Simple Gradient System For Capillary Electrochromatography

Juan Carlos Medina,1 M. Carmen Alonso,2 Damia Barcelo,2 Milton L. Lee3

1 Dow Chemical Co., 2301 N. Brazosport Bl., B-1218, Freeport, Texas, USA2 Department of En�ironmental Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain

3 Department of Chemistry and Biochemistry, Brigham Young Uni�ersity, Pro�o, Utah 84602-5700, USA

Received 6 April 2001; revised 10 July 2001; accepted 19 September 2001

Abstract: A new gradient system for CEC was designed and adapted to ahome-built CEC system. Chromatographic separations are initiated with a prede-termined mobile phase composition and, after a desired time period, a syringepump delivers pure organic solvent at a preset flow rate into a 2 mL vial used asthe inlet buffer reservoir. Mixing is achieved by the use of a microstir bar in thebuffer reservoir, which in turn is driven by an air-actuated stir plate. In this way, aconvex-shaped gradient is generated, and the mobile phase is introduced into theseparation column by electro-osmotic flow. Use of this system significantly reducedanalysis time while still retaining acceptable reproducibility. Nonaqueous CEC was

Ž .used for isocratic and gradient separation � 3.5% RSD in retention times .Demonstrated applications include the analysis of triglycerides in different veg-etable oil samples. This system is expected to be well suited for generating othertypes of gradients, such as pH and ionic strength gradients, in electroseparationtechniques. � 2001 John Wiley & Sons, Inc. J Micro Sep 13: 351�360, 2001

Key words: CEC; mobile phase; sol�ent composition gradient; triglycerides

INTRODUCTIONThe potential of capillary electrochromatogra-Ž .phy CEC to generate separation efficiencies signif-

icantly higher than those obtained in liquid chro-Ž .matography LC is the basis for interest in CEC.

However, because CEC is still in its infancy, there isa significant difference between the level of develop-ment of CEC instrumentation and that achieved bymature techniques such as LC. The capability togenerate a mobile phase gradient is an example ofthis difference. Solvent gradient elution is highlydesirable to shorten analysis time, or to changeselectivity during an analysis and in some instances,it is the only way to elute analytes. Any modern LCinstrument has the ability to perform gradient elu-tion, mixing up to four solvents. In the case of CEC,instrumentation and methods to generate gradients

� �have been reported only recently 1�14 .Several approaches have been used to generate

a gradient in CEC. These fall into one of twocategories, namely, electro-osmotically and pressure

Correspondence to: Milton L. Lee; e-mail: milton lee@byu.edu.

generated gradients. In electro-osmotically driven� �methods 1 , the inlet end of the CEC column is

attached to a tee. Two other capillaries are alsoattached to the tee, and each is connected to vialscontaining solvents that will be mixed to generatethe mobile phase. Two separate high-voltage powersupplies are needed to electro-omostically drive thedifferent liquids through each of the two capillaries.The flows from these capillaries are mixed in thetee, which serves as the source of mobile phase forthe analytical column. The proportion of each com-ponent in the mobile phase is varied by applyingdifferent voltages to the capillaries.

While no mobile phase is wasted in this method,some problems seem obvious. For example, duringinjection the inlet end of the CEC column is discon-nected from the tee and then reconnected to startthe analysis. Bubbles can be easily introduced intothe system, eventually leading to current breakdown.Also, during injection, the outlet ends of the capil-laries used to generate the gradient can dry, therebyeliminating EOF. Perhaps the most serious problemis that the exact composition of the mobile phase isunknown. This is because EOF depends not only onthe applied voltage, but also on other factors such as

Ž . Ž .J. Microcolumn Separations, 13 8 351�360 2001� 2001 John Wiley & Sons, Inc.

351

Medina et al.352

mobile phase relative permittivity or dielectric con-stant, zeta potential, and viscosity. While viscosity isrelatively easy to measure, determination of theother parameters can be quite challenging. The in-ability to reproduce results and transfer methods is aconsequence of the lack of knowledge regardingmobile phase composition in electroosmoticallydriven gradient methods.

