Derivatized layered M

Figure 5.19 Formation of amino acids on ice surfaces irradiated in the laboratory (Nature Nature 416, 403-406 (28 March 2002) doi 10.1038/416403a-permission granted). Data were obtained from analysis of the room temperature residue of photoprocessed interstellar medium ice analogue taken after 6 M HCl hydrolysis and derivatization (ECEE derivatives, Varian-Chrompack Chirasil-L-Val capillary column 12 m x 0.25 mm inner diameter, layer thickness 0.12 pirn splitless injection, 1.5 ml min-1 constant flow of He carrier gas oven temperature programmed for 3 min at 70°C, 5°C min-1, and 17.5 min at 180°C detection of total ion current with GC-MSD system Agilent 6890/5973). The inset shows the determination of alanine enantiomers in the above sample (Chirasil-L-Val 25 m, single ion monitoring for Ala-ECEE base peak at 116 a.m.u.). DAP, diaminopentanoic acid DAH, diaminohexanoic acid a.m.u., atomic mass units.

Evaporate the organic layers of the free and total samples to dryness at 40°C under a gentle stream of nitrogen. Add 10 pi triethylamine and 100 pi of 7% penta-fluorobenzylbromide in acetonitrile and derivatize at room temperature for 15 min. Add 150 pi of 0.5 M HC1 and extract with 1 ml hexane for at least 1 min. Centrifuge the samples for 2 min at 835 xg. Pipette the hexane into a sample vial and evaporate to dryness at 40°C. Add 50 pi hexane and redissolve the residue. Samples are ready for quantification with GC-mass spectrometry (GC-MS). 

Following extraction/cleanup, quinoxaline-2-carboxylic acid can be detected by electron capture, or mass spectrometric techniques, after gas chromatographic separation on capillary or conventional columns. A prerequisite of quin-oxaline-2-carboxylic acid analysis by gas chromatography is the derivatization of the molecule by means of esterification. Esterification has been accomplished with methanol (419, 420, 422), ethanol (421), or propanol (423) under sulfuric acid catalysis. Further purification of the alkyl ester derivative with solid-phase extraction on a silica gel column (422), thin-layer chromatography on silica gel plate (420), or liquid chromatography on Hypersil-ODS, 3 m, column (423), has been reported. 

Separation of enantiomers of etodolac using two different derivitization agents and three chiral stationary phases has been studied [24]. Etodolac was converted to its anilide derivative with either 1,3-dicyclohexyl-carbodiimide or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Etodolac, derivatizing agent, aniline, and dichloromethane were allowed to incubate for 30 minutes, which was followed by addition of 1 M HC1. The organic layer was removed, washed, dried, and then injected into normal phase or reverse phase HPLC. The HPLC system consisted of a 250 x 4.6 mm (5 pm particle size) column packed with chiral stationary phases, and detection was effected by the UV absorbances at 254 and 280 nm. Separation of etodolac enantiomers was achieved on only one of the stationary phases when using 20% 2-propanol in hexane as the mobile phase at a flow rate of 2.0 mL/min. 

This study on the immobilization of glucose oxidase and the characterization of its activity has demonstrated that a bioactive interface material may be prepared from derivatized plasma polymerized films. UV/Visible spectrophotometric analysis indicated that washed GOx-PPNVP/PEUU (2.4 cm2) had activity approximately equivalent to that of 13.4 nM GOx in 50 mM sodium acetate with a specific activity of 32.0 U/mg at pH 5.1 and room temperature. A sandwich-type thin-layer electrochemical cell was also used to qualitatively demonstrate the activity of 13.4 nM glucose oxidase under the same conditions. A quantitatively low specific activity value of 4.34 U/mg was obtained for the same enzyme solution by monitoring the hydrogen peroxide oxidation current using cyclic voltammetry. Incorporation of GOx-PPNVP/PEUU into the thin-layer allowed for the detection of immobilized enzyme activity in 0.2 M sodium phosphate (pH 5.2) at room temperature. 

In this system, amphetamine, 3,4-methylenedioxyamphetamine (MDA) and the A-methyl and A-ethyl analogues of the drugs were derivatized, with 4-chloroamphetamine being used as the internal standard. For powders, solutions were prepared at concentrations of 1 mgml in methanol and a 100 til aliquot blown dry under nitrogen. To the residue was added 50 p.1 of 0.5 M KOH and 500 p,l of toluene. The mixture was then shaken for 30 s and centrifuged, after which the toluene layer was recovered and HFBA (5 til) added. The excess reagent was neutralized with 500 p,l of 10% NaHCOs and an aliquot (1 ttl) analysed by GC-MS. Each of the amphetamines and the A-alkylated derivatives could be separated and identified on the basis of retention index and mass spectral data. 

