Derivatization, analytical reagents

In-situ chemical derivatizations were performed by adding 0.5 to 2 mL of the derivatization reagent, trimethylphenyl ammonium hydroxide (TMPA) in methanol (Eastman Kodak Company, Rochester, NY) directly to the sample cell of an ISCO model SFX extraction unit. The sample and reagent were pressurized with C02 (typically 400 to 500 atm), and heated to an appropriate temperature (typically 80°C). The derivatization was performed in the static SFE mode for 5 to 15 minutes, then the derivatized analytes were recovered by dynamic SFE using a typical flow rate of 0.6 to 1.0 mL/min (measured as liquid C02 at the pump). No other sample preparation of the derivatized extracts was performed prior to GC analysis. All GC analyses were performed using Hewlett-Packard 5890 GCs with appropriate detectors and HP-5 columns (25 m X 250 pm i.d., 0.17 pm film thickness). 

Chapter 7 deals with other types of US assistance to enhance reactions of analytical interest including derivatization and reagent generation, or ultrasound-assisted oxidation or hydrolysis reaotions. 

PHWE-LC systems have been utilized, for example, to determine N-methylcarbamates in food. PHWE was done at 75 °C and a combination of static and dynamic extraction was used. In this system, the effluent from the chromatograph was coupled with a flow injection manifold where the N-methylcarbamates were hydrolyzed and then derivatizatized by reaction with a fluorogenic reagent (o-phthalaldehyde). The derivatized analytes were driven to a fluorimeter equipped with a flow-cell allowing monitoring (31). 

Peroxyoxalate chemiluminescence reactions are analytically important (particularly in HPLC). The most commonly used reagents are bis-(2,4,6-trichlorophenyl)oxalate (TCPO) and bis-(2.4-din-itrophenyl)oxalate (DNPO). Fluorescent compounds (including anthracene, perylene, aminoanthracenes, and aminopyrenes) and suitably derivatized analytes (such as amines, steroids with dansyl chloride thiols with N-[4-(6-dimethylamino-2-benzofuranyl)-phe-nyljmaleimide and catecholamines with fluoresca-mine) can be sensitively detected. 

Luminol can be used to derivatize analytes (for immunoassay or HPLC) through substitution at the primary amine but this results in a 10- to 100-fold decrease in chemiluminescence efficiency. Isoluminol (6-amino-2,3-dihydro-l,4-phthalazinedi-one) is somewhat less efficient than luminol however, it does not suffer a decrease in efficiency upon binding. Aminobutylethylisoluminol (ABEI, (9) is a useful precolumn labeling reagent for amines and carboxylic acids because the hydrocarbon spacer isolates the chemiluminophore from the analyte. 

An acyl chloride such as benzoyl chloride can react with secondary and primary amines and amino acids to form N-substituted benzamide derivatives. Analytical reagents used for precolumn derivatizations are p-nitrobenzene azobenzoyl chloride for LC-UV, o-acetylsalicyloyl chloride for LC-ECD, and 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxa-line-2-carbonyl chloride (DMEQ-COCl) and 4-(4,5-diphenyl-lf/-imidazol-2-yl)benzoyl chloride (DIB-Cl) for LC-FL, respectively. Pentafluorobenzoyl chloride (PFBC) is used as an acylating reagent for GC with electron-capture detection (Figure 4). Many of those described above can also react with hydroxyl and phenol groups. 

Chloroformates are useful analytical reagents for primary, secondary, and tertiary amines. The reaction of these reagents with amines can be performed easily, and the resultant carbamate derivatives are suitable for GC analysis. The carbamate derivatives obtained from primary and secondary amines are determined selectively and sensitively by GC-MS and -BCD. After dealkylation, tertiary amines are derivatized to carbamates in the same manner for primary and secondary amines. The carbamate derivatives obtained with pentafluorobenzyl chloro-formate are determined with high sensitivity by GC-ECD. Chloroformates also react with phenols, thiols, imidazoles, as well as amines. 

Hydroxy compoimds such as alcohols, carbohydrates, steroids, and phenols are known to play important physiological roles. Although many analytical reagents have been developed for determination of hydroxy compounds, some of them can be also applied to amines as described in the previous section (e.g., isocyanates, sulfonyl chlorides, acyl chlorides, halogenobenzofurazans, acyl amides, and chloroformates). The derivatization conditions for hydroxy compounds are relatively more drastic than those for amines. By using fairly different conditions from those for amino compounds. 

See also. Analytical Reagents Specification. Derivat-ization of Analytes. Fluorescence Derivatization Fluorescence Labeling Quantitative Anaiysis. Lipids Fatty Acids. Liquid Chromatography Liquid Chromatography-Mass Spectrometry Pharmaceuticai Applications. Mass Spectrometry Forensic Appiications. Spectrophotometry Derivative Techniques. 

Zacharis, C. K., P. D. Tzanavaras, and D. G. Themelis. 2009. Ethyl-propiolate as a novel and promising analytical reagent for the derivatization of thiols Study of the reaction under flow conditions./. Pharm. Biomed. Anal. 50 384-391. 

For residue analysis, derivatization with perfluorinated reagents (e.g., hep-tafluorobutyric anhydride, perfluorobenzoyl chloride) in association with NCI is important (Knapp, 1979 Pierce, 1982). Besides being easier to chromatograph, the derivatized analytes show high electron affinities due to the high number of halogens and allow a very sensitive detection. 

Table 1. Analyte Functional Groups and Chiral Derivatizing Reagents...

We inferred that these properties might be exploited in a series of unique derivatizing reagents designed specifically for trace analysis of organic compounds using HPLC separation and fluorescence detection. The use of these pyridones for the analytical purposes reported here is based on their acidic properties. Treatment of a lH-2-pyridone with a base converts the pyridone to its salt. 

---