WO2001061335A2 - Method and device for bias-free electrokinetic sample introduction and separation - Google Patents
Method and device for bias-free electrokinetic sample introduction and separation Download PDFInfo
- Publication number
- WO2001061335A2 WO2001061335A2 PCT/EP2001/001822 EP0101822W WO0161335A2 WO 2001061335 A2 WO2001061335 A2 WO 2001061335A2 EP 0101822 W EP0101822 W EP 0101822W WO 0161335 A2 WO0161335 A2 WO 0161335A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- separation
- sample
- separation chamber
- buffer
- cec
- Prior art date
Links
- 238000000926 separation method Methods 0.000 title claims abstract description 109
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000000523 sample Substances 0.000 claims abstract description 89
- 239000000872 buffer Substances 0.000 claims abstract description 56
- 239000012488 sample solution Substances 0.000 claims abstract description 45
- 239000012491 analyte Substances 0.000 claims abstract description 39
- 238000002347 injection Methods 0.000 claims abstract description 35
- 239000007924 injection Substances 0.000 claims abstract description 35
- 239000011159 matrix material Substances 0.000 claims abstract description 25
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 22
- 239000007853 buffer solution Substances 0.000 claims abstract description 17
- 239000000463 material Substances 0.000 claims description 15
- 150000003839 salts Chemical class 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 13
- 239000011534 wash buffer Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 150000002894 organic compounds Chemical class 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 238000000149 argon plasma sintering Methods 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 3
- 230000005494 condensation Effects 0.000 claims description 3
- 230000006911 nucleation Effects 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 229920000858 Cyclodextrin Polymers 0.000 claims description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 2
- 229920001577 copolymer Polymers 0.000 claims description 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 2
- 229940097362 cyclodextrins Drugs 0.000 claims description 2
- 150000002009 diols Chemical class 0.000 claims description 2
- 229920001971 elastomer Polymers 0.000 claims description 2
- 239000000806 elastomer Substances 0.000 claims description 2
- 239000005350 fused silica glass Substances 0.000 claims description 2
- 239000011521 glass Substances 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 125000001165 hydrophobic group Chemical group 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 229910052809 inorganic oxide Inorganic materials 0.000 claims description 2
- 239000003446 ligand Substances 0.000 claims description 2
- 229920002521 macromolecule Polymers 0.000 claims description 2
- 238000012986 modification Methods 0.000 claims description 2
- 230000004048 modification Effects 0.000 claims description 2
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims description 2
- 150000002825 nitriles Chemical class 0.000 claims description 2
- 125000004971 nitroalkyl group Chemical group 0.000 claims description 2
- 229920000620 organic polymer Polymers 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 108091033319 polynucleotide Proteins 0.000 claims description 2
- 102000040430 polynucleotide Human genes 0.000 claims description 2
- 239000002157 polynucleotide Substances 0.000 claims description 2
- 108020003175 receptors Proteins 0.000 claims description 2
- 229940090044 injection Drugs 0.000 claims 4
- 229920000856 Amylose Polymers 0.000 claims 1
- 238000002045 capillary electrochromatography Methods 0.000 abstract 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 10
- 238000005370 electroosmosis Methods 0.000 description 9
- 150000002500 ions Chemical class 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 230000037230 mobility Effects 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- 230000000717 retained effect Effects 0.000 description 7
- 241000894007 species Species 0.000 description 6
- 235000001258 Cinchona calisaya Nutrition 0.000 description 5
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 5
- KZTYYGOKRVBIMI-UHFFFAOYSA-N diphenyl sulfone Chemical compound C=1C=CC=CC=1S(=O)(=O)C1=CC=CC=C1 KZTYYGOKRVBIMI-UHFFFAOYSA-N 0.000 description 5
- 229960000948 quinine Drugs 0.000 description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- 239000012981 Hank's balanced salt solution Substances 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 230000005684 electric field Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229960001680 ibuprofen Drugs 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000088844 Nothocestrum Species 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- -1 polyoxyethylene Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229920001940 conductive polymer Polymers 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000010237 hybrid technique Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 239000011833 salt mixture Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44743—Introducing samples
Definitions
- 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.
