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Real-time fluorescence imaging in analytical chemistry

Johansson, Jonas; Johansson, Thomas; Nilsson, Stefan

Published in:Biomedical Optoelectronics in Clinical Chemistry and Biotechnology

DOI:10.1117/12.229500

Published: 1996-01-01

Link to publication

Citation for published version (APA):Johansson, J., Johansson, T., & Nilsson, S. (1996). Real-time fluorescence imaging in analytical chemistry. In S.AnderssonEagels, M. Corti, N. Kroo, I. Kertesz, T. A. King, R. Pratesi, S. Seeger, H. P. Weber, ... A. Katzir(Eds.), Biomedical Optoelectronics in Clinical Chemistry and Biotechnology (Vol. 2629, pp. 2-9). SPIE. DOI:10.1117/12.229500

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Download date: 31. May. 2018

Real-Time Fluorescence Imaging in Analytical Chemistry

J. Johansson', T. Johansson2 and S. Nilsson2

1Division of Atomic Physics, Lund Institute of Technology,P.O. Box 1 18, 5-221 00 Lund, Sweden.

2Department of Technical Analytical Chemistry, Chemical Center,University of Lund, P.O. Box 124, 5-221 00 Lund, Sweden.

ABSTRACT

A detection system for capillary electroseparation methods based on fluorescence imaging has been developed. In capillaryelectrophoresis (CE) the detection unit is normally placed near the outlet part of the fused silica column where a window isopened in the coating and the fluorescence is recorded over a short distance to maintain a high resolution. Our methodemploys fluorescence imaging of the whole column during separation of various samples. The column is positioned in astraight holder and the outer protective coating of the column is removed to get optical access to the sample. An excimer/dyelaser is used for excitation of the sample and the fluorescence is recorded with an image-intensified CCD detector anddisplayed in real-time. The CCD detector is read out with a rate of about 5 frames per second and the corresponding fullfluorescence line profiles along the column are displayed. Thus, full electropherogram are displayed showing the propagationand gradual separation of the sample fractions. The main advantage of this method is that parameters such as sampleconcentrations, diffusion, wall interaction and sample-to-sample interaction can be studied in real-time over the full length ofthe column, which is crucial for efficient system optimisation. Among several applications, isoelectric focusing,isotachophoresis and enzyme-substrate interactions can be mentioned. Methods for increasing the collection efficiency, suchas fiber optic arrays, have been investigated as well as different methods for computer-assisted signal integration and filtering.A fiber array consisting of 500 optical quartz fibers has been constructed that give a substantial improvement of the opticalcollection efficiency.

Key Words: capillary electrophoresis, chromatography, electrochromatography, laser-induced fluorescence, fluorescenceimaging, CCD

INTRODUCTION

Availability of pure substances for determining thestructure and function of the enormous array ofcomponents encountered in biological system has alwaysbeen a challenge in analytical biochemistry. The fact that asingle cell may contain several thousand proteins, hundredsof RNA's, multiple DNA and polysaccharide componentsmakes the isolation of any single macromolecular species achallenge.' For this reason separation technology andadvancement of biochemistry have been intimately linkedfrom ultracentrifugation (The Swedberg, 30th),electrophoresis (Tiselius & Hjerten, 40th), gaschromatography GC (Martin & Synge ,4Oth) gelfiltrationchromatography (Porath & Flodin, 50th) to highperformance liquid chromatography HPLC (Kirkland &Majors, 70th). Unfortunately, many of the analyticaltechniques upon which life scientists have depended arelabour intensive, slow and/or of low resolving power. Anew candidate, capillary electrophoresis CE2'3, has

developed during the last decade from a promisingtechnique to a method of choice for many applications,developing and growing in all directions.4 A lowerconcentration sensitivity in comparison with liquidchromatography (LC) can be compensated by sampleenrichment and preconcentration such as isotachophoresis(J'p)56 In ITP the sample is injected into the capillaryin-between a leading and a terminating electrolyte. If themobility of the sample is between that of the twoelectrolytes and the concentration of the leadingelectrolyte is higher than that of the sample, a sampleconcentration will occur. After the on-linepreconcentration is achieved an electrophoretic separationcan be initiated by switching electrolytes.

