Hydrodynamic injections

Pure HDF injection can be achieved for sample introduction by using vacuum suction. The sample was sucked through an inserted capillary into a short section of microchannels between three ports. Different amounts could be selected by filling different sections of the microchip [813]. Pressure injection of a DNA sample was also achieved via a transfer capillary sequentially to a five-channel microchip. The use of one capillary allowed for automated sampling necessary for continuous monitoring of an enzymatic DNA restriction digestion experiment [320], 

FIGURE 4.19 Microchip separation of an FITC-labeled synthetic peptide mixture following (a) electrokinetic injection (0.25 s) and (b) diffusion-based injection (0.5 s). Mobile phase 1 mM carbonate buffer (pH = 9.0 separation electric field 300 V/cm 1, FITC-Gly-Phe-Glu-Lys-OH 2, FITC-Gly-Phe-Glu-Lys(FITC)-OH 3, FITC 4, FITC-Gly-Tyr-OH. Analyte concentrations 1 + 2 = 4 = 50 iM [566]. Reprinted with permission from the American Chemical Society. 

FIGURE 4.21 Photograph of the glass microchip (5x2 cm) used for sample injection, separation, and interfacing into the MS system. To minimize the diffusion loss of the sample during separation, the connection between the side channels (leading from Q, R, T, U) and the serpentine separation channel (75 pm deep) was etched to 25 pm (one-ninth of the cross section area of the separation channel) [296]. Reprinted with permission from the American Chemical Society. 

FIGURE 4.22 Sequence of the operation cycle of the microchip A, loading B, injection C, separation D, washing. V, vacuum P, pressure +, separation voltage [296]. Reprinted with permission from the American Chemical Society. 

This feature is possible by applying the hydrodynamic injection technique [338], the principle of which is best explained by referring to Fig. 5.11. By this approach a column of liquid can be exactly metered into a geometrically well-defined conduit (of length L and internal radius R) which is at all times open to other channels, provided that these channels 

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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. 

Hydrodynamic injection uses pressure to force a small portion of the sample into the capillary tubing. To inject a sample hydrodynamically a difference in pressure is applied across the capillary by either pressurizing the sample vial or by applying a vacuum to the destination reservoir. The volume of sample injected, in liters, is given by the following equation... 

A hydrodynamic injection is made by applying a pressure difference of 2.5 X 10 Pa (approximately 0.02 atm) for 2 s to a 75-cm long capillary tube with an internal diameter of 50 Jtm. Assuming that the buffer solution s viscosity is 10 kg m s what volume of sample is injected ... 

Samples of 1-20 nl are usually introduced by electrokinetic rather than hydrodynamic injection (p. 

Hydrodynamic injection, capillary electrophoresis, 4 633-634 Hydrodynamic lubrication regime, 15 210-211... 

Since capillaries have very small diameters, the injection volumes in CE are extremely small. Injection volumes in the order of 10-50 nL are commonly applied (a fog droplet is + lOnL). Several approaches have been applied for the injection of such small volumes of sample into the capillary. These included the use of rotary-, split- and micro-injectors, electrokinetic and hydrodynamic injection. Although all these injection techniques have shown to be quite appropriate, electrokinetic and hydrodynamic injection are mostly applied. All the recent commercial... 

Hydrodynamic injection can also be performed by using gravity to generate AP [41]. This injection mode is also called hydrostatic injection. The inlet of the capillary is placed in the sample vial and this is then raised during a period of time, creating a difference in height (Ah, in cm) between the inlet and the outlet of the capillary. The sample enters the capillary by siphoning. The amount and volume injected are derived from Eqs. 17.46 and 17.47, respectively, after substitution of AP with Eq. 17.48. 

It is important to note that with hydrodynamic injection a volume of sample is injected, which has representative amounts (concentration) of the sample constituents. This characteristic, together with a better precision, makes it the most widely used injection technique in CE. However, owing to the poor detectability in terms of concentration of the widely used detectors (see further), its application is rather limited to the analysis of high-concentration samples. In trace analysis, for example, electromigration is favored over hydrodynamic injection [42],... 