In pressure-driven methods, gradient pumpingsystems already available for LC are used to mixdifferent buffers and then deliver the mixture to the

� �inlet of the CEC column 4�10 . Although the use ofLC gradient pumps allows for accurate control ofdifferent mobile phase compositions, the gradientpumps must be operated at relatively high flow ratesto minimize the delay time for the compositionchange to reach the column inlet. Therefore, mostof the mobile phase provided by the LC pump iswasted by splitting. Also, delivery of the mobilephase to the inlet end of the CEC column mayinduce pressure-driven flow, which decreases effi-ciency compared to that generated by a pure plug-

� �like EOF profile 14 . Finally, voltage isolation toavoid discharge to the metallic components of thepump is required.

Step gradient programming is an alternative ap-� �proach that has not attracted much attention 15�17 .

In this technique, a vial is filled with the initialbuffer and the run is started. At a certain timeduring the analysis, the voltage is suspended, andthe inlet and outlet vials are replaced by other vialswith new mobile phase compositions. A potential isthen reapplied to continue the analysis. This proce-dure can be repeated as many times as desired.While the technique can be carried out with com-mercially available CE instruments, problems associ-ated with analyte diffusion during changes of solventare evident. In addition, changes in selectivity neededto resolve compounds wide-ranging in polarities areachieved by using a range of compositions, instead

� �of just two or three different compositions 18 .� �Tan and Remcho 19 briefly introduced voltage

programming in open tubular column CEC. Thetechnique was more fully developed by Xin and Lee� �20 to shorten analysis times. Voltage programminghas the advantage that it does not impose any spe-cial requirements on voltage isolation and sampleintroduction. However, even though voltage pro-gramming and gradient elution share the commongoal of reducing analysis time, the basis of eachtechnique is different. In gradient elution, changesin mobile phase composition affect partition coeffi-cients and, hence, selectivities. Therefore, the reten-tion time of a late eluting compound is reduced byincreasing the time that it spends in the mobile

phase. In voltage programming, the partition coeffi-cient for a given compound remains the same, how-ever, as the voltage increases, the linear velocity ofmobile phase increases, thereby leading to a reduc-tion in compound retention times.

Gradient elution in CEC is important for sam-ples that contain compounds of widely differing po-larities, such as fats and oils in food products. Oilsand fats constitute one of the three major compo-nents of food products, the others being proteinsand carbohydrates. The use of vegetable oils hasgrown significantly in recent years due to the combi-nation of a pleasant flavor with nutritional benefit.On the basis of this expanding market for vegetableoils, their authenticities have become an importantsubject from both commercial and health perspec-tives. Triglycerides are the main components of oilsand fats. Therefore, monitoring qualitative andquantitative compositions of the triglycerides in veg-etable oils is growing in importance.

The separation of triglycerides has been exten-Ž . � �sively studied by gas chromatography GC 29�31 ,

� �LC 32�36 , and supercritical fluid chromatographyŽ . � �SFC 37�39 . Currently, the primary methods for

� �triglyceride analysis are reversed-phase 32 and sil-� �ver ion complexation 39 chromatography. The first

provides separation according to hydrophobicity,while the latter gives separations according to de-gree of unsaturation. Both separation mechanismshave been applied in micropacked and microbore

� �LC columns for the analysis of triglycerides 40 .� �Recently, Aparicio et al. 41 reviewed different

chromatographic techniques for the authenticationof vegetable oils. However, few papers have reported

� �triglyceride separations using CEC 42 .In this article, we describe the design and con-

struction of a novel gradient system for CEC. Thissystem overcomes problems related to sample intro-duction, isolation of voltage, and generation of EOF.In addition, the composition of the mobile phase atany time during the separation is known. This gradi-ent elution system was applied to the analysis oftriglycerides in different vegetable oil samples. Non-aqueous CEC was used for isocratic and gradientseparations.

