WO2002048673A2 - Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese - Google Patents

Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese Download PDF

Info

Publication number
WO2002048673A2
WO2002048673A2 PCT/US2001/043259 US0143259W WO0248673A2 WO 2002048673 A2 WO2002048673 A2 WO 2002048673A2 US 0143259 W US0143259 W US 0143259W WO 0248673 A2 WO0248673 A2 WO 0248673A2
Authority
WO
WIPO (PCT)
Prior art keywords
injection
sample
electrokinetic
analytes
analyte
Prior art date
Application number
PCT/US2001/043259
Other languages
English (en)
Other versions
WO2002048673A3 (fr
Inventor
James P. Landers
James F. Palmer
Original Assignee
University Of Virginia Patent Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Virginia Patent Foundation filed Critical University Of Virginia Patent Foundation
Priority to EP01988144A priority Critical patent/EP1355858A2/fr
Priority to US10/432,141 priority patent/US7223325B2/en
Priority to AU2002241480A priority patent/AU2002241480A1/en
Publication of WO2002048673A2 publication Critical patent/WO2002048673A2/fr
Publication of WO2002048673A3 publication Critical patent/WO2002048673A3/fr

Links

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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N2030/162—Injection electromigration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • This invention relates to an improved method and system for stacking analytes in electrophoresis, and more particularly an orthogonal analyte stacking/injection system that allows the loading of volumes that exceed that of the separation capillary, channel, well or whatever conduit is used for separation of the analytes (i.e., multiple column volumes).
  • Capillary electrophoresis is a high-resolution technique for separating charged analytes in liquid solutions using electric fields. The micron-scale radial dimensions of the capillary serve to dissipate Joule heating and control convection efficiently at high separation voltages, allowing for plate heights of micrometers or less.
  • FIG. 8 shows an exemplary setup that can be used to practice electrophoresis.
  • Electrophoresis unit 1 is interfaced with data collection/storage unit 14, which contains software/firmware/hardware for control of the instruments and data collection (e.g., processor, personal computer, display monitor and printer, or personal digital assistant (PDA) or the like.
  • Separation channel 2 (shown here as a capillary) has an entrance (inlet) end 4, an exit (outlet) end 6, and a detection window 8. Separation channel 2 is flushed by high pressure or vacuum with three to five capillary volumes of buffer.
  • the outlet reservoir 10 is filled with buffer and holds the exit end 6 of separation channel 2.
  • Inlet reservoir 12 is also filled with buffer and holds the entrance end 4 of separation channel 2.
  • Sample reservoir 11 contains sample for injecting into the separation channel 2.
  • the high voltage power unit 16 has an anode 18 and a cathode 20.
  • Outlet reservoir 10 also holds cathode 20, while inlet reservoir 12 also holds anode 18.
  • An electric field is then applied by high voltage power unit 16 across separation channel 2 from anode 18 to cathode 20, which causes buffer in inlet reservoir 12 to travel through separation channel 2 and into outlet reservoir 10, for electrophoretic conditioning of separation channel 2.
  • channel 2 has been electrophoretically conditioned, anode 18 and entrance end 4 of separation channel 2 are placed within sample reservoir 11.
  • An electric field may be applied across separation channel 2 for electrokinetic injection (injection by electrophoresis instead of by pressure) of a sample plug into separation channel 2.
  • the electric field is applied across separation channel 2 for a period of time sufficient to permit injection of a sample plug of desired length.
  • the plug could be injected into separation channel 2 using a pressure technique known in the art.
  • an analytes zone is located at the detection window 8
  • radiation for example from an ultraviolet (UN) light source 34
  • UN ultraviolet
  • the detector 36 is interfaced with a data collection/storage unit 14 to collect the separation data including the number of analytes zones to traverse the window as well as the relative amount of light that was incident on the detector (which translates to "concentration").
  • FIG.9 is an illustrative setup used to practice electrophoresis.
  • the electrophoretic unit 101 is a T-configuration cross-channel injection microchip.
  • Separation Channel 102 has an entrance end 104, an exit end 106.
  • Outlet reservoir 110 is filled with buffer and is connected to exit end 106 of separation channel 102.
  • Inlet reservoir 112 is also filled with buffer and is connected to entrance end 104 of separation channel 112.
  • Sample channel 140 has an entrance end 142 and an exit end 144.
  • Sample reservoir 111 contains sample for injecting into separation channel 102 and is connected to entrance end 142 of sample channel 140.
  • Waste reservoir 146 is filled with buffer and is connected to exit end 144 of sample channel 140.
  • Sample channel 140 is configured to connect through and cross over separation channel 102 to form a 'T-configuration'.
  • High voltage power unit 116 has an anode connected to inlet reservoir 104 and an anode connected to sample reservoir 111, and a cathode connected to outlet reservoir 110 and a cathode connected to waste reservoir 146.
  • An electric field is applied across separation channel 102 for electrophoretic conditioning. The field is applied from anode connected to inlet reservoir 112 to cathode connected to outlet reservoir 106.
  • an electric field is applied from anode connected to sample reservoir 111 to cathode connected to outlet reservoir 106 for a defined period of time.
  • An electric field is then applied across separation channel 102 causing separation and detection of the various analyte zones. The field is applied from anode connected to inlet reservoir 112 to cathode connected to outlet reservoir 106.
  • micellar electrokinetic chromatography utilized a charged micelle to impart mobility to neutral analytes.
  • electrokinetic chromatography EKC
  • EKC electrokinetic chromatography
  • Recent techniques for stacking neutral analytes in EKC require pressure injections of sample matrixes. For instance, high-salt stacking and sweeping have been applied to afford sample plug lengths up to 60% of the effective capillary length (length from the injection end to the detection point).
  • High-salt stacking utilizes discontinuous buffer conditions (the separation matrix co-ion is different from the sample matrix co-ion).
  • the sample matrix co-ion (the ion with the same charge as the electrokinetic vector, e.g., chloride) must have a higher intrinsic electrophoretic mobility than the electrokinetic vector, and the sample co-ion must be present at a higher concentration in the sample matrix than that of the electrokinetic vector in the separation buffer.
  • sweeping utilizes continuous sample matrix/ separation buffer conditions, i.e., there are no mobility differences between the background electrolyte or buffer, between the sample matrix and the separation buffer.
  • pressure injections are typically low-velocity to diminish mixing at the sample matrix / separation buffer interface and to maintain reproducibility between analyses. While typical separations take as little as 60 seconds, the time necessary to introduce long sample plugs (e.g., 50% of the capillary length, ca. 50- 2000 seconds) by low pressure can, in certain instances, exceed the analysis time by more than an order of magnitude.
  • the electroosmotic flow can be used to inject neutral analytes in EKC without stacking, however, a concomitant stacking of neutral analytes during electrokinetic injection by electroosmotic flow is possible - this is described herein. Furthermore, the electrokinetic injection of high-salt sample matrixes by electroosmotic flow is provided, with the stacking of neutral analytes occurring simultaneously with the injection procedure, in both high-salt stacking and sweeping modes.
  • the present invention method of stacking analytes in electrophoresis comprises: filling a conduit with a separation buffer, the buffer comprising buffer components; orienting a ⁇ first end of said conduit in operative relation with (e.g., articulated with or in synthesis with) the separation buffer; orienting a second end of the conduit in operative relation with (e.g., articulated with or in synthesis with) an analyte sample; applying an electric field across the separation buffer and the analyte sample through the conduit.
  • the present invention method further comprises, adjusting the mobility of either said injection vector or the stacking vector (or both) to allow maximum amount of the injected analyte.
  • the present invention provides an apparatus for stacking analytes in electrophoresis, comprising: means for filling a conduit with a separation buffer, the buffer comprising buffer components; means for orienting a first end of the conduit in operative relation with (e.g., articulated with or in synthesis with) the separation buffer; means for orienting a second end of said conduit in operative relation with (e.g., articulated with or in synthesis with) an analyte sample; means for applying an electric field across the separation buffer and the analyte sample.
  • the present invention further comprises means for adjusting the mobility of either the injection vector or the stacking vector (or both) to allow maximum length of said injected analyte.
  • the present invention electrokinetic injection method is faster than previous injection methods by approximately an order of magnitude.
  • Another advantage of the present invention method is that it allows the loading of multiple-capillary volumes of sample for the first time. Moreover, another advantage of the present invention method is that it provides a higher resolution for injection of large sample plugs in capillary electrophoresis, providing a faster and higher sensitivity injection mode in capillary electrokinetic chromatography.
  • the present invention provides a method and system where stacking can be initiated with injection, rather than post-injection.
  • the present invention further provides electrokinetic injection of neutral analytes with concomitant stacking of neutral analytes can be initiated at the commencement of injection. See Palmer, J.; Burgi, D.S.; Munro, N. J.; Landers, J.P., "Electrokinetic Injection for Stacking Neutral Analytes in Capillary and Microchip Electrophoresis” , Anal. Chem. 2001, 73,725-731.
