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 chemical analysis and in the use of the subject 

technology. 

In addition, SW-846 methods in this manual, with the 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 its own detailed standard operating procedure 

(SOP), either for its own general use or for a specific project application. Performance data 

included in this method are for guidance purposes only 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 instead provides guidance on 

analytical chromatography and describes calibration and QC requirements common to all 

SW-846 chromatographic methods. However, more specific QC requirements provided in the 

applicable determinative method will supersede those noted in Method 8000. Method 8000 

should be applied in conjunction with all SW-846 determinative chromatographic methods. The 

methods include, but are not limited to, the following: 

Method 

Number

Analytes Chromatographic 

Technique (Sec. 1.5)

Detector

6850 Perchlorate HPLC MS, MS/MS

6860 Perchlorate IC MS, MS/MS

7580 White phosphorus (P4) 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
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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 spectrometry 

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 
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October 2012 

1.2 Analytical chromatography is used to separate target analytes from co-extracted 

interferences in samples. Chromatographic methods can be divided into two major categories: 

gas chromatography (GC) and high performance liquid chromatography (HPLC). 

1.2.1 Gas chromatography is the separation technique of choice for organic 

compounds which can be volatilized without being decomposed 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 separation 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 separation 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 have different retention times. Compounds 

that interact strongly with the stationary phase elute slowly (i.e., longer retention times), while 

compounds that remain in the mobile phase elute quickly (i.e., shorter retention times). 

1.3.1 The mobile phase for GC is an inert gas, usually hydrogen or helium, and 

the stationary phases are generally polymer bases. 

1.3.2 In "normal phase" HPLC, the mobile phase is less polar than the stationary 

phase. In "reverse phase" HPLC, the converse is true. Reverse phase HPLC is the 

technique of choice for environmental and waste analyses of non-volatile organic target 

analytes. 

1.3.3 Ion exchange chromatography is used to separate ionic species through 

competition with ions in the mobile phase for oppositely charged exchange sites on a 

stationary phase. Differential selectivities of the ionic species and the mobile phase ions 

for exchange sites are responsible for the chromatographic separation of the ions. 

1.4 A number of specific GC and LC techniques are used 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 analytes listed in that specific procedure. Most 

often, those columns were the ones used by EPA during method development and testing. 

Analysts may change those columns and conditions, provided that they demonstrate 

performance for the analytes of interest that is appropriate 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 list 

of target analytes in a method are needed, the chromatographic conditions and columns may be 

optimized for those analytes). 

1.5.1 Chromatographic performance is demonstrated by the resolution of 

standards and the ability to model the response of the detector during calibration, and by 
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sensitivity, precision, bias, frequency of false positives, and frequency of false negatives 

during analysis. The laboratory must demonstrate that any chromatographic procedure it 

uses provides performance satisfying the analytical requirements of the specific application 

for which it is being used. Such demonstrations should be performed using the procedures 

outlined in Secs. 9.2 to 9.8 of this method and appropriate sections in Chapter One. 

1.5.2 Laboratories must also be cautious whenever the 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 for confirmation of results obtained using 

an SPB-5 column because the stationary phases are not 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 temperature changes the rate of partitioning 

between the mobile and stationary phases, leading to shorter retention times. GC retention 

times can also be changed by selecting a column with a different length, stationary-phase loading 

(i.e., capillary column film thickness or percent loading 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 times 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 phase is easily altered by adjusting the 

composition of the solvent mixture being pumped through the column. In reverse phase HPLC, 

increasing the ratio of water-miscible organic solvent to water generally shortens retention times. 

HPLC retention times can also be changed by selecting a column with a different length, 

alternative bonded phase, or dissimilar particle size (e.g., smaller particles and/or a longer column 

generally increase column resolution, while different bonded phases may resolve specific 

components differently). HPLC methods are also particularly 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 provide conditions that have been 

demonstrated to provide good HPLC separations using specific instruments to analyze a limited 

number of samples. Analysts (particularly those using 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. However, 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, experienced 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 when used for site investigations, including 

coelution of target analytes, false negatives due to 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 or non-selective detectors 

may be appropriate for remediation activities where the analytes of concern are known, of 

limited number, and of significantly greater concentration than potentially interfering 

materials. 
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1.8.3 If the site is not well characterized, and especially 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. Lists in some methods are lengthy; it may not 

be practical or appropriate to determine all the analytes 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 compounds. 

1.10 Analysts should consult the disclaimer statement at the front of the manual and the 

information in Chapter Two for 1) guidance on the intended flexibility in the choice of methods, 

apparatus, materials, reagents, and supplies; and 2) the responsibilities of the analyst for 

demonstrating that the techniques employed are appropriate for the analytes of interest, in the 

matrices of interest, and at the levels of concern.

In addition, analysts and data users are advised that, except where explicitly required in a 

regulation, the use of SW-846 methods is not mandatory in response to Federal testing 

requirements. The information contained in this method 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 application. 

1.11 All of the SW-846 determinative chromatographic methods that reference this 

method are restricted to use by, or under the supervision of, analysts experienced in the use of 

gas or high performance liquid chromatographs and skilled in the interpretation of 

chromatograms. Each analyst must demonstrate the ability to generate an acceptable initial 

demonstration of proficiency (IDP) along with acceptable results according to method 

recommendations and stated project data quality objectives (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 chromatography. 

2.0 SUMMARY OF METHOD 

This method describes general considerations in achieving chromatographic separations 

and performing calibrations. It is to be used in conjunction 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 appropriate procedures for sample preparation, 

extraction, cleanup, and/or derivatization. Consult the specific procedures for additional 

information on these crucial steps in the analytical process. 

2.1 Sec. 4.2 of this method provides general guidance on minimizing contamination, 

including cross-contamination between samples. Sample 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 procedure(s) described in Method 8000 must be 

satisfied. 

2.3 Secs. 4.4 and 4.5 provide information on the effects of chromatographic 

interferences. 

2.4 Sec 6.0 of this method contains generalized specifications for the components of 

both GC and HPLC systems used in SW-846 analyses. 
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2.5 Calibration of the analytical system is another critical step in the generation of quality 

data. Sec. 11.5 discusses specific procedures and calculations for both linear and non-linear 

calibration models. Continued use of any chromatographic procedure necessitates a verification 

of the calibration model, and procedures for such verifications are described in this method as 

well (Sec. 11.7). 

2.6 Identification of target compounds by any chromatographic procedure is based, at 

least in part, on retention times. Sec. 11.6 provides procedures for the determination of retention 

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

2.7 Calculations necessary to derive sample-specific concentrations from the instrument 

responses are common to most of the analytical methods listed in Sec. 1.1. Commonly used 

calculations are summarized in Sec. 11.10. 

2.8 Preventive maintenance and corrective actions are essential to the generation of 

quality data in a routine laboratory setting. Suggestions 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 Chapter 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 all 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 procedures. 

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 analysis. All of these materials must be 

demonstrated to be free from interferences under conditions of analysis by analyzing method 

blanks. Specific selection of reagents and purification 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 high- and low-concentration 

samples are analyzed in sequence. To reduce potential for carryover, the sample syringe or 

purging device must be thoroughly rinsed between samples with an appropriate solvent (including 

water). Purge-and-trap or headspace devices should be thoroughly baked out between 

samples. 
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October 2012 

Where practical, samples with unusually high concentrations of analytes should be followed 

by method blanks or instrument blanks or by analysis of organic-free reagent water to check for 

carryover contamination. If target compounds present in an unusually highly concentrated 

sample are also found to be present in subsequent samples, the analyst must demonstrate that 

the compounds are not affected by carryover contamination. Conversely, if those target 

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 methanol, followed by a distilled water rinse 

and drying in a 105 °C oven between analyses. Detergent solutions may also 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 autosamplers which rinse the vessel(s) between 

runs, may also be employed, provided that the laboratory 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 contamination 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 for 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 results to choose an appropriate dilution factor for 

the GC/MS analysis that will prevent system contamination yet still provide adequate sensitivity 

for the major constituents of the sample. 

4.4 Elevated chromatographic baselines (e.g., baseline 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 quantitative errors. When elevated baselines are 

observed during analysis of blanks and standards, the chromatographic system should be 

considered contaminated. This contamination can result from impure carrier gas, inadequate 

gas conditioning, septum bleed, column oxidation, incomplete 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 maintenance and corrective action. 

4.5 See Sec. 11.11 for suggested preventative maintenance activities that may prevent 

or ameliorate deterioration of chromatographic performance. 

5.0 SAFETY 

This method does not address all safety issues associated 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 chemicals and instrumentation included in this 

method. A reference file of material safety data sheets (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 in this manual is for illustrative 

purposes only, and does not constitute an EPA endorsement or exclusive recommendation for 
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October 2012 

use. Products and instrument settings cited in SW-846 methods represent those products and 

settings used during method development or subsequently evaluated by the Agency. 

Glassware, reagents, supplies, equipment, and settings other than those listed in this manual 

may be employed provided that method performance appropriate for the intended application has 

been demonstrated and documented. 

