


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Comparison of CZE and CEC: Togain some insight into the mechanismof the CEC separation of moderate andstrong bases, CZE was performed usingconditions identical to those used forCEC, except that a 50-µm-I.D. open-tubular fused-silica capillary was used.As shown in Figure 8.3, major changesin selectivity were obtained for the vari-ous solutes at all acetonitrile concen-

trations vs CEC (cf. Figures 8.2 and 8.3). These selectivity differencesinclude changes in separation order,especially at 75% acetonitrile. For CZE, the largest change in migrationrelative to other solutes occurs for qui-nine. Again this phenomenon can be explained by considering that theapparent pH of the mobile phase is nearthe first pKa of quinine. However, other

Figure 8.3: Effect of percent acetonitrile on CZE of basic compounds. Conditions identical toFigure 2 except a 50 µm x 33.5 cm (25 cm length to detector) uncoated fused-silica capillarywas used. Compound key: as per Figure 8.1.


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significant selectivity effects, such asthe reversal in order of amphetamineand methamphetamine, and heroin and noscapine, cannot be explained by pKa considerations. Acetonitrile nothaving a large effect on lowering thepKa is confirmed by the fact that theweak bases diazepam (pKa 3.3) (9) andmethaqualone (pKa 2.5) (9) are clearlycharged at all concentrations of organicmodifier, since they migrate before to inall cases. Therefore, it appears thatprocesses other than free zone electro-phoresis are contributing to the CZEseparations. These other selectivityeffects could be explained by silano-philic and/or hydrophobic interactionswith the silanol groups on the capillarysurface. Although the non-Gaussianpeaks are for the most part triangular in shape (which indicates that electro-dispersion is occurring), there is alsosome evidence of tailing. This latterphenomenon could indicate thatadsorption onto the unbonded silanolgroups is occurring. Hydrophobic inter-actions could also be occurringbetween the epoxide moiety of fusedsilica and the hydrophobic portion of a solute. These effects are minimized by the relatively large amount of aceto-nitrile present in the run buffer.

The large selectivity differencesbetween CEC and CZE indicate that the packed bed is playing a significantrole in the separation process in CEC.The major differences in selectivity that occur between CZE and CEC couldbe explained by larger silanophilicand/or hydrophobic interactions in CEC due to the much larger surfacearea of the packed capillary. In agree-ment with a previous study, (13), theCZE electroosmotic flow (EOF) at a given acetonitrile concentration is 2-3 times higher than the CEC EOF.This effect is not explained by the rela-tive amount of silanol groups present inboth CE columns. It had been previous-ly shown that the EOF is higher for CZEthan CEC when the packing materialwas underivatized silica (14). The mag-nitude of the EOF decreases because ofnonalignment of the flow channels inthe packing material with the capillaryaxis and the lack of electrodrive insidethe particle pores (15)

At all acetonitrile concentrations, signif-icantly higher plate counts wereobtained using CZE vs CEC. In general,the difference in efficiencies betweenthe two techniques increased with anincrease in acetonitrile concentration.


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The increase in efficiency of CZE overCEC ranged from ~20 times at 75% ace-tonitrile to ~7 times at 30% acetonitrile.Despite the lower plate counts for thelatter technique, however, the overallresolution for CEC at a given acetoni-trile concentration was equal or betterthan that obtained via CZE. This isdirectly attributable to the increase in selectivity that is obtained by com-bining a free zone mechanism withenhanced chromatographic phenomenaobtained by using a packed bed.

As expected, in CZE the solutes elutedbefore to at all acetonitrile concentra-tions. The time of the neutral marker(thiourea) significantly increased with adecrease in acetonitrile concentration.

For both CEC and CZE, the meoincreased with increasing acetonitrileconcentration. These results can beattributed to decreases in viscosity (16) and ionic strength at higher ace-tonitrile concentrations. The relativeconductivity (calculated from Ohm's lawfrom CE measurements) and thereforeionic strength decreased with increasingacetonitrile concentration for both CEC and CZE. Again as the acetonitrileconcentration is increased, the relativehydronium ion concentration is lowered,

leading to an increase in apparent pHand an increase in ionization of silanolgroups, which increases the µeo.

There are some conflicting data in theliterature on the effect of acetonitrileconcentration on electroosmotic flow.Some previous CEC studies performedat both decreasing ionic strength (1,17)and constant ionic strength (13,18) also showed that µeo increases withincreased acetonitrile concentration.However, other investigations showed a decrease in µeo with increasing ace-tonitrile concentration and decreasingionic strength (19,20). Similar CZE studies performed at both decreasingionic strength (13,21) and constantionic strength (13,16) conditions found(in contrast to our findings) that the µeodecreased with increasing acetonitrileconcentration. It was also found thatpure acetonitrile/ water mobile phasesµeo exhibited varying behavior withincreases in acetonitrile concentration(21). However, between 20 and 60% acetonitrile, µeo increased with increasing acetonitrile concentration.The reasons for these discrepancies are not clear; however, our studies were performed at a buffer pH of ~2.5vs pH>6 for these other investigations.


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CEC of Basic, Neutral, and Acidic

compounds: Chromatographic conditi-ons similar to those described in Figure8.1C were also viable for the analysis ofweak bases, weak acids, strong acids,and neutral solutes. The simultaneousseparation of the strong bases (amphe-tamine, methamphetamine, procaine),moderate bases (cocaine, heroin, nos-capine, quinine), weak bases (diazepam,

methaqualone), strong acids (∆-9-tetra-hydrocannabinolic acid (pKa1 ~3.0,pKa2 ~13.4 (9)) (pKa values werederived from the structurally similar sal-icylic acid), moderate acids (pheno-barbital (pKa 7.4 (9)), weak acids (∆-9-tetrahydrocannabinol (pKa 10.6 (9)),cannabinol (pKa ~10.6 (9)), and neutralsolutes (testosterone, testosterone pro-pionate) in a single run is shown in

Figure 8.4: CEC step gradient of basic, neutral, and acidic compounds. Initial conditions: (for first minute) acetonitrile/25 mM phosphate buffer pH 2.5 (60:40) with 2µL/mL hexylamine. Final conditions: acetonitrile/25 mM phosphate buffer pH 2.5 (75:25) with 2µL/mL hexylamine. A voltage of 25 kV and temperature of 20°C were used. Other conditions described in Experimental Section. Compound key: Same as Figure 8.1 except (i) Phenobarbital, (j) diazepam, (k) methaqualone, (l) testosterone, (m) cannabinol, (n) testosterone propionate, (o) 9-tetrahydrocannabinol, and (p) ∆-9-tetrahydrocannabinolic acid.


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Figure 8.4. The excellent separation ofall 15 compounds was accomplishedusing a step gradient. This separationcombines the increased resolution of themoderate and strong bases at a loweracetonitrile concentration with the goodresolution and higher speed of analysisof all the other solutes at a higheracetonitrile concentration. As indicatedin Figure 8.4, all solutes except for thestrong and moderate bases elute afterto, indicating that chromatography isplaying a major role in the separationprocess for the weakly basic, acidic, andneutral solutes. This is not surprising,since unlike the moderate and strongbases (which exist as cations at pH 2.2),

these other solutes are either unchargedor partially charged at this pH.

It is interesting that the weak basesdiazepam and methaqualone migratejust before to by CZE, with an elutionorder opposite to that obtained by CEC. These solutes elute just after to byCEC, indicating that they are separatingby a combination of CZE and chromato-graphic phenomena.

Comparison of CEC with MECC:

MECC was also performed on the samemixture of solutes investigated by CEC(see Figure 8.5). A comparison betweenCEC and MECC (cf. Figures 8.4 and 8.5)

Figure 8.5: MECC of basic, neutral, and acidic compounds. Acetonitrile/50 mM SDS/20 mMphosphate buffer pH 8.5 (15:85) with voltage of 25 kV and temperature 20°C. An uncoatedfused-silica capillary (50 µm x 57 cm) (50 cm length to detector)) column is used. Other conditions described in Experimental Section. Compound key: as per Figure 8.4.


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reveals major differences in selectivitybetween the two techniques.

Unlike CEC, where moderate and strongbases elute before to, these solutes eluteafter to (for the most part as a group)via MECC, and (in this case) followingthe elution of phenobarbital and metha-qualone. In addition, the elution orderof the moderate and strong bases isvastly different for both techniques.Similar to CEC, MECC could involve a combination of electrophoretic andchromatographic phenomena (which,for MECC, involves partitioning into themicelle and possible ionic interactionswith the micelle.) Due to differences inthe pH values between the buffers usedin both techniques (pH 2.5 vs pH 8.5),the degree of ionization of the solutescould be different in CEC vs MECC.Changes in pH, partitioning into themicelle, and ion pairing between basicsolutes and the micelle all contribute to the differences in selectivity betweenthe two techniques.

The weak bases methaqualone anddiazepam also exhibit large selectivitydifferences between the two techniques.This is not surprising, considering thatthese solutes are un-ionized in MECC(unlike CEC.)

The weak acids ∆-9-tetrahydrocannabinoland cannabinol, and the neutral com-pounds testosterone and testosteronepropionate, all exhibited similar selectivity in CEC vs MECC; this isbecause they are all uncharged in bothtechniques. This would indicate that the hydrophobic interactions occurringbetween the mobile phase and C8 lig-ands by CEC are similar to the hydro-phobic interactions between the runbuffer and the micelle that occur byMECC. The weak acid phenobarbitaland the strong acid ∆-9-tetrahydrocanna-binolic acid, which are un-ionized viaCEC and ionized using MECC, exhibitdifferent selectivities by both techniques.A contributing factor is the repulsion ofthe negatively charged species by thenegatively charged micelle. Due to thelarge selectivity differences between thetwo techniques, CEC and MECC arecomplementary for the separation ofacidic, neutral, and basic drugs. The useof multiple techniques is clearly helpfulfor drug screening.

One advantage CEC has over MECC isan infinite elution range; for MECC, theelution range is the time of the micelle(tmc) divided by to. It is not uncommonin MECC for multiple solutes to elute at the tmc, especially nonpolar solutes.


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The hydrophobic compounds ∆-9-tetra-hydrocannabinol and cannabinol, whichare well separated by CEC, coelute viaMECC either because of limited elutionrange or by being fully incorporatedinto the micelle. In the presence of anorganic modifier, it is difficult to mea-sure tmc.

Another advantage of CEC over MECCis the solubility of the solutes in aninjection solvent that is compatible withthe separation system. For CEC, theinjection solvent is the mobile phase (orstarting mobile phase for step gradient),which contains a relatively high amount

of acetonitrile. To solubilize the morehydrophobic compounds for injectionby MECC, it is necessary to dissolve thesolutes in methanol. As a result, even atthe lowest pressure injection allowedby the instrument, several solutes suchas amphetamine, methamphetamine,and cocaine exhibit peak splitting (seeFigure 8.5.)

Comparison of CEC with Gradient

HPLC: Gradient HPLC was performedon the same solute mixture using chro-matographic conditions similar to thoseemployed for CEC (∆-9-tetrahydro-cannabinolic acid elutes only if the hold


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at the end of the gradient is extended)(see Figure 8.6). As shown in Figures8.4 and 8.6, major differences in selecti-vities were obtained between the twotechniques for the strong and moderatebases. In comparison to CEC, wherethese solutes eluted before to, thesecompounds eluted after to in HPLC.Electrophoresis plays a major role inthe former technique, while chromato-graphic processes dominate in HPLC.

