The Newsletter for Capillary Electrophoresis

Volume 2 • Issue 1Winter 1998

BECKMAN

Advances in the quantification of HIV-1 viral loadJILL M. KOLESAR AND PETER G. ALLEN

SCHOOL OF PHARMACY, UNIVERSITY OF WISCONSIN

Introduction

Human Immunodeficiency Virus(HIV-1) — the retrovirus that causesAcquired Immunodeficiency Syndrome(AIDS) — is estimated by the Centers forDisease Control (CDC) to affect 612,078individuals in the United States and morethan 20 million worldwide. The fatalityrate of this disease, approaching 100 per-cent at 10 years in some regions, high-lights the magnitude and significance ofthis epidemic.

Recent advances in anti-retroviraltherapy, primarily triple and quadrupledrug therapy, including a proteaseinhibitor, have begun to improve out-comes. These potent drug combinationsare able to decrease viral loads — thesingle best marker of disease progression— to levels undetectable with currentassay methodology.1,2,3

The nucleic acid-based methods usedcurrently to monitor AIDS includereverse transcriptase polymerase chainreaction (RT-PCR), branched-chain signalamplification, and nucleic acid sequence-based amplification (NASBA).4,5

However, variability in amplification andcurrent detection limits are not adequateto monitor contemporary anti-retroviraltherapy. Therefore, the need for moresensitive and reliable assays is present.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can detectmolecules in the attomole range6 and pro-vides a nonradioactive, fast, reproducible,and sensitive method for the analysis of

nucleotide fragments. Previous studieshave used CE-LIF to quantify geneexpression7 and to identify HIV-1 andHTLV-I PCR products8,9; however, theseprocedures still require RT- PCR to gen-erate DNA fragments. A report hasattempted to quantify HIV-1 RNA direct-ly from the plasma without specific HIV-1 probes,10 but plasma samples may con-tain non-HIV-1 viral RNA includingHTLV-1 (human T-cell leukemia virusType-1), and hepatitis A, C, D and Ewhich would eliminate HIV-1 specificity.

Additionally, contamination of plasmawith leukocytes or other cells wouldresult in the presence of nonspecifichuman RNA.

This application note describes amethod to quantify HIV-1 RNA in plas-ma by using a specific, non-radioactiveprobe and CE-LIF. This procedure elimi-nates variability created by the use ofamplification techniques, and provides amore accurate and sensitive means ofquantification, which may help optimizeantiretroviral therapy and prolong sur-vival for patients with HIV.

Experimental

CE-LIF analysisSeparations were performed on a

P/ACE™ 2050 CE system (BeckmanInstruments, Fullerton, CA, USA) withthe temperature held constant at 20ºC.Detection of hybridization samples wasachieved using laser-induced fluores-cence in the reversed-polarity mode(anode at the detector side) at excitationof 488 nm and emission of 520 nm.Samples were introduced hydrodynami-cally by 10 s injections at 0.34 Pa acrossa 65-cm-x-100-mm coated eCAP™

dsDNA capillary filled with replaced lin-ear polyacrylamide (BeckmanInstruments, Fullerton, Calif). The capil-lary was conditioned with eCAP dsDNA1,000 gel buffer containing 60 µL ofLiFluor dsDNA 1000 EnhanCE intercala-tor per 20 µL (Beckman Instruments,Fullerton, Calif.). Separations were per-formed under constant voltage at 7.0 kVfor 15-30 minutes. The capillary wasrinsed with gel buffer for three minutes ▼

THIS PROCEDURE ELIMINATES

VARIABILITY CREATED BY THE USE OF

AMPLIFICATION TECHNIQUES, AND

PROVIDES A MORE ACCURATE AND

SENSITIVE MEANS OF QUANTIFICATION,WHICH MAY HELP OPTIMIZE

ANTIRETROVIRAL THERAPY AND PROLONG

SURVIVAL FOR PATIENTS WITH HIV

prior to each injection. The capillary wascalibrated with the fluorescently labeledprobe and a mixture of RNA molecularmarkers (Ambion, Austin, Texas). Thefive markers ranged in sized from 100 to500 bp. Postrun analysis of data was per-formed using the System Gold® chro-matography data system (BeckmanInstruments, Fullerton, Calif.).

HybridizationRNA was obtained from an HIV-

seropositive patient by centrifugation ofwhole blood at 3,000 xg for 15 minutes at4ºC, and RNA was extracted from plasmasamples by standard procedures.

To ensure specificity, a unique genesequence is probed. The pol region is themost genetically unique of the HIVgenome and a 26-bp sequence in thisregion was selected (GenBank entryU62632). The corresponding probesequence was synthesized and floures-cently labeled. Sample RNA washybridized with the DNA probe, anddigested with RNAase One to yield adouble-stranded DNA/RNA 26mer com-plex for CE-LIF analysis. Further detailson RNA isolation, design and labeling ofthe probe and hybridization conditionscan be found in reference 11.

Results and Discussion

Comparison of Weight to Copy NumberOne copy of HIV cDNA contains

9,000 base pairs (bp). One pcg of DNAcontains 9 x 108 bp, making onepicogram equal to approximately 100,000equivalents and one attogram approxi-mately equal to 0.09 copies of HIV RNA.

Analysis of Hybridization Samples The hydridization product consists of

an RNA/DNA complex 26 bp in length.Hybridizations were carried out on asample obtained from a seropositive HIVpatient and a seronegative normal volun-teer. RNA (0.095 µg/µL) was diluted 10-1

to 10-12, hybridized and analyzed as previ-ously described. The minimum reliablydetectable quantity (CV% 5.6-7.3) was190 attograms, which corresponds to 21copies of HIV. A dilution of 10-7 (19 fem-

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Figures 1, 2, 3:Electropherogramanalysis of hybridiza-tion products. RNAsamples obtainedfrom an HIV seroposi-tive patient and aseronegative volun-teer were hybridizedwith an HIV-specificprobe and analyzedas described inexperimental. [1]HIV RNA/Probe com-plex elutes at 12 min-utes, indicating thepresence of HIV RNAin the patient’sserum. [2]Seronegative volun-teer. [3] Negativecontrol containing allreaction componentsexcept RNA.

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tograms, which is equal to 1,710 copiesof HIV RNA) was determined to be opti-mal, and subsequent hybridizations wereperformed in triplicate on three differentoccasions and analyzed by CE-LIF.(Figures 1a-c.)

Hybridization products were injectedhydrodynamically without further samplepreparation and quantitated by comparingthe peak area obtained to the standardcurve. The intra-day and inter-day migra-tion time precision was 0.18% and 0.22%(n = 9), respectively, the intra-day peakarea precision was 7.3% (n = 9), and theinter-day peak area precision was 5.6% (n= 9), and represents both the hybridiza-tion and capillary electrophoresis por-tions of the assay.

In this approach, cellular RNA ishybridized with an HIV-1 specific probe.A complex is formed if HIV-1 RNA ispresent and unbound RNA is digestedwith RNAase One.