 This section does not list all common laboratory glassware (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 purge-and-trap apparatus is described in 

Method 5030 for water samples and in Method 5035 for soil and other solid samples. See 

Method 5000 for guidance on all forms of sample introduction of volatiles into the GC and 

GC/MS system. 

6.2.2 Semivolatile organics 

Sample extracts containing semivolatile organic compounds 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 maintenance 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 columns (> 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 the flow (and sample) onto the 

head of the analytical column. After a predetermined splitless introduction time the 

split valve is opened so that most of the flow is vented during analysis, thus 

eliminating the solvent tail while maintaining proper flow through the column. The 

initial oven temperature should be below the boiling point of the injection solvent if 

the solvent front interferes with early-eluting analytes or if the solvent effect is 

needed to resolve difficult to separate analytes. 

6.2.2.4 Cool on-column injection and programmable temperature 

vaporizer inlets allow the analysis of labile compounds that degrade on packed 

columns and in split/splitless injectors. 

6.3 GC flow control 

Precise control of the gas mobile phase is necessary to achieve reproducible GC retention 

times. Flow controllers within any GC used for analyses described in SW-846 methods should 

deliver a precisely metered gas flow at a rate appropriate for the GC column mounted in the 

instrument. 
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October 2012 

6.3.1 Most GCs have restrictors built into electronic pressure controllers (EPCs) 

monitored using a digital readout. These restrictors are used to provide precise flow at the 

carrier gas flow rate listed in the method (e.g., use <20 mL/min restrictors for wide-bore 

capillary methods). 

6.3.2 Analysts should ensure that cylinder pressures are regulated properly and 

manifold pressures are sufficiently large that a change in the head pressure of an individual 

instrument does not affect the flow through all instruments. 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 conditioned between the cylinder 

and the GC with a scrubber to remove any residual water, oxygen and hydrocarbons as 

necessary. Gas regulators should contain stainless steel diaphragms. (Neoprene 

diaphragms are a potential source of gas contamination, and should not be used.) 

6.4 GC columns 

Each determinative method in SW-846 provides a description 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 criteria 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 intended application. 

Use of capillary columns has become standard practice in environmental and waste 

analysis. Capillary columns have an inherently greater ability to separate analytes than packed 

columns. However, packed columns can provide adequate resolution of some analytes and are 

most appropriately employed when the list of analytes to be determined is relatively short. 

6.4.1 Narrower columns are more efficient (i.e., can 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; resolution 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., loading) increases column 

capacity and retention times. 

6.5 GC detectors 

Detectors are the transducers that respond to components 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, selective non-MS detectors should be maintained 

at least 20 °C above the highest oven temperature employed to prevent condensation and 

detector contamination. To prevent condensation between the GC and an MS detector, transfer 

lines should be maintained at a temperature above the highest temperature of the oven program, 

or as specified by the instrument manufacturer. 
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6.6 HPLC injectors 

Liquids are essentially uncompressible, so a mechanical device is necessary that allows 

introduction of the sample into a high pressure flow without significant disruption in the flow rate 

and hydraulic pressure. Normally, a 6-port valve is used for this purpose. A sample loop is 

isolated from the flow of the mobile phase and filled 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 turned so that the mobile phase flows through the 

loop. This procedure virtually eliminates dead volume 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 delivering solvent at 5000 psi or above with 

excellent precision. Rate of delivery depends on the column used for the separation. Flow 

rates should be checked by collecting column effluent in a graduated cylinder for a designated 

time period. 

Most pumping systems are capable of changing solvent 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 proportional mixing of the solvents before they are 

pumped. In either case, solvent mixing can cause changes in the solubility of dissolved gases, 

formation of bubbles in the mobile phase, or non-reproducible gradients. 

6.8 HPLC Columns 

 HPLC columns are generally constructed of stainless steel tubing and are sealed with 

compression fittings. These columns should be constructed with minimum dead volume and a 

narrow particle size distribution. Manufacturers provide columns bonded with dissimilar 

functional groups (e.g., C18, cyano, TMS) and have different percent carbon loading. 

6.8.1 Use of high quality columns that are uniformly packed with the appropriate 

particle size and bonded phase will result in optimal chromatographic performance. For 

example, columns with silica-based particles with free 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 column is held at a constant temperature. 

Temperature control ovens capable of maintaining the HPLC column at ± 0.1 °C should be utilized 

to provide consistent retention times throughout the course of an HPLC analysis. Normal oven 

operating temperature should be 3 – 5 °C above ambient laboratory temperature. 

6.10 HPLC detectors 
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Detectors are the transducers that respond to components 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 interface that separates target analytes from the 

aqueous mobile phase. Examples include the thermospray (TSP), electrospray (ESP), and the 

atmospheric pressure chemical ionization (APCI) interfaces. 

6.11 Data systems 

Raw chromatographic data have to be reduced in order 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. Organizations should select the system most 

suitable for their applications. 

6.12 Supplies 

Chromatographers use a variety of supplies. Specific items that should be stocked depend 

on laboratory instrumentation and the analyses performed. 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 need supplies such as high 

purity gases, scrubbers for gas conditioning, gas-tight fittings, capillary cutters, magnifying 

glasses, septa with proper temperature limits, appropriate ferrules, dichlorodimethylsilane 

(for deactivating surfaces), glass wool, spare columns and injection port liners. 

6.12.2 Laboratories performing HPLC analyses need supplies such as high purity 

solvents, column packing material, frits, narrow inner 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 tests. Unless otherwise indicated, it 

is intended that all reagents conform to the specifications of the Committee on Analytical 

Reagents of the American Chemical Society, where such specifications are available. Other 

grades may be used, provided the reagent is of sufficiently high purity to permit its use without 

lessening the accuracy of the determination. Reagents should be stored in glass to prevent 

leaching of contaminants from plastic containers. 

7.2 See specific extraction and determinative methods for the reagents and standards 

needed. 

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

Sample collection, preservation and storage requirements may vary by EPA program and 

may be specified in a regulation or project planning document that requires compliance 

monitoring for a given contaminant. Where such requirements 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 collection, preservation and storage 
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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 assurance (QA) and quality control (QC) 

protocols. When inconsistencies exist between QC guidelines, 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 effort involving collection of analytical data 

should include development of a structured and systematic planning document, such as a Quality 

Assurance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which translates project 

objectives and specifications into directions for those that will implement the project and assess 

the results. 

Each laboratory should maintain a formal quality assurance program. The laboratory 

should also maintain records to document the quality of the data generated. Development of 

in-house QC limits for each method is encouraged, as described in Sec. 9.6. Use of instrument 

specific QC limits is encouraged, provided such limits 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 chromatography 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 analysis is performed, the 

performance of the instrument system should be checked. These checks should be part of 

a formal quality control program that includes analysis of instrument blanks, calibration 

standards, and other QC as appropriate for that method. In addition to these instrument 

QC checks, performance of the entire analytical process (i.e., preparation, cleanup and 

analysis) should be monitored. These additional checks should include method blanks, 

matrix spikes, laboratory control samples, replicate samples and other QC (e.g., the lower 

limit of quantitation (LLOQ)) as appropriate for that method or project. It is generally 

advisable, although not required, that all method QC samples be run at the same time as the 

samples on the same instrument. 

9.2.2 Ongoing data quality checks are compared with established performance 

criteria to determine if the results of analyses meet the performance characteristics of the 

method and/or the measurement quality objectives (MQOs) established for intended 

application. 

9.2.3 In addition to the quantitative measures of comparison described below 

and in the individual methods, analysts should evaluate 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 analyses? 
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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 detector response and laboratory 

contamination should be corrected. Tailing problems 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 place when performance 

changes to the point that the calibration verification acceptance criteria (Sec. 11.7) cannot 

be achieved. Recalibration of the instrument should be performed as required per 

determinative methods. 

9.2.6 Before processing any samples, the analyst should demonstrate that all 

parts of the equipment contacting the sample and reagents 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 chronic laboratory contamination. Consult 

the appropriate 3500 or 5000 series method for specifics 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 frequency of at least 

5%: one method blank for each group of up to 20 samples prepared at the same 

time, by the same procedures. For volatile samples analyzed by purge-and-trap, 

preparation is equivalent to the analysis. Therefore, 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 are analyzed on 

separate instruments or in separate analytical shifts, the method blank associated 

with those samples (e.g., extracted with the samples) must be analyzed on at least 

one of those instruments. A solvent blank should be analyzed on all other 

instruments on which the set of samples was analyzed to demonstrate the 

instrument is not contributing contaminants to the samples. 

9.2.6.3 Unless otherwise described in a determinative 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 cleanup 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 of acceptable blank contamination 

specified in the approved quality assurance project plan (QAPP) or other appropriate 

systematic planning document. 
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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 any samples associated with the 

contaminated method blank. If the method blank results still do not meet the 

acceptance criteria in 9.2.6.5 and re-analysis is not practical, then the data user 

should be provided with the sample results, the method 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 concentration to avoid 

reporting false positive results. 

