ORGANONITROGEN PESTICIDES: METHOD 5601, Issue 1, dated 15 January 1998 - Page 21 of 21
1. By Relative Retention Times (Retention Index).The Retention Index (RI) may vary considerably from column to column and from one set of conditions to another. But it will be reasonably consistent once a set of conditions has been chosen and will be much more reliable for day-to-day comparisons than will absolute retentiontimes. Actual RIs need to be established for each set of conditions. Compounds that are ionic under elution conditions or that interact strongly with polar sites on the column will have the most variable retention times and retention indices. 2. By Second Column. The cyanopropyl stationary phase strongly induces some exchanges in elution orders and alters relative spacings between adjacent analytes in the chromatogram that may be useful in confirmations. 3. By Alternate Solvent. As mentioned earlier (Section B1), methanol as the mobile phase B solvent on a C18 column can be used just as effectively to establish confirmations because methanol interacts differently with the stationary phase than acetonitrile, and so different molecular forces come into play. Significant alterationsin retention order are thus obtained; for some analytes this is more dramatic than with a cyanopropyl column. 4. By Ratio of Two UV Absorption Bands. As long as the UV absorption channels are not saturated, there should be a consistent ratio between the background-corrected absorption bands that is characteristicof each analyte and should reflect the ratio of relative heights of the absorption spectra at their respective UV bands in the UV spectra. This ratio is dependent, however, on the bandwidth of the adsorption bands employed,and consistent bandwidths must be used. The consistency of the ratio of absorption across the HPLC peak is also indicative of the purity of the peak. A constantly changing ratio indicates that the peak may have multiple components. 5. By Matching to Reference UV Spectra. Unknown spectra should not be oversaturated in any portion. They need to be background-corrected properly. If the baseline is rising, a background selected from the backside of the peak may induce losses of absorption in the region below 210 nm. Conversely, one selected from in front of the unknown HPLC peak may add to this region of the spectra. It is better to get an averaged background first. Spectra maxima should match within a few nanometers. Relative absorbance at each maxima may vary, even after background subtraction, depending upon the concentration of the analytes and the characteristics of different scanning UV detectors.
NIOSH Manual of Analytical Methods, Fourth Edition
