WO2001061335A2

WO2001061335A2 - Method and device for bias-free electrokinetic sample introduction and separation

Info

Publication number: WO2001061335A2
Application number: PCT/EP2001/001822
Authority: WO
Inventors: Alexis Michael Bazzanella
Original assignee: Evotec Oai Ag
Priority date: 2000-02-17
Filing date: 2001-02-19
Publication date: 2001-08-23
Also published as: WO2001061335A3

Abstract

A method for a bias-free electrokinetic sample introduction, in particular injection, into a separation device and sample separation comprising the following steps: a) providing a sample solution, consisting of analyte and matrix, with an ionic strength higher than the ionic strength of an injection buffer in a separation chamber of the separation device, b) applying voltage to introduce the sample solution into the separation chamber, c) performing a separation of the sampleand device for capillary electrochromatography (CEC) separation comprising: a CEC separation chamber with at least one inlet (20) and at least one outlet orifice (20), said chamber comprising the stationary phase (80), a first reservoir comprising a buffer solution (90), a second reservoir comprising a sample solution (50), means for applying voltage (10), means (170) for measuring and/or adjusting an ionic strength of the sample solution and/or buffer solution.

Description

Method an device for bias-free elecirokinetic sample introduction and separation

The present invention is related to a method and device lor bias-tree electrokinetic sample introduction, in particular injection, into a separation device and separation.

In the field of chemical and biochemical analysis microseparation processes as e. g. capillary elcctrophoresis (CE). or liquid chromatography (LC), or capillary eiectrochromatography (CEC) play a very important role due lo their high separation efliciencics in the range of everal hundred thousand plates per meter and the low analyte volumes needed.

The general idea of chromatographic separation processes is to dissolve the sample in a mobile phase. This mobile phase is moved tlirough a non-mixable stationary phase which is located in a separation chamber c. g. in a column or a chip channel, or is fixed onto a surface. The two phases have to be chosen in such a way that the diflerent sample compounds have different affinities to the mobile and the stationary phase.

Capillary eiectrochromatography (CEC) is a hybrid technique of liquid chromatography and capillary electrophoresis, combining the advantages of both methods in one technique. In CEC the stationary phase in most cases consists of fused-silica capillaries or a chip channel packed with conventional stationary phase material as generally known in liquid chromatography as e. g. HPLC, such as Hypersil (Separations Group), Nucleosil (Macherey- Nagel Co.) or ISRP GFF1 -S5-80 (Regis Technologies, Tnc.). In the capillary the stationary phase is retained preferably by two frits. With CEC it is possible to separate, charged and neutral molecules simultaneously. For charged analytes, the chromatographic separation based on differential distribution between mobile and stationary phase is superimposed by their electrophoretic migration. The mobile phase flow is generally driven by electroosmosis. The characteristics of the electroosmotic flow (EOF) give rise to the high separation efficiency of CEC and allow the use of stationary phase material with very small diameters (< 20 nm), thereby further improving efficiency without the problem of column backpressure usually occurring in pressure driven chromatography. Typically, efficiencies in the order of several hundred thousand plates per meter licn been observed | Λ. ermaux and P. Sandra, Klectrophorc i 1 999. 20. 3027-1065. J.

Hut .:^•- well .r, for ( Ε on crucial point in ^("1 ⁽. ^' ' ■ l\, ..ample inl iodiictiυn l l i . normally pe lυπncd elcclrυkinetically by application of high voltage between a sample reservoir, e. g. a sample vial, at the capillary inlet and the running buffer reservoir, e. g. buffer vial, at the outlet, or hydrodyπamically. However, hydrodynamic sample introduction, the most common sample introduction method in capillary clcctrophoresis, is much more difficult to use in CEC, as the packed column bed causes a large pressure drop along the capillary. Λs described by equation (1 ), the pressure drop Δp related to a flow velocity u depends on the column length L and is inverse proportional to the square of the diameter d_p of the particles of the packing material.