9.2.6.8 Method and/or solvent blanks may also be used to check for 

contamination by carryover from a high-concentration sample into subsequent 

samples (Sec. 4.2). When analysis of such blanks is not possible, such as when an 

unattended autosampler is employed, the analyst should carefully review the results 

for at least the next sample after the high-concentration 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 evidence that carryover may have 

occurred, then the affected samples should be reanalyzed. 

9.3 Initial demonstration of proficiency (IDP) 

Prior to implementation of a method, each laboratory 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 change to instrumentation or procedure 

occurs, the laboratory must demonstrate that acceptable precision and bias can still be obtained 

by the changed conditions (Sec. 9.3.1). Whenever new staff members are trained, an analyst 

IDP must be performed. (Sec. 9.3.2). 

9.3.1 Demonstration of proficiency for instrument or method changes 

If a major change to the sample preparation procedure is made (e.g., a change in 

solvent), the IDP must be repeated for that preparation procedure by a minimum of four 

spiked reference samples. Alterations in instrumental procedures only, such as changing 

GC temperature programs or HPLC mobile phases or the detector interface, require a new 

calibration but not a new IDP because the preparation procedure is unchanged. Each 

laboratory must have policy for performance and documentation of IDP. 

9.3.2 Demonstration of proficiency for new analysts

Each laboratory should have a training program which 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 that the new analyst is capable of 

successfully following the SOP established by the laboratory. 

 

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 prepare reference samples for a 

representative set of analytes (e.g., the primary analyte mix for Method 8270, or a mix of 

Aroclor 1016 and 1260 for Method 8082) for each preparation method the analyst will be 

performing. The instrument analyst being trained will 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 spiking solution 

containing each analyte of interest. The reference sample concentrate (spiking 

solution) may be prepared from pure standard materials or purchased as certified 

solutions. This reference standard should be made from the same source as the 

calibration standards to eliminate any additional variability due to differences 

between sources. 

 9.3.3.2 Preparation of the reference sample concentrate is dependent 

upon the method being evaluated. Guidance for certain methods is listed in 

Methods 3500 and 5000. In other cases, the determinative methods contain 

guidance on preparing the reference sample concentrate and the reference sample. 

If no guidance is provided, prepare a reference sample 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 of the calibration range for each 

analyte in that matrix. 

 9.3.3.3 Concentrations of target analytes in the reference sample may 

be adjusted to reflect more accurately the concentrations 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 for information on selecting an 

appropriate spiking level. 

9.3.4 Evaluation 

 9.3.4.1 To evaluate the performance of the total analytical process, 

reference samples must be handled in exactly the same manner as actual samples. 

Additional LCS or MS/MSD samples can be omitted. Use a clean matrix for spiking 

purposes (one without any target or interference compounds) 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 replicate 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 (usually a 3500 series method for 

extractable organics or a 5000 series method for volatile 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 results. 

 9.3.4.4 Multiple-laboratory performance data are included in some 

determinative methods and may be used as guidance in 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 data. 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 method. If s and ( for all 

analytes of interest meet the appropriate acceptance criteria, then the system 

performance is acceptable and analysis of actual samples can begin. If any 

individual s value exceeds the precision limit or any value falls outside the range 

for bias, then the system performance may be unacceptable for that analyte. 

NOTE: The large number of analytes in each of the methods presents a substantial 

probability that one or more analyte will fail at least one of the performance 

criteria when all analytes of a given method are determined. 

 9.3.4.5 Performance data in many of the methods are based on 

single-laboratory performance. As with multiple-laboratory 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 outside the limits; however, the 

majority should be within the acceptance limits. 

 9.3.4.6 When one or more of the analytes fail at least 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 interest 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 documenting the effect of the matrix 

on method performance (precision and bias). At a minimum, this check should include the 

analysis of at least one matrix spike and one duplicate unspiked sample or preferably, one 

matrix spike/matrix spike duplicate (MS/MSD) pair with each preparation batch of up to 20 

samples of the same matrix processed together (Chapter One). If samples are expected to 

contain target analytes of concern, laboratories may use one matrix spike and a duplicate of 

an unspiked field sample as an alternative to the MS/MSD pair (Sec. 9.4.3). 

For samples requiring an extraction procedure separate from analysis (e.g., 

semivolatiles by Method 8270), the MS/MSD, or MS and duplicate sample, should be 

extracted with the batch of samples but may be analyzed at any time. Conversely, if 

calibration standards and other analytical QC are processed 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 indicate 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 matrix. In the case of very contaminated 

samples or when the lab does not receive enough samples 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 processed identically to the samples. 
SW-846 Update V 8000D- 17 Revision 4 

October 2012 

In the case of samples that need an extraction procedure separate from analysis 

(e.g., semivolatiles by Method 8270), the LCS should be extracted with the batch of samples 

but may be analyzed at any time. However, if calibration 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 the samples and may also serve as the 

continuing calibration verification (CCV) standard.

NOTE: If an LCS also serves as a CCV, acceptance criteria 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 determined 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 assessment (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 concentration of a specific 

analyte in the sample is being checked against a regulatory concentration limit or 

action level, the spike should be at or below the limit, or 1 - 5 times the background 

concentration (if historical data are available), whichever 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 purposes to ensure that high 

concentrations of target analytes and/or interferences 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, then the analyst may spike the MS or 

MS/MSD sample(s) at the same concentration as the reference sample (Sec. 9.3.3) 

at 20 times the estimated LLOQ in the matrix of interest, or at a concentration near 

the middle of the calibration range. It is suggested 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 source 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 sample, and an MS/MSD pair; or 

analyze the original sample, a duplicate sample, and one spiked sample. If 

samples are not expected to contain the target analytes of concern, then the 

laboratory may use a MS/MSD pair. If samples are expected to contain the target 

analytes of concern, then the laboratory may use one matrix spike and a duplicate 

analysis of an unspiked field sample as an alternative to the MS/MSD pair. 

9.4.2.4 Begin by analyzing one sample aliquot to determine 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 aliquot 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 procedures 

employed for the original sample, and calculate the concentration of each analyte in 

the matrix spike and matrix spike duplicate. Likewise, 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 analytes 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 LCS according to the following 

formula. 

	
  % 

  





  100

where: 

Cs = Measured concentration of spiked sample aliquot 

Cu = Measured concentration of unspiked sample aliquot (use 0 for LCS) 

Cn = Nominal (theoretical) concentration increase that results from 

spiking the sample, or the nominal concentration of the spiked 

aliquot (for LCS). 

MS/MSD recoveries may not be meaningful if the amount 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 difference (RPD) of the 

concentrations (not the recoveries) measured for MS/MSD pairs, or for duplicate 

analyses of unspiked samples. Calculate RPD according to the formula below. 

where:

 C1 = Measured concentration of first sample aliquot

C2 = 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 the MS and MSD results in 

an artificially high RPD when based on concentration. Using 

approximately the same sample size or scaling the spike amount to the 

sample size for the MS/MSD will minimize bias in the 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, laboratories should monitor method 

performance in each matrix, through the use of control charts and other techniques. 

Many methods may not contain recommended acceptance criteria for LCS results. 

The laboratory should use 70 - 130% as interim acceptance 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 in a clean matrix. 

Ideally, the acceptance criteria for MS/MSD recovery and/or duplicate relative % 

difference will be established for the field samples through the DQOs contained in a written 

QAPP. These criteria should be established with consideration given to performance data 

provided in the reference method and/or by the laboratory 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 criteria 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 develop in-house performance criteria based 

on their historical data for use in project planning and for comparison to any relevant 

performance criteria in the reference methods. Development of in-house performance 

criteria is discussed in Sec. 9.6. Where methods do contain performance data for the 

matrix of interest, use Secs. 9.4.4.1 - 9.4.4.3 below 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 same matrix. Given that such 

method performance criteria were developed from multi-laboratory data, they should 

be met by almost all laboratories. See Sec. 9.6.10 for more information on 

comparisons between limits. Performance data include an allowance for error in 

measurement of both the background and spike concentrations. If spiking was 

performed at a concentration substantially lower than 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 the laboratory has sufficient data (a 

minimum of 20 or more MS/MSD samples of the same matrix) to generate their own 

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

October 2012 

precision and bias limits. Alternatively, acceptance 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 calculated MS/MSD, rather than LCS 

recovery limits once sufficient data points have been 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 matrix spike with the LCS data 

(use the average recovery if an MS/MSD were analyzed). If any individual %R in the MS 

and/or MSD falls outside the designated range for recovery, the laboratory should determine 

if there is a matrix effect or a laboratory performance 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 recovery limits. General considerations for 

developing in-house acceptance criteria for surrogate 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 recovery limits, the following procedures 

are necessary. 

9.5.2.1 Check for errors in the calculations, surrogate solutions or 

internal standards. If errors are found, recalculate 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 instrument problem is 

identified, correct the problem and reanalyze the sample or extract. 

