Capillary thermostatting in capillary electrophoresis

Technical Note

Introduction

Forced air cooling in the AgilentCapillary Electrophoresis system ensures: • temperature consistency, • reproducible migration times, • column flexibility, • no maintenance, and • lower cost of ownership without compromising the separation.

This Technical Note focuses on avariety of applications and demonstra-tes the reproducibility, ease of use,

sensitivity, and resolution obtainedusing the Agilent CE system.

Capillary thermostatting is fundamentalto CE instrumentation. The ther-mostatting system serves three distinct purposes. It • ensures high reproducibility by isola-

ting the capillary from changes inambient temperature,

• removes Joule heat generated withinthe capillary, and

• provides a wide experimental andstable temperature range.

Reasons for temperature control in CE

1. High reproducibility Buffer viscosity and therefore migra-tion time are highly dependent on capillary temperature. It has beenreported that migration time varies 2 to 3 % per 1 °C temperature change.1Laboratory temperature fluctuations of 5 °C, for example, alter migrationtime 10 to 15 % in a non-thermostattedsystem.

Isolation of the capillary from changesin ambient temperature is thereforenecessary to obtain high reproducibility.Since ambient temperature canincrease or decrease, a system thatcan cool and heat to maintain con-stancy is beneficial compared withone that functions simply as an oven.The thermostatting system used in thisstudy has this capability and maintainsthe set-point temperature to within ± 0.1 °C.

2. Control of Joule heating In CE power and heat are generatedupon the application of voltage (wherepower is defined by the product of vol-tage and current). Generation of heatwithin the capillary causes radial tem-perature gradients, with a higher tem-perature in the center of the capillary.These gradients can produce parabo-lic flow profiles, zone broadening, andloss of resolution.

Further, high temperature can causedegradation of thermally labile samplecomponents. The effects of heatingcan be greatly limited by thermostat-ting the capillary to remove heat. Evenwith efficient thermostatting, however,it is much more beneficial to limit heatgeneration rather than remove it after-wards.

Heating can be reduced by the expe-dient choice of buffers and buffer con-centration. Even more beneficial is theuse of narrow internal diameter capil-laries since highly conductive buffersand high electric fields can be used.Current generation in capillaries of 25 µm id, for example, will be fourtimes lower than that in 50-µm idcapillaries and 16 times lower thanthat in 100-µm id capillaries.

3. Wide range of temperature control Active control of capillary temperaturecan be a useful parameter in optimi-zing separations. Further, lowering thetemperature increases buffer viscosityand decreases current. This approachcan be particularly useful in high cur-rent situations.

Finally, temperature extremes can beused to affect selectivity in separati-ons based on interactions of analyteswith buffer additives, such as forMEKC and chiral separations. With theAgilent CE system, temperature canbe controlled from 10 °C belowambient to 60 °C.

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High reproducibility

Heating within the capillary is oftenpresented in the form of an Ohm’s Lawplot, which shows current as a func-tion of voltage as demonstrated infigure 1. Excess heat is indicated bynon-linearity of this plot. While someti-mes used to “illustrate” the superiorefficiency of one type of thermostat-ting system over another, in practicecurvature of the Ohm’s law plot can beirrelevant to the separation.

The real issues for the experimenterare whether the required resolution isobtained and if the electropherograms

are reproducible under the conditionsemployed. In this section, examples ofseparations obtained under highpower conditions will be presented todemonstrate the efficiency of the ther-mostatting used in the Agilent CEsystem.

High buffer concentrations are oftenused to limit solute-wall interactions,especially for peptides and proteins.When combined with wide-bore capil-laries (75- to 100-µm id) for micropre-parative use, high currents are gene-rated and efficient heat removal isrequired to maintain high resolutionand reproducible separations.

Thermostatting systems

The overall design of the capillaryelectrophoresis system and the effi-ciency with which it can dissipateheat is as important as the temperatu-re control mechanism itself. Whilethermostatting with liquid is theoreti-cally more efficient than with air, thehigh-velocity, forced air thermostattingdesign used in the Agilent CE hasbeen proven to be as efficient asliquid cooling.

In addition to excellent reproducibility,there are a number of other advanta-ges with high-velocity forced air ther-mostatting: • simplification of the capillary cartrid-

ge so that no gaskets or screws arerequired,

• quick capillary change, • fast equilibration of set-point tempe-

rature (including temperature gradi-ents during an analysis),

• no expensive, volatile cooling fluids,and

• no leakage of coolant fluids. The fol-lowing sections demonstrate the effi-ciency and flexibility of the high-velocity forced air thermostatting inthe Agilent CE system.

