METHOD 8000D DETERMINATIVE CHROMATOGRAPHIC SEPARATIONS .
METHOD 8000DDETERMINATIVE CHROMATOGRAPHIC SEPARATIONSTable of Contents1.0SCOPE AND APPLICATION. 12.0SUMMARY OF METHOD. 63.0DEFINITIONS. 74.0INTERFERENCES/CHROMATOGRAPHIC PERFORMANCE . 75.0SAFETY . 86.0EQUIPMENT AND SUPPLIES . 87.0REAGENTS AND STANDARDS . 128.0SAMPLE COLLECTION, PRESERVATION, AND STORAGE . 129.0QUALITY CONTROL. 1210.0 CALIBRATION AND STANDARDIZATION . 2511.0 PROCEDURE . 2612.0 DATA ANALYSIS AND CALCULATIONS . 5513.0 METHOD PERFORMANCE . 5514.0 POLLUTION PREVENTION . 5515.0 WASTE MANAGEMENT . 5516.0 REFERENCES . 5617.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA . 56APPENDIX A . 56SW-846 is not intended to be an analytical training manual. Therefore, methodprocedures are written based on the assumption that they will be performed by analysts who areformally trained in at least the basic principles of chemical analysis and in the use of the subjecttechnology.In addition, SW-846 methods in this manual, with the exception of required use for theanalysis of method-defined parameters, are intended to be guidance documents. They containgeneral information on how to perform an analytical procedure or technique, which a laboratorycan use as a basic starting point for generating its own detailed standard operating procedure(SOP), either for its own general use or for a specific project application. Performance dataincluded in this method are for guidance purposes only and must not be used as absolutequality control (QC) acceptance criteria for the purpose of laboratory QC or accreditation.1.0SCOPE AND APPLICATIONPlease see Appendix A at the back of this document for a summary of revisions to Method8000C (From Revision 3, March 2003).SW-846 Update VI8000D- 1Revision 5March 2018
1.1Method 8000 is not a determinative method but instead provides guidance onanalytical chromatography and describes calibration and QC requirements common to allSW-846 chromatographic methods. However, more specific QC requirements provided in theapplicable determinative method will supersede those noted in Method 8000. Method 8000should be applied in conjunction with all SW-846 determinative chromatographic methods. Themethods include, but are not limited to, the 28082908310ChromatographicTechnique (Sec. 1.5)HPLCICGCGC, capillary columnGC, packed & capillarycolumnGC, capillary columnGC, packed columnGC, packed columnGC, capillary columnUnderivatized orderivatized; GC, capillarycolumnGC, capillary columnDetectorNitrosaminesOrganochlorine pesticidesPolychlorinated biphenylsNitroaromatics and cyclicketonesPolynuclear aromatichydrocarbonsHaloethersChlorinated hydrocarbonsAniline and selectedderivativesOrganophosphorus pesticidesAcid herbicidesGC, packed columnGC, capillary columnGC, capillary columnGC, capillary columnGC, packed & capillarycolumnGC, capillary columnGC, capillary columnGC, capillary columnGC, capillary columnDerivatized; GC, capillarycolumnNPD, ELCD, TEDECD, ELCDECD, SemivolatilesToxaphene and CongenersDioxins and DibenzofuransDioxins and DibenzofuransPolynuclear aromatichydrocarbonsGC, capillary columnGC, capillary columnNAGC, capillary columnThermal extraction/GCGC, capillary columnGC, capillary columnGC, capillary columnHPLC, reverse phaseMSMSDS/ITMSMSMSNICI/MSLow resolution MSHigh resolution MSUV, FluorescencePerchloratePerchlorateWhite phosphorus (P4)EDB, DBCPNon-halogenated volatilesSW-846 Update VI8000D- 2MS, MS/MSMS, MS/MSNICI/MSECDFIDPID, ELCDNPDECDNPDFID, ECDECDECDECDNPDFPD, NPD, ELCDECDRevision 5March 2018
108430DBCP DS/ITMS ECD EDB ES/ITMS ELCDFIDFPDFT-IRGCHPLC AnalytesCarbonyl compoundsAcrylamide, acrylonitrile,acroleinN-Methyl carbamatesExtractable non-volatilesOrganotin compoundsExtractable non-volatilesNitroaromatics and 2-chloroethyl) etherhydrolysis productsDibromochloropropaneDirect sampling/ion trap massspectrometryElectron capture detectorEthylene dibromideElectrospray ionization/ion trap massspectrometryElectrolytic conductivity detectorFlame ionization detectorFlame photometric detectorFourier transform-infraredGas chromatographyHigh performance liquid chromatographyChromatographicTechnique (Sec. 1.5)Derivatize; HPLCHPLC, reverse phaseDetectorDerivatize; HPLCPLC, reverse phaseHPLC, reverse phaseHPLC, reverse phaseHPLC, reverse phaseHPLC, ion pair, reversephaseHPLC, reverse phaseGC, capillary columnGC, capillary columnFluorescenceTS/MS, UVES/ITMSPB/MS, UVUVUVUVFT-IRFT-IRUVUVMS MS/MS NICI/MS NPDNAPAHsPB/MSPIDTEDTS/MSUVMass spectrometryMass spectrometry/Mass spectrometryNegative Ion Chemical Ionization/Massspectrometry Nitrogen/phosphorous detector Not applicable Polynuclear aromatic hydrocarbons Particle beam mass spectrometry Photoionization detector Thermionic emission detector Thermospray mass spectrometry Ultraviolet1.2Analytical chromatography is used to separate target analytes from co-extractedinterferences in samples. Chromatographic methods can be divided into two major categories:GC and HPLC.1.2.1GC is the separation technique of choice for organic compounds whichcan be volatilized without being decomposed or chemically rearranged.1.2.2HPLC is a separation