One explanation to the success of CE is that it combinesthe rapidity and automation capability of HPLC and theextremely high resolving power of electrophoresis. It isalso possible to transfer some of the knowledge fromHPLC during method development. Secondly, CE usesonly minute amounts of separation media Qil-ml) and

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sample volumes (pl-nl). The extremely small samplevolumes makes it possible to look into a single cell7'8 andanalyse its content of a certain species. CE also produceminute amounts of waste. The use of CE as a tool inanalytical chemistry has rapidly increased in the past fewyears and has found important applications in analysis ofmetabolites, pharmaceuticals and biopolymers and will bethe future work horse in this area. CE may be the tool ofchoice to solve such challenging tasks as to elucidate thehuman genome, where about one billion base pairs have tobe identified.

To extract more information from CE experiments, a newapproach has been proposed by our group9. The systememploys real-time fluorescence-imaging 1 O 1 1 for

a better understanding of the processes acting in capillaryelectrophoresis. Rather than exciting just one point at theend of the capillary column we have chosen to use theentire column as a detection cuvette and thus generateinformation during the main part of the separation with alimit of detection of 5x1O0 M for fluorescein. Manyuseful and unexpected findings have been revealed duringour investigations performed so far. In addition, thistechnique was used to image the process of ITP, displayingthe gradual sample concentration during theisotachophoretic phase prior to CE separation12. Parallelto our work, similar systems have been developedelsewhere utilising both absorption and fluorescencedetection13. An interesting imaging technique employs arectangular channel plate in which the fluorescence isdetected by a fiber optic array perpendicular to the flowdirection and where an injection capillary is scanned acrossthe channel plate14. In this paper we present the principlesof our detection system and discuss the advantages andproblems in relation with conventional CE detectors. Inparticular, we will address the system detection limit andshow how this can be improved. Some preliminarysupporting data will also be shown. We will also show datafrom a few experiments. The system detection limit wasdetermined using fluorescein at different concentrations.One example of isotachophoresis (ITP) will also be shown.

Samples

MATERIAL AND METHODS

Fluorescein standard sodium salt (internal standard, P/N477406, Beckman Instruments, Fullerton, CA) was used atconcentrations 50, 33 and 20 nM for estimation of thelimit of detection. The DNA standard was an HAE IIIrestriction digest of 4X 174 RF DNA (gibco BRL, Div. oflife Technologies, Gaithersburg, MD). The digestconsisted of 11 fragments with the following sizes: 72,

118, 194, 234, 271, 281, 301, 603, 872, 1078 and 1353

base pairs. The nucleotide mixture was ethanolprecipitated and redissolved in distilled water to aconcentration of 40 tg/mL. Rhodamine B (base) (JanssenChimica, Beerse, Belgium) was used for ITP and wasdissolved in ethanol and diluted in 6-aminocaproic acid(EACA) (Fluka, Buchs, Switzerland) to a concentration ofio7 M. The leading electrolyte was EACA and theterminating electrolyte was acetic acid (E. Merck,Darmstadt, Germany). For further details, see refs 10 and12.

Electrophoretic Set-up

The CE equipment used for the ITP experiments was builtin-house. A stabilised power supply (0-30 kV, Zeta-elektronik, Höör, Sweden) was used. The electrode vesselsconsisted of Eppendorf microfuge tubes. The capillary wasfixed in a Plexiglas holder and fixed in a horizontalposition, which allowed the laser light to access thecapillary with the exception of stretches at each end.Injections of the analytes for ITP were made by hand witha 5 j.tL Hamilton micro-syringe. Fluorescein samples wereintroduced electrokinetically for 5 sec at 65 V/cm andanalysed at 195 V/cm. DNA samples were introduced for5 sec at 21 V/cm and separated at 157 v/cm. 100 .tm innerdiameter capillaries (DB-17, J&W Scientific, Folsom, CAin fluorescein experiments and ds DNA 1000, BeckmanInstruments, Fullerton, CA in DNA experiments), wereused. In the case of liT, 100 im inner diameter fusedsilica capillary (Polymicro Technologies, Phoenix, AZ,USA, modified by polyacryl amide coating), were used.The total length was 20 cm, out of which 7 cm werecovered by the camera. The outer protective polyamidecoating on the capillary was removed according to refs 10and 12.