Silanophilic interactions may be playinga larger role in CEC than HPLC, sincethe former phase has a larger amount of unbonded silanol groups (1). Weakbases, weak acids, a moderate acid, astrong acid, and neutral solutes allexhibited similar selectivity via CECand HPLC (cf. Figures 8.4 and 8.6).Except for diazepam and methaqualone,which have pKa's near the pH of themobile-phase buffer, the other solutes

Figure 8.6: HPLC gradient of basic, neutral, and acidic compounds. Initial conditions: acetonitrile/phosphate buffer with 3.4 µL/mL hexylamine pH 2.0 (2:98) with20 min linear ramp. Final conditions: acetonitrile/phosphate buffer with 3.4 µL/mL hexylaminepH 2.0 (65:35) with 10-min hold. Flow rate of 1.5 mL/min with temperature of 20°C. An HPLC Hypersil ODS, 5 m (12.5 cm x 4.0 mm I.D.) column is used. Other conditions described in Experimental Section. Compound key: as per Figure 8.4.


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are uncharged at pH 2.0. This wouldindicate that hydrophobic interactionsbetween the mobile phase and the C8ligands are dominant in both techniquesfor these uncharged solutes. It is also of interest that these additional soluteselute later relative to to using HPLC vsCEC. Due to the significant selectivitydifferences between the two techniques,CEC and HPLC are complementary forthe separation of acidic, neutral, andbasic drugs. Again, the use of multipletechniques are useful for drug screening.

Unlike HPLC, where a full gradient isemployed, only a step gradient isrequired for the separation of acidic,neutral, and basic solutes in CEC. This is a direct consequence of using a mobile phase with a high organic con-tent coupled with free zone electro-phoresis for basic solutes.

The extra peaks present in the HPLCchromatogram are primarily due toimpurities present in the hexylamine. It is of interest to note that the CEC run is devoid of these extraneous peaks.These impurities are soluble at the high-er acetonitrile concentrations used inCEC and thus remain constant duringthe run.

Due to solubility considerations, it was necessary to dissolve the mixtureof solutes in a mobile phase that isstronger than the starting mobile phasefor the gradient HPLC run. This is oftenrequired in gradient HPLC, especiallywhen chromatographing a mixture ofhydrophilic and hydrophobic com-pounds. To avoid adverse effects suchas peak distortion and loss in resolution(especially of the early eluting bands),the amount of sample that can be inject-ed is limited to small volumes (i.e., <10µL) (22).

Acknowledgment

The authors are grateful to AgilentTechnologies for providing CECcolumns. We would also like to thankDr. Mahmoud ElSohly of The Universityof Mississippi for providing ∆-9-tetrahydrocanna-binolic acid A. We are also grateful to Dr. GerardRozing and Dr. David Heiger of AgilentTechnologies for helpful discussions.Presented in part at the Eleventh Inter-national Symposium on High Perfor-mance Capillary Electrophoresis andRelated Microscale Techniques,Orlando, FL, February 1-5, 1998.


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References

1. Dittman, M.M., Rozing, G.P., J. Chromatogr. A., 744, 63-74, 1996.

2. Dittman, M.M., Wienand, K., Bek, F., Rozing, G.P.,LC-GC, 13, 800-814, 1995.

3. Colón, L.A., Guo, Y., Fermier, A., Anal. Chem., 15, 4461A-467A, 1997.

4. Majors, R.E. LC-GC, 16, 96-110, 1998.

5. Smith, N.W., Evans, M.B.,Chromatographia, 41, 197-203, 1995.

6. Smith, N.W., Eleventh International Symposium on High Performance CapillaryElectrophoresis and RelatedMicroscale Techniques, Orlando, FL,February 1-5, 1998, oral presentation.

7. Wei, W., Luo, G., Yan, C., Eleventh International Symposium on High Performance CapillaryElectrophoresis and RelatedMicroscale Techniques, Orlando, FL, February 1-5, 1998,

poster presentation.

8. Lurie, I.S., Meyers, R.E., Conver, T.S.,Anal. Chem., 70, 3255-3260, 1998.

9. Moffat, A.C., Ed. Clarke's Isolation andIdentification of Drugs, 2nd ed.,

The Pharmaceutical Press:

London, 1986.

10. Gill, R., Alexander, S.P., Moffat, A.C., J. Chromatogr., 247, 15-37, 1982.

11. Lurie, I.S., Carr, S.M., J. Liq. Chromatogr., 6, 1617-1630,1983.

12. Lurie, I.S., Allen, A.C., J. Chromatogr., 317, 427-442, 1984.

13. Choudhary, G., Horvath, C., J. Chromatogr. A., 781, 161-183, 1997.

14. Govindaraju, K., Ahmed, A., Lloyd, D. K.,J. Chromatogr. A., 768, 3-8, 1997.

15. Knox, J.H., Grant, I.H.,Chromatographia, 32, 317-328, 1991.

16. Schwer, C., Kenndler, E., Anal. Chem., 63, 1801-1807, 1991.


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References

17. Lelièvre, F., Yan, C., Zare, R.N.,Gareil, P., J. Chromatogr. A., 723,145-156, 1996.

18. van den Bosch, S.E., Heemstra, S,Kraak, J.C., Poppe, H., J. Chromatogr. A., 755, 165-177,1996.

19. Yamamoto, H., Baumann, J., Erni, F.,J. Chromatogr., 593, 313-319, 1992.

20. Yan, C., Schaufelberger, D., Erni, F., J. Chromatogr. A., 670, 15-23, 1994.

21. Wright, P.B.; Lister, A.S.; Dorsey, J. G.,Anal. Chem., 69, 3251-3259, 1997.

22. Snyder, L.R., Kirkland, J.J.,Introduction to Modern LiquidChromatography, 2nd ed.;

John Wiley & Sons, Inc.: New York,Chapter 7, 1979.


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9 88/143Practical Applications of CEC in the Pharmaceutical Industry (page 88-94)

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Melvin R Euerby* and Christopher MJohnson, Astra Charnwood, Pharma-ceutical and Analytical R & D, BakewellRoad, Loughborough, LeicestershireLE11 5RH, UK.

Introduction

This article will illustrate the success-fulness of CEC in the separation ofdiastereo-isomeric steroids, which can-not be resolved by conventionalreverse-phase HPLC. In addition, theuse of step-gradient CEC on commer-cially available instrumentation hasbeen shown to extend the technique’s

role in the analysis of components ofwidely differing lipophilicities.

Experimental

CEC Instrumentation. CEC was performed on a Agilent Technologies(Cheadle Heath, UK) capillary electro-phoresis (CE) system capable of opera-ting at a pressure of up to 12 bar at each end of the capillary as describedpreviously (1). The mobile phase was asstated in the individual Figure legends,all mobile phases were filtered througha 0.2 mm PTFE syringe filter and de-gassed by ultrasonication prior to use.


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Steroids. Steroids seem to be particu-larly amenable to separation by CEC, as shown in the following example(Figure 9.1).

Tipredane, 4 has previously been sepa-rated from its related substances, 1-3and 6 using conventional reverse-phaseHPLC on a variety of C-18 stationaryphases (2,3). However, a survey of over20 different stationary phase chemis-

tries and the use of solvent optimisationprocedures failed to produce baselineseparation of tipredane from its C-17diastereo-isomer 5 (4). Being diastereo-isomers (not enantiomers), their separation should in theory have been

straightforward on conventional achiral columns. However, it has beennecessary to resort to chiral HPLC,using a Chiral AGP (5), β-cyclodextrinstationary phases or a urea solubilised,β-cyclodextrin mobile phase and a

OF

HO

H

H

OF

HOSCH3

H

H

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HOSCH3

H

H

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OF

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SCH3

H

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HOSCH2CH3

SCH2CH3

H

H

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O

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H

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SCH2CH3

Figure 9.1: Structure of tipredane and related substances

9 Practical Applications of CEC in the Pharmaceutical Industry (page 88-94)

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reverse-phase column (6), in order toachieve baseline separation.

In striking contrast, the use of non-optimised, non-pressurised, CEC con-

ditions using a Spherisorb ODS1 packedcapillary resulted in baseline resolutionof tipredane from all its related substan-ces including the C-17 diastereo-isomer(see Figure 9.2).

Time [min]2.5 5 7.5 10 12.5 15 17.5

Absorbance[mAU]

0

10

20

30

40

50

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804 5

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Figure 9.2: Isocratic CEC separation of tipredane (4) from five related substances; for peakassignment see Figure 1. Column 250 mm x 50 µm id 3 µm Spherisorb ODS1 packed capillary.Voltage 15 kV, 15°C, 5kV/15 second injection, unpressurised capillary, analyte 0.16 mg/ml in 8:2ACN /H2O v/v, 240 nm; ACN /TRIS (50 mmol/l, pH 7.8) buffer 80:20 v/v. Reprinted with permission from ref. 9.1.


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Method development simply involvedreplacing the potassium dihydrogenphosphate buffer used in HPLC with the zwitterionic buffer (TRIS). Theresultant elution order observed in CEC was found to be identical to that in HPLC. This suggests that HPLC meth-ods for unionised analytes should bedirectly transferable to CEC.

Use of Step-Gradient CEC withCommercially Available CEInstrumentation

In order to fully realise the potential of CEC, it is necessary to develop the capacity of gradient elution for theseparation of complex mixtures ofwidely differing lipophilicities as inHPLC. Literature contains relatively few examples of true gradient CEC, i.e.electrodriven separations (7) ratherthan voltage assisted pressure drivenseparations (8), however all the exam-ples have utilised "home-built HPLCbased systems”. The results reported sofar are extremely encouraging, however,for the technique to gain in popularity,the approach must be feasible on com-mercially available CE systems. The fea-sibility of employing step-gradient CECon commercially available CE equip-

ment was investigated. The pressurizedCE system used possesses the capacityto perform step-gradients in an auto-mated sequence. The analysis is startedwith the initial mobile phase conditionsthen the voltage is removed and theinlet and outlet buffer vials exchangedfor the final mobile phase conditions;the voltage is re-applied and the analysis continued. Finally, as part ofthe method, after the last peak has eluted, the voltage is terminated, the initial buffer vials replaced and the voltage re-applied to condition the capillary to the initial run conditionsprior to the next analysis.

This concept was investigated using a mixture consisting of a range of sixdiuretics of differing lipophilicitieswhose separation has previously beenreported under isocratic CEC conditi-ons (1). As can be seen from Figure 9.4,the elution time of the first four peakswas comparable to those in the iso-cratic run (Figure 9.3b) indicating thatthey had not experienced the finalmobile phase composition. The twolater eluting peaks are extremely sharpindicating that they had experienced a gradient effect. A reduction in analy-sis time from 35 to 16.5 minutes (25


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minutes including the re-equilibrationstep) was obtained (see Figures 9.3 and 9.4). Three consecutive runs of this

semi-optimized step-gradient methodgave Rt <1% RSD.

Absorbance[mAU]

010203040

Time [min]5 10 15 20 25 30 35

05

101520

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107

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a

b

Figure 9.3. Isocratic CEC separation of chlorothiazide (7), hydrochlorothiazide (8), chlorthalidone (9), hydroflumethiazide (10), bendroflumethiazide (11) and bumetanide (12); 230 mm x 50 µm id 3 µm CEC Hypersil C18 packed capillary, 30 kV, 15°C, 5kV/15 second injection, 8 bar capillary pressurisation, analyte 0.2 mg/ml in 1:1 ACN /H2O v/v, 210 nm; a) ACN /Na2HPO4 (50 mmol/l, pH 2.5) buffer/H2O 60:20:20 v/v/v; b) ACN /Na2HPO4 (50 mmol/l, pH 2.5)buffer/H2O 40:20:40 v/v/v. Reprinted with permission from ref. 9.


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Our preliminary evaluation of step-gra-dient CEC using a commercially avail-able CE system has shown that the con-

cept is feasible and that compoundswith widely differing lipophilicities maynow be analyzed.