Δp =u^*L^*η^*Φ/d_p ² equation (1)

Therefore, in particular for long columns packed with small particles, high sample introduction pressures in the range of several bar have to be applied to the sample vial to perform the introduction of a sample plug. However, under these conditions precise sample introduction is difficult to achieve and the column frits retaining the packing material are exposed to a high mechanical stress, leading to a strongly reduced lifetime of the column.

In electrokinetic introduction the driving force is the electric field produced by the injection voltage applied to the electrode immersed in the sample solution. Unfortunately, the quantity of the species injected is a function of mobility and therefore representative sample introduction can only be expected for neutral solutes. For ions electrokinetic introduction is discriminative. Ions of high mobility are introduced into the capillary at higher rate than ions of low mobility, thus concentrations in the injected sample zone do not reflect the concentrations in the sample reservoir. By selecting the e'ectrode polarity either negative or positive ions even can be entirely excluded from beinr. introduced into the column (mobility bias). For c.ipillai v elect. ophoicsis (CE ) scvei.il technical solutions have been described in the prior art to overcome tins problem ot mobi lity bias in elccirokiiie c sample introduction However, these technical solutions comprise l at her complex approaches anri i cqiiirr specially designed inst i ui iicnIi ioii

One method described in the prior art is the method of isolation of the sample introduction end of the capillary from the electric field by an on-column fracture. The on-column fracture allows ions and thus current to pass, but prevents a buffer How from the ambient buffer reservoir into the fracture. Application of high voltage between the fracture and the capillary outlet induces the sample introduction at the capillary inlet by EOF traction [M. C Einhares and P. T. Kissinger, Anal. Chcm. 1991 , 63, 2076-2078.; H. Wei et al., Anal. Chcm. 1998, 70, 2248-2253.; Q.-S. Pu and Z.-L. Fang, Analylica Chimica Acta 1999, 398, 65-74.]. However, manufacturing of on-column fractures is elaborate and difficult to perform reproducibly. The crucial point in this approach is to achieve ion transport through the fracture without inducing an additional butler volume flow, which would lead to dilution of the fluid in the capillary. In addition, analyte loss can occur al the fracture.

Another method mentioned in prior art is the exhaustive electrokinetic sample introduction from a thin film of sample solution formed on a wire loop al the capillary inlet. The wire is used as electrode for sample introduction [P. K. Dasgupta and K. Surowiec, Anal. Chem. 1996, 68, 4291-4299.]. In this approach all analyte ions present in a small droplet of sample solution are completely injected, and thus no discrimination takes place. However, the exhaustive electrokinetic sample introduction from a droplet lacks a reproducible formation of identical droplet size, which is a prerequisite for quantification of analytes using this method. The size of such a droplet strongly depends on the surface tension of the sample solution which is affected by the sample composition. The wire loop itself is a potential adsorption site for analytes and is prone to cross contamination between subsequent sample solutions. In addition, reproducible manufacturing of the wire loop is difficult to achieve.

Because of these disadvantages these methods are not suited to solve the problem of mobility bias in capillary eiectrochromatography. Therefore, il is an object of the present invention to provide a method for bias-free electrokinetic sample introduction, in particular injection, and separation, which is applicable especially in _^'1_C systems. Additionally a device: for CEC separation is provided.

I he inventive method and device allow reproducible biu -iiee elecliokinetic sample introduction, in particular injection, of any species, especially into a CEC system, irrespective of their charge stale, without compromising the separation efficiency of the column.

In the context of the present invention a sample solution or sample consists of analyte and matrix. Whereas matrix preferably comprises solvent, salts such as inorganic salts or organic compounds comprising ionizablc functional groups, or mixtures thereof; furthermore the matrix may also comprise proteins, other macromolecules or other substances. Analyte preferably comprises one component or a mixture of components to be separated from the matrix and/or from each other.

According to the present invention the sample solution is exclusively drawn inlo the capillary by electroosmotic flow and no active electrophoretic migration of analyte occurs during sample introduction.