EXPERIMENTALMaterials. Porous spherical spherisorb ODS1

˚Ž .particles 3 �m, 80 A were purchased from PhaseŽ .Separations Norwalk, CT, USA . Fused silica capil-

lary tubing was purchased from Polymicro Technolo-Ž . Žgies Phoenix, AZ, USA . Alkylbenzenes benzene to

.n-nonylbenzene and crystal violet were purchasedŽ .from Aldrich Milwaukee, WI, USA , and all chemi-

cals used in the preparation of mobile phases were

Simple Gradient System For Capillary Electrochromatography 353

Ž .purchased from Fisher Fair Lawn, NJ, USA . Veg-Ž .etable oils sunflower, olive and corn were pur-

chased from a local supermarket. SFC-grade carbonŽ .dioxide for column packing was obtained from

Ž .Airgas Salt Lake City, UT, USA . PEEK tubing wasŽobtained from Upchurch Scientific Murrieta, CA,

.USA .Prior to use, all buffers, solvents and samples

were filtered through a 0.22 �m Durapore� mem-Ž .brane filter Millipore, Bedford, MA, USA and

degassed thoroughly. Likewise, samples were de-gassed and filtered through a 0.2 �m polytetrafluo-

Ž . Žroethylene PTFE syringe filter Chromacol, Trum-.bull, CT, USA before use. The mobile phase

consisted of solutions of different percentages ofŽ .acetonitrile and Tris buffer 50 mM, pH 8.2 . The

sample was dissolved in a portion of the mobilephase to a concentration of 2 �L mL�1 per compo-nent.

Triglycerides were extracted from the three veg-etable oils using acetonitrile containing 50 mM am-monium acetate. A 500 �L volume of this organicsolvent was added to 100 �L of oil sample. Themixture was centrifuged for 5 min. Finally, the topsolution layer was filtered through a 0.2 �m PTFEsyringe filter.

Preparation of packed capillary columns. Fused-Ž .silica capillary columns 75 �m i.d.� 360 �m o.d.

were packed using a modified carbon dioxide slurry� �packing method 21 . The pressure used at the be-

ginning of the packing process was 900 psi. Then,Ž �1 .the pressure was raised periodically 75 psi min

to maintain a constant packing rate until the columnwas completely filled. The final packing pressure was4,500 psi. This process was performed with the col-umn placed in a sonicator bath. The column wasthen left to depressurize gradually which requiredapproximately 12 h. Inlet and outlet frits and awindow for on-column UV detection were prepared

Ž .using a resistive heating device InnovaTech, UK� �while water was pumped through the column 22 .

Isocratic CEC system. The CEC system used inthis study was a home-built instrument and is de-

� �scribed in detail elsewhere 23 . Briefly, the com-puter-controlled system was equipped with a Spell-man Model SL60PN30 high voltage power supplyŽ .Hauppauge, NY, USA with voltage capabilities upto plus or minus 60 kV, and a ThermoSeparation

Ž .Model UV 3000 detector Sunol, CA, USA . Theholder assemblies and the CEC column were con-tained in a Plexiglass box to isolate the high voltage.

Gradient CEC system. The inlet end of the CECsystem used for isocratic elution was modified toenable gradient capability. Figure 1 shows aschematic of the inlet end of this system. Two 1�16�

Figure 1. Schematic of the inlet end of the CECgradient system.

holes were drilled in the holder assembly. A sectionŽ � .of PEEK tubing 1�16 o.d.� 380 �m i.d. was

placed in each of these holes in order to allowcapillaries of 365 �m o.d. to pass through and enterthe vial containing the mobile phase. A platinumwire electrode was placed between these two capil-

Žlaries. An open top cap for 2-mL vials Supelco,.Bellefonte, PA, USA was glued to the bottom face

of the holder, and the capillaries and electrode werepassed through the opening of this cap. One of thecapillaries was a packed column used for separation.The second capillary was an open tube of 50 cm �200 �m i.d. attached to a 2.5 mL glass syringeŽ .Hamilton, Reno, NV, USA mounted on a syringe

Žpump stepper motor Harvard Apparatus, Holliston,.MA, USA . The syringe contained pure organic sol-

Ž .vent acetonitrile used to create the gradient.Before the entire system was assembled, experi-

ments were carried out to determine the degree ofmixing achieved by pure diffusion, and by stir-as-sisted methods. A 400 �L volume of pure water wasplaced in a 2-mL vial. An aqueous solution of crystal

Ž .violet a purple dye was pumped at a flow rate of 50�L min�1 through the capillary using the syringepump. From naked-eye observation, it was evidentthat diffusion alone was not enough for good mixing,as the crystal violet remained at the end of thecapillary.