CZE has been employed for the analysis of another set of dyes in foodstuffs. The chemical structures, numbers and names of the dyes included in the investigation are listed in Fig. 3.142. A fused-silica capillary column of 57 cm length (50 cm effective length 75 jum i.d.) was employed for the separations. The capillary was conditioned by 1.0 M NaOH for 20 min followed by 10 min wash with water and 10 min wash with the running buffer. The buffer was prepared by adding NaOH to 10 mM phosphoric acid to reach pH 11.0. The capillary was thermostated at 25°C and the separation voltage was 20 kV. A hydrodynamic injection mode was applied (0.5 psi, 4 s, 21 nl) and spectra of... 

Fig. 3.143. Electropherogram of the dyes studied under optimized conditions. Electrophoretic buffer buffer phosphates 10 mM, pH 11.0 applied voltage, 20 kV, and detection wavelength, 280 nm. Hydrodynamic injection at 0.5 psi for 4 s. All analytes have around 4.5 pg/ml, and the sample has 9 yUg/ml of E-131 and 14.8 pg/ml of E-102. Reprinted with permission from M. Perez-Urquiza et al. [186],...

Figure 9.1 Schematic representation of a basic capillary electrophoresis system. The main components include a capillary (commonly contained within a housing that allows for temperature control), a power supply, and a detector. Automation is achieved through the use of computer-controlled setting of solutions and samples, displacement forces (to replace capillary contents and for hydrodynamic injection), and automatic data collection. (Courtesy of Agilent Technologies.)...

Pressure (or hydrodynamic) injection involves using pressure to push the sample onto the capillary. The sample loaded onto the column is independent of mobility and is indiscriminant with regard to what is loaded onto the capillary. The quantity injected is given by Hagen-Poiseuille equation ... 

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

Injection time-. Most modern instruments have a control function of the injection pressure that automatically corrects for hydrodynamic injection variability through the injected time. An injection time of at least 3 s is needed for this to function properly. Too short injection times decrease precision and too long injection times induce band broadening. Rather increase pressure if possible. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

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. 

Proteins or antibodies (36 pg) were mixed with ampholine pH 3.5—9.5 (final concentration of 5%, Amersham Biosciences, distributed by GE Healthcare, Uppsala, Sweden), p7 markers (Bio-Rad, Hercules, CA), and hydroxypropyl methyl cellulose (final concentration of 0.2% HPMC, Sigma-Aldrich, St. Louis, MO). The final protein concentration was 0.3mg/mL. Figure 17 shows a schematic of the sample preparation. The mixture was mixed thoroughly and was introduced to the capillary (eCAP neutral-coated, 50 micron X 30 cm, Beckman, Fullerton, CA) by hydrodynamic injection. Injections were performed using 20 psi for 99 s. The solution was then separated under an electric field of 25 kV for 10 min. The focused protein was then pushed/pulled out of the capillary through a mobilization process using the cathodic mobilizer (Bio-Rad, Hercules, CA). 

Injection may be hydrodynamic (using a pressure difference between the two ends of the capillary) or electrokinetic (using an electric field to drive sample into the capillary). For hydrodynamic injection (Figure 26-17), the capillary is dipped into a sample solution and the injected volume is... 

U. Backofen, F.M. Matysik and C.E. Lunte, A chip-based electrophoresis system with electrochemical detection and hydrodynamic injection, Anal. Chem., 74 (2002) 4054-4059. 

Berzas Nevado et al. [138] developed a new capillary zone electrophoresis method for the separation of omeprazole enantiomers. Methyl-/ -cyclodextrin was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated to optimize separation and run times. Analysis time, shorter than 8 min was found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM /1-cyclodextrin and 5 mM sodium disulfide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/1 for the omeprazole enantiomers. The method was applied to pharmaceutical preparations with recoveries between 84% and 104% of the labeled contents. 

The analysis of aliphatic acids was performed using a P/ACE MDQ capillary electrophoresis instrument equipped with a 60 cm x 50 pm id fused silica capillary (Beckman Coulter, Fullerton, CA). The samples were filtered through a 0.45-gm cellulose acetate filter (Whatman, Maidstone, UK) prior to hydrodynamic injection at 15 psi for 4 s. The voltage was set to 20 kV at reversed polarity. The electrolyte, composed of 5.0 mM trimellitic acid, 50 mM tris(hydroxymethyl)-aminomethane, 1.0 mM tetradecyl-trimethylammoniumbromide, and 0.5 mM calcium chloride, had a pH of 9.8. Before use, it was filtered through a 0.2-gm cellulose nitrate filter and degassed withhelium. Detection was performedby indirect UV absorption at 220 nm. Succinic acid was used as internal standard. 

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