Samples are then analyzed by CE-LIF.Two peaks elute if HIV-1 RNA is present;the first, eluting at approximately 11 min-utes, is the DNA/DNA unbound probecomplex. The second, the DNA/RNAcomplex, elutes at approximately 12 min-utes. Although the complexes are both26mers, the DNA/RNA complex has adifferent secondary structure, slightlyhigher molecular weight and a subse-quently longer retention time.

Any double-stranded nucleotide com-plex, as well as the fluorescently labeledprobe, will be detected with this

approach. All samples are digested underidentical conditions, and when no com-plex formation (normal volunteer) occursthere is excess RNA present for diges-tion.

The relatively greater quantity ofRNA, (when compared with the HIV-pos-itive sample) may be incompletely digest-ed and nonspecifically interact with thefluorescently labeled probe, providing acluttered response. Additionally, the neg-ative control contains no RNA, and probeis the only substrate for RNAase One. Alarger degree of digestion occurs, leavinga smaller peak area as comparison with-samples containing RNA.

Conclusions

We have used CE-LIF to analyze HIVRNA and are able to reliably and quanti-tatively detect as little as 190ag (21copies per 1 mL of starting plasma) ofHIV RNA. This approach appears to bean efficient and sensitive method toquantitatively analyze HIV RNA, andwarrants further evaluation and compari-son to established HIV assay techniques.

Address Correspondence to:Jill M. Kolesar, Pharm.D., BCPSAssistant ProfessorUniversity of Wisconsin-MadisonSchool of Pharmacy425 N. Charter StreetMadison, WI 53706-1515Email: jmk@pharmacy.wisc.edu

Bibliography

1. D. . Ho, A. U. Neumann, A. S.Perelson, W. Chen, J. M. Leonard and M.Markowitz, Nature, 373 (1995) 123.

2. J. W. Mellors, L. A. Kingsley, C.R. Rinaldo, Jr., J. A. Todd, B. S. Hoo, R. P.Kokka and P. Gupta, Ann. Intern. Med.,122 (1995) 573.

3. J. W. Mellors, C. R. Rinaldo, Jr.,P. Gupta, R. M. White, J. A. Todd and L.A. Kingsley, Science, 272 (1996) 1167.

4. J. Todd, T. Yeghiazarian, B. Hoo,J. Detmer and J. Kolberg, Serodiag. andImmuno. Infect. Dis., 6 (1994) 233.

5. J. Compton, Nature, 350 (1991)91.

6. H. E. Schwartz and K. Ulfelder,Anal. Chem., 64 (1992) 1737.

7. J. M. Kolesar, J. D. Rizzo, and J.G. Kuhn, J. Cap. Elec., 2 (1995) 287.

8. H. E. Schwartz, K. Ulfelder, F. J.Sunzeri, M. P. Busch and R. G. Brownlee,J. Chromatogr. B, 559 (1991) 267.

9. N. Bianchi, C. Mischiati, G.Feriotto, D. Fiorentino, S. DiBiase, N.Apicella and R. Gambari, J. Virol.Methods, 47 (1994) 321.

10. N. Fernandez-Arcas, J. L.Dieguez-Lucena, J. G. Garcia-Villanova,J. Pena, M. Morell-Ocana and A. Reyes-Engel, J. Acq. Immune Def. Synd. HumanRetriov. 12 (1996) 107.

11. J. Kolesar, P. Allen, C. Doran, J.Chromatogr. B, 697 (1997) 189.

To provide a forum for the exchange of ideas, we invite you to sub-mit articles or manuscripts describing novel uses of capillary elec-trophoresis in your laboratory.As a reward, we will give $500 in “CE Cash” to all laboratorieswhose articles are selected. This currency is valid for the purchase ofBeckman capillary electrophoresis supplies and reagents.

Content: You decide. If the subject is of interest to you, it may be interesting to others as well.

Length: Please keep your article to less than 1,200 words.

Use appropriate referencing, where copywrite and trademarks areinvolved.Beckman reserves the right to edit for length/clarity.Send a hard copy version of your article and electronic version(on disk) to:

Beckman Instruments, NAO-BP/ACE Setter Review Group, M/S E-32-C2500 Harbor Blvd.Fullerton, CA 92834-3100or send your article via email to:jdchapman@beckman.com

Submit manuscripts — exchange ideas and earn ‘CE Cash’

CapillaryElectro-chromatog-raphy (CEC)enables the scien-tist to performreverse-phase chro-matography in acolumn of highefficiency, but onethat cannot bepumped with regu-lar HPLC pumps.

If we look at a comparison of HPLCcolumns with the same dimensions as apacked capillary, the separation time isgreater and the pressure drop is prohibi-tive for the packed capillary.

As Table 1 indicates, pumping acrosscomparable separating capillaries is notpossible with normal HPLC pumps.

Where does the pumping come from in CEC?

The pump is electroosmotic flow(EOF); the same mechanism that createsEOF in a fused-silica capillary propelsthe analytes through the reverse-phasemedia. Standard partitioning between sol-vents and packing phase causes a separa-tion. The efficiency results from the smallpacking particle size and the narrow col-umn. Solvent savings and separation effi-ciency are driving the methodology.Popularity of CEC is also partially driven

by our familiarity with HPLC.Figure 4 illustrates the gain in effi-

ciency from using the zonal type, electro-osmotically driven flow of CE withinCEC, as compared with the laminar flowof HPLC.

A simple comparison of the efficiencygained from doing chromatography withina capillary, using EOF as the pump, isshown in Table 2. Note that the HPLCcolumns must be shorter, with decreasingparticle diameter, because of the prohibitivepressure drop across the capillary.

Why isn’t everyone using it?

If CEC is so great, why isn’t everyoneusing it? It is presently restricted to iso-cratic separations with commerciallyavailable hardware. Also it has manypacking preparation problems that arebeing addressed by several companies(Unimicro being one) and other researchgroups.1 It does offer chromatographicseparation of non-aqueous soluble ana-lytes.2 However, few non-aqueous separa-tions exist in the literature. It is alsoattractive as a separation technique inter-faced with mass spectrometry.3 Thecharged capillary surface that creates theEOF pump needs some water for ioniza-tion. It does separate neutral compoundswithout the addition of buffer additives asin micellar electrokinetic chromatography(MEKC), but MEKC has shown greaterversatility than CEC.

Other random thoughts

• Isocratic-only instrumentation isavailable. See Reference 4 for gradi-ent CEC suggestions.

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Tom Bronzert

REBEL’SREVIEW

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Put voltage to work as your HPLC pumpCharacteristic Pump Driven Pump Driven Voltage Driven Voltage Driven

Particle Size 3 µm 1.5 µm 3 µm 1.5 µmColumn length 60 cm 15 cm 30 cm 10 cmElution Time 30 minutes — 15 minutes 5 minutesPressure Drop 6,000 psi 72,000 psi none none

Table 1: Pumping across comparable separating capillaries.▼

Figure 4: Zonal type electroosmotically driven flow within CEC compared with thelaminar flow of HPLC.