9.5.2.4 If no instrument problem is found, the sample should be 

re-extracted and reanalyzed (or another vial reanalyzed 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 "estimated concentration." If the 

recovery is within the limits in the reanalysis, report 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 accordingly. 
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 overcome significant interferences. This 

may result in dilution of the surrogate responses to 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 limits 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 criteria and control charting or similar 

procedures to track laboratory performance cannot be overemphasized. Many data systems 

and commercially available software packages support the use of control charts. 

Procedures for calculating in-house performance criteria for MS/MSD, LCS and surrogate 

recoveries are provided below. 

9.6.1 Once sufficient data have been acquired and the recovery and RPD 

calculated as in Secs. 9.4.3 and 9.5 for a given sample 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; and, 

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 generate 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 laboratory 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 should 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 outside of control limits 

necessitates evaluation by the laboratory such as comparison with the LCS recovery. 

9.6.4.1 If recoveries of analytes in the LCS are outside of the control 

limits, then the problem may lie with application of the extraction and/or cleanup 

procedures applied to the sample matrix, or with analysis. Once the problem has 

been identified and addressed, corrective action may 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 number of analytes in the LCS 

or matrix spike, the statistical probability of a few analytes outside of control limits 

becomes high. Therefore, a number of analytes should be allowed to marginally fail 

the limits without requirement for corrective action. 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 monitoring, the analyst must take 

steps to demonstrate that the analytes of concern can be determined in the sample 

matrix at the levels of interest. 

9.6.5 Once established, control limits should be reviewed regularly and updated 

on a routine basis as established by the laboratory’s quality management plan. Reviewing 

appropriateness of these criteria with respect to generated data is especially important for 

newly implemented procedures or those not in continual 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 those in place at the time of sample 

analysis. Once control limits are updated, they should 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 limits should be established using available 

data or by analogy to similar methods or matrices. 

9.6.7 Results used to develop acceptance criteria should meet all other QC 

criteria associated with the determinative method. For example, matrix spike recoveries 

from a GC/MS procedure should be generated from samples 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 retention 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 criteria for matrix spike recovery and 

precision are often a function of the spike concentration used. Therefore, use caution 

when pooling data in establishing acceptance criteria. Not only should results all be from 

roughly the same type matrix but spiking levels should also be similar. 

9.6.9 Similarly, acceptance criteria for matrix spike, 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 solid 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 acceptance criteria is discarding data 

that do not meet a preconceived notion of acceptable performance. I.e., while professional 

judgment is important in evaluating data used to develop acceptance criteria, do not discard 

specific results simply because they do not meet one'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 without the results considered suspect. 

Then, observe the effect of deleting suspect data. 

9.6.11 In-house QC limits must be examined for reasonableness; it is not EPA's 

intent to legitimize poor recoveries due to incorrect choice of methods or spiking levels. 

In-house limits should also be compared with the DQOs of specific analyses. For 

example, recovery limits (for surrogates, MS/MSD, LCS, 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 of the potential for high bias. 

9.6.12 It may be useful to compare QC limits generated in the laboratory with 

performance data listed in specific determinative methods. However, the analyst must be 

aware that performance data generated from multi-laboratory studies tend to be significantly 

wider than those generated from a single laboratory. 

9.7 Lower Limit of Quantitation (LLOQ) check standard 

 The laboratory shall establish the LLOQ as the lowest point of quantitation, which in 

most cases, is the lowest concentration in the calibration curve. LLOQ verification is 

recommended for each project application to validate quantitation capability at low analyte 

concentration levels. This verification may be accomplished with either clean control material 

(e.g., reagent water, solvent blank, Ottawa sand, diatomaceous 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 control 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 basis with established DQOs, a 

representative matrix-specific LLOQ verification may provide a more reliable estimate of the 

lower quantitation limit capabilities. 

 9.7.1.1 An LLOQ check standard (not part of an initial calibration) is 

prepared by spiking a clean control material with the analyte(s) of interest at the 

predicted LLOQ concentration level(s). Alternatively, a representative sample 

matrix may be spiked with the analytes of interest at the predicted LLOQ 

concentration levels. The LLOQ check is carried through the same preparation 

procedures as environmental samples and other QC samples. 

9.7.1.2 Recovery of target analytes in the LLOQ check standard should 

be within established in-house limits, or other such project-specific acceptance 

limits, to demonstrate acceptable method performance at the LLOQ. Until the 

laboratory has sufficient data to determine acceptance 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 sufficient 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 to 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 additional quality assurance practices 

for use with 8000-series methods. Specific practices 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 the environmental measurements. When 

doubt exists over identification of a peak on the chromatogram, confirmatory techniques such as 

GC with a dissimilar column, element-specific detector, or mass spectrometer (selected ion 

monitoring or full scan) must be used. Whenever possible, the laboratory should analyze 

standard reference materials and participate in relevant performance evaluation studies. 

10.0 CALIBRATION AND STANDARDIZATION 

Refer to the appropriate determinative method for detailed 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 to selection of sample preparation procedures 

to obtain reliable measurements. 

11.1 Extraction 

Individual determinative methods for organic analytes in SW-846 often recommend 

appropriate sample extraction procedures. General guidance on semivolatile extraction 

procedures can be found in Method 3500. Guidance on volatile procedures can be found in 

Method 5000. 

11.2 Cleanup and separation 

Individual determinative methods for organic analytes in SW-846 often recommend 

appropriate cleanup procedures. General guidance on 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 balance 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 columns and conditions are typically those used 

during development and testing of the method. However, other chromatographic systems may 
SW-846 Update V 8000D- 25 Revision 4 

October 2012 

have somewhat different characteristics as analytical instrumentation continues to evolve. 

Therefore, SW-846 methods allow analysts flexibility to change conditions as long as adequate 

performance is demonstrated. 

 Chromatographic performance is demonstrated by resolution of standards and ability to 

model the response of the detector during calibration; also important are sensitivity, bias, 

precision, frequency of false positives, and frequency of false negatives during analysis. For any 

chromatographic procedures or conditions used, the laboratory must demonstrate that the 

performance satisfies the analytical requirements of the specific application for which the 

chromatographic procedure is being used. Such demonstrations should be performed using 

procedures outlined in Secs. 9.2 - 9.5 of this method and in Chapter One. 

11.4 Initial Calibration 

Calibration of an analytical instrument involves delineation of the relationship between 

response of the instrument and amount or concentration 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 calibration, must be established before the 

analyses of any samples. 

Historically, many analytical methods have relied on linear models of the calibration 

relationship, where the instrument response is directly proportional to the amount of a target 

compound. The linear model has many advantages including simplicity and ease of use. 

However, given the advent of new detection techniques 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 methods involves analysis of standards 

containing the target compounds at a minimum of five different concentrations within the working 

range of the instrument. In order to produce acceptable 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 quantitative errors, regardless 

of the calibration model chosen. It may be necessary to prepare calibration standards that cover 

concentration ranges appropriate for specific projects 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 chromatographic 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. 11.5 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), and GC/FT-IR (e.g., Method 8410) are 
SW-846 Update V 8000D- 26 Revision 4 

October 2012 

described in those methods. Some determinative methods may provide specific guidance on 

calibration such as Method 8085, GC/AED with compound-independent calibration. 

Regardless of the specific calibration technique used, introduce calibration standards and 

samples into the instrument by the same technique and at the same volume. Tabulate peak 

responses against the mass or concentration introduced as described in Secs. 11.4.2 - 11.4.4. 

11.4.1 Preparation of calibration standards 

Calibration standards are prepared using specific procedures indicated in the 

determinative methods. However, the general procedure is described here. 

11.4.1.1 For each analyte and surrogate of interest, prepare calibration 

standards at a minimum of five different concentrations 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 of the compounds but 

rather indicate the applicable method(s). Therefore, if an analyte is not 

relevant to a specific project, it need not be included in the calibration 

standards associated with that project. 

11.4.1.2 The lowest concentration calibration standard 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 back to a sample concentration using 

the sample amount, dilution, and final injection volume 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 working range of the 

detector or correspond to the expected range of concentrations found in samples 

that are also within the working range of the detector. 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 from the adjacent standards by a 

constant factor, e.g., 10, 20, 40, 80, and 160 ng. 

However, the relatively wide spacing of upper standards in a geometric 

series can mask the situation where a detector is reaching saturation and the 

instrument responses are leveling off somewhere between the last two standards. 

Therefore, it may be preferable to use a partial arithmetic series in which 

concentrations of the upper standards differ by a constant amount, not a constant 

factor. Using the same overall calibration range as in the example above, one such 

series might be 10, 20, 40, 80, 120, and 160 ng, with 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 purposes only. Both the overall 

calibration range and concentrations or amounts used for the standards 

are a function of the specific instrumentation, demonstrable 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 range and standards for 

their specific circumstances. 

11.4.1.4 For each analyte, at least one of the calibration standards 

MUST correspond to a sample concentration at or below the quantitation levels 

needed for the project; this may include establishing compliance with a regulatory or 

action limit. Given that different limits may be associated with different analytes, the 

same standard should not be expected to fulfill this 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 necessary to prepare several sets 

of calibration standards, each set consisting of different 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 common approaches to calibrations. 

