《美国FDA农残分析手册》（第一卷英文版）Chapter 5 GLC

Pesticide Analytical Manual Vol. I SECTION 501 its volume can be measured by reading both ends of the liquid. Injec- tion is then made. The initial solvent flushes the extract or standard into the chromatograph. This technique is referred to as the"solvent flush or"sandwich"technique 2)The syringe is filled by drawing extract (or standard solution)com- pletely into the barrel (i.e, none is left in the needle). Total volume is measur red by reading both ends of the liquid. Injection is made, with the syringe removed quickly from the inlet. The syringe plunger is with- drawn until whatever volume of liquid remains is completely in the barrel of the syringe, where it is measured as before. The difference in liquid volume before and after injection is the amount actually injected It is important when using this technique to remove the syringe from the heated injection port as quickly as possible after injection to avoid any evaporation of liquid remaining in the syringe 8)The syringe is filled to the desired volume, the volume noted, and the injection made. The volume measured is considered to be the volume injected. This technique introduces error, because it ignores the volume n the needle and the volume that remains after injection. The effective error can be minimized by use of the same solvent for both sample extract and standard solution and by injection of the same volume of Whichever injection technique is chosen, it must be performed reproducibly. Each analyst should choose the injection technique he/ she finds most reproducible and use it routinely Poor precision among chromatograms from repetitive injections may be caused by faulty syringes or poor analyst technique, as well as by inappro- priate solvents or inadequate sample cleanup. Volume of liquid in the syringe should be measured by holding the syringe in the same manner each time while looking toward a light background. The same injection technique must be used for both the sample extract and the reference standard to which it will be com- pare Choice of injection technique is not solely based on personal preference; type of described above can be applied when using packed columns. However, too much olvent can overwhelm the small diameter capillary column, so injection volume must be limited. Several inlet systems and injection options are used with capillary columns to accommodate both column restrictions and volume requirements of residue determination (Section 502 C) Consistently good capillary column results have been achieved with manual injection and the solvent flush technique. The syringe needle should remain in the inlet I sec for each uL injected to allow the urge from vaporization of solvents to dissipate The syringe manufacturer's should be followed. sy checked occasionally by injecting a volume of pure solvent; if the syringe is clean no peaks other than the solvent peak will appear Autoinjectors The best injection performance is achieved using an autoinjector (also called autosampler). Various commercially available autoinjectors can be interfaced with 501-7

Pesticide Analytical Manual Vol. I SECTION 501 501–7 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) its volume can be measured by reading both ends of the liquid. Injec￾tion is then made. The initial solvent flushes the extract or standard into the chromatograph. This technique is referred to as the “solvent flush” or “sandwich” technique. 2) The syringe is filled by drawing extract (or standard solution) com￾pletely into the barrel (i.e., none is left in the needle). Total volume is measured by reading both ends of the liquid. Injection is made, with the syringe removed quickly from the inlet. The syringe plunger is with￾drawn until whatever volume of liquid remains is completely in the barrel of the syringe, where it is measured as before. The difference in liquid volume before and after injection is the amount actually injected. It is important when using this technique to remove the syringe from the heated injection port as quickly as possible after injection to avoid any evaporation of liquid remaining in the syringe. 3) The syringe is filled to the desired volume, the volume noted, and the injection made. The volume measured is considered to be the volume injected. This technique introduces error, because it ignores the volume in the needle and the volume that remains after injection. The effective error can be minimized by use of the same solvent for both sample extract and standard solution and by injection of the same volume of each. Whichever injection technique is chosen, it must be performed reproducibly. Each analyst should choose the injection technique he/she finds most reproducible and use it routinely. Poor precision among chromatograms from repetitive injections may be caused by faulty syringes or poor analyst technique, as well as by inappro￾priate solvents or inadequate sample cleanup. Volume of liquid in the syringe should be measured by holding the syringe in the same manner each time while looking toward a light background. The same injection technique must be used for both the sample extract and the reference standard to which it will be com￾pared. Choice of injection technique is not solely based on personal preference; type of column being used (packed vs capillary) must also be considered. Any technique described above can be applied when using packed columns. However, too much solvent can overwhelm the small diameter capillary column, so injection volume must be limited. Several inlet systems and injection options are used with capillary columns to accommodate both column restrictions and volume requirements of residue determination (Section 502 C). Consistently good capillary column results have been achieved with manual injection and the solvent flush technique. The syringe needle should remain in the inlet 1 sec for each µL injected to allow the pressure surge from vaporization of solvents to dissipate. The syringe manufacturer’s recommendations for use and care of the syringe should be followed. Syringes must be kept free of traces of analyte. This should be checked occasionally by injecting a volume of pure solvent; if the syringe is clean, no peaks other than the solvent peak will appear. Autoinjectors The best injection performance is achieved using an autoinjector (also called autosampler). Various commercially available autoinjectors can be interfaced with

SECTION 501 Pesticide Analytical Manual Vol I GLC systems. For normal use of autoinjectors, extracts and standard solutions are placed in disposable glass vials with vapor-tight septum caps. The autoineccandard the syringe completely and removes air bubbles by pumping extract (or st solution) into the barrel. It then draws a precisely measured volume of solution into the barrel and injects it into the chromatograph. Between injections, the autoinjector flushes the needle with appropriate solvent to clean it. Beyond the improved reproducibility achieved with autoinjectors, their use permits unattended operation of the chromatograph and frees the chromatographer to perform other tasks 501E REFERENCE STANDARDS Section 205 provides on on pesticide standards. The importance of rel able standard solution ate pesticide analyses cannot be overemphasized Solvents used for GLO olutions are subject to the same requirements and limitations listed extracts The quality assurance plan for analyses involving GLC determination should in- clude routine injection of a mixed standard solution. The mixture should include compounds normally used as markers for retention time and response and should also include compounds prone to adsorption or degradation. Vulnerable com- pounds serve as indicators of problems developing in the system; e.g., the presence of p, P-DDT in such a solution serves to alert the analyst when degradation to p,p TDE occurs. GLC systems used for determination of organophosphorus or other polar residues should be checked with a solution that includes, at a minimum methamidophos, acephate, and monocrotophos. Response to acephate may disap- pear in systems that contain too much glass wool, and response to methamidophos may not be seen if it elutes with the solvent front or if column packing is of poor ality; both these situations can be avoided by monitoring the system with rou- tine injection of an appropriate mixed standard Frequency of injection of mixed standard, at least twice during an 8-hr period, should be specified in the quality assurance plan For best quantitative results, reference standards should be dissolved in the same solvent that is used for the final sample extract. In addition, reference standards should be injected within minutes of the sample containing the residue(s) to be uantitated, and responses to residue and standard should match within +25% for accurate quantitation. References [1 Standard Practice for Gas Chromatography Terms and Relationships, ASTM E 355- 77, reapproved 1983, ASTM, Philadelphia, PA 2]Jennings, w.(1987) Analytical Gas Chromatography, Academic Press, Orlando [3] Burke, J.A., and Giuffrida, LA.(1964)J. Assoc Of. Agric. Chem. 47, 326-342 501-8 Transmittal No. 94-1(1/94]

501–8 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) SECTION 501 Pesticide Analytical Manual Vol. I GLC systems. For normal use of autoinjectors, extracts and standard solutions are placed in disposable glass vials with vapor-tight septum caps. The autoinjector wets the syringe completely and removes air bubbles by pumping extract (or standard solution) into the barrel. It then draws a precisely measured volume of solution into the barrel and injects it into the chromatograph. Between injections, the autoinjector flushes the needle with appropriate solvent to clean it. Beyond the improved reproducibility achieved with autoinjectors, their use permits unattended operation of the chromatograph and frees the chromatographer to perform other tasks. 501 E: REFERENCE STANDARDS Section 205 provides information on pesticide standards. The importance of reli￾able standard solutions to accurate pesticide analyses cannot be overemphasized. Solvents used for GLC standard solutions are subject to the same requirements and limitations listed above for extracts. The quality assurance plan for analyses involving GLC determination should in￾clude routine injection of a mixed standard solution. The mixture should include compounds normally used as markers for retention time and response and should also include compounds prone to adsorption or degradation. Vulnerable com￾pounds serve as indicators of problems developing in the system; e.g., the presence of p,p′-DDT in such a solution serves to alert the analyst when degradation to p,p′- TDE occurs. GLC systems used for determination of organophosphorus or other polar residues should be checked with a solution that includes, at a minimum, methamidophos, acephate, and monocrotophos. Response to acephate may disap￾pear in systems that contain too much glass wool, and response to methamidophos may not be seen if it elutes with the solvent front or if column packing is of poor quality; both these situations can be avoided by monitoring the system with rou￾tine injection of an appropriate mixed standard. Frequency of injection of mixed standard, at least twice during an 8-hr period, should be specified in the quality assurance plan. For best quantitative results, reference standards should be dissolved in the same solvent that is used for the final sample extract. In addition, reference standards should be injected within minutes of the sample containing the residue(s) to be quantitated, and responses to residue and standard should match within ±25% for accurate quantitation. References [1] Standard Practice for Gas Chromatography Terms and Relationships, ASTM E 355- 77, reapproved 1983, ASTM, Philadelphia, PA [2] Jennings, W. (1987) Analytical Gas Chromatography, Academic Press, Orlando, FL [3] Burke, J.A., and Giuffrida, L.A. (1964) J. Assoc. Off. Agric. Chem. 47, 326-342

Pesticide Analytical Manual Vol. I SECTION 502 502: COLUMNS 502 A: INTRODUCTION Separations among analytes in GLC are achieved within the column. Although choice of detector dictates which class of analytes can be determined, individual detection and measurement of multiple analytes would not be possible without the separations provided by the column Columns are available in several different physical configurations, each of which offers advantages and disadvantages to pesticide residue determination. The two basic types of GlC columns currently used in pesticide residue determination are 1)packed columns, in which liquid phase is immobilized as a film on particles of fine mesh solid support and packed into 2-4 mm id columns, and(2)open tubular capillary columns, in which liquid phase is immobilized as a film on the interior walls of a capillary tube. Capillary columns are further distinguished by internal diameter: wide bore (0.53 mm id), traditional (0.25-0.32 mm id), and narrow bore(s0.25 mm id). Each type of column requires unique hardware and operating parameters In all GLC columns, identity of the liquid(stationary) phase is the primary factor dictating what separations are achievable. Carrier gas(mobile phase) is also inte- gral to GlC operation and must be included in any discussion of columns. How- ever, only inert gases are used as carrier gases, so few options exist. Operating parameters that affect column efficiency, including column temperature and car rier gas identity and flow rate, provide additional variables that can be adjusted to achieve separations required for the analysis Column Specifications Descriptions of GLC columns and opera ting conditions must specify the following type of column (packed or capillary); its length, in meters(or feet), and internal diameter (id), in mm; identity and amount of liquid phase; identity of solid sup- port, including pretreatments and mesh size (packed columns only); operating temperature; and carrier gas identity and flow rate Liquid phases used in GlC are viscous materials able to be thinly di d solid support or on an internal column wall. Many different liquid phases are available, but relatively few are in routine use for pesticide residue determination because pesticide residues usually either chromatograph on one of these phases or are not amenable to GLC. The chemical structure of the most common phases consists of a polysiloxane backbone with various substituent groups; Figure 502-a illustrates several of these Liquid phase polarity, important to its separation capabilities, varies with polarity and concentration of substituent group(s) on the polysiloxane. Thus, in terms of yk<cyanopropylphenyk<50% phenyl vl. Th 100% methyl-substituted phase, least polar of those in Figure 502-a, is best suited to separation of nonpolar analytes; it has been used for many years as a general purpose phase for a wide variety of pesticide residues. The phase with 50% cyanopropylphenyl-substitution is the most polar of those shown and is a better choice for more polar analytes mittal No.94101/94 m FDA 2905a(6/92 502-1

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 502–1 Pesticide Analytical Manual Vol. I SECTION 502 502: COLUMNS 502 A: INTRODUCTION Separations among analytes in GLC are achieved within the column. Although choice of detector dictates which class of analytes can be determined, individual detection and measurement of multiple analytes would not be possible without the separations provided by the column. Columns are available in several different physical configurations, each of which offers advantages and disadvantages to pesticide residue determination. The two basic types of GLC columns currently used in pesticide residue determination are (1) packed columns, in which liquid phase is immobilized as a film on particles of fine mesh solid support and packed into 2-4 mm id columns, and (2) open tubular capillary columns, in which liquid phase is immobilized as a film on the interior walls of a capillary tube. Capillary columns are further distinguished by internal diameter: wide bore (0.53 mm id), traditional (0.25-0.32 mm id), and narrow bore (≤0.25 mm id). Each type of column requires unique hardware and operating parameters. In all GLC columns, identity of the liquid (stationary) phase is the primary factor dictating what separations are achievable. Carrier gas (mobile phase) is also inte￾gral to GLC operation and must be included in any discussion of columns. How￾ever, only inert gases are used as carrier gases, so few options exist. Operating parameters that affect column efficiency, including column temperature and car￾rier gas identity and flow rate, provide additional variables that can be adjusted to achieve separations required for the analysis. Column Specifications Descriptions of GLC columns and operating conditions must specify the following: type of column (packed or capillary); its length, in meters (or feet), and internal diameter (id), in mm; identity and amount of liquid phase; identity of solid sup￾port, including pretreatments and mesh size (packed columns only); operating temperature; and carrier gas identity and flow rate. Liquid phases used in GLC are viscous materials able to be thinly dispersed on solid support or on an internal column wall. Many different liquid phases are available, but relatively few are in routine use for pesticide residue determination, because pesticide residues usually either chromatograph on one of these phases or are not amenable to GLC. The chemical structure of the most common phases consists of a polysiloxane backbone with various substituent groups; Figure 502-a illustrates several of these. Liquid phase polarity, important to its separation capabilities, varies with polarity and concentration of substituent group(s) on the polysiloxane. Thus, in terms of polarity, methyl<5% phenyl<cyanopropylphenyl<50% phenyl<cyanopropyl. The 100% methyl-substituted phase, least polar of those in Figure 502-a, is best suited to separation of nonpolar analytes; it has been used for many years as a general purpose phase for a wide variety of pesticide residues. The phase with 50% cyanopropylphenyl-substitution is the most polar of those shown and is a better choice for more polar analytes

SECTION 502 Pesticide Analytical Manual Vol I Figure 502-a Polysiloxane Stationary Phases CH2 CH3 CH3 C6H5 CH3 CH2 0- si 0-Si CH3 H3」x C6H5 C6H5 50% methyl 50% cyanopropylphenyl, 50% meth 17 B225 Equivalent products suitable for different column configurations are commercially vailable for most common liquid phases; Table 502-a lists some of these products Although the table refers to liquid phases themselves, most pesticide residue lab- oratories no longer purchase liquid phases as materials for preparing columns in-house. Instead, laboratories that use packed columns usually purchase them prepacked or at least purchase packing material precoated with liquid phase Residue laboratories always purchase commercially prepared capillary columns The liquid phase for a particular analysis is selected to take advantage of differ- ences in chemical and physical properties of the analytes involved. No one liquid phase is universally applicable to the wide range of chemical and physical proper- ties found in pesticide residues, so a variety of liquid phases of different polarities should be available in a residue laborator For packed columns, the amount of liquid phase, often called"liquid load, "is described as a percentage, i.e., weight liquid phase x 100/(weight liquid phase weight solid support). For open tubular columns, the amount of liquid phase is described as film thickness (um) of the layer of liquid phase bonded to the inter- nal wall of the column GLC columns lways heated to a temperature at which analytes remain in the vapor phase th isothermal and temperature-programmed operation are possible. Use of illary columns with increasingly common, but this operation chapter because Fda has not yet validated its use on an interlaboratory basis. Maximum operating temperatures vary with specific stationary phases; information on each is provided by the manufacturer. Increasingly polar stationary phases(e. g cyanopropylphenyl) have significantly lower maximum operating temperatures than nonpolar phases(e.g, 100% methyl). In use, maximum operating temperature is sually 20 C higher for temperature programming than for isothermal work. Column Parameters The following column characteristics or parameters are commonly used to de- scribe chromatographic behavior or to measure column performance; terminal- ogy of these parameters is illustrated in Figure 502-b. Evaluation and comparison 50a-2 Form FDA 2905a (6/92]

SECTION 502 Pesticide Analytical Manual Vol. I 502–2 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Figure 502-a Polysiloxane Stationary Phases CH3 O Si CH3 x 100% methyl (DB-1) CH3 O Si CH3 x x = y C6H5 O Si C6H5 y 50% phenyl, 50% methyl (DB-17) CH3 O Si CH3 x x = y CH2 O Si C6H5 y 50% cyanopropylphenyl, 50% methyl (DB-225) CH2 CH2 CN Equivalent products suitable for different column configurations are commercially available for most common liquid phases; Table 502-a lists some of these products. Although the table refers to liquid phases themselves, most pesticide residue lab￾oratories no longer purchase liquid phases as materials for preparing columns in-house. Instead, laboratories that use packed columns usually purchase them prepacked or at least purchase packing material precoated with liquid phase. Residue laboratories always purchase commercially prepared capillary columns. The liquid phase for a particular analysis is selected to take advantage of differ￾ences in chemical and physical properties of the analytes involved. No one liquid phase is universally applicable to the wide range of chemical and physical proper￾ties found in pesticide residues, so a variety of liquid phases of different polarities should be available in a residue laboratory. For packed columns, the amount of liquid phase, often called “liquid load,” is described as a percentage, i.e., weight liquid phase × 100/(weight liquid phase + weight solid support). For open tubular columns, the amount of liquid phase is described as film thickness (µm) of the layer of liquid phase bonded to the inter￾nal wall of the column. GLC columns are always heated to a temperature at which analytes remain in the vapor phase. Both isothermal and temperature-programmed operation are possible. Use of capillary columns with temperature programming is becoming increasingly common, but this operation will not be described further in this chapter because FDA has not yet validated its use on an interlaboratory basis. Maximum operating temperatures vary with specific stationary phases; information on each is provided by the manufacturer. Increasingly polar stationary phases (e.g., cyanopropylphenyl) have significantly lower maximum operating temperatures than nonpolar phases (e.g., 100% methyl). In use, maximum operating temperature is usually 20° C higher for temperature programming than for isothermal work. Column Parameters The following column characteristics or parameters are commonly used to de￾scribe chromatographic behavior or to measure column performance; terminol￾ogy of these parameters is illustrated in Figure 502-b. Evaluation and comparison

Pesticide Analytical Manual Vol. I SECTION 502 Table 502-a: Common GLC Liquid Phases Used in Pesticide Residue Equivalent Commercially Available Products' Basic Structure, Substitutions Capillary Open Tubular Packe Polysiloxane, 100% methyl DB-l(ht), HP-1, HP-101 OV-101 OV-1 007-1(MS),SP-2100 SP2100,DC200, SPB-1 BP-1 CP-Sil 5CB CP-Sil 5. SE-30 L-150, Rtx-1,SP2100,CB-1,OV-1, PE-1,SE-30,AT-1 Polysiloxane, 50% phenyl DB-17(ht),HP-17,PE-17, OV-17,OV11 50% methyl 007-17(MPS50),AI-50, SP-2250,OV-22 SP2250,Rtx-50,RSL-300 DC710 Polysiloxane, 50% cyanopropyl- DB-225, HP-225, OV-225, OV-225 phenyl, 50% methyl SP-2330 CP-Sil 43CB. RSI-500 Rtx-225,BP-225,CB225, PE225,007-225,AT-225 Polysiloxane, 14% cyano DB-1701. SPB-7 CP-Sil 19CB. Ov-1701 phenyl, 86% meth Rtx-1701,BP-10,CB-1701 OV-1701,PE-1701,007-1701 Polysiloxane, 5% phenyl DB-5(ht), HP-5, Ultra-2, OV-3. OV-79 CP-Sil 8 95 methyl OV-5. SPB-5. Rix-5 CP-Sil SCB. RSL-2000 BP5,CB-5,PE-5,SE-52, 007-2(MPS5),SE54 Polysiloxane, 50% trifluoro- DB-210, RSL-400, SP-2401 Ov-210,SP-2401, propyl, 50% methyl Ov-202,OV215 Polyethylene gly DB-WAX HP-20M. Carbowax Supelcowax 10, CP-WAX 52CB cowan SUPEROX IL. Stabilwax. BP-20 CB-WAX. PE-CW ethylene glycol succinate No equivalent DEGS (no longer cial codes for each material are related to their manufactur 007: Quadrex, New Haven, CT AT RSL SUPEROX: Alltech Associates Inc. Deerfield. IL BP. SGE. Ine Carbowax: Union Carbide Cor CB, CP-Sil, CP-WAX: Chrompak International BV, Middleburg, The Netherlands DC: Dow Corning Corp, Midland, MI DB: J&w Scientific, Folsom, CA DEGS: Analabs Inc. New Haven. CT HP and Ultra: Hewlett-Packard, Co., Wilmington, DE OV: Ohio Valley Specialty Chemical Co., Marietta, OH PE: Perkin Elmer Corp, Norwalk, CT Rtx, Stabilwax: Restek Corp, Bellefonte, P. SE: General electric SP, SPB, and Supelcowax: Supelco, Inc, Bellefonte, PA m FDA 2905a(6/92 502-3

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 502–3 Pesticide Analytical Manual Vol. I SECTION 502 Table 502-a: Common GLC Liquid Phases Used in Pesticide Residue Determination Equivalent Commercially Available Products1 Basic Structure, Substitutions Capillary Open Tubular Packed Polysiloxane, 100% methyl DB-1(ht), HP-1, HP-101, OV-101, OV-1, 007-1(MS), SP-2100 SP-2100, DC 200, SPB-1, BP-1, CP-Sil 5CB, CP-Sil 5, SE-30 Ultra 1, RSL-150, RSL-160, Rtx-1, SP-2100, CB-1, OV-1, PE-1, SE-30, AT-1 Polysiloxane, 50% phenyl, DB-17 (ht), HP-17, PE-17, OV-17, OV-11, 50% methyl 007-17(MPS-50), AT-50, SP-2250, OV-22, SP-2250, Rtx-50, RSL-300 DC-710 Polysiloxane, 50% cyanopropyl- DB-225, HP-225, OV-225, OV-225 phenyl, 50% methyl SP-2330, CP-Sil 43CB, RSL-500, Rtx-225, BP-225, CB-225, PE-225, 007-225, AT-225 Polysiloxane, 14% cyanopropyl- DB-1701, SPB-7, CP-Sil 19CB, OV-1701 phenyl, 86% methyl Rtx-1701, BP-10, CB-1701, OV-1701, PE-1701, 007-1701 Polysiloxane, 5% phenyl, DB-5 (ht), HP-5, Ultra-2, OV-3, OV-73, CP-Sil 8 95% methyl OV-5, SPB-5, Rtx-5, CP-Sil 8CB, RSL-2000, BP-5, CB-5, PE-5, SE-52, 007-2(MPS-5), SE-54 Polysiloxane, 50% trifluoro- DB-210, RSL-400, SP-2401 OV-210, SP-2401, propyl, 50% methyl OV-202, OV-215 Polyethylene glycol DB-WAX, HP-20M, Carbowax, Carbowax 20M, Supelcowax 10, CP-WAX 52CB, Supelcowax 10 SUPEROX II, Stabilwax, BP-20, CB-WAX, PE-CW Diethylene glycol succinate No equivalent DEGS (no longer produced) 1 Commercial codes for each material are related to their manufacturer: 007: Quadrex, New Haven, CT AT, RSL, SUPEROX: Alltech Associates, Inc., Deerfield, IL BP: SGE, Inc., Austin, TX Carbowax: Union Carbide Corp. CB, CP-Sil, CP-WAX: Chrompak International BV, Middleburg, The Netherlands DC: Dow Corning Corp., Midland, MI DB: J&W Scientific, Folsom, CA DEGS: Analabs, Inc., New Haven, CT HP and Ultra: Hewlett-Packard, Co., Wilmington, DE OV: Ohio Valley Specialty Chemical Co., Marietta, OH PE: Perkin Elmer Corp., Norwalk, CT Rtx, Stabilwax: Restek Corp., Bellefonte, PA SE: General Electric SP, SPB, and Supelcowax: Supelco, Inc., Bellefonte, PA