technique useful for semivolatile and non-volatilechemicals or for analytes that decompose upon heating. Successful liquidchromatographic separation requires that the analyte(s) of interest be soluble in thesolvent(s) selected for use as the mobile phase.1.3All chromatographic processes achieve separation by passing a mobile phase overa stationary phase. Constituents in a mixture are separated because they partition differentlybetween the mobile and stationary phases and thus have different retention times.Compounds that interact strongly with the stationary phase elute slowly (i.e., longer retentiontimes), while compounds that remain in the mobile phase elute quickly (i.e., shorter retentiontimes).1.3.1The mobile phase for GC is an inert gas, usually hydrogen or helium, andthe stationary phases are generally polymer bases.SW-846 Update VI8000D- 3Revision 5March 2018
1.3.2In "normal phase" HPLC, the mobile phase is less polar than thestationary phase. In "reverse phase" HPLC, the converse is true. Reverse phase HPLCis the technique of choice for environmental and waste analyses of non-volatile organictarget analytes.1.3.3Ion exchange chromatography is used to separate ionic species throughcompetition with ions in the mobile phase for oppositely charged exchange sites on astationary phase. Differential selectivities of the ionic species and the mobile phase ionsfor exchange sites are responsible for the chromatographic separation of the ions.1.4A number of specific GC and liquid chromatography (LC) techniques are used forenvironmental and waste analyses. Specific techniques are distinguished by thechromatographic hardware and chemical mechanisms used to achieve separations.1.4.1GC methods, including those in SW-846, can be categorized on the basisof the chromatographic columns employed.1.4.2HPLC methods in SW-846 are categorized on the basis of themechanism of separation.1.5SW-846 methods describe columns and conditions that have been demonstratedto provide optimum separation of all or most target analytes listed in that specific procedure.Most often, those columns were the ones used by EPA during method development and testing.Analysts may change those columns and conditions, provided that they demonstrateperformance for the analytes of interest that is appropriate for the intended application. This isespecially true when limited groups of analytes are to be monitored (i.e., if only a subset of thelist of target analytes in a method are needed, the chromatographic conditions and columnsmay be optimized for those analytes).1.5.1Chromatographic performance is demonstrated by the resolution ofstandards and the ability to model the response of the detector during calibration, and bysensitivity, precision, bias, frequency of false positives, and frequency of false negativesduring analysis. The laboratory must demonstrate that any chromatographic procedure ituses provides performance satisfying the analytical requirements of the specificapplication for which it is being used. Such demonstrations should be performed usingthe procedures outlined in Secs. 9.2 to 9.8 of this method and appropriate sections inChapter One.1.5.2Laboratories must also be cautious whenever the use of two dissimilarcolumns is included in a method for confirmation of identification and quantitation. Forinstance, a DB-5 column generally cannot be used for confirmation of results obtainedusing an SPB-5 column because the stationary phases are not sufficiently dissimilar andthe changes in elution order (if any) will not provide adequate confirmation.1.6When GC conditions are changed, retention times and analytical separations areoften affected. For example, increasing the oven temperature changes the rate of partitioningbetween the mobile and stationary phases, leading to shorter retention times. GC retentiontimes can also be changed by selecting a column with a different length, stationary-phaseloading (i.e., capillary column film thickness or percent loading for packed columns), oralternative liquid phase. As a result, two critical aspects of any SW-846 chromatographicmethod are the determination and/or verification of retention times and analyte separation.SW-846 Update VI8000D- 4Revision 5March 2018
1.7HPLC retention times and analytical separations are also affected by changes inthe mobile and stationary phases. The HPLC mobile phase is easily altered by adjusting thecomposition of the solvent mixture being pumped through the column. In reverse phase HPLC,increasing the ratio of water-miscible organic solvent to water generally shortens retentiontimes. HPLC retention times can also be changed by selecting a column with a different length,alternative bonded phase, or dissimilar particle size (e.g., smaller particles and/or a longercolumn generally increase column resolution, while different bonded phases may resolvespecific components differently). HPLC methods are also particularly sensitive to smallchanges in chromatographic conditions, including temperature. HPLC column temperaturecontrol ovens should be used to maintain constant retention times because ambient