Electrokinetic injections

It was found that a 250-pm double-T injector increased in the peak signal as compared to a straight cross-injector [548,620], Although a five-fold increase was expected based on the intersection volume calculation of a 250-pm injector versus a cross-injector, the discrepancy was likely to be caused by the back flow of 

FIGURE 4.3 Repetitive sample injection and separation. Cycle conditions injection time, 5 s dead time, 1 s separation time, 45 s dead time, 1 s. The insets show two separation events on an expanded time scale. Sample fluorescein-labeled phosphorothioate oligonucleotide mixture, poly(dT)10 25. Separation conditions buffer, 100 mM Tris, 100 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.5 Electric field strength, 2300 V/cm separation length, 3.8 cm [547]. Reprinted with permission from the American Chemical Society. 

FIGURE 4.4 Slides (a) and (b) are composed of HF etched channels with powder-blasted access holes in (a). The bonding of the two slides gives a cross-shaped channel layout (c) of injection volume of 27 pL [314]. Reprinted with permission from the American Chemical Society. 

In EK injection, sample introduction is usually biased. However, the sample can be introduced to a microchip in a non-biased manner, as long as the electric voltage was applied for a sufficient time so that the slowest migrating component has passed the intersection and entered the analyte waste channel. Accordingly, the sample composition at the intersection is representative of that in the original sample, though it is not the case somewhere downstream in the waste channel [107,340]. 

EK injection bias still exists in pinched injection, with neutral species injected in a greater amount than anionic species. However, the orthogonal nature of the loading and injecting steps reduced this bias, although it did not completely eliminate it [549]. 

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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. 

Table 9.3 Migration Time Reproducibility (n = 10) for CE-SDS Using 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... 

Electrokinetic injection is useful when the analyte is in the presence of interfering species (with different mobilities), qualitative applications, or when viscous buffers or gels are being used. It is usually not suitable for quantitative applications since the variability caused by conductivity, microenvironments, and matrix differences significantly reduces the reproducibility. Since sample depletion can be a significant issue, it is recommended that different samples are used when repeated injections are needed. 

Electrokinetic injection and Control ionic strength of sample... 

Injection mode-. Hydrodynamic injection is generally more reproducible than electrokinetic injection. The electrokinetically injected amount has a non-linear relationship with the injection time. °... 

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. 

Electrokinetic injection requires a few special considerations. The material used must be very pure. Classic glass vials may give unknown peaks. The water must be very pure and fresh as contamination may be picked up from the air. During the run, the different buffer and rinse vials may be contaminated and need to be replaced more frequently. The vial used for postinjection water must be replaced for every tun. 

Hydrodynamic injection was compared with electrokinetic injection (data not shown). The two injection modes gave comparable percent peak areas. Electrokinetic injection gave slightly higher resolution compared to hydrodynamic injection. For the CE-SDS method, electrokinetic injection is generally recommended. 

FIGURE 4 Chiral CE ESI/MS analysis of five amphetamine derivatives and two pharmaceutical compounds. Total ion current (TIC) and extracted ion currents (XIC) of amphetamine (A), methamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA), and tramadol (TMD) and methadone (MTD) in plasma after LLE with electrokinetic injection. 

Ermakov, S. V., S. C. Jacobson, and J. M. Ramsey. Computer simulations of electrokinetic injection techniques in microfluidic devices. Anal. Chem. 72, 3512-3517 (2000). 

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