Time [min]

0 5 10 15 20

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-5

0

5

10

15

40:20:40MeCN / buffer /

H2O

40:20:40MeCN / buffer / H2O

60:20:20MeCN / buffer / H2O

78

9

10

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12

Figure 9.4. Step-gradient CEC separation, for CEC conditions see Figure legend 9.3, except step-gradient programme used: time 0 - 6.50 minutes ACN /Na2PO4 (50mmol/l, pH 2.5) buffer/H2O40:20:40 v/v/v: time 6.50 - 17.25 minutes, 60:20:20 v/v/v; time 17.25 - 25.00 minutes, 40:20:40 v/v/v.Reprinted with permission from ref. 9.


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References

1. Euerby, M.R., Johnson, C.M., Gillgan, D., Roulin, S., Myers, P. and Bartle, K.D., J. Microcolumn Sep., 9, 373, 1997.

2. Euerby, M.R., Hare J. and Nichols, S.C.,J. Pharm. Biomed. Anal., 10, 269,1992.

3. Euerby, M.R., Johnson, C.M. andNichols, S.C., in Proceeding of theFifth Symposium on the Analysis ofSteroids, Szombathely, Hungary, 1993,S. Görög, Ed., p213, Akadéminiai

Kiadó, Budapest, 1994.

4. Euerby, M.R., Johnson, C.M. and Mole, J., Fisons Pharmaceuticals,1994, unpublished result.

5. Noroski, J., Mayo, D., Kirschbaum, J.J., J. Pharm. Biomed. Anal., 10, 447,1992.

6. Euerby, M.R. and Johnson, C.M.,Fisons Pharmaceuticals, 1994,unpublished result.

7. Yan, C., Dadoo, R., Zare, R.N., Rakestraw, D.J. and Anex, D.S., Anal. Chem., 68, 2726, 1996.

8. Behnke, B. and Bayer, E.,J. Chromatogr. A., 680, 93, 1994.

9. Euerby, M.R., Johnson, C.M., Gillgan, D. and Bartle, K.D., Analyst, 122, 1087, 1997.


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95/143Analysis of Triglycerides (page 95-101)

Ana

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10 Analysis of Triglycerides

P. Sandra and A. Dermaux, University ofGent, Department of Organic Chemistry,Krijgslaan 281 (S.4), B-9000 Gent,Belgium

Introduction

Triglycerides (TGs) are the most abun-dant constituents of oils and fats. Theyconsist of a glycerol molecule in whicheach hydroxyl group is esterified with a fatty acid. This results in an intricateseries of compounds and highly efficientseparation techniques are required tounravel this complexity. TGs are namedby the fatty acid composition; for exam-ple PPO stands for a TG containing twopalmitic and one oleic ester chains. Theseparation of TGs has been extensivelystudied by capillary gas chromatography,(cGC) (1,2,3) liquid chromatography(LC) (4,5,6,7,8) and supercritical fluidchromatography (SFC) (9,10,11). Nowadays the primary methods for TGanalysis are reversed phase LC (RPLC)(4) and silver ion chromatography (SIC)(7). The first provides separations according to the partition number (PN)which is defined as PN = CN-2NDB inwhich CN is the carbon number andNDB is the number of double bonds,

while the latter gives separations according to degree of unsaturation. RP micropacked and microbore LCcolumns were successfully used for thedetermination of the TG composition of vegetable oils (12,13), and it seemedobvious to evaluate capillary electro-chromatography (CEC), providing higherefficiencies, for the same solutes andsamples.

Experimental

An Agilent CE system (AgilentTechnologies, Waldbronn, Germany)equipped with diode array detection(DAD) was used in this study. Fused sil-ica capillary columns with 0.350 mmo.d., and 0.100 mm i.d. packed with 3 µmHypersil ODS particles were applied.The lengths of the packed beds were 25 cm and 40 cm, respectively (AgilentTechnologies, Waldbronn, Germany).The applied voltages ranged between 20and 30 kV at 20°C. Samples were intro-duced either by applying 10 bar or 10 kVduring 3s. The mobile phase was non-aqueous and consisted of acetonitrile/isopropanol/n-hexane in ratio 57/38/5 towhich 50 mM ammonium acetate wasadded.

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Results

In CEC, the non-aqueous mobile phasedeveloped for RPLC could be used withexception of addition of 50 mM ammo-nium acetate which provided a stablecurrent. In Figure 10.1 the performanceof micro-RPLC and RPCEC are com-

pared for the analysis of corn oil. The far better resolution in the sameanalysis time applying CEC is obvious.In the CEC analysis, the EOF was 0.47 mm/s and 71.000 were calculatedfor POP which represents a reducedplate height h of 1.88.

Figure 10.1: Triglyceride analysis of corn oil by micro-LC (A) and CEC (B).

(A) Column: 2 x 25 cm x 1 mm i.d., Stainless Steel (SS). Biosil C18 HL 5 µm. Mobile phase: ace-tonitrile/isopropanol/n-hexane 57/38/5 at 55 µl/min. Detection: evaporative light-scatteringdetection (ELSD) at 50°C, 1.8 bars nitrogen, range 4. Temperature 20°C. (B) Column: 40 cm x 0.1 mm i.d., FSOT, Hypersil ODS 3 µm. Mobile phase: acetonitrile/iso-propanol/n-hexane 57/38/5 – 50 mM ammonium acetate. Detection UV at 200 nm. Temperature 20°C. Voltage 30 kV. Injection 10 kV during 3 s.

Peaks : 1 = LLLn, 2 = LLL, 3 = OLnL, 4 = OLL, 5 = OLnO, 6 = PLL, 7 = POLn, 8 = OLO, 9 = SLL, 10 = PLO,11 = PLP, 12 = OOO, 13 = SLO, 14 = POO, 15 = PLS, 16 = POP.

Analysis of Triglycerides (page 95-101)

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More than 30 samples (oils, margarines,pharmaceutical formulations, etc.) wereanalyzed by CEC and in all cases morecomplete triglyceride profiles wereobtained compared to LC. To illustratethe robustness of CEC for triglycerideanalysis, Table 10.1 compares the

absolute migration time (RT) and rela-tive migration time (RRT) values forsome of the major TGs of corn oil(Figure 10.1B) and walnut oil analyzedsix months later on a new CEC columnselected at random from the AgilentTechnologies production line. RRT

values are excellent allowing their usefor TG structure elucidation.

No differences were observed in thequalitative and quantitative profiles ofthe samples obtained by injecting at 10 kV or 10 bar during 3 s which can be

explained by the neutral character ofthe TGs. Relative standard deviationson migration times of the 8 major TGsfor 10 consecutive runs, however, weresubstantially lower for electrokineticinjection (% RSD ≤ 0.45) compared tohydrodynamic injection (% RSD ≤ 1.32).

RT % RSD RRT % RSDcolumn 1 column 2 column 1 column 2corn oil walnut oil corn oil walnut oil

Isopropanol 14.80 14.64 0.73 0.23 0.23 0.34LLL 44.66 44.49 0.27 0.69 0.69 0.12OLL 53.49 53.22 0.35 0.83 0.83 0.03PLL 56.69 56.47 0.27 0.88 0.88 0.11OLO 64.46 64.11 0.39 1.00 1.00 0.00PLO 68.69 68.38 0.32 1.07 1.07 0.07OOO 78.21 77.75 0.42 1.21 1.21 0.03POO 83.74 83.34 0.34 1.30 1.30 0.05

% RSD for three determinationsConditions as described in figure 10.1B

Table 10.1: Retention parameters for the main triglycerides of corn and walnut oil on two different 40 cm columns.


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Optimised injection time for the analy-sis of TGs is 3 s or shorter. Increasingthe injection time from 3 to 12 s resul-ted in an efficiency drop of 25%.

A pharmaceutical application is shownin Figure 10.2. A formulation consistingof some testosterone esters and a lipid

mixture was analyzed on a 25 cm column. The less hydrophobic testoste-rone esters elute before the lipids. Theelution order of the testosterone esterscould be elucidated by the recordedDAD spectra (insert Figure 10.2) whilethe lipid profile corresponds to peanutoil.

Figure 10.2: Analysis of a pharmaceutical formulation. Conditions: see figure 10.1B except col-umn: 25 cm x 0.1 mm i.d., FSOT, Hypersil ODS 3 µm, detection : UV from 0-9 min at 254 nm (∗ ).

Peaks : 1’ = testosterone phenylpropionate, 2’ = testosterone propionate, 3’ = testosterone isocaproate, 4’ = testosterone decanoate, 1 = LLLn, 2 = LLL, 3 = OLL, 4 = PLL, 5 = PLnP, 6 = OLO, 7 = SLL, 8 = PLO, 9 = OOO, 10 = SLO, 11 = POO, 12 = PLS, 13 = POP, 14 = POS.

Insert: recorded DAD spectra for 1’ and 3’.


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Vegetable oils are relatively simple intheir TG composition because the common fatty acids are palmitic (P),stearic (S), oleic (O), linoleic (L) andlinolenic (Ln) acid. Fish oils, on theother hand, are characterised by abroader range of carbon numbers and ahigher degree of unsaturation. The mostcomplete pictures for fish oils can beobtained with CEC as illustrated in fig-ure 3 for the analysis of SardinaPilchardus oil. More than 100 peaks

were registered and the structurescould be elucidated by combining off-line silver ion supercritical fluidchromatography (SI-pSFC) with CECand electrospray mass spectrometry(ES-MS) (16).

Conclusion

CEC is a very promising technique tounravel the complexity of triglyceridesin vegetable oils and fish oils.

Figure 10.3 : Separation of the triglycerides of the oil of Sardina Pilchardus. Column and con-ditions as described in Figure 10.1B.


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References

1. Christie, W.W., "Gas Chromatography and Lipids. A Practical Guide.”, The Oily Press, Ayr, Scotland, 1989.

2. Geeraert, E. and Sandra, P., J. Am. Oil Chem. Soc., 64, 100, 1987.

3. Garcia Regueiro, J.A., Diaz, I.,David, F. and Sandra, P.,J. High Resol. Chromatogr., 17, 180,1994.

4. Christie, W.W., "High Performance LiquidChromatography and Lipids. A Practical Guide.” Pergamon Press, Oxford, UK, 1987.

5. Frede, E.,Chromatographia, 21, 29, 1986.

6. Aitztmüller, K. and Grönheim, M.,J. High Resol. Chromatogr., 15, 219, 1992.

7. Christie, W.W.,J. High Resol. Chromatogr., 10, 148, 1987.

8. Christie, W.W., Prog. Lipid Res., 33, 9, 1994.

9. Laasko, P. in "Advances in Lipid Methodology”, ed. W.W. Christie, The Oily Press,Dundee, Scotland, 82, 1992.

10. Sandra, P. and David, F. in"Supercritical Fluid Technology inOil and Lipid Chemistry”, ed. J.W. King, and G.R. List,

AOCS Press, Champaign, Illinois, USA, 321, 1996.

11. Demirbücker, M. and Blomberg, L.S.,J. Chromatogr. A., 550, 765, 1991.

12. Ferraz, V. and Sandra, P.,Proc. 16th Int. Symp. Cap. Chrom., P. Sandra (Ed.), Hüthig Verlag,Heidelberg, Germany (Publ.), pp. 1544, 1994.