Detection System

A system utilising laser-induced fluorescence imaging as adetection method was constructed. The novelty of thisdetection method is based on the fact that the fluorescencefrom samples in the capillary is imaged onto a CCD. Bythis technique the sample can be continuously followedthrough the capillary, displaying e.g. a separation, binding,catalysis, isoelectric focusing or a concentration process.The system set-up is shown in Fig. 1 . The excitationsource was an excimer laser pumped dye laser (EstonianAcademy of Science, ELI-76E, Tallin, Estonia and ELTOLtd., VL 2200, Tartu Estonia, respectively) tuneable overthe entire visible region. The output of the dye laser wascoupled into a 1 mm diameter non-fluorescent opticalfiber. The mean power out from the optical fiber was in

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Fig. 1. Set-up for fluorescence imaging of capillary electrophoresis.

the range of 1 mW. The laser was generally operated at 5Hz and the excitation wavelength was either 488 or 500nm. The divergent light from the optical fiber was focusedto a 10 cm linear streak onto the capillary by means ofcylindrical lenses. The capillary was fixed in a straightposition in a Plexiglas holder with exception to the inletand the outlet portion that were dipped into the respectiveelectrolyte vials. The fluorescence was collected with animage intensified CCD camera (Spectroscopy Instruments,ICCD-576E, Gilching, Germany) equipped with a 35 mmcamera objective. The distance from the capillary to theCCD was about 30 cm. The CCD camera was cooled to -20°C to lower the dark current of the detector. In front ofthe CCD a cut-off filter (Schott, GG530, Mainz, Germany)was placed to remove stray light at the laser wavelengthwhile transmitting the fluorescence light. The fluorescenceprofile across the capillary detected by the CCD was readout, digitized, stored and displayed on a PC in real-time.The principle of the recording of a fluorescence profile isshown in Fig. 2. The pixels along a horizontal streak of the

CCD, in the pixels where the fluorescence is recorded,were vertically binned together and shifted downwards tothe CCD shift register and the horizontal fluorescenceprofile was displayed as in the lower part of Fig. 2. Onerun typically lasted for a few minutes producing about500-1000 consecutive fluorescence profiles. In the case ofslower migrating samples the signal from up to 10individual profiles (or scans) were added together toincrease the signal-to-noise ratio.

RESULTS AND DISCUSSION

The work on a multipurpose system for fluorescenceimaging in analytical chemistry, in particular, in CE hasbeen in progress for a few years. The work is divided intotwo parts; the work on optimising the performance of thedetection system and applications in analytical chemistry.One important parameter that has to be taken intoconsideration is the detection limit of the system. In orderto determine the detection limit, a series of CE

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Fig. 2. Principle of data acquisition. The fluorescencefrom the capillary is imaged as a streak onto the CCD. Thesignal is binned and shifted to the shift register and readout to the PC. The fluorescence intensity as a function ofposition along the capillary is displayed on the PC monitorat a repetition rate of the data integration time. (Note thatthe image and profile displayed here are not recorded atthe same time).

experiments with fluorescein were performed. Thefluorescein was electrokinetically injected into thecapillary. One example of such a recording is shown inFig. 3. The time scale starts shortly after injection and afluorescein peak, moving from left to right is followed inthe figure. For clarity, only every 7th fluorescence profilesare shown in Fig. 3. The signal-to-noise (SIN) ratio wascalculated from a mean value of the S/N ratio of theindividual profiles in Fig. 3. Three recordings at differentconcentrations were made and the corresponding S/Nratios are shown in the insert in Fig 3. From this curve thelimit of detection for fluorescein was estimated to be about5x1010. It is important to note, however, that since the

SIN ratio presented in the insert was a mean value, severalindividual profiles had a higher SIN ratio. Thus, when thefull 5 mm. recording is observed, e.g. as a video recording,even smaller concentrations are possible to detect. Thefluctuation in fluorescence intensity between differentelectropherogram is due to fluctuations in laser pulseenergy.

Real-time imaging of capillary electrophoresis givesexcellent opportunities to follow a separation on column.An example of this is shown in Fig. 4a, where selectedelectropherogram from a DNA size ladder separation areshown. The DNA mixture (Hae III restriction digest of X174 RF DNA, lower part) was almost separated alreadywhen it entered the visualised portion of the capillary,which was placed a few cm from the inlet. It is interestingto note that a separation can be achieved in a capillarylength of only a few cm. This was also confirmed with acommercial capillary electrophoresis equipment (BeckmanP/ACE 2050). In Fig 4b a close-up from Fig. 4a is shown.Here, the gradual separation of one of the peaks dividinginto two peaks, is shown. With the present set-up it wasnot possible to visualise the inlet and the first cm of thecapillary although this would have in a better wayillustrated the initial separation of the DNA mixture. Thismatter will be discussed in the next section.

