ORGANONITROGEN PESTICIDES: METHOD 5601, Issue 1, dated 15 January 1998 - Page 17 of 21 3. Analytical Columns a. General: The main analytical column specified in this method is a C18 reverse-phase column. Other columns known to perform well also may be used, following manufacturer recommendations. There are many good columns available [28,33]. b. Base Deactivation: The basic compounds (benomyl, carbendazim, and formetanate) present special challenges that are easily overcome with highly inert or base-deactivated columns [26]. The addition of TEA-PO4 buffer to mobile phase A also greatly improves performance for these compounds [16, 26, 27]. c. Dimensions: A longer column (300 mm) was used in this method in order to improve resolution for a large number of analytes and interferences, expected and unexpected. Long columns have higher operating pressures; therefore, necessary steps should be taken to provide for them, such as thicker-walled (narrower-bore) transfer lines (if polymer tubing is used), etc. Shorter columns may work well if alimited number of analytes or interferences are expected. The diameter (3.9-mm) is not necessarily critical and should be governed by user preference and equipment, adjusting flow rates and otherparameters as necessary. Diameters from 2.0 to 4.6 mm should be expected to perform similarly, as long as the columns are stable and rugged. d. Packing Density: Packing (or ligand) density is a better parameter than carbon loading for comparing columns [21,22]. A column with a ligand density near that of the one used in this method should be expected to perform similarly. 4. Mobile Phase Composition a. Modifier: Because of the high percentage of water in the initial mobile phase, a condition referred to as hydrophobic collapse of the C18 phase occurs [23,24], which results in poor reequilibration and irreproducible retention times and peak shapes for early eluting analytes such as oxamyl and methomyl. The addition of a constant amount of an alcohol to both mobile phases A and B has been reported and found to improve column performance under these conditions The method specifies 2% 1-propanol to be added to both mobile phases A and B. A concentration of 1-propanol between 3% and 4% has been reported as optimum [24,25]; 2% is a compromise made in order to generate retention (capacity) factors for the earliest eluites, oxamyl and methomyl, of greater than 5,as suggested in the literature [26]. Also, 1-propanol has been reported (and found) to reduce the time required for reequilibration at the end of the run [24]. Other alcohols may be used at higher concentrations (except for reducing reequilibration times) [1]. These are isopropanol (at 3% to 5%) and methanol (at 5% to 10%). If early-eluting analytes are to be determined, or if a column is found that gives sufficiently long retention times for the earliest expected eluite with an initial mobile phase composition as high as 5% to 10% acetonitrile in water, the alcohol could be eliminated (from both phases). Table 4 would not apply if such changes are made. b. Mobile Phase B: Pure methanol as mobile phase B results in a steep rise in the baseline UV response, making automatic integrations difficult and UV scanning for confirmation spectra nearly impossible. For this reason acetonitrile has been chosen [28,29]. Methanol is acceptable if these conditions are tolerable and is recommended as an alternate solvent system on a C18 column for confirmation (Section F3); however, precision and LOD values reported in Table 2 would not be applicable. c. Solvent Programming: One of the most serious concerns that may be encountered in trying to make adaptations of this method is an attempt to shorten retention times by using higher concentrations of organic modifier in mobile phase A, higher percentages of mobile phase B in the initial solvent program condition, or faster solvent programming. In any case, these changes will seriously affect the peak shape, sensitivity, and retention time reproducibility of early eluting analytes. There is also a greater possibility of false positives from potential interferences because of poor resolution in the early region of the chromatogram. Retention times for the earliest eluting analyte should be kept at 3 to 6 times the retention time of the solvent front (i.e., of an unretained analyte), which is equivalent to a capacity factor of 2 to 5 [5]. The retention or capacity factor for any analyte can be calculated as follows: Retention or capacity factor = (tr – to) / to where tr = the retention time of the compound and to = the retention time of an unretained analyte (the mobile phase NIOSH Manual of Analytical Methods, Fourth Edition