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Method Particle Size Column length Plates/Column

HPLC 5 µm 50 cm 55,0003 µm 25 cm 45,000

1.5 µm 10 cm 30,000CEC 5 µm 50 cm 115,000

3 µm 50 cm 170,0001.5 µm 50 cm 250,000

Table 2: Comparison of efficiency gained by doing chromatography within acapillary, using EOF as the pump.

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Electroosmotic drivePressure Drive

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• Sample injection is limited to electro-kinetically driven hydrodynamic viainduced flow. Samples cannot be sim-ply pushed or pulled into the capil-lary. Selective ion transfer could be aproblem.

• Detector sensitivity can be limiting ondilute samples. It is difficult to con-centrate through trapping mechanismssince with isocratic elution bandbroadening will be a problem. Usinglarger capillaries affects the resolution,and longer pathlength detector cellsallow multiple analytes into the cell atthe same time.

• Packed capillary availability is gettingbetter. However, unless the capillariesare packed well, pressure must beapplied to both sides of the capillaryduring separation. This is likely due to“hot spots” forming at poorly packedregions. Good temperature control is amust for all CEC applications.

• Charged capillary walls are requiredto induce EOF, but charged surfaces

bind oppositely charged analytes andthe ability to “clean” the capillary isnon-existent in an isocratic separation.

• Additionally, any bound analyteschange the capillary charge, affectingthe consistency of the pump, EOF.

• Cleaning and recharging the packedcapillaries must be done off-line. Thiscapability is not available within cur-rent CE instrumentation.

• Finally, the bottom line is this: CEC ismore complex experimentally thanCE. Electrohydrodynamics is verypoorly understood in packed beds.The effect of buffer ionic strength canbe large. My recommendation is, ifyou want to research into CE, manygood tools are available. However, ifyou want to develop a routine assaynow — use MEKC (micellar electro-kinetic chromatography) — it works.

References

1. Preparation of Continuous BedsDerivatized with One-Step Alkyl andSulfonate Groups for CapillaryElectrochromatography. Liao, Chen,Ericson and Hjerten, Anal. Chem. 1996,68, 3468-3472.

2. Behavior and Use of Non-aqueous Media without SupportingElectrolyte in CE and CEC. Wright,Lister, and Dorsey, Anal. Chem. 1997, 69,3251-3259.

3. Capillary Electrochroma-tography-Electrospray MassSpectrometry: A Microanalysis Technique.Schmeer, Behnke and Bayer, Anal. Chem.1995, 67, 3656-3658.

4. Capillary Electrochroma–tography with Gradient Elution. Huber,Choudhary and Horvath, Anal. Chem.1997, 69, 4429-4436.

Tom Bronzert is a Field MarketingSpecialist with Beckman Instruments.

Journey with us into deepspace. You will leave the HyattOrlando at 6:30 p.m. in a trans-porter reserved for the evening.After a brief trek, you will dis-embark where food, drinks andentertainment await.Explore your inner self

You will have the opportuni-ty to explore the complex sys-tems of the human body, includ-ing how nutrients are utilizedand how the immune systemprotects us from some ofnature’s alien invaders.Spend an evening with the stars

During the evening, you andyour colleagues will soar through spaceand confront the challenging questionsof stars, galaxies, constellations andblack holes via an eight-story, 28,000-watt CineDome.

You be the judgeYour opinions count. You will judge

the Fifth Annual Beckman CEparationof the Year Contest. Help select themost exceptional separation of the year.

By now you should have allreceived your invitations to thisevent and reserved a spot. If youhaven’t yet responded:

RESPOND NOW!

Fax the following informationby Jan. 15 to 714-773-8186:

NameTitleCompany/InstitutionPhonee-mailAddress

MARK YOUR CALENDARSMonday, Feb. 2, 19986:30 – 11:00 p.m.Orlando Science Center(Departure from the Hyatt Orlando)

During HPCE ‘98

Make space on your calendar for P/ACE™ user’s meeting

At the BeckmanTechnical Center,we receive manyquestions thatinvolve the separa-tions of small mole-cules. These includecompounds such aspharmaceuticals,ions, vitamins andsynthetic molecules.Some of the chal-

lenges to analysis include lack of chro-mophore, lack of charge, solubility issuesor “sticky” moleculesthat adhere to the wallsof the capillary. Thebeauty of capillaryelectrophoresis (CE) isthe investigator’s con-trol over the environ-ment in the capillarytube; in other words, Idecide which type ofcapillary, buffers oradditives to utilize.

With many smallmolecules being ana-lyzed, one oftenencounters the waterinsoluble analyte. Thegood news is, CE hasseveral modes, allowingthe user to overcome themost unwilling of participants.

Consider surfactants

One possibility is to use surfactants.Dr. Shigeri Terabe has written extensivelyon the subject of micellar electrokineticchromatography (MEKC) and has writtena primer for Beckman on this topic.

MEKC uses surfactants above theircritical micelle concentration as a bufferadditive. They exhibit both hydrophobicand hydrophilic characteristics. Micellarsolutions may solubilize hydrophobic com-pounds that otherwise may be insoluble inwater. I try to dissolve my analytes in a10% dilution of my running buffer to also

achieve some stacking and concentrationof my sample. I usually use a 75-microncapillary to get as large a mass load as pos-sible because many of the small moleculeswe analyze in the Technical Center have lit-tle UV absorbance.

The MEKC approach is also the methodof choice for separating neutral compounds.You may wish to try the capillary elec-trochromatography approach to separate theneutral compounds, but I prefer the MEKCmethod. Drs. Haleem Issaq and GeorgeJanini from SAIC-Frederick have demon-strated many interesting MEKC techniques

with small molecules. It is also possible to add organic sol-

vents miscible with water to the micellarsolution. According to Dr. Terabe thismanipulates the capacity factors and/or theselectivity. In our lab we do not add morethan 20% organic solvent; we mostly workin the 10% to 15% range. Above 20%organic one could break down the micellarstructure.

Organic additivescan be useful

The choice of organic solvent can alsobe useful in improving the resolution orpartitioning effect being sought. So, it is a

good idea to try different organic additivessuch as acetonitrile, methanol or 2-propanol. We have kept our buffer thesame and simply changed the organic addi-tive with some wonderful effects. However,be careful with phosphate buffers and theamount of organic additive; too high anamount could cause the phosphate to pre-cipitate.

It is always a good idea to make thesolutions and let them rest on the benchtopbefore proceeding.

There have been reports of investiga-tors using 80% to100% methanol withSDS solutions, but Idon’t believe we stillhave micelles at thathigh an organic con-tent.

Another type ofanalysis we are askedto perform quite fre-quently is the analy-sis of small basiccompounds.

I prefer to notuse HPLC, becausethe compounds willoften non-specifical-

ly bind to the sup-port. Peak shapeand recovery areusually poor.