It involves a simple comparison of instrument responses from the sample to the target 

compound responses in the calibration standards. Sample peak responses are compared 

with calibration standard peak responses. The ratio of the detector response to the amount 

(mass) of analyte in the calibration standard is defined as the calibration factor (CF). 

 

CF 

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

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

Advantages of external standard calibration are that it is simple and can be applied 

to a wide variety of specific chromatographic methods. The primary disadvantage is that it 

is greatly affected by stability of the chromatographic detector system and presence of 

chromatographic interferences in a sample or sample extract. 

The CF may also be calculated using the standard concentration rather than mass in 

the denominator of the equation above. However, use of concentration in calculating the 

CF will necessitate changes to the equations used to calculate sample concentration (Sec. 

11.10.3). 

For multi-component analytes (e.g. PCBs and Toxaphene), 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 of instrument responses from the 

target compounds in the sample to responses of other standards added to the sample or 

extract before injection. Response of the target compound 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 sample 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 included 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 relative response factor (RRF), indicating 

that the target compound response is calculated relative 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 to normalizing the peak response of the 

target compound to the response of the internal standard in the sample or extract for that 

injection, the internal standard may be used to calculate the relative retention time (RRT) of 

the target compound. RRT is expressed as a unitless quantity. 

2 

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

"!"$! "$% ' "0 $!"
!#3 /"#!(#
(

 If RRT is used by the laboratory to establish peak identity, the RRT of each target 

analyte in each calibration standard should agree within established limits set by the 

laboratory in their SOP. These limits should be appropriate for reliable identification of the 

target analyte. If this criterion is not met and there are no other indicators of an analyte's

identification (such as a very unique and high probability mass spectral match), that analyte

may not be considered as identified by RRT. 

The RRT of the analyte in the sample should be within 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 and high probability mass spectral 

match), that component may not be considered as identified by RRT. 

RRT evaluation allows the analyst to compensate for modest shifts in the 

chromatographic conditions that can occur due to interferences and day-to-day instrument 

variability. Many methods that employ internal standard 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 calibration are that internal standards 

must be compounds not found in the samples to be analyzed and they must produce an 

unambiguous response on the chromatographic detector system. Many SW-846 methods 

recommend brominated or fluorinated compounds and/or stable isotopically-labeled 

analogs of target compounds (e.g., a compound containing a deuterium atom instead of 

hydrogen, or a 13C atom instead of a 12C atom) as internal standards. Isotopically-labeled 

compounds are most often employed in MS detection methods because the detector can 

differentiate between the target compound and the labeled 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 chromatographically resolve many internal standards 

from the target compounds. 

Internal standards recommended in many SW-846 methods were used during 

development of the method. Analysts may employ other internal standards in place of, or 

in addition to, those recommended. If internal standards are not recommended in the 

method, the analyst should select one or more compounds similar in analytical behavior to 

the analytes of interest and not expected to be found in the samples. Whichever internal 

standards are employed, the analyst should demonstrate (as detailed in the determinative 

method) that measurement of the internal standard is 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 concentrations of the target analytes will vary. 

The internal standard solution may contain more than one internal standard, and their 

relative concentrations may differ within the spiking solution. However, the mass of each 

internal standard added to the samples or sample extracts 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 yield 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 the 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, calculate the RF values for each target 

analyte relative to one of the internal standards as follows. 

4 

5  6

56  

 

 where: 

As = Peak response of the analyte or surrogate 

Ais = Peak response of the internal standard 

Cs = Mass of the analyte or surrogate in the sample aliquot 

Cis = Mass of the internal standard in the sample aliquot 

Response factors for GC/MS methods may also be calculated 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. Therefore, 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 for routine variations in the 

chromatographic separation of target compounds, there 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 internal standards should be chosen to cover the 

expected retention time range of the target compounds. Accordingly, internal standards 

can compensate for small retention time shifts or response 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 compound 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 calibration include acceptance limits for 

responses of the internal standards in the calibration standards, samples, or both. Those 

limits are typically expressed in terms of peak areas 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 present during analysis). Common consensus 

limits are 50 - 200% of the area of the internal standard in the most recent calibration 

standard. Representing a factor of two, these limits are used as a gross diagnostic check 

on addition of the internal standards to the samples 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 internal 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 other sample preparation steps 

such as pH adjustment, drying, or extraction solvent addition. Physical and chemical 

properties of each labeled compound are virtually the same as its unlabeled "native" analog. 

Thus, any losses of the target compound that may occur during sample preparation or 

determinative steps will be mirrored by a similar loss of the labeled standard. Similarities 

between labeled compounds and unlabeled analogs mean that RFs and RRTs for the 

unlabeled compounds are very close to 1.0. 

Labeled compounds are spiked into samples and standards 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 assumption, 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 dilution methods include some limits on the 

apparent recovery of the labeled compounds. However, 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 when the apparent spike recovery of the 

labeled compound is low. Labeled compound recoveries >100% are allowed as well. 

Such recoveries can occur as a result of the inherent variability in calibration of the labeled 

compounds, and are not indicative of contamination or other problems. 

Built-in recovery correction is one of the principal advantages of isotope dilution 

calibration. Isotope dilution requires an MS detection system and isotopically labeled 

analogs of target analytes and generally produces more precise data with lower bias. The 

added cost of isotopically labeled compounds is a disadvantage, but can be offset by higher 

quality data, as well as eliminating some routine QC analyses, such as surrogates and 

MS/MSDs used with internal standard calibration. However, whether or not to add 

surrogate or prepare MS/MSD aliquots should be described in a QAPP and not left to the 

analyst's professional judgment. 

Isotope dilution calibration is often used in conjunction with selected ion monitoring 

(SIM) GC/MS procedures, such as those for polychlorinated dibenzo-p-dioxins and 

polychlorinated dibenzofurans. These procedures, along with a relatively small list of 

target compounds, allow the instrument to be operated 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 labeled analogs; this application 

significantly increases the sensitivity of the method and reduces interferences. Because 

isotope dilution methods have acceptance limits for recovery of the labeled analogs added 

prior to extraction or other sample preparation steps, these methods typically do not contain 

limits on responses of the traditional internal standards used to monitor those recoveries. 

 

An RF is calculated for each target compound relative to its labeled analog and for 

each labeled analog relative to the traditional internal 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, traditional 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 internal standards using the same 

assumptions made for isotope dilution. 

This approach is most helpful when the compounds used as internal standards are 

very closely related to the target compounds. It is similar to the internal standard procedure 

used for volatile organic analyses by purge-and-trap or headspace extraction. 

11.5 Calibration models and acceptance criteria 

SW-846 chromatographic methods allow the use of three 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 also provides suggested criteria for calibration 

models; however, method- or project-specific criteria will always supersede general guidance. 

 

Choice of calibration model may begin with the simplest 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, knowledge of the physics of the detector, or 

specific manufacturer's recommendations. For the calibration 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 methods in SW-846 employ a minimum of five 

standards for average response factor or linear (first-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 proper 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 evaluate each option. In addition to this suggested 

acceptance criteria, two general methods for assessing 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 representative of the model chosen as described 

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

October 2012 

exceed the calibration range must be diluted to fall within the range. Criteria listed in these 

sections are designed for quantitation of trace-level 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 beyond the range of calibration standards is 

not to be inferred. At least five calibration levels should be used to construct the average 

CF or RF model (Sec. 11.5). 

To evaluate linearity of the initial calibration, calculate the mean CF (external 

standard calibration) or RF (internal standard calibration), the standard deviation (SD), and 

the RSD (also called coefficient of variance, CV) as follows: 

:  ;

∑ =46  4>





6?

!  1 :  ;

∑ =46  4>





6?

!  1

%#! 4  4@@@@ 

∑ 46



6?

!

 %#! 4  4@@@@ 

∑ 46



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 range, the slopes of the 

lines for each standard are sufficiently close to one 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. Alternatively, either of the 

two methods described in 11.5.4 may be used to determine 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 analytes that may be 

analyzed in some methods, it is likely that some analytes may exceed the 

acceptance limit for the RSD for a given calibration. In those instances, it is 

recommended, but not required, that corrective actions as described in Sec. 11.5.6.1 

be followed. Sec. 11.5.6.1 also provides alternative 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 in Sec. 11.5.4.1. The calculated 

amount introduced into the instrument, xs, is: 

 

5

4@@@@

 #!(  

5

4@@@@

  

6

5C

@@@@

where: 

Xs = Calculated mass of the analyte or surrogate in the sample 

aliquot introduced into the instrument (in ng) 

As = Peak response of the analyte or surrogate in the sample 

Ais = Peak response of the internal standard in the sample 

Cis = Mass of the internal standard in the sample aliquot introduced 

into the instrument (in ng) 

CF = Average calibration factor from the most recent initial 

calibration 

RF = Average RF from the most recent initial calibration. 

