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High-sensitivity detection cell for the Agilent Capillary Electrophoresis system Introduction Detection sensitivity is often cited as the most significant limitation of capillary electrophoresis (CE). This is a result of both small sample volumes (1–50 nL) and the short detection path length associated with on-capillary detection. Whereas on- capillary detection is simple, the UV-visible detection path length is limited to the internal diameter of the capillary. In practice the path length is further reduced because the capillary has a circular cross section, and the actual path length is approximately 80 % of the internal diameter. 1 To improve sensitivity of CE analyses, Agilent Technologies developed extended path length capillaries, known as bubble cell capillaries. These capillaries increased the internal diameter of the capillary at the point of detection by about a factor of three and thereby increased sensitivity by the same amount. Agilent also developed the high-sensitivity detection cell which effectively decou- ples the detection cell from the rest of the capillary. The high-sensitivity detection cell for the Agilent CE system represents a major advance in detection technology for capillary electrophoresis, increasing sensitivity by an order of magnitude. The high-sensitivity detection cell is available as a kit (Agilent part number G1600-68723) which includes the cell, capillaries, preassembled ferrules, finger-tight fittings and cassette. Technical Note

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Page 1: High-sensitivity detection cell for the Agilent Capillary ... detection cell for the Agilent Capillary Electrophoresis ... the decoupled design ... detection cell for the Agilent Capillary

High-sensitivity detection cell forthe Agilent Capillary Electrophoresissystem

Introduction

Detection sensitivity is often cited as the most significant limitation of capillaryelectrophoresis (CE). This is a result of both small sample volumes (1–50 nL) and theshort detection path length associated with on-capillary detection. Whereas on-capillary detection is simple, the UV-visible detection path length is limited to theinternal diameter of the capillary. In practice the path length is further reducedbecause the capillary has a circular cross section, and the actual path length isapproximately 80 % of the internal diameter.1

To improve sensitivity of CE analyses, Agilent Technologies developed extendedpath length capillaries, known as bubble cell capillaries. These capillariesincreased the internal diameter of the capillary at the point of detection by about a factor of three and thereby increased sensitivity by the same amount.

Agilent also developed the high-sensitivity detection cell which effectively decou-ples the detection cell from the rest of the capillary. The high-sensitivity detectioncell for the Agilent CE system represents a major advance in detection technologyfor capillary electrophoresis, increasing sensitivity by an order of magnitude.

The high-sensitivity detection cell is available as a kit (Agilent part numberG1600-68723) which includes the cell, capillaries, preassembled ferrules, finger-tight fittings and cassette.

Technical Note

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order of magnitude increase in sensi-tivity, but also a significant improve-ment in the linear range and unsur-passed spectral fidelity with the diodearray detector.

The high-sensitivity detection cell canbe fitted in any version of Agilent CEsystems. As shown in figure 1 the cellis incorporated in an optical interfacewhich is nearly identical to that usedfor standard and bubble-cell capillar-ies. This makes capillary and cassetteinstallation the same as for other capillaries. Figure 1 also illustrates the decoupled design which allowscapillary replacement.

The capillaries are attached to the cellby preassembled ferrules and finger-tight fittings. Replacement of capillariescan be performed within 2–3 minutes.

Preservation of peak shape is funda-mental to the design of the cell andcapillary coupling. Because poor cap-illary coupling and alignment wouldresult in peak broadening and distor-tion, the capillary couplings of thehigh-sensitivity detection cell have aunique geometry. The detection cell isdesigned for use with Agilent capillar-ies of 75 µm internal diameter whichare flared to 100 µm at the point ofconnection to the flow cell. This isillustrated in figure 2 which also

The high-sensitivity detection cellThe high-sensitivity detection cell is adecoupled detection cell for direct UV-visible absorbance detection withthe Agilent CE system. The increasedpath length of the high-sensitivitydetection cell provides an order ofmagnitude increase in sensitivity andan increase in linear range while atthe same time enhancing spectralanalysis.

The high-sensitivity detection cell isconstructed using microfabricationtechniques and is designed so that itis fully decoupled from the separationcapillary. The low volume of the high-sensitivity detection cell has negligibleimpact on peak shape, making it idealfor applications which demand sensi-tivity (trace analysis), increased linearrange (chiral excess, impurity analy-sis) and high resolution. The reusablecell body, with replaceable capillaries,makes the high-sensitivity flow cellextremely flexible and reduces thecost of achieving high sensitivity UV-visible absorbance detection in CE.

The high-sensitivity detection cell hasa unique design – constructed fromsilica parts which are fused togetherto form the detection cell. Theschematic in figure 1 illustrates theincreased path length relative to thecapillary dimensions. The high-sensi-tivity detection cell is 100 µm squarewith a path length of 1.2 mm and a vol-ume of 12 nL. The light path throughthe cell is made entirely of black fusedsilica to minimize stray light and todefine the aperture for diode-arraydetection. In addition the reflectiveinterior functions as a light pipe,ensuring almost 100 % transmission ofthe light which enters the detectioncell. The detection cell is flanked byflat clear windows. All these proper-ties combine to provide not only an

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Figure 1Schematic of the new high-sensitivity detection cell for the Agilent Capillary Electrophoresis system.

