By Authority Of
THE UNITED STATES OF AMERICA
Legally Binding Document
By the Authority Vested By Part 5 of the United States Code § 552(a) and
Part 1 of the Code of Regulations § 51 the attached document has been duly
INCORPORATED BY REFERENCE and shall be considered legally
binding upon all citizens and residents of the United States of America.
HEED THIS NOTICE: Criminal penalties may apply for noncompliance.
THE EXECUTIVE DIRECTOR
OFFICE OF THE FEDERAL REGISTER
AOAC: Official Methods of Analysis, 1980
21 CFR 131.150(c)
METHODS OF ANALYSIS
ASSOCIATION OF OFFICIAL
WILLIAM HORWITZ, Editor
THIRTEENTH EDITION, 1980
PUBLISHED BY THE
ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS
PO Box 540, BENJAMIN FRANKLIN STATION
WASHINGTON, DC 20044
Direct inquiries related to the scientific content of Official Methods of Analysis to:
Editor, Official Methods of Analysis
Association of Official Analytical Chemists
Box 540, Benjamin Franklin Station
Washington, DC 20044 USA
Direct inquiries related to the procurement of Official Methods of Analysis, supplements (Changes in Methods). Journal of the
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COPYRIGHT 1920,1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965, BYTHE
ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS
AND 1970, 1975, 1980 BY THE
ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS
The methods of the Association were also copyrighted in 1916, when they
were published in the Journal of the Association of
Official Agricultural Chemists
Library of Congress Catalog Card Number: 20-21343
A copy of the 13th edition of this publication is on file with the Office of the Federal Register. U. S. Government Agencies may apply
to the Director of the Office of the Federal Register for approval to incorporate this edition by reference in their regulations. The
procedures that Federal agencies must follow in applying for the Director's approval are in Title 1, Part 51 of the Code of Federal
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Preface to Thirteenth Edition
The most noticeable physical change in this thirteenth edition
of Official Methods of Analysis of the Association of Official
Analytical Chemists ("The Book of Methods") is its size. A survey
of users of the Book of Methods revealed an overwhelming
desire to maintain the compendium as a single volume. The
easiest way to do this was to increase the size of the page.
Users also expressed a desire for a system that will keep the
same reference number for a given method from edition to
edition. A practical system that will maintain the continuity of
the numbering system and the organizational structure of the
methods within a chapter has not yet been devised.
Approximately 175 new methods have been added during the
current five year period (1974-1978); 83 methods have been
deleted, replaced, or surplused. The approval of an average of
only 35 new methods per year represents a marked decline from
the 70 per year of the four year period of the previous edition
(1970-1973) and the 50 per year of the five year period of the
tenth edition (1965-1969). The decline is undoubtedly the result
of a number of factors. Chief among them are the greater
complexity of modern methods, requiring a large investment in
resources that is not readily mobilized to fit an Associate
Referee's schedule; and the fact that government agencies are
attempting to obtain compliance, especially of the newer stat-
utes, by promulgation of regulations and by auditing rather than
by laboratory examinations.
The greatest activity, as measured by approval of new meth-
ods, is in the field of pesticide formulations, partly as a result
of the active implementation of the cooperative agreement
with the Collaborative International Pesticide Analytical Council
(CIPAC). Other active areas include extraneous materials, vita-
mins and other nutrients, dairy products, and microbiological
methods. In fact, examination for and by biological constituents
(filth, microbiological assays, and examination for food-borne
pathogens) comprises approximately 17% of the new methods
A comparison of the types of methods adopted between this
and the previous edition reveals that gas-liquid chromatography
has overtaken spectrophotometry in its various forms (visible,
ultraviolet, and fluorescent) as the most predominant quanti-
tative technique in Official Methods. High pressure (or perform-
ance) liquid chromatography has spurted from none to 6% of
the adopted methods within the relatively short period of five
years. Electrometric methods (potentiometric, polarographic,
and ion-selective) are now sufficiently numerous to deserve a
separate category. Infrared methods are no longer a major
factor in quantitation; they are now mainly used for identification
and confirmation. A comparison of the present and previous
editions is presented in Table 1. The figures given are only
rough approximations because of the arbitrariness often re-
quired in classifying a method and in deciding when a new
method or revision is sufficiently independent to warrant con-
tributing to the statistics.
Table 1. Classification of new methods approved by the AOAC
in the thirteenth and twelfth editions
Biological and microbiological
High pressure liquid chromatography
Chromatography (thin layer, etc.)
Miscellaneous (physical, qualitative,
The most interesting new collaboratively studied method is
the mass spectrometric method for the detection of adulteration
of honey with high fructose corn sirup. Corn sirups (from a
monocotyledonous plant) have a distinctly different 13C/12
than sirups from most dicotyledonous plants, which are the
source of most honeys. The isotope ratio mass spectrometer
required forthis determination is a highly specialized instrument,
even in the field of mass spectrometry. Despite the rarity of the
instrumentation, sufficient laboratories participated in the col-
laborative study to establish the reproducibility of the method.