Because of the small size of the vial used, micro-stir bars of 2 mm i.d. and 7-mm-long were purchased

Ž .from Fisher Scientific Pittsburgh, PA, USA . Sincethe stirring plate needed to be inside the Plexiglasbox, two factors were considered for its construction:

Medina et al.354

Ž .1 to avoid the use of electricity and metallic partsŽ .in order to prevent potential discharge, and 2 to

avoid using a bulky plate, in order to facilitatehandling and moving during injection. Therefore amicrostir plate with these characteristics was de-

Ž .signed and built see Figure 1 . The main body ofthis stirring unit was a cylinder of nylon with adiameter of 6 cm and a height of 6 cm. It containeda turbine made of black Delrin, to which a smallTeflon-coated stirring bar was attached. Spinning ofthis magnetic bar was achieved by a flow of airdirectly hitting the blades of the turbine. One end ofa piece of 1�8 in. o.d. Teflon tube was connected toa drilled opening 1 cm from the top of the plate,while the other end was connected to a solenoidvalve. In turn, the solenoid valve was attached to anair cylinder which was set at a pressure of one bar.To avoid discharge, the solenoid valve was glued onthe outside wall of the Plexiglas box.

Three programs were written in Microsoft Vi-sual Basic 5.0 to control the gradient system. Theyincluded a program for controlling the power supplyand for setting injection and voltage times, a pro-gram to control time and flow rates for the syringepump, and a program to control the solenoid valve.The syringe pump originally had capability only for asingle flow rate during a run, and no control forduration of delivery. However, by using home-writ-ten software, up to three different flow rates for anydesired time could be achieved in a single run.

The system was operated in the following man-ner. Prior to the beginning of a run, a volume ofmobile phase having the desired initial compositionand a microstir bar were placed in a 2-mL vial. Thevial was screwed into the fixed cap in the assemblyholder, and the run was started. After a predeter-mined time, the syringe pump began to deliver apreset flow of pure organic solvent, while at thesame time the microstir bar began to spin. Theseoperations were controlled by computer. A gradientwas created in the vial reservoir, and the mobilephase was introduced into the separation column byEOF. The system was operated with the groundelectrode at the inlet vial and a negative voltageapplied at the outlet end. Samples were injectedusing electrokinetic injection, by the application of anegative voltage.

RESULTS AND DISCUSSIONTheory of gradient generation. Although this new

gradient system was developed independently, theprinciple behind the generation of the gradient issimilar to that recently reported by MacNair et al.� �20 for ultrahigh-pressure liquid chromatography.Obviously, materials used to build the instruments

were quite different. While the main concern inCEC was to avoid electrical discharge, materialsable to withstand extraordinarily high pressures hadto be considered for ultrahigh-pressure liquid chro-matography. For a mobile phase consisting of just

Žtwo components, A and B A, organic solvent; B,.aqueous buffer , the fraction of organic solvent, XA

Ž .v�v , at any time t during a chromatographic runcan be given by the expression

VAŽ .X A t � ž /VA�B

Ž .V � F � EOF � tiŽ A. Ž A. Ž A. Ž .� , 1Ž .V � F � EOF � tiŽ A�B . Ž A. Ž A�B .

where V is the initial volume of A, F is theiŽ A. Ž A.Žflow into the reservoir flow delivered by the syringe

.pump , EOF is the flow of A out of the reservoirŽ A.due to electroosmosis, V is the initial totaliŽ A�B .volume placed in the reservoir before the start of arun, and EOF is the flow of mobile phase outŽ A�B .of the vial due to electroosmosis.

Typically, an initial volume of 400 �L of mobilephase was placed in the vial, and the flow rate of thesyringe pump which delivered pure organic solvent,F , was 50 �L min�1. Since electro-osmotic flowŽ A.

�1 � �rates are usually in the sub-�L min range 25 ,Ž .the terms EOF and EOF in Equation 1 canŽ A. Ž A�B .

Ž .be eliminated. Therefore, Equation 1 may berewritten as,

V V � F � tA iŽ A. Ž A. Ž .X � t � . 2Ž A. t ž /V V � F � tA�B iŽ A�B . Ž A.