  • the present invention method provides an electrokinetic injection of neutral analytes by electroosmotic flow in the presence of an orthogonal stacking system, i.e., an anionic electrokinetic vector in the separation buffer, constitutes an orthogonal analyte stacking/ injection system (OAS/IS).
  • an orthogonal stacking system i.e., an anionic electrokinetic vector in the separation buffer
  • the requisite conditions are simply that the electrokinetic vector in the separation buffer has a mobility opposite that of the analyte injection force, and that a suitable stacking boundary is formed between the injected sample and the electrokinetic vector in the separation buffer.
  • OAS/IS orthogonal analyte stacking/ injection system
  • FIGS. 1A-1C is theoretical depiction of co-ion boundary movement into a capillary.
  • the lower velocity of the co-ion boundary (CB) allows sample plugs longer than 100% of the effective capillary length.
  • Anode is at the injection end.
  • the boundaries include a sample plug front (SPF) and sample plug back (SPB).
  • SPF sample plug front
  • SPB sample plug back
  • the co-ion boundary velocity is half that of the electroosmotic flow velocity. This is a plausible situation when the co-ion boundary has an anodic mobility.
  • FIG. 1 A depicts an analyte zone (AZ) length for a 100% capillary effective length injection without stacking.
  • IB depicts a 100% capillary effective length injection with stacking occurring at a co-ion boundary with a velocity half that of the electroosmotic flow.
  • FIG. 1C depicts a position of co-ion boundary and stacked analytes after transition of the sample plug through the co-ion boundary.
  • FIG. 2 is an electropherogram where the absorbance at the detector [in milli- absorbance units (mAU)] is plotted versus time after a multiple capillary-length sample plug injection has been initiated followed by separation - resolution of corticosteroid analytes is seen.
  • mAU milli- absorbance units
  • the general conditions are as follows: separation buffer containing 40 mM sodium dodecyl sulfate (SDS), and 240 mM borate; the sample matrix consisting of 270 mM borate and containing four corticosteroids at 50 ppb; and a 33 cm capillary having a 50 ⁇ m i.d. Also provided is a continuous injection from the sample matrix into the separation buffer under normal polarity at 15 kV.
  • FIG.3 is an electropherogram showing absorbance (mAU) versus time when comparing the differences in effectiveness of electrokinetic versus pressure injections.
  • the general conditions are as follows: 150 mM sodium chloride sample matrix, duration of 30 kV or 50 mbar injections as listed in the figure.
  • the separation conditions are: 80 mM sodium cholate, 10% ethanol, 5 mM tetraborate, pH ⁇ 9separation buffer, in a 19 mm (i.d.) by 33 cm capillary; separation at 30 kN.
  • the total analysis times with an 80% effective-length plug injection by pressure was 1928 seconds. With electrokinetic injection, the total analysis time was 250 seconds; peak order for 1-5, cortisone, cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, and progesterone. This graph indicates that electrokinetic injection not only allows for higher-resolution to be obtained when compared to pressure injection, but it is also approximately an order of magnitude faster.
  • FIG. 5. is an electropherogram showing absorbance (mAU) plotted versus time for electrokinetic stacking injection with continuous buffer conditions.
  • Sample matrix was 50 mM borate (approximately the same conductivity of the separation buffer). The parameters are: cortisone, 11-deoxycortisol, and progesterone at 10 ng/ml.
  • the separation buffer comprises: 50 mM SDS, 20% methanol, 5 mM tetraborate, pH ⁇ 9. 50 ⁇ m i.d. by 33 cm capillary, and separation at 30 kN.
  • FIG. 6 an electropherogram showing a graphical representation (absorbance versus time) for electrokinetic stacking injection instigated by a leading co-ion.
  • Sample matrix was 20 mM borate with 4 mM hydrogen phosphate, pH ⁇ 9, cortisone, 11- deoxycortisol, and progesterone analytes at 50 ng/ml, with 30 kN injection for 60 seconds. Separation conditions and peak order as in FIG. 5.
  • FIG. 7 is an electropherogram showing relative fluorescence versus time for electrokinetic stacking injection on a microchip.
  • Sample matrix was 150 mM sodium chloride.
  • FIG. 8 is a schematic representation of an exemplary test set-up used to practice the present invention.
  • FIG. 9 is a schematic representation of an exemplary test set-up used to practice the present invention, wherein electrophoretic unit is a T-configuration cross-channel microchip.
  • FIG. 10 is an electropherogram showing a graphical representation of absorbance (mAU) versus time to illustrate the effect of SDS concentration on analyte migration time.
  • Separation buffer comprises: SDS at concentrations from 5 to 160 mM as shown in the figure, with 20 mM borate. The capillary is 50 ⁇ m by 33 cm. Borate sample matrix equivalent conductivity to each separation buffer. Injection for 40 seconds at 15 kN. The EOF velocity (for 5 to 160 mM SDS buffers) was as follows: 0.288, 0.285, 0.276, 0.265, 0.260, 0.260 cm/sec. The respective length of sample plug is 11.5 to 10.4 cm. Separation was at 15 kN and sample solvent zones are indicated in the figure. Migration time range for the slowest and fastest analytes are indicated by brackets below each electropherogram. Analyte concentrations were 1800 ppb, 1. Cortisol, and 4 Progesterone.
  • FIG. 11 is an electropherogram showing a graphical representation of absorbance (mAU) versus time for depicting the effect of methanol modification of the separation buffer.
  • Separation buffers with 40 mM SDS and 20 mM borate, and augmented with 0, 5, 10, 15, 20, and 25% methanol as stated in the figure.
  • Borate sample matrix was equivalent in conductivity to each separation buffer.
  • Electrokinetic injection was at 30 kN for 20 seconds with separation at 30 kN.
  • Analyte concentration was 1800 ppb.
  • the peaks 1 - 4 cortisol, 11-deoxycortisol, 17 ⁇ -hydroxyprogesterone, and progesterone. Sample solvent zone is indicated by brackets in the figure.
  • FIG. 12 is a graphical representation of peak velocity versus borate concentration for illustrating the effect of background electrolyte concentration on analyte velocity.
  • FIG. 13 is an electropherogram showing a graphical representation (absorbance
  • Separation buffer 40 mM SDS in 240 mM borate at pH ⁇ 9.2. Both injection and separation is at 15 kN. The injections were for 5, 7, 9, and 11 minutes, corresponding to injection plug lengths of 65.7 to 144.5 cm as indicated. Lines above each electropherogram correspond to the length of electrokinetic injection in minutes. The length in cm for each injection is listed above each bracket. The arrow extending downward from the right-side of each line points to the time the inlet vial was switched from the sample matrix to separation buffer.
  • the sample matrix consists of 270 mM borate with analytes at ⁇ 270 ppb. Further included are peaks 1 - 4: cortisol, 11- deoxycortisol, 17 ⁇ -hydroxyprogesterone, and progesterone.
  • the EOF velocity is established at 0.219 cm sec as per the text.
  • FIGS. 14A-14F are schematic depictions of the position of stacked analyte/micelle peak during extended injections.
  • a 33 cm capillary with a 24.5 cm effective length is depicted.
  • the detector position is indicated by vertical arrows.
  • FIG. 14A demonstrates the location of the stacked analyte at the conclusion of a 171 cm injection.
  • the stacked analyte/micelle peak has progressed 20.38 cm into the capillary.
  • 20.38 cm of unstacked sample plug remain to pass into the micelle zone to be stacked.
  • FIG. 14B illustrates the movement of the stacking analyte peak and the sample plug.
  • FIG. 14C shows the position of the analyte/micelle peak at the conclusion of the passage of the sample matrix.
  • the analyte/micelle peak has moved 23.14 cm into the capillary, leaving ⁇ 1.4 cm remaining for separation.
  • the injection-side of the sample plug does not have time to pass through the stacked analyte/micelle peak before the peak has entered the detector.
  • a 196 cm injection is depicted in FIG. 14D.
  • the analyte/micelle peak has moved 23.4 cm into the capillary. The moment the peak has entered the detector window, the length of sample plug that has still not passed through the analyte/micelle peak is 16.7 cm as shown in FIG. 14E.
  • FIG. 15 is an electropherogram of absorbance (mAU) versus time for demonstrating the empirical corroboration of maximum injection length.
  • the 144.5 cm injection does not exceed the. length required for the injection-side of the sample plug to transit the analyte/micelle peak before it reaches the detector, and there is adequate distance remaining within the capillary to allow separation of the analytes following the sample injection.
  • the 171 cm injection exhibits a slightly lower peak height than the 197 cm injection, as explained in the text. Injections beyond 180 cm are predicted to be identical.
  • the Inset depicts injections of 196, 223, 250, and 276 cm, overlaid.
  • the present invention provides a method and system of injecting analytes into a capillary or conduit filled with separation buffer for stacking and separation by electrokinetic chromatography.
  • the method of injection utilizes electroosmotic flow to inject analytes against a stacking front composed of an anionic electrokinetic vector.
  • This method is instituted electrokmetically by injecting the analytes directly from a sample matrix into the capillary or other microchannel electrophoresis apparatus by applying an electric field.
  • the present invention provides the first instance of an orthogonal analyte stacking/injection system (OAS IS) and related method thereof. This system and method and other potential OAS/IS conditions are described herein. As shown in FIG.
  • OFIS orthogonal analyte stacking/injection system
  • orthogonal analyte stacking and injecting can allow for the injection of sample plugs that are equal to or greater than the entire length of the capillary. Or said differently, this technique allows for large sample volumes (large by microanalytical technique standards) to be injected for analysis.
  • the conventional rule or standard with most separations is that no more than 1-5% of the total column volume can be injected as a sample plug.
