**SW-846 Update V 8000D- 1 Revision 4**

**October 2012 METHOD 8000D** 

**DETERMINATIVE CHROMATOGRAPHIC SEPARATIONS**

**SW-846 is not intended to be an analytical training manual. Therefore, method procedures**  **are written based on the assumption that they will be performed by analysts who are formally**  **trained in at least the basic principles of chemica l analysis and in the use of the subject**  **technology.**

**In addition, SW-846 methods in this manual, with th e exception of required use for the**  **analysis of method-defined parameters, are intended to be guidance documents. They contain**  **general information on how to perform an analytical procedure or technique which a laboratory**  **can use as a basic starting point for generating it s own detailed standard operating procedure**  **(SOP), either for its own general use or for a spec ific project application. Performance data**  **included in this method are for guidance purposes o nly and must not be used as absolute quality**  **control (QC) acceptance criteria for the purpose of laboratory QC or accreditation.** 

**1.0 SCOPE AND APPLICATION**

**Please see Appendix A at the back of this document for a summary of revisions to Method**  **8000C (From Revision 3, March 2003).**

**1.1 Method 8000 is not a determinative method but i nstead provides guidance on**  **analytical chromatography and describes calibration and QC requirements common to all**  **SW-846 chromatographic methods. However, more spec ific QC requirements provided in the**  **applicable determinative method will supersede thos e noted in Method 8000. Method 8000**  **should be applied in conjunction with all SW-846 de terminative chromatographic methods. The**  **methods include, but are not limited to, the follow ing:** 

**Method**

**Number Analytes**  **Chromatographic Technique (Sec. 1.5)**  **Detector**

**6850 Perchlorate**  **HPLC MS, MS/MS** 

**6860 Perchlorate**  **IC MS, MS/MS** 

**7580 White phosphorus (P**  **4 )**  **GC NICI/MS** 

**8011 EDB, DBCP**  **GC, capillary column ECD** 

**8015 Non-halogenated volatiles**  **GC, packed & capillary**

**column FID** 

**8021 Volatiles**  **GC, capillary column PID, ELCD** 

**8031 Acrylonitrile**  **GC, packed column NPD** 

**8032 Acrylamide**  **GC, packed column ECD** 

**8033 Acetonitrile**  **GC, capillary column NPD** 

**8041 Phenols**  **Underivatized or derivatized; GC, capillary** 

**column FID, ECD** 

**8061 Phthalates**  **GC, capillary column ECD** 

**8070 Nitrosamines**  **GC, packed column NPD, ELCD, TED** 

**8081 Organochlorine pesticides**  **GC, capillary column ECD, ELCD** **SW-846 Update V 8000D- 2 Revision 4**

**October 2012 Method** 

**Number Analytes**  **Chromatographic Technique (Sec. 1.5)**  **Detector**

**8082 Polychlorinated biphenyls**  **GC, capillary column ECD, ELCD** 

**8091 Nitroaromatics and cyclic** 

**ketones GC, capillary column**  **ECD**

**8100 Polynuclear aromatic** 

**hydrocarbons GC, packed & capillary** 

**column FID** 

**8111 Haloethers**  **GC, capillary column ECD** 

**8121 Chlorinated hydrocarbons**  **GC, capillary column ECD** 

**8131 Aniline and selected** 

**derivatives GC, capillary column**  **NPD**

**8141 Organophosphorus pesticides**  **GC, capillary column FPD, NPD, ELCD** 

**8151 Acid herbicides**  **Derivatized; GC, capillary**

**column ECD** 

**8260 Volatiles**  **GC, capillary column MS** 

**8261 Volatiles**  **GC, capillary column MS** 

**8265 Volatiles**  **NA DS/ITMS** 

**8270 Semivolatiles**  **GC, capillary column MS** 

**8275 Semivolatiles**  **Thermal extraction/GC MS** 

**8276 Toxaphene and Congeners**  **GC, capillary column NICI/MS** 

**8280 Dioxins and Dibenzofurans**  **GC, capillary column Low resolution MS** 

**8290 Dioxins and Dibenzofurans**  **GC, capillary column High resolution MS** 

**8310 Polynuclear aromatic** 

**hydrocarbons HPLC, reverse phase**  **UV, Fluorescence**

**8315 Carbonyl compounds**  **Derivatize; HPLC UV** 

**8316 Acrylamide, acrylonitrile,** 

**acrolein HPLC, reverse phase**  **UV**

**8318 N-Methyl carbamates**  **Derivatize; HPLC Fluorescence** 

**8321 Extractable non-volatiles**  **PLC, reverse phase TS/MS, UV** 

**8323 Organotin compounds**  **HPLC, reverse phase ES/ITMS** 

**8325 Extractable non-volatiles**  **HPLC, reverse phase PB/MS, UV** 

**8330 Nitroaromatics and nitramines**  **HPLC, reverse phase UV** 

**8331 Tetrazene**  **HPLC, ion pair, reverse phase**  **UV**

**8332 Nitroglycerine**  **HPLC, reverse phase UV** 

**8410 Semivolatiles**  **GC, capillary column FT-IR** 

**8430 Bis(2-chloroethyl) ether** 

**hydrolysis products GC, capillary column**  **FT-IR**

**DBCP = Dibromochloropropane DS/ITMS = Direct sampling/ion trap mass spectrometr**  **y ECD = Electron capture detector**  **EDB = Ethylene dibromide ES/ITMS = Electrospray ionization/ion trap mass** 

**spectrometry ELCD = Electrolytic conductivity detector** 

**FID = Flame ionization detector FPD = Flame photometric detector** 

**FT-IR = Fourier transform-infrared GC = Gas chromatography**  **HPLC = High performance liquid chromatography MS = Mass spectrometry**  **MS/MS = Mass spectrometry/Mass spectrometry NICI/MS = Negative Ion Chemical Ionization/Mass** 

**spectrometry NPD = Nitrogen/phosphorous detector**  **NA = Not applicable PAHs = Polynuclear aromatic hydrocarbons**  **PB/MS = Particle beam mass spectrometry**

**PID = Photoionization detector**

**TED = Thermionic emission detector TS/MS = Thermospray mass spectrometry** 

**UV = Ultraviolet SW-846 Update V 8000D- 3 Revision 4** 

**October 2012 1.2 Analytical chromatography is used to separate t**  **arget analytes from co-extracted 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 is the separation techniqu e of choice for organic**  **compounds which can be volatilized without being de composed or chemically rearranged.** 

**1.2.2 High performance liquid chromatography (HPLC) is a separation technique** 

**useful for semivolatile and non-volatile chemicals or for analytes that decompose upon**  **heating. Successful liquid chromatographic separat ion requires that the analyte(s) of**  **interest be soluble in the solvent(s) selected for use as the mobile phase.** 

**1.3 All chromatographic processes achieve separatio n by passing a mobile phase over**  **a stationary phase. Constituents in a mixture are separated because they partition differently**  **between the mobile and stationary phases and thus h ave different retention times. Compounds**  **that interact strongly with the stationary phase el ute slowly (i.e., longer retention times), while**  **compounds that remain in the mobile phase elute qui ckly (i.e., shorter retention times).** 

**1.3.1 The mobile phase for GC is an inert gas, usua lly hydrogen or helium, and**  **the stationary phases are generally polymer bases.**

**1.3.2 In "normal phase" HPLC, the mobile phase is l ess polar than the stationary**  **phase. In "reverse phase" HPLC, the converse is tru e. Reverse phase HPLC is the**  **technique of choice for environmental and waste ana lyses of non-volatile organic target**  **analytes.**

**1.3.3 Ion exchange chromatography is used to separa te ionic species through**  **competition with ions in the mobile phase for oppos itely charged exchange sites on a**  **stationary phase. Differential selectivities of th e ionic species and the mobile phase ions**  **for exchange sites are responsible for the chromato graphic separation of the ions.** 

**1.4 A number of specific GC and LC techniques are u sed for environmental and waste**  **analyses. Specific techniques are distinguished by the chromatographic hardware and chemical**  **mechanisms used to achieve separations.**

**1.4.1 GC methods, including those in SW-846, can be categorized on the basis**  **of the chromatographic columns employed.**

**1.4.2 HPLC methods in SW-846 are categorized on the basis of the mechanism**  **of separation.**

**1.5 SW-846 methods describe columns and conditions that have been demonstrated to**  **provide optimum separation of all or most target an alytes listed in that specific procedure. Most**  **often, those columns were the ones used by EPA duri ng method development and testing.**  **Analysts may change those columns and conditions, p rovided that they demonstrate**  **performance for the analytes of interest that is ap propriate for the intended application. This is**  **especially true when limited groups of analytes are to be monitored (i.e., if only a subset of the lis**  **t of target analytes in a method are needed, the chro**  **matographic conditions and columns may be optimized for those analytes).** 

**1.5.1 Chromatographic performance is demonstrated b y the resolution of**  **standards and the ability to model the response of the detector during calibration, and by** **SW-846 Update V 8000D- 4 Revision 4**

**October 2012 sensitivity, precision, bias, frequency of false po**  **sitives, and frequency of false negatives during analysis. The laboratory must demonstrate t**  **hat any chromatographic procedure it uses provides performance satisfying the analytical**  **requirements of the specific application for which it is being used. Such demonstrations sh**  **ould be performed using the procedures outlined in Secs. 9.2 to 9.8 of this method and app**  **ropriate sections in Chapter One.**

**1.5.2 Laboratories must also be cautious whenever t he use of two dissimilar** 

**columns is included in a method for confirmation of identification and quantitation. For**  **instance, a DB-5 column generally cannot be used fo r confirmation of results obtained using**  **an SPB-5 column because the stationary phases are n ot sufficiently dissimilar and the**  **changes in elution order (if any) will not provide adequate confirmation.** 

**1.6 When GC conditions are changed, retention times and analytical separations are**  **often affected. For example, increasing the oven t emperature changes the rate of partitioning**  **between the mobile and stationary phases, leading t o shorter retention times. GC retention**  **times can also be changed by selecting a column wit h a different length, stationary-phase loading**  **(i.e., capillary column film thickness or percent l oading for packed columns), or alternative liquid**  **phase. As a result, two critical aspects of any SW -846 chromatographic method are the** 

**determination and/or verification of retention time s and analyte separation.** 

**1.7 HPLC retention times and analytical separations are also affected by changes in the**  **mobile and stationary phases. The HPLC mobile phas e is easily altered by adjusting the**  **composition of the solvent mixture being pumped thr ough the column. In reverse phase HPLC,**  **increasing the ratio of water-miscible organic solv ent to water generally shortens retention times.**  **HPLC retention times can also be changed by selecti ng a column with a different length,**  **alternative bonded phase, or dissimilar particle si ze (e.g., smaller particles and/or a longer column**  **generally increase column resolution, while differe nt bonded phases may resolve specific**  **components differently). HPLC methods are also par ticularly sensitive to small changes in**  **chromatographic conditions, including temperature. HPLC column temperature control ovens** 

**should be used to maintain constant retention times because ambient laboratory temperatures**  **may fluctuate throughout the day. SW-846 methods p rovide conditions that have been**  **demonstrated to provide good HPLC separations using specific instruments to analyze a limited**  **number of samples. Analysts (particularly those us ing HPLC/MS) may need to tailor the**  **chromatographic conditions listed in the method for their specific application and/or instrument.** 

**1.8 Chromatographic methods can be used to produce data of appropriate quality for the**  **analysis of environmental and waste samples. Howev er, data quality can be greatly enhanced**  **when the analyst understands both the intended use of the results and the limitations of the**  **specific analytical procedures employed. Therefore , these methods are recommended for use**  **only by, or under the close supervision of, experie nced analysts. Many difficulties observed in**  **the performance of SW-846 methods for the analysis of RCRA wastes can be attributed to the**  **lack of skill and training of the analyst.**

**1.8.1 Methods using selective (e.g., PID, NPD, ELCD ) or non-selective (e.g.,**  **FID) detectors may present serious difficulties whe n used for site investigations, including**  **coelution of target analytes, false negatives due t o retention time shifts, and false positives**  **and quantitation errors due to coeluting non-target sample components.** 

**1.8.2 In contrast, GC methods employing selective o r non-selective detectors**  **may be appropriate for remediation activities where the analytes of concern are known, of**  **limited number, and of significantly greater concen tration than potentially interfering** 

**materials. SW-846 Update V 8000D- 5 Revision 4** 

**October 2012 1.8.3 If the site is not well characterized, and es**  **pecially if large numbers of target analytes are of concern, analysis by GC/MS or HPLC/**  **MS may be more appropriate.**

**1.9 Each chromatographic method includes a list of the compounds recommended for**  **analysis given the procedures described therein. L ists in some methods are lengthy; it may not**  **be practical or appropriate to determine all the an alytes simultaneously. Such analyte lists do**  **not imply a regulatory requirement for the analysis of any or all of the compounds, but rather**  **indicate the method(s) applicable to those compound s.** 

**1.10 Analysts should consult the disclaimer stateme nt at the front of the manual and the**  **information in Chapter Two for 1) guidance on the i ntended flexibility in the choice of methods,**  **apparatus, materials, reagents, and supplies; and 2 ) the responsibilities of the analyst for**  **demonstrating that the techniques employed are appr opriate for the analytes of interest, in the**  **matrices of interest, and at the levels of concern.**

**In addition, analysts and data users are advised th at, except where explicitly required in a**  **regulation, the use of SW-846 methods is not mandat ory in response to Federal testing**  **requirements. The information contained in this me thod is provided by EPA as guidance for the**  **analyst and regulated community in making judgments necessary to generate results meeting the**  **data quality requirements for the intended applicat ion.** 

**1.11 All of the SW-846 determinative chromatographi c methods that reference this**  **method are restricted to use by, or under the super vision of, analysts experienced in the use of**  **gas or high performance liquid chromatographs and s killed in the interpretation of**  **chromatograms. Each analyst must demonstrate the a bility to generate an acceptable initial**  **demonstration of proficiency (IDP) along with accep table results according to method**  **recommendations and stated project data quality obj ectives (DQOs). Method 8000 is intended**  **to be a supplement to but is NOT intended to be a substitute for formal training in**  **the basic principles of gas or high performance liquid chroma**  **tography.**

**2.0 SUMMARY OF METHOD**

**This method describes general considerations in ach ieving chromatographic separations**  **and performing calibrations. It is to be used in c onjunction with all SW-846 determinative**  **chromatographic methods, including, but not limited to, each method listed in Sec. 1.1. Each of**  **these chromatographic methods recommends appropriat e procedures for sample preparation,**  **extraction, cleanup, and/or derivatization. Consul t the specific procedures for additional**  **information on these crucial steps in the analytica l process.** 

**2.1 Sec. 4.2 of this method provides general guidan ce on minimizing contamination,**  **including cross-contamination between samples. Sam ple screening procedures are strongly**  **recommended, and discussed in Sec. 4.3.**

**2.2 Before any sample or blank is introduced into a chromatographic system, the**  **appropriate resolution criteria and calibration pro cedure(s) described in Method 8000 must be** 

**satisfied.**

**2.3 Secs. 4.4 and 4.5 provide information on the ef fects of chromatographic** 

**interferences.**

**2.4 Sec 6.0 of this method contains generalized spe cifications for the components of**  **both GC and HPLC systems used in SW-846 analyses. SW-846 Update V 8000D- 6 Revision 4** 

**October 2012 2.5 Calibration of the analytical system is another**  **critical step in the generation of quality data. Sec. 11.5 discusses specific procedures and c**  **alculations for both linear and non-linear calibration models. Continued use of any chromatog**  **raphic procedure necessitates a verification of the calibration model, and procedures for such v**  **erifications are described in this method as well (Sec. 11.7).** 

**2.6 Identification of target compounds by any chrom atographic procedure is based, at**  **least in part, on retention times. Sec. 11.6 provi des procedures for the determination of retention** 

**times and retention time windows to be used with th e specific methods listed in Sec. 1.1.** 

**2.7 Calculations necessary to derive sample-specifi c concentrations from the instrument**  **responses are common to most of the analytical meth ods listed in Sec. 1.1. Commonly used** 

**calculations are summarized in Sec. 11.10.**

**2.8 Preventive maintenance and corrective actions a re essential to the generation of**  **quality data in a routine laboratory setting. Sugg estions for such procedures are found in Sec.** 

**11.11.**

**2.9 Most of the methods listed in Sec. 1.1 employ a common approach to QC. While**  **some of the overall procedures are described in Cha pter One, Sec. 9.0 describes routinely used**  **procedures for calibration verification, instrument performance checks, demonstrating acceptable**  **performance, etc.**

**2.10 Before performing analyses of specific samples , analysts should work with data**  **users to determine acceptable recovery ranges for a ll target analytes of interest in the type of**  **matrices to be tested. Analysts must also be able to demonstrate that the sensitivity of the**  **procedure employed is appropriate for the intended application. One approach to such a**  **demonstration is to estimate the method sensitivity for the analytes of interest using the**  **procedures in Chapter One or other appropriate proc edures.** 

**3.0 DEFINITIONS**

**Refer to Chapter One, the individual determinative methods, and the manufacturer's**  **instructions for definitions that may be relevant.**

**4.0 INTERFERENCES/CHROMATOGRAPHIC PERFORMANCE**

**4.1 Solvents, reagents, glassware, and other sample processing hardware may yield**  **artifacts and/or interferences during sample analys is. All of these materials must be** 

**demonstrated to be free from interferences under co nditions of analysis by analyzing method**  **blanks. Specific selection of reagents and purific ation of solvents by distillation in all-glass**  **systems may be necessary. Refer to each method to be used for specific guidance on QC**  **procedures and to Chapter Four for general guidance on the cleaning of glassware.** 

**4.2 Contamination by carryover can occur whenever h igh- and low-concentration**  **samples are analyzed in sequence. To reduce potent ial for carryover, the sample syringe or**  **purging device must be thoroughly rinsed between sa mples with an appropriate solvent (including**  **water). Purge-and-trap or headspace devices should be thoroughly baked out between**  **samples. SW-846 Update V 8000D- 7 Revision 4** 

**October 2012 Where practical, samples with unusually high concen**  **trations of analytes should be followed by method blanks or instrument blanks or by analysi**  **s of organic-free reagent water to check for carryover contamination. If target compounds prese**  **nt in an unusually highly concentrated sample are also found to be present in subsequent s**  **amples, the analyst must demonstrate that the compounds are not affected by**  **carryover contamination. Conversely, if those targ et**  **compounds are not present in the subsequent sample(s), then they do**  **not need to be reanalyzed.**

**Purging vessels may be cleaned by rinsing with meth anol, followed by a distilled water rinse**  **and drying in a 105** ° **C oven between analyses. Detergent solutions may a lso be used, but care**  **must be taken to remove the detergent residue from the purging vessel. Other approaches to**  **cleaning purging vessels, such as some modern autos amplers which rinse the vessel(s) between**  **runs, may also be employed, provided that the labor atory can demonstrate that they are effective**  **in removing contaminants.**