Units for the mass of analyte should be the same units 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 accordingly. 

11.5.2 Linear calibration using a least squares regression 

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 the 

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 response 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 calibration 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 instrument 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 x  Cs s
SW-846 Update V 8000D- 34 Revision 4 

October 2012 

changed accordingly. 

Option 1: Xs is the mass of the analyte in the calibration standard aliquot introduced into the 

instrument and Ys is the ratio of response of the analyte to the response of 

internal standard times the mass of the internal standard in the calibration 

standard aliquot introduced into the instrument. 

   #!( G  5 x 

6

56

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

into the instrument to the internal standard mass in 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: 

Cs = Mass of analyte in the volume of calibration standard introduced 

into the instrument. 

Cis = Mass of internal standard in the volume of calibration standard 

injected into the instrument. 

As = Peak response of analyte. 

Ais = Peak response of internal standard. 

A linear least squares regression attempts to construct a linear equation of the form, 

by minimizing the sum of squared differences between the observed response (yi, 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 I6

6  6

J







6?

where: 

Wi = Weighting factor for the i

th calibration standard (w=1 for unweighted 

least squares regression, or 1/x or 1/x2 for weighted least squares 

y  

As

Ais

x  

Cs

Cis

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

October 2012 

regression) 

yi = 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 regression curves will tend to fit points that 

are at upper calibration levels better than those points 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 factor which reduces this tendency can be 

used as compensation. 

11.5.2.1 Do not include an X-value of zero as a calibration point. 

However, most data systems and many commercial software packages will allow the 

analyst to "force" the regression through the origin. 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 calibrations, 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 calibration range demonstrated 

by the analysis of the standards. Results should not be reported at a 

concentration below the LLOQ unless qualified as estimated. 

11.5.2.2 In the specific case of an unweighted linear least squares 

regression, i.e., a regression that varies both a and b, the correlation coefficient (r) 

can be used to measure the "goodness of fit." 


  

! ∑ 66  ∑ 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 the origin or the weighting 

factor is variable, then the coefficient of determination, 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 line 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 r2 value of 1.00 

indicates that all variability in response is due to 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. Alternatively, either of 

the two methods described in Sec. 11.5.4 may be used to determine whether the 

calibration function meets acceptance criteria. It is recommended that the resulting 

calibration curve be inspected by the analyst as described 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 equation is rearranged. 

 External standard calibration allows the mass of the analyte in the sample 

aliquot introduced into the instrument to be calculated. 

For the internal standard method, the calculation will depend on which of 

the two options described in Sec. 11.5.2 is chosen.

U-"$! 1  

V

5 x 6

56 X  Y

#

 U-"$! 2  

 5

56  Y ) x 6

#

where: 

Xs = Calculated mass of the analyte or surrogate in the sample 

aliquot introduced into the instrument (in ng) 

As = Peak response of the analyte or surrogate in the sample 

Ais = Peak response of the internal standard in the sample 

Cis = Mass of the internal standard in the sample aliquot 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

xs  

As- 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 instrument response does not follow a 

linear model over a sufficiently wide calibration range, 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 construct 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 calibration to compensate for detector 

saturation or to avoid proper instrument maintenance. 

 

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 estimate 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 calibration 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 regression can be modified. Weighting in 

a calibration model or forcing through the origin may significantly improve the ability of the 

least squares regression to fit the data at lower concentration 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 analysis of the standards.

11.5.3.1 Linear and non-linear least squares regressions are 

mathematical methods that minimize differences (the residuals) between observed 

instrument response, yi

, and calculated response, yi’, 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 methods described in 11.5.4 may be 
SW-846 Update V 8000D- 38 Revision 4 

October 2012 

used to determine calibration function acceptability. It is recommended that the 

resulting calibration curve be inspected by the analyst, as described in Sec. 11.5.4.1. 

As noted in Sec. 11.5, whichever of these options is employed, an analyte 

or surrogate concentration must fall within the calibration range. Analysts are also 

advised to check both second- and third-order calibration 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 calibration 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 measured 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 

xi = True amount of analyte at calibration level i, in mass or 

concentration units. 

Percent error between the calculated and expected amounts of an analyte 

should be ≤ 30% for all standards. For some data uses, ≤50% may be acceptable 

for the lowest calibration point. 

11.5.4.2 Calculation of Relative Standard Error (RSE - expressed as %) 

:\  100 * aH b6

J  6

6

c

 

6?

d!  -

where: 

xi = 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 calibration 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 should be set at ≤20% for good 

performing compounds and ≤30% for poor performing compounds. 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 for a simple calibration model. For 

example, the response of a flame photometric detector in the sulfur mode is known to be 

proportional to the square of the sulfur concentration. 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 integration) 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 verifications until the next calibration. 

11.5.5.2 The transformation model chosen must be consistent with the 

behavior of the instrument and detector. All data transformations must be clearly 

defined and documented by the analyst and related back to the fundamental 

behavior of the detector. In other words, this approach 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 to the instrument response itself). 

11.5.5.4 When the transformed data are used to develop calibration 

factors, those factors should meet the acceptance criteria described in Sec. 11.5.1, 

and it is recommended that the resulting calibration "curve" be inspected by the 

analyst as described in Sec. 11.5.4. 

11.5.6 Inspecting the calibration model and recommended corrective actions 

Given the potentially large numbers of analytes that 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 invalidated. Information obtained from the initial 

calibration of targeted analytes not meeting the acceptability criteria may have other uses 

such as for screening and for estimation of quantitation (see Sec 11.5.6.1), but those uses 

should still fit the needs of the project objectives. 

Whichever calibration model is selected, it is recommended that the model be 

subjected to an additional check to establish the representativeness of the data that were 

used to produce the model. This check is the refitting 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. Criteria for acceptability based upon the 

additional check would have a similar impact upon the 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 calibration 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 Section11.5.4.1, corrective action may 

be needed. Some recommended courses of action and additional options for 

modifying the calibration ranges follow. More specific corrective actions that are 

provided in the applicable determinative methods will supersede those noted in 

Method 8000. Generally, the calibration should not be used for quantitative 

analyses of that analyte when the calibration criteria (RSD/RSE/r

2and % Error) are 

not met. 

11.5.6.2 For all calibration models the following options are allowed. 

However, if none result in an acceptable calibration, a new initial calibration must be 

performed. 

 11.5.6.3 Generally, the first option is to check the instrument operating 

conditions. The suggested maintenance procedures in Sec. 11.11 may be useful in 

guiding such adjustments. This option will apply in those instances where a linear 

instrument response is expected. It may involve some trade-offs to optimize 

performance across all target analytes. For instance, changes to the operating 

conditions necessary to achieve linearity for problem 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 r2

 < 0.99), the analyst may wish to review the results (proper 

identification, area counts, calibration or RFs, and 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 appears to be associated 

with a single standard, that one standard may be reanalyzed, to rule out problems 

due to random error, and the calibration function may be recalculated and 

reevaluated against the acceptance criteria. Replacing the standard may be 

necessary in some cases. If the criteria still cannot be met, the entire initial 

calibration should be performed again. 

NOTE: An initial calibration should be considered a single event process and a 

reanalysis of a calibration standard should be performed immediately to ensure that 

the reanalysis is still part of the original initial calibration event, and before any 

samples are analyzed. 

 11.5.6.5 A third option is to narrow the calibration 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 calibration range, document the 

calibration linearity, and proceed with analyses. Changes to the upper end of the 

calibration range will affect the need to dilute samples above the range, while 

changes to the lower end may increase the LLOQ. Consider the regulatory limits or 

action levels associated with the target analytes when 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 minimum 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 calibration range by removing 

data points from either extreme end of the range and recalculating the calibration 

function. It is prohibited to remove data points from 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 still be met for the model. 

NOTE: As noted in Sec. 11.4.1.2, the lower limit of quantitation (LLOQ) is 

established by the concentration of the lowest standard analyzed during the initial 

calibration. Hence, narrowing the calibration range 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 specific regulatory limit or action 

level, the analyst should ensure that the LLOQ is at 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 % error 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 meet the % error criteria, but the 

calibration points used to generate the initial curve are retained. The LLOQ 

becomes the lowest end of the adjusted calibration range. The calibration model 

should meet the RSD/RSE/r2

 criteria (Secs. 11.5.1 – 11.5.3) and the minimum 

number of data points (Sec. 11.5.3.1) before this option can be used. 

NOTE: This guidance allows the use of a calibration model 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 the range for usefulness to only 

those data points that refit the model within the criteria 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 calibration. Without reanalysis of 

standards or manipulations of the model, the initial calibration can be used to 

estimate quantitation and information from the calibration can be used to verify the 

identification of target analytes when used to screen samples. 

If the initial calibration does not meet the acceptance criteria, it may not be used for 

quantitative analyses; however, estimates of the quantitation can be made. 

