Reference chromatograms

Theoretically, providing the chromatographic conditions are kept constant, then, the reference chromatogram need only be run once a day. However, it is advisable to run the reference chromatogram at least every two hours and many analysts run a reference chromatogram immediately after each sample. 

Fig. 1 Reflectance scan of a nux vomica extract (A) and a reference chromatogram containing 1 gg each of strychnine and brucine (B). Start (1), brucine (2), strychnine (3), front (4).

Hunt et al.150 proposed the incorporation of LC-MS techniques to eliminate other identity assays required in the QC lab. A LCQ ion trap mass spectrometer was added downstream of the UV spectrometer. In addition to a UV trace, a total ion current chromatogram was generated. Comparison to a reference chromatogram was performed to define identity without extensive interpretation of the mass spectral data. This LC-MS method eliminated the need for other identity assays such as N-terminal sequencing and was validated as a release assay for three proteins.150... 

FIGURE 3 An example of a reference chromatogram useful for troubleshooting. 

The remainder of this paper illustrates that families of reference chromatograms can be developed to determine if a given column combination is the best one to be used with an unknown polymer or polymer mixture, the families of chromatograms being based on the standard Probe Mixtures, one to four column combinations and different plate levels. Extended banks of lower efficiency columns would be required to attain the same degree of resolution of even one of these ULTRASTVRAGEL columns. 

Equilibrate the column with mobile phase A for at least 15 min. Inject 100 (A of the reference solution. The test is not valid unless the chromatogram obtained with each solution is qualitatively similar to the appropriate Ph. Eur. Reference chromatogram of interferon-a.2 digest. The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution. 

Automatic peak detection. If the chromatography system runs unattended, the software must automatically find the desired peaks for the measurement. The predefinition of certain positions in the chromatogram, based on retention times from a reference chromatogram, is not sufficient. It should also be noted that ... 

Repeat step 62 with your reference chromatograms. What you look for are the overall patterns. Match peak ratios and the patterns of a group of peaks. The sample has been subjected to high temperatures but the standards have not, so they won t be exactly the same. However, if an accelerant was used, then you may see some sort of a pattern remaining. 

If everything is OK, inject standard. Check if the obtained chromatogram superimposes with a reference chromatogram are the peak data (area, height, asymmetry) and retention times unchanged If so, your system is ready and you can start your measurements. 

The simplest and safest method for an equipment check is a double injection with a known, stable sample, (control sample. System Suitability Test SST). If the resulting chromatograms are identical with a reference chromatogram which you obtained with the equipment in perfect condition - maybe after a scheduled master check - there is no need for further checks. You know that everything works well (method plus equipment). As criteria for the comparison, you could use retention time, peak area, peak height and asymmetry. Acceptable deviations are to be documented. 

This is riot possible with the vast majority of substances. They must be detected with the help of reference chromatograms or reagents since localisation through i /-values is most unreliable [223]. 

Substances which absorb weakly or not all in the UV region have often been localised with iodine vapour, as already described, or with the help of a reference chromatogram, and then eluted with an appropriate solvent and determined quantitatively through a colour reaction [6, 79, 217, 637]. 

In this way, generally after localisation through the fluorescence of the substances themselves, determinations have been carried out of coumarin derivatives, following chromatographic separation from lemon and grapefruit oils [729] quinoxaline derivatives of 2-ketoacids [651] benzo-(a)-pyrene [599] and vitamin Bg factors [702]. Cortisol [228] and aldosterone [229] which had been extracted from urine and purified by TLC, have been localised with the help of a reference chromatogram and then determined fluorometrically after treatment with sulphuric acid. After extraction from the thin layer, nicotine, nomicotine and anabasine could be determined phosphorimetrically (see p. 141) with a relative standard deviation of 6% [761]. 

Under suitable conditions, micro amounts could be determined coulometrically there is no example so far of this use. Very small amounts of vitamin have been determined with a microbiological turbidity test after they had been locaflsed bioautographically on a reference chromatogram (cf. p. 82) and extracted [128]. The quantitative determination of radioactive substances is treated in the following chapter. 

Fig. 119. Thin-layer chromatogram of an oily vitamin A + D concentrate, viewed in UV light (254 nm). Plate illuminated through a quartz cover in the chamber. Reference chromatogram on the left side...

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