13. Ferraz, V.,"Ph. D. Dissertation”, University of Gent, Gent, Belgium, 1995.


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References

14. Sandra, P., Dermaux, A., Ferraz, V.,Ditmann, M.M. and Rozing, G.P., J. Micro. Sep., 9, 409, 1997.

15. Dermaux, A., M. Ksir, Zarrouck, K.F.F. and Sandra, P., J. High Resol. Chromatogr., 21, 545, 1998.

16. Dermaux, A., Medvedovici, A., Ksir, M., Van Hove, E., Talbi, M. and Sandra, P.,J. Micro. Sep., (in press).


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11 102/143Analysis of Fatty Acids and Derivatives (page 102-108)

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11 Analysis of Fatty Acids and Derivatives

A.Dermaux and P. Sandra, University ofGent, Department of Organic Chemistry,Krijgslaan 281 (S.4), B-9000 Gent, Belgium.

Introduction

The most important fatty acids naturallyoccurring in the triglycerides of vegeta-ble oils are palmitic acid (16:0), stearicacid (18:0), oleic acid (18:1), linoleicacid (18:2) and, linolenic acid (18:3).The number of double bonds is repre-sented by :x and they all have the cisconfiguration.

In chapter 10 we presented our CECresults for the analysis triglycerides invegetable and fish oils and, comparedto micro LC, the separations were muchbetter in terms of efficiency and speedof analysis (1). In this contribution,experiments with CEC for the analysisof free fatty acids (FFAs), of fatty acidmethyl esters (FAMEs) and of fatty acidphenacyl esters (FAPEs), originatingfrom the above mentioned oils, are pre-

sented. CEC data are compared tothose obtained with micro LC (2,3).

Experimental

The same instrumental setup was used as described in chapter 10 exceptthat the mobile phase consisted ofacetonitrile/50 mM MES pH 6 (90/10 v/v).The preparation of FFAs, FAMEs andFAPEs is described in reference 2.

Results

In HPLC with ODS phases, the separa-tion of FFAs, FAMEs and FAPEs takesplace according to the partition number(PN) which is defined as PN = CN-2NDBin which CN is the carbon number andNDB is the number of double bonds.The separation occurs in increasingorder of CN while one double bond inthe chain reduces the retention time by the equivalent of two carbon num-bers. Two components with the samePN number are by definition criticalpairs.


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Figure 11.1 compares the isocratic sepa-rations of the FAPEs 18:3, 18:2, 18:1,16:0 and 18:0, obtained by micro LC ona FSOT column (30 cm x 0.32 mm i.d.)packed with 3 µm ODS particles (RoSilC18 HL from BioRad) with methanol/wa-ter (95/5) at a velocity of 0.7 mm/s (A),and by CEC on a 25 cm x 0.1 mm i.d. (B)and a 40 cm x 0.1 mm i.d. (C) FSOT col-umn both packed with 3 µm HypersilODS particles.

For the mobile phase used the platenumbers on the 25 and 40 cm columnare 31000 (h=2.7) and 52000 (h=2.6),respectively. For the same particle size, CEC performed much better than micro LC for which N was 28500(h=3.5) at a velocity of 0.7 mm/s. Forthe CEC columns the mobile phasevelocities were 1.9 mm/s for the 25 cmand 1.1 mm/s for the 40 cm column. The currents generated were 5.8 µA and

Figure 11.1: Separation of FAPEs by micro-LC (A), CEC on a 25 cm (B) and on a 40 cm (C) column(A) Column: 30 cm L. x 0.32 mm i.d., FSOT, RoSil C18 HL 3 µm.

Mobile Phase: methanol/water (95/5 v/v). Injection volume: 60 nl. Detection: UV at 242 nm.

(B) Column: 25 cm L. x 0.1 mm i.d., FSOT, Hypersil C18 3 µm. Mobile phase: acetonitrile/50 mM MES pH 6 (90/10). Injection 10 kV during 3s.Detection: UV at 242 nm. Temperature: 20°C. Voltage: 30 kV.

(C) Conditions as described in figure 11.1B except column: 40 cm L. x 0.1 mm i.d., FSOT,Hypersil C18 3 µm. Peaks: 1 = 18:3; 2 = 18:2cc; 3 = 16:0; 4 = 9c-18:1; 5 = 18:0

Analysis of Fatty Acids and Derivatives (page 102-108)

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4.4 µA, respectively. A better criterionto express the resolving power is theseparation number. The SN between16:0 and 18:0 is 12.7, 16.0 and 22.4 forthe micro-LC, and the 25 and 40 cm CECcolumns, respectively. The SN/min valuesare 0.20, 0.85 and 0.49, respectively,illustrating the superior performance ofCEC compared to micro-LC.

Figure 11.2 shows a typical CEC separa-tion of the FFAs (A) and the FAPEs (B)from soya oil on the 40 cm column. The lack of suitable chromophores in the FFAs (and also in FAMEs-see fur-ther) makes UV detection difficult andlow wavelengths have to be applied.Because of the short detection pathwayin electrodriven separation methods,

Figure 11.2: Separation of the FFAs (A) and FAPEs (B) of Soya oil by CEC on a 40 cm column(A) Conditions as described in 11.1C except detection: UV at 200 nm.(B) Conditions as described in 1C. Peaks: 1 = 18:3, 2 = 18:2, 3 = 18:1, 4 = 16:0 and 5 = 18:0


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operating at 200 nm or below is feasible.Nevertheless, for quantitative purposesthe analysis of FFAs and FAMEs makeno sense. On the one hand, the responsefactor for unsaturated fatty acids differtoo much to be applied for quantitativeanalysis, and on the other hand the sa-turated fatty acids can not be detected.

In the FAPE analysis the fatty acidsidentified are 18:3, 18:2, 18:1, 16:0 and18:0. Their % composition is 6.4, 53.4,23.2, 13.0 and 4.0, respectively. This cor-responds with quantitative dataobtained with other techniques, like

capillary gas chromatography analysisof the methyl esters.

In order to obtain more insight in thefatty acid composition of fish oils, butabove all to evaluate the performanceof CEC, because capillary gas chroma-tography (4) is still the best techniqueto analyze the fatty acid composition of lipids, the oil of Sardina Pilcharduswas hydrolyzed and the free fatty acids (FFAs) –A, the methyl esters(FAMEs) -B and the phenacyl esters(FAPEs) -C derivatives were analyzed.


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The chromatograms are shown inFigure 11.3. The highest efficiency isobtained for the analysis of the FFAs.

Most probably the charged species arefocused by their electrophoretic mobi-lity towards the positive electrode.

Figure 11.3: Separation of FFAs (A), FAMEs (B) and FAPEs (C) of the oil of Sardina Pilchardus. (A) Column: 40 cm L. x 0.1 mm i.d., FSOT, Hypersil C18 3 µm.

Mobile phase: acetonitrile/50 mM MES pH 6 (90/10). Detection: UV at 200 nm. Temperature:20°C. Voltage: 30 kV. Injection 10 kV during 3 s.

(B) Conditions as described in figure 11.3A.Peaks: 1 = 18:4, 2 = 20:5, 3 = 22:6, 4 = 16:1, 5 =18:1

(C) Conditions as described in figure 3A except detection: UV at 242 nm.Peaks: 1 = 18:4, 2 = 20:5, 3 = 22:6, 4 = 16:1, 5 = 14:0, 6 = 18:1, 7 = 16:0, 8 = 20 :1 and 9 = 18:0


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For the FAPE derivatives the responsefactors are close to one and the norma-lization method can be used whereasthis is not the case for the FFAs andFAMEs as discussed above. The amountsof 18:4, 20:5, 22:6, 16:1, 14:0, 18:1, 16:0,20:1 and 18:0 are 4.5, 22.5, 20.9, 9.6, 8.7,10.3, 18.6, 1.6 and 3.3 %, respectively.Fish oil is characterized by a high

degree of unsaturation mainly carriedby eicosapentaenoic acid (20:5) anddocosahexaenoic acid (22:6). Thesefatty acids are the active principles with metabolic action in the constitutionof cellular membranes and synthesis

of prostaglandins, thromboxanes andleucotrienes (5,6). An interesting fea-ture of the CEC-DAD analysis is thatDAD data can be used to calculate thenumber of double bonds in the fattyacid moiety of the FAPEs. The plot ofthe ratio of the extinction coefficients240 over 210 nm versus the number ofdouble bonds is shown in Figure 11.4.

The absorbance ratios ε240/ε210 for 6, 5, 4, 3, 2, 1 and 0 double bonds were0.42, 0.51, 0.64, 0.92, 1.40, 1,83 and 2.06,respectively. The principle was appliedto identify the peaks 4 and 5. The ratioswere 1.82 and 2.04, respectively, which

0,1

1,0

10,0

0 1 2 3 4 5 6

Number of double bonds

ε 240/ε

210

Figure 11.4 : Absorbance ratio (ε240/ε210) versus number of double bonds


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means that peak 4 is 16:1 and peak 5 is14:0. This was confirmed for the latterby plotting the log k versus the carbonnumber for the saturated fatty acids.The plot is linear which also illustratesthat the mobile phase flow created byelectroosmosis is constant during theanalysis.

Conclusion

CEC is a viable technique for the analy-sis of FAPEs in vegetable and fish oils.Compared to micro-LC, better and fasterseparations are obtained.

References

1. Sandra, P., Dermaux, A., Ferraz, V.,Ditmann, M.M. and Rozing, G.P., J. Micro. Sep., 9, 409-419, 1997.

2. Dermaux, A., Ferraz V. and Sandra, P.,Electrophoresis, in press.

3. Dermaux, A., Ksir, M.,Zarrouck, K.F. and Sandra, P.,J. High Resol. Chromatogr., 21, 545, 1998.

4. Van Landuyt, N., Denoulet, B.,David, F. and Sandra, P.,Int. Anal., 1, 28, 1987.

5. Dyerberg, J., Bang, H.O. and Aagaard, O.,Lancet, 1, 199, 1980.

6. Cambien, F., Jacquesson, A.,Richard, J.L., Am. J. Epidemiol., 124, 624, 1986.


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12 Elucidation of cis/trans C18:1 Ratios in Margarines

A.Dermaux and P. Sandra, Departmentof Organic Chemistry, University ofGent, Krijgslaan 281 (S4), B-9000 Gent,Belgium.

Introduction

The potential of CEC, for the analysis of the fatty acids in the triglycerides ofvegetable and fish oils, was illustratedin chapter 11. Derivatization of the fattyacids as phenacyl esters (FAPEs) wasrequired to obtain reliable qualitativeand quantitative data, on the fatty acidcomposition in vegetable and fish oils.Unsaturated free fatty acids (FFAs),however, can be analyzed by CEC with-out any sample preparation by detec-tion at 200 nm. This does not sufficethough to quantify unsaturated FFAs inoils because the response depends onthe degree of unsaturation.Nevertheless, when the degree of unsat-uration is the same e.g. positional andgeometrical isomers of 18:1, quantita-tion becomes possible. The principle is illustrated with theanalysis of the cis/trans 18:1 ratio in an experimental margarine.

Experimental

The same instrumental setup and oper-ating conditions as described in chapter10 and 11 were applied. The preparationof FFAs from margarine samples isdescribed in reference 1.

Results

Hydrogenation of vegetable oils can result in substantial amounts ofpositional and geometrical isomers.Best known is the formation of thetrans isomer of 9-18:1 i.e. elaidate as well as cis- and trans-isomers withthe double bonds in 6 and 11 positions.The elucidation of the cis/trans ratio of 18:1 is of utmost importance. Trans fatty acids in dairy products are classified as cholesterol raising(increasing LDL while decreasing HDL)and are not metabolized (2).

Determination of the cis/trans ratio can be performed by micro LC on ODSwith Ag+ loaded mobile phases afterformation of the FAPE derivatives. The stability constants of the π-Ag+

12 Elucidation of cis/trans C18:1 Ratios in Margarines (page 109-111)

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complexes are more stable for the cisthan for the trans isomers and bothgroups can be differentiated with thecis isomers eluting first (3).