- capillary elcctrophoresis CE
- liquid chromatography LC
- capillary eiectrochromatography CEC
- chromatographic separation processes 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 is a hybrid technique of liquid chromatography and capillary electrophoresis, combining the advantages of both methods in one technique.
- 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.).
- HPLC such as Hypersil (Separations Group), Nucleosil (Macherey- Nagel Co.) or ISRP GFF1 -S5-80 (Regis Technologies, Tnc.).
- the stationary phase is retained preferably by two frits. With CEC it is possible to separate, charged and neutral molecules simultaneously.
- 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.
- EEF electroosmotic flow
- electrokinetic introduction the driving force is the electric field produced by the injection voltage applied to the electrode immersed in the sample solution.
- the quantity of the species injected is a function of mobility and therefore representative sample introduction can only be expected for neutral solutes.
- 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.
- concentration bias 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.
- 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.
- 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.
- a sample solution or sample consists of analyte and matrix.
- 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.
- the sample solution is exclusively drawn inlo the capillary by electroosmotic flow and no active electrophoretic migration of analyte occurs during sample introduction.
- the analyte in a high ionic strength matrix.
- 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.
- the sample solution is introduced into the separation chamber by the pumping action of the electroosmotic flow (EOF) only and no mobility bias occurs.
- EEF electroosmotic flow
- 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.
- Said salts may also be buffer salts and may additionally contribute to pi I.
- 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.
- physiological sample solutions such as blood, serum, plasma, urine, etc. which normally contain salts at about 1% concentration.
- salts, or organic compounds, or macr ⁇ inolecules, proteins or other substances of the matrix are retained at the stationary phase of the separation chamber.
- 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.
- 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).
- inventive method in combination with the CEC device according to the present invention.
- inventive method it is most preferable to use the inventive method in combination with the CEC device according to the present invention.
- inventive method in combination with the CEC device according to the present invention.
- 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.
- FIG. 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).
- FIG. 1 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).
- 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 (10 ⁇ 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.
- a high voltage power supply 10 ⁇ 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).
- 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.
- 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 miecti ⁇ buf fer solution
- n is prclcrred to adjust only the ionic strength of the sample solution to achieve the desired ratio of the ionic strength.
- At least one detection means (190) lo characterise the composition of the analyte qualitatively and optionally quantitatively may be adapted to the separation device.
- 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.
- inventive device may also comprise a means for automatic exchange of the reservoirs.
- the stationary phase in the CEC separation chamber may comprise, preferably consist of, porous and/or non-porous support material.
- 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.
- the surface of said support mater ial is preferably modified with hydrophilic, hydrophobic, ionic, chiral, and/or affinity residues.
- hydrophobic residues are especially alkyl chains of O - Cso, preferably between C and C 22 , 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.
- 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.
- outlet orifice of the separation chamber is adapted to function as an electrospray means.
- 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.
- 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).
- 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.
- ion t he (T.C ,epa ⁇ ai ⁇ on . 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.
- 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.
- microfluidic devices are known in the prior art and therefore are not described in more detail.
- 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).
- analyte and matrix are both retained at the beginning of the separation chamber.
- the separation chamber e. g. CEC column
- a stationary phase capable ol revc. sed-phase like retention of analyte e. g.
- 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.
- 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).
- 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.
- the ionic strength of the sample solution is det .rrnined and/or adjusted prior to injection.
- 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.
- the separat ion o l the samj.lo is pre fer ably
- a first buffer solution washing buffer
- second buffer solution separation buffer
- 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 ,C ⁇ , C
- 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.
- the preferably used high amount of acetonitrile may be replaced by alternative organic solvents such as methanol.
- 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.
- composition of the analyte is not known it is preferred to use so called universal buffers, which are optimized for such analytes.
- 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.
- the washing buffer was identical to the injection buffer.
- 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.
- 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.
- the injection buffer reservoir was exchanged by a reservoir containing the respective sample solution.
- the sample reservoir was replaced again by a buffer reservoir containing the injection washing buffer to remove the matrix.
- 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.
- the column was reconditioned according to a) prior to the next sample injection. . .
- Figuics 4 a) to 4 c) demonstrate the feasibility of bias-free electrokinetic sample introduction according to the present invention.
