Derivatisation of samples

There are many compounds which cannot be readily analysed by GC, either because they are not sufficiently volatile or because they tail badly and are too strongly attracted to the stationary phases. Derivatisation before analysis 

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Table 4.15 fists the many possibilities for solid sampling for GC analysis. In general, sample preparation should be considered in close conjunction with injection. Robotic sample processors have been introduced for automatic preparation, solvent extraction and injection of samples for GC and GC-MS analyses. Usually, facilities are included for solvent, reagent, and standard additions and for derivatisation of samples. 

Derivatisation of samples in HPLC is undertaken principally for two reasons. First, there is no detector for HPLC that has universally high sensitivity for all solutes hence a suitable chemical transformation of the solute can greatly extend the sensitivity and versatility of a selective detector. Second, sample derivatisation may be undertaken to enhance the detector response to sample bands relative to overlapping bands of no analytical interest. This experiment involves the pre-column derivatisation of sample with dinitro-fluorobenzene so enhancing the spectrophotometric response of the sample, that is, the amino acids from a protein hydrolysate. The conversion of the amino acids to their apolar DNP derivatives allows them to be analysed using reverse phase chromatography. Thus, polar DNP-amino acids, such as DNP-aspartate, will elute early whereas apolar DNP-amino acids, such as DNP-alanine, will elute later. The DNP-amino acids are detected by an... 

The configuration can be expanded by adding other sample preparation instruments to facilitate automating other preparative steps that may intervene between SFE and the analytical instrument, e.g. solvent exchange, internal standard addition, serial dilutions for calibration curve generation, SPE for further cleanup of the extract output by SFE, derivatisation of components within the SFE extract, and many other (currently) manual-human intervention techniques. 

Hyphenation in capillary electrophoresis is still in its infancy. Critical aspects of CE hyphenation include the minute volumes of sample injected (typically a few nL) and small flow-rates (in the order of nLmin-1). Interfaces are not commercially available. CZE-UV can be used for the analysis of higher polyamide oligomers in HF1P solution [859]. A solvent elimination design with nebuliser has been described for CE-FTIR and CEC-FTIR coupling absolute detection limits are hundreds of pg [860]. An advantage of CE-FTIR is that analytes may be detected and identified without derivatisation. CE(C)-NMR [861-863] is advancing rapidly. 

Wahlberg et al. [20] determined NPEO in Blue mussels by GC-ECD after derivatisation of the phenols with PFBC1. To this end, samples were homogenised in acetone/hexane (5 2), followed by extraction with hexane/diethylether (9 1). Lipids were removed by L/L extraction with acetonitrile in 0.1 M NaOH followed by L/L extraction in TMP/sulphuric acid. Recoveries of 93, 34, 65 and 100% were reported for NP, NPEOi, NPEO2 and NPEO3, respectively. 

Tabor and Barber examined the 2800 km reach of the Mississippi river for the occurrence and fate of LAS in waters and bottom sediments [3], Dissolved LAS was concentrated on reversed-phase (RP)-Cis solid-phase extraction (SPE) cartridges, while sediment-bound LAS was extracted with methanol in three 24-h cycles on a rotating mixer. After derivatisation of the extracts to yield the trifluoroethyl esters, analyses were performed by gas chromatography mass spectrometry (GC-MS). Upon enrichment of 900 mL samples, detection limits of 0.1p.gL 1 were achieved. LAS was identified in 21% of the water samples at concentrations ranging from 0.1 to 28 p,g L-1 and was detected at all of the Mississippi river sediment samples at concentrations ranging from 0.01 to 20 mg kg-1 with a mean value of 0.83 mg kg-1. The concentrations of LAS sorbed onto sediment particles generally did not correlate with... 

All analytical methods involve some kind of sample preparation and thus the type of factors selected for a HPLC method as shown in Table 5.2 are similar for all methods. However, different stages in the preparation will be more critical for some methods. For instance, derivatisation will be more common for gas chromatography methods and hence more critical. 

A so-called plasmalogen extract. This is a pool of extracted and derivatised erythrocyte samples. It is used solely to check for the detector response of the plasmalogens, thereby identifying active spots in the injection system. 

The procedure of sample preparation for faecal BA analysis and derivatisation is an adaptation of the method of Czubayko et al. [17]. The internal standard (125 pg HDCA) is added to the aqueous phase of extraction. The sample is saponified with 200 pi 10 mol/1 sodium hydroxide at 120°C for 120 min and then acidified to pH 1 with hydrochloric acid. After extraction of BAs with diethyl ether (4 x 1 ml), the solvent phases are pooled and evaporated under a stream of nitrogen. The residue is... 

Figure 9. A HRTEM micrograph of sample A stained with the cluster crown compound showing strong contrast on the surface of the particles indicating the clusters to be bound predominently to the outer surface of the derivatised MCM-41 particles.

Research in this area of resolution methods was first in g.l.c. However, the need to derivatise many of the compounds of interest to make them sufficiently volatile for analysis tended to increase the complexity of sample preparation. High performance liquid chromatography does not have this drawback (p. 232) and research activity in this field is now extensive. One illustrative example of the elegance of this technique is noted here257 but the reader s attention is directed to an excellent summary to 1983 of both these chromatographic methods.258... 

