Pesticide Analytical Manual Vol. I SECTION 601 LSC is useful for separation of isomers and classes of compounds differing in polarity and number of functional groups. It works best with compounds that have relatively low or intermediate polarity. Highly polar compounds may irreversibly adsorb on the column. Poor LSC separations are usually obtained for chemicals containing only nonpolar aliphatic substituents Liquid-Liquid Chromatography LLC, also called partition chromatography, involves a solid support, usually silica gel or kieselguhr, mechanically coated with a film of an organic liquid. A typical system for NP LLC is a column coated with B, B -oxy dipropionitrile and a nonpolar solvent like hexane as the mobile phase. Analytes are separated by partitioning between the two phases as in solvent extraction Components more soluble in the tationary liquid move more slowly and elute later. lLC has now been replaced by BPC for most applications Bonded Phase Chromatography BPC uses a stationary phase that is chemically bonded to silica gel by reaction of silanol groups with a substituted organosilane. Unlike LLC, the stationary phase is not altered by mobile phase development or temperature change. All solvents can be used, presaturation of the mobile phase with the stationary phase is not re- quired, and gradient elution can be used to improve resolution Specialized applications of BPC have been developed for ionized compounds which are highly water soluble and generally not well retained on RP BPC col- umns. Retention and separation can be increased by adding an appropriate pH buffer to suppress ionization (ion suppression chromatography) or by forming a lipophilic ion pair(ion pair chromatography) between the analyte and a counter ion of opposite charge. The resultant nonionic species are separated by the same column techniques used for naturally nonionic organic molecules Ion suppression is the preferred method for separation of weak acids and bases for which the ph of the mobile phase can be adjusted to eliminate analyte ioniza- tion while remaining within the pH 2-8 stability range of bonded silica phases. The analyte is chromatographed by RP HPlC, usually on a C-18 column, using metha- nol or acetonitrile plus a buffer as the mobile phase. The technique is often preferred over IEC (see below) because C-18 columns have higher efficiency equilibrate faster, and are generally easier to use reproducibly compared to ion exchange phases. Strong acids and bases are usually separated on an ion exchange column or by ion pair chromatography Ion pair chromatography is used to separate weak or strong acids or bases as well as other types of organic ionic compounds. The method involves use of a C-18 column and a mobile phase buffered to a ph value at which the analyte is com- pletely ionized (acid pH for bases, basic pH for acids)and taining an appro- priate ion pairing reagent of opposite charge. Trialkylammonium salts are com monly used for complex acidic analytes and alkylsulfonic acids for basic analytes The ion pairs separate as if they are neutral polar molecules, but the exact mecha- nism of ion pair chromatography is unclear. Retention and selectivity are affected by the chain length and concentration of the pairing reagent, the concentratio of organic solvent in the mobile phase, and its pH. Retention increases up to a point as the chain length of the pairing reagent or its concentration increases hen decreases or levels off [ 1lSECTION 601 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 601–3 Pesticide Analytical Manual Vol. I LSC is useful for separation of isomers and classes of compounds differing in polarity and number of functional groups. It works best with compounds that have relatively low or intermediate polarity. Highly polar compounds may irreversibly adsorb on the column. Poor LSC separations are usually obtained for chemicals containing only nonpolar aliphatic substituents. Liquid-Liquid Chromatography LLC, also called partition chromatography, involves a solid support, usually silica gel or kieselguhr, mechanically coated with a film of an organic liquid. A typical system for NP LLC is a column coated with ß,ß'-oxy dipropionitrile and a nonpolar solvent like hexane as the mobile phase. Analytes are separated by partitioning between the two phases as in solvent extraction. Components more soluble in the stationary liquid move more slowly and elute later. LLC has now been replaced by BPC for most applications. Bonded Phase Chromatography BPC uses a stationary phase that is chemically bonded to silica gel by reaction of silanol groups with a substituted organosilane. Unlike LLC, the stationary phase is not altered by mobile phase development or temperature change. All solvents can be used, presaturation of the mobile phase with the stationary phase is not re￾quired, and gradient elution can be used to improve resolution. Specialized applications of BPC have been developed for ionized compounds, which are highly water soluble and generally not well retained on RP BPC col￾umns. Retention and separation can be increased by adding an appropriate pH buffer to suppress ionization (ion suppression chromatography) or by forming a lipophilic ion pair (ion pair chromatography) between the analyte and a counter ion of opposite charge. The resultant nonionic species are separated by the same column techniques used for naturally nonionic organic molecules. Ion suppression is the preferred method for separation of weak acids and bases, for which the pH of the mobile phase can be adjusted to eliminate analyte ioniza￾tion while remaining within the pH 2-8 stability range of bonded silica phases. The analyte is chromatographed by RP HPLC, usually on a C-18 column, using metha￾nol or acetonitrile plus a buffer as the mobile phase. The technique is often preferred over IEC (see below) because C-18 columns have higher efficiency, equilibrate faster, and are generally easier to use reproducibly compared to ion exchange phases. Strong acids and bases are usually separated on an ion exchange column or by ion pair chromatography. Ion pair chromatography is used to separate weak or strong acids or bases as well as other types of organic ionic compounds. The method involves use of a C-18 column and a mobile phase buffered to a pH value at which the analyte is com￾pletely ionized (acid pH for bases, basic pH for acids) and containing an appro￾priate ion pairing reagent of opposite charge. Trialkylammonium salts are com￾monly used for complex acidic analytes and alkylsulfonic acids for basic analytes. The ion pairs separate as if they are neutral polar molecules, but the exact mecha￾nism of ion pair chromatography is unclear. Retention and selectivity are affected by the chain length and concentration of the pairing reagent, the concentration of organic solvent in the mobile phase, and its pH. Retention increases up to a point as the chain length of the pairing reagent or its concentration increases, then decreases or levels off [1]