Evaluation of the chromatogram

After completing the detection procedure the various separated solutes on the TLC plate are marked with the help of a sharp needle (e.g., pithing needle) subsequently, their evaluation may be carried out either qualitatively or quantitatively, as stated below  

The Rf value (Retention Factor) various separated solutes is determined accurately. The Rf value represents the differences in rate of movement of the components duly caused by their various partition coefficients i.e., their different solubility in the mobile and stationary phases. In order words, the Rf value (relate to front) is- the ratio between the distance starting point-centre of spot and distance starting point-solvent front , thus it may be expressed as  

Distance of centre of spot from starting point 

Distance of solvent front from starting point 

Important Points (/) Due to the always longer path of the solvent front, the Rf value is invariably lesser than 1. 

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For this GC determination with ECD, use only a sample test solution which has been adequately subjected to cleanup by silica gel column chromatography or by other means. Eluates 4 and 5 frequently contain considerable amounts of co-extractives, which may affect the evaluation of the chromatograms. 

Normal procedures for the qualitative and quantitative evaluation of the chromatograms are employed. Table 10.5 lists normal values for plasma amino acids as determined by this procedure. 

Almost always, compounds of high polarity and low volatility tend to undergo adsorption on the chromatographic support or decomposition on contact with it. These phenomena usually result in peak tailing and the quantitative evaluation of the chromatograms is difficult or even impossible. A wellknown example is the GC analysis of cholesterol, which can be analysed as such or as the TMS derivative (Fig. 1.1). If the support is not modified, free cholesterol provides a wide, tailing peak which can be evaluated quantitatively only with difficulty, whereas the TMS ether provides a sharp, symmetric peak the retention time of which is, however, substantially shorter [1]. 

S.4.4 Anomalies During Method Development. Further evaluation of the chromatograms in Figure 8-24 revealed that some late eluting peaks were... 

The Quantitative Evaluation of the Chromatogram Peak Area Measurements Peak Height Measurements Quantitative Analytical Methods for GC and LC Quantitative Analysis by TEC... 

K describes the relative affinity of a component for the two phases and hence relates to the distance and speed with which it moves through the plate on elution. Distribution coefficients and rates of migration, however, are difficult to evaluate, certainly in routine analysis, and a more practical evaluation of the chromatogram is required. In TLC and paper chromatography the results obtained are described by quoting the... 

An evaluation of the chromatograms upon which these values are based fails to support these weight fraction values. 

Installation of a GC/MS requires an area of 150 x 80 cm of laboratory bench space, and it is therefore essential that the location should allow for the heat evolved by the gas chromatograph. For the evaluation of the chromatograms, a writing desk with space for a computer and printer should be provided. The equipment should be accessible from all sides for maintenance purposes. If no central gas supply exists, space for a helium bottle is also required. A total floor area of approximately 8 should be sufficient for the installation of a GCZMS. 

Gas chromatographic analyses are likely to become automated, with automatic injection and computer evaluation of the chromatograms. Computer treatment is of even greater importance in combined gas chromatographic-mass spectrometric analysis. These improvements will probably be available in many laboratories within the next few years. 

Quantitative Analysis. The voltage output of the detector is related to the mole fraction of the material being detected in the vapor, so there is a correlation between the relative areas under the peaks in the chromatogram and the relative amounts of each of the components in the mixture. The quantitative evaluation of the chromatogram thus requires rehable methods for determining these peak areas. 

A multiple purpose template, made of perspex, for marking, sample apphcation and evaluation of the chromatograms (Fig. 19). 

If the starting material is a Sperry extract [202], 1 ml of extract is concentrated to 0.14 ml and the amount of this concentrated extract to be applied is calculated from the equation ml lipid concentrate = 1.67/mg-% ester cholesterol in the serum. Immediately after the TLC-separation, the 200 X 38 mm plate is sprayed evenly with antimony(ni)chloride reagent and then heated ca. 5 min at 110°C. The characteristic coloured reaction product changes from red to blue at a rate which varies from fraction to fraction correction factors must be thus determined for the particular technique used, with the help of standard substances. Table 123 contains such factors for the Beckmann spectrophotometer DU G 4700 at 575 nm. Some electrophoresis scanners with adequate light intensity are also suitable for photometric evaluation of the chromatograms (cf. also p. 139). 

Evaluation of the chromatogram 3. Filtration 4. Addition of a precipitant 5. Precipitation of sulfate 6. Crystallization of the precipitate over night 7. Filtration and rinsing of the precipitate 8. Ashing of the filter 9. Glowing of the residue 10. Weighing and evaluation... 

Other possibilities are related to methods based on chemical reactions. These methods are more generally used for scanning chromatograms in the reflectance mode or absorbance mode. In the visible spectral range, simple and inexpensive instruments with filter photometer can also be used. Direct fluorimetric evaluation of the chromatograms after treatment with a suitable fluorogenic reagent is the most sensitive method. Three different variables of fluorimetric evaluation can be done ... 

Step 3 Visual evaluation of the chromatograms and, if necessary, the UV spectra of the chromatograms. Decision to fine-tune optimization... 

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