According to the invention it is preferable to provide the analyte in a high ionic strength matrix. Preferably, the ionic strength of the matrix is 5 to 500, more preferably 10 to 200 fold, higher than in the buffer solution in the CEC separation chamber when the injection takes place (injection buffer). Whereas in CE this would cause a lot of disadvantages such as serious zone broadening and deterioration of the sample during separation.

Due lo the difference in ionic strength between the sample solution and the CEC buffer in the separation chamber the electric field strength in the sample zone is strongly reduced and thus active migration of analyte is suppressed. The sample solution is introduced into the separation chamber by the pumping action of the electroosmotic flow (EOF) only and no mobility bias occurs. Thcrclorc, it might be useful according to the present invention first to measure the ionic sli engih ol the sample solution and compare it to that of the injection buflci , which is known or has lo I v dclei inincd a.s well. I n case the ionic strength ol the sample .solution is loo low an ap|.ropriatc amount or solutions ol either inorganic salts 01 organic compounds comprising loni iublc luncliυiial groups, or mixtures thcrcυl arc added to the sample solution in order to achieve the required ratio of ionic strength. Said salts may also be buffer salts and may additionally contribute to pi I.

Alternatively, it is also possible to use a sample solution without measuring the ionic strength and/or adding any salt or organic compound or solutions thereof, if the ionic strength is known and is high enough compared to the injection buffer in the separation chamber. This is e. g. the case for physiological sample solutions such as blood, serum, plasma, urine, etc. which normally contain salts at about 1% concentration.

According to the present invention it is also possible lhat the matrix after sample introduction is not retained at the stationary phase in the separation chamber.

It may also be preferable that one or more of the salts, or organic compounds, or macrυinolecules, proteins or other substances of the matrix are retained at the stationary phase of the separation chamber.

Furthermore, it is preferred after introduction of the sample to use CEC buffers (washing buffers) preferably with low ionic strength that provide rapid elution of the matrix and/or strong retention of the analyte at the stationary phase. According to the present invention the washing buffer is preferably the same as the injection buffer. Separation and elation of the analyte is subsequently achieved through changing the CEC buffer solutions to higher elution strength (separation buffer).

Generally, it is most preferable to use the inventive method in combination with the CEC device according to the present invention. Hereinafter, [. referred embodiments of the inventive device will be described in view of the attached drawings.

Figui e 1 shows a fii sl embodiment o l t he sample introduct ion and capi l lar . eiectrochromatography device.

Figure 2 shows another embodiment of the sample introduction and capillary eiectrochromatography device.

Figure 3 also shows an embodiment according to the present invention.

Figures 4a), 4b) and 4c) show electropherograms performed with different sample introduction methods: electrokinetic introduction of a low salt sample (a), elektrokinetic introduction of a sample containing an additional salt concentration of 150 m HBSS (b), and hydrodynamically introduction of a low salt sample (c).

One preferred embodiment of the sample introduction device addressed by this invention is shown in Figure 1. It comprises a separation chamber containing the stationary phase (80) and at least two openings (20) which are spaced apart from each other, whereby the openings provide liquid contact to buffer reservoir (90) and sample reservoirs (50) and electrical contact to the high voltage power supply (10). The separation chamber shown here is a CEC capillary (60). As shown, the sample (150) to be introduced into the electrochromatographic separation chamber via one of the openings (20) in the sample reservoir (50) and the buffer liquid (120) in the buffer reservoir (90) are in contact with a conductive means (30). This means provides the electrical contact to a high voltage power supply (10χ such as an electrode or an integrated contact in the buffer and in the sample reservoir, said conductive means are made of either metal, carbon, or a conductive polymer. Connected to the sample reservoir is a means to measure and/or adjust the ionic strength of the sample (170). Optionally additional sample and/or buffer reservoirs may be included. It is also preferable to replace reservoirs by other ones, such as the sample reserv oir may be replaced by another sample reservoir or a buffer reservoir containing e. g. injection buffer, washing buffer or separation buffer. In another embodiment a means to measuie and optionally adjust the ionic strength of a solution, may also be connected to the buffer reservoir, to determine and optionally adjust the ionic strength ol the used buffer .solution, picieiably of the miectiυπ buf fer solution

According lo the present invention n is prclcrred to adjust only the ionic strength of the sample solution to achieve the desired ratio of the ionic strength.