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Fluorescenceline profile

Fig. 3. Selected electropherogram from an electrophoreticrun with fluorescein. The concentration was 20 nM. Theelectropherogram are presented as fluorescence intensityvs. pixel and are separated by 15 sec. in time. The insertshows the calculated S/N for the run in Fig. 3 and 2additional runs (not shown here).

Pixel

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Fig. 4. (a) Fluorescence profiles from an electrophoreticseparation ofa DNA mixture (Hae Ill restriction digest of

X 174 RF DNA). Each electropherogram is presentedasfiuorescence intensity vs. pixel and are separated by 15sec. along the time axis. (b) Close-up from (a) showingthe gradual separation of the peakfirst appearing at 1.25mm movingfrom the left to the right.

One limitation of CE is the lower concentration sensitivitycompared with HPLC. This limitation can, however, beovercome by different preconcentration methods. A verypowerful preconcentration method is isotachophoresis(ITP). Real-time fluorescence imaging is an excellent toolfor illustrating the dynamics of ITP. An example of real-time fluorescence imaging of ITP is shown in Fig. 5.Rhodamin B was chosen as a test sample for theexperiments and was dissolved in ethanol and diluted in 6-aminocaproic acid (EACA) to a concentration of iO M.The leading electrolyte was EACA and the terminatingelectrolyte was acetic acid. As can be seen, the samplestarts to accumulate in the left of the detection window.This is the location of the border between the sample zoneand the terminating electrolyte. As the whole train movesfrom left to right, more and more Rhodamin is "collected"from the sample zone and is concentrated in the border.Note also that the fluorescence from the Rhodamin in the

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Fig. 5. Isotachophoretic preconcentration ofRhodamin Bin ethanol/EACA. . The fluorescence intensity is shown asa function ofposition along the capillary and time afterinjection.

sample zone that is not yet collected can be observed as abroad continuum to the right in the first profiles at earlytimes. The Rhodamin will eventually be collected by thepeak if the ITP is allowed to proceed for long enoughtime. This clearly illustrates the advantage of real-timeimaging as a detection method for preconcentration. If theITP is terminated too early only a portion of the sample iscollected. On the other hand, the liT must not beterminated too late for practical reasons. With thistechnique we can easily determine the optimal time toterminate the ITP and initiate a CE. In this experiment wedid not include the CE since it was not the object of thisinvestigation.

SYSTEM DEVELOPMENTS

Two very important parameters for a successfulelectrophoresis as for all other analytical techniques, areresolution and detection limit. We have shown here, e.g. inFig. 5, an excellent resolving power of the imagingsystem. In the liT measurements the resolution waslimited by the pixel size and number of pixels (576) perrow of the CCD. In the future, a CCD camera with twiceas many or even more pixel per row, will be used. Thiswill facilitate an even better system resolution. However, itshould be pointed out, that optical resolution andcollection efficiency are related parameters. Hence, it isvery difficult to simultaneously achieve a high resolutionand an efficient collection of the fluorescence. If opticswith a low f# is chosen to increase the light collection, theoptical resolution will be accordingly decreased. Note thatthis is in analogy with the width of the detection window

200Pixel

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Fig. 6. Set-up of optical fiber array for fluorescencecollection. The array consists of 400, 100 .t m diameteropticalfibers. Thefibers were arranged to a single row atthe input and rearranged to six rows at the output of thearray as indicated in the Fig. The fluorescence wasimaged onto the CCD using two /2 lenses. A shield waspositioned close to the capillary to lower the amount ofautofluorescencefrom thefiber array.

for conventional CE. In other words, if a high collectionefficiency of the optics is achieved, precautions must betaken in order not to deteriorate the optical resolution ofthe system.

The main challenge of the imaging system is to image along section of the capillary. As the length of the imagedsection increases, the lower the solid angle of detection hasto be to avoid aberrations and, thus, a poor resolution willresult. One approach to this problem is to use an array ofoptical fibers.