Estimates of quantitation can be useful when screening for the level of 

contamination and determining the degree of dilutions that may be necessary when 

high levels of contamination are encountered. If quantitation estimates for a 

positively identified analyte are not within the scope of the project DQOs, then an 

acceptable initial calibration should be prepared for that analyte. 

If information from the initial calibration will be used to verify the identification of a 

targeted analyte for screening purposes, there should be sufficient sensitivity at the 

screening level to verify identification. Reasonable 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 identification of target compounds. Absolute 

retention times are used for compound identification in all GC and HPLC methods that do not
SW-846 Update V 8000D- 42 Revision 4 

October 2012 

employ internal standard calibration. Retention time windows are established to compensate for 

minor shifts in absolute retention times as a result of sample loadings and normal 

chromatographic variability. Width of the retention time window should be carefully established 

to minimize the occurrence of both false positive and false negative results. Tight retention time 

windows may result in false negatives and/or may cause 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 may not be confirmed. 

The following subsections describe one approach that may be used to establish retention 

time windows for GC and HPLC methods. Other approaches 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 that the system conditions are optimized 

for the target analytes and surrogates in the sample matrix to be analyzed. Make three 

injections of all single component standard mixtures 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 windows that are too tight. 

11.6.2 Record the retention time (in minutes) for each single component analyte 

and surrogate to three decimal places. Calculate the mean and standard deviation of the 

three absolute retention times for each single component analyte and surrogate. For 

multi-component analytes, choose three to five major peaks (see the determinative methods 

for more details) and calculate the mean and standard deviation of those peaks. 

11.6.3 If the standard deviation of the retention times for a target compound is 

0.000 (i.e., no difference between the absolute retention times), then the laboratory may 

either collect data from additional injections of standards or use a default standard deviation 

of 0.01 minutes. (Recording retention times to three 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 window 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, the 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, other 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 the surrogates may be diluted out of 

certain sample extracts, their retention times in the calibration standards may be useful in 

tracking retention time shifts. Whenever the observed retention time of a surrogate is 

outside of the established retention time window, the analyst is advised to determine the 

cause and correct the problem before continuing analyses. 
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 calibration verification applies to both external 

standard and internal standard calibration techniques, 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 which samples are analyzed using a calibration 

verification standard prepared at the appropriate level of concern. (Some methods may specify 

more frequent verifications and recommended standard 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 calibration) or % Drift (for all other types of 

calibration) of an analyte is within ±20% of the expected concentration or amount based on the 

initial calibration, then the initial calibration is considered still valid, and the analyst may continue 

to use the calibration curve to quantitate sample results. The ±20% criterion may be superseded 

in certain determinative methods. 

Except where the determinative method contains alternative calibration verification criteria, 

if the % Drift or % Difference is >± 20%, the initial calibration relationship may no longer be valid.

NOTE: The process of calibration verification is fundamentally 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 sample 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 criteria, perform any necessary instrument 

maintenance, and inject another aliquot of the calibration verification standard. If the response 

for the analyte is still not ± 20%, then a new initial 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 

CFv  CF

CF

* 100%  

RFv  RF

RF

* 100

where: 

 CFv = calibration factor calculated for the calibration verification standard 

RFv = response factor calculated for the calibration verification standard 

CF = mean calibration factor from the initial calibration 

RF = mean response factor from the initial calibration 

 If >10% of the analytes in a multi-analyte method exceed the calibration verification 

criteria, and instrument maintenance does not correct 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 analytes 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 exceeds the lower acceptance criteria (e.g., 

<-20%), a sensitivity verification standard at or below the LLOQ should be analyzed in the 

analytical batch. The analyte should be detected in the LLOQ standard and meet all of the 

qualitative identification criteria that the laboratory routinely uses (for example, qualifier ions 

of columns, signal to noise, etc). In any event, the limitation to no more than 10% of 

analytes exceeding the calibration verification criteria applies to both detected and 

non-detected analytes. 

11.7.2 Verification of non-linear calibration 

Calibration verification of a non-linear calibration 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 near 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 using 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 further check on the initial calibration. 

Concentrations of these standards should generally reflect those observed in samples. 

11.7.4 Additional analyses of the midpoint calibration 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 accurate measurement is dependent 

on the detector and the sample matrix. Very sensitive detectors that operate in the 

sub-nanogram range are generally more susceptible to changes in response caused by 

column contamination and changes in ambient conditions. Therefore, more frequent 

verification of calibration (i.e., after every 10 samples) may be necessary for some types of 
SW-846 Update V 8000D- 45 Revision 4 

October 2012 

detectors (i.e., electron capture, electrochemical conductivity, photoionization, fluorescence 

detectors). 

Sec. 11.8.2 states that samples analyzed using external standards must be 

bracketed by periodic analyses of standards that meet the QC acceptance criteria (e.g., 

calibration and retention time). Therefore, more frequent analyses of standards will 

minimize the number of sample extracts to be reinjected if the QC limits are violated for the 

standard analysis. Results from these bracketing standards should meet the calibration 

verification criteria in Secs. 11.7.1 and 11.7.2 and 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 shift, 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 preparative methods (Methods 3500 

and 3600) may be run immediately after the calibration verification analyses to confirm that 

laboratory contamination does not cause false positive results, or at any other time during 

the analytical shift. If the method blank indicates 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 into the GC or HPLC varies, 

depending on the physical and chemical properties of 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 include 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 prevent overloading and contamination of 

the purge-and-trap system. Semivolatile and non-volatile 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 the 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 large 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 made specifically for this technique. 

The analyst should ensure that all of the QC requirements 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. Larger volumes may be injected if 

better sensitivity is needed; however, chromatographic performance may be 

affected. 

11.8.1.5 Automated injection (HPLC) 

Inject 10 – 100 μL. Laboratories should demonstrate that the injection 

volume is reproducible. Larger volumes may be injected if greater sensitivity is 

needed; however, the solvent of the standards and samples should be matched to 

the initial mobile phase to avoid chromatographic performance degradation. 

11.8.2 All analyses, including field samples, duplicates, MS/MSDs, LCS, method 

blanks, and any other QC samples are performed during an analysis sequence. The 

sequence begins with instrument calibration, which is followed by the analysis of sample 

extracts. Verification of calibration and retention 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 standard 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 between every set of 10 samples is 

strongly recommended, especially for highly sensitive GC and HPLC detectors at 

sub-nanogram concentrations. Frequent analysis of calibration verification standards 

helps ensure that chromatographic systems are performing acceptably and that false 

positives, false negatives and poor quantitation are minimized. Samples analyzed using 

external standard calibration should be bracketed by the analyses of calibration standards 

that meet the QC limits for verification of calibration and retention times. If criteria are 

exceeded, corrective action should be taken (Sec. 11.11) to restore the system and/or a 

new calibration curve prepared for that compound and the samples reanalyzed. 

Certain methods may also include QC checks on column resolution, analyte 

degradation, mass calibration, etc., at the beginning of a 12-hour analytical shift. 

11.8.3 Sample concentrations are calculated by comparing 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 extract (or sample) and reanalyze. Extracts should 

be diluted so that all peaks are on scale, as overlapping 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 presence 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 necessary when the composition of samples is 

not well characterized. Confirmation techniques include 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, or by other recognized confirmation techniques. 

For HPLC/UV methods, analyte confirmations at two different 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 identification of a target analyte using an 

HPLC/UV method may be confirmed with a different type of detector such as a mass-selective 

detector or a fluorescence detector at different excitation 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 composition of the sample matrix is well 

established by prior analyses as when a pesticide known to be produced or used in a facility is 

found in a sample from that facility. 

Many chromatographic interferences result from coelution of one or more compounds with 

the analyte of interest, or may be the result of the 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 quantitative results from two dissimilar columns. 

Therefore, even when the identification has been confirmed on a dissimilar column, the analyst 

should evaluate the agreement of the quantitative results on both columns, as described in Sec. 

11.10.4. 

11.10 Calculations 

Calculation of sample results depends on the type of calibration (external or internal 

standard) and the calibration model employed (linear or non-linear). Calculations of the mass of 

the analyte in the sample aliquot introduced into the instrument can be found in Secs. 11.5.1.3, 

11.5.2.3, and 11.5.3. The following sections describe 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 purposes only. Various dilution schemes 

and conventions for defining final volumes and injection 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 

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w1

x





yz

y6

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

October 2012 

where: 

Xs = Calculated mass of analyte (in ng) in sample aliquot introduced into 

instrument. Type of calibration model used determines derivation of 

xs. See Secs. 11.5.1.3, 11.5.2.3, and 11.5.3. 

Vt

 = Total volume of concentrated extract (in μL). For purge-and-trap 

analysis, Vt

 is the purge volume and will be equal to Vi. Thus, units 

other than μL may be used for purge-and-trap analyses. 

D = Dilution factor, if sample or extract was diluted prior to analysis. If no 

dilution, D=1. Always dimensionless. 

Vi

 = Volume of extract injected (in μL). The nominal injection volume for 

samples and calibration must be the same. For aqueous 

purge-and-trap analysis or direct injection of a liquid sample into a GC 

or HPLC, Vi

 will be equal to Vt. 