  • sample plug volumes equivalent to and exceeding 100% of the column volume i.e., multiple column volumes
  • Equation (1) describes analyte loading by pressure injections:
  • Q is the moles of analyte, i that are injected, 1,- is the length of the capillary that analyte i occupies at the conclusion of injection, A is the cross sectional area of the capillary, and is the molarity of analyte i in the injection vial.
  • Equation (2) describes electrokinetic injections in continuous systems (i.e., in the absence of an isotachophoresis (ITP) effect):
  • V B is the bulk electroosmotic flow velocity
  • x is the length of the sample plug injected that is expressed as a fraction of the total capillary length
  • v s is the velocity of the electroosmotic flow solely in the presence of sample matrix
  • v sep is the velocity of the electroosmotic flow solely in the presence of separation buffer.
  • electrokinetic stacking injection of neutral analytes in electrokinetic chromatography ⁇ KC
  • Electroosmotic flow is used to inject long sample plugs under continuous and discontinuous conditions.
  • the simultaneous stacking of neutral analytes at a co-ion interface with the electrokinetic vector in the separation buffer during injection is described.
  • the decreased analysis times using electrokinetic injection, as well as the enhanced detection limits and reproducibility versus pressure injection are shown.
  • the results are derived from the use of neutral analytes, with an anionic electrokinetic vector, in the presence of electroosmotic flow, under normal polarity (with the anode at the inlet).
  • a comprehensive outline of potential OAS/IS method and system conditions are provided for alternative embodiments.
  • the instant disclosure describes the potential of OAS/IS methods and systems for anionic and cationic analytes, and cationic and neutral electrokinetic vectors. Also provided is the potential of orthogonal analyte stacking and injecting methods and systems in the absence of EOF, and the potential of orthogonal analyte stacking and injecting under reversed polarity. While the length of the sample plug injection increases proportionally with the duration of the injection multiplied by the EOF rate (inj time x EOF), the analytes are stacked at the pseudo steady-state boundary that is moving against the EOF. This feature allows stacking to be initiated at the capillary inlet at the beginning of the injection. As injection continues, the boundary moves into the capillary because the electrophoretic mobility of the boundary is less than the electrophoretic mobility of the EOF.
  • the effective capillary length remaining for separation at the conclusion of injection can be determined by:
  • ⁇ ⁇ OF is the electrophoretic mobility of EOF
  • E is the applied field
  • t is the number of seconds of injection
  • Lj is the length of the injection
  • ⁇ psb is the electrophoretic mobility of the pseudo steady-state boundary
  • t t r a n s is the amount of time it takes the pseudostationary boundary to completely transit the sample plug
  • Ltra ns is distance into the capillary the stacking boundary has moved upon completely transiting the sample plug.
  • FIG. 2 is an electropherogram where the absorbance at the detector [in milli- units(mAU)] is plotted versus time for a continuous injection from the sample matrix into the separation buffer.
  • the electroosmotic flow indicator is shown at 2.2 minutes, while the analyte peaks do not appear until 12.8 minutes. This indicates a sample plug length of approximately 191 cm in a capillary that is only 33 cm long.
  • the peak height of the analytes is large ( ⁇ 70 MAU) for an analyte concentration of only 50 ppb, indicating a limit of detection in the parts per trillion (ppt) range. This sensitivity has never before been reported for neutral analytes in capillary electrophoresis.
  • FIG. 1 is an electropherogram where the absorbance at the detector [in milli- units(mAU)] is plotted versus time for a continuous injection from the sample matrix into the separation buffer.
  • the electroosmotic flow indicator is shown at 2.2 minutes, while the analyt
  • FIG. 3 is an electropherogram showing absorbance (mAU) versus time that illustrates the differences in effectiveness of using electrokinetic injection compared to pressure injection, whereby electrokinetic injection can be up to an order of magnitude faster than conventional art.
  • mAU absorbance
  • a negatively-charged electrokinetic vector such as a micelle is commonly used.
  • a neutral analyte acquires an effective mobility when it interacts with the electrokinetic vector, in this case a micelle.
  • the micelle is mobilized against the EOF.
  • Stacking techniques in this mode using pressure injection of the sample matrix have been developed based on the difference in mobility of neutral analytes in a sample matrix, devoid of a micelle, with either equivalent conductivity (See Quirino, J.P.; Terabe, S.
  • the minimum velocity for a neutral analyte is the velocity of the micelle (neglecting the size and mass of the analyte) if an analyte is completely complexed with the micelle.
  • the velocity of the micelle is simply a sum of the anodic mobility of the micelle and the mobility of the bulk fluid toward the cathode (EOF) multiplied by the electric field.
  • neutral analytes are prepared in a sample matrix containing the same background electrolyte or buffer (e.g., borate) as the separation buffer.
  • buffer e.g., borate
  • SDS sample matrix co-ion
  • borate as the background electrolyte
  • the borate concentration in the sample matrix was adjusted to provide equivalent conductivity to the separation buffer that contained both borate and the charged micelle. This condition is referred to as "continuous conductivity" to distinguish it from the use of a discontinuous sample matrix co-ion (i.e., high-mobility) such as chloride.
  • the neutral analytes chosen as model compounds for the study disclosed herein are members of the vertebrate corticosteroid metabolic pathway, step-wise hydroxylated from progesterone to 17 ⁇ -hydroxyprogesterone, 11-deoxycortisol, and finally to cortisol.
  • the step- wise hydroxylation of each analyte decreases the hydrophobic interaction with the micellar phase.
  • the background electrolyte, borate does not act as an electrokinetic vector for the selected analytes.
  • the migration order of the analytes under normal polarity electrophoresis with SDS as the electrokinetic vector indicates that elevated hydrophobic interactions between the analytes and SDS cause an increase in analyte migration time, i.e., the more hydrophobic analytes display longer migration times.
  • L max is the maximum injectable plug length
  • P EOF is the velocity of EOF
  • » ekv is the velocity of the analyte due to interaction with the elecfrokinetic vector (charged micelle)
  • L et is the length of the capillary to the detector.
  • One approach to increase injection length would be to augment EOF by utilizing pressure or a capillary surface coating.
  • all components are affected by bulk flow, including the analyte/ electrokinetic vector complex.
  • Increasing the EOF would simply move all components toward the detector and reduce capillary length remaining for separation of analytes. Reducing EOF is undesirable because it would lengthen the amount of time required to inject a sample plug. It is apparent that the j>a/ekv should be reduced without an equivalent decrease in P EOF to afford an increased sample plug length as well as to maintain injection speed.
  • the present invention method and system minimizes analyte/micelle complex velocity without substantially reducing the EOF velocity.
  • Separation buffer parameters including the concentration of the micellar species, organic modifier, and background electrolyte were examined herein.
  • the sample matrix conductivity was adjusted to the equivalent separation buffer conductivity by addition of the same background electrolyte used in the separation buffer (borate).
  • borate background electrolyte
  • the present invention provides for the electrokinetic injection of neutral analytes by elecfroosmotic flow under normal polarity in electrokinetic chromatography with a negatively-charged electrokinetic vector. Under these conditions, the electrokinetic vector is moving opposite, or orthogonally to the electroosmotic flow.
  • This is the first demonstration of orthogonal analyte stacking/ injection systems (OAS/IS).
  • OAS/IS orthogonal analyte stacking/ injection systems
  • Conditions for OAS/IS methods and systems by electrokinetic and pressure injections are outlined below in Tables 1-6, with directions given for different polarity, electrokinetic vector charge, and analyte charge, as well as positive and negative pressure.
  • Elecfrokinetic vector can include a neutral species that affects the electrophoretic mobility of a charged species, such as an entanglement mafrix.
  • Table 1.I.A.1 A capillary or microchannel known to those skilled in the art is filled with a separation buffer containing an anionic electrokinetic vector such as described in the "Examples" section of this instant disclosure, e.g., sodium dodecyl sulfate or sodium cholate. Typical concentration is about 5- 300 mM.
  • a buffering agent such as sodium borate or sodium phosphate (Na 2 HPO 4 , NaH 2 PO4, H 3 PO4), or
  • Tris/TRIZMA Tris/TRIZMA, or combinations thereof or other buffering agents to obtain a pH that allows the formation of electroosmotic flow under an applied electric field is used.
  • a sample matrix without the electrokinetic vector is prepared with either the same buffering agent as found in the separation buffer, or a different salt such as sodium chloride or other salt to maintain a similar or higher conductivity than the separation buffer.
  • the sample matrix may also comprise a biological or other sample containing a native salt concentration, which may be used "as is" or augmented with an appropriate salt or buffer to allow the formation of a stacking boundary necessary to allow subsequent electrokinetic stacking.
  • the sample matrix may also contain a combination of a buffering agent and a higher-mobility co-ion such as phosphate, sulfate, charged cyclodextrins, fluoride, chloride, bromide, or iodide or other high-mobility co-ions. Neutral analytes of interest are dissolved in the sample matrix.
  • a buffering agent such as phosphate, sulfate, charged cyclodextrins, fluoride, chloride, bromide, or iodide or other high-mobility co-ions.