laboratorytemperatures may fluctuate throughout the day. SW-846 methods provide conditions that havebeen demonstrated to provide good HPLC separations using specific instruments to analyze alimited number of samples. Analysts (particularly those using HPLC/MS) may need to tailor thechromatographic conditions listed in the method for their specific application and/or instrument.1.8Chromatographic methods can be used to produce data of appropriate quality forthe analysis of environmental and waste samples. However, data quality can be greatlyenhanced when the analyst understands both the intended use of the results and the limitationsof the specific analytical procedures employed. Therefore, these methods are recommendedfor use only by, or under the close supervision of, experienced analysts. Many difficultiesobserved in the performance of SW-846 methods for the analysis of RCRA wastes can beattributed to the lack of skill and training of the analyst.1.8.1Methods using selective (e.g., PID, NPD, ELCD) or non-selective (e.g.,FID) detectors may present serious difficulties when used for site investigations, includingcoelution of target analytes, false negatives due to retention time shifts, and false positivesand quantitation errors due to coeluting non-target sample components.1.8.2In contrast, GC methods employing selective or non-selective detectorsmay be appropriate for remediation activities where the analytes of concern are known, oflimited number, and of significantly greater concentration than potentially interferingmaterials.1.8.3If the site is not well characterized, and especially if large numbers oftarget analytes are of concern, analysis by GC/MS or HPLC/MS may be more appropriate.1.9Each chromatographic method includes a list of the compounds recommended foranalysis given the procedures described therein. Lists in some methods are lengthy; it may notbe practical or appropriate to determine all the analytes simultaneously. Such analyte lists donot imply a regulatory requirement for the analysis of any or all of the compounds, but ratherindicate the method(s) applicable to those compounds.1.10 Analysts should consult the disclaimer statement at the front of the manual and theinformation in Chapter Two for 1) guidance on the intended flexibility in the choice of methods,apparatus, materials, reagents, and supplies; and 2) the responsibilities of the analyst fordemonstrating that the techniques employed are appropriate for the analytes of interest, in thematrices of interest, and at the levels of concern.In addition, analysts and data users are advised that, except where explicitly required in aregulation, the use of SW-846 methods is not mandatory in response to Federal testingrequirements. The information contained in this method is provided by EPA as guidance forthe analyst and regulated community in making judgments necessary to generate resultsmeeting the data quality requirements for the intended application.SW-846 Update VI8000D- 5Revision 5March 2018
1.11 All of the SW-846 determinative chromatographic methods that reference thismethod are restricted to use by, or under the supervision of, analysts experienced in the use ofgas or high-performance liquid chromatographs and skilled in the interpretation ofchromatograms. Each analyst must demonstrate the ability to generate an acceptable initialdemonstration of proficiency (IDP) along with acceptable results according to methodrecommendations and stated project data quality objectives (DQOs). Method 8000 is intendedto be a supplement to, but is not intended to be a substitute for, formal training in the basicprinciples of GC, GC/MS, LC, LC/MS or HPLC.2.0SUMMARY OF METHODThis method describes general considerations in achieving chromatographic separationsand performing calibrations. It is to be used in conjunction with all SW-846 determinativechromatographic methods, including, but not limited to, each method listed in Sec. 1.1. Each ofthese chromatographic methods recommends appropriate procedures for sample preparation,extraction, cleanup, and/or derivatization. Consult the specific procedures for additionalinformation on these crucial steps in the analytical process.2.1Sec. 4.2 of this method provides general guidance on minimizing contamination,including cross-contamination between samples. Sample screening procedures are stronglyrecommended and discussed in Sec. 4.3.2.2Before any sample or blank is introduced into a chromatographic system, theappropriate resolution criteria and calibration procedure(s) described in Method 8000 must besatisfied.2.3Secs. 4.4 and 4.5 provide information on the effects of chromatographicinterferences.2.4Sec 6.0 of this method contains generalized specifications for the components ofboth GC and HPLC systems used in SW-846 analyses.2.5Calibration of the analytical system is another critical step in the generation ofquality data. Sec. 11.5 discusses specific procedures and calculations for both linear and nonlinear calibration models. Continued use of any chromatographic procedure necessitates averification of the calibration model, and procedures for such