**4.3 In addition to carryover of compounds from one sample to the next, the analysis of**  **high-concentration samples can lead to contaminatio n of the analytical instrument itself.**  **Eliminating this contamination can cost significant time and effort that cannot be spent analyzing**  **samples. The most reliable procedure for ensuring minimum down time is to screen samples by**  **a higher level technique. Samples to be analyzed f or volatiles can be screened using an**  **automated headspace sampler (Method 5021) connected to a GC/PID/ELCD detector (Method**  **8021) or by analyzing large (** e.g. **, 100-fold) dilutions of the samples on the GC/MS. Samples to be**  **analyzed for semivolatiles can be screened using GC /FID. Other screening methods are also**  **acceptable. The analyst should use screening resul ts to choose an appropriate dilution factor for**  **the GC/MS analysis that will prevent system contami nation yet still provide adequate sensitivity**  **for the major constituents of the sample.**

**4.4 Elevated chromatographic baselines (e.g., basel ine humps) should be minimized or**  **eliminated during these analyses by application of appropriate sample clean-up (Method 3600),**  **extract dilution, use of pre-columns and/or inserts , or employing a selective detector. Integration**  **of "hump-o-grams" can result in significant quantit ative errors. When elevated baselines are**  **observed during analysis of blanks and standards, t he chromatographic system should be** 

**considered contaminated. This contamination can re sult from impure carrier gas, inadequate**  **gas conditioning, septum bleed, column oxidation, i ncomplete elution of non-target interferences,**  **and/or pyrolysis products in the injector or column . Such contamination is unacceptable and**  **must be addressed through a program of preventive m aintenance and corrective action.** 

**4.5 See Sec. 11.11 for suggested preventative maint enance activities that may prevent**  **or ameliorate deterioration of chromatographic perf ormance.** 

**5.0 SAFETY**

**This method does not address all safety issues asso ciated with its use. The laboratory is**  **responsible for maintaining a safe work environment and a current awareness file of OSHA**  **regulations regarding the safe handling of the chem icals and instrumentation included in this**  **method. A reference file of material safety data s heets (MSDSs) should be available to all**  **personnel involved in these analyses.**

**6.0 EQUIPMENT AND SUPPLIES**

**6.1 Mention of trade names or commercial products i n this manual is for illustrative**  **purposes only, and does not constitute an EPA endor sement or exclusive recommendation for** **SW-846 Update V 8000D- 8 Revision 4**

**October 2012 use. Products and instrument settings cited in SW-**  **846 methods represent those products and settings used during method development or subseque**  **ntly evaluated by the Agency. Glassware, reagents, supplies, equipment, and setti**  **ngs other than those listed in this manual may be employed provided that method performance ap**  **propriate for the intended application has**

**been demonstrated and documented.**

**This section does not list all common laboratory g lassware (e.g., beakers and flasks) that**  **might be used.**

**6.2 GC inlet systems**

**6.2.1 Volatile organics**

**Volatile organic analytes are introduced into a GC through a purge-and-trap system,**  **by direct injection, or other technologies. The pu rge-and-trap apparatus is described in**  **Method 5030 for water samples and in Method 5035 fo r soil and other solid samples. See**  **Method 5000 for guidance on all forms of sample int roduction of volatiles into the GC and**  **GC/MS system.**

**6.2.2 Semivolatile organics**

**Sample extracts containing semivolatile organic com pounds are introduced into a**  **GC with a syringe that passes through a septum into an injection port. The injection port**  **allows the sample extract to be vaporized prior to being flushed onto the GC column, hence**  **the term "gas" chromatography. Correct setup and m aintenance of the injector port is**  **necessary to achieve acceptable performance with GC methods.** 

**6.2.2.1 An injection port septum and liner should be installed in the GC**  **inlet as appropriate for the system.**

**6.2.2.2 Packed columns and wide-bore capillary colu mns (> 0.32-mm**  **ID) should be mounted in 1/4-inch injectors.**

**6.2.2.3 Narrow-bore capillary columns (≤ 0.32 mm ID ) should be**  **mounted in split/splitless (Grob-type) injectors. Split/splitless injectors should** 

**have automated valve closures that direct most of t he flow (and sample) onto the**  **head of the analytical column. After a predetermin ed splitless introduction time the**  **split valve is opened so that most of the flow is v ented during analysis, thus**  **eliminating the solvent tail while maintaining prop er flow through the column. The**  **initial oven temperature should be below the boilin g point of the injection solvent if**  **the solvent front interferes with early-eluting ana lytes or if the solvent effect is**  **needed to resolve difficult to separate analytes.**

**6.2.2.4 Cool on-column injection and programmable t emperature**  **vaporizer inlets allow the analysis of labile compo unds that degrade on packed**  **columns and in split/splitless injectors.**

**6.3 GC flow control**

**Precise control of the gas mobile phase is necessar y to achieve reproducible GC retention**  **times. Flow controllers within any GC used for ana lyses described in SW-846 methods should**  **deliver a precisely metered gas flow at a rate appr opriate for the GC column mounted in the** 

**instrument. SW-846 Update V 8000D- 9 Revision 4** 

**October 2012 6.3.1 Most GCs have restrictors built into electron**  **ic pressure controllers (EPCs) monitored using a digital readout. These restricto**  **rs are used to provide precise flow at the carrier gas flow rate listed in the method (e.g., u**  **se <20 mL/min restrictors for wide-bore capillary methods).** 

**6.3.2 Analysts should ensure that cylinder pressure s are regulated properly and**  **manifold pressures are sufficiently large that a ch ange in the head pressure of an individual**  **instrument does not affect the flow through all ins truments. Toggle valves that allow** 

**instruments to be isolated are recommended for all multi-instrument gas delivery systems.** 

**6.3.3 Carrier gas should be of high purity, and con ditioned between the cylinder**  **and the GC with a scrubber to remove any residual w ater, oxygen and hydrocarbons as**  **necessary. Gas regulators should contain stainless steel diaphragms. (Neoprene**  **diaphragms are a potential source of gas contaminat ion, and should not be used.)** 

**6.4 GC columns**

**Each determinative method in SW-846 provides a desc ription of a chromatographic column**  **or columns with associated column specifications. Other GC columns may be substituted in**  **SW-846 methods to improve performance if (1) the cr iteria described in Sec. 9.3 are satisfied, and**  **(2) target analytes are sufficiently resolved from one another and from co-extracted interferences**  **to provide data of appropriate quality for the inte nded application.** 

**Use of capillary columns has become standard practi ce in environmental and waste**  **analysis. Capillary columns have an inherently gre ater ability to separate analytes than packed**  **columns. However, packed columns can provide adequ ate resolution of some analytes and are**  **most appropriately employed when the list of analyt es to be determined is relatively short.** 

**6.4.1 Narrower columns are more efficient (i.e., ca n resolve more analytes) but**  **have a lower capacity (i.e., can accept less sample without peak distortion).** 

**6.4.2 Longer columns can resolve more analytes; res olution increases as a**  **function of the square root of column length. Run- time is also increased.** 

**6.4.3 Columns with greater film thickness (i.e., lo ading) increases column**  **capacity and retention times.**

**6.5 GC detectors**

**Detectors are the transducers that respond to compo nents eluting from a GC column and**  **produce the electrical signal used for quantitative determinations. SW-846 analyses in this**  **manual are conducted using the detectors listed in Sec. 1.1. Except where otherwise**  **recommended by the instrument manufacturer, selecti ve non-MS detectors should be maintained** 

**at least 20** ° **C above the highest oven temperature employed to pr event condensation and**  **detector contamination. To prevent condensation be tween the GC and an MS detector, transfer**  **lines should be maintained at a temperature above t he highest temperature of the oven program,**  **or as specified by the instrument manufacturer. SW-846 Update V 8000D- 10 Revision 4** 

**October 2012 6.6 HPLC injectors** 

**Liquids are essentially uncompressible, so a mechan ical device is necessary that allows**  **introduction of the sample into a high pressure flo w without significant disruption in the flow rate**  **and hydraulic pressure. Normally, a 6-port valve i s used for this purpose. A sample loop is**  **isolated from the flow of the mobile phase and fill ed with a sample extract. (Larger sample loops**  **may be used to increase sensitivity; however, they may degrade chromatographic performance).**  **The extract is then injected by the valve being tur ned so that the mobile phase flows through the**  **loop. This procedure virtually eliminates dead vol ume in the injector and is fully compatible with**  **automated operation.**

**6.7 HPLC pumps**

**The mobile phase used for HPLC should be accurately pressurized before it enters the**  **injector. HPLC pumps are generally capable of deliv ering solvent at 5000 psi or above with**  **excellent precision. Rate of delivery depends on t he column used for the separation. Flow**  **rates should be checked by collecting column efflue nt in a graduated cylinder for a designated**  **time period.**

**Most pumping systems are capable of changing solven t concentration during an analysis**  **(i.e., gradient elution). Gradients are generated by either high**  **pressure mixing of two streams between the pump and the injector or by proportiona**  **l mixing of the solvents before they are pumped. In either case, solvent mixing can cause c**  **hanges in the solubility of dissolved gases, formation of bubbles in the mobile phase, or non-re**  **producible gradients.**

**6.8 HPLC Columns**

**HPLC columns are generally constructed of stainles s steel tubing and are sealed with**  **compression fittings. These columns should be cons tructed with minimum dead volume and a**  **narrow particle size distribution. Manufacturers p rovide columns bonded with dissimilar**  **functional groups (e.g., C 18**  **, cyano, TMS) and have different percent carbon loa ding.** 

**6.8.1 Use of high quality columns that are uniforml y packed with the appropriate**  **particle size and bonded phase will result in optim al chromatographic performance. For**  **example, columns with silica-based particles with f ree silol groups show less tailing of polar**  **materials (e.g., amines).**

**6.8.2 A smaller particle (and pore) size generally gives better resolution, higher**  **back pressure, and smaller sample capacity.**

**6.8.3 Lifetime and performance of HPLC columns can be improved through**  **proper maintenance. Analysts should filter sample extracts and use compatible guard** 

**columns.**

**6.9 HPLC column temperature control ovens**

**HPLC retention times are more reproducible if the c olumn is held at a constant temperature.**  **Temperature control ovens capable of maintaining th e HPLC column at ± 0.1**  ° **C should be utilized to provide consistent retention times throughout th**  **e course of an HPLC analysis. Normal oven operating temperature should be 3 – 5**  ° **C above ambient laboratory temperature.**

**6.10 HPLC detectors SW-846 Update V 8000D- 11 Revision 4** 

**October 2012 Detectors are the transducers that respond to compo**  **nents eluting from a HPLC column and produce the electrical signal used for qualitative**  **and quantitative determinations. SW-846 analyses are conducted using selective detectors or**  **mass spectrometers listed in Sec. 1.1. HPLC/MS involves the use of a sophisticated interfa**  **ce that separates target analytes from the aqueous mobile phase. Examples include the thermos**  **pray (TSP), electrospray (ESP), and the atmospheric pressure chemical ionization (APCI) int**  **erfaces.**

**6.11 Data systems**

**Raw chromatographic data have to be reduced in orde r to provide the quantitative**  **information needed by analysts. Sophisticated data systems are strongly recommended for**  **SW-846 chromatographic methods because the ability to store and re-plot chromatographic data**  **is invaluable during data reduction and review. Or ganizations should select the system most**  **suitable for their applications.**

**6.12 Supplies**

**Chromatographers use a variety of supplies. Specif ic items that should be stocked depend**  **on laboratory instrumentation and the analyses perf ormed. At a minimum, laboratories need**  **PTFE tape, stainless steel regulators, acid-washed copper tubing, syringes, and replacement**  **parts for instruments.**

**6.12.1 Laboratories performing GC analyses also nee d supplies such as high**  **purity gases, scrubbers for gas conditioning, gas-t ight fittings, capillary cutters, magnifying**  **glasses, septa with proper temperature limits, appr opriate ferrules, dichlorodimethylsilane**  **(for deactivating surfaces), glass wool, spare colu mns and injection port liners.** 

**6.12.2 Laboratories performing HPLC analyses need s upplies such as high purity**  **solvents, column packing material, frits, narrow in ner diameter tubing, appropriate ferrules,**  **solvent filtration apparatus, and solvent degassing equipment.** 

**7.0 REAGENTS AND STANDARDS**

**7.1 Reagent grade chemicals should be used in all t ests. Unless otherwise indicated, it**  **is intended that all reagents conform to the specif ications of the Committee on Analytical**  **Reagents of the American Chemical Society, where su ch specifications are available. Other**  **grades may be used, provided the reagent is of suff iciently high purity to permit its use without**  **lessening the accuracy of the determination. Reage nts should be stored in glass to prevent**  **leaching of contaminants from plastic containers.**

**7.2 See specific extraction and determinative metho ds for the reagents and standards** 

**needed.**

**8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE**

**Sample collection, preservation and storage require ments may vary by EPA program and**  **may be specified in a regulation or project plannin g document that requires compliance**  **monitoring for a given contaminant. Where such req uirements are specified in the regulation,**  **they must be followed. In the absence of specific regulatory requirements, use the information in**  **Chapter Four as guidance in determining sample coll ection, preservation and storage** **SW-846 Update V 8000D- 12 Revision 4**

**October 2012 requirements. Additional information may be found**  **in some of the individual sample extraction, preparation, and determinative methods.** 

**9.0 QUALITY CONTROL**

**9.1 General Guidance**

**Refer to Chapter One for guidance on quality assura nce (QA) and quality control (QC)**  **protocols. When inconsistencies exist between QC g uidelines, method-specific QC criteria take**  **precedence over both technique-specific and Chapter One criteria; technique-specific QC criteria**  **take precedence over Chapter One criteria. Any eff ort involving collection of analytical data**  **should include development of a structured and syst ematic planning document, such as a Quality**  **Assurance Project Plan (QAPP) or a Sampling and Ana lysis Plan (SAP), which translates project**  **objectives and specifications into directions for t hose that will implement the project and assess** 

**the results.**

**Each laboratory should maintain a formal quality as surance program. The laboratory**  **should also maintain records to document the qualit y of the data generated. Development of**  **in-house QC limits for each method is encouraged, a s described in Sec. 9.6. Use of instrument**  **specific QC limits is encouraged, provided such lim its will generate data appropriate for use in the**  **intended application. All data sheets and quality control data should be maintained for reference**  **or inspection.**

**9.2 Evaluating chromatographic performance**

**The analyst's expertise in performing chromatograph y is a critical element in the successful**  **performance of chromatographic methods. Successful generation of data demands selection of**  **suitable preparative and determinative methods and an experienced staff to use these methods.** 

**9.2.1 For each 12-hour period during which analysi s is performed, the**  **performance of the instrument system should be chec ked. These checks should be part of**  **a formal quality control program that includes anal ysis of instrument blanks, calibration**  **standards, and other QC as appropriate for that met hod. In addition to these instrument**  **QC checks, performance of the entire analytical pro cess (i.e., preparation, cleanup and**  **analysis) should be monitored. These additional ch ecks should include method blanks,**  **matrix spikes, laboratory control samples, replicat e samples and other QC (e.g., the lower**  **limit of quantitation (LLOQ)) as appropriate for th at method or project. It is generally**  **advisable, although not required, that all method Q C samples be run at the same time as the**  **samples on the same instrument.**

**9.2.2 Ongoing data quality checks are compared wit h established performance**  **criteria to determine if the results of analyses me et the performance characteristics of the**  **method and/or the measurement quality objectives (M QOs) established for intended**  **application.**

**9.2.3 In addition to the quantitative measures of comparison described below**  **and in the individual methods, analysts should eval uate chromatograms and instrument**  **operation. Questions that should be asked include the following:** 

**Do the peaks look normal (Gaussian)? Is the response obtained comparable to the response**  **from previous calibrations? Are non-target peaks present in calibration analyse**  **s? SW-846 Update V 8000D- 13 Revision 4** 

**October 2012 Are contaminants present in the blanks?**  **Is the injector leaking (e.g., does the GC injector septum need replacing)?**  **Do the column fittings need tightening? Does the HPLC guard column need replacement?** 

**9.2.4 Significant peak tailing, leaks, changes in d etector response and laboratory**  **contamination should be corrected. Tailing problem s are generally traceable to active sites**  **on the column, cold spots in a GC, improper choice of HPLC mobile phase, the detector**  **inlet, or leaks in the system.**

**9.2.5 Recalibration of the instrument must take pla ce when performance**  **changes to the point that the calibration verificat ion acceptance criteria (Sec. 11.7) cannot** 

**be achieved. Recalibration of the instrument shoul d be performed as required per** 

**determinative methods.**

**9.2.6 Before processing any samples, the analyst sh ould demonstrate that all**  **parts of the equipment contacting the sample and re agents are interference-free. This is**  **accomplished through the analysis of a method blank . Each time samples are extracted,**  **cleaned up, and analyzed, a method blank should be prepared and analyzed for the**  **compounds of interest as a safeguard against chroni c laboratory contamination. Consult**  **the appropriate 3500 or 5000 series method for spec ifics of the preparation of method**  **blanks. The following general guidelines apply to the interpretation of method blank** 

**results.**

**9.2.6.1 Method blanks should be prepared at a frequ ency of at least**  **5%: one method blank for each group of up to 20 sam ples prepared at the same**  **time, by the same procedures. For volatile samples analyzed by purge-and-trap,**  **preparation is equivalent to the analysis. Therefo re, one purge-and-trap method**  **blank must be analyzed with each group of up to 20 samples analyzed on the same**  **instrument during the same analytical shift.**

**9.2.6.2. When samples that are extracted together a re analyzed on**  **separate instruments or in separate analytical shif ts, the method blank associated**  **with those samples (e.g., extracted with the sample s) must be analyzed on at least** 

**one of those instruments. A solvent blank should b e analyzed on all other**  **instruments on which the set of samples was analyze d to demonstrate the**  **instrument is not contributing contaminants to the samples.** 

**9.2.6.3 Unless otherwise described in a determinati ve method, the**  **method blank may be analyzed immediately after the calibration verification**  **standard to ensure that there is no carryover from the standard or at another point in**  **the analytical shift.**

**9.2.6.4 When sample extracts are subjected to clean up procedures,**  **the associated method blank must also be subjected to the same cleanup**  **procedures.**

**9.2.6.5 Results of the method blank should be less than the LLOQ**  **(Sec. 9.7) for the analyte or less than the level o f acceptable blank contamination**  **specified in the approved quality assurance project plan (QAPP) or other appropriate**  **systematic planning document. SW-846 Update V 8000D- 14 Revision 4** 

**October 2012 9.2.6.6 If the method blank results do not meet the**  **acceptance criteria above, the laboratory should take corrective action**  **to locate and reduce the source of the contamination and re-extract and reanalyze a**  **ny samples associated with the**