Fiber Array Construction

An optical fiber array, 50 mm wide was constructed of 400optical fibers (100 jtm core, 110 m cladding, 125 .tmcoating), each 30 cm long. The fibers were glued withepoxy between two pieces of floatglass (55 mm wide, 8mm thick, 25 mm length), which were bevelled to an angleof 20 degrees. These are used as a fixture during thepolishing procedure. The output was divided into six lineargroups, where each group was glued with epoxy to amicroscope slide and these packets were glued on top of

each other. Precautions were taken to make sure that theorder of the fibers were the same at the collecting and theoutput sides. After the epoxy was cured, the ends of thefibers were covered with Norland Optical Adhesive no. 68.This adhesive is harder than epoxy and more suitable topolish. The collecting end is polished with the floatglass asbearer and a 20 degrees edge will occur in the middle ofeach fiber. A free hand polishing will smoothen the edgeand the rounded shape will enhance the collection of thefluorescence light from the inner of the capillary. Theoutput end is polished flat. The fiber array and its positionin the overall set-up is shown in Fig. 6.

Data Acquisition and Results

The emission from the fluorophores in the capillary wascollected with the linear fiber array. The collecting endwas fixed as close as possible to the capillary and thereforeit was necessary to shield the fiber array from theexcitation light with a metal plate, carefully adjusted toavoid fluorescence emission from the collecting end. Theoutput end, divided into six smaller arrays, was arrangedto a quadratic configuration to produce a more compactarray. The output was imaged on to the 576 x 384 pixelimage-intensified CCD camera through a lens system andcut-off a filter, which prevents the scattered excitationlight to enter the camera. The compact arrangement of thefiber array output facilitates the use of small diameteroptics between the fiber array and the CCD camera andavoids the problem of minifying the image of thecapillary. In the case of the fiber array, the CCD wasbinned into 6 groups producing 6 line profiles in contrastto the previous arrangement that yield single profiles foreach laser shot. A complex electropherogram using theoptical fiber array arrangement is shown in Fig. 7. Thesample was Rhodamin B at a concentration of 1x10° M.Each of the six line profiles display a section of thecapillary and these profiles can easily be post-processed(added together) to form a complete electropherogram.

The gain of using the fiber array arrangement is twofold.First, it yields an improvement in terms of opticalcollection efficiency in relation to the camera objectivepreviously used. A theoretical calculation showed that asmuch as a factor of 30 can be gained by the fiber arrayarrangement in comparison with the camera objectiveapproach. The result of the calculation is graphicallypresented in Fig. 8, where the solid angle of the opticalfibre is shown as a function of distance from the capillary.A value of 1 corresponds to the collection efficiency equalto that of the camera objective arrangement. The results ofour preliminary studies were also in accordance with thecalculations. However, it should be pointed out here, that

friput of array

Output ofarray

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orescenceLens coupling

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Fig. 7. Fluorescence line profiles using the fiber arrayarrangement. The sample was 1x101° Mfiuorescein. Allsix profiles were simultaneously recorded at 35 secondspost injection.

the camera objective arrangement would probably bepossible to improve in terms of collection efficiency. Onthe other hand, if a capillary with a smaller diameter isused, the gain factor of the fiber array arrangement wouldalso increase. Secondly, with the fiber array arrangement,light is preferentially collected from the inside of thecapillary, which is the region where the fluorescence fromthe sample is produced. Thus, the fiber array arrangementadditionally contributes to an increase of the signal-to-background fluorescence ratio. Also this has beenexperimentally verified.

Distance (mm)

Fig. 8. Theoretical calculation of the useful solid angle inrelative units (compared with the camera objectiveapproach) as a function of distance from the capillaryouter wallfor thefiber array.

signal from a certain sample would be assumed to move acertain number of pixels between each recorded frame.The advantage would then be that the signal would beadded for all frames, whereas stationary fluorescencebackground from e.g. the capillary and adsorbedfluorophores would cancel out, and the S/N ratio wouldthus be enhanced. Early results using this method suggestsa substantial improvement in limit of detection.

In this paper the experimental set-up for a fluorescenceimaging detector is shown. We have also shown a fewexamples of the use of the detector and discussed some ofthe advantages with this system. The strength of thismethodology lies in the fact that the whole or a substantialpart of the capillary may be monitored simultaneously,with applications to new system developments but maybealso for pure analytical work. In addition to the workpresented here, measurements were performed withapplication to capillary chromatography andelectrochromatographyl5. This methodology can be usedfor many different kinds of analytical techniques whereoptical access can be obtained to a column or a plate.Valuable information can be obtained from measurementsof the injection site or from transition zones such as fits inpacked columns and real-time imaging may be the methodof choice for system optimisation in many applications.