Experimental conditions

Capillary electrophoresis experimentswere performed on an Agilent CEsystem with built-in diode-array detec-tor and ChemStation software. Thecapillary thermostatting systememployed high-velocity forced air, cir-culating at 10 m/sec. A Peltier devicewas used to control the temperaturebetween 10 °C below ambient and 60°C, with a precision of 0.1 °C.

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Figure 1Ohm’s plot demonstrating high-velocity forced air thermostatting.

Voltage [kV]

Liquid thermostatting

10 m/s air thermostatting – Agilent CE

No thermostatting

Current[µA]

5

150

100

200

250

300

50

10 15 20 25 300

Figure 2 shows the separation of atryptic digest of human growth hormo-ne (hGH) using 105 mM phosphatebuffer in a 75-µm id capillary. Uponapplication of 25 kV, a current of 130µA developed, yielding a power gene-ration of 4.0 W/m. As can be seen inthe figure, even under such excessiveheating conditions excellent separati-on and reproducibility were obtained.In fact, the reproducibility was suffi-cient to obtain multiple fraction collec-tions of each peptide peak for furtheranalysis by sequencing and massspectrometry.

Another example of the efficiency andstability of the thermostatting systemis shown in figure 3 which illustratesthe use of 100-µm id capillaries with ahighly conductive phosphate buffer.While current generation in such widebore capillaries is often excessive,their use is desirable since single-runfraction collection can often containsufficient sample for further analysis.Again, excellent separation was obtai-ned and each peak indicated in theelectropherogram was collected andidentified.

Figure 2Demonstration of capillary thermostatting efficiency and system stability under high power condi-tions (Voltage=25 kV, Current=130 µA, Power=4.0 W/m).

Reproducibility (% RSD)

Peak 1 Peak 2 Peak 3Migration time 0.36 % 0.60 % 0.33 %Area 1.63 % 1.89 % 2.09 %*

Time [min]10 15 20 25

250

0

mAU 200 nm

*not completely resolved

1

2

3

Chromatographic conditionsSample: hGH tryptic digest (2 mg/mL)Running buffer: 105 mM phosphate, pH 2.0Effective/total length: 72/80.5 cmInternal diameter: 75 µm BF 3Injection: 150 mbar x sElectric field: 310 V/cmCurrent: 130 µADetection wavelength: 200/20 nmTemperature: 35 °C

Figure 3Use of 100 µm id capillaries for micro-preparative separation and sub-ambient temperaturewas used to limit heating. Peaks indicated (*) were successfully collected and sequenced.

Peak 4 reinjected

*

04035202

0

80

mAU 200 nm

35

* * **

*

*

*

Time [min]

Chromatographic conditionsSample: Tryptic digest of bacterial chaperonin GroES Protein (7 mg/mL)Running buffer: 105 mM phosphate, pH 2.0Effective/total length: 87.5/96 cmInternal diameter: 100 µmInjection: 150 mbar x sElectric field: Gradient to 250 V/cmCurrent: 155 µADetection wavelength: 200/20 nm

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Control of Joule heating

Applications where extremely highionic strength buffers are used canpresent problems for any thermostat-ting system. By using a very narrow,25-µm id capillary, the current genera-ted can be restricted. The sensitivityof this narrow bore capillary can beimproved five-fold over standard 25-µm id capillaries by using an exten-ded light path capillary (”bubble cell”)which has a 125 mm pathlength, bub-ble factor BF5. This combination ofnarrow bore capillary with extendeddetection light path is ideal whenusing extremely high buffer concen-trations.

Figure 4 shows the use of 150 mMphosphate buffer with 200 mM ammo-nium sulfate, pH 7.0 to separate pro-teins while minimizing their interactionwith the capillary wall. The rapidseparation required the use of a highelectric field (400V/cm). Under theseextreme conditions, current generationwas limited to 138 µA by use of 25-µm idcapillaries and capillary thermostattingat 15 °C. These conditions could not bereplicated with 50-µm or even 75-µmid capillaries since current generationwould exceed 500 and 1200 µA res-pectively.