The same principle could not be appliedin CEC because the addition of Ag+ions, for unknown reasons, resulted invery unstable currents. Some positionaland geometrical isomers can be separa-

ted by CEC on a C18 column but thisseparation is hardly applicable becauseof overlaps of cis-trans isomers and oftrans isomers with 16:0 (1).

To our surprise, the 18:1 cis/trans ratioin margarine’s can be measured in theCEC analysis of the FFAs because 16:0is not detected at 200 nm. Figure 12.1shows the analysis of the FFAs in an

Figure 12.1: Separation of FFAs of margarine by CEC on a 40 cm column. Column : 40 cm L. x 0.1 mm I.D., FSOT, Hypersil C18 3 µm. Mobile phase: acetonitrile/50 mM MES pH 6 (90/10). Injection 10 kV during 5 s. Detection: UV at 200 nm. Temperature: 20°C. Voltage : 30 kV. Peaks : 1 = 18:3, 2 = 18:2, 3 = all-c-18:1, 4 = all-t-18:1


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experimental margarine and the cisgroup can nicely be distinguished fromthe trans group. Because both groupshave the same response factors the cal-culated cis/trans ratio is 1.3 which wasconfirmed by the micro-LC/ODS/Ag+technique of the FAPEs (3).

Conclusion

The potential of CEC is illustrated forthe determination of the cis/trans ratioof 18:1 in margarine without the need toderivatize the FFAs.

References

1. Dermaux, A., Ferraz, V. and Sandra, P.,Electrophoresis, in press.

2. Mensink, P.R. and Katan, M.B., N. Engl. J. Med., 323, 439, 1990.

3. Correa, R.C., Ferraz, V., Cerne, K. and Sandra, P., in Proceedings of the TwentiethInternational Symposium on CapillaryChromatography, P. Sandra, Ed., CD Rom, IOPMS, Kennedypark 20, B-8500 Kortrijk, Belgium, 1998.


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13 Analysis of Nucleosides

Thomas Helboe, Jette Tjørnelund andSteen Honoré Hansen*, Department of Analytical and PharmaceuticalChemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark.

Introduction

The analysis of nucleosides, nucleotidesand modified nucleosides is very usefulin the diagnosis of several serious disea-ses and metabolic disorders. Profilingof nucleosides in urine, serum or plasmashows noticeable differences betweenhealthy subjects and individuals withvarious types of cancer (1). The levelsof nucleosides and modified nucleo-sides have therefore been proposed as cancer marker (2) and have alsobeen proposed as diagnostic markers of HIV (3). Further, the level of nucleo-sides and nucleotides can be used toestablish myocardial cellular energy sta-tus, and they are also useful in thestudy of energy metabolism in cardiactissue (4, 5).

Several methods have been developedfor the separation of nucleosides andnucleotides. The vast majority of theseseparations have been performed, by

using either reversed phase HPLC withgradient elution [2-4, 6] or using reversedphase ion-pair chromatography [1,5]. Inthis paper we explore the possibility ofusing capillary electrochromatography(CEC) for the separation in order toavoid the use of gradient elution or ion-pairing reagents.

Experimental

Apparatus: An Agilent Technologies CE capillary electrophoresis system (Waldbronn, Germany) fitted with a 100 µm I.D. column with a packed bedlength of 25 cm (CEC-Hypersil C18, 3 µmobtained from Agilent Technologies),was used for all the experimental work.The total column length was the lengthof the packed bed plus 8.5 cm of poly-imide coated fused silica tubing. Thecolumn was conditioned with everynew background electrolyte for at least2 hours before any samples were injected. Conditioning was done by applying 20 kV over the column. Both inlet and outlet were pressurizedat 10 bar during conditioning and analy-sis. No pair of background electrolytevials (inlet and outlet) was used formore than a total of 2 hours run-time.

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Results and discussion

All CEC experiments were performedusing a mixture of the six nucleosides;adenosine, cytidine, guanosine, inosine,thymidine and uridine (Figure 13.1).

A background electrolyte (5 mM aceticacid, 2 mM triethylamine pH 5.0)-acetonitrile (90:10 v/v) that provided a reasonable separation was found from initial experiments.

As seen from Figure 13.1 only cytidineand adenosine have pKa-values in therange from 2 - 8. Thus only these twonucleosides were affected by changes inpH in the range from 4 to 6. The fastestseparation of the six nucleosides was

obtained at pH 5. The separation wasalso affected when changing the con-centration of the buffer electrolytes.Increasing the concentration of aceticacid decreased the electro-osmotic flow

Figure 13.1: Structure and pKa-values (7,8) of the six nucleosides.

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(EOF) but did not affect the separation,whereas increasing the triethylamineconcentration decreased the EOF but also improved the separation. An increasing percentage of acetonitrilewas found to increase the EOF but alsoled to co-elution of the nucleosides. Increasing the temperature or voltageincreased the EOF but did not affect the separation. The final backgroundelectrolyte consisting of (5 mM aceticacid, 3 mM triethylamine pH 5.0)-acetonitrile (92:8 v/v) provided a goodseparation of the six nucleosides at20°C and 25 kV (Figure 13.2).

The method was validated with respectto limit of detection (1 µg/mL) andquantitation (approximately 5 µg/mL)and long and short-term repeatability.As seen from Table 13.1 the area repeatability was not satisfactory, butby using an internal standard therepeatability was considerably improved.This indicated that the injected volumewas not repeatable and that the use ofan internal standard was necessary,especially when quantitative analysiswas performed. This has also beendescribed in other CEC-separations(9,10) and the area repeatability needsto be improved before CEC can be usedas a standard analytical technique.

Figure 13.2 : Baseline separation of 6 nucleosides. EOF was marked by thiourea (not shown in this chromatogram) at 6.5 minutes. Peak identification: 1) cytidine, 2) uridine, 3) inosine, 4) guanosine, 5) thymidine, 6) adenosine. Conditions: (5 mM acetic acid, 3 mM triethylamine pH 5)-acetonitrile (92:8 v/v), 20 kV and 20°C.


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Conclusions

The work presented demonstrates that the 6 nucleosides can be baselineseparated in a relatively short time (< 13 minutes) by using CEC. The con-ventional HPLC methods require abouttwice that time to separate the nucleo-sides. The separation was performedwithout the use of a gradient or ion-pairing reagents. The method developedshows good repeatability of the retention time. However, the arearepeatability is insufficient without the use of an internal standard.

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Acknowledgements

The authors are grateful to AgilentTechnologies for providing the CEC-col-umn.

RSD%Short term Long term

tR RTR Area RArea tR RTR Area RArea

Cytidine 0.09 0.86 9.70 3.05 0.28 1.08 20.63 2.03Uridine 0.59 0.34 11.46 2.39 0.69 0.42 21.46 1.50Inosine 0.82 0.11 11.53 1.22 0.94 0.16 21.25 4.99Guanosine 0.84 0.08 11.41 1.69 0.96 0.15 22.42 2.24Thymidine 0.92 0.00 12.72 0.00 1.09 0.00 21.88 0.00Adenosine 0.29 0.66 10.53 2.42 0.38 0.77 20.69 6.50

Table 13.1 : Short term repeatability (n=10) and long term repeatability (day 1, n=10; day 4,n=10; day 7, n=10) of the retention time (tR), relative retention time (RtR), area and relativearea (Rarea) of the nucleosides. The relative retention time and relative area were calculatedwith respect to thymidine.


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References

1. Werner, A.,Chromatographia, 31, 401, 1991.

2. Liebich, H.M., Di Stefano, C., Wixforth, A., Schmid, H.R., J. Chromatogr., 763, 193, 1997.

3. Pane, F., Intrieri, M., Calcagno, G.,Izzo, E., Liberti, A., Salvatore, F.,Sacchetti, L.,J. Liq. Chromatogr., 16, 1229, 1993.

4. Fürst, W., Hallström, S.,J. Chromatogr., 578, 39, 1992.

5. Childs, K.F., Ning, X.H., Bolling, S.F., J. Chromatogr. B., 678, 181, 1996.

6. Simek, P., Jegorov, A., Dusbábek, F.,J. Chromatogr. A., 679, 195, 1994.

7. Kortüm, G., Vogel, W., Andrussow, K.,Dissociation constants of organic acidsin aqueous solution, International

union of pure and applied chemistry,Butterworths, London, 1961.

8. Luckenbach, R.,Beilsteins Handbuch der OrganischenChemie, Beilstein-Institut für

Literatur der Organischen Chemie,

Springer-Verlag, Heidelberg, 1981.

9. Lurie, I.S., Meyers, R.E., Conver, T.S.,Anal. Chem., 70, 3255, 1998.


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14 Analysis of Norgestimate and i ts Potential Degradation Products

Jian Wang, Daniel E Schaufelberger and Norberto A Guzman. R.W. JohnsonPharmaceutical Research Institute,Route 202, P.O. Box 300, Raritan, NJ 08869, U.S.A.

Introduction

Norgestimate is a synthetic steroid usedin a variety of treatments, e.g. in the

treatment of acne vulgaris. The parentdrug and its related impurities havequite different hydrophobicities, and thestructural isomers (syn- and anti-) aredifficult pairs to separate in HPLC(Figure 14.1). Here we describe thedevelopment of a CEC method for theanalysis of these compounds anddescribe some factors in the validationof the method.

Analysis of Norgestimate and its Potential Degradation Products (page 117-124)

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Figure 14.1: Chemical structures of norgestimate and degradation products.


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Materials and Methods

CEC experiments were performed onan Agilent CE system fitted with a diodearray detector. The column used was a fused silica capillary 25cm effectivelength (33.5cm total) x 100µm I.D.packed with 3 µm CEC-Hypersil C18particles. The temperature was main-tained at 25°C. For each new column or buffer used in the system, the col-umn was equilibrated by applying a stepwise voltage gradient (5 to 30kv)over 30 minutes with 8 bar pressureapplied to the inlet vial only. The systemwas subsequently run with a pressure of 8 bar, applied to both inlet and outletvials. Tris(hydroxymethyl)methylamine-HCl salt was purchased from Sigma (St Louis, MO), acetonitrile (ACN), tetra-hydrofuran (THF), methanol (MeOH)and 2-propanol (IPA), all HPLC-grade,were obtained from Fisher Scientific(Pittsburgh, PA). Norgestimate, norge-strel, norgestrel oxime and norgestrelacetate were obtained from an in housereference standard group at the R.W.Johnson Pharmaceutical ResearchInstitute. Deionized water (18.2 MW/cm)was purified by a milliQ plus water system (Bedford, MA.). A stock solutionof 25mM Tris-HCl (pH 8.0) was preparedin de-ionized water. From this stock

solution the various mobile phases wereprepared by mixing the appropriate vol-umes of stock solution with acetonitrileand THF organic solvents. Mobile phas-es were filtered through a 0.2 µm PTFEfilter (Fisher Scientific, Pittsburgh, PA)and degassed by ultrasonication undervacuum prior to use. Analytes were dis-solved in a methanol / 25 mM Tris-HCl(pH 8.0) mixture (80:20).

Method Development

Organic solvent composition effect

on CEC separation. In the CEC separation of these compounds, no single organic modifier was found togive satisfactory results. However, in isocratic HPLC, by using a binaryorganic modifier system, a separationeffect similar to that of gradient elutionmay be obtained. Therefore, in theabsence of a commercially availablegradient CEC system, binary combina-tions of acetonitrile with THF, IPA andMeOH were investigated. Figure 14.2shows the separations of norgestimateand its degradation impurities using 3binary systems that have the same iso-elutropic strength (1). The combinationof ACN with IPA or MeOH slowed theseparation considerably compared tothe ACN/THF system. The ACN/THF


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Figure 14.2: Solvent composition effect on CEC separation of norgestimate and its relateddegradation impurities. Mobile phase condition were: (A) ACN-THF-Tris-HCl (pH 8.0) –H2O (35:20:20:25) (B) ACN-IPA-Tris-HCl (pH 8.0) –H2O (35:21:20:24) (C) ACN-MeOH -Tris-HCl (pH 8.0) –H2O(35:29:20:16). Tris-HCl pH 8.0 was 25mM. Other conditions were, voltage 30 kV, temperature 25°C, injection 3s, 10 kV, detection UV at 225nm; pressure 8 bar both sides. Peaks: 1, norgestrel; 2, syn-norgestrel oxime; 3, anti- norgestrel oxime; 4, norgestrel acetate;5, syn- norgestimate; 6, anti-norgestimate.


