SECTION 601 Pesticide Analytical Manual Vol I Compounds not ionized at the operative ph will not pair with the reagent, but they may still be strongly retained by a C-18 column depending on their alkyl structure. In this case. however. retention will not increase with the addition of an ion pairing reagent, and some decrease in retention may occur, probably due to reagent competition for the stationary phase [1] Ion Exchange Chromatography IEC is used to separate ionic compounds. Microparticulate insoluble organic pol mer resin or silica gel is used as the support. Negatively charged sulfonic acid oups chemically bound to the support produce strong acid catio on exchange (SCX) phases. Positively charged quaternary ammonium ions bound to the sup- port produce strong base anion exchange(SAX) phases. The most widely used resin support is cross-linked copolymer prepared from styrene and divinylbenzene Mobile phases are aqueous buffers. Separations in IEC result from competition between the analytes and mobile phase ions for sites of opposite charge on the stationary phase. Important factors control- ling retention and selectivity include the size and charge of the analyte ions, the type and concentration of other ions in the buffer system, pH, temperature, and the presence of organic solvents Ion chromatography, a subcategory of IEC, has been used primarily for separa tions of inorganic cations or anions. Because a conductivity detector is usually employed, some means is required to reduce the ionic concentration and, hence, the background conductance of the mobile phase. A second ion exchange sup- pressor column to convert mobile phase ions to a nonconducting compound may be used. Alternatively, a stationary phase with very low exchange capacity may be used with a dilute, low conductance mobile phase containing ions that interact strongly with the column Size Exclusion Chromatography SEC separates molecules based on differences in their size and shape in solution SEC cannot separate isomers. SEC is carried out on silica gel or polymer packings having open structures with solvent-filled pores of limited size range. Small analyte molecules can enter the pores and spend a longer amount of time passing through the column than large molecules, which are excluded from the pores. Ideally, there should be no interaction between the analytes and the surface of the station- ry phase Two important subdivisions of SEC are gel permeation chromatography(GPC) and gel filtration chromatography (GFC). GPC uses organic solvents for organic polymers and other analytes in organic solvents. GFC uses aqueous systems to separate and characterize biopolymers such as proteins and nucleic acids The chemist developing an HPLC method must first consider the properties of the analytes of interest and choose an HPLC separation method that best takes advantage of those properties. Many of the references in the bibliography(Section 608)offer guidance to making these choices. A general, simplified guide for select ng an HPLC mode according to the properties of the analyte(s)is illustrated in Figure 601-C; the guide is based on the principles of Snyder and Kirkland [2 FormSECTION 601 Pesticide Analytical Manual Vol. I Transmittal No. 94-1 (1/94) 601–4 Form FDA 2905a (6/92) Compounds not ionized at the operative pH will not pair with the reagent, but they may still be strongly retained by a C-18 column depending on their alkyl structure. In this case, however, retention will not increase with the addition of an ion pairing reagent, and some decrease in retention may occur, probably due to reagent competition for the stationary phase [1]. Ion Exchange Chromatography IEC is used to separate ionic compounds. Microparticulate insoluble organic poly￾mer resin or silica gel is used as the support. Negatively charged sulfonic acid groups chemically bound to the support produce strong acid cation exchange (SCX) phases. Positively charged quaternary ammonium ions bound to the sup￾port produce strong base anion exchange (SAX) phases. The most widely used resin support is cross-linked copolymer prepared from styrene and divinylbenzene. Mobile phases are aqueous buffers. Separations in IEC result from competition between the analytes and mobile phase ions for sites of opposite charge on the stationary phase. Important factors control￾ling retention and selectivity include the size and charge of the analyte ions, the type and concentration of other ions in the buffer system, pH, temperature, and the presence of organic solvents. Ion chromatography, a subcategory of IEC, has been used primarily for separa￾tions of inorganic cations or anions. Because a conductivity detector is usually employed, some means is required to reduce the ionic concentration and, hence, the background conductance of the mobile phase. A second ion exchange sup￾pressor column to convert mobile phase ions to a nonconducting compound may be used. Alternatively, a stationary phase with very low exchange capacity may be used with a dilute, low conductance mobile phase containing ions that interact strongly with the column. Size Exclusion Chromatography SEC separates molecules based on differences in their size and shape in solution. SEC cannot separate isomers. SEC is carried out on silica gel or polymer packings having open structures with solvent-filled pores of limited size range. Small analyte molecules can enter the pores and spend a longer amount of time passing through the column than large molecules, which are excluded from the pores. Ideally, there should be no interaction between the analytes and the surface of the station￾ary phase. Two important subdivisions of SEC are gel permeation chromatography (GPC) and gel filtration chromatography (GFC). GPC uses organic solvents for organic polymers and other analytes in organic solvents. GFC uses aqueous systems to separate and characterize biopolymers such as proteins and nucleic acids. The chemist developing an HPLC method must first consider the properties of the analytes of interest and choose an HPLC separation method that best takes advantage of those properties. Many of the references in the bibliography (Section 608) offer guidance to making these choices. A general, simplified guide for selecting an HPLC mode according to the properties of the analyte(s) is illustrated in Figure 601-c; the guide is based on the principles of Snyder and Kirkland [2]