Processing the chromatogram

In HPLC-TLC coupling, the crucial aspect is the maintenance of the chromatographic integrity during the deposition process. The chromatogram is preserved after LC separation, and is available for further separation and/or investigation. LC-TLC coupling increases the separation efficiency, and allows detection modes which are incompatible with LC (e.g. spectroscopic techniques... 

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. 

Let the distance between the injection point and the peak maximum (the retention distance on the chromatogram) be (y) cm and the peak width at the points of inflexion be (x) cm. If a computer data acquisition and processing system is employed, then the equivalent retention times can be used. 

Using equation (10), the efficiency of any solute peak can be calculated for any column from measurements taken directly from the chromatogram (or, if a computer system is used, from the respective retention times stored on disk). The computer will need to have special software available to identify the peak width and calculate the column efficiency and this software will be in addition to that used for quantitative measurements. Most contemporary computer data acquisition and processing systems contain such software in addition to other chromatography programs. The measurement of column efficiency is a common method for monitoring the quality of the column during use. 

Microchemical reactions These can be carried out either with universal reagents [11] or with such substances which react with particular functional groups (group-characterizing reagents). If the separation process ensures that only one component occurs at a particular spot on the chromatogram, then this can be detected sub-stance-specifically . But specificity in an unequivocal sense can only be produced by a combination of the separation and the detection process. (The same is true of other forms of detection.)... 

This effect, which can also be produced if fluorescent substances are applied to the chromatogram by spraying or dipping after development, is an absorption effect and not a quenching process in the true sense of the word. It is correct to refer to fluorescence or phosphorescence diminishing. The more absorbant sample molecules there are present in the zone the darker this will appear (Fig. 4B). 

The chromatogram is observed and documented as soon as the spots are readily visible. The iodine can then be allowed to evaporate from the chromatogram (fume cupboard ). The chromatogram can then be subjected to further reactions or processes after this reversible reaction. 

Every chromatographic investigation begins with the preparation of the sample and the chromatographic system. This is followed by the crux of the separation process (development of the chromatogram) which is in turn followed by the visualization of the separated substances and the preservation of the chromatogram and finally by the analysis of the results. 

Each of these steps must be so documented that it can always be repeated simply from the protocol. The most important steps will be discussed below, paying special attention to the processing of the chromatogram after development. 

A precise mastery of the chromatographic process also requires that the relative humidity be controlled. There are sufficient examples demonstrating that reproducible development is only possible if temperature and relative humidity are maintained constant. The influence of the latter on chromatographic behavior can be investigated using the Vario KS chamber (Fig. 59). When the relative humidity IS altered it is possible that not only the zone behavior will be changed but also the order of the zones on the chromatogram (Fig. 60). 

Note The full fluorescence intensity usually only develops about 30 min after the dipping process it then remains stable for several days if the chromatograms are stored in the dark (1, 5]. Fluorescein sodium can be employed in the reagent in place of 2, 7 -dichlorofluorescein [5]. The detection limits lie in the lower nanogram to picogram range [1, 5]. 

The mixture is identical in each example. The peaks are shown separated by 2, 3, 4, 5 and 6 (a) and it is clear that a separation of 6a would appear to be ideal for accurate quantitative results. Such a resolution, however, will often require very high efficiencies which will be accompanied by very long analysis times. Furthermore, a separation of 6o is not necessary for accurate quantitative analysis. Even with manual measurements made directly on the chromatogram from a strip chart recorder, accurate quantitative results can be obtained with a separation of only 4a. That is to say that duplicate measurements of peak area or peak height should not differ by more than 2%. (A separation of 4a means that the distance between the maxima of the two peaks is equal to twice the peak widths). If the chromatographic data is acquired and processed by a computer, then with modem software, a separation of 4a is quite adequate. 

---