Optionally, at least one detection means (190) lo characterise the composition of the analyte qualitatively and optionally quantitatively may be adapted to the separation device.

Generally the inventive device comprises : a CEC separation chamber with at least one inlet (20) and al least one outlet orifice (20), said separation chamber comprising the stationary phase (80), a first reservoir comprising a buffer solution (90), a second reservoir comprising a sample solution (50), means for applying voltage (10), means (170) for measuring and/or adjusting an ionic strength of the sample solution and/or buffer solution.

The device may also comprise a detection means (190).

The detection means may be a masspectrometer and/or optical detector, especially light scattering detector, UV-detector and/or fluorescence detector and/or refractive index detector and/or condensation nucleation light scattering detector.

In a preferable embodiment the inventive device may also comprise a means for automatic exchange of the reservoirs.

According lo the present invention the stationary phase in the CEC separation chamber may comprise, preferably consist of, porous and/or non-porous support material. Wherein Ihe support material preferably comprises, more preferably is made of. an inorganic oxide, preferably silica and/or an organic polymer and/or copolymer, in particular polystyr l/diveny I benzene.

According to Ihe present invention the surface of said support mater ial is preferably modified with hydrophilic, hydrophobic, ionic, chiral, and/or affinity residues. Whereas hydrophobic residues are especially alkyl chains of O - Cso, preferably between C and C₂₂, and/or aryl residues, preferably phcnyl and benzyl groups; hydropliilic groups are especially hydroxyl, diol, amine, amide, nitrile, cyano or nitroalkyl, polyoxyethylene, polyethylene glycol; chiral residues are especially cyclodextrins, proteins, a ylose derivatives, Pirkle-type, diphenylethyldiaminc; affinity ligands are especially proteins, antibodies, Fab-fragments, molecular imprinted polymers, receptors, oligo- or polynucleotides.

Furthermore, it may be preferred that the stationary phase in the CEC separation chamber is a chromatographic restricted access material and/or comprises, any porous support material with different surface regions comprising different surface modifications.

In another preferred embodiment the outlet orifice of the separation chamber is adapted to function as an electrospray means.

Alternatively, the separation chamber may be a part (100) of an integrated separation device (300) on a chip, as shown in Figure 2, or a combination of a CEC capillary with an integrated separation device on a chip, shown in Figure 3. As shown in Figure 2 the electrochromatographic separation chamber is in liquid contact with a separate sample reservoir (290), also connected to a high voltage power supply via a conductive means (30). Whereas according to the present invention the sample reservoir is connected and the buffer reservoir optionally may be connected to a means for measuring and adjusting the ionic strength to find or create optimal conditions for performing the introduction of the sample into the separation chamber.

In the case of the integrated separation device (Figure 2), or a CEC capillary coupled to a microfluidic device (chip) as especially shown in Figure 3, additional reservoirs (90) are optionally integrated, also in liquid c ontact with ( e eparat i n ehambei . The latter inav contain t ! ^• (. buf fers of dif ferent composit ion, e g lor gradient format ion

Accoidni!' the piesenl invent ion t he (T.C ,epaι ai ιon . liambci may be ,t capillai v column or pa.i t of a channel system on a chip, wherein the capillary column or ihe chip may comprise, preterably consist of, polymer and/or glass and/or fu.sed silica and/or ceramics and/or elastomer.

It i.. further preferable that at Ieasl two CEC separation chambers are coupled via a capillary system or a channel system or several CEC separation chambers, preferably 2 to 50, more preferably 2 lo 16 are arranged in parallel and/or two-dimcnsionally.

Generally, microfluidic devices (chips) are known in the prior art and therefore are not described in more detail.