**contaminated method blank. If the method blank res ults still do not meet the**  **acceptance criteria in 9.2.6.5 and re-analysis is n ot practical, then the data user**  **should be provided with the sample results, the met hod blank results, and a**  **discussion of the corrective actions undertaken by the laboratory. Qualification of**  **the samples may be needed.**

**9.2.6.7 The laboratory should not subtract the results of the method**  **blank from those of any associated samples. Such " blank subtraction" is**  **inappropriate for the GC and HPLC methods addressed here and may lead to**  **negative sample results.**

**NOTE : The analyst may raise the LLOQ to 5x the blank co**  **ncentration to avoid**

**reporting false positive results.**

**9.2.6.8 Method and/or solvent blanks may also be us ed to check for**  **contamination by carryover from a high-concentratio n sample into subsequent**  **samples (Sec. 4.2). When analysis of such blanks i s not possible, such as when an**  **unattended autosampler is employed, the analyst sho uld carefully review the results**  **for at least the next sample after the high-concent ration sample. If analytes in the**  **high-concentration sample are not present in the subsequent sample, then lack of**  **carryover has been demonstrated. If there is evide nce that carryover may have**  **occurred, then the affected samples should be reana lyzed.** 

**9.3 Initial demonstration of proficiency (IDP)**

**Prior to implementation of a method, each laborator y must perform an IDP consisting of at**  **least four replicate reference samples spiked into a clean matrix taken through the entire sample**  **preparation and analysis. Whenever a significant c hange to instrumentation or procedure**  **occurs, the laboratory must demonstrate that accept able precision and bias can still be obtained**  **by the changed conditions (Sec. 9.3.1). Whenever n ew staff members are trained, an analyst**  **IDP must be performed. (Sec. 9.3.2).**

**9.3.1 Demonstration of proficiency for instrument o r method changes** 

**If a major change to the sample preparation procedu re is made (e.g., a change in**  **solvent), the IDP must be repeated for that prepara tion procedure by a minimum of four**  **spiked reference samples. Alterations in instrumen tal procedures only, such as changing**  **GC temperature programs or HPLC mobile phases or th e detector interface, require a new**  **calibration but not a new IDP because the preparati on procedure is unchanged. Each**  **laboratory must have policy for performance and doc umentation of IDP.** 

**9.3.2 Demonstration of proficiency for new analysts**

**Each laboratory should have a training program whic h documents that a new analyst**  **is capable of performing the method, or portion of the method, for which the analyst is**  **responsible. This demonstration should document th at the new analyst is capable of**  **successfully following the SOP established by the l aboratory.** 

**For example, when analysts are trained for a subset of analytes for an 8000 series** **SW-846 Update V 8000D- 15 Revision 4**

**October 2012 method, the new sample preparation analyst should p**  **repare reference samples for a representative set of analytes (e.g., the primary a**  **nalyte mix for Method 8270, or a mix of Aroclor 1016 and 1260 for Method 8082) for each pre**  **paration method the analyst will be performing. The instrument analyst being trained w**  **ill need to analyze prepared samples (such as semi-volatile extracts).** 

**9.3.3 Preparation of reference samples**

**9.3.3.1 Reference samples are prepared from a spik ing solution**  **containing each analyte of interest. The reference sample concentrate (spiking**  **solution) may be prepared from pure standard materi als or purchased as certified** 

**solutions. This reference standard should be made from the same source as the**  **calibration standards to eliminate any additional v ariability due to differences** 

**between sources.**

**9.3.3.2 Preparation of the reference sample concen trate is dependent**  **upon the method being evaluated. Guidance for cert ain methods is listed in**  **Methods 3500 and 5000. In other cases, the determi native methods contain**  **guidance on preparing the reference sample concentr ate and the reference sample.**  **If no guidance is provided, prepare a reference sam ple concentrate in methanol (or**  **any water-miscible solvent) at a concentration such that the spike will provide a** 

**concentration in the clean matrix near the middle o f the calibration range for each**  **analyte in that matrix.**

**9.3.3.3 Concentrations of target analytes in the r eference sample may**  **be adjusted to reflect more accurately the concentr ations to be analyzed by the**  **laboratory. If the concentration of an analyte is being evaluated relative to a**  **regulatory limit or action level, see Sec. 9.4.1 fo r information on selecting an**  **appropriate spiking level.**

**9.3.4 Evaluation**

**9.3.4.1 To evaluate the performance of the total a nalytical process,**  **reference samples must be handled in exactly the sa me manner as actual samples.**  **Additional LCS or MS/MSD samples can be omitted. U se a clean matrix for spiking**  **purposes (one without any target or interference co mpounds) such as organic-free**  **reagent water for aqueous matrices and organic-free sand or soil for solid matrices.** 

**9.3.4.2 Prepare and analyze at least four replicat e aliquots of the**  **well-mixed reference samples by the same procedures used to analyze actual**  **samples (procedure section for each of the methods) . This will include a**  **combination of the sample preparation method (usual ly a 3500 series method for**  **extractable organics or a 5000 series method for vo latile organics) and the**  **determinative method (an 8000 series method).**

**9.3.4.3 Calculate the mean recovery (**  **and the standard deviation of the recovery (**  s **) for each analyte of interest using the four resul ts.** 

**9.3.4.4 Multiple-laboratory performance data are i ncluded in some**  **determinative methods and may be used as guidance i n evaluating performance in a**  **single laboratory. However, comparison with single -laboratory performance data is**  **much more indicative regarding expectations of how any individual laboratory will**  **perform, than in comparison with multi-laboratory d ata. Compare s and**  **for each SW-846 Update V 8000D- 16 Revision 4** 

**October 2012 analyte with the corresponding performance data for**  **precision and bias given in the performance table at the end of the determinative m**  **ethod. If s and (**  **for all analytes of interest meet the appropriate acceptanc**  **e criteria, then the system performance is acceptable and analysis of actual sa**  **mples can begin. If any individual s value exceeds the precision limit or a**  **ny** **value falls outside the range for bias, then the system performance may be unacce**  **ptable for that analyte.**

**NOTE : The large number of analytes in each of the meth**  **ods presents a substantial**

**probability that one or more analyte will fail at l east one of the performance**  **criteria when all analytes of a given method are de termined.** 

**9.3.4.5 Performance data in many of the methods ar e based on**  **single-laboratory performance. As with multiple-la boratory data, the criteria in**  **those methods may be used as guidance when evaluating laboratory performance.**  **When comparing your laboratory data to performance data developed from**  **single-laboratory data, certain analytes may be out side the limits; however, the**  **majority should be within the acceptance limits.**

**9.3.4.6 When one or more of the analytes fail at l east one of the**  **performance criteria, the analyst should repeat the test only for those analytes that**  **fail to meet criteria. Repeated failure, however, will confirm a general problem with**  **the measurement system. If this occurs, locate and correct the source of the**  **problem and repeat the test for all compounds of in terest beginning at Sec. 9.3.4.** 

**9.4 Matrix spike and laboratory control samples**

**9.4.1 General Discussion**

**The laboratory must also have procedures for docume nting the effect of the matrix**  **on method performance (precision and bias). At a m inimum, this check should include the**  **analysis of at least one matrix spike and one dupli cate unspiked sample or preferably, one**  **matrix spike/matrix spike duplicate (MS/MSD) pair w ith each preparation batch of up to 20**  **samples of the same matrix processed together (Chap ter One). If samples are expected to**  **contain target analytes of concern, laboratories ma y use one matrix spike and a duplicate of**  **an unspiked field sample as an alternative to the M S/MSD pair (Sec. 9.4.3).** 

**For samples requiring an extraction procedure separ ate from analysis (e.g.,**  **semivolatiles by Method 8270), the MS/MSD, or MS an d duplicate sample, should be**  **extracted with the batch of samples but may be anal yzed at any time. Conversely, if**  **calibration standards and other analytical QC are p rocessed identically to the field samples**  **(e.g., volatiles by Method 8260), the MS/MSD, or MS and duplicate sample, should be**  **prepared and analyzed concurrently with the samples**

**.**

**When the results of the matrix spike analysis indic ate a potential problem due to the**  **sample matrix itself, the laboratory control sample (LCS) results are used to verify that the**  **laboratory can perform the analysis in a clean matr ix. In the case of very contaminated**  **samples or when the lab does not receive enough sam ples to perform a single matrix spike,**  **an LCS and LCS duplicate (LCSD) may be performed to document precision and bias. An**  **LCS should be included with each preparation batch. The LCS is an aliquot of the same**  **clean (control) matrix used for the method blank(s) and of a similar weight or volume as the**  **method blank and field samples. The LCS is spiked with similar analytes at the same**  **concentrations as in the matrix spike and is proces sed identically to the samples.** **SW-846 Update V 8000D- 17 Revision 4**

**October 2012 In the case of samples that need an extraction proc**  **edure separate from analysis (e.g., semivolatiles by Method 8270), the LCS shoul**  **d be extracted with the batch of samples but may be analyzed at any time. However, if calib**  **ration standards and other analytical QC are processed identically to the field samples (e.g**  **., volatiles by Method 8260), the LCS**  **should be prepared and analyzed concurrently with t he samples and may also serve as the**  **continuing calibration verification (CCV) standard.**

**NOTE : If an LCS also serves as a CCV, acceptance crite**  **ria of the CCV should be used.**

**9.4.2 Spiking procedure for the MS/MSD and LCS**

**The solution used to fortify a sample and/or an LCS should contain all of the target**  **analytes and their concentration levels should be d etermined as described in Secs. 9.4.1**  **and 9.4.2. For those methods that apply to a large list of analytes or that contain**  **compounds that may interfere with an accurate asses sment (i.e., coeluting or multi-peak**  **analytes), a smaller subset of analytes may be used (see the specifics directions in the**  **appropriate individual method).**

**9.4.2.1 If, as in compliance monitoring, the concen tration of a specific**  **analyte in the sample is being checked against a re gulatory concentration limit or**  **action level, the spike should be at or below the l imit, or 1 - 5 times the background**  **concentration (if historical data are available), w hichever concentration is higher.** 

**If historical data are not available, a background sample of the same matrix**  **from the site may be submitted for matrix spiking p urposes to ensure that high**  **concentrations of target analytes and/or interferen ces will not prevent calculation of** 

**recoveries.**

**9.4.2.2 If the concentration of a specific analyte in a sample is not**  **being checked against a limit specific to that analyte, t**  **hen the analyst may spike the MS or MS/MSD sample(s) at the same concentration as the r**  **eference sample (Sec. 9.3.3)**
**at 20 times the estimated LLOQ in the matrix of int erest, or at a concentration near**  **the middle of the calibration range. It is suggest ed that a background sample of the**  **same matrix from the site be submitted as a sample for matrix spiking purposes.** 

**NOTE : Preparing the spiking solution from the same sou**  **rce as the calibration standards helps minimize additional variability due**  **to differences between**

**sources.**

**9.4.2.3 To develop precision and bias data for the spiked compounds,**  **the analyst has two choices: analyze the original s ample, and an MS/MSD pair; or**  **analyze the original sample, a duplicate sample, an d one spiked sample. If**  **samples are not expected to contain the target anal ytes of concern, then the**  **laboratory may use a MS/MSD pair. If samples are e xpected to contain the target**  **analytes of concern, then the laboratory may use on e matrix spike and a duplicate**  **analysis of an unspiked field sample as an alternat ive to the MS/MSD pair.** 

**9.4.2.4 Begin by analyzing one sample aliquot to de termine the**  **background concentration of each analyte. Prepare a matrix spike concentrate**  **according to one of the options described in Sec. 9 .4.2.1 or 9.4.2.2.** 

**9.4.2.5 Prepare a matrix spike sample by adding the appropriate**  **volume of the matrix spike concentrate to another a liquot of the sample to yield the** **SW-846 Update V 8000D- 18 Revision 4**

**October 2012 desired concentration (Secs. 9.4.2.1 and 9.4.2.2).**  **If MS/MSD analysis will be performed, prepare a matrix spike duplicate sample**  **from a third aliquot of the sample.** 

**9.4.2.6 Analyze the MS/MSD samples using the same p rocedures**  **employed for the original sample, and calculate the concentration of each analyte in**  **the matrix spike and matrix spike duplicate. Likew ise, analyze the LCS samples**  **using the same procedures employed for the original sample, and calculate the**  **concentration of each analyte in the LCS.**

**9.4.3 MS/MSD, Duplicate and LCS calculations**

**9.4.3.1 Calculation of % recovery (bias)**

**Bias is estimated from the recovery of spiked analy tes from the matrix of**  **interest. Laboratory performance in a clean matrix is estimated from the recovery**  **of analytes in the LCS. Calculate the recovery of each spiked analyte in the matrix**  **spike, matrix spike duplicate (if performed) and LC S according to the following** 

**formula.**

	
  %  
  
 


  100

**where:**

C

s **= Measured concentration of spiked sample aliquot**

C

u **= Measured concentration of unspiked sample aliquo t (use 0 for LCS)** 

C

n **= Nominal (theoretical) concentration increase tha t results from** 
**spiking the sample, or the nominal concentration of the spiked** 
**aliquot (for LCS).**

**MS/MSD recoveries may not be meaningful if the amou nt of analyte in the**  **sample is large relative to the amount spiked.**

**9.4.3.2 Calculation of relative percent difference (precision)** 

**Precision is estimated from the relative percent di fference (RPD) of the** 
**concentrations (** not

**the recoveries) measured for MS/MSD pairs, or for duplicate** 
**analyses of unspiked samples. Calculate RPD accord ing to the formula below.** 

**where:**

C 1 **= Measured concentration of first sample aliquot**

C 2 **= Measured concentration of second sample aliquot**

**.**

 |      |       2   100**SW-846 Update V 8000D- 19 Revision 4**

**October 2012 NOTE**  **: A difference in the amount of sample used for th e MS and MSD results in** 
**an artificially high RPD when based on concentratio n. Using** 
**approximately the same sample size or scaling the s pike amount to the** 
**sample size for the MS/MSD will minimize bias in th e RPD calculation for** 

**MS/MSD.**

**9.4.4 Recommended QC acceptance criteria for matrix spike samples and LCS** 

**The laboratory should develop performance data for precision and bias in the**  **matrices of interest (Sec. 9.6). In addition, labo ratories should monitor method**  **performance in each matrix, through the use of cont rol charts and other techniques.** 

**Many methods may not contain recommended acceptance criteria for LCS results.**  **The laboratory should use 70 - 130% as interim acce ptance criteria for recoveries of spiked**  **analytes, until in-house LCS limits are developed ( Sec. 9.6). Where in-house limits have**  **been developed for matrix spike percent recoveries, the LCS results should be similar to or**  **tighter than those limits, as the LCS is prepared i n a clean matrix.** 

**Ideally, the acceptance criteria for MS/MSD recover y and/or duplicate relative %**  **difference will be established for the field sample s through the DQOs contained in a written** 
**QAPP. These criteria should be established with co nsideration given to performance data**  **provided in the reference method and/or by the labo ratory in order to avoid overly**  **conservative expectations. In the absence of site- or project-specific acceptance criteria**  **for matrix spike and duplicate QC samples, these cr iteria should be based on in-house**  **performance data generated by the laboratory or on the performance data in the reference** 

**method.**

**Even when the project QAPP or determinative methods provide performance criteria**  **for matrix spikes and LCS, laboratories must develo p in-house performance criteria based**  **on their historical data for use in project plannin g and for comparison to any relevant**  **performance criteria in the reference methods. Dev elopment of in-house performance** 

**criteria is discussed in Sec. 9.6. Where methods d o contain performance data for the**  **matrix of interest, use Secs. 9.4.4.1 - 9.4.4.3 bel ow as guidance in evaluating data**  **generated by the laboratory.**

**9.4.4.1 When multi-laboratory performance data for the matrix of**  **interest are provided in the determinative method, compare the recovery for each**  **analyte with the method performance data of the sam e matrix. Given that such**  **method performance criteria were developed from mul ti-laboratory data, they should**  **be met by almost all laboratories. See Sec. 9.6.10 for more information on**  **comparisons between limits. Performance data inclu de an allowance for error in**  **measurement of both the background and spike concen trations. If spiking was**  **performed at a concentration substantially lower th an the level used to generate the**  **recovery data in the reference method, the recovery data in the method may not be**  **appropriate for assessing the quality of the sample results, and criteria generated**  **from in-house data may be more relevant.**

**9.4.4.2 When a method is initially established in a laboratory, the LCS**  **limits may be applied to the matrix spikes until th e laboratory has sufficient data (a**  **minimum of 20 or more MS/MSD samples of the same ma trix) to generate their own** 

**statistical limits. These data should be used as t he basis for determining MS/MSD** **SW-846 Update V 8000D- 20 Revision 4**

**October 2012 precision and bias limits. Alternatively, acceptan**  **ce criteria based on historical LCS data may continue to be used for evaluating bias in**  **matrix spike recovery and may be more sensitive to matrix effects than acceptance**  **limits based on MS/MSD data. It is generally preferable to use statistically cal**  **culated MS/MSD, rather than LCS recovery limits once sufficient data points have be**  **en collected (i.e., ≥ 20 MS/MSD samples). See Secs. 9.6.1 - 9.6.3 for calculating**  **in-house performance criteria for LCS, MS/MSD and surrogate recoveries.** 

**9.4.5 Also compare the recovery data from the matr ix spike with the LCS data**  **(use the average recovery if an MS/MSD were analyze d). If any individual %R in the MS**  **and/or MSD falls outside the designated range for r ecovery, the laboratory should determine**  **if there is a matrix effect or a laboratory perform ance problem. A matrix effect is indicated if** 

**the LCS data are within limits but the MS/MSD data exceed the limits. Surrogate recovery**  **data (Sec. 9.5) should also be used to evaluate the data. Recoveries of both matrix spike**  **compounds and surrogates outside of the acceptance limits suggest more pervasive**  **analytical problems in the batch and/or instrument than problems with the recoveries of**  **either matrix spikes or surrogates alone.**

**9.5 Surrogate recoveries**

**9.5.1 It is necessary that the laboratory evaluate surrogate recovery data from**  **individual samples versus in-house surrogate recove ry limits. General considerations for**  **developing in-house acceptance criteria for surroga te recoveries are described in Sec. 9.6.** 

**9.5.2 Surrogate recovery is calculated below.**

	  %  !!"
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 #%&!"  '&!(

!!"
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 #%&!"  #((( ) * 100

**If recovery is not within in-house surrogate recove ry limits, the following procedures** 

**are necessary.**

**9.5.2.1 Check for errors in the calculations, surro gate solutions or**  **internal standards. If errors are found, recalcula te the data accordingly.** 

**9.5.2.2 Examine chromatograms for interfering peaks and proper peak**  **integration.**

**9.5.2.3 Check instrument performance. If an instru ment problem is**  **identified, correct the problem and reanalyze the s ample or extract.** 

**9.5.2.4 If no instrument problem is found, the samp le should be**  **re-extracted and reanalyzed (or another vial reanal yzed for volatiles).** 

