CA1309058C

CA1309058C - Laser-excitation fluorescence detection electrokinetic separation

Info

Publication number: CA1309058C
Application number: CA000518429A
Authority: CA
Inventors: Richard N. Zare; Ernst Gassmann
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 1985-09-18
Filing date: 1986-09-17
Publication date: 1992-10-20
Anticipated expiration: 2009-10-20
Also published as: DE3650483D1; EP0216600A3; US4675300A; JPH11326277A; EP0216600A2; JPS6273138A; JPH07111398B2; EP0485370A3; EP0216600B1; EP0485370A2; DE3650483T2; JP2002139475A

Abstract

LASER-EXCITATION FLUORESCENCE DETECTION
ELECTROKINETIC SEPARATION

Abstract of the Invention An electrokinetic process and apparatus employing coherent radiation-excited fluorescence for detection is disclosed. Specifically, the detection of electro-kinetically-transported target species as said species pass in a support electrolyte through a detection volume can be carried out with improved efficiency when the detection event associated with the passage of the species through the detection volume is a change in emitted light, in particular fluorescence, which emitted light has been generated by a beam of electromagnetic radiation supplied on-column by a coherent source. The use of coherent radiation source permits the radiation of a well-defined wavelength to be delivered on-column to the sample without hazard and without appreciable loss of intensity and without unwanted interference from scattered light, as compared to incoherent light sources. The use of coherent radiation increases sensitivity because interference from Raman and Rayleigh scattering is minimized. This detection system allows mixtures of compounds to be analyzed and/or separation with improved efficiency. It is possible, using the detection system of this invention, to detect amount of targets in the range of a femtomole (10-15 moles) or less.

Description

LASER-EXCITATION FLUORESCE~CE DETECTION
~LECT~OKINETIC SEPA~ATION

BACKGRo~c~ OF TH~ I~VENUIoN

Field of the Invention Thi~ invention relates to the field of elec~rophoresi~. More particularly, it concern~ an i~proved proces~ and appacatus for ca~rying out and detecting electrokine~ic separation6 in open-tubular capillarie~.
Brief Descciption of ~he Drawin In the drawings:
Figure 1 is a cros6-sectional view of a liquid-filled tube illustrating the proce~s of electeoos~otic pumping.
FiguLe 2 i6 a cross-sec~ional view of a liquid-~illed tube illustrating the pcoces~ of electrokinetic ~eparation.
. Figure 3 i8 a partially schematic block diagram and partially cross-sectional view o~ one type of apparatu~ for ca~rying GUt the pre~ent inven~ion.
Figure 4 i~ a c~os6-sectional view of an on colu~n optical fluore6ce~ce-~ea~uring cell for use in accord with the invention.
Figures 5a and 5b are two repre~en~ative el~ctropherogram6 showing sepacations achieved and detected u~in~ the pce~ent i~vention.
esceiption of ~ackqround Info~ation In 197~, Pretorius, et al. (J. Chro atoqr., 99, 23) described the concept of electroosmo~is which they ~tated to be the flow of a liquid, in contact with a ~olid ~ur~ace. under the in~}uence of a tan~entially applied elect~ic ~ield. ~hey attributed the electroosmo~ic ~low to the for~ation Oe an electric - 2 ~3~) ~3~

double layer, at the 601id-liquid inter~ace, due to the preferential ad~orption of ion~ on the ~ur~ace. Thi~
~ra!16pQrt proce~6 can be vi6uali~ed wi~h raference ~o Figure 1. In Figure 1, a ~all bore double open-ended tube 10 i6 ~hown in cu~ away CLOS6 ~ect~on. The tube is filled with a conductive liquid 11 someti~es referred to herein as a "6upport electrolyte~'. The wall of tube 10 contaàn6 prefe~entially ad~orbed po~i.tive ions 12.
(Depending upon the ~aterial of tube~ 10, the adsorbed charge could be negative, in~ead.) Po~itive ion~ 12 attract anion6 13 from conductive liquid 11 and ~et up an ele~tlic double layer 13. Thi~ preferen~ial attraction of anions to the wall re~ults in a net exces6 po6itive cha~ge in the body of li~uid 11. Thu6, when an electric potential i6 applied, such a~ a 30 kV potential between electode6 15 and 16, located at the ends o:E the colu~n of liquid 11 contained within tube 10, the po~itively charged liquid move~ toward the cathode.
Pretor iU6 et al. propo~ed the use of this proce6~ in ~hin-layer and high ~peed liquid chromatogra~hy 6ettings.
In 1979, Mikker~, et al. ~J. Chromato~r. 169, 11) desc ibed the use of narrow-~ore (e.g. 0.2-0.35 mm i.d.) tubes for hi~h perfor~ance zone electrophoresi6.
~o~e Lecently, J.W. Jo~gen60n ~d K.D. Lukac6 have reported (J. C~ro~a~oq. 2~8 11981), Z09: Anal. Che~. 53 (1981), 129B: and Science 222 (1983), 266) the use of 75 ~m glass capillarias to carry out ~uch ~epara~ions. Tsuda, et al, reported ~imilar work in J.
Chromatoq. 248 (1982~, 241 and J. Chro~ato~. 264 (19B3~, 385. An advantage to the u~e of capillary channels i~
that joule heating effects which distulb the ~ample flow are minimiæed.
The ~eparation proces~ relies upon the elec~roosmo~is e~fect j~t de6cribed and upon the differential effect of ~he elect~ic ~ield on ~olutes in the liquid mediu~ depending upon their posi~i~e, neutral or ~egative charge. These ~elated ~ffacts may be _ 3 ~3~

vi~ualized with refecenc2 to Figure 2. Figu~e 2 i~ a copy of Figure ~ but with ~ariou~ charged ~pecie~ 18 and 19 in llquid 11. Cationic species 18 i~
elect~o~horetically drawn toward cathode 16. Anionic specie6 19 i6 electropho~e~ically r~pelled by cathode 16. A~ hown in Figure 2, and a~ is usually ~he case, the velocity of the liquid 11 i8 larger than the elect~ophocetic velocitie6 of the 6pecie~ in ~olution ~uch that all the ~pecies can be 6een to move in the direction of the electLoosmotic flow but at dif~ering rateg. The co~ination of electroo6motic flow and electrophoretic ~ovemQnt i~ referred ~o in the lite~atu~e and herein a~ electrokinetic movement, an~ a sepa~ation which relies upon these two effect~ i~
cefereed to a~ an electrokinetic 6eparation.
~ oreove~, electeoo6motic flow has plug flow characteri6tc~ as oppo~ed to laminar flow characteri~tics. This favo~s high re601ution separations. One can, in theory, ufie an electrokinetic ~eparation to provide ~eparation of species in solution, and one should in principle be able to detect ~he~e ~eparation~. However, a6 stated by Jorqenson and Lukac6 in the conclu6ion o~ their Science ~eview article, "ThP
grea~est obstacle to ~ur~her development and utilization of capillaries tin ~uch separation methods] i8 the requi~ement of ex~remely 6ensitive de~ection, and ~ore types of detection with higher ~en6itivity are greatly needed." The ~ypes of detector6 used heretofore to indicate the presence of ~pecie~ a6 they move through electrokinetic ~eparatio~ column6 have i~cluded W
absorption and conductivity u6ed by ~ikkers, et al;
on-column fluorescence detection with lamp excitatîon u~ed by Jorgen60n and ~ukac~ and W ab~orption detection u~ed by T~uda, et al. David, et al, of the Oak aidge National La~orato~y in re6eacch report ORNL/TM-9141 in contract W-7405-eng-26 have di~clo6ed an on-colum~
lamp-excited fluoreecence detector 8y8tem and it~ use in ~3~

