Hydrodynamic injection, method

The injection volumes in CE are extremely small because of the use of capillaries with very small diameters. Typical injection volumes are in the order of 10—50nE (a fog droplet is +10 nL). Injection of such small volumes of sample into the capillary is very challenging and requires specific approaches including 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 methods are mostly applied. Recent commercial instruments are usually equipped with these two injection modes as standard methods.Chapter 3 provides more details on the different injection modes. 

Hydrodynamic injection methods are based on the creation of a pressure difference across the separation column with the column inlet located in the sample vial by any of three general methods raising the height of the sample vial above the level of the column outlet by overpressure of the sample vial by application of a vacuum to the detector end of the capillary. The average length of the sample zone, Li j, introduced is given by... 

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. 

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. 

Two introduction methods are commonly employed in capillary electrophoresis. Hydrodynamic injection is based on siphoning, or gravity feeding the sample into the anodic end of the capillary. The anodic end is removed from the buffer reservoir and placed in the sample solution. The capillary end is then raised so that the liquid level in the sample vial is at a height Ah above the level of the cathodic buffer, and is held in this position for a fixed time t. This process has been automated for reproducibility, and the hydrodynamic flow rate has been shown to obey Eq. 12.9 ... 

The separation capillary is prepared by polymerizing 6%T, 5%C monomers with 30% formamide and 7 M urea inside a 37 cm long, 50 pm i.d. fused silica capillary. Electrokinetic injection at 200 V/cm for 30 s was used, since hydrodynamic injection does not work with gel-filled capillaries. Separation on the gel occurs by seiv-ing, so that the shortest fragments elute first. Figure 12.12 shows the data obtained using a TAMRA dye label that is excited at 543.5 nm and emits at 590 nm. This method has a detection limit of 2 zmol (1 zmol = 10 21 mol) for each fragment. 

In addition to publications detailing criteria for method development, numerous examples of the use of CD-based CE have appeared in the literature. To illustrate method performance, a paper by Zhou et al. details the development and validation of methodology for the determination of enantiomeric purity of the compound shown in Figure 8a. An optimized separation (Fig. 8b) was obtained using 1.5% (w/w) sulfated-/3-CD in pH 2.5 sodium phosphate buffer (25 mM), a 63-cm (56 cm effective length) x 75- xm capillary, detection at 200 nm, a temperature of 30 °C, and an electric held strength of —15 kV. Sample solutions of 0.5 mg/ml in 90/10 (v/v) water/acetonitrile were introduced using hydrodynamic injection at 50 mbar for 3 s. 

Hydrodynamic loading is consistently reported to be the more precise injection method. Hettiarachchi and Cheung have concluded that hydrodynamic loading should always be used for high-precision quantitative applications because it is less dependent on the ionic concentration of the sample buffer (62), but running buffer is the recommended sample diluent. 

Figure 5.13. Determination of chloride by the mercury thiocyanate method with the hydrodynamic injection manifold in Fig. 5.12a, where C was 100 cm long and the volume of conduit L was 25 xL. The pumping rate x = z was 1.1 mL/min and the aspiration rate y was 3.0 mL/min, operated for 12 s. The detector was tuned at 490 nm. (a) Standard calibration run of samples in the range 10-50 ppm Cl (b) stopped-flow experiment with the 40 ppm Cl standard recorded at high paper speed to demonstrate the fast rate of reaction and (c) monitoring of the content of chloride in a solution of NaCl in which the analyte concentration was changed intermittently and measured at fixed time intervals by the system.

Die injection system must be capable of reproducibly introducing very small sample volumes into the capillary. The volume of the whole capillary is only in the order of p,L. To minimise band broadening, sample plugs must be as short as possible. Hence, not more than a few nL of sample are introduced into the capillary. Two injection methods are commonly used (1) electrokinetic injection and (2) hydrodynamic injection. 

While the above method of electrokinetic injection requires only a single step, it is possible to couple electrokinetic injections with a brief hydrodynamic injection of water. The column is initially full of BGE, followed by a hydrodynamic injection of water. Analyte is then electrokinetically injected until the measured current through the column is approximately 70-90% of the column when filled... 

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