After setting the ionic strength of the sample solution to 5 to 500 fold, preferably 10 to 200 fold, that of the CEC injection buffer either by hand or automatically, the sample solution is electrokinetically introduced from a sample reservoir (50) or (290) into the separation chamber (60) oi (100) by applying high voltage between the sample reservoir (50) or (290) in liquid contact with a first opening (20) of the CEC separation chamber and another opening (20) of the CEC separation chamber spaced apart from the first opening (20).

In one preferred embodiment analyte and matrix are both retained at the beginning of the separation chamber. For performing the CEC separation it may be necessary to remove the matrix from the separation chamber, e. g. CEC column, to create suitable conditions for a successful CEC separation.

It is also an object of the present invention to provide CEC conditions during the sample introduction and the initial phase of the separation, causing sufficient retention of the analyte but not causing considerable retention of the matrix. This way the matrix is rapidly eluted out of the CEC separation chamber, whereas the analyte is retained and transferred into the lower conductive CEC buffer environment. To achieve this it may be preferred to use a stationary phase capable ol revc. sed-phase like retention of analyte, e. g. C|χ-πιodified silica in combination wit h .111 almost aqueous buffer of very low elution strength a.s mobile phase Inciea mg the elut ion .strength of the CEC mobile plia.se. e. g. by increasing t he organic modifier c ontent , the analyte. which compπ .cs at least one component, is subsequent ly separated and clulc i

Serious analyte zone broadening due to electrodi.spersion, which commonly occurs on sample introduction of highly conductive sample solutions in CE, is prevented, and thus high separation efficiency is maintained.

According to the invention for removal of the matrix and the subsequent separation, high voltage is applied between a buffer reservoir (90) containing preferably washing buffer which is in liquid contact with one of the openings (20) of the CEC separation chamber and a buffer reservoir containing buffer, which is in liquid contact to a second opening (20) of said CEC separation chamber spaced apart from the first opening.

In case gradient separation is preferred, after the removal of the matrix as described above high voltage is applied between different buffer reservoirs (90) staying in liquid contact with a first opening (20) of the CEC separation chamber and at least one buffer reservoir staying in liquid contact with a second opening of said CEC separation chamber, either subsequently (step gradient) or simultaneously (continuous gradient).

In general, the inventive method comprise the following steps: a) providing a sample solution, consisting of analyte and matrix, with an ionic strength higher than the ionic strength of an injection buffer in the separation chamber of the separation device, b) applying voltage to introduce the sample solution into the separation chamber, c) performing a separation of the sample.

Optionally, the ionic strength of the sample solution is det .rrnined and/or adjusted prior to injection. Whereas the ionic strength of the sample soluti m is adjusted to 5 to 500 fold preferably 10 to 200 fold, higher than the ionic strength of the injection buffer in the sepnralion chamber by addit ion of an appropriate amount or solutions of cither inorganic salts or organic compounds comprising' ionizable functional groups, or mixtures (hereof.

According lo the im ention the separat ion o l the samj.lo is pre fer ably |.erfυπncd with capillarv eiectrochromatography and comprises ihe following steps: injecting a first buffer solution (washing buffer) into the separation chamber to elutc the matrix of the sample solution out of the separation chamber, injecting a second buffer solution (separation buffer) into the separation chamber to separate and elutc the analyte, wherein prior to injection of the first buffer solution the sample reservoir may be exchanged against a buffer reservoir.

According to the invention it is preferred to measure after scpcration of the analyte its composition and/or the concentration of the at least one of its components.

The injection and separation method according to the present invention is not restricted to special CEC separation chambers, e. g. columns, and buffers. All kinds of CEC columns comprising reversed-phase type, e. g. C ,Cβ, C|₈ or phenyl, stationary phases may be used with all common buffers such as phosphate, boratc. TlvlS, formiatc or acetate.

The injection and the washing buffer preferably contain a low amount of organic solvent in order to minimize the elution strength of the buffer with respect to the analyte. For the separation buffer and elution buffer the preferably used high amount of acetonitrile may be replaced by alternative organic solvents such as methanol.

