Volume-Based Injection Procedures

Although the rotary valves used in FIA might be designed and mechanically manufactured differently, the function of this type of valve is essentially that schematically shown in Fig. 5.1a. The valve has two po- 

A six-port valve (Fig. 5.2) with six holes on the rotor matching six holes on the stator provides a wide range of the configurations employed in the FIA techniques described in Chapter 4. The valve can be used for simultaneous sample and/or reagent injection, as well as to direct streams into different sections of the manifold (cf. Fig. 4.41). Erickson et al. have recently reviewed and summarized these options [5.1], which are outlined in the following. 

The six-port valve shown in Fig. 5.3 was incorporated into a FIA microconduit (Section 4.12 cf. also Fig. A Alb), constructed by bolting a Telfon disk with six evenly spaced holes on a PVC plate, 1 cm thick. Matching holes are drilled through the plate and the disk, and channels 

The standard six-port arrangement shown in Fig. 5.2 can be formed by simply connecting pairs of ports on the rotor (or on the plate). Alternatively for a simple injection the valve can be configured as shown in Fig. 5.4. 

The valve scheme shown in Fig. 5.5 allows simultaneous sample/re-agent injection after filling the two loops (merging zones). Note that the two carrier inlets may be propelled by a single pump tube, or by separate 

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Common for the time-based injection procedures is that the sample volume is metered as a function of time, the most important feature of this... 

Sidnei, G. S. Fabio, P.R (2010). A flow injection procedure based on solenoid micropumps for spectrophotometric determination of free glycerol in biodiesel. Talanta. Volume 83, Issue 2,15 December 2010, Pages 559-564. doi 10.1016/j.talanta.2010.09.061. 

BARS concentration by some 5-fold to 15-fold, based on the calculations that the intracellnlar concentration is around 20 //g/ml and on the assumption that 5-10% of the cell volume is injected. Prior to injection, the protein is mixed with 0.4 mg/ml fluorescein isothiocyanate (FITC)- or tetramethyl-rhodamine B isothiocyanate (TRITC)-labeled dextran (Molecnlar Probes) as a marker for the microinjected cells (Bonazzi et al., 2005). To give an example, in stndies of basolateral and apical transport (using the vesicular stomatitis virus glycoprotein and p75, respectively), the proteins were microinjected 1 h after the beginning of the 20° incubation in the transport assay. After injection, the cells were allowed to recover for 1 h before proceeding with the experimental protocol (Bonazzi et al, 2005). The BARS (p50-2) and dynamin (DYN2) antibodies were injected at 4.5 mg/ ml, 3 h before farther experimental procedures. 

Typical protein precipitation procedures use one volume of plasma plus three to six volumes of acetonitrile or methanol (or a mixture) with the internal standard at an appropriate concentration for the assay. Poison et al.102 reported that protein precipitation using acetonitrile eliminates at least 95% of the proteins after filtration or centrifugation, the supernatant can often be directly injected into the HPLC/MS/MS system. Usually this step is performed using 96-well plates that are ideal for semi-automation of sample preparation. Briem et al.103 reported on a robotic sample preparation system for plasma based on a protein precipitation step and a robotic liquid handling system that increased throughput by a factor of four compared to a manual system. 

An external standard method is used when the standard is analyzed on a separate chromatogram from the sample. Quantitation is based on a comparison of the peak area/height (HPLC or GC) of the sample to that of the reference standard for the analyte of interest. The external standard method is more appropriate for samples with a single target analyte and narrow concentration range, where there is a simple sampling procedure, and for the analysis of hydrocarbon fractions. The calculation requires an accurate extract final volume and constant injection size. The peak area of an analyte is compared with that from a standard or standard curve and corrected for volume ... 

Kawahara [156] introduced pentafluorobenzyl esters, prepared by the following procedure. A mixture of four acids (0.8 mg of each) was dissolved in 100 ml of acetone, and 250 mg (25-fold excess) of a-bromo-2,3,4,5,6-pentafluorotoluene and 50 mg (10-fold excess of potassium carbonate were added (it can be replaced with an ethanolic solution of potassium hydroxide). After refluxing for 3 h, the mixture was diluted with 500 ml of diethyl ether and 20 ml of ethyl acetate, washed with 10 ml of water and dried with 8 g of anhydrous sodium sulphate. After filtration, the sulphate and the filter were washed with 50 ml of diethyl ether, the solvent was removed and the residue was dried at 40°C and 50 mmHg it was further dissolved in 100 ml of -hexane and, after an additional 100-fold dilution, 6 /il were injected. The ECD response to pentafluorobenzoate was almost the same as that to aldrin. A method for the preparation of p-substituted benzyl esters of lower monocarboxylic acids on the micro-scale [157] is based on the same reaction scheme. A 10-pl volume of an ethanolic solution of carboxylic acids (ca. 1 pg/pl)... 

A more complex biosensor for acetylcholine has been developed by Larsson et al. [154]. Three enzymes, AChE, ChOX, and HPR, have been coimmobilized in an Os-based redox polymer on solid graphite electrodes. After a careful optimization of the immobilization procedure, the biosensor, inserted into a flow cell of very small volume, was integrated into a flow injection system, and some samples of microdialysate, taken from rat brains before and after stimulation with KCl, were analysed. Even if a clear increase in signal could be noted, it was not possible to distinguish whether it was due to an increase in choline or in acetylcholine, since the biosensor responded to both metabolites. 

Pharmacokinetics After Oral and Intravenous Administration. For proper characterization of an inhalation drug, information on the systemic pharmacokinetic properties needs to be provided. One of the major challenges for such studies is to provide a suitable formulation for injection, especially because new drug candidates are often very lipophilic. The resulting parameters of such studies (systemic clearance, volume of distribution, half-life, mean residence time) can then easily be extracted from concentration-time profiles after IV administration and subsequent standard pharmacokinetic analysis by noncompartmental approaches. In addition, a detailed compartmental analysis based on concentration-time profiles will be useful in evaluating the systemic distribution processes in sufficient detail. This will be especially important if deconvolution procedures (see later) are included for the assessment of the pulmonary absorption profiles. 

To alleviate these drawbacks, alternative methodologies relying on the continuous provision of fresh extractant volumes to the solid sample under mvestigation have been developed, characterized, and contrasted with the classical end-over-end extraction procedures. The fundamental principles of these novel, dynamic (nonequilibrium) strategies, based primarily on the use of continuous-flow analysis (Ruzicka and Hansen, 1988), flow injection analysis (Ruzicka and Hansen, 1988 Trojanowicz, 2000 Miro and Frenzel, 2004b), or sequential injection analysis (Ruzicka and Marshall, 1990 Lenehan et al., 2002), are described in detail below, and their advantageous features and limitations for fractionation explorations are discussed critically. 

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