Figure 5 shows the use of 25 µm BF5capillaries when using highly sulpho-nated cyclodextrins. These chiral sel-ectors have associated high currentsin solution, due to the high number ofsubstituted sulphonic acid groups andthe associated sodium counter-ions.The excellent control of joule heatingis demonstrated by the Ohm's Lawplot (V vs I) linearity. This conformsalmost perfectly to theoretical expec-tations and the stability of thermostat-ting is further demonstrated by thereproducibility of the peak migration.

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Figure 4Use of 25 µm id capillaries to limit heating with highly conductive buffers.

40

50

30

20

10

0

mAU

Time [min]0 5 10 15 20

1

2 3

4

5

Chromatographic conditionsSample Protein mixture: 1 trypsin inhibitor, 2 bovine milk ß-lactoglobulin B,

3 bovine milk ß-lactoglobulin A, 4 horse skeletal muscle myoglobin 5 bovine pancreatic ribonuclease A, (approximately 1 mg/mL, each

Running buffer: 150 mM phosphate, 200 mM ammonium sulfate, pH 7.0Effective/total length: 41/49.5 cmInternal diameter: 25 µm BF 5Injection: 250 mbar x sElectric field: 400 V/cmCurrent: 138 µADetection wavelength: 210/16 nmTemperature: 15 °C

Figure 5Ohm’s law plot for highly sulphonated cyclodextrins, 25-µm id BF 5 capillary.

Voltage [kV]0 10 20 30 40

0

5

10

15

20

25

30

Current[µA]

r2 = 0.999 Absorbance[mAU]

Time [min]2 4 6 8 10 12 14

0

50

0.30%0.31%

Chromatographic conditionsBuffer: 25 mM phosphate/TEA, pH 3.3, 5 % 9 W/v), heptakis-6 sulfato-cyclodextrin,Effective/total length: 40/48.5 cmInternal diameter: 25 µm BF 5Injection: 3000 mbar x sVoltage: 30 kVTemperature: 20 °C

Wide range of temperaturecontrol

Decreasing the capillary temperatureis another effective way of minimizingthe effects of heating. Since bufferviscosity increases as temperature isreduced, current and therefore hea-ting are also reduced. In addition, thisincreases the time the solutes remainin the capillary, having a similar bene-fit to increasing the capillary lengthwhile holding the electric field con-stant. The benefit of this approach isshown in the chiral separation in figu-re 6, where decreasing the temperatu-re from 35 °C to 15 °C improved theseparation. In the case of chiral sepa-rations, the lower temperature mayalso increase the difference in com-plexation of one of the enantiomerswith the chiral selector.

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Figure 6Optimization of capillary temperature for chiral analysis.

Time [min]8.2 9.2 10.2 11.2 12.2 13.2

mAU

0

10

20

30

40

5035 ˚C

30 ˚C25 ˚C

20 ˚C15 ˚C

Chromatographic conditionsSample: Epinephrine (10 µg/mL)Running buffer: 50 mM phosphate-Tris, pH 2.4, 20 mM dimethyl-bcyclodextrinEffective/total length: 56/64.5 cmInternal diameter: 50 µmInjection: 100 mbar x sElectric field: 465 V/cmDetection wavelength: 214/10 nm

Conclusions

The efficiency, reproducibility and stability of the high velocity forced airthermostatting used in the Agilent CEsystem was demonstrated. High reso-lution and reproducible separationswere shown using capillaries from 25-µm id to 75- and 100-µm id capilla-ries with highly conductive buffers.The benefit of sub-ambient capillarytemperatures was also shown toimprove chiral separations. Despiteexcellent thermostatting efficiency, itis still a well known fact that currentand heat generation should always beminimized. The prudent selection ofbuffers and the use of narrow borecapillaries should always be the goal.

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References

1. S.Hjertén, “Zone Broadening inElectrophoresis with SpecialReference to High PerformanceElectrophoresis in Capillaries: AnInterplay Between Theory andPractice,” Electrophoresis 11, 665-690,1990.

2.R.Grimm, and M.Herold,“Micropreparative Single Run FractionCollection of Peptides Separated byCZE for Protein Sequencing,” J. Cap.Elec., 1, 79-82, 1994.

3.“Diode-array Detection in Capillaryelectrophoresis using AgilentExtended Light Path Capillaries”,Agilent Technologies Technical Note,Publication Number 5990-3410EN,2009.

www.agilent.com/chem/ce

© Agilent Technologies, Inc., 1994-2009Published March 1, 2009Publication Number 5990-3407EN