Generally, the use of a buffer is dependent on the nature and the properties of the analyte to be injected and separated and the stationary phase in the separation chamber. In case the composition of the analyte is not known it is preferred to use so called universal buffers, which are optimized for such analytes. Comparison of different sample introduction methods using a sa ple mixture consisting of thiourca, quinine, ibuprofcπ and diphenylsulfon

Employed instrumentation, materials and procedures

A HP^3D CE Capillary Electrophoresis System (Agilent Technologies, Waldbronn, Germany) with UV diode array detection has been employed for all experiments. As appropriate detection wavelength for all test compounds 210 nm has been employed. For capillary eiectrochromatography CEC Cap Hypersil Cis 3 μm columns with an inner diameter of 100 μm, a total length of 32 cm and an effective length (packed section to the detection window) of 23.8 cm (Agilent Technologies) were used.

The experiments were conducted according to the following procedures:

For the experiments performed, the washing buffer was identical to the injection buffer.

a) Preconditioning of the column

The column was preconditioned with the injection/washing buffer comprising 5 mM ammonium acetate (Fluka) in water with 5% (v/v) acetonitrilc (Baker), pH 4.7. Both, (cathode and anode) buffer vessels were filled with this injection/washing buffer and the column was rinsed by voltage application (15 kV) for 10 in.

b) Sample preparation

Stock solutions of the individual compounds (thiourea, quinine, ibuprofen (Sigma) and diphenylsulfon (Aldrich)) with concentrations of 10 mg/ml respectively, were prepared in a mixture of 25% water and 75% acetonitrile.

Subsequently, for electrokinetic and hydrodynamic sample introduction according to the state of the art (corresponding to Figure 4 a) and 4c)), 100 μl of each stock was mixed together

with 600 itl 25%

acclot.iti il to yield a final concentration of 1 mg/ml for the individual analytes.

f or bias fi ec electrokinet ic sample int roduction a _cotdm.'_ lo (he [. resent invent ion ( see 1 J LMH C 4b)) the final sample solution was prepared from the slock solution with 25% water/75% acctonitrile containing Hanks balanced salt solution (HBSS, Sigma) to yield a salt concentration of 150 mM in the sample (corresponding fo 0, 185 g/1 calcium chloridc*H₂0; 0,0976 g/1 magnesium sulfate (anhydrous), 0,4 g/1 potassium chloride; 0,06 g/1 potassium hydrυgene phosphate (anhydrous); 8 g/1 sodium chloride; 0,0479 g/I sodium dihydrogenc phosphate (anhydrous), 1 g/1 D-glucosc).

c) Sample injection

For sample injection the injection buffer reservoir was exchanged by a reservoir containing the respective sample solution.

Electrokinetic injection (Figure 4a) and 4 b)): Voltage 5 kV for 3 s. Hydrodynamic injection (Figure 4c)): Inlet pressure 10 bar for 10 s.

d) Column washing

The sample reservoir was replaced again by a buffer reservoir containing the injection washing buffer to remove the matrix. For the washing step 15 kV were applied for 5 min.

e) Analyte separation

For separation and elution of the analyte in the sample a buffer comprising 5 mM ammonium acetate with 60% (v/v) acetonitrile at pH 4.7 (separation buffer) was employed. The buffer reservoirs containing injection/washing buffer were replaced with reservoirs containing this separation buffer and a voltage of 15 kV was applied. After the separation was completed, the column was reconditioned according to a) prior to the next sample injection. _{. .}

Description ol the experiments and the results

Figuics 4 a) to 4 c) demonstrate the feasibility of bias-free electrokinetic sample introduction according to the present invention. An analyte conij.ri.sing quinine as a cation, diphenylsulfon as neutral analyte species, ibuprofen a.s an aniυn and thiouiea as EOF marker wa injected into a 1 0 μm i.d. CEC column packed with common Ciβ-modified silica using three different sample iniroduction conditions. As CEC injection buffer 5 mM ammonium acetate al pH 4.7 with 5% (v/v) acetonitrile was used. After sample introduction, the clectrochromatographic separation chamber was washed with the same buffer for 5 in at 15 kV to remove the matrix according lo the present invention. Under these conditions, analyte is strongly retained, whereas the matrix quickly clute from the column. As an exception, thiourca elutes directly with (he EOF, as this analyte species is not retained on the stationary phase. In a second step, the buffer reservoirs were replaced with buffer reservoirs containing 5 mM ammonium acetate at pH 4.7 with 60% (v/v) acetonitrile. The high acctonitrile content of this buffer induces the separation and elution of the analyte species.