Instead, I use an amine-coated capillarywith CE. This gives a static positive chargeto the capillary wall, which preventsadsorption at a wide range of pHs andreverses the electroosmotic flow. CE alsoprovides very fast analyses with high reso-lution. Another advantage to this approach:We are free to experiment with differentbuffers, pHs and additives.

Ion analysis as an option

Most of the methods we develop arefor pharmaceutical companies who wish todetermine metal ions, small amines, simpleorganic acids, anion or cation composition.We primarily use indirect UV detection for

Resolving small-molecule analysis problems

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Mark Flocco

NOTES FROMTHE LAB

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Figure 5: Schematic of the separation principle of MEKC. The detector window isassumed to be positioned near the negative electrode.

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Figure 6:Effect of PEOtreatment onmigrationtimerepeatability.

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these types of analyses. This detectionmode is useful for analytes havingpoor or no UV absorbance. Oneuses a strongly absorbing buffersuch as phthalate or imidazole. Theanalytes’ response is a negative peakthat the software turns over for easi-er integration.

There are a wide variety of appli-cations, and an excellent source ofinformation is the new Beckmanprimer on ion analysis by Kevin D.Altria of Glaxo Research andDevelopment. There are also severalcommercially available ion analysissolutions, which can be purchasedfrom a number of vendors that willprovide a quick start-up.

Non-aqueous CE (NACE)

After listening to several talks atthe Frederick Conference by Drs.Gyula Vigh and Kevin Altria on thesubject, I decided to give non-aque-ous CE (NACE) a try. I had severalsmall molecules to analyze thatdecided that water was not theirfavorite environment. Also, I waslooking for low-level contaminants,which meant I wanted to get as muchof the compound into solution as Ihumanly could. I attempted a methodthat was described by Kevin Altria:50/50 MeOH, MeCN with 10mMAcetate pH 9.3.

I had great success with thismethod but I took great care to auto-matically switch “buffer sets” every10 runs to maintain repeatability. Thesoftware’s increment option in MDQmade this quite easy. By changing thebuffer, I maintained my current pro-file run to run and assured the ionicstrength of my buffer did not changemy EOF profile.

Small molecules do not pose aserious problem with CE because ofthe flexibility of the technology. Infact, small problems will be no prob-lem with some careful thought and awillingness to experiment.

Mark Flocco is a Field MarketingSpecialist with Beckman Instruments.

A fused-silicacapillary wall con-tains silanol groupsthat, above pH 1.5,will contribute anegative charge tothe surface. At abuffer pH of 2.5, theelectroosmotic flow(EOF) is relativelysmall, but increasesrapidly with increas-ing pH until approxi-

mately pH 11.0, when the EOF is at amaximum. Phosphate is one of the mosteffective and popular buffers at this lowpH and is used in many CE analyses. It isalso well-documented that the phosphatebuffer ions interact with the silica sur-face.1,2 Dynamic interaction between phos-phate ions in the buffer and the silanolmoieties on the silica surface eventuallyestablishes equilibrium. For example, afreshly installed capillary in phosphatebuffer (at pH 2.5) would initially yield amuch greater EOF than after several runsof equilibration.

As a practical example we noticed thatwhen separating enantiomers using highlysulfated cyclodextrins in a phosphatebuffer at pH 2.5, the migration times ofthe reference and chiral species was sub-stantially longer in a freshly prepared cap-illary than that of a capillary with extend-ed use. This note essentially recognizesour observation of the dynamics of migra-

tion time changes experienced when usinga “new” or un-seasoned capillary. Moreimportantly, we propose a practical solu-tion to yield reproducible runs even with-out extensive equilibrations.

Figure 6 illustrates the migration timechanges that occur within a freshly pre-pared capillary as seen over the first 10injections. The trend that migration timedecreases with the increasing number ofruns is consistent with the reduction ofEOF. On the 11th injection we rinsed thecapillary with a solution of polyethyleneoxide (PEO, Part No. 477497) 20 psi for 1minute, which coats the capillary surface.The results are dramatic; we are able toeffectively reduce EOF and improve thereproducibility of the migration time.

Of course, if you treat the capillaryfirst with PEO, prior to buffer equilibra-tion, the stability of migration times isseen from injection 1 (data not shown).

References

1) Emerick, R.J. Amer. Instit. Nutr.1987, 22, 1925-1928

2) Mitsyuk, B.M., Russ. J. Inorg. Chem.1972, 17, 471-473

Dr. Fu Tai Albert Chen is Manager ofProbe Technology, BiotechnologyDevelopment Center for BeckmanInstruments.

Also contributing to this article wasRamon Evangelista of BeckmanInstruments.

The PEO equilibration experience

Fu-Tai A. Chen

DR. CHEN’SCORNER

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MT

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The P/ACE™

System MDQ isdesigned to be themost flexible andadaptable capillaryelectrophoresisinstrument on themarket, allowingyou to more easilytransfer methodolo-gy through yourorganization to a

quality-control environment.However, when transferring methodologybetween instrument types, one must considerthe differences created from the new hard-ware and software that you will be using.

With a thoughtful and reasonedapproach, any methods transfer can beanticipated. In this article we will high-light the transfer of an isoelectric focusingmethod from the P/ACE 5000 series tothe P/ACE System MDQ. We will pointout some of the “common traps” that youwill want to avoid, by trouble-shootingthrough this methods transfer with you.

IEF methodology was chosen because itadds an element of challenge to the trans-fer process. These guidelines should beconsidered regardless of the instrumentfrom which you are transferring.

Figure 7 illustrates the isoelectricfocusing of a “proprietary glycoprotein”performed on a P/ACE 5000 instrument.

An efficient separation ofthe various glycoforms ofthe protein is achieved. Thisparticular method employschemical mobilization asoriginally developed by Zhu,et al. Briefly, the protocolinvolves the following steps:

1) The capillary is filledwith a solution containingan electro-endosmotic flowmodifier, i.e. hydroxy-propyl-methyl cellulose(HPMC), ampholytes, theproteins of interest andTEMED. The TEMED isused to form a chemical“cushion” so the pH gradient can form onthe appropriate side of the detector.Solutions of phosphoric acid and sodiumhydroxide are used as anolyte andcatholyte, respectively.

2) The pH gradient within the capil-lary is set up and proteins are initiallyfocused for a short time by applying astrong electric field. A neutral-coated

capillary, in combination withthe HPMC, is used to suppresselectroosmotic flow.

3) After the focusing period,the cathode is moved to a bufferreservoir containing a chemical-mobilizing agent, usually noth-ing more than a salt solution.This causes a shift in the pHgradient, resulting in a bulkmigration past the detector.