In addition to the above mentioned improvement of thelimit of detection, methods for computer assisted signalfiltration and integration have been taken intoconsideration. It is clear, that with the knowledge of themigration speed of the samples, the individual peaks canbe followed and a signal integration over a longer timeperiod (e.g. 1 mm.) can be employed. In such a method the

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ACKNOWLEDGEMENTS

Valuable discussions with J.M. Mesaros and A.G. Ewingare gratefully acknowledged. Martin Andersson isgratefully acknowledged for valuable computer support.This work was supported by the Swedish Research Councilfor Engineering Sciences, the Swedish Natural ScienceResearch Council, the Carl Trygger Foundation, theCrafoord Foundation, the Royal Physiografic Society ofLund and the Magnus Bergwall Foundation.

REFERENCES

1 . S. Nilsson ENZYMES; Separation and Determination inPhysiological Samples, in: Encyclopedia of AnalyticalScience. Academic Press Limited UK Chap. 46 (1995)

2. J.W. Jorgenson, and K.D. Lukacs, High-ResolutionSeparations Based on Electrophoresis and Electroosmosis,J. Chromatogr. 218, 209-216 (1981)

3. 5. Hjertén, High-Performance Electro-phoresis TheElectrophoretic Counterpart of High-Performance LiquidChromatography, J. Chromatogr. 270, 1-6 (1983)

4. C.A. Monning, and R.T. Kennedy, CapillaryElectrophoresis, Anal. Chem. 66, 280R- 314R, (1994)

5. M. E. Swartz and M. Merion, On-line samplepreconcentration on a packed-inlet capillary for improvingthe sensitivity of capillary electrophoretic analysis ofpharmaceuticals, J. Chromatogr., 632, 209-213 (1993)

6. F. Foret, E. Szökö and B.L. Karger, On-columntransient and coupled column isotachophoreticpreconcentration of protein samples in capillary zoneelectrophoresis, J. Chromatogr. 608, 3-12 (1992)

7. T.M. Olefirowicz, and A.G. Ewing, CapillaryElectrophoresis in 2 and 5 mm Diameter Capillaries:Application to Cytoplasmic Analysis, Anal. Chem. 62,1872-1876 (1990)

8. B.L. Hogan, and ES. Yeung, Determination ofIntracellular Species at the Level of a Single Erythrocytevia Capillary Electrophoresis with Direct and IndirectFluorescence Detection, Anal. Chem. 64, 2841-2845(1992)

9. 5. Birnbaum, J. Johansson, P.-O. Larsson, A.Miyabayashi, K. Mosbach, S. Nilsson, S. Svanberg andK.-G. Wahiund, Detektorer for separationsprocesser,Swedish patent application (March 17, 1992), Methodand detector for separation processes, PCT/SE93/00305(1993).

10. S. Nilsson, J. Johansson, M. Mecklenburg, S.Birnbaum, S. Svanberg, K.-G. Wahlund, K. Mosbach, A.Miyabayashi and P.-O. Larsson, Real-time fluorescenceimaging of capillary electrophoresis, J. Cap. Elec. 2, 46-52(1995).

11 . S. Nilsson, Selective electroseparations in capillaries,Proc. Kemistdagar i analytisk kemi (eng: Chemists days inanalytical chemistry), Junel4-18, 1993, Lund, Sweden

12. J. Johansson, S. Nilsson, D.T. Witte and MaritaLarsson, Real-time fluorescence imaging ofisotachophoresic preconcentration for capillaryelectrophoresis, submitted to Anal. Chem.

13. J. Wu and J. Pawliszyn, Imaging detection methods forcapillary isoelectric focusing, American Laboratory oct,48-52 (1994)

14. J.M. Mesaros, G. Luo, J. Roeraade and A.G. Ewing,Continuous electrophoretic separations in narrow channelscoupled to small-bore capillaries, Anal. Chem. 65, 33 13-3319 (1993)

15. 5. Birnbaum and S. Nilsson, Protein-based capillaryaffinity gel electrophoresis for the separation of opticalisomers, Anal. Chem. 64, 2872-2874 (1992)

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