**9.5.2.5 If upon re-analysis (in either 9.6.2.2 or 9 .6.2.4), the recovery is**  **again not within limits, report the data as an "est imated concentration." If the**  **recovery is within the limits in the reanalysis, re port the reanalysis data for the**  **samples. If the method holding time for the sample has expired prior to**  **re-extraction and/or reanalysis, qualify the data a ccordingly.** **SW-846 Update V 8000D- 21 Revision 4**

**October 2012 9.5.2.6 Some samples may need dilution to bring one**  **or more target analytes within the calibration range or to overcom**  **e significant interferences. This may result in dilution of the surrogate responses t**  **o the point that recoveries cannot be measured. If surrogate recoveries are available**  **from a less-diluted (or undiluted) aliquot of the sample or sample extract,**  **those recoveries may be used to demonstrate that the surrogates are within the QC l**  **imits and no further action is**

**needed.**

**9.6 Generation of performance criteria for MS/MSD, duplicates, surrogates, LCS, and** 

**IDP**

**Usefulness of developing in-house performance crite ria and control charting or similar**  **procedures to track laboratory performance cannot b e overemphasized. Many data systems**  **and commercially available software packages suppor t the use of control charts.** 

**Procedures for calculating in-house performance cri teria for MS/MSD, LCS and surrogate**  **recoveries are provided below.**

**9.6.1 Once sufficient data have been acquired and t he recovery and RPD**  **calculated as in Secs. 9.4.3 and 9.5 for a given sa mple matrix, the following statistics should**  **be used to calculate acceptance criteria.**

**9.6.1.1 Mean percent recovery (** **) and standard deviation (** s **) for:**

**1) Each added target compound in the MS/MSD samples ;**  **2) Each added target compound in the LCS samples; a nd,**  **3) Each added surrogate in the field samples.**

**9.6.1.2 Mean RPD and standard deviation for MS/MSD or duplicate QC**  **samples.**

**A minimum of 20 data points should be used to gener ate meaningful criteria.** 

**Inclusion of additional data should result in more robust criteria that better describe variance**  **in method performance and result in fewer outliers. If the lower limit of the acceptance**  **range is calculated to be <10%, it should be set to 10%. However, an alternative lower**  **acceptance limit may be established by the laborato ry or at the project level through the** 
**DQOs in a QAPP.**

**9.6.2 Calculate the upper and lower control limits for % recovery of each target or**  **surrogate compound in LCS, MS/MSD and field samples using the respective**  **and** s **values calculated in Sec. 9.6.1.**

**Acceptance range =** **(** mean percent recovery) ± 3s **(standard deviation)**

**Upper control limit =** **+** 3s

**Lower control limit =** - 3s

**9.6.3 Calculate the upper control limit for the RPD for the MS/MSD using the** 

**mean RPD value +** 3s

**of the RPDs of historical MS/MSD pairs. RPD shoul d be calculated**  **based on the concentration or amount, not the spike recovery.** 

**NOTE : The RPD limit only has a maximum value, because**  **perfect agreement between C1**

**and C2 would result in a RPD of 0. Refer to Sec. 9 .4.3.2 for the calculation.** **SW-846 Update V 8000D- 22 Revision 4**

**October 2012 9.6.4 Any matrix spike or surrogate recovery outsid**  **e of control limits necessitates evaluation by the laboratory such as c**  **omparison with the LCS recovery.**

**9.6.4.1 If recoveries of analytes in the LCS are ou tside of the control**  **limits, then the problem may lie with application o f the extraction and/or cleanup**  **procedures applied to the sample matrix, or with an alysis. Once the problem has**  **been identified and addressed, corrective action ma y include reanalysis of samples,**  **or extraction and analysis of new sample aliquots, including new matrix spike**  **samples and LCS. However, when there are a large n umber of analytes in the LCS**  **or matrix spike, the statistical probability of a f ew analytes outside of control limits**  **becomes high. Therefore, a number of analytes shou ld be allowed to marginally fail**  **the limits without requirement for corrective actio n. Laboratories should have a**  **documented procedure to assess and qualify marginal exceedance limits.** 

**9.6.4.2 When LCS results are within control limits but matrix spike**  **results are not, the problem may either be related to the specific sample matrix or to**  **an inappropriate choice of extraction, cleanup, and /or determinative method. If**  **results are to be used for regulatory compliance mo nitoring, the analyst must take**  **steps to demonstrate that the analytes of concern c an be determined in the sample** 

**matrix at the levels of interest.**

**9.6.5 Once established, control limits should be re viewed regularly and updated**  **on a routine basis as established by the laboratory ’s quality management plan. Reviewing**  **appropriateness of these criteria with respect to g enerated data is especially important for**  **newly implemented procedures or those not in contin ual use. The laboratory should**  **monitor trends in both analyte recovery performance and also in the control limits. Control**  **limits used to evaluate sample results should be th ose in place at the time of sample**  **analysis. Once control limits are updated, they sh ould apply to all subsequent analyses of**  **new samples.**

**9.6.6 For analytes, methods, and matrices with very limited data (e.g., unusual**  **analytes or matrices not analyzed often), interim l imits should be established using available**  **data or by analogy to similar methods or matrices.**

**9.6.7 Results used to develop acceptance criteria s hould meet all other QC** 

**criteria associated with the determinative method. For example, matrix spike recoveries**  **from a GC/MS procedure should be generated from sam ples analyzed after a valid tune and**  **initial calibration that includes the matrix spike compounds. Analytes in GC or HPLC** 

**methods should fall within the established retentio n time windows in order to be used to**  **develop acceptance criteria.**

**9.6.8 Laboratories are advised to consider effects of spiking concentration on**  **matrix spike performance criteria. Acceptance crit eria for matrix spike recovery and**  **precision are often a function of the spike concent ration used. Therefore, use caution**  **when pooling data in establishing acceptance criter ia. Not only should results all be from**  **roughly the same type matrix but spiking levels sho uld also be similar.** 

**9.6.9 Similarly, acceptance criteria for matrix spi ke, LCS and surrogate results**  **should all be generated using the same combination of extraction, cleanup, and analysis**  **techniques. For example, do not mix results from s olid samples extracted by ultrasonic**  **extraction with those extracted by Soxhlet. SW-846 Update V 8000D- 23 Revision 4** 

**October 2012 9.6.10 Another common error in developing acceptanc**  **e criteria is discarding data that do not meet a preconceived notion of acceptabl**  **e performance. I.e., while professional judgment is important in evaluating data used to de**  **velop acceptance criteria, do not discard specific results simply because they do not meet on**  **e's expectations. This practice results in a censored data set, which when used to develop**  **acceptance criteria, will lead to unrealistically narrow criteria. Rather, employ a**  **statistical test for outlier values, or at least calculate the acceptance limits both with and witho**  **ut the results considered suspect. Then, observe the effect of deleting suspect data.** 

**9.6.11 In-house QC limits must be examined for reas onableness; it is not EPA's**  **intent to legitimize poor recoveries due to incorre ct choice of methods or spiking levels.** 

**In-house limits should also be compared with the DQ Os of specific analyses. For**  **example, recovery limits (for surrogates, MS/MSD, L CS, etc.) that include allowance for a**  **relatively high positive bias (e.g., 70 - 170%) may be appropriate for determining that an**  **analyte is not present in a sample. However, they would be less**  **appropriate for analysis of samples near but below a regulatory limit because o**  **f the potential for high bias.**

**9.6.12 It may be useful to compare QC limits genera ted in the laboratory with**  **performance data listed in specific determinative m ethods. However, the analyst must be**  **aware that performance data generated from multi-la boratory studies tend to be significantly**  **wider than those generated from a single laboratory**

**.**

**9.7 Lower Limit of Quantitation (LLOQ) check standa rd** 

**The laboratory shall establish the LLOQ as the low est point of quantitation, which in**  **most cases, is the lowest concentration in the cali bration curve. LLOQ verification is**  **recommended for each project application to validat e quantitation capability at low analyte**  **concentration levels. This verification may be acc omplished with either clean control material**  **(e.g., reagent water, solvent blank, Ottawa sand, d iatomaceous earth, etc.) or a representative**  **sample matrix, free of target compounds. Optimally , the LLOQ should be less than the desired**  **regulatory action levels based on the stated DQOs.**

**9.7.1 Determination of LLOQs using spiked clean co ntrol material represents a**  **best-case scenario and does not evaluate potential matrix effects of real-world samples.**  **For application of LLOQs on a project-specific basi s with established DQOs, a**  **representative matrix-specific LLOQ verification ma y provide a more reliable estimate of the**  **lower quantitation limit capabilities.**

**9.7.1.1 An LLOQ check standard (not part of an ini tial calibration) is**  **prepared by spiking a clean control material with t he analyte(s) of interest at the**  **predicted LLOQ concentration level(s). Alternative ly, a representative sample**  **matrix may be spiked with the analytes of interest at the predicted LLOQ** 
**concentration levels. The LLOQ check is carried th rough the same preparation**  **procedures as environmental samples and other QC sa mples.** 

**9.7.1.2 Recovery of target analytes in the LLOQ che ck standard should**  **be within established in-house limits, or other suc h project-specific acceptance**  **limits, to demonstrate acceptable method performanc e at the LLOQ. Until the**  **laboratory has sufficient data to determine accepta nce limits, the LCS criteria ± 20%**  **may be used for the LLOQ acceptance criteria. This acknowledges the poorer**  **overall response at the low end of the calibration curve. Historically-based LLOQ** **SW-846 Update V 8000D- 24 Revision 4**

**October 2012 acceptance criteria should be determined as soon as**  **practical once sufficient data points have been acquired.** 

**9.7.2 In-house acceptance criteria for recovery of the LLOQ check standard for a**  **particular sample matrix can be calculated when suf ficient data points exist. The laboratory**  **should have a documented procedure for establishing in-house acceptance ranges; if the**  **lower limit of the acceptance range is calculated t o be <10%, it should be set to 10%.**  **However, an alternative lower acceptance limit may be established by the laboratory or set**  **at the project level through the DQOs in a QAPP.**

**9.8 It is recommended that the laboratory adopt add itional quality assurance practices**  **for use with 8000-series methods. Specific practic es that are most productive depend upon the**  **needs of the laboratory, nature of the samples, and project-specific requirements. Field**  **duplicates may be analyzed to assess precision of t he environmental measurements. When**  **doubt exists over identification of a peak on the c hromatogram, confirmatory techniques such as**  **GC with a dissimilar column, element-specific detec tor, or mass spectrometer (selected ion**  **monitoring or full scan) must be used. Whenever po ssible, the laboratory should analyze**  **standard reference materials and participate in rel evant performance evaluation studies.** 

**10.0 CALIBRATION AND STANDARDIZATION**

**Refer to the appropriate determinative method for d etailed calibration and standardization**  **procedures and the general guidance as noted in Sec . 11.0.** 

**11.0 PROCEDURE**

**Extraction and cleanup are critical for successful analysis of environmental samples and**  **wastes. Analysts should pay particular attention t o selection of sample preparation procedures** 

**to obtain reliable measurements.**

**11.1 Extraction**

**Individual determinative methods for organic analyt es in SW-846 often recommend**  **appropriate sample extraction procedures. General guidance on semivolatile extraction**  **procedures can be found in Method 3500. Guidance o n volatile procedures can be found in** 

**Method 5000.**

**11.2 Cleanup and separation**

**Individual determinative methods for organic analyt es in SW-846 often recommend**  **appropriate cleanup procedures. General guidance o n cleanup procedures can be found in**  **Method 3600. While some relatively clean matrices (e.g., groundwater samples) may not need**  **extensive cleanups, the analyst should carefully ba lance time savings gained by skipping**  **cleanups against potential increases in instrument downtime and loss of data quality that can** 

**occur as a result.**

**11.3 Chromatographic Performance**

**Recommended chromatographic columns and instrument conditions are described in each**  **determinative method. As noted earlier, these colu mns and conditions are typically those used**  **during development and testing of the method. Howe ver, other chromatographic systems may** **SW-846 Update V 8000D- 25 Revision 4**

**October 2012 have somewhat different characteristics as analytic**  **al instrumentation continues to evolve. Therefore, SW-846 methods allow analysts flexibilit**  **y to change conditions as long as adequate performance is demonstrated.** 

**Chromatographic performance is demonstrated by res olution of standards and ability to**  **model the response of the detector during calibrati on; also important are sensitivity, bias,**  **precision, frequency of false positives, and freque ncy of false negatives during analysis. For any**  **chromatographic procedures or conditions used, the laboratory must demonstrate that the**  **performance satisfies the analytical requirements o f the specific application for which the**  **chromatographic procedure is being used. Such demo nstrations should be performed using**  **procedures outlined in Secs. 9.2 - 9.5 of this meth od and in Chapter One.** 

**11.4 Initial Calibration**

**Calibration of an analytical instrument involves de lineation of the relationship between**  **response of the instrument and amount or concentrat ion of an introduced analyte. The graphical**  **depiction of this relationship is often referred to as the calibration curve. To perform quantitative**  **measurements, this relationship, termed initial cal ibration, must be established before the**  **analyses of any samples.**

**Historically, many analytical methods have relied o n linear models of the calibration**  **relationship, where the instrument response is dire ctly proportional to the amount of a target**  **compound. The linear model has many advantages inc luding simplicity and ease of use.**  **However, given the advent of new detection techniqu es and because many methods cannot be**  **optimized for all the analytes to which they may be applied, the analyst is increasingly likely to** 

**encounter situations where the linear model neither applies nor is appropriate.** 

**Initial calibration for SW-846 chromatographic meth ods involves analysis of standards**  **containing the target compounds at a minimum of fiv e different concentrations within the working**  **range of the instrument. In order to produce accep table sample results, instrument response**  **must be within the range established by the initial calibration.** 

**Extrapolation of the calibration to concentrations above or below those of the actual** 

**calibration standards is not appropriate and may lead to significant quantitati**  **ve errors, regardless of the calibration model chosen. It may be necessa**  **ry to prepare calibration standards that cover concentration ranges appropriate for specific proje**  **cts or types of analyses. For instance, the analyst should not necessarily expect to perform a**  **calibration appropriate for sub-ppb level analyses and use the same calibration data for high**  **-ppb or ppm-level samples. Preparation of calibration standards is described in general terms**  **in Sec. 11.4.1.**

**SW-846 methods in this manual for quantitative chro matographic analysis rely on one of**  **three commonly used calibration approaches:**

• **External standard calibration,**

• **Internal standard calibration, and**

• **Isotope dilution calibration.**

**These approaches are described in general terms in Secs. 11.4.2 - 11.4.4.** 

**General calibration criteria are provided in Sec. 1 1.5 for GC and HPLC procedures using**  **non-MS detection. Calibration procedures for GC/MS (e.g., Methods 8260, 8270, 8276, 8280,**  **and 8290), HPLC/MS (e.g., Methods 8321 and 8325), a nd GC/FT-IR (e.g., Method 8410) are** **SW-846 Update V 8000D- 26 Revision 4**

**October 2012 described in those methods. Some determinative met**  **hods may provide specific guidance on calibration such as Method 8085, GC/AED with compou**  **nd-independent calibration.**

**Regardless of the specific calibration technique us ed, introduce calibration standards and**  **samples into the instrument by the same technique a nd at the same volume. Tabulate peak**  **responses against the mass or concentration introdu ced as described in Secs. 11.4.2 - 11.4.4.** 

**11.4.1 Preparation of calibration standards**

**Calibration standards are prepared using specific p rocedures indicated in the**  **determinative methods. However, the general proced ure is described here.** 

**11.4.1.1 For each analyte and surrogate of interest , prepare calibration** 

**standards at a minimum of five different concentrat ions with an appropriate solvent.**  **Alternatively, provided they meet the objectives of the intended application,**  **prepared standards may be purchased from commercial suppliers.** 

**NOTE : As previously mentioned in Sec. 1.9, it may not**  **be practical or appropriate**

**to attempt to determine simultaneously all analytes listed in a given**  **method. Analyte lists in the determinative methods do not imply a**  **regulatory requirement for analysis of any or all o f the compounds but**  **rather indicate the applicable method(s). Therefor e, if an analyte is not**  **relevant to a specific project, it need not be incl uded in the calibration**  **standards associated with that project.**

**11.4.1.2 The lowest concentration calibration stand ard analyzed during**  **an initial calibration will generally establish the lower limit of quantitation (LLOQ).**  **(See Sec. 9.7 for suggested LLOQ establishment and verification.) The analyte** 

**concentration in the lowest standard is related bac k to a sample concentration using**  **the sample amount, dilution, and final injection vo lume used for the specific analysis.**  **Thus, changes to specific sample amounts, dilutions , and volumes employed will be**  **reflected in the LLOQs for samples.**

**11.4.1.3 Higher concentrations should define the wo rking range of the**  **detector or correspond to the expected range of con centrations found in samples**  **that are also within the working range of the detec tor. Standards prepared by serial**  **dilution of a stock solution will typically form a geometric series in which the**  **concentrations or amounts of each standard vary fro m the adjacent standards by a**  **constant factor, e.g., 10, 20, 40, 80, and 160 ng.**

**However, the relatively wide spacing of upper stand ards in a geometric** 

**series can mask the situation where a detector is r eaching saturation and the**  **instrument responses are leveling off somewhere bet ween the last two standards.**  **Therefore, it may be preferable to use a partial ar ithmetic series in which**  **concentrations of the upper standards differ by a c onstant amount, not a constant**  **factor. Using the same overall calibration range a s in the example above, one such**  **series might be 10, 20, 40, 80, 120, and 160 ng, wi th a constant difference of 40 ng**  **between the top four standards, and resulting in a six-point calibration that will better**  **define the instrument response.**

**NOTE : Amounts shown above are for illustrative purpose**  **s only. Both the overall**

**calibration range and concentrations or amounts use d for the standards**  **are a function of the specific instrumentation, dem onstrable working range** **SW-846 Update V 8000D- 27 Revision 4**

**October 2012 of instrumentation, and intended application of the**  **specific method. Therefore, labs must determine the calibration rang**  **e and standards for their specific circumstances.** 

**11.4.1.4 For each analyte, at least one of the cali bration standards**  **MUST correspond to a sample concentration at or bel ow the quantitation levels**  **needed for the project; this may include establishi ng compliance with a regulatory or**  **action limit. Given that different limits may be a ssociated with different analytes, the**  **same standard should not be expected to fulfill thi s requirement for all analytes.** 

**11.4.1.5 Given the large number of target compounds addressed by**  **some of the methods listed in Sec. 1.1, it may be n ecessary to prepare several sets**  **of calibration standards, each set consisting of di fferent analytes. Initial calibration**  **will then involve analysis of each of these sets of standards.** 

**11.4.2 External standard calibration**

**External standard calibration is one of the most co mmon approaches to calibrations.**  **It involves a simple comparison of instrument respo nses from the sample to the target**  **compound responses in the calibration standards. S ample peak responses are compared**  **with calibration standard peak responses. The rati o of the detector response to the amount**  **(mass) of analyte in the calibration standard is de fined as the calibration factor (CF).** 