Figure 8 illustrates the ini-tial results achieved when thesame method is applieddirectly on a P/ACE SystemMDQ. Why the big differ-ence? There is a simple

answer: One must consider closely thevariables, which define the method.These include the following:

Capillary temperature

This is a common trap; the programmedtemperature is of the temperature control

medium, not the capillary. The improveddesign of the MDQ cartridge yields moreefficient cooling, resulting in more effec-tive temperature control of the capillary.In fact, it is not uncommon to witness a3- to 4-degree temperature differencebetween the P/ACE 5000 series and theP/ACE System MDQ. Of course on lessefficient, air-cooled CE instruments, thedifference is even more pronounced. Inturn, temperature affects viscosity, whichwill affect movement through the capil-lary during both sample loading andmobilization.

As a practical matter, if differentresults are obtained on P/ACE systemMDQ as compared with another instru-ment, you should probably raise the tem-perature on the MDQ to counterbalance.A good guideline is to choose a tempera-ture that generates the same current underidentical field strengths, as this will moreeffectively mimic the other instruments’“capillary temperature.” Additionally, it isnot uncommon to see greater than 10˚ Cdifferences between air and liquid tem-perature control systems.

Field strength

This is probably the most commonlymade mistake. Do not simply transferapplied voltage in your method, butensure you are transferring the same field

Alex Szabo

BETWEEN 2ELECTRODES

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Transferring IEF methodology: the adventure

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Figure 7: Isoelectric focusing of a ‘proprietary gly-coprotein’ performed on a P/ACE 5000 instrument.

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Figure 8: Isoelectric focusing of a ‘proprietary glyco-protein’ performed on a P/ACE System MDQ.

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Time: 25.877 Minutes. Amp: 0.000254 Au

Time: 25.959 Minutes. Amp: -0.001642 Au

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strength. The distance from the detectorwindow to the outlet in the P/ACE™ sys-tem MDQ is 10 cm compared with 7 cmon the P/ACE 5000. When transferringmethods between instrument models orbrands, this difference in length needs tobe taken into account so that the samefield strength (measured in V/cm) isapplied.

Length of focusing period

With IEF, an alternative to increasingvoltage, may be to increase the durationof focusing prior to mobilization. Figure9 is an illustration of the effect thatincreasing the length of the focusing had(as long as 12 minutes). Although in

this case it did not improve our results, itdid provide an important clue as to themajor source of our failed separation.

Electroosmotic flow

And so the mystery reveals itself. Theneutral capillary and addition of HPMCwere used to reduce the EOF, unfortu-nately it did not eliminate it. Notice thateven though the switch to the chemicalmobilizer did not occur until 12 minutes,the impurity peak had already begun tomigrate past the detector, suggesting thepresence of an electroosmotic flow (thecapillary used in this example is from amanufacturer other than Beckman). Toquote the immortal words of the fictionalSherlock Holmes: “Elementary, my dearWatson.” The glycoprotein of interest was

reaching the detector prior tobeing fully focused or per-haps even being pushed pastby the EOF and focusing onthe other side of the detector.

This was re-enforced byrunning the entire experi-ment at maximum voltagewithout a switch to mobiliz-er (Fig. 10). Note that, inthis case, the protein stillreaches the detector.

The interpretation

Although the capillarylength to the detector was

the same in both systems, thetotal length of capillary dif-fers. Because the percentageof TEMED applied wasfixed, the effective length ofthe pH gradient was differentbetween the two instruments.Since the MDQ has a longerlength from detector to out-let, the pH gradient begins toform nearer to the detectorthan on the P/ACE 5000 (badTEMED can also cause thiseffect). For basic proteins inparticular (the pI of the pro-tein of interest in this case isin excess of 9.0) there may

not be sufficient time to focus prior toreaching the detector or once again thefocus may be on the other side of thedetection window.

The solution

The solution in this caseis to do nothing more thanincrease the concentration ofTEMED. As can be observedin Fig. 11, this extends thepH gradient on the properside of the window, allowingeffectively more “visible”time for focusing. The resultis a separation equal to orsuperior to that achieved pre-viously.

It is important to under-

stand all the variables that make up yourseparation. With IEF, the concentration ofTEMED is critical for controlling thelength of the pH gradient, which will bedifferent from one instrument design toanother. Although obvious with hindsight,it may be very easy to miss when blindlytransferring methodology.

Other issues. For a further discussionof these issues as they relate to broaderCE applications, call 1-800-742-2345 (1-800-387-6799 in Canada) for a copy ofBeckman Technical Information Note T-1839A, “Translating Methodologies fromP/ACE 2000 and 5000 to P/ACE MDQ,”by Harry Whatley and Ben Archer.

Alex Szabo is a Field MarketingSpecialist with Beckman Instruments.

Also contributing to this article wasDr. Harry Whatley, a CE MarketingSpecialist for Beckman’s BiotechnologyDevelopment Center.

Figure 9: The effect of increasing the length of focusing.▼

Figure 10: Result of running entire experiment at max-imum voltage without a switch to mobilizer.

▼

Figure 11: Result of increasing TEMED concentration.▼

Time: 0.092 Minutes. Amp: -2.5e-005 Au

Time: 5.744 Minutes. Amp: -0.00114 Au

Time: 0.097 Minutes. Amp: 0.000106 Au

▲10

ALEX SZABOBECKMAN INSTRUMENTS

For seven years, the FrederickConference on Capillary Electrophoresishas highlighted some of the most excitingresearch in the field. This year’s eighthannual meeting proved to be no excep-tion.

The scientific committee, led byGeneral Chairman Haleem Issaq puttogether a wonderful program that gave aglimpse of the future of this increasinglypowerful technique and also illustratedthe breadth of its current application tothe chemical and pharmaceutical indus-tries. A description of some of the highpoints follows:

Capillaryelectrochromatography

Several of the presentations were ded-icated to CEC, which uses the pump ofelectroosmotic flow to drive analytesthrough a chromatography bed. One ofthe primary limitations to the broaderadoption of CEC has been the prepara-tion of suitable columns.

Csaba Horvath showed a variety ofinnovative approaches he has takentoward overcoming these obstacles. Theseinclude the use of capillaries containingan interior tube to improve the electroos-motic flow profile (“Tube in the Tube”),and formation of the bed by scinteringfused silica rather than packing it underpressure. Horvath demonstrated that earlyversions of columns of this design havecomparable performance to more classi-cal packed beds.

Ziad El Rassi described some earlysteps in the application of CEC to thestudy of carbohydrates. The chief obsta-cles to overcome in this case are theirpolar nature — which makes separationusing octadecyl-silica difficult — andtheir lack of absorption in the UV region.Both obstacles can be overcome by theuse of derivitizing agents. With thesereagents, he was able to demonstrate effi-ciencies on the order of 100,000 plates

per meter using glucose polymers as ana-lytes. A secondary problem is the genera-tion of anomers from reducing polysac-charides when separations are performedin aqueous solution. El Rassi pointed outthat this problem can be overcome by theaddition of borate, which complexes dif-ferentially with the “a” and “b” forms.

Micellar electrokineticchromatography

Of a more immediate impact upon thepharmaceutical industry may be the tech-nique of Micellar ElectrokineticChromatography (MEKC), invented byShigeru Terabe. This technique is alreadypracticed extensively using BeckmanP/ACE™ systems and other commercialinstruments.