- CEC injection buffer 5 mM ammonium acetate al pH 4.7 with 5% (v/v) acetonitrile was used.
- 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.
- 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.
- 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 hydrodv ⁇ nmic sample intioductio ⁇ Ibupiofcn is sliongiy disciiminated in elccliokinctic sample introdu.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Electrostatic Separation (AREA)
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 dp of the particles of the packing material.
Δp =u*L*η*Φ/dp 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 HP3D 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.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18309000P | 2000-02-17 | 2000-02-17 | |
US60/183,090 | 2000-02-17 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001061335A2 true WO2001061335A2 (en) | 2001-08-23 |
WO2001061335A3 WO2001061335A3 (en) | 2002-07-18 |
Family
ID=22671399
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2001/001822 WO2001061335A2 (en) | 2000-02-17 | 2001-02-19 | Method and device for bias-free electrokinetic sample introduction and separation |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2001061335A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105067686A (en) * | 2015-07-23 | 2015-11-18 | 常州大学 | Selectivity identification of poly L-glutamic acid/amylose-modified glassy carbon electrode on tryptophan enantiomer |
CN110501402A (en) * | 2019-09-24 | 2019-11-26 | 山东农业大学 | A kind of electrochemical sensor and its detection method of super sensitivity detection sulphathiazole |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0620432A1 (en) * | 1993-04-15 | 1994-10-19 | Ciba-Geigy Ag | Method for controlling sample introduction in microcolumn separation techniques and sampling device |
WO1996033405A1 (en) * | 1995-04-17 | 1996-10-24 | Mayo Foundation For Medical Education And Research | Sample preprocessor |
WO1997003352A1 (en) * | 1995-07-13 | 1997-01-30 | Beckman Instruments, Inc. | Electrophoresis capillary tube with a conductive tip |
WO1998005950A1 (en) * | 1996-08-02 | 1998-02-12 | Dionex Corporation | Electromigration injection from a microreservoir-electrode in capillary separation systems |
-
2001
- 2001-02-19 WO PCT/EP2001/001822 patent/WO2001061335A2/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0620432A1 (en) * | 1993-04-15 | 1994-10-19 | Ciba-Geigy Ag | Method for controlling sample introduction in microcolumn separation techniques and sampling device |
WO1996033405A1 (en) * | 1995-04-17 | 1996-10-24 | Mayo Foundation For Medical Education And Research | Sample preprocessor |
WO1997003352A1 (en) * | 1995-07-13 | 1997-01-30 | Beckman Instruments, Inc. | Electrophoresis capillary tube with a conductive tip |
WO1998005950A1 (en) * | 1996-08-02 | 1998-02-12 | Dionex Corporation | Electromigration injection from a microreservoir-electrode in capillary separation systems |
Non-Patent Citations (5)
Title |
---|
CIKALO M G ET AL: "CAPILLARY ELECTROCHROMATOGRAPHY" ANALYST, LONDON, GB, vol. 123, July 1998 (1998-07), pages 87R-102R, XP000956125 * |
GUO W ET AL: "Sensitive, universal detection for capillary electrochromatography using condensation nucleation light scattering detection" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 849, no. 2, 23 July 1999 (1999-07-23), pages 587-597, XP004174305 ISSN: 0021-9673 * |
LANE S J ET AL: "AN AUTOMATED ELECTRO-OSMOTIC SAMPLE INTRODUCTION/SEPARATION INTERFACE FOR AUTOMATED ELECTRO-OSMOTIC FLOW FLOW-INJECTION MASS SPECTROMETRY, INFUSION MASS SPECTROMETRY AND CAPILLARY ELECTROCHROMATOGRAPHY MASS SPECTROMETRY" RAPID COMMUNICATIONS IN MASS SPECTROMETRY, HEYDEN, LONDON, GB, vol. 12, 1998, pages 947-954, XP002929949 ISSN: 0951-4198 * |
TAYLOR M R ET AL: "Gradient capillary electrochromatography of drug mixtures with UV and electrospray ionisation mass spectrometric detection" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 768, no. 1, 18 April 1997 (1997-04-18), pages 89-95, XP004064146 ISSN: 0021-9673 * |
