Impurity peak elution order

Gradient elution places special demands on solvent purity. Only carefully purified solvents should be used, and, it is recommended that prior to use they be passed over activated alumina or silica (7). The column acts as a collector of impurities which may elute as sharp peaks at a certain eluent composition and can be mistaken for sample components. It is therefore advisable to run the gradient first without injecting the sample in order to recognize the impurity peaks. 

A similar CRF may be used for the separation of more than two peaks if it is known that there will always be one critical compound which elutes closest to the main compound of interest. This may be the case for example in a method for determining the purity of a compound when there is one impurity which is very closely structurally related to the compound of interest and therefore much more difficult to resolve from the peak for the main component of the sample than anything else. In other cases, however, it may be equally important to separate all the components from one another. In such instances there must be a high weighting on solutions where the number of peaks observed is the same as the number of components in the sample. Another feature of such examples is that when experimental variables are changed, the elution order of the compounds may change. In some optimisation procedures this is ignored but more frequently the peaks are tracked . This may be done, for... 

Chromatographic Separation. With respect to chromatographic techniques, specificity can be demonstrated by a sufficient separation of the substances present. For the assay, appropriate separation means an adequate resolution between the peak of interest and other peaks (e.g., impurities, placebo or matrix components), which need not to be separated from each other. In contrast, universal procedures for the determination of impurities require a sufficient separation of all relevant impurity peaks. The required resolution is strongly dependent on the difference in the size of the corresponding peaks as well as on their elution order. Therefore, if separation factors are determined, the typical concentration levels or the specification limits (as worst case) of the impurities should be maintained. Resolution factors can be calculated according to EP [Eq. (3)] and USP [Eq. (4)] at half height and at the baseline, respectively. However, this is only sensible for baseline-separated peaks. The USP approach is less sensitive toward tailing, but more complex to determine. 

Using split injection mode, inject 1 to 5 xL of the standards into the chromatograph. Determine the peak areas by electronic integration. Plot peak area against concentration for each analyte corrected for the blank to construct a standard curve. Determine the concentration of additives and byproducts by comparison to the standard curve. The sum of the concentrations of the impurities and stabilizers is less than 1.0%. The order of elution and approximate retention times, in minutes, are as follows methyl chloride 2.8 vinyl chloride 3.0 ethyl chloride 3.5 propylene oxide 4.1 2-methyl-2-butene 4.5 vinylidene chloride 4.6 dichloromethane 5.3 trans-1,2-dichloroclhylene 5.9 chloroform 8.7 cyclohexane 10.5 and carbon tetrachloride 12.0. 

In many cases, however, the sample composition makes it necessary to use gradient elution in order to elute all compounds of interest within a reasonable time and with sufficient resolution. In gradient elution it is important to use very pure solvents since, otherwise, impurities from the solvents may accumulate on top of the column as the gradient runs with low solvent strength, to be eluted as interfering peaks as the solvent strength is increased. 

We assume this is not a peak from the last run. Be happy that the excluded "impurities elute early and do not disturb the chromatogram. However, if you would like to analyze these solutes, you have to use a GPC column for polymers or proteins or an ion exclusion/ion exchange column for ions or salt or change the eluent in order to alter the solvation of these strong ions (see Chapter 5 Retention of lonizable Components in Reversed-Phase HPLC). 

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