  • Injection is performed by subjecting the inlet end of the capillary in contact with the sample matrix and the outlet end of the capillary in contact with the separation buffer.
  • the inlet end of the capillary contains the anode
  • the outlet end of the capillary contains the cathode.
  • An electric field is applied, typically but not exclusively between about 10 and 1000 volts per centimeter. This causes the injection of neutral analytes by elecfroosmotic flow (EOF) into the capillary.
  • EEF elecfroosmotic flow
  • the anionic electrokinetic vector in the separation buffer forms a boundary with the sample matrix co-ion(s), and the solvent in the sample matrix passes through the boundary as the solvent moves toward the cathode (outlet).
  • a typical injection can allow more than 5 capillary volumes of sample matrix to be introduced into the capillary (See Palmer etal. Analytical Chemistry 2001). Injection is terminated by ceasing the electric field, removing the sample matrix from contact with the capillary inlet, and placing separation buffer and the anode at the capillary inlet. An electric field is again applied, typically between about 10 and 1000 volts per centimeter, to allow the subsequent separation and detection of the neutral analytes.
  • Table 1.I.A.2 These same conditions (1.I.A.1) can be used to stack and detect cationic analytes dissolved in the sample mafrix.
  • Table 1.I.A.3 These same conditions (1.I.A.1) can be used to stack and detect cationic analytes, in this case, with an uncharged (neutral) or zwitterionic elecfrokinetic vector in the separation buffer instead of an anionic electrokinetic vector.
  • a typical capillary or microchannel is filled with a separation buffer containing cationic electrokinetic vector. Typical concentration is about 5- 300 mM.
  • a buffering agent such as sodium borate or sodium phosphate (Na 2 HPO 4 , NaH 2 PO4, H 3 PO4), or Tris/TRIZMA, or combinations thereof or other buffering agents or proprietary compounds to obtain conditions that allow for reversed electroosmotic flow under an applied electric field is used.
  • a sample matrix without the electrokinetic vector is prepared with either the same buffering agent as found in the separation buffer, or a different salt such as sodium chloride or other salt to maintain a similar or higher conductivity than the separation buffer.
  • the sample matrix may also comprise of a biological or other sample containing a native salt concentration, which may be used "as is” or augmented with an appropriate salt or buffer to allow the formation of a stacking boundary necessary to allow subsequent electrokinetic stacking.
  • the sample matrix may also contain a combination of a buffering agent and a higher-mobility co-ion such as lithium, sodium, potassium, charged cyclodextrins, or other high-mobility co-ions. Neutral analytes of interest are dissolved in the sample matrix.
  • Injection is performed by subjecting the inlet end of the capillary in contact with the sample matrix and the outlet end of the capillary in contact with the separation buffer.
  • the inlet end of the capillary contains the cathode
  • the outlet end of the capillary contains the anode.
  • An electric field is applied, typically but not exclusively between about 10 and 1000 volts per centimeter. This causes the injection of neutral analytes by reversed electroosmotic flow into the capillary.
  • the cationic electrokinetic vector in the separation buffer forms a boundary with the sample matrix co-ion(s), and the solvent in the sample matrix passes through the boundary as the solvent moves toward the anode (outlet).
  • Injection is terminated by ceasing the electric field, removing the sample matrix from contact with the capillary inlet, and placing separation buffer and the cathode at the capillary inlet.
  • An electric field is again applied, typically between about 10 and 1000 volts per centimeter, to allow the subsequent separation and detection of the neutral analytes.
  • Table 1.I.B.2 These same conditions (l.I.B.l) can be used to stack and detect anionic analytes dissolved in the sample matrix.
  • Table 1.I.B.3 These same conditions (1.I.B.1) can be used to stack and detect anionic analytes, in this case, with an uncharged (neutral) or zwitterionic electrokinetic vector in the separation buffer instead of a cationic electrokinetic vector.
  • Table 2.II.A.1 The same conditions as found in (1.I.A.1) can be used with the injection vector, electroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 2.II.A.2 The same conditions as found in (1.I.A.2) can be used with the injection vector, elecfroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 2.II.A.3 The same conditions as found in (1.I.A.3) can be used with the injection vector, electroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 2.H.B.1 The same conditions as found in (l.I.B.l) can be used with the injection vector, electroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 2.II.B.2 The same conditions as found in (1.I.B.2) can be used with the injection vector, electroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 2.II.B.3 The same conditions as found in (1.I.B.3) can be used with the injection vector, electroosmotic flow, augmented by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the elecfrokinetic injection step.
  • Table 2.II.C.1 The same conditions as found in (1.I.A.1) can be used with the injection vector, electroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 2.II.C.2 The same conditions as found in (1.I.A.2) can be used with the injection vector, electroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 2.II.C.3 The same conditions as found in (1.I.A.3) can be used with the injection vector, elecfroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 2.II.D.1 The same conditions as found in (1.I.B.1) can be used with the injection vector, elecfroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 2.II.D.2 The same conditions as found in (1.I.B.2) can be used with the injection vector, elecfroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 2.II.D.3 The same conditions as found in (1.I.B.3) can be used with the injection vector, electroosmotic flow, augmented by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.A.1 The same conditions as found in (2.II.A.1) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 3.III.A.2 The same conditions as found in (2.II.A.2) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the elecfrokinetic injection step.
  • Table 3.III.A.3 The same conditions as found in (2.II.A.3) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 3.III.B.1 The same conditions as found in (2.II.B.1) can be used in an absence of or reduced elecfroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 3.III.B.2 The same conditions as found in (2.II.B.2) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the elecfrokinetic injection step.
  • Table 3.III.B.3 The same conditions as found in (2.II.B.3) can be used in an absence of or reduced elecfroosmotic flow with the injection vector provided by applying positive pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the inlet during the electrokinetic injection step.
  • Table 3.III.C.1 The same conditions as found in (2.II.C.1) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.C.2 The same conditions as found in (2.II.C.2) can be used in an absence of or reduced elecfroosmotic flow with the inj ection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.C.3 The same conditions as found in (2.II.C.3) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.D.1 The same conditions as found in (2.II.D.1) can be used in an absence of or reduced elecfroosmotic flow with the injection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.D.2 The same conditions as found in (2.II.D.2) can be used in an absence of or reduced electroosmotic flow with the injection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • Table 3.III.D.3 The same conditions as found in (2.II.D.3) can be used in an absence of or reduced elecfroosmotic flow with the injection vector provided by applying negative pressure, typically but not exclusively about 1 to 500 mbar above atmospheric pressure, at the outlet during the electrokinetic injection step.
  • HPLC-grade water (Fisher Company, St. Louis, MO) was used for all separation buffers and sample matrixes.
  • Sodium tetraborate, sodium hydroxide, sodium chloride, and punctilious ethanol were obtained from Sigma Company (St. Louis, MO).
  • Corticosteroids were obtained from Steraloids, Inc. (Newport, RI).
  • BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-propanol) was obtained from Molecular Probes Company(Eugene, Oregon), and sodium fluorescein was obtained from Acros Company, New Jersey. Sodium dodecyl sulfate was obtained from Bio-Rad Laboratories (Hercules, California).
  • Capillary temperature was maintained at 20°C and detection was by UV absorption at 242 nm with a 20 nm bandwidth except as noted.
  • Stacking of a fluorescent neutral compound (BODIPY) was performed with a Beckman P/ACE 5510 using a P/ACE system laser module 488 equipped with a 27 ⁇ m by 37 cm capillary.
  • the separation buffer vial volumes were 0.7 ml.
  • the sample matrix volumes were 100 ⁇ l.
  • the separation buffer consisted of 80 mM sodium cholate, 5 mM tetraborate, and 10% punctilious ethanol in HPLC-grade water (pH range 8.9-9.1), or sodium dodecyl sulfate, at concentrations as noted, with 5 mM tetraborate and 20% methanol. Separation buffers were degassed by manual decompression in a syringe and passed through a 0.2 ⁇ m pore- diameter filter before use. Sample matrixes were prepared with sodium chloride or sodium tetraborate at stated concentrations in HPLC-grade water.
  • Crystalline corticosteroids were dissolved in punctilious ethanol at 1.00 ⁇ M (316 to 362 ⁇ g/ml). Aliquots were dried down and re-suspended at stated dilutions. Standards were stored at 4°C when not in use. Analyte concentrations in the sample matrixes were as stated in each experiment.
  • the capillary length was 33 or 48.5 cm with an i.d. of 50 ⁇ m except as noted.
  • New capillaries were conditioned by flushing with 1.0 M sodium hydroxide, water, and separation buffer, in order. The same procedure was used to flush the capillaries at the start of each day's experiments.
  • the capillary was first flushed by high pressure (-950 mbar) with a three- to five- capillary volume of fresh separation buffer (1-5 min). Separations were carried out at 30 kV. The separation buffer was replaced after no more than two hours total running time and the capillaries were reconditioned daily by flushing with 1.0 M sodium hydroxide and water.