Figure 4a) shows an elcctropherogram of the compounds of the analyte. The sample introduction was performed electrokinetically without pretreatmcnt of the sample solution.

Figure 4b) shows an elcctropherogram of the compounds of the analyte. The sample introduction was performed electrokinetically with a sample containing an additional salt concentration of 150 M. The commercially available salt mixture ITBSS (Hanks balanced salt solution) was used for this purpose.

Figure 4c) shows an electropherogram of the compounds of the analyte. The sample introduction was performed hydrodynamically, using a sample introduction pressure of 10 bar. The ratios of the peak areas of the different compounds of the analyte obtained from this chromatogram reflect ihe non-discriminative situation, as pressure driven sample introduction is intrinsically bias-free.

For all three sample introduction approaches, the peak area ra io of diphenylsulfon (peak 3) to thiourea (peak 1) is identically about 0.27, as both analyte >pecies are neutral and are not affected by mobility bias effects. Electrokinetic sample introduction of the not pretreated sample solution results in strong sample introduction of quinine (peak 2). the peak aiea ratio diphenylsulfon/qumme is reduced lo 07 as compared lo 233 for hydrodvπnmic sample intioductioπ Ibupiofcn is sliongiy disciiminated in elccliokinctic sample introdu. lion and thus the peak aiea ratio diphcnyFulfonΛbupioIci is incicisinc to 1 ^{] ι} eυπij.aicd to i 1 lor h drodjnainic sample introduction.

1 he electrokinetic sample introduction according to the present invention reduces the effect of mobility bias for ionic species. In this case, the peak area ratios for diphcnylsulfon/quinine and diphcnylsulfon/ibuprofen are 2.25 and 1.26, respectively

Claims

! A method for a bi.ts-licc cleαrokinei ic sa'iψlc ini ioduclion m paπ ieulai micction mio a separat ion evice and sample • en n t ion . < >ni|-" i m the follow ι <i >tep. aj pi υv iding a sample solution, consisting ol analyte and matrix, with an ionic strength higher than the ionic strength of an injection buffer in a separation chamber of the separation device, b) applying voltage to introduce the sample solution into the separation chamber, c) performing a separation of the sample.

2. The method according to claim 1, wherein the ionic strength of the sample solution is determined and/or adjusted prior to introduction.

3. The method according lo claim 2, wherein the ionic strength of the sample solution is adjusted by addition of an appropriate amount or solutions of cither inorganic salts or organic compounds comprising ionizablc functional groups, or mixtures thereof.

4. The method according to any of claims 1 to 3, wherein the ionic strength of the sample solution is adjusted to 5 to 500 fold, preferably 10 to 200 fold, higher than the ionic strength of the injection buffer in the separation chamber.

5. The method according to any of claims 1 to 4, wherein the matrix contains inorganic salts and/or organic compounds comprising ionizable functional groups and/or proteins and/or macromolecules.

6. The method according to any of claims 1 to 5, wherein said separation of the sample solution in step c) is performed with capillary eiectrochromatography and comprises the following steps: injecting a first buffer solution (washing buffer) into the separation chamber to elute the matrix of the sample solution out of the separation chamber, injecting a second buffer solution (separation buffer) into the separation chamber to separate and elute the analyte.

7. he method according to any of claims I to 6. wherein after separation of the analvtc its composition and/or the concentration of the at least one of its components is measured by a detection means.