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system reduced the separation time bya factor of two compared with theHPLC method used in-house.

Since the mobile phase is moved throughthe column by virtue of the EOF, andthis is related to the zeta potential, rela-tive and vacuum permitivities, electricfield strength and solvent viscosity, anychanges in organic solvent compositionwhich changes these parameters, willresult in a change in the EOF. Thereforethe mobile phase linear velocity willchange. Whereas, in HPLC, the iso-elutropic theory is used to exploit iso-elutropic mixtures to enhance selectivitywhile keeping the migration timesroughly the same, because the EOF andlinear flow velocity is also dependentupon the organic modifiers used in themobile phase, the theory cannot beapplied to CEC.

Effect of Organic solvent strength

on CEC separation. Since the THFconcentration was fixed only the ACNconcentration could be varied in orderto observe improvements in the sepa-ration. Although, some variation in the EOF was encountered when thechanges were very small compared with the earlier composition changes in the mobile phase. Increasing the ace-

tonitrile content leads to a decrease inretention factors similar to LC. In addi-tion, because the background electro-lyte in CEC and the mobile phase in LCare very similar in composition, thisfacilitates the method transfer betweenthese two techniques.

Effect of Buffer concentration on

CEC separation. The influence of thebuffer ionic strength on the separationwas investigated over the range 2.5 to10 mM by varying the Tris-HCl bufferconcentration in the mobile phases. Thethickness of the double layer increaseswith decreasing ionic strength andtherefore the zeta potential increases,as does the EOF. At buffer concentra-tions greater than 5 mM, the retentiontimes for all compounds increased withbuffer concentration, as would beexpected. However at 2.5 mM Tris-HCLthe results did not follow this trend.This is probably because the final pH of the mobile phase was unadjusted,and at low buffer concentration thebuffering capacity was not able to main-tain a pH close to pH 8.0.

It has been demonstrated in earlier CEC studies (2-5) that low buffer con-centration produces higher separationefficiency. However in order to obtain


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reproducible data and to prevent iondepletion a reasonable ionic strengthand buffer capacity should be used. Inour experiment, the use of 5.0 mM Tris-HCl at pH 8.0 produced reproducibleand highly efficient separations.

In this CEC experiment the plate numbers obtained were 118,000/m for syn-norgestimate, 115,00/m for anti-norgestimate, 107,000/m for norgestrel,108,000/m for syn-norgestrel oxime and94,700 for anti-norgestrel oxime. It isobvious that the highly efficient natureof CEC played a key role in this rapidseparation. Further more, as shown in the literature (2) better column effi-ciency was obtained at higher voltage;thus, shorter analysis time could beobtained if there were no 30-kV high-voltage limit for the CE instrument anda higher voltage could be applied. In

addition, although many publicationsshowed excellent work on CEC withouta pressurized system, our experimentsfound a pressurized system to be essen-tial for developing a robust and repro-ducible CEC method.

Repeatability, linearity and detection

limit. With a standard solution, contai-ning 51 µg/ml of syn-norgestimate, 7 µg/ml of anti-norgestimate, 186 µg/mlof norgestrel, 46 µg/ml of syn-norgestreloxime, 69 µg/ml of anti-norgestrel oximeand 128 µg/ml of norgestrel acetate, sixreplicates were performed and theresults for retention factor and peakareas shown in table 14.1. In all casesthe RSDs were better than 2% whichwas comparable with the HPLC methodand much better than those obtainedfrom CZE or MEKC.

syn- antiNorgestrel Norgestrel Norgestrel syn- anti

Norgestrel Oxime Oxime Acetate Norgestimate Norgestimate

Mean k' 1.22 1.33 1.45RSD (%) 1.30 1.40 1.30Mean PeakArea (mAU) 0.25 0.06 0.13RSD (%) 1.70 1.90 1.90Table 14.1: Repeatability Study of CEC Separation of Norgestimate and Its DegradationImpurities


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The linearity was determined for allcompounds and found to be linear over2 orders of magnitude in each case withr2 >0.995. The detection limit test was

performed by sequentially diluting astandard mixture until the signal-to-noiseratio approached 3:1 (Figure 14.3). A lower detection limit is anticipated

Figure 14.3 : Detection limits for norgestimate and its related degradation impurities. 1, norgestrel (9.28 µg/ml); 2, syn-norgestrel oxime norgestrel (3.07 µg/ml); 3, anti-norgestrel oxime norgestrel (4.61 µg/ml); 4, norgestrel acetate norgestrel (6.4 µg/ml); 5, syn-norgestimate norgestrel (2.56 µg/ml); 6, anti-norgestimate norgestrel (3.84 µg/ml).


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with optimization of the electrokineticinjection. However, as shown in Figure14.4, the CEC method can separate and

quantitate 0.1% degradation impuritieswhen spiked in the norgestimate drugsubstance.

Figure 14.4: CEC profile of norgestimate drug substance spiked with 0.1% pf degradationimpurties. CEC conditions as described infigure 2 with ACN-THF. Peaks: 1, norgestrel; 2, syn-norgestrel oxime; 3, anti-norgestrel oxime; 4, norgestrel acetate; 5, syn-norgestimate; 6, anti-norgestimate.


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Conclusions

A rapid and reliable CEC method was developed for the separation ofnorgestimate and its related degradationproducts. The total analysis time wasreduced by more than 50% when com-pared with our HPLC method. Using abinary solvent composition for methodoptimization compensated the lack of practical gradient elution in CEC.The method clearly demonstrates theseparation and quantitation of 0.1%degradation impurities in a simple man-ner. The results of repeatability, lineari-ty and detection limit determinationsalso demonstrated that by using com-mercially available instrumentation andpacked capillary columns, the CECtechnique has the potential to be usedon a routine basis in pharmaceuticalanalysis.

References

1. Schoenmakers, P.J., Billiet, H.A.H. and de Galan, L., J. Chromatogr., 205, 13-30, 1981.

2. Yan, C., Schaufelberger, D. and Erni, F., J. Chromatogr., 670, 15-23, 1994.

3. Smith, N.W. and Evans, M.B. Chromatographia, 38, 649-657, 1994.

4. Smith, N.W. and Evans, M.B. Chromatographia, 41, 197-203, 1995.

5. Dittman, M.M., Keinand, K., Bek, F. and Rozing, G.P., LC-GC, 13, 800-814, 1995.


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15 Analysis of Phenols inMainstream and Sidestream Tobacco Smoke

M.Saeed1* / M.Depala1 / D.H.Craston1/I.G.M.Anderson2

1 LGC (Teddington Ltd), Queens Road,Teddington, Middlesex TW11 OLY, UK2 British American Tobacco, RegentsPark Road, Millbrook, Southampton,S015 8TL, UK

Introduction

Phenolic compounds, namely hydroxy-quinone, resorcinol, catechol, phenol, and o-, m-, and p-cresols, have beenidentified in both the inhaled (main-stream) and the emitted (sidestream)components of tobacco smoke. In sepa-rate studies these species has beenquantified by trapping the particulateson glass fibre filters and then analysingliquid extracts from the filters using gaschromatography (GC) [1-5] or high per-formance liquid chromatography(HPLC) [6-8].

Since phenols are polar and of highboiling point, GC methods use a derivatisation step using agents such asbis-N,O-trimethylsilyl-trifluroacetamide(BSTFA) [2,3]. This procedure is timeconsuming and if done poorly may lead

to incomplete conversion to derivatisedform and potential low results. However,with or with out derivatisation, GC ana-lysis offers high resolution, sensitivityand specificity when combined withselective detectors (GC-MS): henceanalysis by GC is usually favoured overHPLC, where the benefits of faster sam-ple preparation are offset by the loss inseparation resolution and measurementsensitivity. Capillary electrophoresis, an alternative and higher resolutiontechnique for separating components in liquid samples, has been applied to the measurement of phenols [9];however since the compounds are neu-tral, separation requires the addition ofmicellar reagents into the electro-phoretic buffer (micellar electrokineticcapillary chromatography - MECC) (9).Results published to date suggest thatMECC methods are unlikely to be suffi-ciently reproducible or sensitive for analysing phenols in tobacco smoke.

In this paper a CEC method is describedthat has been developed to providereproducible and quantitative analysisof individual phenols in tobacco smokewith minimal sample preparation.

Analysis of Phenols in Mainstream and Sidestream Tobacco Smoke (page 125-134)

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Experimental

Materials. Samples of tobacco smokecondensate, from the mainstream andsidestream smoke of cigarettes wereprovided by British American Tobaccoand the LGC Tobacco analysis group.Disodium hydrogen orthophosphate(Analytical reagent 99%) was obtainedfrom Merck (Poole, UK), and acetonitrile(Far UV grade S) was purchased fromRathburn Chemicals Ltd, Walkerburn,Scotland. All buffers were preparedusing ultra pure water (resistivitygreater than 18 MWcm). All buffers,standards and samples were filteredusing Pro-Mem 0.45 _m PTFE 25 mmsyringe filters (Radleys, hydrophobicsolvent resistant), and stored in a refrig-erator at 4 °C prior to use.

Preparation of CEC mobile phase.

Buffers were prepared from a 10 mMsolution of phosphate salt by adjustmentusing 10% (concentrated) phosphoricacid. The mobile phases used consistedof a mixture of buffer’s and acetonitrilein defined ratios, and were filtered priorto use.

Preparation of samples and stan-

dards. Tobacco smoke condensate sam-ples were supplied on filter pads.

Phenols were extracted from these by immersion in acetonitrile (20 ml)with mechanical stirring for a period of 25 mins. All sample extracts werestored in a refrigerator at 4°C and fil-tered prior to injection.

Standards were prepared from a stocksolution (100 mg/ml-1 of each mono-and dihydroxy phenol) in 100% acetoni-trile using the appropriate dilutions.The prepared standards were stored ina refrigerator at 4 °C prior to use.

Instrumentation set-up and meth-

ods. Samples, standards and CEC mobile phases were sonicatedusing an ultrasonic bath for 5 min atroom temperature to remove any airbubbles prior to use. Capillary electro-chromatography (CEC) was performedon a Agilent Technologies CE unit(Waldbronn, Germany). Separationswere carried out using a commerciallyavailable C18 capillary column (CEC-Hypersil C18, 3 µm, 33.5 cm (effectivelength 25 cm) x 50 µm I.D.), an appliedvoltage of 25 kV (758 v.cm-1). Both ends of the capillary were pressurisedat 8 bar to prevent air bubble formation.Samples were introduced into the anod-ic end (inlet) of the capillary by the application of an electrokinetic


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injection (5kV@4s), which was followedby a similar injection of mobile phasebefore ramping the voltage to therequired operating level over 0.2 min.The column temperature was kept con-stant under airflow at 25 °C. Data wascollected directly from the diode arraydetector using a measurement wave-length of 200 nm and a reference wave-length of 350 nm. The measurement ratewas 20 Hz.