Organic solvents

One of the more interesting develop-ments was the use of organic solventswith no background electrolyte. Thisapproach is attractive for a number ofreasons, particularly because it potential-ly provides a means for the direct cou-pling of CE with MS for hydrophobiccompounds such as combi-chem prod-ucts.

John Dorsey showed that the elec-troosmotic flow in a variety of organicsolvents, such as acetonitrile, methanoland formamide, is of a velocity compara-ble to water. Because of their low con-ductivity, much higher voltages canpotentially be applied using organic sol-vents prior to Joule heating becoming aproblem. The consequent gain in efficien-cy derived from these increased voltagesmakes this an attractive approach to chro-matographers.

Chiral CE

One of the most elegant talks wasreserved for those attendees who stuck itout until the final day. Gyula Vigh’s pre-

sentation also reminded us that it is diffi-cult to decide which is more impressive— the quality of his research or his abili-ty to liven up a presentation with hissense of humor.

Vigh illustrated the separation powerof a new set of sulfated cyclodextrins aschiral selective agents. His presentationincluded numerous examples of the reso-lution of enantiomers, many in the rangeof 20 to 40. These are “separations sogood that, even in Texas, [one] can drivebetween the peaks.” Since these reagentsare charged, they can also be used for theseparation of neutral enantiomeric com-pounds, a class of molecules that has pre-viously been refractory to separation withnative (neutral) cyclodextrins.

Vigh also illustrated an extension ofhis approach for systematizing chiralmethods development. Typically, one ofthe most difficult steps in this process isto identify conditions that permit someinitial resolution. Vigh demonstrated thatthrough an approach involving multiplesample/marker injection it is possible todetermine whether conditions are promis-ing, even if only peak-broadening, notactual resolution of the underlying enan-tiomers, is achieved.

Beckman is in the process of com-mercializing a series of highly sulfatedcyclodextrins and is looking for users ofP/ACE System MDQ to evaluate them.For more information about this program,see P/ACE Setter Vol. 1, Issue 2.

Student presentations

A session of the conference wasreserved for presentations by graduatestudents. As has been the case in pastmeetings, these talks were some of themost exciting in the meeting and gaveattendees a glimpse into the expandingfuture of CE. At Beckman we are proudof being a sponsor to this session in ourcontributions to student travel.

Yelena Lyubarskaya from BarryKarger’s laboratory at NortheasternUniversity gave examples of the coupling

Frederick Conference: Now and tomorrow of CE

▼

▲11

of the P/ACE™ instrumentwith mass spectrometryfor pharmaceutical analy-sis. This combination ofinstrumentation is findingincreasing usefulness inthe analysis of combi-chem products as well asbiopharmaceuticals.

Employing the antibiot-ic vancomycin and a set ofpeptides as an example,Lyubarskaya demonstratedthat it is possible to useCE-MS to determine solu-tion phase affinitiesbetween analytes. This typeof approach will likely gaingreater use as the FDAincreasingly emphasizessolution phase approachesamong its Points toConsider guidelines.

Adam Fermier ofSUNY Buffalo describedthe progress he is makingtoward overcoming one of the chief limi-tations to capillary electrochromatogra-phy — the ability to easily and repro-ducibly pack very-narrow-bore columns.Fermier demonstrated the operation of acentripetal packing device of his owndesign (fashioned from, among otherthings, a used tractor tire).

With respect to pressure packing,centripetal packing of columns offersseveral advantages: the capacity to havemultiplex packing and impact velocitythat is not dependent upon the diameterof the column. Fermier’s centripetallypacked columns gave theoretical platecounts that exceeded that of pressure-packed columns.

Capillary electrophoresis offers manyadvantages for the large-scale sequenc-ing of nucleic acids. Among these areenhanced resolution with respect to slabgel systems, shorter run times and thepossibility to automate the entireprocess, including sample injection.

Indu Kheterpal from Richard Mathies

lab at U.C. Berkeley illustrated thepotential of this approach by demonstrat-ing an experimental sequencing devicecapable of simultaneously performing upto 1,000 sequencing runs.

By combining this instrumentationwith new fluorescence energy transferlabels to enhance signals and reducespectral overlap, she hopes to improvethe productivity of sequencing laborato-ries.

Other students presenting includedJoerg Kutter, Dorothea Jeffery, PankajSinghal, Li Tao, and Zheru Zhang.

CE validation for thepharmaceutical industry

Following the main session, KevinAltria of Glaxo-Welcome Pharmaceu-ticals led an afternoon workshop in vali-dation of CE methods. Through numer-ous examples from Glaxo and otherpharmaceutical companies, Altriademonstrated that CE is fast becoming a

technique that can rival HPLC for druganalysis.

A number of parameters are uniqueto the performance of capillary elec-trophoresis and must be rigorously testedand documented. These include lot-to-lotvariation in capillaries, electrolyte stabil-ity, separation voltage and sample load-ing. The use of buffer arrays for robust-ness testing of pH effects is important.Altria gave useful illustrations of proto-cols to test these various factors and theirpotential interplay.

Of course, the application of CE tothe pharmaceutical industry is not limit-ed to Glaxo. A recent survey of P/ACESetter readers revealed that a large num-ber of companys have already developedvalidated methods using their BeckmanCE instruments.

Alex Szabo is a Field MarketingSpecialist with Beckman Instruments.

▼

Figure 12: Participants of the Frederick Conference stop by the Beckman displays.

▲12

Ever wonder what scientific instru-ments do when they are not busy? Aconversation was overheard inBeckman’s Sommerset, New Jersey, CEapplication lab, where only instrumentswere present. A Demo P/ACE SystemMDQ was heard talking to the otherCE instruments that make upBeckman’s P/ACE product line.The conversation went somethinglike this:

“Man, in the last severalmonths I have traveled extensive-ly in the East. Starting in lateJune I appeared at theWashington, DC, CE User’sForum, where I heard lecturesfrom Dr. Mark Richards withthe USDA, and MarkFlocco and TomBronzert withBeckman. Afterthis successfulforum I decidedto attend twoothers. The firstwas in Bostonwith fellow speak-ers Jeff Chapman ofBeckman, talking about new strategiesin chiral methods development; Dr. JinPing Liu of Bristol Myers Squibb,speaking on affinity CE; and Dr.Michael Fasco, with the New YorkState Department of Health, pre-senting data on quantitative RNAPCR.

“The next forum took menear Montreal (the lovelycity of Pointe Claire),Quebec, where I listened tothe lectures of Dr. Karen Waldron onCE and MECC, and Dr. Andrea Versariaddressing the separation of phenols byCE. I even got to practice a littleFrench.