connection with a capillary electrophoresis system. The selection of a suitable detection system is rendered more difficult by the practical considerat:ion of operator safety when high voltages are present~ With electric potentials in the range of several tens of thousands of volts passing through the sample as it is being measure, the detector must be reliable and require no operator manipulation in or directly around the sample.
It is an object of the present invention to provide an improved detection method and system sought by the art.
It is a further object of this invention to provide new and more sensitive electrokinetic assay methods by employing this improved detection method and system.

STATEMENT OF THE INVENTION

According to one aspect of the present invention there is provided a fluoroassay method for detecting the presence of a target species in an electroosmotically pumpable fluorescible liquid sample which comprises~
a. placing said sample into one end of an electroosmotically pumpable-liquid-full narrow bore double open ended walled channel at least a section of which is translucent;
b. applying an effective electroosmstic pumping potential to said pumpable sample and pumpable liquid thereby transporting the sample through the channel;
c. irradiating the sample with coherent radiation of a wavelength e~fective to excite fluorescence in said sample; and d. detecting a change in the fluorescence emitted through the translucent section o~ the channel as the target species moves past the translucent section.
It has been found that the detection of .",,;

~,3~ ~&~

electrokinetically-transported target species as said species pass in a support electrolyte through a detection volume can be carried out with improved efficiency when the detection event associated with the passage of the species through the detection volume is a change in emitted light, in particular fluorescence, which emitted light has been generated by a beam of electromagnetic radiation supplied on-column by a coherent source. The use of a coherent radiation source - permits the radiation of a well-defined wavelength to be delivered on-colu~n to the sample without hazard and without appreciable loss of intensity and without unwanted interference from scattered light, as compared to incoherent light sources. The use of coherent radiation increases sensitivity because interference from Raman and Rayleigh scattering is minimized. This detection system allows 1 ~ixtures of compounds to be analyzed and/or separated with improved efficiency. It is possible, using the detection system of this invention to detect amounts of target in the range of a femtomole (10 15 moles) or less.
In an additional aspect, this invention provides a detector for indicating the presence of fluorescence species in a liquid sample comprising:
a. a narrow bore double open ended walled channel for containing the sample at least a section of which channel is translucent;
b. means for irradiating the sample with coherent J ~ ratiation of a wavelength ef~ective to excite ~luorescence in - the fluorescent qpecies; and c. means for collecting through the translucent section fluorescence emitted by the fluorescent species.
This application also discloses the electrokinetic separation of racemic mixtures into their optically active constituents which occurs when an optically active (i.e.
chiral) support liquid is used in the electrokinetic -5a-separation process.
Thus, there is disclosed a process for separating chiral compounds which comprises:
a. placing said compounds in an electroosmotically pumpable chiral support electrolyte, in an electrokinetic zone; and b. applying an effective electrokinetic potential to the suport electrolyte in the zone for a period effective to separate the chiral compounds.
In one embodiment, the process separates and detects the separation of mixed chiral compounds into enantiomers by the use of a cltiralsupport electrolyte. The chiral support electrolyte has the property of containing one or more chiral species that will preferentially interact with one member of the mixture of enantiomers causing it to preferentially acquire a different electrokinetic mobility than the other members of the mixtureO The modification of electrokinetic mobility can take the form of preferentially associating a charged species with one enantiomer. It can also take the form of changing the charge density o one o~ several charged enantiomeric species by preferentially associating unchanged bulking groups with it or preferentially associating groups which vary one enantiomer's ability to co~bine with or attract charged species from the support electrolyte.

Descri tion of Preferred Embodiments P __ _ _ The present invention will be first described by the following examples. These examples are provided to illu~teate one mode ~or prac~icing the pre~ent invention and are not to be con6trued a6 limiting the 6cope of the invention a~ defined by the a~pended claim6.

F~xamvle I
An electrokinetic 6epara~ion sy6tem emploring a la6er-excited fluoLescence detec~or wa~s construc~ed.
This ~ytem will be described with reference to Figures 3 and 4. The system included a fu6ed-s1Llica capillary 30 (~ewlett-Packard Co.) which was 7S c~ in ~otal length and which had a 75 ~m in6ide diameter. The capillary had an opaque polyimide protective coating 31 on its outer surface, a section of which was removed wi~h flame to give a translucent section 32. Capillary 30 was liquid-filled with a support electrolyte containing 5 mM
l-histidine, Z.5 mM CuS04 5H20 and 10 mM
am~onium acetate adiusted to pH 7-8 by the addition of NH40H. Feed container 34 and outflow container ~5 contained suppor~ electrolyte a~ well, ~o that liquid-filled capillary 30 ceeated a continuous liquid and electrical connection between them. A -30 kV
potential from poWe ~upply 36 was applied acros6 electrode~ 37 and 39 by means of wire~ 40 and 41, respectlvely, and gave a complete electrical circuit.
The inner surface of capillary 30 was such as to preferentially adsorb po6itive ions: ~his cau6ed cations in the electrolyte to be preferentially drawn to the capillary ~all as a double layer and in turn imparted a net positive charge to the body of the support electrolyte in capillary 30. When a -30 kV potential wa6 applied to the liquid in outflow conductor 35 by electrode 39, it cau6ed thi~ positively ~harg~d liquid to be electroosmotically drawn from capillary 30 into containe~ 35 and to draw additional electrolyte out of ~3~
.