Derivatization and Extraction. Modifications of the procedures of Ebeler et al. (49) were used for all aldehyde analyses. Briefly, 3.0 mL of wine were mixed with 60 pL of internal standard (10 mg 2,4,5-trimethylthiazole/mL in 10% aqueous ethanol) and 1 mL of 0.03 M aqueous cysteamine (pH 8.5) the pH was adjusted with HCl or NaOH (pH s from 2-10 were evaluated as discussed below). Following reaction at room temperature for 1 hour, the pH was re-adjusted to 8.5 and the solution was extracted two times with 1.5 mL of chloroform the chloroform layer was removed each time and then combined to give a total of 3.0 mL of extract. Samples were injected onto a gas chromatograph fitted with either a mass spectrometer or nitrogen phosphorous detector. Peak area ratios of the internal standard to the analyte were used for all quantitative calculations. 

PMMA itself does not possess ready-to-use fiinctional groups for covalent binding with biological molecules. The amine-terminated PMMA were often produced by immersing the freshly cleaned PMMA substrate into a 1.0 M ethylenediamine in dimethyl sulfoxide (DMSO) solution for 15 min at room temperature (115) or coated with a thin layer of polyethyleneimine (PEI) or polyallylamine hydrochloride (PAH). This was first treated in 1 N sodium hydroxide (NaOH) solution at 55 C for 30 min and then immersed in a PEI or PAH solution (0.2%, pH 7) at room temperature for 1 h (117). Tsai and Lin (2005) demonstrated that PEI-derivatized PMMA was used for the determination of alpha-fetoprotein by quartz crystal microbalance (QCM) (118). Furthermore, the amine-terminated PMMA could be generated by reacting with 10% hexamethylene diamine (HMD) (reaction shown in scheme 8.2) or 1,3-diaminopropane (DAP) in 100 mM borate buffer pH 11.5 for 2 h (119) or exposing to -lithioethylenediamine (120) (reaction shown in scheme 8.3). 

Another popular host for molecular recognition studies in monolayers is thiol-derivatized cyclodextrin. Kaifer and coworkers have prepared monolayers from fi-cyclodextrin 19 in which all primary hydroxyls are replaced by SH groups. This compound can form up to seven gold-sulphur chemical bonds on the surface. These monolayers are incomplete to cover the gold surface completely, they were treated successively with ferrocene (to block the cyclodextrin cavities) and pentanethiol (to fill in the spaces between cavities). Cyclic voltammetry showed that the layers obtained bind ferrocene. Ferrocene trapped in the cyclodextrin cavities can be replaced by another known guest, electroinactive m-toluic acid444. 

Sample preparation 5 mL Urine + 2 mL 500 mM pH 7.0 phosphate buffer -I- 0.5 mL 20 mg/mL p-bromophenaaqueous layer and add it to 100 p-L 2% tributylphosphine in MeOH, heat at 50°for 30 min, wash with 6 mL hexane, add 200 p,L 0.2% N-(4-dimethylamino-3,5-dinitrophenyDmaleimide in acetone to the aqueous layer, mix, let stand at room temperature for 5 min, wash with 6 mL hexane, discard the hexane layer, acidify the aqueous layer with about 200 pL 2 M HCl, extract twice with 6 mL portioiis of benzene (Caution Benzene is a ceircmogen ). Combine the organic layers and add 10 pg IS, evaporate to dryness imder reduced pressure, reconstitute the residue in 200 pL MeOH, inject a 5-20 pL aliquot. (Free captopril is derivatized as its p-bromophenaQrl bromide adduct then oxidized captopril is reduced and derivatized as its N-(4-dimethylamino-3,5-dinitro-phenyl)maleimide adduct.)... 

Second method 0.5 g sample was digested with a 5 mL mixture of 10% HBr and 60% methanol and stirred for 1 h. Extraction was carried out by addition of 10 mL of 0.05% tropolone in methanol, stirring for 30 min and then centrifuging for 5 min. The organic layer was derivatized with 0.2 mL of 4% NaBH4 solution. Clean-up was performed with Florisil followed by elution with hexane. Separation was by GC (column of 5 m length, 0.32 mm internal diameter, DB-1 as stationary phase He as carrier gas at 5 mL min injector temperature of 250 °C column temperature ranging from 80 to 250 °C). Detection was by FPD (detector temperature of 350°C). 

N-hydroxysuccinimide ester )but not by the chloramine-T or lactoperoxidase procedures (24, 25). This observation suggested to us that VPg does not contain accessible Tyr residues, (iii) VPg-RNA can be labeled with ( h)tyrosine 5 vivo. Digestion of ( tyrosyl-5fi)VPg-pUp with Pronase yielded (tyrosyl-5H)Y with almost no loss of radioactivity (24). These results.are at odds with the iodination data. It is known, that 0 - derivatized tyrosine residues cannot be iodinated by the chloramine-T procedure (26). Failure to iodinate YPg by using chloramine-T or lactoperoxidase can be explained if one assumes that "VPg contains only one t osine residue whose phenolic group is blocked, (iv) Digestion of ( P ) TPg-pUp with 5.6 M HC1 at IIO C for 2 hr yields (52p o4-(3t phospho-51-uridylyl)tyrosine (Tyr- 4 pUp) whose structure was identified by degradation with enzymes and thin-layer chromatography (24). 

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