Documentation on the Chromatogram

The required exposure times are difficult to estimate. They are best found by trial and error. Documentation of fluorescence quenching at A = 254 nm usually only requires one trial. The exposure time found to be adequate here is normally suitable for all following exposures of fluorescence quenching if the exposure conditions are maintained constant (camera type, film type, distance of objective and lamp, aperture etc.). The exposure time required for fluorescent chromatograms is primarily dependent on the intensity of the fluorescence and, therefore, has to be optimized for each chromatogram. It is best to operate with a range of exposure times, e.g. aperture 8 with exposures of 15,30,60,120 and 240 seconds. Experience has shown that one exposure is always optimal. 

The unusual feature of the programs is the automatic processing based on sample class. The operator provides the relevant parameters to the system of programs using the program SETUP, which contains several fill-in-the-form screens. These parameters Include the "class" of the specimen and the required automatic processing, as described below, and other pertinent information such as Mark-Houwink parameters, vial number, injection number, and documentation of the experimental conditions under which the chromatogram is to be measured. 

The chemical aspects of these studies focus primarily on the chemical characterization of the test substance and/or mixture. The identity of the test chemical should be proven, and the analytical procedures used, such as gas or liquid chromatography, nuclear magnetic resonance spectrometry, or nass spectroscopy, should be available for audit. This would include the chromatograms or spectra from these analyses. It is imperative that raw data be left intact as they emerge from an instrument to maintain data integrity. Chro-natographic printouts are to remain attached and in sequence. If some data points are not used in the final report, the reason is to be documented and those not used are to remain with the stud/ file. 

Ink analysis of the writing on each page of the agreement and the notes, showed that the same ink formulation was used on the documents. Figure 6 shows the similarity among these chromatograms. In addition, the findings revealed that the documents could not have been in existence in 1958, because a unique dye identified in the ink was first synthesized by Ciba Chemical Corp. in 1959 and the ink formulation was not produced until 1960. 

Direct copying on Ozalid or Ultrarapid blue print paper has also been employed for documentation [25—27]. For this purpose the chromatograms are laid layer-... 

Practical Tip for stabilizing colored zones On their way from the spray cabinet or plate heater to a documentation system, colored chromatograms should be covered with a clean glass plate to minimize reactions with oxygen. 

Documentations obtained when evaluation is performed with a flat-bed scanner are always dependent on the software used and also, of course, on the scanned object. As mentioned in Section 7.4, a flat-bed scanner can be directly used for processing colored chromatograms only. The first investigations were done with the Pharmacia system, which was also used for the evaluation of DNA electrophoresis gels. 

The analyst enters the date of completion of the chromatogram and also his or her signature in the spaces provided, and any additional information sheets are also dated. The checker initials this form and the last of the additional sheets on completion of all the documentation that applies to this plate (sheets with pasted-on photographs, attached scanner printouts, videoprints etc.). 

It has been almost seven years since the publication of the first English Edition of my book on TEC The following improvements in technology over the years have made it necessary for me to update the first edition new precoated layers for both existing and new fields of applications, a new generation of equipment for safe operations and reproducible results, new devices such as the Diode Array Detector and Bioluminescence Analyzer, new methods of interface between TEC and analysis methods, especially the use of digital cameras for the documentation of thin layer chromatograms. For the reader s benefit, I have updated my description of available products on the market. 

The essential recording that is obtained for each separation is called a chromatogram. It corresponds to a two-dimensional diagram traced on a chart paper or a screen that reveals the variations of composition of the eluting mobile phase as it exits the column. To obtain this document, a sensor, of which there exists a great variety, needs to be placed at the outlet of the column. The detector signal appears as the ordinate of the chromatogram while time or alternatively elution volume appears on the abscissa. 

Figure 1.10 Effect of column length on the resolution. Chromatograms obtained with a GC instrument illustrating that by doubling the length of the capillary column, the resolution is multiplied by 1.41 or. ffl (adapted from a document of SGE Int. Ltd).

Autoradiography. Autoradiography is one of the oldest methods used for semiquanti-tative measurement of radioactivity. The chromatogram is placed on an x-ray film and exposed in the dark for usually less than 1 h (a detailed procedure is given elsewhere) (Theobald 1994). This method is no longer in use since it is time-consuming and inaccurate. Visualization of the radioactivity distribution on a film may be useful for documentation purposes. 

Fig. 71. Examples of ways of documenting thin-layer chromatograms, a chromatogram traced on transparent paper (transferred back to ordinary paper) h documentation by photostating c as rectangles of the same area as the planimetrically measured spot areas. Experimental details separation of bromoureides using cyclohexane-chloroform-pyridine (20 + 60 -f 5) on silica gel G layers. 8 start ...

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