0.78

0.80

0.82

0.84

0.86

0.88

0.90

0.92

0.94

0 2 4 6 8 10

time (min)

Xo

rg

Figure 2. Gradient used for separation of a mixture( ) (of alkylbenzenes solid line and linear gradient dashed

) ( )line . The solid line was generated using Equation 2with the following �alues: V � 320 �L; V �i( A) i( A� B)

400 �L; F � 50 �L min� 1.( A)

Simple Gradient System For Capillary Electrochromatography 355

The syringe pump flow rate, F , needed for aŽ A.

particular application is found by using the followingequation:

V � X � ViŽ A�B . f iŽ A. Ž .F � , 3Ž A. Ž .1 � X � tf

where X is the desired final fraction of A in thefmobile phase, and t is the total time needed tocomplete the gradient.

Figure 2 depicts the shape of a gradient used toperform an analysis of a mixture of alkylbenzenes.The initial volume in the vial was 400 �L, contain-

ing 80% A. The flow delivered by the syringe pumphad a rate of 50 �L min�1, and in 10 min, theconcentration of A in the vial increased to 91%.The dark-line plot in Figure 2 was generated using

Ž .Equation 2 . The dotted line represents a lineargradient with a percentage of organic solvent Aincreasing from 80 to 91% in 10 min. As the solidline in Figure 2 shows, the shape of the gradientgenerated by this system is convex. This may seem tobe a drawback, as most systems used in liquid chro-matography generate linear gradients. The degree ofdeviation from linearity depends on the initial andfinal concentrations of organic solvent, and the timerequired to achieve that gradient.

( ) ( )Figure 3. CEC chromatograms of a mixture of alkylbenzenes benzene to n-nonylbenzene using A isocratic and( )B gradient conditions. Experimental conditions: 25 cm � 75 �m i.d. fused-silica capillary packed with 3 �m

( )porous spherisorb ODS1; ACN�Tris buffer 50 mM, pH 8.2 mobile phase, UV detection at 210 nm; �5 kV, 1 s( ) ( ) ( ) ( ) ( )injection, �20 kV applied �oltage. A ACN�Tris 80:20 ; B ACN�Tris 80:20 initially, then gradient to 91:9

in 10 min according to the profile depicted by the solid line in Figure 2.

Medina et al.356

A convex shape reaches a specific concentrationof organic solvent faster than a linear gradient.Samples having compounds that elute together inbands and produce empty spaces in between thosebands, and separations of homologous oligomers

� �have been reported 26,27 as examples of separa-tions in which convex gradients are advantageouscompared to linear gradients.

E�aluation of the CEC gradient elution system.The new gradient system was tested by observing

Žseparations of a mixture of 10 alkylbenzenes ben-.zene to n-nonylbenzene . This sample contained

compounds of increasing hydrophobicities due toregular increase in alkyl chain length on the ben-zene ring. Figure 3 shows a separation achievedunder isocratic conditions compared to one usingthe gradient depicted in Figure 2. The analysis timewas decreased by ca. 40%, while preserving excellentresolution.

Because the 2-mL inlet vial was only partiallyfilled with 400 �L of mobile phase and, during thecourse of a run, the volume increased to a finalvolume of 900 �L, there was always headspace inthe vial. Therefore, a negligible increase in pressureat the inlet end of the column was produced, whichavoids potential distortion of the flat flow profiletypical of electro-osmotic flow. Furthermore, it wasfound that by fully degassing the mobile phase andconditioning the column, no pressure was requiredto avoid bubble formation.

The system performed consistently, as demon-strated by reproducibility measurements. A samplewas run six times both in the isocratic mode and inthe gradient elution mode. Relative standard devia-tions presented in Table I ranged between 1.6 and2.5% for isocratic conditions, and from 2.3 to 3.5%for gradient operation. Reported RSD values for

Table I. Reproducibility of retention times inisocratic and gradient CEC.