  • the velocity of sample plug injection by low-pressure and electrokinetic injection was determined for 33 cm capillaries with i.d.'s of 19, 27, and 50 ⁇ m using 150 mM sodium chloride sample matrix, and 80 mM sodium cholate with 10% ethanol and 40 mM borate separation buffer (pH ⁇ 9).
  • Triplicate injections were made under reverse pressure (-50 mbar) or reverse voltage (-30 kV) from the outlet (sic) vial, which contained the high-salt matrix.
  • time of absorbance shift at the detection point under these reversed conditions was recorded to determine injection velocity.
  • sample matrixes consisting of 0, 25, 50, 75, 100, 150, 200 and 300 mM sodium chloride were examined for stacking effect by elecfrokinetic injections at 30 kV for 40 seconds.
  • optimal salt concentration for stacking 150 mM sodium chloride
  • 5 10, 20, 40, and 80 second injections were examined to verify linearity of injection time with peak response of embarkral analytes at the detector. Peak area was examined to determine linearity of analyte injection with injection duration.
  • Triplicate electrokinetic injections from six identical high-salt samples, with separation mode using a single set of separation buffer vials were utilized to determine the robustness of the separation buffer.
  • a simple T-configuration cross-channel injection microchip (Alberta Microelectronics Corporation, Edmonton, Alberta, Canada) was utilized to demonstrate stacking of a neutral analyte on a microchip format.
  • the separation channel was 7.6 cm from the junction of the sample cross channel to the outlet (O), with a hemispherical cross section 50 ⁇ m wide by 20 ⁇ m deep.
  • the sample T-channel had the same cross-section dimensions as the separation channel, extending perpendicular to the separation channel for 0.6 cm to the sample well (S) and waste well (W).
  • the microchip channels were conditioned by flushing with 1.0 M sodium hydroxide, water, and separation buffer, in order.
  • the apparatus for pressure- flushing the microchip channels was previously described in copending US Application No. 09/418,659 by Palmer et al.
  • the sample well was flushed with 150 mM sodium chloride, then filled with the sample matrix consisting of 150 mM sodium chloride with BODIPY at 67 nM.
  • Wells I, O, and W were filled to equivalent levels with separation buffer.
  • the separation channel was conditioned by inducing a field between I and O (+500/ -2000) for 100 seconds while holding S and W at ground to reduce cross- channel leakage.
  • Electrokinetic injection was performed by applying an electric field between S and O (+250/ -1000) for 20, 40 60, 80, 100 and 120 seconds, while floating W and I.
  • the stacking efficiency in this mode is dependent on the concentration of salt in the sample matrix.
  • the co-ion in the sample matrix (chloride in this case) must be present at a concenfration sufficient to reduce its velocity to less than that of the electrokinetic vector in the separation buffer. This causes the formation of a pseudo-steady state field boundary that forces an increased concentration of the electrokinetic vector in the separation buffer at the cathode-side of the co-ion boundary.
  • a "soft boundary" between zones of different conductivity can occur.
  • a soft boundary is caused by differences in the zonal electroosmotic flow intrinsic mobilities that form a pressure barrier, leading to a Pousieulle-flow profile that detracts from the stacking effect of analytes encountering the boundary.
  • ⁇ s and ⁇ ev are the electrophoretic mobility of the co-ion in the sample matrix and separation buffer, respectively, and Es and E v are the electric field strengths in the sample co-ion zone and the electrokinetic vector zone.
  • chloride having a postulated intrinsic mobility of roughly 1.5-3 times that of the cholate micelle under these conditions. This is based on the necessity of supplying chloride ion in the sample matrix at a concentration of 1.5-3 times that of the electrokinetic vector in the separation buffer for stacking to occur. Absolute mobility is difficult to interpret due to variations in ionic constituents that induce ITP effects as well as field differences induced by zonal conductivity differences.
  • capillary has a low-pressure injection velocity of 0.081 cm per second, or an electroosmotic flow injection velocity of 0.372 cm per second (with an applied voltage of 30 kV). Elecfrokinetic injection is 4.6 times faster at this capillary length and internal diameter. However, a 33 cm capillary with an i.d. of 19 ⁇ m has a low-pressure injection velocity of 0.012 cm per second, or an elecfroosmotic flow injection velocity of 0.295 cm per second, representing an injection rate 25-times faster when utilizing elecfroosmotic flow. With longer capillaries, low-pressure injection is slowed by an increase in flow resistance proportional to the capillary length. Likewise with electrokinetic injection, the field is reduced proportional to capillary length.
  • the total analysis times for electrokinetic versus pressure injection of long sample plugs can be compared by summing the injection and analysis times.
  • elecfrokinetic injection is approximately 50% faster (ca. 2.5 minute total analysis versus ca. 3.5 minutes with pressure injection).
  • total analysis times can be decreased by roughly an order of magnitude with the use of electrokinetic injection. For example, an 80 % effective capillary length injection takes 1808 seconds by low-pressure injection with a 33 cm x 19 ⁇ m capillary. The same injection can be made by a 73 second electrokinetic injection at 30 kV.
  • the total analysis time with electrokinetic injection is approximately 9 times faster (FIG.3).
  • capillary injections of more than 100% of the effective capillary length can be made (FIG. 1), with capillary length still remaining for separation of the stacked analytes.
  • an electrokinetic injection length 170% of the effective capillary length ( ⁇ 42 cm sample plug length with an effective capillary length of 24.5 cm) can be made. The resulting resolution is similar to that observed when using a pressure injection of -30 cm with a capillary length of 80.5 cm with capillaries of 50 ⁇ m i.d.
  • sodium dodecyl sulfate was utilized as the electrokinetic vector for corticosteroid analytes.
  • the sample matrix was 10 mM tetraborate with a leading co-ion hydrogen phosphate at 5 mM.
  • Electrokinetic injection at 30 kV was made for 60 seconds directly from the sample vial. Separation was carried out at 30 kV.
  • Three corticosteroids are resolved in less than two minutes at 50 ng/ ml. While the signal to noise with electrokinetic injection under sweeping conditions (FIG.5) is similar to that already reported, the results (FIG. 6) show approximately an order of magnitude better detection for the progesterone peak, with a theoretical plate number of 2,000,000 per meter.
  • the 11 -deoxycorticosterone exhibited a plate number of 1 , 100,000 per meter, while the cortisone peak is not as well sharpened. This stacking mechanism is thus approximately an order of magnitude more sensitive, as well as an order of magnitude faster than that previously reported in the presence of conventional electroosmotic flow.
  • Electrokinetic Stacking Injections On A Microchip Field-amplified stacking of charged analytes on a microchip has been previously demonstrated. Since UN detection is difficult to accomplish on the microchip, the demonstration of high-salt elecfrokinetic stacking injection on a microchip will have to exploit monral, fluorescent analytes that can be excited by an argon ion laser. Consequently, BODIPY, a neutral and fluorescent analyte, was utilized to demonstrate stacking on a microchip. As shown in FIG.
  • the analyte was dissolved in the separation buffer and injected for 20 to 120 seconds, with the 80 second injection shown (note the control had twice the initial concenfration of analyte as used in the stacking experiments) (FIG. 7).
  • the injection field was 50% that of the separation field.
  • the linear increase in analyte peak height with injection time is clear evidence that the stacking of the tortral analyte is occurring.
  • the 100 second injection has a fluorescence response approximately 10-times greater than the analyte injected in separation buffer (non-stacking injection).
  • the non-stacking conditions had twice the concenfration of BODIPY, hence, a 20-fold peak height improvement is evidenced by utilizing a high-salt stacking injection.
  • Electrokinetic injection has the potential to provide a convenient mode for stacking injections with capillary arrays, and might also be exportable to other methods that utilize electrophoretically-active stacking junctions, such as pH-mediated stacking.
  • electrophoretically-active stacking junctions such as pH-mediated stacking.
  • elecfrokinetic injection allows the translation of neutral analyte stacking in high-salt samples from the capillary to the microchip format.
  • Many methods for gating injections in the microchip format have been devised. In this case, a chemical boundary is used to maintain an interface between the sample matrix and separation buffer during injection.
  • New capillaries were conditioned by flushing at -1 bar with 1.0 M sodium hydroxide for sixty minutes, followed by water for 30 minutes. Capillaries were re-conditioned at the start of each day's experiments by flushing with 1.0 M sodium hydroxide and water for five minutes each.
  • the separation buffer vial volume was 700 ⁇ l.
  • Separation buffers consisted of SDS and borate at concentrations as stated. Borate was prepared from sodium tetraborate, and concentrations are stated as borate (4 times the tetraborate concentration). The pH of separation buffers was -9.0 except as stated. Anhydrous methanol was added to the separation buffer at stated percentages (v/v). Separation buffers were degassed by decompression in a syringe and passed through a 0.2 ⁇ m pore-diameter filter before use. The capillary was flushed with fresh separation buffer at -1 bar for two minutes before each experiment.
  • Separation polarity was normal (the anode was at the inlet). Separations occurred at 15 or 30 kV as stated. Separation buffer was in the inlet and outlet vials for all separations.