8. i he method according to any of claim 7, wherein ihe detection means is a masspeclromelcr and/or optical detector, especially light scattering detector, U V-deteclor and/or fluorescence detector and/or refractive index detector and/or condensation nucleation light scattering detector.

9. Device for capillary eiectrochromatography (CEC) separation comprising: a CEC separation chamber with at least one inlet (20) and at least one outlet orifice (20), said chamber comprising the stationary phase (80), a first reservoir comprising a buffer solution (90), a second reservoir comprising a sample solution (50), means for applying voltage (10), means (170) for measuring and/or adjusting an ionic strength of the sample solution and/or buffer solution.

10. The device according lo claim 9, with at least one additional reservoir comprising buffer solution or sample solution.

1 1. The device according to claim 9 and /or 10, comprising a means for automatic change of the reservoirs.

12. The device according to any of claims 9 to 1 1, wherein the CEC separation chamber is coupled to a detection means (190).

13. The device according to any of claim 12, wherein the detection means is a masspectrometer and/or optical detector especially light scattering detector, UV-detector and/or fluorescence detector and/or refractive index detector and/or condensation nucleation light scattering detector.

14 1 he device according to any of claims 9 (o 13, wherein the stalionary phase in (he CEC .separation chamber comprises porous and/or non-porous support material.

1 5 fhe device according to claim 14. wherein the support mater ial comprises an inorganic oxide, preterably silica and/or an organic polymer and/or a copolymer, in particular polystyro 1/diveιιylbeπzene.

16. ^'Fhe device according to any of claims 14 and/or 15, wherein the support material has a surface which is modified with hydrophilic, hydrophobic, ionic, chiral, and/or affinity residues.

17. The device according to claim 16, wherein hydrophobic residues arc alkyl chains of Cj - C .o, prefciably between C and C₂₂, and/or aryl residues, preferably phenyl and benzyl groups; hydrophilic groups are hydroxyl, diol, aminc, amide, nitrile, cyano or nitroalkyl, polyoxycthylene, polyethylene glycol; chiral residues are cyclodextrins, proteins, amylose derivatives, Pirkle-type, diphenylethyldiaminc affinity ligands are proteins, antibodies. Fab- fragments, molecular imprinted polymers, receptors, oligo- or polynucleotides.

1 . The device according to any of claims 14 to 17, wherein the stationary phase in the CEC separation chamber is a chromatographic restricted access material and/or comprises any porous support material with different surface regions comprising different surface modifications.

19. The device according to any of claims 9 to 18, wherein the CEC separation chamber is a capillary column.

20. The device according any of claims 9 to 19, wherein the CEC separation chamber is part of a channel system on a chip.

21. The device according to any of claims 9 to 20, wherein the capillary column or the chip comprise polymer and/or glass and/or fused silica and/or ce amics and/or elastomer.

22. I he device according any of claims 9 to 21 , w herein at least two CIX^' .sepai atiυu chambers di coupled via a capillary system or a channel system

23 The device according to any of claims ° lo 22 w herein several CEC separatum chambers prcfciably 2 to 50, more picier ably 2 to 16 are arranged in parallel and/or two- dimcnsionally.

24. The device according to any of claims 9 to 23, wherein the outlet orifice is adapted to function as an electrospray means

,„„„ _Λ O 01/61335 20

Summary

A method for a bias-free electrokinet ic sa ple introduction, lii particular inject ion, mio a separation device and sample separat ion comprising the following slcps: a) providing a sample solution, consisting of analyte and matrix, with an ionic strength higher than the ionic strength of an injection buffer in a separation chamber of the separation device, b) applying vollage to introduce the sample solution into the separation chamber, c) performing a separation of the sample.

and device for capillary eiectrochromatography (CEC) separation comprising: a CEC separation chamber with at least one inlet (20) and at least one outlet orifice (20), said chamber comprising the stationary phase (80), a first reservoir comprising a buffer solution (90), a second reservoir comprising a sample solution (50), means for applying voltage (10), - means (170) for measuring and/or adjusting an ionic strength of the sample solution and/or buffer solution.