container 3~ into capillary 30. The current flow wa~
30-33 ~A. The linear velocity of liquid ~hrough capillary 30 was abou~ m/second.
CapillaLy 30 pas~ed through :Elow cell 42 and wa6 held in position by fittings 44 amd 45 with translucent sec~ion 3~ which defined a detecti~n volume in the c2nter of flow cell 42.
Flow cell 42 included an on-column fluorescence detector which used a helium-cadmium laser 46 (Liconix.
Model 4240B, Sunnyvale, CA) having a 5 mW continuous wave output at 325 nm wavelength a6 excitation source.
The ~iltered output of the laser was focused via lens 47 on optical eiber 49 (B0 ~m ~used silica material) which carried the ~eam of laser light into ~low cell 42. Fiber 49 was held in po~ition by a 3-axis positioner head 50 with its output focused on translucent section 32 of capillary 30. Fluore~cence emanating from the fluid being transported through the detection ~olume was collected perpendicular to the excitation beam by a 0.6 mm fused 6ilica optical fiber 51. Fiber 51 was held in po6ition by 3-axi~ po6itioning head 5~. The fluoLe~cence collected with fibec 51 was passed through a high pa~s cut off filter 53 and a fast monochcomator 5~ (Centronic Model Q 424gB) which served ~ ~elect a variable wavelength bandpass, and a photomultiplier 55, ~he outpu~ of which was amplified by ~eans of a Keithly Instru~ent~ Inc. Model 480 picoammeter (not shown) and fed through line 56 to stcipchart recorder 57.
In use, a sample was injected in~o capillary 30. ~his was accompli~hed by dipping the anode end of capillary 30 into the liquid sample contained in container 5~, connecting anode lead 40 to electrode 59 and turning on the high voltage ~or a sho~t peciod ~5-10 13Q~B

seconds) ~t 6 kV. This caused a defined 1 to 5 mm long "plug" of sample to be drawn into column 3G.
A first ~ample, made up of 1(1 M of each of each of the ~ollowing dan~yla~ed amino acids:
d-Tyr l-Tyr d-Phe l-Phe d-Asp l-~sp d-Glu l-Glu was prepaLed in the ~ame liquid used as the support electLolyte. The labeled amino acids weee purcha~ed from Sigma Chemical Co., St. Louis, M0, or prepared by the methods o~ Tapuhi, et al. (I), Anal. Biochem. 115, 123 ~1981), and Tapuhi, et al. (II), J. Chromato~., 205, 325 (1981). The support electrolyte liquid contained a copper-II comple~ wi~h l-hi~tidine. This compIex i~
optically active and thus the suppor~ electrolyte was a chiral ~upport electrolyte. ~5 the four optically active-pair6 moved through column 30, they were separated from one another. Al60, separation occurred between the members of each pair of optical i~omers. ~s each of the eight separatea ~pecies passed through the detection voll~me they were detected. In this case, their dansyl labels emitted fluorescence which wa~
detected. The cesults are 6hown in Figu~e 5a a~ an electropherogram, in this case a plot o~ laser-excited fluo~escence signal ver~us ti~e. Then one half of the l-histidine in the support liquid was replaced with d-his~idine 80 as to yield a 1:1 mixture of d- and l-histidine of equal total concentra~ion to that used in the previous experiment. The experiment was repeated, 13 ~ ~ q~ ~ ~
g using this nonchiral support electrolyte and no separa~ion of the d and 1 isomer6 was observed. Four individual peaks were ~ound, one ~or each a~ino acid, as shown in Figure 5b. Similarly, when the l-histidine was completely replaced with d-histidine, ~eparation occurred with the order of the dansyla~ed d and l amino acid isomers being reversed.
These three experiments thus illustrated tha broad aspect of this inven~ion that measurement of changes in fluorescence excited on-column by a coherent (i.e. laser) energy &ource i6 an effective and ef~icient method to detect ~he pre6ence o~ species separated in electroosmotically pumpable li~uids in narrow bore channels by electrokinetic processe~. The6e experiments used a detec~ion volume of about 0.5 nanoliters. The specie6 being detec~ed had concentrations in the electrokinetically-pumpable support electrolyte of about lO moles per liter. The signal to noi~e ratios observed were greater than lO0:1. Combining these factors, one finds that the present detection sy&tem can detect 5 x lO moles (that i~, les~ than a femtomole) of target species.
These experiments also illustrate another aspect of this invention which i6 that electrokinetic separation processes can be used to separate optical isomers. It i~ believed that this separation of optical isomers is unprecedented both in terms of speed and in terms of sensitivity.

Example 2 The e~periment of Example l was repea~ed using a wider range of amino acids. The am;no acids were used in various combinations. The results are listed in Table l.

-lQ-Table 1: Migration times ~td, t~ t values ~see Eq. (1)~, and relative peak areas (~d~ ~1) for some d,l-dansyl-amino acids, taken under the condi~ions explained in the text at pH ~Ø The Leproducibility for migration ti~es i8 betteL than ~3% relative standaLd deviation (R. S .D~ ) units arld for relative peak areas is about i5% R.S.D.
~mino Acid td(min) tl(min) ~t x la Ad . . _ _ 10di-DNS-Ty~ 6.30 6.36 -0.95 1.5 1.8 DNS-Met 6.75 6.71 0.63 1.6 1.6 DNS-~B 6.83 6.75 1.2 1.3 1.0 D~S-Phe 6.80 6.91 -1.6 0.18 0.36 DNS-Ser 7.00 7.00 0.0 0.46 DNS-Val 7.40 7.32 1.1 2.1 1.8 di-DNS-Cys7.90 8.00 -1.3 0.37 0.39 DNS-~sp 9.B0 9.95 -l.S 0.18 0.24 DNS-Glu 10.30 L0.10 1.9 1.71 1.38 DNS-Cys-~cid 10.40 10.70 -2.9 0.15 O.Z9 N-dansyl-~-aminobutyric acid N-dansyl-cysteic acid The excellent signal-to-noise ratios observed in Example 1 and illustrated in Figures 5a and 5b were again observed with this wider range of samplesu The sig~al-to-noise ratio indicated that this wider range of labeled amino acids could be de~ected at femtomole (10 mole) or lower le~els by the pre~ent simple experimental arrangement. I~ is 6een that baseline resolution is pos~ible when the absolute magnitude of the quantity ~t _ (td-t~ 2 (~d+tl)3 exceeds 0.01, where ~d and tl are the migration times of the d and 1 optical isomers. Replacement of l-histidine by d-his~idine in the supp~rt electLoly~e rever6e6 the ~igration order of the d ,and 1 amino acid~, i.e., it changes the sign of ~. Figure 5a also ~hows that the fluorescence ~ignals differ for the two optical i~omers of ~he sa~e dansyl-amino acid. In Table 1 ~he mig~ation times, ~t values, and ~ela~ive peak areas referred to l-arginine as an in~ernal standard for ten di~fe~ent d,l-dansyl-amino acids, are given. In all cases except d,l-serine, eesolution is achie~ed, bu~ the signs of ~t varies with the amino acid. ~lthough the migration order of the amino acids ob6erved differs rom that found in HPLC (see Lam, et al., J. Chromatog~, 199, 295 (~980), and J. Chromatog., 239, 451 (1982), the migration order of the enantiomers, as shown by the sign of ~t, is the same.
~lthough not wi~hing to be bound by any particular theoy of how the present invention works, chiral recogni~ion with the Cu~II) l-histidine support electroly~e can be explained by mixed chalate complexation to form diasteromeric ternary complexes.
The following explanation is consistent with all the present data. Amino acids bound to Cu(II) l-histidine migrate faster ~han fre2 amino acids bacause the Cu(II) l-histidine carries positive charge under the pH
condition~ u~ed. However, amino acids bound more strongly ~o Cu(II) l-hi~tidine show a ~eaker fluor2scence signal causad by quenching ~rom association with the copper ion. Hence, tha more complexed enantiome~ migrates ~a6ter but show~ a lower ~.3~

fluorescence signal caused by quenching from association with the copper ion (see Figure 5a and Table 1).