  • EOF migration time was affirmed by the following two separate methods: distance the sample solvent moved through the capillary (24.5 cm to the detector, and 33 cm to the outlet) versus time of UV signal of the sample solvent at the detector, and the return to background current as the sample solvent exited the outlet 11 , e.g., 105.6 seconds to the detector (24.5 cm), as determined by UV absorption, and 143.1 seconds as determined by the return to background current at the end of the capillary (33 cm).
  • a two second pressure injection of separation buffer containing 10% organic (ethanol) was used to allow conductivity changes to be observed by the exit of the sample mafrix solvent from the capillary under electrophoresis with the various separation buffers tested.
  • the two methods corroborated EOF velocity with a precision between 99.8 and 98.7% for all buffers, with an average of 0.65% difference.
  • Analyte migration time was adjusted for electrokinetic injection duration (e.g., a 20-second electrokinetic injection contributed to the movement of the sample solvent into the capillary by 20 s times the EOF velocity).
  • Sample matrix vial volume was 150 to 700 ⁇ l. Sample matrixes were prepared with tetraborate, and concentrations were stated as borate.
  • sample matrixes The pH of sample matrixes was -9.0 to 9.2. Crystalline corticosteroids were dissolved in punctilious ethanol at 316 to 362 ⁇ g/ml in accordance with their molecular weight. Standards were stored at 4° C when not in use. Aliquots were dried down to remove the organic solvent and dissolved in sample matrixes at stated concentrations. The elecfrokinetic injection polarity was normal (the anode was at the inlet). Injections were performed at 15 or 30 kV as stated. Injections were undertaken with the sample vial at the inlet, and the separation buffer vial used in the subsequent separation was at the outlet. Conductivity of solutions was determined with a Model 35 conductance meter (YSI Scientific, Yellow Springs, OH).
  • sample mafrix 10% ethanol/water with varied concentrations of SDS in the separation buffer were examined for minimum peak velocity in combination with minimum peak width.
  • Borate was included at 20 mM in separation buffers to maintain pH -9.
  • the analytes exhibited decreasing velocity with increasing SDS concentration from 10 to 160 mM.
  • Elecfropherograms from separations with a pressure-injected sample plug (20 seconds at 50 mbar, -1.6 cm) were used to identify the individual analyte peaks with 40 mM SDS in the separation buffer (data not shown).
  • separation buffers contained 20 mM borate and SDS at 5, 10, 20, 40, 80, and 160 mM. Sample matrixes were adjusted with borate to maintain equivalent conductivity with the respective separation buffers. Selectivity was restricted by the use of SDS/borate separation buffers without further modification (e.g., cyclodextrins or other organic modifiers to improve peak resolution). This led to a poor resolution of the individual analytes.
  • An organic modifier can increase the viscosity of the aqueous solvent, causing a decrease in the elecfroosmotic flow velocity.
  • an increase in viscosity is not productive toward improving the efficiency of stacking efficiency because charged species in the separation buffer (i.e., the micelle) experience a decrease in mobility similar to that of the electroosmotic flow. It was considered whether the addition of an organic modifier might increase the affinity of the analytes for the micelle, thus allowing extended injection lengths.
  • methanol a simple organic modifier, was included at 0, 5, 10, 15, 20, and 25% in a separation buffer containing 40 mM SDS and 20 mM borate.
  • Separation buffers containing borate from 5 to 80 mM with 40 mM SDS were examined, with the borate concenfration in respective sample matrixes adjusted to maintain equivalent conductivity with the separation buffer. There was a slight decrease in analyte velocity with increasing borate concenfration from 5 to 80 mM (data not shown). The decrease in analyte velocity suggested the examination of higher-borate buffers.
  • the effects of borate at concentrations of 80, 120, 160, 200, and 240 mM in the separation buffer with 40 mM SDS were examined, with sample matrix conductivity adjusted with borate to equivalent conductivity with the separation buffer.
  • the minimum analyte velocity is simply the sum of the elecfroosmotic flow electrophoretic mobility plus the electrophoretic mobility of the elecfrokinetic vector (SDS), times the applied electric field.
  • SDS electrophoretic mobility of the elecfrokinetic vector
  • t r ans is the amount of time it takes for the SDS to transit the sample plug (dependent on the sample plug length)
  • ⁇ o F and ⁇ ekv are the intrinsic mobilities of the EOF and the micelle
  • E is the applied field.
  • V a/ekv is the velocity of the analyte/electrokinetic vector complex.
  • the velocity of the analyte/electrokinetic vector complex, a/ekv can be determined by observing the time of appearance of the analyte peak at the detector under continuous electrokinetic injection from the sample mafrix vial through the detector.
  • L plug is determined by the velocity of the EOF multiplied by the duration of the injection in seconds.
  • Equation (10) was used to predict the maximum plug length injection with the 40 mM SDS/240 mM borate separation buffer:
  • the migration time for the progesterone peak averaged 15.62 minutes for injections ranging from 15 to 21 minutes (197 to 276 cm injected plug length). This gave a velocity of 0.0261 cm/sec for the progesterone/SDS complex.
  • the ratio of Va/ ekv to V EOF times the length of the capillary to the detector (24.5 cm) gives the distance the stacked progesterone/SDS peak moves into the capillary for each effective capillary-length injection. The distance in this case is 2.93 cm.
  • the transition of the analyte/electrokinetic vector complex into the capillary with different length injections is depicted in FIGS. 14A-14F.
  • ⁇ EOF electrophoretic mobility of electroosmotic flow
  • ⁇ a/ekv apparent electrophoretic mobility of the analyte/electrokinetic vector complex.
  • the ⁇ E OF was determined by dividing the velocity of the sample solvent by the applied electric field.
  • the ⁇ a/e k v was determined with continuous sample matrix injections into separation buffer until the analyte/electrokinetic vector complex appeared at the detector window.
  • the distance the analyte/electrokinetic vector complex traveled "upstream" of elecfroosmotic flow to the detector window was calculated by multiplying the elecfroosmotic flow velocity by the time (in seconds) required for the complex to reach the detector window.
  • Equation (18) is simply a transformation of equation (10), utilizing the same data to determine results.
  • the distance the progesterone/micelle complex traveled from the injection point (capillary inlet) to the detection window is the same as the maximum injectable plug length, 180 cm.
  • the negatively-charged progesterone/micelle complex was moving against elecfroosmotic flow for 15.62 minutes (937 sec), during which time 180 cm of sample solvent was traversed.
  • the electrophoretic mobility of the analyte/micelle complex can now be determined by:
  • EXAMPLE NO. 2 CONCLUSIONS Extreme attenuation of analyte zones is obtained by extended electrokinetic stacking injection in EKC. Separation buffer parameters were investigated to allow extended injection plug lengths with continuous conductivity sample matrixes. As analytes are injected by elecfroosmotic flow into the separation buffer, each analyte exhibits a decrease in velocity due to the interaction with the micelles in the separation buffer. To determine maximum-length injections, the velocity of each analyte/electrokinetic vector complex must be considered. Equations have been introduced to determine the maximum injection length per individual analyte/micelle complexes.
  • L max is the injection length at which the injector-end of the sample plug arrives at the detector simultaneous to the conclusion of stacking
  • ⁇ j n j is the electrophoretic mobility of the analyte during the injection process
  • ⁇ or t h is the electrophoretic mobility of the analyte in the stacking process as it encounters a counter-mobility vector.
  • This type of system is referred to as an orthogonal analyte stacking/ injection system (OAS/IS).
  • OFS/IS orthogonal analyte stacking/ injection system
  • An advantage of the present invention elecfrokinetic injection method is that it is faster than previous injection methods by approximately an order of magnitude.
  • Another advantage of the present invention method is that it allows the loading of multiple-capillary volumes of sample for the first time.
  • another advantage of the present invention method is that it provides a higher resolution for injection of large sample plugs in capillary electrophoresis, providing a faster and higher sensitivity injection mode in capillary elecfrokinetic chromatography.
  • long sample plugs could be injected into a capillary by pressure, with post-injection stacking of analytes occurring due to electrophoretic phenomena.
  • the present invention provides that stacking can be initiated with injection, rather than post-injection.
  • the present invention further provides that electrokinetic injection of neutral analytes with concomitant stacking of shore analytes can be initiated at the commencement of injection.
  • the present invention method provides an electrokinetic injection of monral analytes by electroosmotic flow in the presence of an orthogonal stacking system, e.g., an anionic elecfrokinetic vector in the separation buffer, constitutes an orthogonal analyte stacking/ injection system (O AS/IS).
  • an orthogonal stacking system e.g., an anionic elecfrokinetic vector in the separation buffer
  • O AS/IS orthogonal analyte stacking/ injection system
  • the primary conditions are simply that the elecfrokinetic vector in the separation buffer has an opposing mobility to the analyte injection force, and that a suitable stacking boundary is formed between the injected sample and the elecfrokinetic vector in the separation buffer.
  • OAS/IS an extensive list of conditions for OAS/IS is provided (e.g., Tables 1-6). With properly instituted OAS/IS conditions, it is actually possible to inject sample plugs that are longer than the entire capillary.
  • the present invention provides a method of stacking injections for neutral analytes on the microchip format, with all the advantages of the OAS/IS described for the capillary format translatable to the microchip format without modification, including cationic and anionic analytes, as described in Tables 1 through 3.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