Figure 2Schematic of capillary connection.

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gical samples with conventional capil-laries. A possible solution may be toload more sample, however, this iscomplicated further when complex,multicomponent biochemical samples,such as tryptic digests of proteins,must be analyzed. The use of largevolume injections to overcome sensiti-

vity limitations can cause losses inresolution and eliminate any benefitsobtained from using a high resolutiontechnique like CE.

Figure 4 compares the analysis of atryptic digest of myoglobin using astandard capillary and using the high-

shows how the outer edge of the capillary is beveled to complete theno-dead-volume connection to theflow cell.

PerformanceThe high-sensitivity detection cell canbe used for both CE and CEC applicationsand is especially useful where increasedsensitivity or linear dynamic range isrequired. The application areasdemanding these attributes includechiral analysis in the pharmaceuticalindustry, analysis of dilute biologicalsamples, analysis of small polar com-pounds of environmental interest andin trace analysis of components in biochemical analysis.

Naphthalene sulfonic acids are ofinterest in environmental analysiswhere they occur at very low concen-trations. The previous lack of sensitivityassociated with CE made such ananalysis extremely problematic.However with the increase in sensitivityavailable using the high-sensitivitydetection cell, these analytes can bequantified easily. Figure 3 shows theanalysis of naphthalene sulfonic acidson a 75 µm id standard capillary. Thesignals obtained are minimal althoughthe separation exhibits a reasonableresolution. Figure 3 also shows a comparison with the high-sensitivitydetection cell coupled to a 75 µmcapillary of the same length and usingthe same injection parameters. Thesignal-to-noise ratio (S/N) obtainedwith the high-sensitivity detection cellis about 10 times greater than with the75 µm id standard capillary. With res-pect to resolution, a slight reductionmay be found when the interpeakvolume is smaller than the cell volume(about 12 nL). These losses are oftenminimal, as shown here, and the sepa-ration can usually be optimized for usewith the high-sensitivity detection cell.

The sensitivity limit of CE is oftenexceeded when analyzing dilute biolo-

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Figure 3Comparison of the high-sensitivity detection cell and a standard capillary for the CZE analysis ofnaphthalene sulfonic acids.

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Figure 4Analysis of dilute sample of 5 µM myoglobin tryptic digest using the high-sensitivity detection cell.

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Figure 5 shows the chiral separationof epinephrine on a 75 µm id standardcapillary, using 20 mM dimethyl-ß-cyclodextrin in a 50 mM tris-phosphatebuffer at pH 2.4. The S/N ratio obtainedfor a 75 mbar·s injection (about 20 nL)of a 100 µM solution of (+/-) epinephri-ne was 62.5. The S/N ratio using thehigh-sensitivity detection cell increa-sed to 650, giving a true increase insensitivity of more than tenfold.

Conventionally the linear range of CEis limited to 300–500 mAU because ofstray light associated with the smallsize, curvature and transparent wallsof the capillary. With the high-sensiti-vity detection cell the construction ofthe cell body from black fused silicaeliminates stray light and the signal islinear beyond 1300 mAU with less than1 % deviation and to more than 2000mAU with less than 3 % deviation from

sensitivity detection cell. The analysiswas performed at low pH with a 72 cmeffective-length capillary and detec-tion was performed at 200 nm wherethe peptide bond has maximumabsorbtion. With a sample concentra-tion of about 5 pmol/µL the 75 µm idstandard capillary was unsuitablefor analysis of such a dilute sample(despite the low detection wave-length). Using the high-sensitivitydetection cell under similar conditionsclearly improved the sensitivity of theanalysis. The three peaks that migra-ted at about 24 minutes illustrate howwell resolution was preserved evenwhen transferring a method directlyfrom a standard capillary to the high-sensitivity detection cell.

Analysis of chiral drug compounds iseasily performed by CE because thechiral selector (for example, cyclodex-trin) is dissolved in the buffer ratherthan bound to a stationary phase as inhigh-performance liquid chromatogra-phy (HPLC). This enables more rapidmethod development, improved effi-ciency and resolution, and reducedcost of both analysis and methoddevelopment. CE is frequently themethod of choice for chiral analyses.Regulatory requirements demand thatpharmaceutical manufacturersdemonstrate the chiral purity of anychiral drug substance and the accep-table criteria for presence of excessenantiomer is about 0.1–0.5 %. Thisrequirement therefore demands thatthe analysis not only has adequatesensitivity but also that the linearrange is such that the minor compo-nent may be quantitatively reported as(area/area) percent of the main com-ponent. To date, the success of CE forthis purpose has been limited.