### CF -#. 
/-!/ ' "0 /"#!(#
( %-&!(

%#// ' "0 %-&!(  !#!1
#%/ 

**Advantages of external standard calibration are tha t it is simple and can be applied**  **to a wide variety of specific chromatographic metho ds. The primary disadvantage is that it**  **is greatly affected by stability of the chromatogra phic detector system and presence of**  **chromatographic interferences in a sample or sample extract.** 

**The CF may also be calculated using the standard co ncentration rather than mass in**  **the denominator of the equation above. However, us e of concentration in calculating the**  **CF will necessitate changes to the equations used t o calculate sample concentration (Sec.**  **11.10.3).**

**For multi-component analytes (e.g. PCBs and Toxaphe ne), see the appropriate**  **determinative method for information on which peaks to employ for CF calculation.** 

**11.4.3 Internal standard calibration**

**Internal standard calibration involves comparison o f instrument responses from the**  **target compounds in the sample to responses of othe r standards added to the sample or**  **extract before injection. Response of the target c ompound is normalized to the response of** 

**the other standard. This other standard is called an**  internal **standard because it is contained within the aliquot of the sample or sampl**  **e extract injected into the**

**instrumentation.**

**A constant amount of the internal standard is added to all samples or extracts. That** 

**same amount of the internal standard is also includ ed in each of the calibration standards.**  **In the sample or sample extract, the peak response ratio of the target compound to the** **SW-846 Update V 8000D- 28 Revision 4**

**October 2012 internal standard is compared with a similar ratio**  **derived for each calibration standard. This ratio is termed the response factor (RF) or re**  **lative response factor (RRF), indicating that the target compound response is calculated rel**  **ative to that of the internal standard.**

**Advantages of internal standard calibration include that it can account for routine**  **change in response of the chromatographic system as well as variation in the volume of the**  **introduced sample or sample extract. In addition t o normalizing the peak response of the**  **target compound to the response of the internal sta ndard in the sample or extract for that**  **injection, the internal standard may be used to cal culate the relative retention time (RRT) of**  **the target compound. RRT is expressed as a unitles s quantity.** 

2 "!"$! "$% ' "0 #!#3"

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**If RRT is used by the laboratory to establish peak identity, the RRT of each target**  **analyte in each calibration standard should agree w ithin established limits set by the**  **laboratory in their SOP. These limits should be app ropriate for reliable identification of the**  **target analyte. If this criterion is not met and th ere are no other indicators of**  an analyte's **identification (such as a very unique and high prob ability mass spectral match), that**  analyte **may not be considered as identified by RRT.**

**The RRT of the analyte in the sample should be with in the RRT limits of the analyte** 

**in the standards. If this criterion is not met and there are no other indicators of a**  **component’s identification (such as a very unique a nd high probability mass spectral**  **match), that component may not be considered as ide ntified by RRT.** 

**RRT evaluation allows the analyst to compensate for modest shifts in the**  **chromatographic conditions that can occur due to in terferences and day-to-day instrument**  **variability. Many methods that employ internal sta ndard calibration use more than one**  **internal standard; target compounds are related to the internal standards based on similarity**  **of their respective chromatographic retention times or physical and chemical properties.** 

**Principal disadvantages of internal standard calibr ation are that internal standards**  **must be compounds not found in the samples to be an alyzed and they must produce an**  **unambiguous response on the chromatographic detecto r system. Many SW-846 methods**  **recommend brominated or fluorinated compounds and/o r stable isotopically-labeled**  **analogs of target compounds (e.g., a compound conta ining a deuterium atom instead of**  **hydrogen, or a 13**  **C atom instead of a 12**  **C atom) as internal standards. Isotopically-labele d**  **compounds are most often employed in MS detection m ethods because the detector can**  **differentiate between the target compound and the l abeled internal standard based on its**  **added mass even when the two compounds elute at the same retention time. In general,** 

**internal standard calibration is not as useful for GC and HPLC methods with non-MS**  **detectors because of the inability to chromatograph ically resolve many internal standards**  **from the target compounds.**

**Internal standards recommended in many SW-846 metho ds were used during**  **development of the method. Analysts may employ oth er internal standards in place of, or**  **in addition to, those recommended. If internal sta ndards are not recommended in the**  **method, the analyst should select one or more compo unds similar in analytical behavior to**  **the analytes of interest and not expected to be fou nd in the samples. Whichever internal**  **standards are employed, the analyst should demonstr ate (as detailed in the determinative**  **method) that measurement of the internal standard i s not affected by target analytes,**  **surrogates, or matrix interferences. SW-846 Update V 8000D- 29 Revision 4** 

**October 2012 When preparing calibration standards, add the same**  **amount of the internal standard solution to each calibration standard. Therefore,**  **the internal standard concentration is the same in each calibration standard, whereas concentr**  **ations of the target analytes will vary. The internal standard solution may contain more tha**  **n one internal standard, and their relative concentrations may differ within the spiki**  **ng solution. However, the mass of each internal standard added to the samples or sample ex**  **tracts immediately before injection**

**must be the same as in each calibration standard. The volume of the internal standard**  **solution spiked into a sample extract should cause minimal dilution of the extract.** 

**An ideal internal standard concentration would yiel d a response factor (RF) of 1 for**  **each analyte. However, this is unlikely to be the case when dealing with more than a few**  **target analytes. Therefore, as a general rule, the internal standard should produce an**  **instrument response ≤ 100 times that produced by th e least responsive target analyte** 

**associated with the internal standard. This should result in a minimum RF of approximately**  **0.01 for the least responsive target compound.**

**For each of the initial calibration standards, calc ulate the RF values for each target**  **analyte relative to one of the internal standards a s follows.** 



### 4 5
 

###   6



### 5 6 

###  



**where:**



### A s **= Peak response of the analyte or surrogate**



### A is **= Peak response of the internal standard**



### C s **= Mass of the analyte or surrogate in the sample a liquot** 



### C is **= Mass of the internal standard in the sample aliq uot** 

**Response factors for GC/MS methods may also be calc ulated using sums of the**  **areas of two ions (expressed as mass over charge,** m/z **) for each target analyte and internal**

**standard.**

**Note that in the equation above, RF is unitless. T herefore, units such as ng or μg**  **may be used for amounts of the analyte, surrogate, and internal standard, provided that they** 

**are uniform.**

**Because internal standards are used to compensate f or routine variations in the**  **chromatographic separation of target compounds, the re is a significant advantage to using**  **more than one internal standard when dealing with a large number of target compounds or**  **when those compounds elute over a long time frame. When multiple internal standards are**  **employed, target compounds are associated with the internal standards on the basis of their**  **respective retention times. Therefore, the interna l standards should be chosen to cover the**  **expected retention time range of the target compoun ds. Accordingly, internal standards**  **can compensate for small retention time shifts or r esponse changes in the portion of the**  **chromatographic run in which they occur. Ideally, the analyst will employ enough internal**  **standards to result in a RRT for each target compou nd in the range 0.80 - 1.20, though other**  **RRT ranges may be appropriate as well. SW-846 Update V 8000D- 30 Revision 4** 

**October 2012 Many methods that utilize internal standard calibra**  **tion include acceptance limits for responses of the internal standards in the calibrat**  **ion standards, samples, or both. Those limits are typically expressed in terms of peak are**  **as because the concentration of the internal standard cannot be measured directly (e.g.**  **, one has to assume that the entire mass injected into the sample or sample extract is prese**  **nt during analysis). Common consensus**

**limits are 50 - 200% of the area of the internal st andard in the most recent calibration**  **standard. Representing a factor of two, these limi ts are used as a gross diagnostic check**  **on addition of the internal standards to the sample s or extracts and injection of the sample**  **aliquot into the instrument.**

**11.4.4 Isotope dilution calibration**

**Isotope dilution calibration is a special case of i nternal standard calibration. In**  **isotope dilution, the internal standards are stable isotopically-labeled analogs of the target**  **analytes and are added to the sample prior to extraction or othe**  **r sample preparation steps such as pH adjustment, drying, or extraction solven**  **t addition. Physical and chemical properties of each labeled compound are virtually t**  **he same as its unlabeled "native" analog. Thus, any losses of the target compound that may oc**  **cur during sample preparation or determinative steps will be mirrored by a similar l**  **oss of the labeled standard. Similarities between labeled compounds and unlabeled analogs mea**  **n that RFs and RRTs for the unlabeled compounds are very close to 1.0.** 

**Labeled compounds are spiked into samples and stand ards at a constant amount.**  **RFs developed from the calibration standards assume that all of the labeled compounds**  **added to the sample reach the instrument. This ass umption, termed recovery correction,** 

**allows for correction to observed concentrations of the target compound relative to its**  **labeled counterpart.**

**The degree to which the labeled compounds meet this assumption is monitored by** 

**use of traditional internal standards added to the sample extract immediately prior to**  **injection. Separate RFs relate the concentrations of the labeled compounds to the**  **traditional internal standards. Most isotope dilut ion methods include some limits on the**  **apparent recovery of the labeled compounds. Howeve r, those limits are often consensus**  **limits that may be overly conservative. As long as responses for both native and labeled**  **compounds can be distinguished from the background instrumental noise, isotope dilution**  **calibration can provide excellent results, even whe n the apparent spike recovery of the**  **labeled compound is low. Labeled compound recoveri es >100% are allowed as well.** 

**Such recoveries can occur as a result of the inhere nt variability in calibration of the labeled**  **compounds, and are not indicative of contamination or other problems.** 

**Built-in recovery correction is one of the principa l advantages of isotope dilution**  **calibration. Isotope dilution requires an MS detec tion system and isotopically labeled**  **analogs of target analytes and generally produces m ore precise data with lower bias. The**  **added cost of isotopically labeled compounds is a d isadvantage, but can be offset by higher**  **quality data, as well as eliminating some routine Q C analyses, such as surrogates and** 

**MS/MSDs used with internal standard calibration. H owever, whether or not to add**  **surrogate or prepare MS/MSD aliquots should be desc ribed in a QAPP and not left to the**  **analyst's professional judgment.**

**Isotope dilution calibration is often used in conju nction with selected ion monitoring**  **(SIM) GC/MS procedures, such as those for polychlor inated dibenzo-**  p **-dioxins and polychlorinated dibenzofurans. These procedures, a**  **long with a relatively small list of target compounds, allow the instrument to be operat**  **ed in a mode that detects only those SW-846 Update V 8000D- 31 Revision 4** 

**October 2012 ions (**  m/z **) corresponding to the target compounds and their l abeled analogs; this application**  **significantly increases the sensitivity of the meth od and reduces interferences. Because**  **isotope dilution methods have acceptance limits for recovery of the labeled analogs added**  **prior to extraction or other sample preparation ste ps, these methods typically do not contain**  **limits on responses of the traditional internal sta ndards used to monitor those recoveries.** 

**An RF is calculated for each target compound relati ve to its labeled analog and for**  **each labeled analog relative to the traditional int ernal standard added immediately prior to**  **injection. These calculations may involve areas of more than one ion (**  m/z **) for each compound (e.g., Methods 8280 and 8290).** 

**11.4.5 Extracted internal standards**

**Extracted internal standard calibration is a hybrid of internal standard calibration and**  **isotope dilution calibration. In this approach, tr aditional internal standards are added to the**  **samples before extraction instead of just prior to analysis. Results for the target**  **compounds can be corrected for recovery of the inte rnal standards using the same**  **assumptions made for isotope dilution.**

**This approach is most helpful when the compounds us ed as internal standards are**  **very closely related to the target compounds. It i s similar to the internal standard procedure**  **used for volatile organic analyses by purge-and-tra p or headspace extraction.** 

**11.5 Calibration models and acceptance criteria**

**SW-846 chromatographic methods allow the use of thr ee different calibration models:**  **average calibration factor or response factor (Sec. 11.5.1), linear regression (Sec. 11.5.2), and**  **non-linear regression (Sec. 11.5.3). Any of these models can be applied to either external or** 

**internal standard calibration data. This section a lso provides suggested**  **criteria for calibration models; however, method- or project-specific criter**  **ia will always supersede general guidance.**

**Choice of calibration model may begin with the simp lest approach, the average calibration**  **factor or response factor model, and then progress through linear and then non-linear regression**  **until the calibration acceptance criteria are met. Another appropriate approach is to choose a**  **calibration model based on previous experience, kno wledge of the physics of the detector, or**  **specific manufacturer's recommendations. For the c alibration model to be usable, it must be**  **continuous and monotonic throughout the calibration range. More calibration points are required**  **for more complex models. The chromatographic metho ds in SW-846 employ a minimum of five**  **standards for average response factor or linear (fi rst-order) calibration models, six standards for a**  **quadratic (second-order) model, and seven standards for a cubic (third-order) model.** 

**NOTE : The option of using non-linear calibration may be**  **necessary to address specific**

**instrumental techniques. However, it is not EPA's intent to allow non-linear calibration**  **to compensate for detector saturation or avoid prop er instrument maintenance.**  **Regardless of the calibration model chosen, an** X **value of zero should not be included as a calibration point.** 

**The following sections describe various options for initial calibration evaluation and provide**  **the calibration acceptance criteria used to evaluat e each option. In addition to this suggested**  **acceptance criteria, two general methods for assess ing the accuracy of the calibration curve for**  **all allowed curve models are presented in 11.5.4. It is further recommended that each calibration**  **model be inspected to ensure that the data are repr esentative of the model chosen as described** 

**in Sec. 11.5.6. Whichever calibration model is sel ected, samples with concentrations that** **SW-846 Update V 8000D- 32 Revision 4**

**October 2012 exceed the calibration range must be diluted to fal**  **l within the range. Criteria listed in these sections are designed for quantitation of trace-lev**  **el concentrations of the analytes of interest. If data of lower quality will satisfy project-specific**  **data needs, less stringent criteria may be employed provided they are documented and approved**  **in a QAPP.**

**11.5.1 Linear calibration using average calibration or response factor** 

**As calculated in Sec 11.4, each CF or RF represents the slope of the line between**  **the origin and the given standard response. If the relative standard deviation (RSD) of**  **variation in the factors is ≤ 20%, the linear model is generally representative over the range**  **of calibration standards. Representativeness beyon d the range of calibration standards is** 

**not to be inferred. At least five calibration leve ls should be used to construct the average**  **CF or RF model (Sec. 11.5).**

**To evaluate linearity of the initial calibration, c alculate the mean CF (external**  **standard calibration) or RF (internal standard cali bration), the standard deviation (SD), and**  **the RSD (also called coefficient of variance, CV) a s follows:** 



### : ; ∑ =4

6 

###  4 >   6?


### !  1 : ; ∑ =4

6 

###  4 >   6?


### !  1

%#! 4  4

@ @ @ @ ∑ 4

6  6?



### ! %#! 4  4 @ @ @ @ ∑ 4

6  6?



### !

: :

4 @ @ @ @  100 : :

4 @ @ @ @  100

**Where** n **is the number of calibration standards and RSD is expressed as a percentage (%).** 

**11.5.1.1 If the RSD is ≤20% over the calibration ra nge, the slopes of the**  **lines for each standard are sufficiently close to o ne another that the use of the linear** 
**model is generally appropriate** over the range of standards that are analyzed **;** 

### 4 @ @ @ @ **or**



### 4 @ @ @ @ **may be used to determine sample concentrations. A lternatively, either of the**  **two methods described in 11.5.4 may be used to dete rmine calibration function**  **acceptability.**

**NOTE : The RSD approach is equivalent to a**  

A B **weighted linear least square**

**regression line that is forced through the origin.**

**11.5.1.2 Given the potentially large numbers of ana lytes that may be**  **analyzed in some methods, it is likely that some an alytes may exceed the**  **acceptance limit for the RSD for a given calibratio n. In those instances, it is**  **recommended, but not required, that corrective acti ons as described in Sec. 11.5.6.1**  **be followed. Sec. 11.5.6.1 also provides alternati ve uses for initial calibrations that**  **do not meet their criteria of acceptability.**

**11.5.1.3 Calculation of sample amounts SW-846 Update V 8000D- 33 Revision 4** 

**October 2012 If all the conditions in Secs. 11.5.1.1 and 11.5.1.**  **2 are met, the** 

### 4 @ @ @ @ **or** 

### 4 @ @ @ @
**may be used to determine sample concentrations, as described in Sec. 11.10. It is** 
**recommended that the curve generated by the** 

### 4 @ @ @ @ **or** 

### 4 @ @ @ @ **be examined for acceptability using the refitting check described i**  **n Sec. 11.5.4.1. The calculated amount introduced into the instrument, x** 

**s , is:** 



### 
 

### 5



### 4 @ @ @ @ #!( 

 

### 5



### 4 @ @ @ @   6



### 5
C 

### @ @ @ @

**where:**



### X s **= Calculated mass of the analyte or surrogate in t he sample** 
**aliquot introduced into the instrument (in ng)**



### A s **= Peak response of the analyte or surrogate in the sample** 



### A is **= Peak response of the internal standard in the sa mple** 

C is **= Mass of the internal standard in the sample aliq uot introduced** 
**into the instrument (in ng)**

CF **= Average calibration factor from the most recent i nitial** 

**calibration**



### RF **= Average RF from the most recent initial calibrat ion.** 

**Units for the mass of analyte should be the same un its used to calculate the** 

**CFs or RFs. If different units are used for amount (e.g., μg/L), these calculations**  **and those found in Sec. 11.10 should be adjusted ac cordingly.** 

**11.5.2 Linear calibration using a least squares reg ression** 

**A linear calibration model based on a least squares regression may be employed**  **based on past experience or** a priori **knowledge of the instrument response. Based on th e**  **professional judgment of the analyst, this approach also may be used for analytes that do** 

**meet the RSD criteria in Sec. 11.5.1 This is most easily achieved by performing a linear**  **least squares regression of the instrument response versus the mass of the**  **chromatographed analyte. Treat the instrument resp onse as the dependent variable (**  y **) and the amount as the independent variable (**  x **). This is a statistical requirement and is not simply a graphical convention. At least five calib**  **ration levels should be used to construct the linear regression model (Sec. 11.5).** 

**For external standard calibration,** x **is the mass of the analyte in the sample aliquot**

**introduced into the instrument and** y **is the instrument response.**

**For an internal standard calibration,** 

### x **and** 

### y **can be assigned in various ways**

**where** 

### x **is the amount of the analyte introduced into the in strument and**  

### y **is the instrument response to that analyte. Two options are provided**  **here using the mass introduced into the instrument. If other assignments for**  

### x **and** 

### y **are used, e.g., concentration, subsequent equations used for calculating mass of the analyte**  **introduced into the instrument must be** 

### y  A s x  C s**SW-846 Update V 8000D- 34 Revision 4**

**October 2012 changed accordingly.** 

**Option 1:** X

s **is the mass of the analyte in the calibration stan dard aliquot introduced into the** 

**instrument and** Y

s **is the ratio of response of the analyte to the res ponse of** 

**internal standard times the mass of the internal st andard in the calibration**  **standard aliquot introduced into the instrument.**



### 
 

### 
 

### #!( G

 

### 5
 

### x  6



### 5 6

**Option 2:** x **is the ratio of the analyte mass in the calibration standard aliquot introduced** 

**into the instrument to the internal standard mass i n the calibration standard**  **aliquot introduced into the instrument and** y **is the ratio of response of the analyte to the response of internal standard.** 

**where:**



### C

s **= Mass of analyte in the volume of calibration stan dard introduced** 

**into the instrument.**



### C is **= Mass of internal standard in the volume of calibr ation standard** 

**injected into the instrument.**



### A s **= Peak response of analyte.**



### A i s **= Peak response of internal standard.**

**A linear least squares regression attempts to const ruct a linear equation of the form,** 

**by minimizing the sum of squared differences betwee n the observed response (**  y i **, the instrument response) and the predicted response (**  y' i , **the response calculated from the constructed equation) at each calibration level.** 

**Weighting the sum of the squares of the differences may significantly improve the**  **ability of the least squares regression to fit the linear model to the data, especially at the low**  **end of the calibration curve. The general form of the sum of the squares of the differences**  **containing the weighting factor is:**



### H I

6 

###  6 

###  6

J 

###   

6?