Dans ce procédé d'électrophorèse capillaire électrocinétique, les analytes sont injectés par flux électroosmotique directement à partir d'une matrice d'échantillons dans un tampon de séparation qui contient un vecteur électrocinétique présentant une mobilité opposée. Les analytes peuvent alors être injectés à la vitesse du flux électro-osmotique mais sont retenus au niveau de l'interface entre les co-ions de la matrice d'échantillons et les zones des micelles du tampon de séparation, sous forme de complexes analyte/micelle. La manipulation de la force d'injection et de la force de tassement opposée permet d'augmenter dans une mesure importante la durée ou le volume d'injection. Les concentrations de micelles, de méthanol et de borate dans le tampon de séparation sont calculées de manière à augmenter au maximum la durée d'injection des analytes neutres. La réduction de la vitesse des analytes dans le tampon de séparation sans réduction notable de la vitesse d'injection de l'analyte à partir du micro-flacon d'échantillon, permettent d'allonger fortement les durées d'injection du volume d'échantillon. Ce procédé permet en outre l'injection de volumes de solvants d'échantillon correspondant à environ vingt fois le volume effectif des capillaires. L'invention concerne également de équations et des algorithmes décrivant ce procédé d'injection et les durées d'injection maximales associées à ce procédé de tassement utilisé en chromatographique capillaire électrocinétique. Ce procédé permet une injection associée à un tassement électrocinétique maximal convenant pour une large variété d'analytes et de systèmes de séparation.
PCT/US2001/043259 1999-10-15 2001-11-19 Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese WO2002048673A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP01988144A EP1355858A2 (fr) 2000-11-17 2001-11-19 Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese
US10/432,141 US7223325B2 (en) 1999-10-15 2001-11-19 Method for orthogonal analyte stacking/injection systems in electrophoresis
AU2002241480A AU2002241480A1 (en) 2000-11-17 2001-11-19 Method for orthogonal analyte stacking/injection systems in electrophoresis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24961100P 2000-11-17 2000-11-17
US60/249,611 2000-11-17

