Chromatograms applications

F. Dondi, A. Betti, L. Pasti, M.C. Pietrogrande and A. Felinger, Fourier analysis of multicomponent chromatograms — application to experimental chromatograms. Anal. Chem., 65 (1993) 2209-2222. 

Davis, J.M., Giddings, J.C. (1985). Statistical method for estimation of number of components from single complex chromatograms application to experimental chromatograms. Anal. Chem. 57, 2178. 

Zissis, K.D., Dunkerley, S., and Brereton, R.G. 1999. Chemometric techniques for exploring complex chromatograms Application of diode array detection high performance liquid chromatography electrospray ionization mass spectrometry to chlorophyll a allomers. Analyst 124 971-979. 

Statistical Method for Estimation of Number of Components from Single Complex Chromatograms Application to Experimental Chromatograms, J. M. Davis and J. C. Giddings, Anal. Chem., 57, 2178 (1985). 

Massa, V., Susplugas, P., Salabert, J. Microdetermination of alkaline earth cations by fluo-rometry of their thin-layer chromatograms. Application to the analysis of vegetable ash. 

Direct property prediction is a standard technique in drug discovery. "Reverse property prediction can be exemplified with chromatography application databases that contain separations, including method details and assigned chemical structures for each chromatogram. Retrieving compounds present in the database that are similar to the query allows the retrieval of suitable separation conditions for use with the query (method selection). 

Examples of the application of HPLC to the analysis of (a) acetaminophen, salicylic acid, and caffeine (b) chlorinated pesticides (c) tricyclic antidepressants and (d) peptides. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Column design involves the application of a number of specific equations (most of which have been previously derived and/or discussed) to determine the column parameters and operating conditions that will provide the analytical specifications necessary to achieve a specific separation. The characteristics of the separation will be defined by the reduced chromatogram of the particular sample of interest. First, it is necessary to calculate the efficiency required to separate the critical pair of the reduced chromatogram of the sample. This requires a knowledge of the capacity ratio of the first eluted peak of the critical pair and their separation ratio. Employing the Purnell equation (chapter 6, equation (16)). 

In practice a few iodine crystals are usually placed on the bottom of a dry, closed trough chamber. After the chamber has become saturated with violet iodine vapor the solvent-free plates are placed in the chamber for 30 s to a few minutes. The iodine vapor condenses on the TLC layers and is enriched in the chromatogram zones. Iodine vapor is a universal detector, there are examples of its application for all types of substances, e.g. amino acids, indoles, alkaloids, steroids, psychoactive substances, lipids (a tabular compilation would be too voluminous to include in this section). 

In addition, information must be provided concerning the enrichment and clean up of the sample. If possible the sample solution prepared should be adjusted to a particular concentration, so that the application of the chosen volume gives a preliminary idea of the amounts in the chromatogram produced. 

Fig. 1 Fluorescence plot (A) of the chromatogram track of an unpurified extract of sinapis seed (application 2 pi of a 1% solution in methanol) and (B) of a reference track with 1 pg sinigrin per chromatogram zone.

Many proteins and polymers have been analyzed on SynChropak GPC and CATSEC columns. Table 10.6 lists some of the published applications. The use of a surfactant to analyze the caseins in milk is illustrated in Eig. 10.12. Viruses have also been analyzed on SynChropak GPC columns, as seen in the chromatogram from Dr. Jerson Silva of the University of Illinois (Pig. 10.13). Dr. Nagy and Mr. Terwilliger analyzed cationic polymers on a series of CATSEC columns using differential viscometry as detection (Pig. 10.14) (9). 

Gianesello et al. (120) described the determination of the bronchodilator brox-aterol in plasma by on-line LC-GC. After deproteination and extraction, the LC separation was carried out by using a mixture of -pentane and diethyl ether (55 45 (vol/vol) as mobile phase. A small cut of the LC chromatogram (shown in Figure 11.9(a)) was introduced at 85 °C into the GC via so-called concurrent solvent evaporation. Figure 11.9(b) demonstrates that a detection limit of about 0.03 ng/ml was obtained. A fully automated LC-GC instrument was described by Munari and Grob (121) and its applicability was demonstrated by the determination of heroin metabo-... 

Comprehensive two-dimensional GC has also been employed for the analysis of pesticides from serum, which, although not strictly a forensic analytical problem , provides an example of the promise of this technique to forensic applications, such as the analysis of drugs of abuse (40). Two-dimensional gas chromatograms of a 17-pesticide standard and an extract from human serum are shown in Figure 15.13. The total analysis time of about 5 min, high peak capacity and the separation of all... 

Procedure. Pour the developing solvent into the chromatographic tank to a depth of about 0.5 cm and replace the lid. Take a prepared plate and carefully spot 5 pL of each indicator on the origin line (see Section 8.6, under Sample application) using a micropipette. Allow to dry, slide the plate into the tank and develop the chromatogram by the ascending solvent for about 1 h. Remove the plate, mark the solvent front and dry the plate in an oven at 60 °C for about 15 min. Evaluate the R value for each of the indicators using the equation... 

The refractive index detector, in general, is a choice of last resort and is used for those applications where, for one reason or another, all other detectors are inappropriate or impractical. However, the detector has one particular area of application for which it is unique and that is in the separation and analysis of polymers. In general, for those polymers that contain more than six monomer units, the refractive index is directly proportional to the concentration of the polymer and is practically independent of the molecular weight. Thus, a quantitative analysis of a polymer mixture can be obtained by the simple normalization of the peak areas in the chromatogram, there being no need for the use of individual response factors. Some typical specifications for the refractive index detector are as follows ... 

Figure 3.15 Reconstructed ion chromatograms obtained for ions in the background mass spectrum from the LC-MS analysis of a pesticide mixture From applications literature published by Micromass UK Ltd, Manchester, UK, and reproduced with permission.

---