**where:**



### W i **= Weighting factor for the** i th **calibration standard (** w=1 **for unweighted**

**least squares regression, or 1/x or 1/x 2**  **for weighted least squares** 

### y A s
A is x C s
C is

y  ax  b**SW-846 Update V 8000D- 35 Revision 4**

**October 2012 regression)** 



### y i **= Observed instrument response for the** i th **calibration standard.**


### y' i **= Predicted (or calculated) response for the** i th **calibration standard.**



### n **= Total number of calibration standards.**

**Mathematics used in least squares regression favors numbers of larger value over**  **numbers of smaller value. Thus, unweighted regress ion curves will tend to fit points that**  **are at upper calibration levels better than those p oints at lower calibration levels. If** 

**concentrations of concern are at lower calibration levels, an unweighted regression curve**  **tends to give less accurate results. A weighting f actor which reduces this tendency can be**  **used as compensation.**

**11.5.2.1 Do not include an X-value of zero as a cal ibration point.**  **However, most data systems and many commercial soft ware packages will allow the**  **analyst to "force" the regression through the origi n. Forcing the curve through the**  **origin is not the same as including the origin as a fictitious point in the calibration. In**  **essence, if the curve is forced through the origin, the intercept is set to 0**  before **the regression is calculated, thereby setting the bias**  **to favor the low end of the calibration range by ”pivoting“ the function around**  **the origin to find the best fit and resulting in one less degree of freedom. It may be**  **appropriate to force an unweighted regression though the origin for some ca**  **librations, but not when the regression is weighted.** 

However, forcing the regression through the origin may NOT be used as a rationale for reporting results below the cali bration range demonstrated by the analysis of the standards. **Results should not be reported at a concentration below the LLOQ unless qualified as es**  **timated.**

**11.5.2.2 In the specific case of an unweighted line ar least squares**  **regression,** i.e. **, a regression that varies both a and b, the correl ation coefficient (**  r **) can be used to measure the "goodness of fit."** 



### ! ∑  6 6 

###  ∑  6 

### ∑ 6  6?  6?  6? 

### O

! ∑  6   6? 

###  = ∑  $

 6? 

### >  

### ) = P

! ∑ 6   6? 

###  ∑ 6

 6? 

###   

### >

**The value of** 

### r **is such that**



### - 1≤ r ≤ 1

**.**

**The instrument data system will typically calculate** 

### r

. **An** 

### r



### - **value of +1.00 indicates a positive perfect correlation; an**  r

- **value of -1.00 indicates a negative perfect correlation; an**  r

- **value of 0 indicates no correlation.**

**However, if the regression line is forced through t he origin or the weighting**  **factor is variable, then the coefficient of determi nation, more often termed**  r 2

, **should be used to measure the "goodness of fit”, such that**  **0 ≤** r2 **≤ 1. This shows the strength of the association between**  x **and** y **. The** r

2 **value allows the analyst to determine the percent of the data closest to the li**  **ne of best fit. For consistency, it is SW-846 Update V 8000D- 36 Revision 4** 

**October 2012 acceptable to use**  r 2 **for linear unweighted curves as well. An** r 2 **value of 1.00 indicates that all variability in response is due t**  **o variation in concentration.**

**In order for the linear regression model to be used for quantitative** 

**purposes,** r

**or** r 2 **should be ≥ 0.995, and 0.99, respectively. Altern atively, either of**  **the two methods described in Sec. 11.5.4 may be use d to determine whether the**  **calibration function meets acceptance criteria. It is recommended that the resulting**  **calibration curve be inspected by the analyst as de scribed in Sec. 11.5.4.1.** 

**11.5.2.3 To calculate the mass (** x **) of the analyte in the sample aliquot introduced into the instrument, the regression equa**  **tion is rearranged.**

**External standard calibration allows the mass of t he analyte in the sample**  **aliquot introduced into the instrument to be calcul ated.** 

**For the internal standard method, the calculation w ill depend on which of**  **the two options described in Sec. 11.5.2 is chosen.**



### U-"$! 1 

 

### V  5
 

### x  6 

### 5 6 

### X  Y

#

U-"$! 2 
 

###  5
 

### 5 6 

###   Y ) x 

6



### #

**where:**



### X s **= Calculated mass of the analyte or surrogate in th e sample** 
**aliquot introduced into the instrument (in ng)**



### A s **= Peak response of the analyte or surrogate in th e sample** 



### A is **= Peak response of the internal standard in the s ample** 



### C is **= Mass of the internal standard in the sample aliq uot introduced** 

**into the instrument (in ng).**

**Units for analyte mass should be the same as those used to determine the**  **regression equation. If alternative units such as concentrations are used, calculations for**  

### x  y

- b 

a

x s  A s - b 

a**SW-846 Update V 8000D- 37 Revision 4**

**October 2012 the final sample concentrations found in Sec. 11.10**  **should be adjusted accordingly.**

**11.5.3 Non-linear calibration**

**In situations where the analyst knows the instrumen t response does not follow a**  **linear model over a sufficiently wide calibration r ange, or when other approaches described**  **here have not met acceptance criteria, a non-linear calibration model may be employed. At** 

**least six calibration levels are recommended to con struct a quadratic (second-order)**  **calibration curve, and at least seven levels should be used for a cubic (third-order) curve**  **(Sec. 11.5).**

**NOTE : It is not EPA's intent to allow non-linear calibr**  **ation to compensate for detector**

**saturation or to avoid proper instrument maintenanc e.** 

**When a calibration model for quantitation is used, the curve must be continuous:**  **continuously differentiable and monotonic over the calibration range. The model chosen**  **should have no more than four parameters,** i.e.

**,**

'#, Y, , (, 

**where** f **indicates a function with up to four parameters,** a – d **, and** x **is the independent variable. If the model is polynomial,**  **it may be no more than third-order, as in the equation:** 

# [  Y     (

**When the linear regression model is used to estimat e model parameters for the**  **calibration data, the instrumental response (** y **) must be treated as the dependent variable, and the amount of the calibration standard (**  x **) must be the independent variable. An**

x **-value of zero should not be included as a calibrat ion point, although the curve may either**  **be weighted or forced through the origin as long as calibration criteria are met.** 

**Model estimates from the regression must be used as calculated, e.g., no term (**  a, b, c, or d **) calculated as a result of the least squares regre ssion can be modified. Weighting in**  **a calibration model or forcing through the origin m ay significantly improve the ability of the**  **least squares regression to fit the data at lower c oncentration levels.**  However, forcing the regression through the origin may NOT be used as a rationale for reporting results below the calibration range demonstrated by the ana lysis of the standards.

**11.5.3.1 Linear and non-linear least squares regres sions are**  **mathematical methods that minimize differences (the residuals) between observed**  **instrument response,** y i , **and calculated response,** y i ’ **, by adjusting coefficients of the polynomial (**  a, b, c, and d **) to obtain the polynomial best fitting the data.**

**The coefficient of determination** r **2 may be used as a measure of goodness** 

**of fit. See Sec. 11.5.2.2 for the definition of** r **2**

**.**

**11.5.3.2 Under ideal conditions, i.e., a "perfect" fit of the model to the**  **data, the** r

**2 will equal 1.00. In order to be an acceptable non**  **-linear calibration, the** r **2**
**must be ≥ 0.99. Alternatively, either of the two m ethods described in 11.5.4 may be** **SW-846 Update V 8000D- 38 Revision 4**

**October 2012 used to determine calibration function acceptabilit**  **y. It is recommended that the resulting calibration curve be inspected by the ana**  **lyst, as described in Sec. 11.5.4.1.**

**As noted in Sec. 11.5, whichever of these options i s employed, an analyte**  **or surrogate concentration must fall within the cal ibration range. Analysts are also** 

**advised to check both second- and third-order calib ration models to ensure that all**  **tangents to the curve within the calibration range are of the same sign and no**  **tangent is zero. Samples with concentrations that exceed the calibration range**  **must be diluted to fall within the range.**

**11.5.4 Acceptance criteria independent of calibrati on model** 

**Either of the two procedures described in Secs. 11. 5.4.1 and 11.5.4.2 may be used**  **to determine calibration function acceptability for linear and non-linear curves. These**  **include refitting the calibration data back to the model. Both % Error and Relative Standard**  **Error (RSE) evaluate the difference between the mea sured and the true amounts or** 

**concentrations used to create the model.**

**11.5.4.1 Calculation of the % Error**



### % \

 A ]^ A _ ]

A ] 

### x 100

**where:**



### x’ i **= Measured amount of analyte at calibration level** i **, in mass or**

**concentration units**



### x i **= True amount of analyte at calibration level** i **, in mass or**

**concentration units.**

**Percent error between the calculated and expected a mounts of an analyte** 

**should be ≤ 30% for all standards. For some data u ses, ≤50% may be acceptable**  **for the lowest calibration point.**

**11.5.4.2 Calculation of Relative Standard Error (RS E - expressed as %)** 



### :\  100 * a

H b  6 J 

###   6



###  6 

### c  

6? 

### !  -

d

**where:**



### x i **= True amount of analyte in calibration level** i **, in mass or**

**concentration units**



### x ́ i **= Measured amount of analyte in calibration level** i **, in mass or**

**concentration units**



### p **= Number of terms in the fitting equation**

**(average = 1, linear = 2, quadratic = 3, cubic = 4) SW-846 Update V 8000D- 39 Revision 4** 

**October 2012** 

### n **= Number of calibration points.**

**The RSE acceptance limit criterion for the calibrat ion model is the same as** 

**the RSD limit for** 

### 4

@ @ @ @ **or** 

### 4 @ @ @ @ **in the determinative method. If the RSD limit is not**  **defined in the determinative method, the limit shou ld be set at ≤20% for good**  **performing compounds and ≤30% for poor performing c ompounds. A list of known**  **poorly performing compounds can be found in Sec. 16 of this document.** 

**11.5.5 Data transformations**

**An understanding of the fundamental behavior of the detector may be used to** 

**choose a data transformation that will then allow f or a simple calibration model. For**  **example, the response of a flame photometric detect or in the sulfur mode is known to be**  **proportional to the square of the sulfur concentrat ion. Therefore, using the data system to**  **take the square root of the instrument response or peak height allows for a calibration factor**  **approach rather than a polynomial calibration curve . Instrument response may be**  **transformed prior to any calculations (including in tegration) subject to the following** 

**constraints:**

**11.5.5.1 Any parameters used in the transformation must be fixed for the**  **calibration and all subsequent analyses and verific ations until the next calibration.** 

**11.5.5.2 The transformation model chosen must be co nsistent with the** 

**behavior of the instrument and detector. All data transformations must be clearly**  **defined and documented by the analyst and related b ack to the fundamental**  **behavior of the detector. In other words, this app roach may not be used in the**  **absence of specific knowledge about the behavior of the detector.** 

**11.5.5.3 No transformations should be performed on areas or other**  **results (e.g., the transformation must be applied t o the instrument response itself).** 

**11.5.5.4 When the transformed data are used to deve lop calibration**  **factors, those factors should meet the acceptance c riteria described in Sec. 11.5.1,**  **and it is recommended that the resulting calibratio n "curve" be inspected by the**  **analyst as described in Sec. 11.5.4.**

**11.5.6 Inspecting the calibration model and recomme nded corrective actions** 

**Given the potentially large numbers of analytes tha t may be analyzed in some**  **methods, it is likely that some analytes may exceed acceptance limits for a given calibration.**  **If the criteria is not met by a target analyte, the acceptability of the initial calibration for other**  **analytes that have met their criteria is not invali dated. Information obtained from the initial**  **calibration of targeted analytes not meeting the ac ceptability criteria may have other uses**  **such as for screening and for estimation of quantit ation (see Sec 11.5.6.1), but those uses**  **should still fit the needs of the project objective s.** 

**Whichever calibration model is selected, it is reco mmended that the model be**  **subjected to an additional check to establish the r epresentativeness of the data that were**  **used to produce the model. This check is the refit ting of the calibration data back to the**  **model or the comparison of the calculated amount of each of the standards against the**  **expected amount, as described in Sec. 11.5.4. Crit eria for acceptability based upon the**  **additional check would have a similar impact upon t he usability of a calibration for**  **quantitation as is discussed in the above paragraph**

**. SW-846 Update V 8000D- 40 Revision 4** 

**October 2012 11.5.6.1 Corrective action may be needed if the cal**  **ibration criteria (**  

### RSD/RSE/ r

2 **and % Error) are not met. If** any **analyte for** any

**calibration standard has a percent error > ±30% as described in Section1**  **1.5.4.1, corrective action may**

**be needed. Some recommended courses of action and additional options for**  **modifying the calibration ranges follow. More spec ific corrective actions that are**  **provided in the applicable determinative methods wi ll supersede those noted in**  **Method 8000. Generally, the calibration should not be used for quantitative**  **analyses of that analyte when the calibration crite ria (**  

### RSD/RSE/ r 2 **and % Error) are**

**not met.**

**11.5.6.2 For all calibration models the following o ptions are allowed.**  **However, if none result in an acceptable calibratio n, a new initial calibration must be**  **performed.**

**11.5.6.3 Generally, the first option is to che ck the instrument operating**  **conditions. The suggested maintenance procedures i n Sec. 11.11 may be useful in**  **guiding such adjustments. This option will apply i n those instances where a linear**  **instrument response is expected. It may involve so me trade-offs to optimize**  **performance across all target analytes. For instan ce, changes to the operating**  **conditions necessary to achieve linearity for probl em compounds may cause the**  **RSD for other compounds to increase, but as long as all analytes meet the RSD**  **limits for linearity, the calibration is acceptable**

**.**

**If the initial calibration for any analyte does not meet the acceptance criteria (**  e.g.

**, RSD/RSE > 20% or r**  **2 < 0.99), the analyst may wish to review the result**  **s (proper identification, area counts, calibration or RFs, an**  **d RSD/RSE) for those analytes to ensure that the problem is not associated with just**  **one of the initial calibration**

**standards.**

**11.5.6.4 As a second option, if the problem app ears to be associated**  **with a single standard, that one standard may be re analyzed, to rule out problems**  **due to random error, and the calibration function m ay be recalculated and**  **reevaluated against the acceptance criteria. Repla cing the standard may be**  **necessary in some cases. If the criteria still cann ot be met, the entire initial**  **calibration should be performed again.**

**NOTE : An initial calibration should be considered a sin**  **gle event process and a reanalysis of a calibration standard should be perf**  **ormed immediately to ensure that the reanalysis is still part of the original initia**  **l calibration event, and before any samples are analyzed.** 

**11.5.6.5 A third option is to narrow the calibra tion range by replacing one** 

**or more of the calibration standards with standards that cover a narrower range. If**  **linearity can be achieved using a narrower calibrat ion range, document the**  **calibration linearity, and proceed with analyses. Changes to the upper end of the**  **calibration range will affect the need to dilute sa mples above the range, while**  **changes to the lower end may increase the LLOQ. Co nsider the regulatory limits or**  **action levels associated with the target analytes w hen adjusting the lower end of the**  **range. Replacing one or more of the standards is** NOT **to be confused with discarding results from a given standard. The mini**  **mum number of standards**

**described in Sec. 11.5.3.1 should still be used for calibration.** **SW-846 Update V 8000D- 41 Revision 4**

**October 2012 11.5.6.6 A fourth option is to narrow the calib**  **ration range by removing data points from either extreme end of the range an**  **d recalculating the calibration function. It is prohibited to remove data points f**  **rom within a calibration range while still retaining the extreme ends of the calibration**  **range. The minimum number of**

**calibration levels described in Sec. 11.5 should st ill be met for the model.** 

**NOTE : As noted in Sec. 11.4.1.2, the lower limit of qua**  **ntitation (LLOQ) is**  **established by the concentration of the lowest stan dard analyzed during the initial**  **calibration. Hence, narrowing the calibration rang e by changing the concentration**  **of the lowest standard will, by definition, change the LLOQ. When the purpose of**  **the analysis is to demonstrate compliance with a sp ecific regulatory limit or action**  **level, the analyst should ensure that the LLOQ is a t least one calibration point below**  **the regulatory limit or action level.**

**If criteria for RSD/RSE/** r **2 has been met for the calibration model but the % e**  **rror of one or more of the individual calibration points at**  **the extreme ends of the calibration range exceeds the criteria described in Sec. 11.5.4**  **.1, the usable range of the calibration may be narrowed to the standards that m**  **eet the % error criteria, but the calibration points used to generate the initial cur**  **ve are retained. The LLOQ becomes the lowest end of the adjusted calibration**  **range. The calibration model**

**should meet the RSD/RSE/r 2**  **criteria (Secs. 11.5.1 – 11.5.3) and the minimum number of data points (Sec. 11.5.3.1) before this o**  **ption can be used.**

**NOTE : This guidance allows the use of a calibration mod**  **el constructed using all of the data points (with the exception of the highest**  **or lowest point, which may be dropped, but will change linear range) but limits t**  **he range for usefulness to only those data points that refit the model within the c**  **riteria set in Sec. 11.5.5.1, i.e., <**

**30% difference.**

**11.5.6.7 A fifth alternative is available for target analytes that do not**  **meet the acceptance criteria for the initial calibr ation. Without reanalysis of**  **standards or manipulations of the model, the initia l calibration can be used to**  **estimate quantitation and information from the cali bration can be used to verify the**  **identification of target analytes when used to scre en samples.** 

**If the initial calibration does not meet the accept ance criteria, it may not be used for**  **quantitative analyses; however, estimates of the qu antitation can be made.**  **Estimates of quantitation can be useful when screen ing for the level of**  **contamination and determining the degree of dilutio ns that may be necessary when**  **high levels of contamination are encountered. If q uantitation estimates for a**  **positively identified analyte are not within the sc ope of the project DQOs, then an**  **acceptable initial calibration should be prepared f or that analyte.** 