Publications (2)

Publication Number Publication Date
WO2002048673A2 true WO2002048673A2 (fr) 2002-06-20
WO2002048673A3 WO2002048673A3 (fr) 2002-08-15

Family

ID=22944247

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/043259 WO2002048673A2 (fr) 1999-10-15 2001-11-19 Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese

Country Status (3)

Country Link
EP (1) EP1355858A2 (fr)
AU (1) AU2002241480A1 (fr)
WO (1) WO2002048673A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7718046B2 (en) * 2004-06-10 2010-05-18 The United States Of America, As Represented By The Secretary Of Commerce, The National Institute Of Standards & Technology Micellar gradient focusing
CN108562637A (zh) * 2018-04-18 2018-09-21 福州大学 一种基于毛细管电泳的极性物质电动提取方法
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5340452A (en) * 1991-02-01 1994-08-23 Beckman Instruments, Inc. On-column preconcentration of samples in capillary electrophoresis
US5800691A (en) * 1996-12-23 1998-09-01 Guest Elchrom Scientific Ag Electrophoresis gels containing sample wells with enlarged loading area
US5843294A (en) * 1988-11-29 1998-12-01 Isco, Inc. Capillary electrophoresis sample injection technique
US6010607A (en) * 1994-08-01 2000-01-04 Lockheed Martin Energy Research Corporation Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis
US6045676A (en) * 1996-08-26 2000-04-04 The Board Of Regents Of The University Of California Electrochemical detector integrated on microfabricated capilliary electrophoresis chips
US6214191B1 (en) * 1998-05-22 2001-04-10 Lynx Therapeutics, Inc. Electrophoresis apparatus and method
US6319379B1 (en) * 1999-08-23 2001-11-20 The Regents Of The University Of California Modified electrokinetic sample injection method in chromatography and electrophoresis analysis

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843294A (en) * 1988-11-29 1998-12-01 Isco, Inc. Capillary electrophoresis sample injection technique
US5340452A (en) * 1991-02-01 1994-08-23 Beckman Instruments, Inc. On-column preconcentration of samples in capillary electrophoresis
US6010607A (en) * 1994-08-01 2000-01-04 Lockheed Martin Energy Research Corporation Apparatus and method for performing microfluidic manipulations for chemical analysis and synthesis
US6045676A (en) * 1996-08-26 2000-04-04 The Board Of Regents Of The University Of California Electrochemical detector integrated on microfabricated capilliary electrophoresis chips
US5800691A (en) * 1996-12-23 1998-09-01 Guest Elchrom Scientific Ag Electrophoresis gels containing sample wells with enlarged loading area
US6214191B1 (en) * 1998-05-22 2001-04-10 Lynx Therapeutics, Inc. Electrophoresis apparatus and method
US6319379B1 (en) * 1999-08-23 2001-11-20 The Regents Of The University Of California Modified electrokinetic sample injection method in chromatography and electrophoresis analysis

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7718046B2 (en) * 2004-06-10 2010-05-18 The United States Of America, As Represented By The Secretary Of Commerce, The National Institute Of Standards & Technology Micellar gradient focusing
US11207677B2 (en) 2018-03-07 2021-12-28 University Of Virginia Patent Foundation Devices, systems, and methods for detecting substances
CN108562637A (zh) * 2018-04-18 2018-09-21 福州大学 一种基于毛细管电泳的极性物质电动提取方法

Also Published As

Publication number Publication date
EP1355858A2 (fr) 2003-10-29
WO2002048673A3 (fr) 2002-08-15
AU2002241480A1 (en) 2002-06-24

Similar Documents

Publication Publication Date Title
Quirino et al. Sweeping: concentration mechanism and applications to high-sensitivity analysis in capillary electrophoresis
Hirokawa et al. High‐sensitive capillary zone electrophoresis analysis by electrokinetic injection with transient isotachophoretic preconcentration: Electrokinetic supercharging
US7223325B2 (en) Method for orthogonal analyte stacking/injection systems in electrophoresis
US5453382A (en) Electrochromatographic preconcentration method
Kim et al. On-line sample preconcentration techniques in micellar electrokinetic chromatography
Vaher et al. Non-aqueous capillary electrophoresis in acetonitrile using lonic-liquid buffer electrolytes
Reinhoud et al. Strategy for setting up single-capillary isotachophoresis—zone electrophoresis
Breadmore Unlimited‐volume stacking of ions in capillary electrophoresis. Part 1: Stationary isotachophoretic stacking of anions
Thabano et al. Capillary electrophoresis of neurotransmitters using in-line solid-phase extraction and preconcentration using a methacrylate-based weak cation-exchange monolithic stationary phase and a pH step gradient
Bessonova et al. Electrophoretic determination of albumin in urine using on-line concentration techniques
Masár et al. Determination of chloride, sulfate and nitrate in drinking water by microchip electrophoresis
Terabe Peer reviewed: micellar electrokinetic chromatography
Shi et al. Determination of chlorophenols in water using dispersive liquid–liquid microextraction coupled with water‐in‐oil microemulsion electrokinetic chromatography in normal stacking mode
Britz-Mckibbin et al. Sensitive and high-throughput analyses of purine metabolites by dynamic pH junction multiplexed capillary electrophoresis: a new tool for metabolomic studies
Vaher et al. Specific background electrolytes for nonaqueous capillary electrophoresis
Belder et al. Directed control of electroosmotic flow in nonaqueous electrolytes using poly (ethylene glycol) coated capillaries
EP1355858A2 (fr) Procede convenant pour des systemes de tassement/injection orthogonaux d'analytes dans une electrophorese
Moreira et al. High performance separation of quaternary amines using microchip non-aqueous electrophoresis coupled with contactless conductivity detection
Saavedra et al. Development of a frit-free SPE-based in-column preconcentration system for capillary electrophoresis
Haraga et al. Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis
Yang et al. Determination of chromium (VI) and lead (II) in drinking water by electrokinetic flow analysis system and graphite furnace atomic absorption spectrometry
Britz-McKibbin et al. Velocity-difference induced focusing of xanthine and purine metabolites by capillary electrophoresis using a dynamic pH junction
Belder et al. Use of coated capillaries for nonaqueous capillary electrophoresis
Priego‐Capote et al. Dual injection capillary electrophoresis: Foundations and applications
Koch et al. Hydrophobic interaction electrokinetic chromatography for the separation of polycyclic aromatic hydrocarbons using non-aqueous matrices

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 10432141

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2001988144

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 2001988144

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2001988144

Country of ref document: EP

NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP