Pesticide Analytical Manual Vol. I Regulatory Operations eneral Analytical Operations and Information Chapter 3 Chapter 4 Multiclass Selective MRMS MRMS Chapter 5 GLC Chapter 6 HPLC Table of Contents 501: General Information 501A: Principles 501-11/94 01 B: Equipment for glC Gas Chromatographic Components 501-1 Other A. 501-21/94 R 501-31/94 501 C: Residue Methodology for GLC Determination 501-9 1/ 1/94 Reagent Blanks 501-5 Choice of soly 501 D: Injection Techniques 501-6 Manual injection 5016 1/94 0171/94 01 E: Reference standards 501-81/94 Refe 501-8 l/94 m60 500-1
Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I SECTION 500 500–1 Table of Contents page date 501: General Information 501 A: Principles 501-1 1/94 501 B: Equipment for GLC 501-1 1/94 Gas Chromatographic Components 501-1 1/94 Other Apparatus 501-2 1/94 Reagents and Gases 501-3 1/94 501 C: Residue Methodology for GLC Determination 501-3 1/94 Cleanup 501-4 1/94 Reagent Blanks 501-5 1/94 Choice of Solvent 501-6 1/94 501 D: Injection Techniques 501-6 1/94 Manual Injection 501-6 1/94 Autoinjectors 501-7 1/94 501 E: Reference Standards 501-8 1/94 References 501-8 1/94 Chapter 1 Regulatory Operations Chapter 2 General Analytical Operations and Information Chapter 3 Multiclass MRMs Chapter 5 GLC Chapter 4 Selective MRMs Chapter 6 HPLC
Pesticide Analytical Manual Vol I 502: Columns 50211/9 Column Specifications 502-1 1/94 Column parameters 50221/94 502 B: Packed Columns 502-6 /94 Components of Packed Columns 502-6 1/94 Preparation of Packed Columns 5028 1/94 Installation of Packed column 502-9 1/94 Conditioning of Packed columns 502101/94 Rejuvenation of Packed Columns 502-101/94 Criteria for Acceptable Packed Columns 502-111/94 for Packed Colume ating Procedures 502-121/94 502 C: Open Tubular Capillary Columns 502-131/9 Column Description 502141/94 502-161/94 Capillary column Systems 502-161/94 Installation and Conditioning of 502201/94 Capillary columns Rejuvenation of capillary columns 502211/94 Recommended Operating Procedure for 502-22 1/94 Wide bore Columns(Isothermal) Apparatus and Reagents 502221/94 System Startup and Inspection 502251/94 References 502251/94 Detectors 503 A: Introduction 50311/94 Definitions of Detector Characteristics 503-11/94 503 B: Electron Capture Detector 50321/94 Principles 50331/94 Design 50331/94 pparatus and reagents 50331/94 Detector Characteristics 50341/94 Other Influences on Detector Performance 503-7 1/94 d Operating Procedures /9 503 C: Flame Photometric Detector 50391/94 Principle 50391/94 Design 503-91/94 Apparatus and Reagents 503101/94 Detector Characteristics 503101/94 Other Influences on Detector Performance 508-11 1/94 Procedu 503121/9 Troubleshooting 503131/94 503 D: Electrolytic Conductivity Detector 503-141/94 Principles 503141/94 5002 Form FDA 2905a(6/92)
Transmittal No. 96-E1 (9/96) 500–2 Form FDA 2905a (6/92) SECTION 500 Pesticide Analytical Manual Vol. I page date 502: Columns 502 A: Introduction 502-1 1/94 Column Specifications 502-1 1/94 Column Parameters 502-2 1/94 502 B: Packed Columns 502-6 1/94 Components of Packed Columns 502-6 1/94 Preparation of Packed Columns 502-8 1/94 Installation of Packed Columns 502-9 1/94 Conditioning of Packed Columns 502-10 1/94 Rejuvenation of Packed Columns 502-10 1/94 Criteria for Acceptable Packed Columns 502-11 1/94 Recommended Operating Procedures 502-12 1/94 for Packed Columns 502 C: Open Tubular Capillary Columns 502-13 1/94 Column Description 502-14 1/94 Injection onto Capillary Columns 502-16 1/94 Capillary Column Systems 502-16 1/94 Installation and Conditioning of 502-20 1/94 Capillary Columns Rejuvenation of Capillary Columns 502-21 1/94 Recommended Operating Procedure for 502-22 1/94 Wide Bore Columns (Isothermal) Apparatus and Reagents 502-22 1/94 System Startup and Inspection 502-25 1/94 References 502-25 1/94 503: Detectors 503 A: Introduction 503-1 1/94 Definitions of Detector Characteristics 503-1 1/94 503 B: Electron Capture Detector 503-2 1/94 Principles 503-3 1/94 Design 503-3 1/94 Apparatus and Reagents 503-3 1/94 Detector Characteristics 503-4 1/94 Other Influences on Detector Performance 503-7 1/94 Recommended Operating Procedures 503-8 1/94 503 C: Flame Photometric Detector 503-9 1/94 Principles 503-9 1/94 Design 503-9 1/94 Apparatus and Reagents 503-10 1/94 Detector Characteristics 503-10 1/94 Other Influences on Detector Performance 503-11 1/94 Recommended Operating Procedures 503-12 1/94 Troubleshooting 503-13 1/94 503 D: Electrolytic Conductivity Detector 503-14 1/94 Principles 503-14 1/94
Pesticide Analytical Manual Vol. I Design 503-141/94 EICD-X 503-161/94 Principles 503-161/94 Apparatus and ro 503-161/94 Detector characteristics 503-171/94 Other Influences on Detector Performance 503-19 1/94 Recommended Operating Procedures 503-211/94 System Suitability Test 503-211/94 Troubleshooting 503221/94 EICD-N 503-231/94 Apparatus and Reagents 503-231/94 etector Characteristics 503-231/94 Other Influences on Detector Performance 503-24 1/94 Recommended Operating Procedures 503-251/94 System Suitability 503251/94 ableshooting 503-251/94 General Precautions for eIcds 503261/94 503 E: Nitrogen/Phosphorus Detector 503-271/94 Principles 503-271/94 503-271/94 Apparatus and Reagents 503-281/94 Detector Characteristics 503-281/94 Other Influences on Detector Performance 508-30 1/94 Recommended operating Procedures 503-311/94 References 503-321/94 504: Quantitation 504 A: Introduction 504-1 1/94 504 B: Manual Quantitation 1/94 Measurement of peak height Measurement of Area by Triangulation 504-21/94 504 C: Electronic Integration 504-31/94 504 D: Special Considerations for Complex 504-6 1/94 Chromatograms BHC 50461/94 Chlordane 5048 1/94 PCBs 504-101/94 504-141/94 504-151/94 505: Bibliography General Texts 05-11/94 Inlets 05-1 1/94 Columns 05-1 1/94 Detectors 05-1 1/94 m60 500-3
Transmittal No. 96-E1 (9/96) Form FDA 2905a (6/92) Pesticide Analytical Manual Vol. I SECTION 500 500–3 page date Design 503-14 1/94 ElCD-X 503-16 1/94 Principles 503-16 1/94 Apparatus and Reagents 503-16 1/94 Detector Characteristics 503-17 1/94 Other Influences on Detector Performance 503-19 1/94 Recommended Operating Procedures 503-21 1/94 System Suitability Test 503-21 1/94 Troubleshooting 503-22 1/94 ElCD-N 503-23 1/94 Principles 503-23 1/94 Apparatus and Reagents 503-23 1/94 Detector Characteristics 503-23 1/94 Other Influences on Detector Performance 503-24 1/94 Recommended Operating Procedures 503-25 1/94 System Suitability Test 503-25 1/94 Troubleshooting 503-25 1/94 General Precautions for ElCDs 503-26 1/94 503 E: Nitrogen/Phosphorus Detector 503-27 1/94 Principles 503-27 1/94 Design 503-27 1/94 Apparatus and Reagents 503-28 1/94 Detector Characteristics 503-28 1/94 Other Influences on Detector Performance 503-30 1/94 Recommended Operating Procedures 503-31 1/94 References 503-32 1/94 504: Quantitation 504 A: Introduction 504-1 1/94 504 B: Manual Quantitation 504-2 1/94 Measurement of Peak Height 504-2 1/94 Measurement of Area by Triangulation 504-2 1/94 504 C: Electronic Integration 504-3 1/94 504 D: Special Considerations for Complex 504-6 1/94 Chromatograms BHC 504-6 1/94 Chlordane 504-8 1/94 PCBs 504-10 1/94 Toxaphene 504-14 1/94 References 504-15 1/94 505: Bibliography General Texts 505-1 1/94 Inlets 505-1 1/94 Columns 505-1 1/94 Detectors 505-1 1/94
Pesticide Analytical Manual Vol I Fi igures Polysiloxane Stationary Phases 50221/9 502-b GLC Column parameters 50241/94 02-c Ferrules for Connecting Glass and Metal 5029 1/94 502-d Capillary column Cross-Section 502151/94 02-e Inlet Adapters for Capillary Columns 502-171/94 02-f Van Deemter Curves 502191/94 502-g Capillary Column Inlet System 502-2 1/94 503-a Two EC Detector Designs 503-3 1/94 603-b Reproducibility 503-5 1/94 03C Dynamic Response Ranges 50361/9 503-d Linear Response Range 50361/94 508-e Effect of Detector Temperature 50371/94 503-f Effect of Carrier Gas Flow Rate 50371/94 503-g Single- Flame FPD 50391/94 0%-h Dual-Flame fpd 503101/94 508-i Block diagram of the elcd 503-151/94 3-j ElCD Reactor and Conductivity Cell 503-151/94 508-k N/P Detector Components 503281/94 03-1 N/P Detector Configurations 5032 1/94 504-a Manual Peak measurement 504-21/94 504-b Triangulation of Peak on Sloping Baseline 1/94 504-c Technical Chlordane 50481/94 504-d Chlordane, Heptachlor, Heptachlor Epoxide 504-9 1/94 504-e PCBs in chinook salmon 504-131/94 504-f Toxaphene 504141/94 Tables 02-a: Common GlC Liquid Phases Used in Pesticide 502-3 1/94 Residue determination 502-b: Operating Conditions for Packed Columns 502-18 1/94 504-a: Effects of Changing Electronic Integrator 5046 1/94 Setting 504-b: Response of Two Detectors to Four 50471/94 BHC Isomers 504-c: Weight Percent Factors for Individual Gas 504121/94 Chromatographic Peaks in Aroclor Reference Standard 50o4 Form FDA 2905a(6/92)
Transmittal No. 96-E1 (9/96) 500–4 Form FDA 2905a (6/92) SECTION 500 Pesticide Analytical Manual Vol. I page date Figures 502-a Polysiloxane Stationary Phases 502-2 1/94 502-b GLC Column Parameters 502-4 1/94 502-c Ferrules for Connecting Glass and Metal 502-9 1/94 502-d Capillary Column Cross-Section 502-15 1/94 502-e Inlet Adapters for Capillary Columns 502-17 1/94 502-f Van Deemter Curves 502-19 1/94 502-g Capillary Column Inlet System 502-23 1/94 503-a Two EC Detector Designs 503-3 1/94 503-b Reproducibility 503-5 1/94 503-c Dynamic Response Ranges 503-6 1/94 503-d Linear Response Range 503-6 1/94 503-e Effect of Detector Temperature 503-7 1/94 503-f Effect of Carrier Gas Flow Rate 503-7 1/94 503-g Single-Flame FPD 503-9 1/94 503-h Dual-Flame FPD 503-10 1/94 503-i Block Diagram of the ElCD 503-15 1/94 503-j ElCD Reactor and Conductivity Cell 503-15 1/94 503-k N/P Detector Components 503-28 1/94 503-l N/P Detector Configurations 503-29 1/94 504-a Manual Peak Measurement 504-2 1/94 504-b Triangulation of Peak on Sloping Baseline 504-3 1/94 504-c Technical Chlordane 504-8 1/94 504-d Chlordane, Heptachlor, Heptachlor Epoxide 504-9 1/94 504-e PCBs in Chinook Salmon 504-13 1/94 504-f Toxaphene 504-14 1/94 Tables 502-a: Common GLC Liquid Phases Used in Pesticide 502-3 1/94 Residue Determination 502-b: Operating Conditions for Packed Columns 502-13 1/94 504-a: Effects of Changing Electronic Integrator 504-6 1/94 Settings 504-b: Response of Two Detectors to Four 504-7 1/94 BHC Isomers 504-c: Weight Percent Factors for Individual Gas 504-12 1/94 Chromatographic Peaks in Aroclor Reference Standards
Pesticide Analytical Manual Vol. I SECTION 501 501: GENERAL INFORMATION Multiresidue methodology by definition requires determinative steps capable of separating analytes from one another so each can be detected and measured individually. Both gas-liquid chromatography (GLC) and high performance liquid chromatography(HPLC) provide these capabilities, and both are used in modern laboratories GLC has been the predominant determinative step in pesticide multiresidue methodology for over 30 years. Because GlC involves interaction between a vapor phase and liquid phase, its application is restricted to analytes that can be vapor ized without degradation. For heat-labile chemicals, HPLC offers a variety of alter native schemes for separating analytes according to chemical or physical charac minative step of choice for residues to which it is applicable t remain the deter- teristics, but GLC's relative simplicity and ruggedness cause it 501 A: PRINCIPLES paration in glc is achieved by differences in distribution of analytes between mobile and stationary phases, causing them to move through the column at dif ferent rates and from it at different times [1]. A measured aliquot of solution is injected into a gas chromatographic column through an inlet heated to a suffi- gas that forms the mobile phase sweeps analytes through the cole flow of inert ciently high temperature that analytes are vaporized. In this state, th this movement is the analyte's solubilization in the liquid phase. e n: retarding passag through the column, analytes that were injected in the same solution separate from one another because of their different vapor pressures and selective interac- tions with the liquid phase [2]. When analytes elute from the column and enter a detector, the detector responds to the presence of a specific element or func- tional group within the molecule. The detector's response causes a change in electronic signal, which is proportional to the amount of residue; the signal is amplified and recorded as a chromatogram Analytes are identified by the time it takes them to pass through a column of specific liquid phase (retention time), at a specified temperature and gas flow Quantities are calculated from the detector response. Both retention time and response are compared to values obtained for a reference standard solution in- jected into the same system 501 B: EQUIPMENT FOR GLC Gas Chromatographic Components The basic gas chromatograph consists of an inlet system, column, detector, elec- tronic equipment to amplify the detector signal, and a recorder or other data- handling device. Carrier gas(es), with appropriate pneumatic system(s), are also integral to the glc system. The inlet system, column, and detector are maintained in temperature-controlled environment The following are desirable features in GLC hardware: 1)Inlet, column oven, and detector should be individually heated and temperature-controlled. Temperature should be maintained to #O1C 501-1
Pesticide Analytical Manual Vol. I SECTION 501 501–1 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 501: GENERAL INFORMATION Multiresidue methodology by definition requires determinative steps capable of separating analytes from one another so each can be detected and measured individually. Both gas-liquid chromatography (GLC) and high performance liquid chromatography (HPLC) provide these capabilities, and both are used in modern laboratories. GLC has been the predominant determinative step in pesticide multiresidue methodology for over 30 years. Because GLC involves interaction between a vapor phase and liquid phase, its application is restricted to analytes that can be vaporized without degradation. For heat-labile chemicals, HPLC offers a variety of alternative schemes for separating analytes according to chemical or physical characteristics, but GLC’s relative simplicity and ruggedness cause it to remain the determinative step of choice for residues to which it is applicable. 501 A: PRINCIPLES Separation in GLC is achieved by differences in distribution of analytes between mobile and stationary phases, causing them to move through the column at different rates and from it at different times [1]. A measured aliquot of solution is injected into a gas chromatographic column through an inlet heated to a sufficiently high temperature that analytes are vaporized. In this state, the flow of inert gas that forms the mobile phase sweeps analytes through the column; retarding this movement is the analyte’s solubilization in the liquid phase. During passage through the column, analytes that were injected in the same solution separate from one another because of their different vapor pressures and selective interactions with the liquid phase [2]. When analytes elute from the column and enter a detector, the detector responds to the presence of a specific element or functional group within the molecule. The detector’s response causes a change in electronic signal, which is proportional to the amount of residue; the signal is amplified and recorded as a chromatogram. Analytes are identified by the time it takes them to pass through a column of specific liquid phase (retention time), at a specified temperature and gas flow. Quantities are calculated from the detector response. Both retention time and response are compared to values obtained for a reference standard solution injected into the same system. 501 B: EQUIPMENT FOR GLC Gas Chromatographic Components The basic gas chromatograph consists of an inlet system, column, detector, electronic equipment to amplify the detector signal, and a recorder or other datahandling device. Carrier gas(es), with appropriate pneumatic system(s), are also integral to the GLC system. The inlet system, column, and detector are maintained in temperature-controlled environments. The following are desirable features in GLC hardware: 1) Inlet, column oven, and detector should be individually heated and temperature-controlled. Temperature should be maintained to ±0.1° C
SECTION 501 Pesticide Analytical Manual Vol I Control of detector temperature usually is not as critical but should be well controlled, constant, and not affected by such things as line voltage fluctuations 2)Temperature readout should be available for column, detector, and inlet. Check accuracy of instrument temperature indicators with accurate py 3)Instrument design should be simple enough to facilitate troubleshooting and repairs. Design should permit easy removal or inspection of either column or detector without affecting the temperature of the other. 4)System should be designed to prevent or minimize contact between sample injection and any metal parts; system should be all-glass (or as near as possible Several sizes of packed and open tubular capillary columns are used in residue analysis, and hardware for inlet and column must accommodate configurations that will be needed. Section 502, Columns, includes direc tions for adapting equipment. 5) Certain detectors may require multiple heated zones, including combus- tion furnaces For flexibility, designs that permit ready access for servicing nd maintenance are preferred. Section 503 provides details on various detectors used in pesticide residue determination 6) Electrical signal monitoring equipment is usually one of two designs: (1) amplifier with 1 or 10 mV output, compatible with strip chart recorder, d (2)amplifier with I or 10 V output, compatible with data processing by either electronic integrator or computer. Other remote devices such as autosamplers can be easily adapted to any of these systems Other Apparatus Gas Regulators. Two-stage gas pressure regulators with stainless steel diaphragms Poo required for all GLC determinations of trace residues. Regulators with a sec. ary stage maximum pressure of 80 psi are acceptable, but those with 200 psi fer more flexibility. If a hydrogen purifier is used(below), the latter type of regulator is required, because higher pressure is needed Gas lines that connect gas tanks to the chromatograph must be clean and free of components that contain oil or gas-purgeable elastomers; refrigeration grade copper(i.e, cleaned of all oil) is preferred. Tubing (even refrigeration grade) should be sequentially rinsed with methylene chloride and acetone before use Plastic and nylon lines must be avoided to reduce the likelihood of air contami- nating the gas. Syringes. The most common syringes for injection of food extracts into a chro- matograph are 5 and 10 uL fixed needle syringes with 22 bevel points; some other sizes may be needed for special purposes. Hamilton syringes or equivalent are available from all chromatography suppliers. Plunger "guides"are available as options to minimize bending the plunger during injection. 501-2 Transmittal No. 94-1(1/94]
501–2 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) SECTION 501 Pesticide Analytical Manual Vol. I Control of detector temperature usually is not as critical but should be well controlled, constant, and not affected by such things as line voltage fluctuations. 2) Temperature readout should be available for column, detector, and inlet. (Check accuracy of instrument temperature indicators with accurate pyrometer.) 3) Instrument design should be simple enough to facilitate troubleshooting and repairs. Design should permit easy removal or inspection of either column or detector without affecting the temperature of the other. 4) System should be designed to prevent or minimize contact between sample injection and any metal parts; system should be all-glass (or as near as possible). Several sizes of packed and open tubular capillary columns are used in residue analysis, and hardware for inlet and column must accommodate configurations that will be needed. Section 502, Columns, includes directions for adapting equipment. 5) Certain detectors may require multiple heated zones, including combustion furnaces. For flexibility, designs that permit ready access for servicing and maintenance are preferred. Section 503 provides details on various detectors used in pesticide residue determination. 6) Electrical signal monitoring equipment is usually one of two designs: (1) amplifier with 1 or 10 mV output, compatible with strip chart recorder, and (2) amplifier with 1 or 10 V output, compatible with data processing by either electronic integrator or computer. Other remote devices such as autosamplers can be easily adapted to any of these systems. Other Apparatus Gas Regulators. Two-stage gas pressure regulators with stainless steel diaphragms are required for all GLC determinations of trace residues. Regulators with a secondary stage maximum pressure of 80 psi are acceptable, but those with 200 psi offer more flexibility. If a hydrogen purifier is used (below), the latter type of regulator is required, because higher pressure is needed. Gas lines that connect gas tanks to the chromatograph must be clean and free of components that contain oil or gas-purgeable elastomers; “refrigeration grade” copper (i.e., cleaned of all oil) is preferred. Tubing (even refrigeration grade) should be sequentially rinsed with methylene chloride and acetone before use. Plastic and nylon lines must be avoided to reduce the likelihood of air contaminating the gas. Syringes. The most common syringes for injection of food extracts into a chromatograph are 5 and 10 µL fixed needle syringes with 22° bevel points; some other sizes may be needed for special purposes. Hamilton syringes or equivalent are available from all chromatography suppliers. Plunger “guides” are available as options to minimize bending the plunger during injection
Pesticide Analytical Manual Vol. I SECTION 501 Some specialty products exist to facilitate injection and minimize aggravation, and each has found favor with some analysts. For example, syringes with removable needles permit replacement of needles on which"burrs" have formed that destroy septa; removable needles with a"side port point"do not shred the septa as do standard bevel point needles; and syringes with plungers and needles made of a titanium alloy cannot be bent Reagents and gases Reagents associated with GlC include column liquid phases and solid supports gases used for mobile phase and for detector reactions, and certain other reagents relevant to detector operation. Most of these reagents are discussed further in pertinent sections of this chapter; only gases, including filters used to remove contaminants from gas flow, are included in this introductory section Helium, hydrogen, and nitrogen are most commonly used as column carrier gases Purity is always critical to avoid damage to the column, and more stringent purity requirements may be imposed by the detector. Purity specifications of the instru- ment manufacturer should always be followed Helium and hydrogen requirements range from 99.999-999999%o pailable from arity, depend ing on the detector. Even with the highest purity, oxygen traps, chromatography suppliers, are recommended; traps that change color when per meated with oxygen are ideal for alerting the analyst to potential problems Purchase of ultra high purity helium and hydrogen may not be necessary if spe- cially designed purifiers are used. Purifiers are available that permit use of com- purifier gtade gases(99.995%)at a much lower price, justifying the cost of the r.Different purifiers are needed for helium and hydrogen; they are not interchangeable. FDA has had successful experiences with hydrogen purifiers: Model 560, AADCO Instruments, Inc, Clearwater, FL; Model 8372V. Consolidated Technologies. Inc.. West Chester. PA helium purifiers: Product HP, Valco Instrument Co., Houston, TX Model 2-3800, Supelco, Bellefonte, PA Nitrogen is used as a carrier gas only for packed columns(Section 502 B). Either nitrogen or argon/methane (95+5 or 90+10)is also required as a carrier and/or makeup gas for the electron capture detector(Section 503 B). Commercial grades of these gases are acceptable if oxygen and moisture traps are used between the as tank and the chromatograp 501 C RESIDUE METHODOLOGY FOR GLC DETERMINATION Applications of analytical methodology require consideration of many factors to assure compatibility of method steps. The following factors related to extraction and cleanup of food samples profoundly influence accuracy and reliability of glC 501-3
Pesticide Analytical Manual Vol. I SECTION 501 501–3 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Some specialty products exist to facilitate injection and minimize aggravation, and each has found favor with some analysts. For example, syringes with removable needles permit replacement of needles on which “burrs” have formed that destroy septa; removable needles with a “side port point” do not shred the septa as do standard bevel point needles; and syringes with plungers and needles made of a titanium alloy cannot be bent. Reagents and Gases Reagents associated with GLC include column liquid phases and solid supports, gases used for mobile phase and for detector reactions, and certain other reagents relevant to detector operation. Most of these reagents are discussed further in pertinent sections of this chapter; only gases, including filters used to remove contaminants from gas flow, are included in this introductory section. Helium, hydrogen, and nitrogen are most commonly used as column carrier gases. Purity is always critical to avoid damage to the column, and more stringent purity requirements may be imposed by the detector. Purity specifications of the instrument manufacturer should always be followed. Helium and hydrogen requirements range from 99.999-99.9999% purity, depending on the detector. Even with the highest purity, oxygen traps, available from chromatography suppliers, are recommended; traps that change color when permeated with oxygen are ideal for alerting the analyst to potential problems. Purchase of ultra high purity helium and hydrogen may not be necessary if specially designed purifiers are used. Purifiers are available that permit use of commercial grade gases (99.995%) at a much lower price, justifying the cost of the purifier. Different purifiers are needed for helium and hydrogen; they are not interchangeable. FDA has had successful experiences with: hydrogen purifiers: Model 560, AADCO Instruments, Inc., Clearwater, FL; Model 8372V, Consolidated Technologies, Inc., West Chester, PA helium purifiers: Product # HP, Valco Instrument Co., Houston, TX; Model 2-3800, Supelco, Bellefonte, PA Nitrogen is used as a carrier gas only for packed columns (Section 502 B). Either nitrogen or argon/methane (95+5 or 90+10) is also required as a carrier and/or makeup gas for the electron capture detector (Section 503 B). Commercial grades of these gases are acceptable if oxygen and moisture traps are used between the gas tank and the chromatograph. 501 C: RESIDUE METHODOLOGY FOR GLC DETERMINATION Applications of analytical methodology require consideration of many factors to assure compatibility of method steps. The following factors related to extraction and cleanup of food samples profoundly influence accuracy and reliability of GLC determinative steps
SECTION 501 Pesticide Analytical Manual Vol I Cleanup Solvent extraction of pesticide residues also extracts food constituents ("co-extrac tives")from the sample Cleanup steps are included in residue analytical method to remove co-extractives that can interfere in the determinative step of the analysi or cause damage to the column and/or detector. For many years, predominant use of the nonselective electron capture(EC)detec- tor caused justifiable concern about potential detector response to nonpesticidal co-extractives. In addition, documented cases in which sample co-extractives dam aged GLC columns and caused subsequent breakdown of injected residues sup ported the need for extensive cleanup prior to GlC determination [3] More recently, several factors have reduced emphasis on cleanup. The more selec tive glC detectors now in use have decreased the likelihood that sample or re- agent artifacts might be mistaken for pesticide residues. In addition, use of cap llary columns, which are more efficient than equivalent packed columns, result in increased peak height response for the same amount of analyte. The amount of extract injected can thus be reduced without changing the level of quantitation and this in turn reduces the likelihood of damage to the GlC system. Inlet liners and adapters used with capillary columns(Section 502 C)also provide the column with some degree of protection from damage caused by co-extractives. Finally, there are many incentives to perform more analyses with the same or fewer re- sources and to minimize the volume of solvents that must be purchased and disposed of. These factors contribute to a trend toward performing only minimal cleanup of sample extracts during routine surveillance analyses, with the intention of cleanup with applicable step(s)if an extract is found to contain interfering materials Despite these compelling reasons to reduce cleanup glc systems that are not protected from co-extractives deteriorate faster than those into which only cleaned up extracts are injected. The column and/or detector may be damaged by injec tion of insufficiently cleaned up samples, especially when the method and the chromatograph are used repeatedly. Such detrimental effects can occur even when the chromatogram appears to be clean enough for residue identification and measurement. Experience with a variety of sample types should make the analyst aware of these occurrences Detector response to sample co-extractives(artifacts)is still possible even with element-selective detectors. Although a selective detector is less likely to to chemically unrelated artifacts than the nonselective EC detector, artifacts con- analysis. This occurs most often with nitrogen-selective detectors because of the number of nitrogenous chemicals in foods, but it can occur with any detector Likelihood of interferences and potential for mistaken identity increase with de creasIng cleanup. Insufficiently clean extracts may also affect quantitative accuracy when determin- ing residues that are polar or otherwise subject to adsorption by active sites in a GLC column. Such chemicals usually exhibit poor chromatography when standard solutions are injected, because adsorption delays or inhibits the chemical during its passage through the column. Peak tailing and/or changes in retention times are caused by adsorption. The net effect is an apparently diminished detector 501-4 Transmittal No. 94-1(1/94]
501–4 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) SECTION 501 Pesticide Analytical Manual Vol. I Cleanup Solvent extraction of pesticide residues also extracts food constituents (“co-extractives”) from the sample. Cleanup steps are included in residue analytical methods to remove co-extractives that can interfere in the determinative step of the analysis or cause damage to the column and/or detector. For many years, predominant use of the nonselective electron capture (EC) detector caused justifiable concern about potential detector response to nonpesticidal co-extractives. In addition, documented cases in which sample co-extractives damaged GLC columns and caused subsequent breakdown of injected residues supported the need for extensive cleanup prior to GLC determination [3]. More recently, several factors have reduced emphasis on cleanup. The more selective GLC detectors now in use have decreased the likelihood that sample or reagent artifacts might be mistaken for pesticide residues. In addition, use of capillary columns, which are more efficient than equivalent packed columns, result in increased peak height response for the same amount of analyte. The amount of extract injected can thus be reduced without changing the level of quantitation, and this in turn reduces the likelihood of damage to the GLC system. Inlet liners and adapters used with capillary columns (Section 502 C) also provide the column with some degree of protection from damage caused by co-extractives. Finally, there are many incentives to perform more analyses with the same or fewer resources and to minimize the volume of solvents that must be purchased and disposed of. These factors contribute to a trend toward performing only minimal cleanup of sample extracts during routine surveillance analyses, with the intention of cleanup with applicable step(s) if an extract is found to contain interfering materials. Despite these compelling reasons to reduce cleanup, GLC systems that are not protected from co-extractives deteriorate faster than those into which only cleaned up extracts are injected. The column and/or detector may be damaged by injection of insufficiently cleaned up samples, especially when the method and the chromatograph are used repeatedly. Such detrimental effects can occur even when the chromatogram appears to be clean enough for residue identification and measurement. Experience with a variety of sample types should make the analyst aware of these occurrences. Detector response to sample co-extractives (artifacts) is still possible even with element-selective detectors. Although a selective detector is less likely to respond to chemically unrelated artifacts than the nonselective EC detector, artifacts containing an element to which the detector responds can still interfere with residue analysis. This occurs most often with nitrogen-selective detectors because of the number of nitrogenous chemicals in foods, but it can occur with any detector. Likelihood of interferences and potential for mistaken identity increase with decreasing cleanup. Insufficiently clean extracts may also affect quantitative accuracy when determining residues that are polar or otherwise subject to adsorption by active sites in a GLC column. Such chemicals usually exhibit poor chromatography when standard solutions are injected, because adsorption delays or inhibits the chemical during its passage through the column. Peak tailing and/or changes in retention times are caused by adsorption. The net effect is an apparently diminished detector
Pesticide Analytical Manual Vol. I SECTION 501 response, which is especially evident if peak height measurements are used rather than peak area. In contrast, when an uncleaned extract containing the same analyte is injected into the GlC system, co-extractives compete for the columns active sites, and the analyte moves through the column in a tighter chromatographic band. Analyte concentration (per unit time) entering the detector thus increases, and detector response (peak height) is greater. Quantitation by the usual practice (i.e,com- parison of detector responses to residue and reference standard) results in calcu- lation of an inaccurately high residue level, especially if peak heights are com- pared. Quantitative accuracy can be improved for such chemicals by employing more rigorous cleanup of the extract or by using a GlC column with fewer active Sites An appropriate balance is needed between efficiency in processing samples and accuracy in determining residues. Every injected extract should be sufficiently clean that it(1)does not jeopardize the column beyond the point that it can be easily repaired;(2)does not introduce substances that will degrade co-injected or subsequently injected residues; (8)does not foul any part of the detector, includ- ing combustion tube, flame, radioactive source, etc; (4)minimizes introduction of artifacts to which the detector will respond; and (5)does not cause a dispropor tionate response enhancement of the residue in the extract. Reagent Blanks The analyst must ascertain that no interference from reagents and/or glassware occurs during residue analysis. Scrupulous attention is required to eliminate all such contaminants, and routine analysis of reagent blanks should be specified in the laboratory quality assurance plan(Section 206) Contaminants can be introduced from a variety of sources. Studies with the EC detector have identified interferences from impure solvents, adsorbents, so- dium sulfate, glass wool, Celite, blender gaskets, laboratory air filters, and polyeth ylene containers. The more nonselective the detector, the more likely it is to respond to interferences introduced by reagents or the environment. A thorough examination of the reagent blank is also necessary for methods that use a relatively selective detector. One example demonstrated that chemicals extracted by petro leum ether from a polyethylene squeeze bottle caused response by both an EC and a halogen-selective detector [8]. Contaminants can even be pesticides themselves, present on glassware or microliter syringes used in prior analyses, or present in the laboratory environment because of pest control treatment. When interferences are discovered and the source (s)identified, every effort must be made to reduce or eliminate the problem. Solvents can be purchased to meet requirements or may be redistilled. Solids frequently can be washed and/ or heated prior to use Section 204 provides purity tests and procedures for purifying certain commonly used reagents; other reagent purification procedures cluded in ertinent method descriptions in Chapters 3 and 4. Sometimes the method cleanup step removes interferences added to the sample during previous step ps, but whether this is accomplished must be determined by a complete investigation of the method reagent blank Equipment should be washed thoroughly and rinsed with solvent as soon as pos- sible after use. Syringe plungers and needles should be wiped with lint-free wipers 501-5
Pesticide Analytical Manual Vol. I SECTION 501 501–5 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) response, which is especially evident if peak height measurements are used rather than peak area. In contrast, when an uncleaned extract containing the same analyte is injected into the GLC system, co-extractives compete for the column’s active sites, and the analyte moves through the column in a tighter chromatographic band. Analyte concentration (per unit time) entering the detector thus increases, and detector response (peak height) is greater. Quantitation by the usual practice (i.e., comparison of detector responses to residue and reference standard) results in calculation of an inaccurately high residue level, especially if peak heights are compared. Quantitative accuracy can be improved for such chemicals by employing more rigorous cleanup of the extract or by using a GLC column with fewer active sites. An appropriate balance is needed between efficiency in processing samples and accuracy in determining residues. Every injected extract should be sufficiently clean that it (1) does not jeopardize the column beyond the point that it can be easily repaired; (2) does not introduce substances that will degrade co-injected or subsequently injected residues; (3) does not foul any part of the detector, including combustion tube, flame, radioactive source, etc.; (4) minimizes introduction of artifacts to which the detector will respond; and (5) does not cause a disproportionate response enhancement of the residue in the extract. Reagent Blanks The analyst must ascertain that no interference from reagents and/or glassware occurs during residue analysis. Scrupulous attention is required to eliminate all such contaminants, and routine analysis of reagent blanks should be specified in the laboratory quality assurance plan (Section 206). Contaminants can be introduced from a variety of sources. Studies with the EC detector have identified interferences from impure solvents, adsorbents, sodium sulfate, glass wool, Celite, blender gaskets, laboratory air filters, and polyethylene containers. The more nonselective the detector, the more likely it is to respond to interferences introduced by reagents or the environment. A thorough examination of the reagent blank is also necessary for methods that use a relatively selective detector. One example demonstrated that chemicals extracted by petroleum ether from a polyethylene squeeze bottle caused response by both an EC and a halogen-selective detector [3]. Contaminants can even be pesticides themselves, present on glassware or microliter syringes used in prior analyses, or present in the laboratory environment because of pest control treatment. When interferences are discovered and the source(s) identified, every effort must be made to reduce or eliminate the problem. Solvents can be purchased to meet requirements or may be redistilled. Solids frequently can be washed and/or heated prior to use. Section 204 provides purity tests and procedures for purifying certain commonly used reagents; other reagent purification procedures are included in pertinent method descriptions in Chapters 3 and 4. Sometimes the method cleanup step removes interferences added to the sample during previous steps, but whether this is accomplished must be determined by a complete investigation of the method reagent blank. Equipment should be washed thoroughly and rinsed with solvent as soon as possible after use. Syringe plungers and needles should be wiped with lint-free wipers
SECTION 501 Pesticide Analytical Manual Vol I dipped in an appropriate solvent(e. g, acetone), and the barrel should be cleaned ware or syringes previously in contact with high concentrations of pesticide<.e by drawing solvent gh the needle and out the top by a vacuum applied to th top. Particular care Id be taken to assure elimination of residues from glass- Choice of solvent The solvent in which the final extract is dissolved must be compatible with the detector(s)in the GlC determinative step(s). The most basic requirement is that the solvent not contain elements to which the detector responds. Specifically, no amount of chlorinated solvent, such as methylene chloride, can remain in extracts being examined by an EC or halogen-selective detector, and no trace of aceton- tile can be present in extracts examined with nitrogen-selective detectors Other effects besides element selectivity cause incompatibility between detectors and solvents. For example, acetonitrile has an unexplained adverse effect on re sponse of the EC detector, and aromatic and halogenated solvents may increase detector response of the n/p detector and eventually render it useless Solvent volatility must also be considered when using a detector that requires a solvent venting time. For these detectors, the most volatile practical solvent in which residues are soluble should be chosen to minimize length of venting time and avoid potential loss of early eluting analytes Solvent volatility has another practical effect related to the ease with which the extract can be concentrated. Final volume of concentrated extract must be suffi- ciently small that the volume injected into the GlC system contains sufficient equivalent sample weight necessary to reach the targeted level of quantitation (Section 105). Sensitivity of a particular detector to residues of interest governs how much sample equivalent must be injected, and column type and arrangement limit the volume that can be injected. In cases where a very small final extract volume is needed, or where the concentration step begins with a very large solvent volume, practicality dictates the choice of a volatile solvent to minimize time needed for concentration 501 D: INJECTION TECHNIQUES The technique used to inject extracts and reference standards into the chromato- graph is critical to system performance. Improper syringe handling can lead to nation, but manual injection is sall practice cred pes and nonreproducible reten- myriad problems, including asymmetrical peak sh tion times or responses. Autoinjectors are in Manual injection If extracts and standards are injected manually, it is imperative that each analyst develop and follow good technique in syringe handling and sample introduction This can be achieved through practice and care. Several methods presently in use for filling syringes and injecting include 1)A volume of solvent greater than or equal to needle volume is drawn into the syringe, followed by a small amount of air. The extract (or reference standard solution) is then drawn completely into the syringe barrel, when 501-6 Transmittal No. 94-1(1/94]
501–6 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) SECTION 501 Pesticide Analytical Manual Vol. I dipped in an appropriate solvent (e.g., acetone), and the barrel should be cleaned by drawing solvent through the needle and out the top by a vacuum applied to the top. Particular care should be taken to assure elimination of residues from glassware or syringes previously in contact with high concentrations of pesticides. Choice of Solvent The solvent in which the final extract is dissolved must be compatible with the detector(s) in the GLC determinative step(s). The most basic requirement is that the solvent not contain elements to which the detector responds. Specifically, no amount of chlorinated solvent, such as methylene chloride, can remain in extracts being examined by an EC or halogen-selective detector, and no trace of acetonitrile can be present in extracts examined with nitrogen-selective detectors. Other effects besides element selectivity cause incompatibility between detectors and solvents. For example, acetonitrile has an unexplained adverse effect on response of the EC detector, and aromatic and halogenated solvents may increase detector response of the N/P detector and eventually render it useless. Solvent volatility must also be considered when using a detector that requires a solvent venting time. For these detectors, the most volatile practical solvent in which residues are soluble should be chosen to minimize length of venting time and avoid potential loss of early eluting analytes. Solvent volatility has another practical effect related to the ease with which the extract can be concentrated. Final volume of concentrated extract must be sufficiently small that the volume injected into the GLC system contains sufficient equivalent sample weight necessary to reach the targeted level of quantitation (Section 105). Sensitivity of a particular detector to residues of interest governs how much sample equivalent must be injected, and column type and arrangement limit the volume that can be injected. In cases where a very small final extract volume is needed, or where the concentration step begins with a very large solvent volume, practicality dictates the choice of a volatile solvent to minimize time needed for concentration. 501 D: INJECTION TECHNIQUES The technique used to inject extracts and reference standards into the chromatograph is critical to system performance. Improper syringe handling can lead to myriad problems, including asymmetrical peak shapes and nonreproducible retention times or responses. Autoinjectors are increasingly used for residue determination, but manual injection is still practiced. Manual Injection If extracts and standards are injected manually, it is imperative that each analyst develop and follow good technique in syringe handling and sample introduction. This can be achieved through practice and care. Several methods presently in use for filling syringes and injecting include: 1) A volume of solvent greater than or equal to needle volume is drawn into the syringe, followed by a small amount of air. The extract (or reference standard solution) is then drawn completely into the syringe barrel, where
