Electrokinetic injection, capillary

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

Electrokinetic injections are made by placing both the capillary and the anode into the sample vial and briefly applying an electric fleld. The moles of solute injected into the capillary, nj, are determined using... 

The improved design of the gating interface resulted in precise alignment of the two capillaries. A colored dye solution was added to the HPLC eluent to allow for du cct observation of the flow gating and injection processes. Through observation of the movement of the dye through the interface, it was possible to ensure that the electrokinetic injections were performed correctly. Troubleshooting had been a... 

Fig. 3-3. Comparison of the values of enantiomeric resolution of different DNP-D,L-amino acids at different deconvolution stages of a cyclic hexapeptide sublibrary. Resolution values in a cyclo(Arg-Lys-X-X-X-P-Ala) sublibrary, in the first line, are compared to those obtained in sublibraries with a progressively increasing number of defined positions. All the sublibraries were 30 mM in the running buffer while the completely defined cyclo(Arg-Lys-Tyr-P-Tyr-P-Ala) peptide is used at 10 mM concentration. Conditions cyclopeptide sublibrary in 20 mM sodium phosphate buffer, pH 7.0 capillary, 50 pm i.d., 65 cm total length, 57 cm to the window V = -20 kV, I = 40 electrokinetic injection, -10 kV, 3 s detection at 340 nm. (Reprinted with permission from ref. [75]. Copyright 1998, American Chemical Society.)...

Fig. 15. Electrochromatograms obtained in columns coated with sol-gel composites (A) TEOS and (B) C8-TEOS/TEOS. (Reprinted with permission from [80]. Copyright 1999 American Chemical Society). Separation conditions fused silica capillary, 12 pm i.d., 60 cm total length, 40 cm active length, mobile phase 60/40 methanol/1 mmol/1 phosphate buffer, voltage 30 kV, electrokinetic injection 5 s at 6 kV, UV detection at 214 nm. Peaks toluene (1), naphthalene (2), and biphenyl (3)...

The washing of capillaries with dilute alkaline solution is advisable before analysis. The alkaline solution can be followed by deionized water and buffer. Capillaries can be washed between runs too. Samples can be introduced into the capillary by hydrodynamic and electro-kinetic methods. The hydrodynamic method applies a pressure difference (5-10 sec) between the two ends of the capillary. The pressure difference can be achieved by overpressure, vacuum or by creating a height difference between the levels of the buffer and sample reservoirs. In the case of electrokinetic injection, the injection end of the capillary is dipped into the sample for a few seconds and a voltage of some thousand volts is applied. 

Fig. 3.172. Non-aqueous capillary electrophoresis with electrochemical detection of a dye mixture containing (a) 1.7 jUg/ml malachite green, (b) 0.70 jug/ml crystal violet, (c) 4.3 /ig/ml rhodamine B, and (d) 9.1 X 10-6 M ferrocene. Experimental conditions capillary dimensions, 95 cm X 75 pm i.d. running electrolyte, acetonitrile containing 1 M HAc and 10 mM NaAc electrokinetic injection, 20 s 5 kV separation voltage 20 kV applied detection potential, 1.55 V. Reprinted with permission from F.-M. Matysik [206].

In electrophoretic injection, the capillary inlet is immersed in the sample solution and a voltage is applied for a determined period of time. The amount of sample introduced into the capillary depends on the voltage and the time it was applied. Sample injection is a compromise between detection and resolution, and its parameters are often best determined experimentally. If detection is not a problem, resolution can be greatly improved by maintaining the sample plug as narrow as possible. If EOF is present, sample ions will be introduced by a combination of electrophoretic mobility and EOF under these conditions, this injection mode is generally termed electrokinetic injection. 

Electrokinetic injection involves using voltage to inject sample onto the capillary. The sample serves as a buffer reservoir (Figure 1). A voltage is applied and the analyte(s) migrate onto the capillary. The amount of material injected is dependent on the mobility of the analyte(s). The quantity injected is given by... 

The different modes of injection, i.e., gravity injection, pressure or vacuum injection, and electrokinetic injection, are addressed. Also, several practical experimental issues are described, such as correct treatment of electrolyte solutions and the importance of a rigorous capillary rinsing procedure. 

Samples are introduced into the capillary by either electrokinetic or hydrodynamic or hydrostatic means. Electrokinetic injection is preferentially employed with packed or monolithic capillaries whereas hydrostatic injection systems are limited to open capillary columns and are primarily used in homemade instruments. Optical detection directly through the capillary at the opposite end of sample injection is the most employed detection mode, using either a photodiode array or fluorescence or a laser-induced fluorescence (LIF) detector. Less common detection modes include conductivity [1], amperometric [2], chemiluminescence [3], and mass spectrometric [4] detection. 

For electrokinetic injection, the capillary is dipped in the sample and a voltage is applied between the ends of the capillary. The moles of each ion taken into the capillary in t seconds are... 

J. Palmer, N. J. Munro, and J. P. Landers, A Universal Concept for Stacking Neutral Analytes in Micellar Capillary Electrophoresis, Anal. Chem. 1999, 71, 1679 J. Palmer, D. S. Burji, N. J. Munro, and J. P. Landers, Electrokinetic Injection for Stacking Neutral Analytes in Capillary and Microchip Electrophoresis, Anal. Chem. 2001, 73, 725 J. P. Quirino, S. Terabe, and... 