XIONG B ET AL: "CAPILLARY ELECTROCHROMATOGRAPHY WITH MONOLITHIC POLY(STYRENE-CO-DIVINYLBENZENE-CO-METHACRY LIC ACID) AS THE STATIONARY PHASE" JOURNAL OF HIGH RESOLUTION CHROMATOGRAPHY, WILEY VCH, WEINHEIM, DE, vol. 23, no. 1, January 2000 (2000-01), pages 67-72, XP000935790 ISSN: 0935-6304 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105067686A (en) * | 2015-07-23 | 2015-11-18 | 常州大学 | Selectivity identification of poly L-glutamic acid/amylose-modified glassy carbon electrode on tryptophan enantiomer |
CN105067686B (en) * | 2015-07-23 | 2017-09-08 | 常州大学 | The Selective recognition of poly- L glutamic acid/amylose modified glassy carbon electrode tryptophan enantiomer |
CN110501402A (en) * | 2019-09-24 | 2019-11-26 | 山东农业大学 | A kind of electrochemical sensor and its detection method of super sensitivity detection sulphathiazole |
Also Published As
Publication number | Publication date |
---|---|
WO2001061335A3 (en) | 2002-07-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Robson et al. | Capillary electrochromatography: A review | |
US10403488B2 (en) | Solid phase extraction with capillary electrophoresis | |
Tagliaro et al. | A brief introduction to capillary electrophoresis | |
US8241476B1 (en) | Sol-gel coatings for on-line preconcentration in capillary electrophoresis | |
Dadoo et al. | Advances in capillary electrochromatography: Rapid and high-efficiency separations of PAHs | |
NAKAGAWA et al. | Electrokinetic chromatography for drug analysis. Separation and determination of cefpiramide in human plasma | |
Tagliaro et al. | Capillary electrophoresis: principles and applications in illicit drug analysis | |
US6998040B2 (en) | Sol-gel open tubular ODS columns with charged inner surface for capillary electrochromatography | |
Yang et al. | Two-dimensional capillary electrophoresis involving capillary isoelectric focusing and capillary zone electrophoresis | |
US20060042948A1 (en) | Microfluidic electrophoresis chip having flow-retarding structure | |
Collins et al. | A review of recent advances in electrochemically modulated extraction methods | |
Nakagawa et al. | SEPARATION AND DETERMINATION OF CEFPIRANIDE IN HUMAN PLASMA BY ELECTROKINETIC CHROMATOGRAPHY WITH A MICELLAR SOLUTION AND AN OPEN TUBULAR-FUSED SILICA CAPILLARY | |
Wei et al. | Enantiomer separations of phenylephrine and synephrine by capillary electrochromatography on bare silica stationary phase using hydroxypropyl‐β‐cyclodextrin as a mobile phase additive | |
Martınez et al. | Strategies for determining environmental pollutants at trace levels in aqueous samples by capillary electrophoresis | |
WO2001061335A2 (en) | Method and device for bias-free electrokinetic sample introduction and separation | |
Kuldvee et al. | Nonconventional samplers in capillary electrophoresis | |
Brunner et al. | High‐Performance Capillary Electrophoresis in the Pharmaceutical Sciences | |
US6569305B1 (en) | Method for injection and stacking of analytes in high-conductivity samples | |
Otsuka et al. | Micellar electrokinetic chromatography | |
Byrne et al. | Influence of the unpacked section on the chromatographic performance of duplex strong anion-exchange columns in capillary electrochromatography | |
Ahmed et al. | pH-assisted in-line pseudophase microextraction and separation with tridodecylmethylammonium chloride admicelles in open-tubular capillary-based separations | |
Mark | Analytical approaches to the analysis of small samples and Hyphenation of fast capillary electrophoresis to other instrumental techniques | |
Lister | Evaluation of nonaqueous solvents as carrier phases in capillary electrophoresis and capillary electrochromatography | |
Perlatti et al. | Sample Stacking: A Versatile Approach for Analyte Enrichment in CE and Microchip‐CE | |
張洛紅 | Development of in-capillary sample clean-up and preconcentration methods for capillary electrophoresis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): GM JP MW MZ US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): GM JP MW MZ US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase in: |
Ref country code: JP |