**If information from the initial calibration will be used to verify the identification of a**  **targeted analyte for screening purposes, there shou ld be sufficient sensitivity at the**  **screening level to verify identification. Reasonab le responses found at the lowest**  **level of the calibration standards may be used as a verification of identity at that level** 

**of concentration.**

**11.6 Retention time windows**

**Retention time windows are crucial to the identific ation of target compounds. Absolute**  **retention times are used for compound identificatio n in all GC and HPLC methods that do**  not**SW-846 Update V 8000D- 42 Revision 4**

**October 2012 employ internal standard calibration. Retention ti**  **me windows are established to compensate for**

**minor shifts in absolute retention times as a resul t of sample loadings and normal**  **chromatographic variability. Width of the retentio n time window should be carefully established**  **to minimize the occurrence of both false positive a nd false negative results. Tight retention time**  **windows may result in false negatives and/or may ca use unnecessary reanalysis of samples**  **when surrogates or spiked compounds are erroneously not identified. Overly wide retention time**  **windows may result in false positive results that m ay not be confirmed.** 

**The following subsections describe one approach tha t may be used to establish retention**  **time windows for GC and HPLC methods. Other approa ches may be employed, provided the**  **analyst can demonstrate performance appropriate for the intended application.** 

**11.6.1 Before establishing retention time windows, make sure that the**  **chromatographic system is operating reliably and th at the system conditions are optimized**  **for the target analytes and surrogates in the sampl e matrix to be analyzed. Make three**  **injections of all single component standard mixture s and multi-component analytes (such as**  **PCBs) over the course of a 72-hour period. Serial injections or injections over a period of**  **less than 72 hours may result in retention time win dows that are too tight.** 

**11.6.2 Record the retention time (in minutes) for e ach single component analyte**  **and surrogate to three decimal places. Calculate t he mean and standard deviation of the**  **three absolute retention times for each single comp onent analyte and surrogate. For**  **multi-component analytes, choose three to five majo r peaks (see the determinative methods**  **for more details) and calculate the mean and standa rd deviation of those peaks.** 

**11.6.3 If the standard deviation of the retention t imes for a target compound is**  **0.000 (i.e., no difference between the absolute ret ention times), then the laboratory may**  **either collect data from additional injections of s tandards or use a default standard deviation**  **of 0.01 minutes. (Recording retention times to thr ee decimal places rather than only two** 

**should minimize the instances in which the standard deviation is calculated as 0.000).** 

**11.6.4 Width of the retention time window for each analyte, surrogate, and major**  **constituent in multi-component analytes is defined as ±3 times the standard deviation of the**  **mean absolute retention time established during the 72-hour period or 0.03 minutes,**  **whichever is greater.**

**11.6.5 Establish the center of the retention time w indow for each analyte and**  **surrogate by using the absolute retention time for each analyte and surrogate from the**  **calibration verification standard at the beginning of the analytical shift. For samples run**  **during the same shift as an initial calibration, th e retention time of the midpoint standard of** 

**the initial calibration should be used.**

**11.6.6 If the instrument data system is not capable of employing**  **compound-specific retention time windows, then the analyst may choose the widest window**  **and apply it to all compounds. As noted above, oth er approaches may also be employed,**  **but must be documented by the analyst.**

**11.6.7 Surrogates are added to each sample, blank, and QC sample and are also**  **contained in each calibration standard. Although t he surrogates may be diluted out of**  **certain sample extracts, their retention times in t he calibration standards may be useful in**  **tracking retention time shifts. Whenever the obser ved retention time of a surrogate is**  **outside of the established retention time window, t he analyst is advised to determine the**  **cause and correct the problem before continuing ana lyses.** **SW-846 Update V 8000D- 43 Revision 4**

**October 2012 11.7 Calibration verification** 

**The calibration relationship established during the initial calibration (Sec. 11.5) must be**  **verified at periodic intervals. The process of cal ibration verification applies to both external**  **standard and internal standard calibration techniqu es, as well as to linear and non-linear** 

**calibration models.**

**As a general rule, the initial calibration in a SW- 846 method must be verified at the**  **beginning of each 12-hour analytical shift during w hich samples are analyzed using a calibration**  **verification standard prepared at the appropriate l evel of concern. (Some methods may specify**  **more frequent verifications and recommended standar d concentrations). The 12-hour analytical**  **shift begins with the injection of the calibration verification standard (or the MS tuning standard in**  **MS methods). The shift ends after the completion of the analysis of the last sample or standard**  **that can be injected within 12 hours of the beginning of the shift.** 

**If the % Difference (when using average RF calibrat ion) or % Drift (for all other types of**  **calibration) of an analyte is within ±20% of the ex pected concentration or amount based on the**  **initial calibration, then the initial calibration i s considered still valid, and the analyst may conti**  **nue to use the calibration curve to quantitate sample r**  **esults. The ±20% criterion may be superseded**

**in certain determinative methods.**

**Except where the determinative method contains alte rnative calibration verification criteria,**  **if the % Drift or % Difference is >± 20%, the initi al calibration relationship may no longer be valid.** 

**NOTE : The process of calibration verification is funda**  **mentally different from the approach called**

**"continuing calibration" in some methods from other sources. As described in those**  **methods, the calibration factors or RFs calculated during continuing calibration are used**  **to update the calibration factors or RFs used for s ample quantitation. This approach,**  **while employed in other EPA programs, is equivalent to a daily single-point calibration,**  **and is neither appropriate nor permitted in SW-846 chromatographic procedures for**  **trace environmental analyses.**

**If the calibration does not meet the acceptance cri teria, perform any necessary instrument**  **maintenance, and inject another aliquot of the cali bration verification standard. If the response**  **for the analyte is still not ± 20%, then a new init ial calibration may be necessary.** 

**11.7.1 Calibration verification criteria**

**Use the equations below to calculate % Drift or % Difference, depending on the**  **procedure described in the determinative method.**

% Drift Measured Amount

True Amount x 100

**where:**

**Measured amount = mass or concentration determined by the calibration** 

**model True amount = prepared mass or concentration of the**  **analyte in the**

**standard. SW-846 Update V 8000D- 44 Revision 4** 

**October 2012** % Difference CF
v  CF

CF * 100% RF
v  RF

RF * 100

**where:**

**CF**

**v = calibration factor calculated for the calibratio**  **n verification standard**

**RF**

**v = response factor calculated for the calibration v**  **erification standard**

CF **= mean calibration factor from the initial calib ration** 

RF **= mean response factor from the initial calibrat ion** 

**If >10% of the analytes in a multi-analyte method exceed the calibration verification**  **criteria, and instrument maintenance does not corre ct the problem, then a new initial**  **calibration is necessary. If ≤10% of the analytes exceed the calibration verification criteria,**  **then the initial calibration may still be used, but any detected analytes exceeding the limit**  **must be reported as estimated. Non-detected analyt es may be reported if the calibration**  **verification for that specific analyte exceeds the upper acceptance criteria (**  e.g. **. >+20%). In order to report non-detected analytes that excee**  **ds the lower acceptance criteria (** e.g.

**, <-20%), a sensitivity verification standard at or b**  **elow the LLOQ should be analyzed in the analytical batch. The analyte should be detected i**  **n the LLOQ standard and meet all of the qualitative identification criteria that the labora**  **tory routinely uses (for example, qualifier ions of columns, signal to noise, etc). In any event, t**  **he limitation to no more than 10% of analytes exceeding the calibration verification cri**  **teria applies to both detected and non-detected analytes.** 

**11.7.2 Verification of non-linear calibration**

**Calibration verification of a non-linear calibratio n is performed using the % Drift**  **calculation and criteria described in Sec. 11.7.1, above.** 

**It may also be appropriate to employ two standards at different concentrations to**  **verify the calibration. One standard should be nea r the quantitation limit or action limit.**  **Choice of specific standards and concentrations is generally a method- or project-specific** 

**consideration.**

**11.7.3 Calibration verification may be performed us ing both high and low** 

**concentration standards from time to time. This is particularly true when the ECD or ELCD is** 

**used. These detectors drift and are not as stable as FID or FPD, and periodic use of the**  **high and low concentration standards serves as a fu rther check on the initial calibration.**  **Concentrations of these standards should generally reflect those observed in samples.** 

**11.7.4 Additional analyses of the midpoint calibrat ion verification standard during**  **a 12-hour analytical shift are strongly recommended for methods involving external** 

**standard calibration. The same evaluation criteria described in Sec. 11.7.1 should be** 

**used.**

**Frequency of verification necessary to ensure accur ate measurement is dependent**  **on the detector and the sample matrix. Very sensit ive detectors that operate in the**  **sub-nanogram range are generally more susceptible t o changes in response caused by**  **column contamination and changes in ambient conditi ons. Therefore, more frequent**  **verification of calibration (i.e., after every 10 s amples) may be necessary for some types of** **SW-846 Update V 8000D- 45 Revision 4**

**October 2012 detectors (**  i.e. **, electron capture, electrochemical conductivity, p hotoionization, fluorescence**  **detectors).**

**Sec. 11.8.2 states that samples analyzed using exte rnal standards must be**  **bracketed by periodic analyses of standards that me et the QC acceptance criteria (e.g.,**  **calibration and retention time). Therefore, more f requent analyses of standards will**  **minimize the number of sample extracts to be reinje cted if the QC limits are violated for the**  **standard analysis. Results from these bracketing s tandards should meet the calibration** 

**verification criteria in Secs. 11.7.1 and 11.7.2 an d the retention time criteria in Sec. 11.6.**  **However, if the standard analyzed after a group of samples exhibits a response for an**  **analyte that is above the acceptance limit, i.e., >20%, and the analyte**  **was not detected in**  **any of the previous samples during the analytical s hift, then the sample extracts do not need**  **to be reanalyzed, as the verification standard has demonstrated that the analyte would have**  **been detected were it present.**

**11.7.5 Any method blanks described in the preparati ve methods (Methods 3500**  **and 3600) may be run immediately after the calibrat ion verification analyses to confirm that**  **laboratory contamination does not cause false posit ive results, or at any other time during**  **the analytical shift. If the method blank indicate s contamination, then it may be appropriate**  **to analyze a solvent blank to demonstrate that the contamination is not a result of carryover**  **from standards or samples.**

**11.8 Chromatographic analysis of samples**

**11.8.1 Introduction of samples or sample extracts i nto the GC or HPLC varies,**  **depending on the physical and chemical properties o f the compound and the solvent matrix.**  **Volatile organics are primarily introduced by purge -and-trap techniques (Method 5030,**  **water and Method 5035, soils). Other techniques in clude azeotropic distillation (Method**  **5031), vacuum distillation (Method 5032), headspace (Method 5021), or direct aqueous**  **injection. Use of Method 5021 or another headspace technique may be advisable for**  **screening volatiles in some sample matrices to prev ent overloading and contamination of**  **the purge-and-trap system. Semivolatile and non-vo latile analytes are introduced by direct**  **or split/splitless injection.**

**11.8.1.1 Manual injection (GC)**

**Inject a small volume (** i.e. **, 0.5 – 5 μL) of the sample extract. However, other injection volumes may be used if the analyst**  **can demonstrate appropriate performance for the intended application. Use of t**  **he solvent flush technique is necessary for packed columns.** 

**11.8.1.2 Automated injection (GC)**

**Automated injectors can provide volumes both larger and smaller than 1-2**  **μL. The analyst should ensure that the appropriate injector design is used for the**  **volume to be injected and that the injection volume is reproducible. Other injection**  **volumes may be used if the analyst can demonstrate appropriate performance for**  **the intended application.**

**Large Volume Injection (LVI) is the injection of la rge volumes (greater than**  **5 μL) into cooled inlets that allow the solvent to be vented while retaining less volatile**  **analytes. LVI is used to increase the sensitivity of the analysis, either to decrease** **SW-846 Update V 8000D- 46 Revision 4**

**October 2012 LLOQs or to decrease the amount of sample extracted**  **, or extraction solvent used. This procedure is typically performed with inlets m**  **ade specifically for this technique. The analyst should ensure that all of the QC requir**  **ements of both the preparation**

**and determinative methods are met.**

**11.8.1.3 Purge-and-trap**

**Refer to Methods 5000, 5030, or 5035 for details.**

**11.8.1.4 Manual injection (HPLC)**

**Inject 10 – 100 μL. This is generally accomplished by overfilling the**  **injection loop of a zero dead-volume injector. Lar ger volumes may be injected if**  **better sensitivity is needed; however, chromatograp hic performance may be** 

**affected.**

**11.8.1.5 Automated injection (HPLC)**

**Inject 10 – 100 μL. Laboratories should demonstrat e that the injection**  **volume is reproducible. Larger volumes may be inje cted if greater sensitivity is**  **needed; however, the solvent of the standards and s amples should be matched to**  **the initial mobile phase to avoid chromatographic p erformance degradation.** 

**11.8.2 All analyses, including field samples, dupli cates, MS/MSDs, LCS, method**  **blanks, and any other QC samples are performed duri ng an analysis sequence. The**  **sequence begins with instrument calibration, which is followed by the analysis of sample** 

**extracts. Verification of calibration and retentio n times is necessary no less than once**  **every 12-hour analytical shift. The sequence ends when the set of samples has been**  **injected or when qualitative and/or quantitative QC criteria are exceeded. As noted in**  **Secs. 11.5. and 11.7, when employing external stand ard calibration, run a calibration**  **verification standard at the end of the sequence to bracket the sample analyses.**  **Acceptance criteria for the initial calibration and calibration verification are described in** 

**Secs. 11.5 – 11.7.**

**Analysis of calibration verification standards betw een every set of 10 samples is**  **strongly recommended, especially for highly sensiti ve GC and HPLC detectors at**  **sub-nanogram concentrations. Frequent analysis of calibration verification standards**  **helps ensure that chromatographic systems are perfo rming acceptably and that false**  **positives, false negatives and poor quantitation ar e minimized. Samples analyzed using** 

**external standard calibration should be bracketed b y the analyses of calibration standards** 
**that meet the QC limits for verification of calibra tion and retention times. If criteria are**  **exceeded, corrective action should be taken (Sec. 1 1.11) to restore the system and/or a**  **new calibration curve prepared for that compound an d the samples reanalyzed.** 

**Certain methods may also include QC checks on colum n resolution, analyte**  **degradation, mass calibration, etc., at the beginni ng of a 12-hour analytical shift.** 

**11.8.3 Sample concentrations are calculated by comp aring sample responses**  **with the initial calibration of the system (Sec. 11 .5). If sample response exceeds the limits**  **of the initial calibration range, dilute the extrac t (or sample) and reanalyze. Extracts should**  **be diluted so that all peaks are on scale, as overl apping peaks are not always evident when**  **peaks are off scale. When overlapping peaks cause errors in peak area integration, the**  **use of peak height measurements is suggested. SW-846 Update V 8000D- 47 Revision 4** 

**October 2012 11.8.4 If chromatographic peaks are masked by the p**  **resence of interferences, further sample cleanup or dilution may be necessary**  **. See Method 3600 for guidance.**

**11.9 Compound Identification**

**Tentative identification of an analyte occurs when a peak from a sample extract falls within**  **the daily retention time window. Confirmation is n ecessary when the composition of samples is**  **not well characterized. Confirmation techniques in clude further analysis using a second column**  **with dissimilar stationary phase, GC/MS (full scan or SIM) or HPLC/MS (if concentration permits),**  **GC or HPLC with two different types of detectors, o r by other recognized confirmation techniques.**  **For HPLC/UV methods, analyte confirmations at two d ifferent UV wavelengths with a UV or UV**  **diode array detector is not recommended because of the broadband nature of UV absorption**  **spectra of many organic compounds. Positive identi fication of a target analyte using an**  **HPLC/UV method may be confirmed with a different ty pe of detector such as a mass-selective** 

**detector or a fluorescence detector at different ex citation and emission wavelengths.** 

**When confirmation is made on a second column, that analysis should meet all of the QC**  **criteria described above for calibration, retention times, etc. Confirmation is not needed with** 

**GC/MS and HPLC/MS methods.**

**Confirmation may not be necessary if the compositio n of the sample matrix is well**  **established by prior analyses as when a pesticide k nown to be produced or used in a facility is**  **found in a sample from that facility.**

**Many chromatographic interferences result from coel ution of one or more compounds with**  **the analyte of interest, or may be the result of th e presence of a non-analyte peak in the retention**  **time window of an analyte. Such coelution problems affect quantitation as well as identification,**  **and may result in poor agreement between the quanti tative results from two dissimilar columns.**  **Therefore, even when the identification has been co nfirmed on a dissimilar column, the analyst**  **should evaluate the agreement of the quantitative r esults on both columns, as described in Sec.** 

**11.10.4.**

**11.10 Calculations**

**Calculation of sample results depends on the type o f calibration (external or internal**  **standard) and the calibration model employed (linea r or non-linear). Calculations of the mass of**  **the analyte in the sample aliquot introduced into t he instrument can be found in Secs. 11.5.1.3,**  **11.5.2.3, and 11.5.3. The following sections descr ibe the calculations necessary to obtain the**  **concentrations of analytes in the original sample, based on its volume or weight.** 

**These calculations are provided for illustrative pu rposes only. Various dilution schemes**  **and conventions for defining final volumes and inje ction volumes exist and they all cannot be**  **addressed here. The analyst must clearly document and verify all of the calculations that are**  **employed. Specific determinative methods may also contain additional information on how to**  **perform these calculations.**

**11.10.1 Sample concentration by volume (μg/L), e.g. , for aqueous samples** 

!!"
#"$! $! w1

x  

  y z   

 y 6  y
 **SW-846 Update V 8000D- 48 Revision 4**

**October 2012 where:** 

X

s **= Calculated mass of analyte (in ng) in sample ali quot introduced into**  **instrument. Type of calibration model used determi nes derivation of** 

**x s**  **. See Secs. 11.5.1.3, 11.5.2.3, and 11.5.3.**

V t **= Total volume of concentrated extract (in μL). F or purge-and-trap**  **analysis, V t**  **is the purge volume and will be equal to V i**  **. Thus, units other than μL may be used for purge-and-trap analys**  **es.**

D **= Dilution factor, if sample or extract was dilute d prior to analysis. If no**  **dilution, D=1. Always dimensionless.**

V i **= Volume of extract injected (in μL). The nominal injection volume for**  **samples and calibration must be the same. For aque ous**  **purge-and-trap analysis or direct injection of a li quid sample into a GC**  **or HPLC, V i**  **will be equal to V t** 

**.**

V

s **= Volume of aqueous sample extracted or purged (in mL). If units of**  **liters (L) are used for this term, multiply results by 1000 mL/L.** 

**Using the units listed here for these terms will re sult in a concentration in units of**  **ng/mL, which is equivalent to μg/L.**