Prior to each days work the installedC18 column was equilibrated by flush-ing with electrolyte mobile phase at 9bar from the inlet end for 15 min, andthen applying 25 kV across the columnlength for 30 min under a; capillary inletand outlet pressures of 8 bar. The CEC capillary column wasthen tested by injecting a 50 µg/ml phe-nol standard, and separating the compo-nents using the method describedabove.


Optimisation of CEC electrolyte

mobile phase. A preliminary investiga-

tion was performed varying the pH andorganic component of the electrolytemobile phase to optimise the separation.Initially a pH 9.0 buffer and 70% aceto-nitrile was used to resolve a 50 µg/mlmono- and dihydroxyphenol standard.While all the peaks eluted within 3 minutes the peaks were not fullyresolved and tailing was observed for a number of compounds. This tailingwas attributed to partial ionisation ofthe phenols which have pKa`s in therange 9.0 - 10.1 [10]. This problem ofpartial ionisation was overcome by per-forming the separation [11] using a mix-ture with 70% acteonitrile at pH 2.5where the individual phenols are neutraland resolution of four phenols wasachieved in 5 min at a voltage of 30kV.

Further work was performed to deter-mine the correct voltage and pH forbaseline separation of phenols using amixture containing 60% acteonitrile.From previous work this was found toprovide the best separation at low pH[12]. A mixture containing an organiccomponent of 60% acetonitrile and avoltage run of 25 kV was found to pro-


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vide the optimum separation for all thephenol peaks (Figure 15.1).

Reproducibility, linearity and limit

of detection (l.o.d) for mono- and

dihydroxy phenols. A calibration plotwas set-up for mono- and dihydroxyphenols covering the range 1 µg/ml - 50µg/ml. The linearity and variance wasdetermined from six injections at sixdifferent concentrations of phenols. Theuse of electrokinetic injection was foundto be more reproducible when a sample

injection was followed by a similarinjection of electrolyte mobile phase.

Variance of peak height and migrationtime is given in Table 15.1. A coefficientof correlation of 0.999 for linear regres-sion was obtained for all compounds up to 50 µg/ml (Table 15.2). The limit of detection, evaluated from six inde-pendent blanks which were spiked atdifferent levels until the peak heightwas close to three times the baselinenoise, was determined to be 0.5 µg/ml.

Figure 15.1: Typical electrochromatogram for the separation of a 50 µg/ml phenol standardusing an electrolyte mobile phase; acetonitrile-10mM phosphate pH2.5 (60:40 %v/v) at a 25kV (750v.cm-1) voltage, (1) hydroxy quinone (2) resorcinol (3) catechol (4) phenol (5) p-cresol, m-cresol (7) o-cresol


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Peak Area 50 25 12.5 6.25 3.0 1.0

1 1.83 3.04 1.60 1.16 3.00 2.942 1.65 2.23 2.64 2.85 1.11 5.193 1.22 1.26 1.63 4.92 4.13 4.574 1.26 0.58 2.62 4.51 2.11 3.705 *0.46 *2.11 *0.45 *3.22 *0.25 *1.946

Peak Height 50 25 12.5 6.25 3.0 1.0

1 0.9 2.34 6.98 0.97 2.22 7.522 1.11 1.99 6.03 2.07 0.43 3.103 0.76 7.99 2.32 3.00 2.50 4.324 0.51 0.48 2.61 2.70 2.28 4.355 0.62 1.22 2.07 3.28 0.81 4.356 0.76 0.12 2.06 2.60 1.76 4.56

Peak Migration time50 25 12.5 6.25 3.0 1.0

1 0.42 0.34 0.15 0.06 0.12 0.202 0.42 0.34 0.17 0.03 0.06 0.093 0.40 0.36 0.09 0.04 0.18 0.184 0.40 0.36 0.09 0.04 0.18 0.185 0.42 0.33 0.07 0.02 0.05 0.106 0.40 0.33 0.04 0.05 0.03 0.03

1. hydroxyquinone, 2. resorcinol, 3. catechol, 4. phenol, 5. m+p-cresol, 6. 0-cresol, n = 6 for each std *total cresols (m+p+o-cresols).

Table 15.1: Variance (%CV) peak area, peak height and migration time for a calibration plot inthe range 50 µg.ml-1 - 1 µg.ml-1 for day 1


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Sample analysis. Once optimum CECconditions had been obtained a total of44 mainstream and sidestream tobaccocondensate samples were analysed.Each tobacco condensate sample wasprepared from 5 smoked cigarettesextracted from a single filter pad with20 ml of 100% acetonitrile. Analysis ofthe samples was performed over threedays and consisted of three calibrationruns, 6 blank samples (a blank extrac-tion obtained from a non-smoked

single filter pad) and 6 control samplesof known phenol concentration todetermine consistency in the analysis.

A consistent profile for both sidestreamsmoke and mainstream smoke conden-sate samples (Figure 15.2 and 15.3) wasobtained. Differing levels of phenolswere observed between mainstreamand sidestream tobacco smoke conden-sate samples (Table 15.2, Table 15.3).

Figure 15.2: Typical electrochromatogram for phenols in mainstream tobacco smoke


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Figure 15.3: Typical electrochromatogram for phenols in sidestream tobacco smoke

Hydroxy- Resorcinol Catechol Phenol M,p-Cresol o-Cresolquinone

Mean 3.30 0.20* 3.04 1.46 0.49* 0.28*

Standard devation 0.26 0.06 0.29 0.13 0.05 0.04* results below the level of the lowest std (0.59 µg/mg TPM), the above results rep-resent a mean for five cigarettes.

Table 15.2: Determination of individual phenols in mainstream smoke (µg/mg total particulate matter (TPM))


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Long term measurement stability.

The CEC column was flushed with elec-trolyte mobile phase of a slightly higherorganic content (acetonitrile/10 mMphosphate pH 2.5, 70:30 v/v) at the endof a day's run to remove any extraneousmaterial at the column inlet. During theanalysis it was found that tobaccosmoke extracts gave poor quantitativeresults for extended CEC runs. Sincethe concentration of phosphate bufferused was low (10 mM) this can lead to buffer depletion [14] by electrolysisresulting in a pH gradient which affectsmigration time, peak efficiency andselectivity. Migration time and peak areawere much improved when the electro-

lyte mobile phase was replenished everysix injections followed by an electro-phoretic run of 5 min.

During the initial experimental work,disintegration of the frit was observedwhen a high concentration of acetoni-trile was used in either the samplematrix or electrolyte mobile phase. Toprevent disintegration of the inlet fritthe column ends were immersed in avial containing 100% ultra pure water atthe end of a day's run. The softwarepackage on the Agilent CE systemallows all of these actions to be auto-mated. Using the above precautionsreproducible quantitation of individual

Hydroxy- Resorcinol Catechol Phenol m+p-Cresol o-Cresolquinone

Mean 2.13 0.05* 1.59 5.36 0.87 0.67Standard devation 0.23 0.01 0.16 0.56 0.09 0.06* results below the level of the lowest std (0.59 µg/mg TPM), the above results rep-resent a mean for five cigarettes.

Table 15.3: Determination of individual phenols in sidestream smoke (µg.mg total particulate matter (TPM))


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phenols (Table 15.4) in tobacco smokewas possible over a three day period.

Conclusions

The above work is unique as it repre-sents the first known analysis of indivi-dual phenols in tobacco mainstreamand sidestream smoke from the samecigarette by CEC.

The work performed also shows thatreproducible quantitation can beachieved by CEC over a long period of time employing real samples withminimal sample preparation, providedprecautions are taken to prevent fritand column deterioration.

Despite the short detection path lengthused a limit of around 0.5 µg.ml-1 wasobtained.

The results of this work show that CECis an effective separation method for

the determination of phenols in complexmatrices. This CEC technique providesa highly efficient separation with goodprecision, short analysis, minimal samplepreparation, and reduced operating costscompared to the current chromatogra-phic methods used for tobacco smokephenol analysis.

Acknowledgements

Elements of work carried out in thispaper was supported under contractwith Department of Trade and Industryand British American Tobacco as partof the Analytical Innovation Program.The authors would also like to thankH.Lomax of Hypersil for the kind dona-tion of the CEC columns.

Hydroxy- Resorcinol Catechol Phenol m+p-Cresol o-Cresolquinone

Main-stream 1.18 0.6 1.37 0.88 0.32 0.28Side- stream 4.16 0.24 2.14 9.00 1.34 0.92* results represent a SD of for five cigarettes.

Table 15.4: Standard deviation of individual phenols (mg.cig)


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References

1 Deutsch, L.J., Gladden, J.D. and Williams, D.R., CORESTA Meeting 6-11 September1987, Bournemouth. UK.

2 Nanni, E.J., Lovette, M., Hicks, R.D.,Powler, K.W. and Borgerding, M.F., J. Chromatogr. A., 505, 365, 1990.

3 White, E.L., Uhrig, M.S., Johnson, T.J.,Gordon, B.M., Hicks, R.D. and Borgerding, M.F., J. Chrom.Sci., 28, 393, 1990.

4 Z. Mingyue and Z. Fuchen, CORESTA CONGRESS : Smoke StudyGroup, 1992, Jerez De La Frontera.

5 Clark, J.T. and Bunch, J.E., J. Chrom.Sci., 34, 272, 1990.

6 Jeanty, G., Masse, J.,Bercot, P. and Coq, F.,Beitr. Takakforsch., 12, 245, 1984.

7 Risner, C.H. and Cash, S.L., J. Chrom. Sci., 28, 239, 1990.

8 Andres, M.D., Canas, B., Izquierdo, R.C.,Alarcon, P. and Polo, L.,J.Chromatogr. A., 507, 399, 1990.

9 Crego, A.L. and Marina, M.L., J.Liq. Chrom & Rel Technol., 20, 1, 1997.

10 Weast, R.C., CRC Handbook of Chemistry and Physics, Publishers CRC Press, Boca Raton Florida, D 102, 1988.

11 Gillott, N.C., Euerby, M.R., Johnson, C.M., Barret, D.A. and Shaw, P.N., Anal Comm., 35, 217, 1998.

12 Reilly, J. and Saeed, M.,J.Chromatogr. A., 829, 175

13 K. Cooksy, J and W Scientific Capillary

Electrophoresis Appl. 9, 4, 1993.

14 Kelly, M.A., Altria, K.D. and Clark, B.J., J. Chromatogr. A., 768, 73, 1997.


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16 135/143Practical Aspects of Coupling Capillary Electrochromatography with Mass Spectrometry (page 135-139)

16 Practical Aspects of Coupling Capillary Electrochromatography with Mass Spectrometry

R J. Boughtflower and C J. Paterson.Separation Science Group,GlaxoWellcome, Gunnels Wood Road,Stevenage, Herts, SG1 2NY, UK.

Introduction

In CEC experiments, the layout of thepacked and open sections of a one-piececolumn normally dictate that detectionis performed through the open section,immediately after the second retainingfrit, and also that the electric field isapplied across the entire length of thecolumn. There is growing evidence that flow profile disruption (1) occursat the interface of the packed and opentube, causing significant extra peakdispersion. This restricts opportunitiesfor connecting one-piece CEC-UVcolumns to MS simply by extending the unpacked length of capillary andconnecting to the MS (2).

The authors have measured this disper-sion (3) by varying distances from thefrit and to the detection point and com-

paring the peak volume variances. Thiswork suggests that it is not reasonablypossible to use one-piece columns withsignificant open tube lengths of thesame diameter without incurring intole-rable losses to peak efficiency. If smallerdiameter connection capillaries must beused, it is clear that this will involve aphysical join between the packed CECcolumn and the connecting tubing tothe MS. Preferably, UV detection wouldbe achieved at the joining interface.Such a joint also provides a convenientpoint for a suitable electrical groundconnection. This will create a pressure-derived flow in the second connecting tube,which should allow calculation of theexpected dispersion in this tube. If normal dispersion processes withinthe packed section only are assigned avalue of 1, then the ratio of total columnto packed tube dispersion can be calcu-lated for various connecting tubedimensions (table 16.1). This suggeststhat the dispersive effect of addingextra lengths of open tube is critically

Diameter (µm) 50 40 30 25 20 15 12 10Dispersion factor 3.71 2.11 1.35 1.17 1.07 1.02 1.00 1.00

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Table 16.1: The calculated effect of open tube diameter on the dispersion factor for equallengths of packed and open tubes.