verifications are described in thismethod as well (Sec. 11.7).2.6Identification of target compounds by any chromatographic procedure is based, atleast in part, on retention times. Sec. 11.6 provides procedures for the determination ofretention times and retention time windows to be used with the specific methods listed in Sec.1.1.2.7Calculations necessary to derive sample-specific concentrations from theinstrument responses are common to most of the analytical methods listed in Sec. 1.1.Commonly used calculations are summarized in Sec. 11.10.2.8Preventive maintenance and corrective actions are essential to the generation ofquality data in a routine laboratory setting. Suggestions for such procedures are found in Sec.11.11.SW-846 Update VI8000D- 6Revision 5March 2018
2.9Most of the methods listed in Sec. 1.1 employ a common approach to QC. Whilesome of the overall procedures are described in Chapter One, Sec. 9.0 describes routinely usedprocedures for calibration verification, instrument performance checks, demonstratingacceptable performance, etc.2.10 Before performing analyses of specific samples, analysts should work with datausers to determine acceptable recovery ranges for all target analytes of interest in the type ofmatrices to be tested. Analysts must also be able to demonstrate that the sensitivity of theprocedure employed is appropriate for the intended application. One approach to such ademonstration is to estimate the method sensitivity for the analytes of interest using theprocedures in Chapter One or other appropriate procedures.3.0DEFINITIONSRefer to Chapter One, the individual determinative methods, and the manufacturer’sinstructions for definitions that may be relevant.4.0INTERFERENCES/CHROMATOGRAPHIC PERFORMANCE4.1Solvents, reagents, glassware, and other sample processing hardware may yieldartifacts and/or interferences during sample analysis. All of these materials must bedemonstrated to be free from interferences under conditions of analysis by analyzing methodblanks. Specific selection of reagents and purification of solvents by distillation in all-glasssystems may be necessary. Refer to each method to be used for specific guidance on QCprocedures and to Chapter Four for general guidance on the cleaning of glassware.4.2Contamination by carryover can occur whenever high- and low-concentrationsamples are analyzed in sequence. To reduce potential for carryover, the sample syringe orpurging device must be thoroughly rinsed between samples with an appropriate solvent(including water). Purge-and-trap or headspace devices should be thoroughly baked outbetween samples.Where practical, samples with unusually high concentrations of analytes should befollowed by method blanks, instrument blanks, or by analysis of organic-free reagent water tocheck for carryover contamination. If target compounds present in an unusually highlyconce
1.2 Analytical chromatography is used to separate target analytes from co-extracted interferences in samples. Chromatographic methods can be divided into two major categories: GC and HPLC. 1.2.1 GC is the separation technique of choice for organic compounds which can be volatilized without being decomposed or chemically rearranged.
interferences in samples. Chromatographic methods can be divided into two major categories: gas chromatography (GC) and high performance liquid chromatography (HPLC). 1.2.1 Gas chromatography (more properly called gas-liquid chromatography) is the separation technique of choice for organic compounds which can be volatilized without being
chromatography, but now is commonly referred to as hi gh performance liquid chromatography. 1.3 All chromatographic processes achieve separation by passing a mobile phase over a stationary phase. Constituents in a mixture are separated because they partition differently between
Chapter 26: An Introduction to Chromatographic Separations Column Chromatography Migration Rates – Distribution Contstants – Retention Times – Selectivity Factor Zone Broadening & Column Efficiency Optimizing Performance Resolution Intro to Chromatography
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The general elution problem in chromatography. 26E Summary of chromatographic relationships . The number of quantities, terms, and relationships employed in chromatography is large and often confusing. Tables 26 -4 and 26-5 summarize the most important definitions and equations
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Please cite this article as: Deepika N et al., A Review on Bioanalytical Chromatographic Method Development for Quantification & Validation of Cysteinyl Leukotriene Receptor-Antagonists in Plasma Matrices. American Journal of PharmTech Research 2018. A Review on
Bribery and Corruption – V05.0 UPR GV12 - Effective: 9 June 2020 Bribery and Corruption . UPR GV12 version 05.0 . Policies superseded by this document . This document replaces version 04.0 of UPR GV12, with effect from 9 June 2020. Summary of significant changes to the previous version . The threshold for personal gifts in section 8.2.2 has been raised from 20 to 60. References to HEFCE .
