SECTION 601 Pesticide Analytical Manual Vol I resolution. Choice of mobile phase is second only to the choice of operating mode in determining the suitability of the system to produce the desired separa- HPLC had limited use for routine trace multiresidue analysis in the absence of sensitive element-selective detectors. Early development work relied primarily on refractive index(RI)or fixed wavelength UV absorbance detectors. Neither detec- tor demonstrated sufficient sensitivity or selectivity for use in trace residue analysis In the mid-1970s, the fluorescence detector was shown to provide the needed be chemically labeled with a fluorophore. This resulted in the first practical appa sensitivity and specificity for pesticides that are naturally fluorescent or car cation of HPlC to multiresidue pesticide determination (see method for N-methylcarbamates, Section 401) More recently, scientists have investigated photoconductivity and electrochemical detectors and certain applications of the newer multiwavelength UV detectors This research indicates that these detectors can also fulfill the sensitivity and selectivity requirements for determination of certain pesticides at residue levels 601 B: MODES OF OPERATION Figure 601-b HPLC Modes of Operation Separations by HPlC are achieved using the five basic operational modes Figure 601-b). The mode chosen for C a particular application will depend on the properties of the analyte(s) to be separated and determined For residue LLC determination, as for hplc ar BPC SAX SCX SEC in general, BPC is the most widely used suppression pair There are two variations within the five operational modes of HPLC operation; these distinctions are based on the relative polarities of stationary and mobile ases 1)normal phase(NP) chromatography: stationary phase is more polar than the mobile phase; the least polar analytes elute first; analyte retention increased by decreasing mobile phase polarity 2)reverse phase(RP)chromatography: stationary phase is less polar than the mobile phase; the most polar analytes elute first; analyte retention is Liquid-Solid Chromatography LSC, also called adsorption chromatography, uses an adsorbent, usually uncoated silica gel. The basis for separation is the selective adsorption of polar compound presumably by hydrogen bonding, to active silanol (SioH)groups by orientation and on the surface of the silica gel. Analytes that are more polar will be attracted more strongly to the active silica gel sites. The solvent strength of the mobile phase determines the rate at which adsorbed analytes are desorbed and eluted 61-2 FormSECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–2 Form FDA 2905a (6/92) resolution. Choice of mobile phase is second only to the choice of operating mode in determining the suitability of the system to produce the desired separa￾tions. HPLC had limited use for routine trace multiresidue analysis in the absence of sensitive element-selective detectors. Early development work relied primarily on refractive index (RI) or fixed wavelength UV absorbance detectors. Neither detec￾tor demonstrated sufficient sensitivity or selectivity for use in trace residue analysis. In the mid-1970s, the fluorescence detector was shown to provide the needed sensitivity and specificity for pesticides that are naturally fluorescent or can be chemically labeled with a fluorophore. This resulted in the first practical appli￾cation of HPLC to multiresidue pesticide determination (see method for N-methylcarbamates, Section 401). More recently, scientists have investigated photoconductivity and electrochemical detectors and certain applications of the newer multiwavelength UV detectors. This research indicates that these detectors can also fulfill the sensitivity and selectivity requirements for determination of certain pesticides at residue levels. 601 B: MODES OF OPERATION Separations by HPLC are achieved using the five basic operational modes (Figure 601-b). The mode chosen for a particular application will depend on the properties of the analyte(s) to be separated and determined. For residue determination, as for HPLC analyses in general, BPC is the most widely used. There are two variations within the five operational modes of HPLC operation; these distinctions are based on the relative polarities of stationary and mobile phases: 1) normal phase (NP) chromatography: stationary phase is more polar than the mobile phase; the least polar analytes elute first; analyte retention is increased by decreasing mobile phase polarity. 2) reverse phase (RP) chromatography: stationary phase is less polar than the mobile phase; the most polar analytes elute first; analyte retention is increased by increasing mobile phase polarity. Liquid-Solid Chromatography LSC, also called adsorption chromatography, uses an adsorbent, usually uncoated silica gel. The basis for separation is the selective adsorption of polar compounds, presumably by hydrogen bonding, to active silanol (SiOH) groups by orientation and on the surface of the silica gel. Analytes that are more polar will be attracted more strongly to the active silica gel sites. The solvent strength of the mobile phase determines the rate at which adsorbed analytes are desorbed and eluted. Figure 601-b HPLC Modes of Operation HPLC LSC LLC BPC IEC SEC Ion suppression Ion pair SAX SCX GPC GFC