Injection deactivation procedures

Procedure Use a gas chromatograph equipped with a flame-ionization detector, a cold-on-column injector, a suitable deactivated precolumn, and a 10-m x 0.32-mm (id) capillary column coated with an apolar stationary phase 0.12-p.L film thickness. Program the column to heat to 60°, hold for 1 min, heat to 300° at 20°/min, and hold for 3 min. Set the flame-ionization detector to 320°. Chromatograph five injections of the Standard Preparation. Measure the responses. The relative standard deviation for each peak should be below 2%. The peaks for brassicasterol and campesterol should be baseline resolved (Rs > 1.0) and show no tailing. Measure the response of the Internal Standard Solution and all the individual sterols eluting in the relative retention window of 0.98 to 1.13. 

For the assay of piperine in pepper, a 25 m by 0.5 mm I.D. glass capillary deactivated by high temperature silanization and coated with 0V-1 was used. The samples were injected by the on-column injection technique at 100°C, as described by Grob and Grob Jr.. The standard deviation of the whole procedure, sample preparation and chromatographic analysis, was 2.5 %. 

BaA and BaP is added and the entire sample is extracted with methylene chloride (250-, 100-, and 100-mL portions). The extracts are combined, 10 mL of isooctane added, and the solution evaporated to about 10 mL. When the solution approaches 10 mL, a second 10-mL volume of isooctane is added. This is repeated three times. The isooctane solution is made up to 75 mL and extracted with three 100-mL portions of dimethyl sulfoxide (DMSO) that contains 20% phosphoric acid. The DMSO extracts are each washed with three 30-mL portions of fresh isooctane, combined and diluted with 500 mL of water. This solution is extracted with three 80-mL portions of isooctane. The isooctane extracts are combined, rinsed once with water, and evaporated to 25 mL. At this point, a 1-mL portion of the solution is removed for intermediate radioassay and a UV scan. Usually the concentrate is sufficiently free from interferences and is evaporated down to about 0.05 mL and an aliquot injected into the GC. If further purification is indicated, the solution is chromatographed on a 1.1-cm X 90-cm column containing 75 g of Woelm Neutral Alumina deactivated with 2% water. Before deactivation, the alumina is dried in an oven for 1 hr at 150°C. The solvent elution schedule is as follows 25 mL cyclohexane prewash, 25-mL sample in cyclohexane, 100 mL cyclohexane, 100 mL cyclohexane-methylene chloride (9 1), 100 mL cyclohexane-methylene chloride (1 1), 100 mL methylene chloride. After the first 125 mL has eluted, 10 cuts are then radioassayed and combined into a single PNA fraction. This fraction is evaporated to about 0.05 mL in preparation for the GC-UV finishing step. Figure 2 is a diagram of the wastewater procedure. 

Various precolumn concentration procedures used in conjunction with thermal focusing are desirable for biochemical applications. Novotny and Farlow [65] developed a simple technique, where an off-line injection of a relatively large dilute sample onto a small precolumn results in effective concentration. In this procedure, a small volume of deactivated solid support is packed into the glass liner of an injection system. After the volatile solvent is removed, the liner is quickly introduced into the injection port, and the desorbed sample is trapped for several minutes in the cool column. Chromatograms obtained through the following temperature program-... 

Potentiometry is the most common method of electrochemical detection used in flow injection analysis. This methodology creates more favorable conditions for potentiometric measurements when compared to batch procedures. In FIA measurements, it is easier to avoid incidental contamination or that resulting from leakage of the solution from the reference electrode. Moreover, deactivation of the sensing surface of the indicator electrode due to adsorption, precipitation, or corrosion is minimized greatly as a consequence of the short contact time with the sample... 

Split injection is the oldest, simplest, and easiest injection technique to use. The procedure involves injecting 1 /xL of the sample by a standard syringe into a heated injection port that contains a deactivated glass liner. The sample is rapidly vaporized, and only a fraction, usually 1-2%, of the vapor enters the column (see Fig. 6.8). The rest of the vaporized sample and a large flow of carrier gas passes out through a split or purge valve. 

Such agents function less instantaneously than diazomethane. They react faster at quite high temperatures. One way to achieve those has been to mix them with the injection solution. Upon flash vaporization in the hot injection port or the start of the column, the derivatization reaction is nearly instantaneous and complete. This procedure is known as on-column derivatization. Such a mode of introduction of this reagent also achieves the purpose of reacting with and deactivating the free -Si-OH, active site groups in the column, as described at the very end of Section 11.5. This treatment is called silanization. 

The injection method is by far the most used method to microencapsulate enzymes, due to the simple procedure. One of the main drawbacks of the other two methods is the prolonged contact between the enzyme molecule and the organic solvent that contributes to the enzyme deactivation. 

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