“After the CE user forums my fre-quent traveler mileage really started toadd up. I attended Dr. Gallo’sBiotechnology meeting in Baltimore,emphasizing my high-throughput anti-

sense oligomer monitoring. Then it wason to the NIH Research Week for scien-tific posters and the tent exhibit, where Idisplayed my cIEF, SDS molecular

weight determinationand peptide mapping prowess. Headingnorth took me to the next stop at Dr.Haleem Issaq’s Frederick CEConference. 350 attendees saw my newhighly sulfated cyclodextrin reagentintroduced via multimedia CD-ROM. Istood alone in the ability to handle thisheat, generated from this highly success-ful chiral separating reagent.

“Then on to Boston, where thou-sands of pharmaceutical scientists were

attending the AAPS symposium. Heremy 96-well-plate format with the HSCDchiral reagent and CD-ROM were againa big hit.

“My well-deserved week of vacationled right into the Eastern AnalyticalSymposium in Somerset. Thousands ofanalytical chemists attended. I tookthis opportunity to present validationof CE methods at the Monday morn-ing CE workshop. I stressed analytemobility for identification, and nor-malized area/concentration for quan-tification. With these techniques you

guys (P/ACE 5500s, other MDQs)can use my methods and peak

tables as platform-indepen-dent measurements in

CE.“Finally, one

more trip backEast to a chi-ral separationworkshop inWashington,DC, led tothe holidaybreak inDecemberand myreturn home

to the lab tocontinue runningsamples.

“The trip wasexhausting, yet exhilarating. I

expect the rest of you toget ready for the next

trip, a winter vacation in Orlando,Florida, at HPCE ‘98; hope to see youall there. You will also want to check outour International P/ACE User’s meeting;I hear it’s being held at the newlyopened Orlando Science Center.”

MDQ Language translation courtesyof Tom Bronzert of BeckmanInstruments.

P/ACE™ System MDQ earns frequent-traveler mileage

▲13

Automation, with the development ofintegrated laboratory robotic systems, hasplayed a significant role in the evolutionof drug discovery strategies. Of course,processes of synthesis have been greatlyadvanced with the use of combinatorialchemistry, creating even greater demandsfor simplified high-throughput screeningand automated analytical characterization.

Essentially, once ideal compounds havebeen synthesized, screened, identified andconsolidated, a bottleneck generates intheir subsequent re-handling for characteri-zation. An important step in processimprovement becomes the development ofa common format that can be used fromsynthesis to final analysis.

The gap between synthesis, handling,screening and analysis is bridged with theuse of the 96-well plate, an industry stan-dard in automation. This need for automa-tion drives Beckman’s product develop-ment and is considered key in the design ofall the company’s new analytical systems.The most recent example of the “integra-tion of analytical tools” approach is seenwith the release of the P/ACE™ SystemMDQ.

The P/ACE System MDQ is a CE sys-tem developed with the 96-well plate at thecenter of its design. The P/ACE System

MDQ is a pharmaceutical CE developedto assist in drug discovery, and is theonly CE system designed from theground up to streamline methodsdevelopment, methods validation andmethods transfer to QC.

CE has come a long way in thelast nine yearsand is nowenjoyed as avery robustand repro-ducible technology usedextensively for analysisof highly polar com-pounds.

CE has excelled inthe analysis and charac-terization of proteins,including those that areglycosylated; in the sep-aration and quantitationof nucleic acids; and theanalysis of highly polarcompounds such as ionsand those notoriouslydifficult basic pharma-

ceuticals. Additionally, the free-solutionapproach of CE has been ideal for creatinga milieu of chirality and represents the bestanalytical tool for the resolution of chiralenantiomers.

In summary, the P/ACE System MDQutilizes the 96-well plate as a commonsampling platform, allowing an easy transi-tion from screening and consolidation toanalyte characterization. Although thissampling format is currently off-line fromour integrated laboratory robotics, our goalis to integrate these analytical tools asplug-and-play modules, enhancing greatlythe degree of laboratory automation.

Plug-and-play lab automation is not that far off

Figure 14: PACE System MDQ.▼

Figure 13 :In the future, the ORCA® arm will move 96-wellplates from the Biomek® to the P/ACE System MDQ.

▼

THE GAP BETWEEN SYNTHESIS,HANDLING, SCREENING AND

ANALYSIS IS BRIDGED WITH THE

USE OF THE 96-WELL PLATE

▲14

Happy newyear! If you made aresolution toadvance your pro-fessional knowl-edge, we havesome greatresources to helpyou.

Our primersand handbooksprovide an excel-lent survey of prac-

tices and considerations for particular CEapplications.

To establish a good overview andfoundation, try our customer trainingcourses.

Tom Erbach is a Field Training andDevelopment Manager with BeckmanInstruments.

Tom Erbach

ABOUTTRAINING

▼

Advance your knowledge through training

Introduction to Capillary Electrophoresis P/N 149844 $335 (US)This one-day lecture course provides a sound theoretical basis for capillary electrophoresis and an overview of current chemistries andapproaches to methods validation. 8:30 a.m. to 4:30 p.m.1998 training schedule: Somerset, NJ: Jan. 27, April 21, July 28, Nov. 3

Schaumburg, IL: March 10, June 23, Sept. 22, Dec. 1Palo Alto, CA: Feb. 10, May 12, Aug. 11, Nov. 10

P/ACE™ Station Software Operation P/N 149846 $335 (US)This is a one-day class covering advanced software functions and instrument operation. Hands-on exercises at PC workstations utilizepractice data sets provided by the instructor. 8:30 a.m. to 4:30 p.m.NOTE: To ensure maximum benefit for all students, this course requires that all attendees complete the P/ACE Station SoftwareTutorial Workbook prior to attendin and bring exercise results with them.1998 training schedule: Somerset, NJ: Jan. 28, April 22, July 29, Nov. 4

Schaumburg, IL: March 11, June 24, Sept. 23, Dec. 2Palo Alto, CA: Feb. 11, May 13, Aug. 12, Nov. 11

Troubleshooting CE Methods and Systems P/N 149845 $335 (US)In this one-day course the student learns to troubleshoot method and electropherogram problems as well as common system problems.This class includes a troubleshooting workshop and hands-on system maintenance practice. 8:30 a.m. to 4:30 p.m.1998 training schedule: Somerset, NJ: Jan. 29, April 23, July 30, Nov. 5

Schaumburg, IL: March 12, June 25, Sept. 24, Dec. 3Palo Alto, CA: Feb. 12, May 14, Aug. 13, Nov. 12

Course descriptions/Training schedule

Primers/Handbooks

To enroll, call 1-800-742-2345 (USA) • 1-800-387-6799 (CANADA)

Volume Part No. Title

I 360643 Introduction to Capillary Electrophoresis

II 266923 Introduction to CE of Proteins and Peptides

III 266924 Micellar Electrokinetic Chromatography

IV 726388 Introduction to the Theory and Practice of Chiral CE

V 727484 Separation of Proteins and Peptides by CE: Applicationto Analytical Biotechnology

VI 538703 Introduction to Quantitative Applications of CE in Pharmaceutical Analysis

VII 607397 Separation of DNA by CE

VIII 725036 High-Performance CE of Carbohydrates

IX IONS-1 Quantitative Analysis of Small Ions by CE

BR7739B Handbook of Applied Electrochemistry (This is a goodreference for buffer pH measurement)

▲15

Capillary elec-trophoresis is verysimple in concept.An electrically con-ductive liquid isplaced in a thin silicatube. A plug of sam-ple is pushed in. Thewhole thing is put inan electrical field,which causes thingsto move at different

rates. The peak of interestis detected as it passes the window, thenthe whole thing gets flushed out and theprocess is repeated. Nothing could beeasier. So why do I get so many phonecalls, faxes, and e-mails?