Gradient elutionŽ . Ž .Isocratic n � 6 n � 6Ž . Ž .n-Alkylbenzene RSD % RSD %

Benzene 2.5 3.5Toluene 2.3 3.3Ethylbenzene 2.2 2.7n-Propylbenzene 2.2 2.9n-Butylbenzene 1.9 3.0n-Amylbenzene 1.9 3.0n-Hexylbenzene 1.7 2.8n-Heptybenzene 1.6 2.5n-Octylbenzene 1.6 2.5n-Nonylbenzene 1.9 2.3

different CEC gradients ranged from as low as 0.1%� � Ž .to 10% 1�10 . The lowest values 0.1�0.6% were

� �reported by Alexander et al. 8 for a mixture of sixcompounds separated in 10 min using a complexpressure-assisted system.

Unfortunately, only a few papers have reportedRSD values for both isocratic and gradient condi-tions so that the net effect of the gradient could beevaluated. Variations in retention time in CEC aredue to several factors such as changes in ionicstrength and pH during a run. Data in Table I showthat there was an extra 1% increase in RSD valuesdue to the gradient system compared to isocraticelution.

The new gradient system was also tested byanalyzing real samples. Triglycerides from differentvegetable oil samples were analyzed using acetoni-trile�isopropanol�n-hexane as the initial mobilephase in the v�v ratio 60:35:5 to which 50 mMammonium acetate was added. Figures 4�6 comparetriglyceride separations under both isocratic andgradient elution conditions for corn, olive, and sun-flower oils, respectively. The initial volume of mobilephase in the inlet vial was 400 �L. Addition of

Ž .isopropanol�n-hexane 35:5 at 30 �L�min was usedfor gradient elution in all cases. As is easily seen, theanalysis time was significantly reduced for eachsample.

Finally, it should be mentioned that this systemfeatures several advantages over other publishedgradient systems. For example, the time required forre-equilibration between runs was typically about 5min, which is significantly shorter than the 30 minneeded for re-equilibration as reported for an elec-

� �trically driven CEC gradient system 3 . BecauseŽmost of the systems for gradient generation either

.electrically or pressure generated use some kind ofmixer at the head of the analytical column, part ofthe final mobile phase composition remains in the

� �mixer after each run 8,9,24 . Additional time andsolvent are required to rinse these mixers before thenext run to avoid contamination. In our system, thevial used as the mobile phase reservoir was changedafter each run; thus, this system does not require adelay time to rinse the mobile phase remaining inthe mixer after each run, or when changing organicsolvents. This new system is also simple and inex-pensive.

Perhaps the main advantage of this system is itspotential for automation. For example, by adding acarrousel containing vials with sample, rinsing buffer,and initial buffer composition, a large number ofanalyses can be carried out on a continuous basis.The ability to change the initial and final percent-ages of organic solvent in the mobile phase provides

Simple Gradient System For Capillary Electrochromatography 357

( ) ( )Figure 4. CEC Chromatograms of corn oil triglycerides using A isocratic and B gradient conditions.Experimental conditions: 25 cm � 75 �m i.d. fused-silica capillary packed with 3 �m porous ODS1; UV detection

( ) (at 200 nm; �2 kV, 2 s injection, �15 kV applied �oltage. A ACN�isopropanol�n-hexane 50 mM ammonium) ( ) ( ) ( ) ( )acetate 60:35:5 ; B ACN�isopropanol�n-hexane 50 mM ammonium acetate 60:35:5 initially, then 30 �L

( )per minute of isopropanol�n-hexane 35:5 .

Medina et al.358

( ) ( )Figure 5. CEC chromatograms of oli�e oil triglycerides using A isocratic and B gradient conditions. Experimen-tal conditions: same as for Figure 4.

Simple Gradient System For Capillary Electrochromatography 359

( ) ( )Figure 6. CEC chromatograms of sunflower oil triglycerides using A isocratic and B gradient conditions.Experimental conditions: same as for Figure 4.

flexibility in the generation of gradients. The gradi-ent profile can be accurately known and controlledby the flow rate set by the syringe pump.

Another potential use of the system developedin this study is the creation of a pH gradient, which

� �could be useful in CE separations 28 . Elec-trophoretic mobilities for most analytes depend onthe pH of the medium. For a sample containing avariety of compounds, resolution could be improvedby creating a pH gradient that decreases elec-trophoretic mobilities of poorly resolved compounds,and increases mobilities of late-eluting compounds.

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