**11.10.2 Sample concentration by weight (μg/kg), e.g ., for solid samples and**  **non-aqueous liquids**

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  y z   

 y 6  I
 

**where:**

X

s **= Calculated mass of analyte (in ng) in sample aliq uot introduced into**  **instrument. Type of calibration model used determi nes derivation of** 

**x s**  **. See Secs. 11.5.1.3, 11.5.2.3, and 11.5.3.**

V t **= Total volume of concentrated extract (in μL). Fo r purge-and-trap**  **analysis where an aliquot of solvent (methanol, wat er, etc.) extract is**  **added to reagent water and purged, V t**  **is total volume of solvent extract. Also includes any contribution from water**  **present in samples prior to solvent extraction (Sec. 11.10.5).** 

D **= Dilution factor, if sample or extract was dilute d prior to analysis. If no**  **dilution, D=1. This value is always dimensionless.**

V i **= Volume of extract injected (in μL). The nominal injection volume for**  **samples and calibration standards must be the same. For**  **purge-and-trap analysis where an aliquot of solvent (methanol, water,**  **etc) extract is added to reagent water and purged, V**  **i is the volume of**  **solvent extract added to reagent water just prior t o purging. Dilutions** 

**made to the initial volume of solvent extract are a ccounted for in**  D

**. SW-846 Update V 8000D- 49 Revision 4** 

**October 2012** W

s **= Weight of sample extracted or purged (in grams). If kg units are used**  **for this term, multiply results by 1000 g/kg.**

**Using the units listed here for these terms will re sult in a concentration in units of**  **ng/g, which is equivalent to μg/kg. See Sec. 11.10 .5 for situations in which calculated**  **concentrations may need to be corrected based on th e solvent/water dilution effect for**  **extracted volatile organics.**

**11.10.3 Sample concentration when** X

s **is expressed as concentration during**

**calibration**

**As noted in Sec. 11.4, the analyst may develop the calibration using the**  **concentration of analyte and internal standard inst ead of mass. Using such an approach**  **usually involves expressing concentrations as mass of the analyte or internal standard in the**  **volume injected into the instrument (i.e., ng/μL). Thus, calculations for the final**  **concentration of an analyte in a sample in Secs. 11 .10.1 and 11.10.2 must be modified to**  **include the injection volume, V i**  **, into the term** X

s **. Therefore, the equation for sample concentration by volume becomes:** 

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x  

  y z   

 y
 

**And the equation for sample concentration by weight becomes:** 

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1  

  y z   

 I
 

**where** V

t , D, V

s **, and** W

s **are the same as in Secs. 11.10.1 and 11.10.2 and**

X

s **= Calculated concentration of analyte (ng/μL) in t he sample. Type of** 

**calibration model used determines derivation of** X

s **. See Secs. 11.5.1.3, 11.5.2.3, and 11.5.3.** 

**Using the units listed here for these terms will re sult in concentrations in ng/mL,**  **which is equivalent to μg/L, or in ng/g, which is e quivalent to μg/kg. See Sec. 11.10.5 for**  **situations in which calculated concentrations may n eed to be corrected based on the** 

**solvent/water dilution effect for extracted volatil e organics.** 

**11.10.4 Comparison between results from different columns or detectors** 

**When sample results are confirmed using two dissimi lar columns or with two**  **dissimilar detectors, the agreement between the qua ntitative results should be evaluated**  **after the identification has been confirmed. Large differences in the numerical results from**  **the two analyses may be indicative of positive inte rferences with the higher of the results,**  **which could result from poor separation of target a nalytes, or the presence of a non-target**  **compound. However, they may also result from other causes. Thus, in order to ensure that**  **the results reported are appropriate for the intend ed application, the analyst should make a**  **formal comparison, as described below. SW-846 Update V 8000D- 50 Revision 4** 

**October 2012 Calculate the relative percent difference (RPD) bet**  **ween the two concentrations using the formula below.** 

 |      |       2   100

**where C 1**  **and C 2**  **are concentrations on the two columns and the vert ical bars in the** 

**numerator indicate the absolute value of the differ ence. Therefore, RPD is always a positive** 

**value.**

**11.10.4.1 If one result is significantly higher (e .g., >40%), check the**  **chromatograms to see if an obviously overlapping pe ak is causing an erroneously**  **high result. If no overlapping peaks are noted, ex amine the baseline parameters**  **established by the instrument data system (or opera tor) during peak integration. A**  **rising baseline may cause the incorrect integration of the peak for the lower result.** 

**11.10.4.2 If no anomalies are noted, review the ch romatographic**  **conditions. If there is no evidence of chromatogra phic problems, it may be**  **appropriate to report the lower result.**

**Regardless of the presence or absence of chromatogr aphic problems, the**  **data user MUST be advised of the disparity between the two results, because the**  **user, not the laboratory, is responsible for ensuri ng that the most appropriate result**  **is reported or utilized. Under some circumstances, including those involved in**  **monitoring compliance with an action level or regul atory limit, further cleanup of the**  **sample or additional analyses may be needed when th e two values in question span**  **the action level or regulatory limit.**

**11.10.5 Moisture-corrected reporting**

**Results for solid samples may be reported on the ba sis of wet weight (as received)**  **or dry weight (moisture-corrected) sample concentra tion. There are merits to either**  **approach; however, many regulatory limits associate d with solid wastes and solid samples** 

**are based on the form of the waste** as generated **, which rarely involves oven-dry solids. As a result, there is no default preference for one fo**  **rm or the other.**

**The choice of "as received" or moisture-corrected r eporting is ALWAYS a**  **project-specific decision that must be based on kno wledge of intended use of the data.** 

**When moisture-corrected reporting is required, conc entration results for solid**  **samples calculated in Secs. 11.10.2 and 11.10.3 may be converted to moisture-corrected** 

**results as follows:**

{$/"&
 

"( !!"
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$	(" !!"
#"$! 

 100  % %$/"&
   100**SW-846 Update V 8000D- 51 Revision 4**

**October 2012 where % moisture is determined as described in the**  **specific sample preparation or determinative method, typically by drying an aliquo**  **t of the sample at 105 o**  **C overnight.**

**Percent moisture is calculated as follows:**

% {$/"&
  1 ' "as received" /#%-3 

  1 ' (
 /#%-3 

1 ' /#%-3  100

**The % moisture determination may also be called % s olids in some methods. In this**  **case, percent solids should be subtracted from 100, in order to attain % moisture as noted in** 

**the above moisture-corrected calculation. Units fo r the final results, e.g., μg/kg or mg/kg,**  **will be the same, regardless of the % moisture calc ulation.** 

**Except when the sample is completely dry ( i.e., th e % moisture equals 0),**  **moisture-corrected results will always be higher th an "as received" results. In the absence**  **of project-specific requirements, it may be most ap propriate to report results on the "as**  **received" basis of the sample AND provide the % moi sture for each sample. This will allow** 

**the data user to convert the results from one form to another, as needed. The approach**  **used must be clearly described for the data user.**

**In volatile organic analysis, solid samples with si gnificant moisture content (>10%)**  **that are extracted prior to analysis in a water-mis cible solvent such as methanol, are diluted**  **by the total volume of the solvent/water mixture. The total mixture volume can only be**  **calculated based on the sample moisture present as determined by the % moisture**  **determination. This total volume is then expressed as V**  **t in the sample concentration**  **calculations provided in Secs. 11.10.2 and 11.10.3. Therefore, in order to report results for**  **volatile analysis of samples containing significant moisture content on either "as received"**  **or “moisture-corrected” basis, the calculated conce ntration must be corrected using the total**  **solvent/water mixture volume represented as V t**  **. This total solvent/water volume is**

**calculated as follows:**

μx /3	!"

#"
y

z   %x ' /3	!"    % {$/"&
 x 1 ' /#%-3 

100  x 1000 μx/%x

**When the sample moisture content is >10%, it is rec ommended that the calculated**  **concentrations of volatile samples that are extract ed in a water-miscible solvent such as** 

**methanol be corrected for the solvent/water dilutio n effect. Potential underreporting of**  **volatile concentrations is more pronounced as % moi sture increases.** 

**11.11 Suggested chromatographic system maintenance**

**Following is a list of corrective measures that may be employed to prevent or ameliorate the**  **deterioration of chromatographic performance. This list is by no means comprehensive, and**  **analysts should develop expertise in troubleshootin g their specific instruments and analytical**  **procedures. Manufacturers of chromatographic instr uments, detectors, columns, and**  **accessories generally provide detailed information regarding the proper operation and limiting**  **factors associated with their products. Reading an d reviewing this information cannot be**  **overemphasized. SW-846 Update V 8000D- 52 Revision 4** 

**October 2012 11.11.1 GC preventive maintenance and corrective ac**  **tion**

**To prevent or ameliorate deterioration of chromatog raphic performance, analysts**  **should perform routine maintenance activities on th e GC inlet, column, and gas delivery**  **system.**

**11.11.1.1 Inlet maintenance**

**Appropriate injector liners should be installed and replaced as necessary to**  **maintain chromatographic performance. Injection po rt septa should also be**  **changed frequently enough to prevent retention time shifts of target analytes and**  **peak tailing. Over-tightening the septum nut can c ause the inlet to leak. The**  **schedule for changing inlet liners and septa is dep endent on the operation of the**  **injection system, the nature of samples and paramet ers tested, and acceptance** 

**criteria in the reference method.**

**If chromatographic performance or ghost peaks are s till a problem after**  **performing these inlet maintenance steps, replacing the seal in the bottom of the**  **inlet or cleaning and deactivating the metallic sur faces of the injection port itself may**  **be necessary. Deactivation of the injection port n ecessitates the use of toxic**  **reagents and should only be performed by knowledgea ble personnel according to** 

**the instrument manufacturer’s instructions.**

**11.11.1.2 Column maintenance**

**Capillary columns are reliable and easy to use, but overheating and**  **exposure to oxygen can cause damage. Install and c ondition the column as**  **recommended by the manufacturer, and flush the colu mn with carrier gas before**  **conditioning. Avoid contact between the capillary column and the metal surfaces in**  **the GC oven or heating above the maximum column tem perature.** 

**Poor chromatographic performance may also be observ ed when the head**  **of the GC column is contaminated with high-boiling material. Removing as much as**  **0.5 - 1 m from the injector side of the capillary c olumn may restore chromatographic**  **performance. If clipping the head of the column do es not restore performance,**  **replacement of the column may be necessary. Using a guard column may extend** 

**column life.**

**11.11.1.3 Gas delivery system maintenance**

**Analysts should periodically ensure that proper flo w control is maintained.**  **A search for leaks using an electronic leak detecto r or by isolating and pressure**  **testing various parts of the delivery system may be conducted; static pressure tests**  **may also be performed, or other appropriate measure s taken. Electronic pressure**  **controller flow rates should also be checked regula rly (with both the injector and the**  **oven heated) using a bubble meter or other appropri ate device. A leak in the gas**  **delivery system and/or change in delivery pressure can lead to retention time shifts**  **of the target analytes in the GC chromatograms, so if this is observed Monitoring**  **retention times in standards over time can help to ensure that** **SW-846 Update V 8000D- 53 Revision 4**

**October 2012 11.11.2 HPLC preventive maintenance and corrective**  **action**

**Band broadening occurs whenever there is a dead vol ume between the injector and**  **detector. Therefore, plumbing connections should b e of minimum length and diameter,**  **and ferrules should be properly positioned on the t ubing to minimize dead volume.** 

**11.11.2.1 Injection port maintenance**

**Filtration of extracts and injection of solvent mix tures miscible with the**  **mobile phase can help minimize solvent-related prob lems. Otherwise,**  **contamination of subsequent injections may occur wh en the extract contains**  **material that is not soluble in the mobile phase. Injectors also need maintenance,**  **as the surfaces that turn past each other tend to w ear down over time, potentially**  **causing leaks. Injection loops are easily changed, but analysts must ensure that**  **the compression fittings are properly installed to prevent leaks.** 

**11.11.2.2 Column maintenance**

**Use of high quality columns that are uniformly pack ed with the appropriate**  **particle size and bonded phase will result in optim al chromatographic performance.**  **Column temperatures may be regulated by the use of temperature control ovens to**  **ensure reproducibility of retention times.**

**Lifetime and performance of HPLC columns can be imp roved through**  **proper maintenance. Sample extracts should be filt ered prior to analysis, and care**  **should be taken to ensure that storage conditions (** e.g. **, freezing) do not cause subsequent precipitation of solids in the extracts**  **prior to analysis. Guard columns should be used when dirty samples are analyzed beca**  **use HPLC columns can become contaminated with particulates or insoluble**  **materials. If degradation of resolution or changes in back pressure are observed**  **, the replacing the guard column if one is installed may restore performance.**  **Columns should not be stored dry or containing strong buffers, and they should b**  **e replaced when performance degrades (e.g., significant band broadening, peak s**  **plitting, or loss of chromatographic resolution occurs).** 

**11.11.2.3 Mobile phase and pump maintenance**

**Pumping systems should deliver reproducible gradien ts at a uniform flow**  **rate. Pumping flow rates can be checked by collect ing solvent into a graduated**  **cylinder for a designated time period.**

**Air bubbles tend to cause an erratic baseline and, in the case of** 

**low**

**- pressure mixing, bubbles can cause the pump to cavi**  **tate. Therefore, HPLC solvents should be degassed prior to use.** 

**Non-reproducible gradients can result from deterior ation in pump**  **performance and can cause unacceptable variation in retention times from run to**  **run. Mobile phase solvents should be filtered to r emove particles that cause pump**  **piston wear. Seals in the HPLC pumps should be rep laced regularly. Use of**  **strong buffers or solvents such as tetrahydrofuran can significantly shorten the**  **lifetime of pump seals and should be avoided where practical.** 

**Small changes in the composition or pH of the mobil e phase can have a** **SW-846 Update V 8000D- 54 Revision 4**

**October 2012 significant effect on retention times. Buffering t**  **he mobile phase may help make the pH more reproducible from preparation to preparatio**  **n, as long as it doesn’t conflict with the method or cause other problems with the an**  **alysis. Precise measurement of reagents and care in mixing the mobile phase may**  **help ensure consistency from one preparation to the next. A solvent mixer may b**  **e the best way to ensure reproducibility of the mobile phase over time.** 

**12.0 DATA ANALYSIS AND CALCULATIONS**

**12.1 See Sec. 11.0 and the appropriate determinativ e method for information regarding**  **data analysis and calculations.**

**12.2 Results must be reported in units commensurat e with their intended use and all**  **dilutions must be taken into account when computing final results.** 

**13.0 METHOD PERFORMANCE**

**13.1 Performance data and related information are p rovided in SW-846 methods only as**  **examples and guidance. Data do not represent requi red performance goals for users of the**  **methods. Instead, performance goals should be deve loped on a project-specific basis and the**  **laboratory should establish in-house QC performance criteria for the application of this method.**  **These performance data are not intended to be and m ust not be used as absolute QC acceptance**  **criteria for purposes of laboratory accreditation.**

**13.2 Refer to individual determinative methods for performance data examples and**  **guidance.**

**14.0 POLLUTION PREVENTION**

**14.1 Pollution prevention encompasses any technique that reduces or eliminates the**  **quantity and/or toxicity of waste at the point of g eneration. Numerous opportunities for pollution**  **prevention exist in laboratory operation. The EPA has established a preferred hierarchy of**  **environmental management techniques that places pol lution prevention as the management**  **option of first choice. Whenever feasible, laborat ory personnel should use pollution prevention**  **techniques to address their waste generation. When wastes cannot be feasibly reduced at the**  **source, the EPA recommends recycling as the next be st option.** 

**14.2 For information about pollution prevention tha t may be applicable to laboratories and** 

**research institutions consult** Less is Better: Laboratory Chemical Management for Waste

Reduction **, a free publication available from the American Ch emical Society (ACS), Committee on**  **Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229**  **0/pdf/WPCP_012290.pdf**

**.**

**15.0 WASTE MANAGEMENT**

**The EPA requires that laboratory waste management p ractices be conducted consistent**  **with all applicable rules and regulations. Laborat ories are urged to protect air, water, and land by**  **minimizing and controlling all releases from hoods and bench operations, complying with the**  **letter and spirit of any sewer discharge permits an d regulations and complying with all solid and**  **hazardous waste regulations, particularly the hazar dous waste identification rules and land**  **disposal restrictions. For further information on waste management, consult**  The Waste**SW-846 Update V 8000D- 55 Revision 4**

**October 2012** Management Manual for Laboratory Personnel **available from the American Chemical Society at**

**the address listed in Sec. 14.2 above.**

**16.0 REFERENCES**

**16.1 For further information regarding these method s, review Methods 3500, 3600, 5000,**  **the individual sample preparative, cleanup and dete rminative methods, and Chapter One.** 

**16.2 Two references for poorly-performing compounds in general are shown below.** 

**Department of Defense: Appendix G of DoD “**  Quality Systems Manual for Environmental Laboratori es **”, Version 4.2 http://www.denix.osd.mil/edqw/upload/QSM-V4-2-Final**  **-102510.pdf**

**and**

**U.S.E.P.A Contract Laboratory Program SOM01.2 http://www.epa.gov/superfund/programs/clp/som1.htm** 

**16.3 A list of quantitation limits for poorly-per forming**  volatile **compounds can be found in Table App D XIII-1 at:**  **http://www.epa.gov/region1/oeme/appendc.pdf**

**16.4 A list of quantitation limits for poorly-per forming**  semivolatile **compounds can be found in Table App E XIII-1 at:**  **http://www.epa.gov/region1/oeme/appendd.pdf**

**17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION D ATA** 

**There are no tables or figures associated with this method.** **SW-846 Update V 8000D- 56 Revision 4**

**October 2012 Appendix A:** 

**Summary of Revisions to Method 8000C (From Revision 3 March 2003)** 

**1. In Sec. 9.3.3, additional text to indicate tha t the reference samples used for IDP analyses**  **should be prepared from the same source as the cali bration standards. This change is** 

**now consistent with Methods 8260 and 8270. The IDP section was also expanded to**  **include separate language for preparation and extra ction chemists, as well as instrument**  **chemists. This only requires analysts to perform a n IDP on the portion of the procedure**  **that they actually do.**

**2. References to the method quantitation limit (M QL) have been replaced with the lower limit**  **of quantitation (LLOQ).**

**3. Inclusion of Relative Standard Error (RSE) in Se c. 11.5.4.2.** 

**4. Improved overall method formatting for consisten cy with new SW-846 methods style**  **guidance. The format was updated to Microsoft Word 2007.docx.** 

**5. Many minor editorial and technical revisions wer e made throughout to improve method**  **clarity.**

**6. The revision number was changed to 4 and the dat e published was changed to October** 

**2012.**

**7. This appendix was added showing changes from the previous revision.** 

**8. Items regarding instrument maintenance in Secs. 4 and 6 were condensed and moved to** 

**Sec. 11.11.**