Sometimes things do go wrong, and,when they do, someone has to figure outwhy and make things right. Many timesthe answer is right there on the PC screen— the clues are usually held in the cur-rent trace. Lets examine the fundamen-tals, the most important being Ohm’sLaw, V = IR (voltage = current x resis-tance). In the majority of CE methods wehold the voltage constant. If we also holdthe resistance constant the equation pre-dicts that current must be a constant. Sohow do we control resistance?

The resistance, R, is a measure ofhow easily electric current passes througha conductor. In CE, this conductor is thebuffer in the capillary. Some of the criti-cal factors that determine the conductivi-ty of a buffer are:• The concentration (higher concentra-

tion decreases R)• The nature of the buffer ions (phos-

phate is considered a lower resistancebuffer than is borate)

• Other components of the buffer(organics, such as methanol, tend toincrease R; inorganic salts tend todecrease R)

• pH (which can have different effectsdepending on the ion and where youare on the pH scale)The size of the electrical conductor is

also critical. Just as the resistance of cop-per wire drops as you increase the diame-ter of the wire, the resistance of a buffer-filled capillary drops as you increase itsdiameter. Because the cross-sectionalarea of the capillary (or the wire) is theimportant factor, current will increasewith the square of the radius. If you dou-ble the diameter of the capillary, the cur-rent should increase by a factor of four.Resistance increases in direct proportionto capillary length.

Temperature is a factor that affectsthe resistance of our buffer-filled cap-illaries. Generally, as the temperaturegoes up, the resistance goes down.Ohm’s law tells us that current goesup as resistance falls. Other equa-tions tell us that as currentincreases, the production of Jouleheat increases. Here we have acase where increasing tempera-ture causes increasing current, which fur-ther increases temperature until the sys-tem spirals out of control. (Of course, anadequate cooling system can control thisprocess and stop this thermal spiral.)

When something goes wrong with aCE separation, the first thing to do iscompare the current in the bad run withthe current in a good one. If everythingwas done exactly the same in the two

runs the current should be the same. Achange of more than 2% to 3% in currentbetween the good and bad runs meansthat something has changed. Has the cur-rent gone up or gone down?

If the current has changed, the resis-tance has changed. This is usually causedby one of three factors: The capillarydimensions have changed, the tempera-ture has

changed, or the buffer composition haschanged. The most common of thesethree, in my experience, is the buffercomposition. This can be caused byimproper formulation, a change in the lotof a material used in the buffer, a changein the water supply, or improper pHadjustment (future columns will deal withthese issues). Some methods are inher-ently sensitive to small changes, so that a

Diagnosing Problems 1: Staying current with current

Harry Whatley

CURRENTAFFAIRS

▼

▼Figure 15: Ohm’s Law Plot showing non-linearity when temperature is not constant.

— Ideal Ohm’s Law - - - - Exceeds Ohm’s Law

▼

WHEN SOMETHING GOES WRONG

WITH A CE SEPARATION, THE

FIRST THING TO DO IS COMPARE

THE CURRENT IN THE BAD RUN

WITH THE CURRENT

IN A GOOD ONE

BECKMANBeckman Instruments, Inc. ¥ 2500 Harbor Boulevard, Box 3100 ¥ Fullerton, California 92834-3100

Sales: 1-800-742-2345 ¥ Service: 1-800-551-1150 ¥ Internet: http://www.beckman.com ¥ Telex: 678413 ¥ Fax: 1-800-643-4366

Beckman Instruments, Inc. (Canada) ¥ 6733 Mississauga Rd., Suite 604 ¥ Mississauga, Ontario L5N 6J5Sales/Service: 1-800-387-6799 ¥ Internet: http://www.beckman.com ¥ Fax: 1-905-819-1485

BA98-5004 © 1998 Beckman Instruments, Inc Printed in U.S.A.

* All trademarks are property of their respective owners

Pharmaceuticals with asymmetriccarbons that exist as enantiomers pose adifficult challenge for analyticalchemists in designing methodology fortheir separation.

Because these stereoisomers arephysically and chemically identical, onehas to focus on the construction of chiralenvironments to facilitate their separa-tion. A significant advance to thisprocess was accomplished with thedevelopment of the well-characterizedhighly sulfated cyclodextrins. Thesereagents, when coupled in solution withcapillary electrophoresis, provide anideal format for developing chiralmethodology.

To help simplify the processes ofchiral methods development, Beckman

Instruments, Inc. has developed an edu-cation program titled “Reflections onChirality.” This program involves a semi-nar, demonstration workshop and a mul-timedia CD-ROM presentation.

Beckman also is supporting a testprogram for your evaluation of thesenew highly sulfated cyclodextrins. If youare interested in co-sponsoring either aseminar or demonstration workshop atyour facility, or are interested in being atest site for our new chiral reagents, con-tact Jeff Chapman at 714-773-8885 orsend an e-mail to jdchapman@beck-man.com.

To receive a free copy of the“Reflections on Chirality” multimediapresentation, call Beckman Instrumentsat 1-800-808-5067.

‘Reflections onChirality’ CD

small error in buffer composition canhave a drastic effect on the separation.These problems may show up as achange in resistance and, hence, achange in current.

Temperature control is often blamedfor separation problems. If a CE systemhas a high heat dissipation capacity,such as the P/ACE™ liquid cooling sys-tem, a change in temperature is indica-tive of some mechanical problem. Mostcommonly the addition of a few milli-liters of liquid coolant solves the prob-lem. In instruments with less efficientcooling systems, methods that are tem-perature-sensitive will be much moreproblematic, as changes in resistancemay push you into the thermal spiraldescribed above.

In summary, if a method is notworking like it once did, check the cur-rent. If higher or lower than in a previ-ous good run, try to identify the causeof the change in resistance. Has themethod changed (voltage and tempera-ture settings)? Was the buffer preparedproperly? Has the lot of any buffercomponent changed between good andbad runs? Is the water supply stillacceptable? Is the pH meter workingproperly? Is the capillary of the samedimensions (length and diameter) asbefore? Is the cooling system working?

In the next issue I’ll discuss a morecomplex current issue: Why does thecurrent change during a run?

Check out Harry’s web site athttp://www.neptune.net/~whatley/

Dr. Harry Whatley is a CEMarketing Specialist for theBiotechnology Development Center ofBeckman Instruments.

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