Chromatographic separations chromatograms

Development of the Chromatogram. The term development describes the process of performing a chromatographic separation. There are several ways in which separation may be made to occur, eg, frontal, displacement, and elution chromatography. Frontal chromatography uses a large quantity of sample and is usually unsuited to analytical procedures. In displacement and elution chromatography, much smaller amounts of material are used. 

Fig. 1 Schematic diagram of the chromatographic separation (A) and the fluorescence scan (B) of a sugar mixture containing 1 pg substance per chromatogram zone. Lactose (1), fructose (2), arabinose (3), xylose (4), rhamnose (5), mixture (G).

When John Phillips, in 1991, presented the practical possibility of acquiring a real comprehensive two-dimensional gas chromatographic separation (33), the analytical chemists in the oil industry were quick to pounce upon this technique. Venkatramani and Phillips (34) subsequently indicated that GC X GC is a very powerful technique, which offers a very high peak capacity, and is therefore eminently suitable for analysing complex oil samples. These authors were able to count over 10 000 peaks in a GC X GC chromatogram of a kerosine. Blomberg, Beens and co-workers... 

The sulfanes are soluble in carbon disulfide, benzene, tetrachloromethane, and dry diethylether (decreasingly so in that order) while alcohols and aqueous systems initiate rapid decomposition. For this reason a report on the chromatographic separation of the sulfanes H2S by reversed-phase HPLC using methanol as an eluent [35] was shown to be in error The peaks observed in the chromatogram have to be assigned to bismethoxy oligosulfanes... 

Funk et al. have used a low-pressure mercury lamp without filter to liberate inorganic tin ions from thin-layer chromatographically separated organotin compounds these were then reacted with 3-hydroxyflavone to yield blue fluorescent chromatogram zones on a yellow fluorescent background [22]. Quantitative analysis was also possible here (XoK = 405 nm, Xji = 436 nm, monochromatic filter). After treatment of the chromatogram with Triton X-100 (fluorescence amplification by a factor of 5) the detection limits for various organotin compoimds were between 200 and 500 pg (calculated as tin). 

Reactions can be exploited more speciHcally if it is known that particular functional groups are present [cf. Chapter 2]. They still do not allow direct identification, but they increase the specificity of the evidence. The chromatographic separation carried out before detection also contributes to this. This reduces the number of potential components. However, this does not exclude the possibility that there might be several substances in the particular part of the chromatogram involved. This not only applies to thin-layer chromatography but also applies with equal force to other microanalytical separation methods (GC, HPLC). 

Note When combined with thin-layer chromatographic separation the reagent provides a specific detection method for nitrate and nitrite. The color development is often completed within a few minutes on silica gel plates. In the absence of ammonia vapor traces of oxides of nitrogen in the laboratory atmosphere can slowly cause the background to become reddish-brown. The simultaneous presence of the following ions in the chromatogram zones interferes with the detection of nitrate/nitrite I , 10J, IO4, MoO and H2PO2. 

Fig. 1 (A) Chromatographic separation of sugars. Track 1 fructose, 2 sucrose, 3 glucose, 4 mixture of the substances in tracks 1-3, 5 mixture of substances in tracks 1-3 and 6, 6 Fructo-oligosaccharides, 7 1-kestose, 8 mixture of glucose, maltose, maltotriose and maltotetraose. (B) Absorption scan of track 5 with 200 ng each substance per chromatogram zone 1 = fructosyl-nystose, 2 = nystose, 3 = 1-kestose, 4 = fructose, 5 = sucrose, 6 = glucose.

The NMR spectrum is recorded during the chromatographic separation. Data are collected as in a 2D experiment, the two dimensions being the chemical shift and the retention time of the chromatogram. 

The chiral recognition ability of a CSP is quantitatively evaluated from the results of chromatographic separation of enantiomers. Figure 3.4 shows a chromatogram of the resolution of benzoin (19) on cellulose tris(3,5-dimethylphenylcarbamate). The (+)-isomer elutes first followed by the (—)-isomer complete baseline separation is achieved. The results of the separation can be expressed by three parameters—capacity factors (k1), separation factor (a), and resolution factor (Rs)—defined as follows ... 

RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HC1 (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HC1 at 35°C for 16h. Chromatographic separation was performed in an ODS column (150 X 4.6mm i.d. particle size 5/.an). Solvents A and B were water-acetic acid (97 3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in lQmin (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188],... 

Chromatogram the resultant electrical output of sample components passing through a detection system following chromatographic separation. A chromatogram may also be called a trace. 

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