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dependent upon its diameter (withfourth power) and only weakly depen-dent upon its length.

These calculations suggest that 25 µmconnection tubing would be an accep-table diameter, both from a theoreticaland also a practical perspective. Smallertubing is more likely to block. The fol-lowing work utilises connection of the CEC column to 25-µm i.d connec-tion tubing to interface CEC-UV-MS.

Experimental

Instrumentation. All runs were performed on a Agilent CE instrument(Agilent Technologies, Waldbronn,

Germany) with 12 bar of pressureapplied to both buffer vials. This wascoupled to a Agilent MSD (AgilentTechnologies, Waldbronn, Germany)benchtop single quadrupole mass spectrometer fitted with a CapillaryElectrophoresis interface.

Experimental design. Two columnarrangements were used to couple a 100 µm i.d CEC column to the massspectrometer (Figure 1). In both casesthe packed column was joined to a 75 cm length of 25 µm i.d open tube.The CEC column was cut at the retain-ing frit and this end was polished usinga simple home-made polishing unit.This column end connected to

To MS

UV Detection

25um i.d open tube

Join in teflon tubing

Join in teflon tubing

A

BTo MS

UV Detection

100um i.d CEC column Graphitepaste

Brass plateto makeelectricalearth

Retaining frit

Figure 16.1: Schematic diagram of column arrangements.

Practical Aspects of Coupling Capillary Electrochromatography with Mass Spectrometry (page 135-139)

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the open tube section, which was alsopolished at the connection end. The connections were made by a smallpiece of PTFE tubing of slightly smalleri.d than the capillary o.d This facilitateda ‘tight’ fit but care was taken to ensureno swarf from inside the PTFE tubingblocked the connection of the capillaryends.

The electrical connection was made at the connection point. The open tube part of the capillary was paintedwith a graphite paste, which coveredthe end face of the capillary and a suit-able length of the outside tube, to a point where a convenient electricalearth could be connected via a brassconnector.

Results and discussion

An eight component pharmaceuticalmix containing thiourea, caffeine, testosterone, phenytoin, prednisolone,amoxicillin, methyl-prednisolone and cefatrizine was run on columnarrangement A. Figure 16.2 shows theUV chromatogram. All of the compo-nents were clearly detected in the massspectrometer. However, the UV detec-tion is limited by the small pathlengthavailable in the 25 µm i.d open tube.The later eluting peaks are not evenseen in the UV chromatogram. Clearlymore sensitive UV detection is needed,than is provided by in capillary detec-tion using 25 µm connection capillary.

min2 4 6 8 10 12 14

mAU

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Figure 16.2 : UV Chromatogram of eight component pharmaceutical mix using column arrange-ment A. 1.Thiourea, 2.Caffeine, 3.Testosterone, 4.Phenytoin, 5.Prednisolone, 6.Amoxicillin and7.Methyl-prednisolone, 8.Cefatrizine


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Work has been performed using a higher-sensitivity cell connected between thepacked CEC column and connectingtube. Much better UV sensitivities areobtained, however practical problemsin optimising capillary/cell connections

cause extra problems with peak disper-sion.

Figure 16.3 shows a UV chromatogramand the extracted ion chromatograms of a 3 component mix containing pred-

min1 2 3 4 5 6

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min1 2 3 4 5 6

min1 2 3 4 5 6

50000100000150000200000250000

1000020000300004000050000

10000200003000040000

Caffeine

Prednisolone

Dexamethasone

Figure 16.3: UV and extracted ion chromatograms of a 3 component test mix using a columnarrangement B. Mobile Phase: 60%MeCN/20mM NH4OAc (pH=4). Voltage across CEC column:25kV. Injection: 10kV for 5s.


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nisolone (MH+361), dexamethasone(MH+393) and caffeine (MH+195) usingcolumn arrangement B. There is a 20-30% loss in efficiency in the MS sig-nals. This is due to some losses in thesmall section of 100 µm i.d open tube,just after the frit, but prior to the con-nection, and the predictable lossesassociated with transport through the25µm section of connecting tube.

Conclusions

This work demonstrates that using con-ventional CEC arrangements of one- piece columns with UV post-fritdetection, it is imperative to detect asclose as practically possible to the frititself. Even adopting this strategy, thereis likely to be significant dispersionintroduced. If connection to a massspectrometer is desirable it is stronglyadvised that the diameter of the connec-ting tubing is at least a quarter of theseparation column diameter, smaller ifpractically possible. It is possible tocombine both these strategies to couplethe CEC separation to UV detection in a higher sensitivity cell and subsequentdetection in the mass spectrometer.Current UV cell designs would benefitfrom changes to enable more effectiveconnections for flat-ended capillariesand the possibility to make the electrical

earth at the outlet connection. Theseminor modifications should allow CEC-UV-MS to become a routinely used technique.

References

1. Rathore, A.S., Horvath, Cs., Anal. Chem., 70, 3069-77, 1998.

2. Lane, S.J., Boughtflower, R.J.,Paterson, C.J., Underwood, T.,Rapid Communications in Mass

Spec, 9, 1995.

3. Boughtflower, R.J., Knox, J.H.,Paterson, C.J., paper in preparation


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140/143Summary, Conclusions and Outlook (page 140-143)

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Gordon A. Ross and Gerard P. Rozing,Agilent Technologies GmbH, 76337 Waldbronn, Germany

This primer is intended to provide start-up help to novice users of the new technique of capillary electrochro-matography. Basic theory and practicalaspects of CEC are discussed in chapters 1-6. In the following chapters,applications from practitioners in thefield, from both academic and industrialgroups, have been collected to illustratethe potential of the technique in practice.

CEC is new in the sense that almost allthe research work has been publishedduring the last decade; most of it in thelast five years (1994-1998). Therefore anew entrant to the field will find manyareas of possible application where noprior knowledge is available. Althoughthis may hamper progress initially, per-severance will be rewarded in signi-ficantly improved solutions to practicalproblems. This also means that CECprovides an exciting field of study withopportunities to break new grounds inseparation science.

The editors believe that this primerillustrates a number of advantages ofCEC over conventional µHPLC and CE.These can be characterized as follows:

a. Significantly improved separationefficiency compared to µHPLC, whichis preserved in practical applications.

b. CEC is optimally suited for the simul-taneous separation of neutral, weaklyacidic and basic solutes. No solubilityrestriction like in CE due to the highproportion of organic solvents usedin the separation. Feasibility for sepa-ration of strong bases, neutrals andacids in one analysis is demonstrated.

c. A wide range of selectivity is availablethrough the use of HPLC type station-ary phases.

d. Similar retention mechanism for neutrals as in reversed phase HPLC.Therefore providing a simplermethod development due to theinherent higher efficiency and theexisting knowledge pool of HPLCseparations. In many cases, an HPLC gradient separation can bedone by isocratic CEC.

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e. On-line sample preconcentration isfeasible and enhances detection.

f. CEC experiments can be performedusing standard CE equipment.

g. Potential for coupling to MS with nolimitations due to buffer compositionand/or solubility of the solutes as inMECC.

h. Real micro analytical technique andtherefore low chemical waste

This primer also reveals some techno-logical aspects and inherent propertiesof CEC, which require investigation anddemand further development.

The robustness of columns must be improved (capillaries are expensive)Recently published work by, Tanaka(1,2), Hjerten (3), Novotny (4), Horvath(5) and Svec (6,7) and our group (8)have shown feasibility of so-calledmonolithic type of columns which do not require terminating frits. Usingan external flow cell e.g. high sensitivitycell means that the capillary is notweakened by the presence of a detec-tion window.

There are a limited number of availablestationary phases specifically designed

for CEC. Development of such stationaryphases, with properties that enhancethe electro-osmotic flow and improvemass transfer kinetics by providing EOFthrough the pores, is considered to bean area having a high future potential.

There is no instrumental control of theEOF. The actual flow rate generated isdependent upon parameters such as sol-vent composition, temperature, voltageand the stationary phase selected. Workby Behnke (9), Eimer (10) and Rozing(8) has shown though that a combina-tion of hydraulic flow and applied elec-tric field will preserve the high efficien-cy and can enhance selectivity ofcharged compounds by adding elec-trophoretic mobility as a parameter tooptimize the separation.

There is no dedicated, commercialequipment that supports specificrequirements of CEC (high hydraulicpressure and/or gradient elution).Maximum voltage 30 kV. Initial feasibilityof coupling CEC with MS has beendemonstrated but no integrated com-mercial solution yet.

These statements are largely in linewith results from interviews with variousCEC experts in a recent issue of LC.GCmagazine (11).

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Therefore one can envision intensivedevelopment work on CEC, in academiaand industry, specifically focusing onaddressing the current shortcomingsformulated above. This work with nodoubt will be fascinating and rewarding.

The main issue though is going to bewhether the user interest will support a large enough business volume thatmakes investments in developments by the instrument manufacturers suffi-ciently attractive. Therefore CEC as an instrumental technique currentlymay be in the "catch 22" situation whereno user interest means no instrumentaldevelopment which itself restricts widerinvestigation, application and furtherinterest.

Significant progress in the understandingof the technique and further demonstra-tion of its abilities to solve practicalproblems better than existing CE orHPLC solutions will be required inorder to generate a breakthrough.

Acknowledgement. The editors wishto thank all contributors for makingtheir work available for this primer.Also our colleagues Gabi Lichtenbergerand Norbert Schenk in the MARCOMdepartment of the Agilent TechnologiesWaldbronn Analytical Division, for theircreativity, diligence and persistence tohelp finish this primer and our localmanagement for making the resourcesavailable to prepare this primer.

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List of references

1. Minakuchi, H., Nakanishi, K.,Soga, N., Ishizuka, N., Tanaka, N.,J. Chromatogr. A., 797(1-2), 121-131, 1998.

2. Ishizuka, N., Minakuchi, H.,Nakanishi, K., Soga, N. Tanaka, N.,J. Chromatogr. A., 797(1-2), 133-137, 1998.

3. Ericson, C., Liao, J.L., Nakazato, K., Hjerten, S.,J. Chromatogr. A., 767(1-2), 33-41, 1997.

4. Palm, A., Novotny, M.V., Anal. Chem., 69(22), 4499-4507, 1997.

5. Asiaie, R., Huang, X.,Farnan, D., Horvath, C.,J. Chromatogr. A., 806(2), 251-263, 1998.

6. Peters, E.C., Petro, M.,Svec, F., Frechet, J.M.J.,Anal. Chem., 70(11), 2288-2295, 1998.

7. Peters, E.C., Petro, M.,Svec, F., Frechet, J.M.J., Anal. Chem., 70(11), 2296-2302, 1998.

8. Dittmann, M.M., Rozing, G.P., Ross, G.A., Adam, Th. and Unger, K.K., J. Cap. Electrophoresis, 4(5), 201, 1998.

9. Behnke, B., Bayer, E.,J. Chromatogr. A., 680(1), 93 -98Fafa, 1994.

10. Eimer, T., Unger, K.K.,van der Greef, J.,Trends-Anal-Chem., 15(9), 463-468, 1996.

11. Majors, R.,LC-GC Magazine, 16(2), 96 -110, 1998.

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capilary electrophoresis manual

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