In 1990, Bushey and Jorgenson developed the first automated system that coupled HPLC with CZE (19). This orthogonal separation technique used differences in hydrophobicity in the first dimension and molecular charge in the second dimension for the analysis of peptide mixtures. The LC separation employed a gradient at 20 (xL/min volumetric flow rate, with a column of 1.0 mm ID. The effluent from the chromatographic column filled a 10 pU loop on a computer-controlled, six-port micro valve. At fixed intervals, the loop material was flushed over the anode end of the CZE capillary, allowing electrokinetic injections to be made into the second dimension from the first. 

Figure 9.7 Schematic illustration of the flow-gating interface. A channeled Teflon gasket was sandwiched between two stainless steel plates to allow for flow into the electrophoresis capillary, either from the flush buffer reservoir or from the LC microcolumn during an electrokinetic injection.

Eberle et al. [134] separated the enantiomers of omeprazole and structurally related drugs by capillary zone electrophoresis with bovine serum albumin as chiral selector. The separations were carried out on a fused silica column (60 cm x 50 pm, 50 cm to detector) with a buffer consisting of 100-/zM-bovine serum albumin and 7% 1-propanol in 10 mM potassium phosphate pH 7.4. Electrokinetic injection was at 5-8 kV for 7 s. An applied voltage of 300 V/cm was used. Detection was at 290 nm. Detection limits were 0.04 mg/ml for the analytes studied. 

Figure 7.7 Electrochromatograms of noradrenaline and dopamine, respectively. Electrokinetic injection at 20kV/cm for 2 seconds followed by separation at 30 kV/cm, L = 54 cm (total length of capillary), 1 = 40 cm (length of capillary up to detector) [22].

Palmer, J., Burgi, D.S., Munro, N.J., Landers, J.P., Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis. Anal. Chem. 

The small dimensions associated with CE preclude the injection of large volumes. The sample may be introduced to the capillary either by a diplacement technique (i.e., pressure, vacuum, or siphoning) or via electrokinetic injection. The majority of commercial instruments apply a pressure differ-... 

The introduction of the samples onto the capillary column can be carried out by either displacement techniques or electrokinetic migration. Three methods of displacement or hydrostatic injection are available a) direct injection, or pressure b) gravity flow, or siphoning and c) suction. The electrokinetic injection method arose from findings that electroosmosis act like a pump (80). Both methods have advantages and disadvantages. For example, a bias has been reported in electrokinetically injected... 

Fig. 10.1. Separation of polycyclic aromatic hydrocarbons (PAHs) on columns packed with Spherisorb ODS particles. Conditions (A) 35(43) cm x 50 pm i.d. fused silica capillary column packed with 3 pm Spherisorb ODS-1 particles (B) 41(53) cm x 75 pm i.d. fused-silica capillary column packed with 5 pm Spherisorb ODS-1 particles 30 kV applied voltage 5 kV, 5 s electrokinetic injection acetonitrile-50 mM Tris buffer, pH 8.1 (80 20 v/v). Peak identifications 1, benzene 2, naphthalene 3, acenaphthylene 4, fluorene 5, acenaphthene 6, phenanthrene 7, anthracene 8, fluoranthene 9, pyrene 10, benz[n]anthracene 11, chrysene 12, benzo[6]fluoranthene 13, benzo[fc]fluoranthene 14, benzo[a]pyrene 15, dibenz[n,/i]anthracene 16, indeno[7,2,3-af]pyrene 17,...

Fig. 10.2. Separation of a mixture of PAHs on reversed-phase capillaries (a) without and (b) with silicate entrapment. Conditions 75 pm i.d. fused-silica capillary packed with 5 pm Nucleosil ODS particles column effective lengths 25 cm for the non-entrapped column and 17 cm for the entrapped column. Both electrochromatograms were obtained under the same conditions mobile phase, acetonitrile-0.1 M acetate buffer, pH 3.0, 80 10 (v/v) applied voltage 30 kV UV detection at 254 nm 20°C pressure 9 bar applied to both vials electrokinetic injection, 10 kV for 10s. Reproduced with permission from Chirica and Remcho [10].

Fig. 10.17. Capillary electrochromatography of PTH-amino acids with gradient elution. Column, 207 (127) mm x 50 pm i.d. packed with 3.5 pm Zorbax ODS particles, 80 A pores. Starting eluent (A), 5 mM phosphate, pH 7.55, 30% acetonitrile gradient former (B), 5 mM phosphate, pH 7.55, 60% acetonitrile flow-rate (through inlet reservoir), 0.1 ml/min gradient, 0-100% B in 20 min voltage 10 kV current, 1 pA temperature, 25°C UV detection at 210 nm electrokinetic injection, 0.5 s, 1 kV. Peaks in order of elution formamide PTH-asparagine PTH-glutamine PTH-threonine PTH-glycine PTH-alanine PTH-tyrosine PTH-valine PTH-proline PTH-tryptophan PTH-phenyialanine PTH-isoleucine PTH-leucine. The concentration of the PTH-amino acids dissolved in the mobile phase was 30-60 pg/ml. Reprinted with permission from Huber et al. [68]. Copyright 1997 American Chemical Society.

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