ExamPle 3 Bovine insulin was ]abeled with fluorescein isothiocyanate (FITC). Ten milligrams oE the in6ulin (Sigma Chemical Co.~ weLe dissolved in 6 ml of 0.5 M
carbonate-bicarbonate buffeL (pH 9.5). After the addition of 1.~ mg of FITC (Sigma Chemical Co.~ the mixture was stirred overnight at 4~C to couple the FITC
to the in6uli~.
The reaction mixture was placea on a 1.5 ~ 40 cm SephadexTn C-25 silica gel ~iltration column and eluted with 0.01 M phosphate-buffered saline of pH 7.4 at a flow rate of about 1 ml/minute. Unreacted FITC
adsorbed to the æilica gel. A yellow band containing FITC-labeled insulin wa~ taken off the column and diluted with 5 parts of water. The zwi~terionic buffer CHES ~2-~N-cyclohexyla~ino]ethane ~ulfonic acid~ was added to a 10 mM level which brought the pH of the solution to 9.
The ~olu~ion of labeled insulin was used in the appartu~ of Example 1. Sample was placed on the capillary column by applying a SkV potential for 1 ~econds. Then the ~ample was electLokinetically tzansported through the capillary u6ing a 30kV
poten~ial. Current flow was 5.6 milliampe~es.
On-column lasar excitation was at 325 nm- Emission was monitored at 510 nm. Between 5.5 and B.O minute~, ; 3U a group of ~eaks corre~ponding to the FITC labeled insulin were noted.

E~-!D~
The experiment of Example 3 is repeated twice with changes. In Example 4, in place of labeled insulin, the pro~.ein FITC-labeled concanavilin A
(available from Sigma Chemical Co. or Fluka Chemical Corp., Hauppauge, N.Y.) is used. The feed mixture placed on the capillary column contains 0.5 ~g/ml of the protein and 20 ~M of a buffer which holds the pH to 8.
Peaks corresponding to the electrokinetically labelled protein would be observed 5 to ~0 minutes after the electrokinetic voltage is applied.
In Example 5, in place of labelled insulin, FITC-conjugated goa~ antihu~an gamma globulin in phosphate buffered saline (pH 7.2) (available from Sigma Chemical Co.) is used. ~gain, fluorescent peaks caused by the transpo~t of this immunoglobulin through the detection zone would be observed using the on column laser excitation method of this invention.
The invention i8, of cour~e, not limited to the embodiments just depicted. It can employ any configura~ion of narrow bore elongate walled channel in place o~ the capillary described above. In general term~, it is preferred to Ufie a channel that i~ not more than about 500 ~m across. Wider channels can give ri&e to excessive heating when the electrokinetic potential is applied. Preferred channels contain o~e or a plurality of channels each of which is up to about 500 ~m acro~s, especially from about S ~m to about 350 ~m acrcss. It will be appreciated, that the smaller the cross section of the channel, the ~maller the volume in the detection æone and ~hus the greater need for sen~itivity of the det0ction sy~t0m~. ~ecause of their easa of construc~ioQ, circular cross-section capillaries are preferred.

~L3~

The channel ~hould be of a length that is effective to achie~e separation of ~pecies under ~he electro~inetic ~orces. It will be appreciated that the longer the channel the greater the ti~le a sample will take to move through the channel and the greater the distance that the variolls specie~ will be separated fLom one another. ~e the same time band broadening takes place 80 that resolution is not improved by adding langth. These factors sugge~t pcactical limits to the channel length, although longer or shorter lengths could be used if de~ired. For example, good results are achieved with channel lengths as short as about 5 cm.
Similarly, the transpoct time through a channel becomes inconveniently long for many eoutine analytical settings with channel lengths longer than several meters.
Generally, channel lengths of from about 10 cm to about 200 cm, and especially from about 40 cm to 150 cm are preferred. Of course, longer and shortee channels, ~or example up to 4 or 5 meters or down to about 5 cm, can be used without depaL~ing from the SpiLit of this invention.
The elongate channel is constLucted of a material that has the properties of being durable and retaining its physical integrity in contact with the su~ort electrolyte, of being sub~tantially nonconductive 80 as to conduct negligible electricity and to generate negligible heat as the electrokinetic potential is applied to it, and of being able to take on a positive or negative charge on its inner surface. A
suitable ~aterial will have a conductance ~uch that preferably at least about 95% of ~he conductance of the channel and its contained elec~rolyte i6 through the electrolyte. In addition, it is neces6ary that a portion o~ the channel is ~ranslucent so a~ to pe~mit ~3~
.- -15-fluore~cence to be emitted from the ~a~ing liquid contained in ~he channel fo~ de~ection. The ~ranslucent portion of ~he channel can al50 be u~ed, if de~ired, a~
a port for inputting the coherent ~xcitiation energy in~o ~he sample. It i~ pos~ible to employ a 6eparate translucent detection zone section ~o which the channel is joined, but this require6 ~hat the connection of the channel ~o the translucent sec~ion be carried out in a manner tha~ doe~ not lead ~o exce~ive heating o~ arcing when the electrokina~ic ~oltage i~ a~plied across the connection or ~hat doe6 not lead to a disturbance i~ the liquid flow. One can avoid these problems by u~ing a continuou~ channel with a translucent section inherent therei~. Inor~anic materials such as quartz, glas~, and fused silica and organic materials such as Teflon*
(~olytetra~luoro~thylene and fluorinated ethyleneJ
propylene polymer~), polychlorotrifluoroethylene, aramide, nylon (polyamide), poly~inylchloride, polyvinylfluoride, polystyrene, polyethylene and the like may be employed.
~ pointed out herein i~ the Background of the Invention sec~ion, electroo~motic flow is achieved when ~he inner surface of the channel carries or ad60rb~
charged 6pecies. The inner sur~ace can be modified in order to vary its charge such as by contacting the surface with an acidic liquid so as to impart more posi~ive charges, or by contacting the surface with a basic ~aterial 60 a~
to impar~ moee negative char~es o~ by contactin~ the ~urface with a sylylating agent 80 as to redu~e the number of charges. (See Anal~ticaI Chem ~try, 53, ~o. 8, July 1981, 1298 for a description of the u~e of a tri~ethylsilane ~o reduce the charge density on the walls of a narrow chan~eI electrophore6i~ zon~ and thus to vary the tran~poct through the zo~e.) OtheL sureace (*) Trademark ~3~

modification technique~ that a~e hx~n to the ~ ~ay ~e u~ed a6 well.
The voltage applied across the sample should be , a voltage e~ective to cause di~cernable electLokinetie ¦ 5 motion without excessive heating. Voltages below about 1000 volts are generally too low and voltage6 above about 100 kV are not commonly found in conventional high voltage power supplies. Based on the~e practical limits, voltages from abou~ 3 kV to about 90 kV, and especially about 5 kV to about 60 kV, are preferred.
The polarity of the electric potential determines the direction that the elec~rically charged specie~ ~ove.
It i~ preferred for safety reasons to ha~e a~ much of the analy~is system at ground potential as po~ible.
' 15 The channel is ~illed with an j electroosmotically pu~pable support liquid. A liquid is electroosmotically pumpable when it is an elec~rolyt~, that iæ, when it con~ains or carries enough electrically ¦ charged species ~o conduc~ an electric current. Typical ~ 20 electroo6motically pumpable 6upport liquids contain, ; for axample, at lea~t about 0.0005 mole~ per liter of ionic specie~ and preferably from abou~ 0.001 to about 10 moles per liter of ionic 6pecies. Such level~
provide high rates of electrokinetic transfer. Most co~monly the ~upport liguid i~ water-ba~ed oc based on a ~ixed aqueous-organic liquid sy~tem. A ~ixed sy6te~ can be useful to help ~olubilize or ~uspend organic target ~aterials which have limi~ed solubili~y in water alone.
~ neat or~anic liquid that i8 ca~able of conducting elec~Licity can also be u~ed. Repre~entative material~
fOI U82 in the ~upport elect~otype include ~ater and mixed ~olve~ts mad~ up of water admixed with on* or more wa~ec-mi~cible organis ma~erials ~uch a~ lower (e.g~ 1 to 4 carbon ato~ alkanoic acids such aa acetic acid.

~ 3 ~

propionic acid, chloroacetic acid and the like; lower primary and ~econdary alkyl amines such as methyl amine, lower alcohol~ ~uch as e~hanol, methanol,and propanol;
lower polyols such as the lower alkane diol~; nitrogen containing liquids including acetonitLile, pyridine, piperidine and quinoline, lower ketone~ such as acetone and me~hyl ethyl ketone; lower alkyl amides such a~ DMF, N~methyl and N-ethyl for~amide, N-ethyl acetamide and the like. These mateeials are merely representative.
In practice any liquid which i8 itself an electrolyte or which can carry ionic species so as to be conductive may be usec1. ~ith any of these liquids, the 6upport liquid may contain added ionic matecials such as 6alt6, chelates and other complexes, acids, bases, buffers and the like. It is often p~eferred to use added ionic species which are æwitterions at the pH at which the liquid is pas6ing through the electLokinetic channel.
Representative matecials include alkali metal and alkaline ear~h metal and ~ransaction ~etal sal~s of inorganic acids: similar ~alts of organic acids, ammonium and organic base salt6 of such acids: halogen acids, organic acids, and other acid~: metal acids and hydroxide~, amines and other ba~es, and the like.
Typical ~witterions include amino acids and the Good's ~uf~ers marketed by Sigma Chemical Company, St. ~ouis, MO. These added ionic or ionizable materials may be selected from these broad classes generally at will 80 lonq a~ they aLe compatible with the other component~ of the sample and the support electrolyte as their primary ~unction is to increase the conductivity of the support electrolyte.
In one application, the presen~ invention separates and detects the separation of mixed chiral compounds into enantiomers by the use of a chiral ~3~

.
~lB-~upport elec~rolyte. A chiral support el~c~rolyte i6 a liquid which meets the above-described cri~eria but also has the proper~y of contaîning one or mo~e chiral 6pecies which will preferentially interact with one member of the mixture of enantio~ers causing it to preferentially acquire a different elec~rokine~ic mobility ~han the other ~embers sf the ~ix~ure. The m~dification of electrokinetic mobility can take the form of preferentially a~sociating a charged species with one enan~iomer. It can also take the form of changing the charge den6ity of one of several charged enantiomeri~ ~pecie~ by preferentially as60ciating uncharged bulking groups with it or preferentially associating groups which vary one enantiomer'~ ability to combine with or attract charged specie~ from the su~port electeoly~e. A variety of materials useful as chiral ~upport electrolyte components have been discribed in o~her ~etting~ in the li~erature. See, for example, the book Enantiomers, Raeema~e6, and Resolutions by J. Jacques, et al., John Wiley ~ Sons, New York, 19al, which is incorpo ated herein by reference. Suitable chiral ~pecies for inclusion in a chiral support electrolyte include, chiral anions, for example, anion~ of (~) camphor-10-sul~onic acid, (~) camphoric acid, (-) diben20yltartaric acid, (1) and (-) d-~2,~,5,7-tetranitrofluorenylideneaminooxy)propionic acid (TAPA, diace~onekelogulonic a~id, (~) and (-3 ~andelic ~cid, (-3 malic acid (~) and (-) tartaric acid, (~) and (-) 3-b~omo~amphor-9-sulfonic acid and the like;
~hilal cations, ~or axample cations of brucine, quinine, strychnine, (+) and t-) ~phedeine, (-~-2-a~ino-l-butanol (~) and (-) d~*hyI~ylam~ne, (~) and t-) ephedrine and the like; chiral complexes such as the copeer II and zinc II comple~es of l-aspartyl-l-phenylalanine methyl ester (the commercially available artiEicial sweetener, a6partame), copper II complexes with l-proline, l-histidine and l-pipecolic acid and zinc II complexes of L-2-alkyl-4-octyldiethylene~riamine, and the like.
The foregoing list of chiral specie~ for inclusion in a chiral support electrolyte is merely repre~entative. Any other chiral specie~ which preferentially in~eract6 with one member of ~he group of materials souqht to be resolved and ~hereby varies differentially the electrokinetic mobility of the members of the group of material~ may be employed as well. In selecting chiral ~pecie~ for inclusion one can often advantageously follow tha teachings in the art relating to resolution of enantiomeric mixtures, in particular the teaching relating to such resolutions by formation of diastereoisomers.
The present in~ention employs detection of laser-excited fluQLescence to determine the presence o~
electrokinetically-transported target ~pecies. The terms "fluorescent" and "fluoescence" are used bLoadly he~ein so as to include long- and ~hot-lived photolumine6cent spe~ies--that is, to include materials which might be thought of as "pho~p~ore~cent" or "fluore6cent", and to include emis~ions which might be con~idered to be "phosphorescence" of "~luorescence".
The change in emitted fluorescence which is detec~ed can be an increa~e in fluore~cence as would oc5ur if a fluorescing target specie~ traverses the detection zone. The change can be a decrease in fluorescence as would occur if a quenching or "transparent" spQcies tra~erses the detection zone in ~he pre6ence of a fluorescing background ~See, H. Small, et al., ~nal. Chem., 54 (1982), 46~-4~9.) It could also be a change in the ~pectral or temporal characteristics ~ A3~

of the fluorescing specie6. The change in emi~ted fluore~cance spec~rum which is detected can be a change in the intensity of fluorescence in a particular acceptance wavelength band resulting from a target specie~ transiting the de~ec~ion zone and having a fluorescence which has been shifted into or out of the particular acceptance wavelength zone. Such a wavelength ~hift can ce~ult from intramolecular a~ociation6 within the taeget ~pecias and the like. As shown in Fig. 3 this acceptance band can be easily defined by a wavelength band pa~s filter such as a high pa~s cut-off filter and a ~ast (high light throughpu~) monochromator. A change in temporal characteListic~ can be an increase or decrea6e in fluorescence lifetime, for example. It could al60 be a change in the polarizatio~
or angular distribution of the fluorescence which is shown to be a analytically significant effect by M.
Jolley, J. Anal. Tox., 5 (Sept/Oct 1981), 236.
While any of the above-de6cribed change~ in fluorescence or their equivalents can be used as the detected event, the ~08~ commonly studied change in fluore~cence - and thus, ~he change that i8 preferred in the present invention - is the increase in fluorescence that occurs when a fluorescent species traverses the detection zone. In some cases the target 6pecies being measured may be inherently fluo~escent, but generally a fluorescent label i6 covalently attached or otherwise as~ocia~ed wi~h the target specie~. The fluorescent label can be at~ached oe associated with the ta~get ~pecies when the sample i6 placed in the electrokinetic channel. Alternatively, the ~luore~cent label and the target species could interact during the species' passage ~hrough the electrokinetic channel. This interaction during pas~age can involve reac~ion of the ~3~

target specie~ with a material in the support liquid or wi~hin the elect~okinet;c channel wall~ could also result f~om an elect~ochemical ~eac~ion involving the target ~pecies. In any event, it i8 within the purview 5 of thi~ invention ~o u~e any combi~ation of ta~get~, - labels and conditions ~o long a~ tha target electrokinetically moves th~ough a detection 20ne and the detection eYent i6 a change in cohe~en~-radiation-~xcit~d fluorescence.
A wide eange of fluorescent label6 are well known. Repre~enta~ive common fluorescen~ labels in~lude materials containing ~uch prima~y functionalities as 1-and 2 aminonaphthalene, p,p'-diaminostilbe~es, pyrenes, quate~nary phenanthridine 6alt~, 9-aminoacridines, 15 anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bi~-p-oxazolyl benzene, l,Z-benzophenazin, retinol, bis-3-aminopyridini~m ~alts, hellebriganin, tetracycline, ste~ophenol, b~nzimidazolylphenylamine.
20 2-oxo-3-chromen, indole, xanthene~ ~-hydroxycoumarin, : phenoxazine, salicylate, strophanthidin, porphyrin~, tciarylmethane~, rare earth metal chelates, and flavin.
Individual fluorescent co~pound6 which have functionali~ies for linking or can be ~odified ~o incorporate such functionalitie6 include dan~yl chloride, fluoresceins 6uch a~ fluoro~cein and fluoroscein isothiocyanate, 3,6-dihydroxy-9-phenylxanthhydrol, rhodaminei~othiocyanate, ~-ph~nyl 30 2-amino-6-~ulfonylnaphthalen~, 4-ace~amido-4-isothiocyanatostilbene-2,2'-disulfonic acid, pyrene-3-~ulfonic acid, ~-toluidinonaphthalene-~-~ulfonate, N-phenyl, N-methyl 2 a~inonaphthalene-6-~ulfonate~ ethidium bromide, atebrine, auromine-0, 2-(9'-anthroyl~palmitate, dan~yl phospha~idyl-ethanolamine, N,N'-dioctadecyl oxacarbocyanine, N,N~-dihexyl oxacarbocyanine, mecocyanine, 4-(3'-pyrenyl)butyrate, d-3-aminodesoxyequilenin, 12-~9~-anthroyl)stearate, 2-methylanthracene, 9-vinylanthracene, 2~2'-(vinylene-p-phenylene)-bi~-benzoxazole, p-bi~[2-(~-me~hyl-t-pheyloxazolyl)]benzene, ~-dimethylamino-1,2-benzophenazin, retinol 10 bis(3'-aminopyLidinium) l,10-decandiyl diiodide, sulfanaphtheylhydrazone of hellebrigenin, chlortetracycline, N-(7-dimethylamin-4-methyl-Z-oxo-3-chromenyl) maleimide, N-~p-(2-benzimidazolyl)-phenyl]maleimide, 15 N-(4--~luoranthyl~ maleimide, bis(homovanillic acid), Lesazarin, 4-chlor-7-nitro-2,1,3-benzooxadiazole, merocyanine 540, resorufin, rose bengal, polyamine polyacid complexes of europium and tecbium, and 2,4-diphenyl-3(2H)-furanone.
De~irably, fluorescing species should ab~orb light above about 200 nm, preferably above 300 nm and more preferably above about 400 ~m, u~ually emitting at wavelengths greatee than 10 nm higher than the wavelength of the coherent light absorbed. The 25 fluorophore can be joined to the target covalently or noncovalently, directly or indirectly. When bonded covalently, the particular linking group will depend upon the nature of the two moieties to be bonde~ and their re~pective functions. A large number of linking 30 groups and methods for linking are taught in ~he literature.
Binding can also be achie~ed by the u~e of receptors. For instanee, an antigen fluorophore may be bound to a tacget thcough the intermediacy of a ~3~

eeceptor, e.g., antibody, for the antîgen. The receptor in ~ucn may be bound covalently oc noncovalently, e.g., through ad60~ption.
The target molecules ~hich are sepa~ated and 5 mea~ured using the pLe6ent invention c~n be selected virtually without limitation f~o~ the materials which can be fluorescently labeled and ~u6pended o~ dissolved in the support liquid. Mo~t commonlyO target species will be material o~ biological or eco]Logical o~ chemical lo interest The target moleculefi can be macromolecules such as polyamino acid~, i.e., polypeptides and proteins, ~oly~acchaeides: nucleic acids and oligonucleotides such as RNA, DNA and DNA fragments, and combinations 15 thereof. Such combinations of as6emblages include bacte~ia, vieu6es, chromo60me~, genes, mitochondria, nuclei, cell membrane~, and the like.
The wide variety of proteins and ~olypep~ides grouped according to ~imilar ~truc~ural feature~, 20 peoteins having particul~r biological function~, ~roteins related to ~peci~ic ~icroorganism6, particularly di~ease-causing microorgani6ms, etc.
Th~ following are ~lasse6 of protein~ rela~ed by ~truc~ure: protamine~, histone6, albumin~, 25 globulin~, ~clerop~o~eins, pho~phoproteins, mucop~ot~ins, chromoprotein~, lipoproteins, nucleopro~eins, glycoprotein~, proteoglycan~
unclas~ified proteins, e.g., somatot~opin, prolactin, in~lin, and pep~in.
There are, of course, numerous po~ential target protein~ found ln the human plasma which are important clini~ally and include: prealb~in, albumin, lipopeotein, thyroxin-binding globulin, Gc-globulin ~Gc 1-1, Gc 2~ c 2-2), cholinesterase, ~L3~ 8 myoblobin, transferrin, fibrinogen, immunoglobulin G
(IgG), immunoglobulin ~ (IgA), immunoglobulin ~ (IgM~, im~unoglobulin E (IgE) o~ yE-globulin (yE), complement ~actor~, blood clotting ~ac~ors, peptide and p~otein ho~mones including, ~or example, para~hyroid hocmone Sparathromone), insulin, glucagon, somatotLopin ~gro~th ho~mone), follicla stimulating hormone, luteini~ing hormone (inters~i~ial cell-stimulating hormone), gonadotropin, secretin, and gastcin.
Other macromolecular target materials of inte~es~ are mucopoly&accharides and ~olysaccharides derived from or present in ~icroorganisms such as coliform bacteria, saimonellae, shigellae, proteus species, pasteurellae, brucellae, aerobic spore-forming baccilli, anaerobic spore-fo~ming bacilli, mycobacte~ia, actinomycetes (fungus-like bacteria~, spirochetes, mycoplasmas, and the like.
O~her target species can include: eickett6ia (bacteria-like parasites), chlamydia, fungi, and viLuses, including adenoviruses, pox viruses, myxoviru6es, reoviruses Types 1-3, hepatitis viruses, and tumor viruses.
The monomeric or smaller targets will generally be from about 75 to 20,000 molecular weight, more usually from 100 to 3,000 molecular weight. The targets of interest include drugs, metabolites, pesticides, pollutants, and the like. Included among them are the alkaloids such as morphine alkaloids (morphine, codeine, heroin, cocaine, ben~oyl ecgonine, etc.), ergot alkaloids, ~teroid alkaloids, and the like.
Other drugs of interest include 6teroids, which include ~he estrogens and androgens: andrenocortical ste~oids; bile acid~; cardiotonic glycosides; and aglycones, which include digoxin and digoxigenin: the bacbiturates, e.g~, phenobarbital and secoba~bital;
aminoalkylbenzene~, which include the amphetamines:
cannabinal and tetrahydrocannabinol, vita~ins~
prostaglandins, antibiotic6, nucleo6id~as and nucleotides.
~nother group of taeget compounds is amino acids and small peptides which include polyiodothyronine&, e.g., thyro2ine, and triiodo~hyronine, oxytocin, ACTH, angiotensin, met- and leu-inkephalin, their metabolites and derivative6.
The fluorophore can be a~tached to the target species by replacement of a hydrogen or other replacaable unctionality on the target with a bond or linking group. The groups on the target can include hydroxyl, amino, aryl, thio, olefin, etc. The linking grou~ will normally have ~rom 1-20 atoms other than hydrogen. The6e atoms are generally carbon, oxygen, sulfur, nitcogan, and halogens of atomic number 17-35.
The linking functionalities preæent in the linking groups include carbonyl, both oxo and non-oxo, active halogen, diazo, mercapto, ethylene, particularly activated ethylene, amino, and the like. The number of heteroatom~ (i.e., not hydrogen or carbon atoms) will generally range from about 0-6, more usually fro~ about 1-6, and preferably from about 1-4.
For the most part, the lin~ing group~ will be aliehatic, although with diazo groups, aromatic group6 are involved. Generally, the linking group is a divalent chain having about 1-10, more u~ually ~rom about 1-6 a~oms in the chain. oxygen will noemally be present a~ oxo or oxy, bonded to carbon and hydrogen, preferably bonded 801ely to carbon, while nitrogen will normally be peesent as amino, bonded 501ely to carbon, or amido, while &ulfu~ would be analogou6 to oxygen.

J ~
, Common functionalitie6 in forming the covalent bond between the linking gLOUp and the molecule to be conjugated are alkylamine, amide, amidine, thioamide, urea, thiourea, guanidine, and diazo.
Linking g~oup6 which find particular application with conjugation to polypeptide~ are those involving carbo~ylic acids which may be used in conjunc~ion with diimide~, or a5 mixed anhydrides wi~h carbona~e monoe6ters, or as active carboxylic esters, e.g., N-hydroxy succinimide or p-ni~rophenyl. Nitrogen a~alogs may be employed a6 imidoe6ters. Aldehyde~ can be u~ed to form imines under reductive aminations conditions, e.g., in the presence of borohydrides, to produce alkylamines. Other non--oxo carbonyl groups which may be e~ployed include isocyanates and i60thiocyanates. In addition, active acyl groups may be employed, pa~ticularly bromoacetyl gLoups.
In most instance6, ~ha target will h~ve one or ~ore functional groups which may be employed as the site or linking the linking group. Particularly, h~droxy, amino and aLyl groups, particularly activated aryl groups, find use. Also, oxime6 may be prepared from oxo unctionalities and the hydroxyl used as a site for joining to a linking group, such a6 carboxymethyl.
The choice of linking group will vay widely, depending upon the functionalities which are present in the fluorophore, in the compound to which the fluorophoLe is to be conjugated, the nature and length of the linking group desired, and the like.
The pLesent invention b~ing~ about ~eparation of specie6 ba~ed on their relative motion in an electrokinetic field. Species having the sa~e charge as the net exce~ charge of the support electroly~e (~ee Background of the Invention) will tend to move fa6ter 1 3~
-than the support elect~olyte. Uncharged species will move at the velocity of the support electrolyte, and materials of opposite charge will move ~lower ~han the support electrolyte.
The conditions of the separation are very nonevasive. Thi~ make6 it possible to use other propensities of ~pecies to achieve separation~. For example, one can selecively agfiociate a species with another charged ~pecies 60 as to impar~ charge~ to ~he fir6t species. Thi~ can be carried out by matching hydrophobicity of the two species, for axample, by or~ing a micellular dispersion of a first charged material and then preferentially associated the second uncharged material with the micelles. This ~ould, in effect, impart charge to the second material. In another technique, one could vary the pH of the mixture so a~ to preferentially protonate or deprotonate a species in the mixture and thus vary its electrokinetic mobility.
The invention uses a SoULce of coherent radiation -- i.e., a laser -- delivered on column to excite the fluorescent species. A continuous or pulsed ; laser can be used. Good results are achieved wi~h low to modeLate power laseLs such as up to about ~0 wat~s.
Higher power lasers can be used, if desired, but are not seen to offee advantages and potentially have the disadvantage of unnecessarily heating the ~ample. The wavelength of ~he coherent light source should be matched to the excitation wavelength of the fluorescing species being measu~ed -- that is, it 6hould be a wavelength effec~ive to excite fluorescence.
The use of coherent light offers significant advantage~ in that it can be efficiently convenien~ly del;vered diLectly to the sample channel by lenses and mir~or~ but, more importantly, by fiber optic~, a6 well.
The beam of coherent exci~a~ion energy can be ~pplied to the sample ~cro6~ the ~a~ple ~low or, if de~iced, it can be applied axially with or again~t the direction of liquid flow. The measueement o~
fluo~escence i~ carried out u6ing conventional ~ea~uring methods. The~e ca~ be continuous measurements or in~ermitten~, i.e., time-gated, mea~urements. These ~easurements are carried out at fiome ~elected wavelength of the fluore~cent emi6~ion. The mea~urement may be carried out a~ the ~ame poin~ on t~e ~ample channel a6 the excitation occur~ or down6tream from the point of excitation. ~easurement may be advantaqeously downstream in the case o~ long-lived fluorophores (e.g., ehosphore~cent matecials) or when the excitation is supplied copropagating or counterpropagating with the flow. The a~gl,e6 for excitation and detection may be coplanar or may be varied a~ de~ired ~o eliminate inteeference, reflections, and the like.
The signal generated by the fluorescence detector may be Lecorded and/or it ~ay be used a6 a con~rol ~ignal. Recording can be carried out by standard char~ recorder~, and the like. The control ~i~nal could be u~ed, for example, ~o o~en or clo~e a valve 80 a~ to trap or collect tha fluore~cent ~pecie~
in a preparative en~ironment.

,~ .
" .

Claims

1. A fluoroassay method for detecting the presence of a target species in a fluorescible sample which comprises:
a. placing said sample into one end of a narrow bore channel having a cross-section dimension of not more that 500 um and having at least a section of which is translucent with said channel defining a separation zone;
b. applying an electric potential to said sample;
c. irradiating the sample with radiation of a wave-length effective to excite fluorescence in said sample; and d. detecting a change in the fluorescence emitted through the translucent section of the channel as the target species migrates past the translucent section.

2. A fluoroassay method for detecting the presence of a target species in an electroosmotically pumpable fluorescible liquid sample which comprises:
a. placing said sample into one end of an electro-osmotically pumpable-liquid-full narrow bore double open ended walled channel having a cross-section dimension of not more than 500 um and having at least a section of which is translucent;
b. applying an effective electroosmotic pumping potential to said pumpable sample and pumpable liquid thereby transporting the sample through the channel;
c. irradiating the sample with radiation of a wave-length effective to excite fluorescence in said sample; and a. detecting a change in the fluorescence emitted through the translucent section of the channel as the target species moves past the translucent section.

3. The method of either claim 1 or 2 wherein said irradiating takes place through the translucent section of the channel.

4. The method of either claim 1 or 2 wherein said radiation is supplied to the sample as a beam by a fiber optic device.

5. The method of claim 2 wherein the electro-osmotically pumpable liquid is an aqueous liquid.

6. The method of either claim 1 or 2 wherein said channel is tubular.

7. The method of either claim 1 or 2 wherein said target species is fluorescent and wherein the detected change is an increase.

8. The process for detecting the presence of each of a plurality of fluorescence-change-inducing target species in a fluorescible sample which comprises:
a. placing said sample into one end of a narrow bore channel defining an electrophoretic separation zone, said channel having a cross-section dimension of not more than 500 um, said channel having a length sufficient to effect electrophoretic separation of the plurality of species from one another and having after said length at least a section which is translucent;
b. applying an electric potential to said sample thereby electrophoretically separating the plurality of target species from one another;
c. irradiating the sample as the sample moves past the translucent section with radiation of a wavelength effective to excite fluorescence in said sample; and d. detecting a change in fluorescence in said sample emitted through said translucent section as the individual separated target species migrate past the translucent section of the channel.

9. The process for detecting the presence of each of a plurality of fluorescence-change-inducing target species in an electroosmotically pumpable fluorescible liquid sample which comprises:
a. placing said sample into one end of an electro-osmotically pumpable liquid-full narrow bore double open ended walled channel having a cross-section dimension of not more than 500 um and having a length sufficient to effect electrokinetic separation of the plurality of species from one another and having after said length at least a section which is translucent;
b. applying an effective electroosmotic pumping and electrophoretic separating potential to said pumpable sample and pumpable liquid thereby moving the sample through the channel and electrokinetically separating the plurality of target species from one another;
c. irradiating the sample as the sample moves past the translucent section with radiation of a wavelength effective to excite fluorescence in said sample; and d. detecting a change in fluorescence in said sample emitted through said translucent section as the individual separated target species migrate past the translucent section of the channel.

10. The method of either claim 8 or 9 wherein said irradiating takes place through the translucent section of the channel.

11. The method of either claim 8 or 9 wherein said radiation is supplied to the sample as a beam by a fiber optic device.

12. The method of claim 9 wherein the electro-osmotically pumpable liquid is an aqueous liquid.

13. The method of either claim 8 or 9 wherein said walled channel is tubular.

14. me method of either claim 8 or 9 wherein each of the target species is fluorescent and wherein the detected change is an increase in emitted fluorescence.

15. A detector for indicating the presence of fluorescent species in a liquid sample comprising:
a. a narrow bore channel having a cross-section dimension of not more than 500 um for containing the sample at least a section of which channel is translucent;
b. means for irradiating the sample with irradiation of a wavelength effective to excite fluorescence in the fluorescent species; and c. means for collecting through the translucent section fluorescence emitted by the fluorescent species.

16. The detector of claim 15 wherein said channel defines an electrokinetic zone.

17. The detector of claim 16 wherein said channel is a tubular walled channel.

18. The detector of claim 16 wherein said means for irradiating comprise means for delivering a beam of radiation through the translucent section.

19. A system for separating and detecting a plurality of fluorescence-change-inducing species in a sample which comprises:
a. narrow bore channel having a cross-sectional dimension of not more than 500 um and that is connected to and in communication with a translucent detection section;
b. means for feeding said sample into said channel;
c. means for applying an electric potential along said channel and through said detection zone;
d. means for delivering to said sample as it passes through the detection zone radiation of a wavelength effective to excite fluorescence in said sample; and e. means for detecting through the translucent section changes in emitted fluorescence as said fluorescence change inducing species transit the detection zone.

20. A system for separating and detecting a plurality of fluorescence-change-inducing species in a sample including an electroosmotically pumpable liquid which comprises:
a. narrow bore double open ended walled channel for containing an electroosmotically pumpable liquid said channel having a cross-sectional dimension of not more than 500 um and being connected to and in communication with a translucent detection section;
b. means for feeding said sample into said channel;
c. means for supplying osmotically pumpable liquid to said channel before and after said sample is fed;
d. means for applying an effective electrokinetic potential along said channel and through said detection zone;
e. means for delivering to said sample as it passes through the detection zone radiation of a wavelength effective to excite fluorescence in said sample; and f. means for detecting through the translucent section changes in emitted fluorescence as said fluorescence change inducing species transit the detection zone.