Vs = 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 result 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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.1 



yz

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I

where: 

Xs = Calculated mass of analyte (in ng) in sample aliquot introduced into 

instrument. Type of calibration model used determines derivation of 

xs. See Secs. 11.5.1.3, 11.5.2.3, and 11.5.3. 

Vt = Total volume of concentrated extract (in μL). For purge-and-trap 

analysis where an aliquot of solvent (methanol, water, etc.) extract is 

added to reagent water and purged, Vt

 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 diluted prior to analysis. If no 

dilution, D=1. This value is always dimensionless.

Vi

 = 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, Vi

 is the volume of 

solvent extract added to reagent water just prior to purging. Dilutions 

made to the initial volume of solvent extract are accounted for in D. 
SW-846 Update V 8000D- 49 Revision 4 

October 2012 

Ws = 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 result 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 the solvent/water dilution effect for 

extracted volatile organics. 

11.10.3 Sample concentration when Xs 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 instead 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, Vi

, into the term Xs. Therefore, the equation for sample 

concentration by volume becomes: 

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x





yz

y



And the equation for sample concentration by weight becomes: 

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w1

1





yz

I



where Vt

, D, Vs, and Ws are the same as in Secs. 11.10.1 and 11.10.2 and 

Xs = Calculated concentration of analyte (ng/μL) in the sample. Type of 

calibration model used determines derivation of Xs. See Secs. 11.5.1.3, 

11.5.2.3, and 11.5.3. 

Using the units listed here for these terms will result in concentrations in ng/mL, 

which is equivalent to μg/L, or in 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 the 

solvent/water dilution effect for extracted volatile organics. 

11.10.4 Comparison between results from different columns or detectors 

When sample results are confirmed using two dissimilar columns or with two 

dissimilar detectors, the agreement between the quantitative 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 interferences with the higher of the results, 

which could result from poor separation of target analytes, 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 intended 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) between the two concentrations 

using the formula below. 

 

|  

|



  

2



  100

where C1 and C2 are concentrations on the two columns and the vertical bars in the 

numerator indicate the absolute value of the difference. 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 peak is causing an erroneously 

high result. If no overlapping peaks are noted, examine the baseline parameters 

established by the instrument data system (or operator) 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 chromatographic 

conditions. If there is no evidence of chromatographic problems, it may be 

appropriate to report the lower result. 

Regardless of the presence or absence of chromatographic problems, the 

data user MUST be advised of the disparity between the two results, because the 

user, not the laboratory, is responsible for ensuring that the most appropriate result 

is reported or utilized. Under some circumstances, including those involved in 

monitoring compliance with an action level or regulatory limit, further cleanup of the 

sample or additional analyses may be needed when the 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 basis of wet weight (as received) 

or dry weight (moisture-corrected) sample concentration. There are merits to either 

approach; however, many regulatory limits associated 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 form or the other. 

The choice of "as received" or moisture-corrected reporting is ALWAYS a 

project-specific decision that must be based on knowledge of intended use of the data. 

When moisture-corrected reporting is required, concentration 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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"( !!"
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  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 aliquot of the sample at 105 oC overnight. 

Percent moisture is calculated as follows: 

% {$/"&
 

1 ' "as received" /#%-3  1 ' (
 /#%-3

1 ' /#%-3  100

The % moisture determination may also be called % solids 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 for the final results, e.g., μg/kg or mg/kg, 

will be the same, regardless of the % moisture calculation. 

Except when the sample is completely dry ( i.e., the % moisture equals 0), 

moisture-corrected results will always be higher than "as received" results. In the absence 

of project-specific requirements, it may be most appropriate to report results on the "as 

received" basis of the sample AND provide the % moisture 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 significant moisture content (>10%) 

that are extracted prior to analysis in a water-miscible 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 Vt

 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 concentration must be corrected using the total 

solvent/water mixture volume represented as Vt

. This total solvent/water volume is 

calculated as follows: 

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 %x ' /3	!" 

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

100  x 1000 μx/%x

When the sample moisture content is >10%, it is recommended that the calculated 

concentrations of volatile samples that are extracted in a water-miscible solvent such as 

methanol be corrected for the solvent/water dilution effect. Potential underreporting of 

volatile concentrations is more pronounced as % moisture 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 troubleshooting their specific instruments and analytical 

procedures. Manufacturers of chromatographic instruments, detectors, columns, and 

accessories generally provide detailed information regarding the proper operation and limiting 

factors associated with their products. Reading and reviewing this information cannot be 

overemphasized. 
SW-846 Update V 8000D- 52 Revision 4 

October 2012 

11.11.1 GC preventive maintenance and corrective action 

To prevent or ameliorate deterioration of chromatographic performance, analysts 

should perform routine maintenance activities on the 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 port septa should also be 

changed frequently enough to prevent retention time shifts of target analytes and 

peak tailing. Over-tightening the septum nut can cause the inlet to leak. The 

schedule for changing inlet liners and septa is dependent on the operation of the 

injection system, the nature of samples and parameters tested, and acceptance 

criteria in the reference method. 

If chromatographic performance or ghost peaks are still a problem after 

performing these inlet maintenance steps, replacing the seal in the bottom of the 

inlet or cleaning and deactivating the metallic surfaces of the injection port itself may 

be necessary. Deactivation of the injection port necessitates the use of toxic 

reagents and should only be performed by knowledgeable 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 condition the column as 

recommended by the manufacturer, and flush the column 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 temperature. 

Poor chromatographic performance may also be observed 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 column may restore chromatographic 

performance. If clipping the head of the column does 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 flow control is maintained. 

A search for leaks using an electronic leak detector 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 measures taken. Electronic pressure 

controller flow rates should also be checked regularly (with both the injector and the 

oven heated) using a bubble meter or other appropriate 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 volume between the injector and 

detector. Therefore, plumbing connections should be of minimum length and diameter, 

and ferrules should be properly positioned on the tubing to minimize dead volume. 

11.11.2.1 Injection port maintenance 

Filtration of extracts and injection of solvent mixtures miscible with the 

mobile phase can help minimize solvent-related problems. Otherwise, 

contamination of subsequent injections may occur when 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 wear 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 packed with the appropriate 

particle size and bonded phase will result in optimal 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 improved through 

proper maintenance. Sample extracts should be filtered 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 because 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 be replaced when performance 

degrades (e.g., significant band broadening, peak splitting, or loss of 

chromatographic resolution occurs). 

11.11.2.3 Mobile phase and pump maintenance 

Pumping systems should deliver reproducible gradients at a uniform flow 

rate. Pumping flow rates can be checked by collecting 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 cavitate. Therefore, HPLC 

solvents should be degassed prior to use. 

Non-reproducible gradients can result from deterioration in pump 

performance and can cause unacceptable variation in retention times from run to 

run. Mobile phase solvents should be filtered to remove particles that cause pump 

piston wear. Seals in the HPLC pumps should be replaced 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 mobile phase can have a 
SW-846 Update V 8000D- 54 Revision 4 

October 2012 

significant effect on retention times. Buffering the mobile phase may help make the 

pH more reproducible from preparation to preparation, as long as it doesn’t conflict 

with the method or cause other problems with the analysis. 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 be 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 determinative method for information regarding 

data analysis and calculations. 

12.2 Results must be reported in units commensurate 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 provided in SW-846 methods only as 

examples and guidance. Data do not represent required performance goals for users of the 

methods. Instead, performance goals should be developed 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 must 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 generation. Numerous opportunities for pollution 

prevention exist in laboratory operation. The EPA has established a preferred hierarchy of 

environmental management techniques that places pollution prevention as the management 

option of first choice. Whenever feasible, laboratory 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 best option. 

14.2 For information about pollution prevention that 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 Chemical Society (ACS), Committee on 

Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_012290.pdf. 

15.0 WASTE MANAGEMENT 

The EPA requires that laboratory waste management practices be conducted consistent 

with all applicable rules and regulations. Laboratories 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 and regulations and complying with all solid and 

hazardous waste regulations, particularly the hazardous 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 methods, review Methods 3500, 3600, 5000, 

the individual sample preparative, cleanup and determinative 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 Laboratories”, 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-performing 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-performing 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 DATA 

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 that the reference samples used for IDP analyses 

should be prepared from the same source as the calibration standards. This change is 

now consistent with Methods 8260 and 8270. The IDP section was also expanded to 

include separate language for preparation and extraction chemists, as well as instrument 

chemists. This only requires analysts to perform an IDP on the portion of the procedure 

that they actually do. 

 

2. References to the method quantitation limit (MQL) have been replaced with the lower limit 

of quantitation (LLOQ). 

3. Inclusion of Relative Standard Error (RSE) in Sec. 11.5.4.2. 

4. Improved overall method formatting for consistency with new SW-846 methods style 

guidance. The format was updated to Microsoft Word 2007.docx. 

5. Many minor editorial and technical revisions were made throughout to improve method 

clarity. 

6. The revision number was changed to 4 and the date 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.