Various other workers have reported on the determination of volatile organic compounds in soils [186,187] and landfill soils [188]. Soil fumigants such as methyl bromide have also been determined by this technique [189]. Trifluoroacetic acid is a breakdown product of hydrofluorocarbons and hydrochlorofluorocarbon refrigerant products in the atmosphere and, as such, due to the known toxicity of trifluoroacetic acid, it is important to be able to determine it in the atmosphere, water and in soil from an environmental point of view [190]. In this method the trifluoroacetic acid is extracted from the soil sample by sulfuric acid and methanol, which is then followed by the derivatisation of it to the methyl ester. The highly volatile methyl ester is then analysed with a recovery of 87% using headspace gas chromatography. Levels of trifluoroacetic acid in soil down to 0.2 ng/g can be determined by the procedure. 

When examining the first peak (see curve a in Fig. 5) it was found that it was proportional to the hydroperoxide contents as measured by FTIR, after NO derivatisation of un-aged films. When the sample was treated with S02, to decompose the hydroperoxides, the peak disappeared as shown (see curve b in Fig. 5). The CL curve of the S02 -treated sample has the typical sigmoidal appearance, just like an oxidising polypropylene sample. It was thus concluded that the first peak in the CL curve of the oxidising polyethylene in this study is from hydroperoxides already present in the material before the measurement starts. The second peak is the auto-oxidation. It might be... 

The variety of sampling methods that are available, ie dilute and concentrated samples, suspensions, solids, surfaces and combination with chromatographic methods, such as that used in the high performance liquid chromatography separation of o-phthalyl dialdehyde derivatised amino acids in natural and sea water samples. 

One of the most classic examples of chiral expression in thermotropic liquid crystals is that of the stereospecific formation of helical fibres by di-astereomers of tartaric acid derivatised either with uracil or 2,6-diacylamino pyridine (Fig. 9) [88]. Upon mixing the complementary components, which are not liquid crystals in their pure state, mesophases form which exist over very broad temperature ranges, whose magnitude depend on whether the tartaric acid core is either d, l or meso [89]. Electron microscopy studies of samples deposited from chloroform solutions showed that aggregates formed by combination of the meso compounds gave no discernable texture, while those formed by combinations of the d or l components produced fibres of a determined handedness [90]. The observation of these fibres and their dimensions makes it possible that the structural hypothesis drawn schematically in Fig. 9 is valid. This example shows elegantly the transfer of chirality from the molecular to the supramolecular level in the nanometer to micrometer regime. 

Often in identification systems, one pre-treatment procedure is applied to samples, but various end determination techniques (GC, HPLC and, increasingly rarely, TLC). Because of the high sensitivity and broad range of linearity (often encompassing three orders of magnitude) of coupled techniques (GC-MS or LC-MS), some analysts apply one method of sample preparation (extraction and derivatisation) to all analytes, in spite of the low efficiency of the process of isolation of acid compounds subjected to extraction from an alkaline medium. The most extensive method to date, enabling detection and identification of over... 

Figure 5 Quantitative derivatisation of 50 fmol [Glul] fibrinopeptide B with a C-5 quaternary linked tag. The derivatisation was performed on the peptide sample already spotted onto the target slide.

Often it is required to detect compounds with no or only very weak chromophores such as sugars and amino acids. Refractive index detectors and mass sensitive detectors can be used but they are relatively insensitive in the context of biological sample concentrations. Indirect detection using a UV or fluorescent eluent can also be employed. However, the most common approach is the use of derivatisation. Derivatisation of some chemically reactive moiety on the analyte can be performed in two modes. In post-column derivatisation the sample is separated first and then reacted with a flowing stream of derivatising reagent being pumped into... 

Of the two detection techniques mentioned above, fluorescence is preferred in general because it suffers from fewer operational difficulties. Many compounds do not display a native fluorescence however, this can be overcome if the analyte can be converted by chemical reaction into a fluorescent compound. This process is known as derivatisation and can be accomplished by either derivatising the sample prior to injection (precolumn) or after chromatographic separation (post-column). 

With the exception of on-line trace enrichment and/or derivatisation systems, the form of injection is almost exclusively by use of a sample loop with rotary valve (Rheodyne or similar). Normally the size of sample loop is used to decide sample size rather than injection volume. As in other types of laboratory, septum type injection systems in environmental laboratories have long been superseded. 

For measuring at low concentrations, the derivatisation of the extracted organotin compound with sodium tetraethylborate is an established procedure to make a more sensitive gas chromatographic analysis possible. Derivatisation can be combined with the extraction as prescribed in the German method but can also be carried out after the extraction. Selective detection is possible using GC-MS. For organo-metallic compounds atomic emission or atomic absorption detection is not only very sensitive, but very selective. The steps necessary for the chromatographic separation and detection are described in ISO/CD 17353 (2001). Application of this well-described method for soil samples does not mean that the difficulties with the extraction have been overcome. 

Gas chromatography of the methyl esters of fatty acids on two or more columns is most commonly used for final quantification. Liquid chromatography after derivatisation of the acids to form UV sensitive or fluorescent compounds is an alternative approach which at present has not been refined to the extent of gas chromatography but, with improvements in the performance of HPLC columns, may in future offer a rapid method for screening samples. 

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