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75 µm id standard capillarysignal-to-noise ratio = 62.5

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High-sensitivity detection cellsignal-to-noise ratio = 650

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Figure 5Increased sensitivity with the high-sensitivity detection cell.

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linearity (figure 6). This is of the sameorder of magnitude as found in HPLC.

This combination of extended linearrange and increased sensitivity makesthe high-sensitivity detection cellextremely well suited for the quantita-tive analysis of chiral excess. Figure 7shows the analysis of S-form of abasic drug in the presence of traceamounts of the R-form. The R enantio-mer is present at a levels of about 0.05 % area/area with S/N ratio of > 3.

Another advantage of the rectangularcell design is excellent compatibilitywith diode-array detection, allowingthe spectral analysis of components.Spectra may even be obtained frompeaks with very low S/N ratio asshown in figure 7 where the excessenantiomer can be clearly identifiedby its absorption spectra. The high-sensitivity detection cell allows fulluse of the functionality of the diode-array detector, including the use of spectral libraries for analyte identification and peak purity determinations even at such low trace concentrations.

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Figure 6Extended linear range with the high-sensitivity detection cell.

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Figure 7Chiral excess determination with the high-sensitivity detection cell.

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capillary. However, the noise is alsoincreased and therefore the realincrease in S/N ratio is about tenfold.The Z-cell provides a similar increasein S/N ratio of about 10, however,because of light leakage its linearrange is limited to about 600 mAU. Thehigh-sensitivity detection cell is linearto more than 2000 mAU, allowingdetection and quantitative analysisover four orders of magnitude, and itsrectangular shape improves its spec-tral qualities over that of a standard

capillary. Further, the Z-cell is integralto the separation capillary while thedecoupled high-sensitivity detectioncell can be reused many times withonly low-cost replacement of capilla-ries.

Table 1 compares the high-sensitivitydetection cell with a standard capil-lary and with another available optionfor improvements in CE sensitivity, theZ-cell.2 The Z-cell is integral to thecapillary and is created by bendingthe capillary longitudinally for 3 mmand using this section for detection.Whereas the Z-cell is used extensivelyin HPLC, its use in CE has been some-what limited.

Both the high-sensitivity detection celland the Z-cell have an increased cellvolume of 12 nl, therefore in bothcases peaks must travel about 3 mmapart in order to preserve resolution.The expected path length of a stan-dard capillary is the internal diameterof 0.075 mm, however, because of thecapillary curvature the effective pathlength is reduced to about 0.06 mm.The expected path length of the Z-cell is 3 mm, but for the samereasons this produces an effectivepath length of only 1.07 mm.2 The rec-tangular design of the high-sensitivitydetection cell means that its expectedand effective path length is the same,that is, 1.2 mm. This means that it willprovide a full 20-fold increase in signalcompared to a standard 75 µm id

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Capillary type Standard 75 µm id High-sensitivity Z-cellcapillary detection cell

Cell volume 3 nL 12 nL 12 nLExpected detection pathlength 0.075 mm 1.2 mm 3 mmEffective detectionpathlength 0.06 mm 1.2 mm 1.07 mmSignal increase - 20-fold 14.3-foldNoise increase - 2-fold 1.4-foldS/N increase - 10-fold 10-foldLinearity < 500 mAU at 1 % 1400 mAU at 1 % ~ 600 mAU

deviation from linearityResolution - 3 mm between zones 3 mm between zones

to maintain resolution to maintain resolutionSpectral fidelity - Improved over standard Incompatible with

capillary diode-array detector

Table 1Comparison of high-sensitivity detection cell, standard capillary and Z-cell.

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References

1.Heiger, D.N., Kaltenbach, P., Sievert,H.-J.P., “Diode-array detection incapillary electrophoresis”,Electrophoresis, 15, 1234–1247, 1994.

2.Moring, S.E., Reel, R.T., van Soest,R.E., “Optical improvements of a Z-shaped cell for high sensitivity UVabsorbance detection in capillaryelectrophoresis”, Anal. Chem., 65,3454–3459, 1993.

Conclusion

The high-sensitivity detection cell is asignificant advance in the efforts toincrease the sensitivity and utility ofCE and is a major response to the con-cerns of users in all analytical areas.

• The high-sensitivity detectioncell provides a true increase in sen-sitivity of an order of magnitude asindicated by increases in S/N, allo-wing the use of CE in trace analysis.

• The improved linear range beyond2000 mAU rivals that of HPLC andallows determination of low levels of impurities and chiral excess.

• The decoupled capillary design allows capillaries to be replacedindependently of the cell body thusreducing costs.

• Micromachined flared and beveledcapillaries maintain peak shape byensuring proper alignment and coupling to the cell body.

• The materials and design of the high-sensitivity detection cell enhancespectral analysis by diode-arraydetection.

• The high-sensitivity detection cell isdesigned to be used in any version ofthe Agilent CE system.

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www.agilent.com/chem/ce

© Agilent Technologies, Inc., 1997-2008

Published November 1, 2008Publication Number 5989-9808EN