A problem that has arisen is how to handle the numerous
individual instruments of diverse design and manufacture that
have been developed to automate a particular determination.
Even if this problem is solved by incorporating all the available
instruments into the initial collaborative study, the problem
returns with the first "new and improved" modification. Different
instrument designs and their subsequent modifications have
been handled in the infrared determination of milk constituents
by providing performance specifications which must be met by
the basic instrument in general, when compared to a reference
method or reference sample. In addition, the user must satisfy
himself that his particular instrument also meets the perform-
ance specifications by frequent comparisons with the reference
method or sample. This requirement eliminates the need for
repeated collaborative studies every time a manufacturer rede-
signs or modifies his basic equipment, and in addition provides
a continuous quality control technique on the performance of
the instrument, method, and laboratory.
It should not be overlooked that automation is not confined
to physical and chemical determinations. The microbiological
chapter contains three collaboratively studied instrumental
methods for somatic cell count and an instrumental method for
distributing a liquid sample for plate counting. Biochemical
diagnostic kits have also been evaluated collaboratively for their
effectiveness in identifying Salmonella and other enteric isolates.
A criticism that is often leveled at the AOAC validation
mechanism is that it is too slow to keep up with the pace of
requirements for methods by regulatory agencies and the
regulated industry. This objection overlooks the point that the
speed with which a method is validated is almost completely in
the hands of those needing the validated method, rather than
in the hands of the AOAC. The method must be tested for
ruggedness, and the directions tested for clarity; samples must
be prepared and distributed; laboratories must analyze the
samples and report the results. Dubious results must be inves-
tigated and occasionally samples must be reanalyzed. The data
must be tabulated and analyzed, and a report written. There is
no way to short-cut the process of obtaining intra- and interlab-
oratory variability except to conduct the necessary experimental
work and perform the necessary statistical calculations. Only
then is the recommendation subject to the time restrictions of
the AOAC mechanism. To ensure that the AOAC mechanism of
annual approval is not holding up use of needed methods, a
new temporary class of methods has been introduced entitled
"Interim First Action." These are methods which, together with
their supporting studies, have been received between annual
meetings and have been sent through the customary reviewing
procedure. To be designated Interim First Action, a method
must have been approved by the appropriate Referee and
Subcommittee, and by the Chairman of the Committee on
Official Methods. The method only lacks the formal vote by
those AOAC members having regulatory authority over the
commodity involved. Such formal approval is usually provided
at the next annual meeting, at which time the method becomes
"First Action." Approximately five interim methods are approved
annually. Several such methods appear in this volume, depend-
ing upon the time of their submission and review in relation to
the editorial status of the chapter to which they are assigned.
The current status of these Interim First Action methods, as well
as the status of any method, can be found by consulting the
latest cumulative index to Changes in Methods, which appears
as the final pages of each March issue of the Journal.
For those users of AOAC methods who may not be familiar
with the procedures by which a method is included in this
volume, we are reprinting a paper prepared for the Joint
International Symposium, "The Harmonization of Collaborative
Studies," held in London, England 9-10 March 1978. The
document was drafted by the AOAC Committee on Collaborative
Studies: Elwyn D. Schall, Chairman; Charles W. Gehrke, William
Horwitz, Anthony J. Malonski, James P. Minyard, Jr., Forrest W.
Quackenbush, and Ernest S. Windham. This paper appeared in
Analytical Chemistry (March 1978), 50, 337A-340A.
The publication of this "Book of Methods" is possible only
because of the extensive cooperation of thousands of analysts
who have volunteered to direct, participate in, and review the
numerous collaborative studies that form the basis for inclusion
of the several thousand methods that appear herein. Special
recognition is due to Mrs. Betty Johnson who has prepared an
entirely new index for this edition.
28 September, 1979
William Horwitz, Ph.D.
Preface to First Edition
"In presenting this revision of the official and tentative methods of analysis of the Association of Official Agricultural Chemists, it
is appropriate to give a brief statement of the organization of the Association, its purpose, and the procedure by which the methods
"Membership in the Association is institutional and includes the State Departments of Agriculture, the State Agricultural Colleges
and Experiment Stations, the Federal Department of Agriculture, and the Federal, State, and City offices charged with the enforcement
of food, feed, drug, fertilizer, insecticide and fungicide control laws.
"The Association was founded at Philadelphia, Pa., September 9, 1884, by the following representative agricultural chemists of that
time, the organization being the result of a series of informal meetings held the immediately preceding years:
"Prof. H. W. Wiley, Chemist of the Department of Agriculture, Washington, D.C.
Mr. Clifford Richardson, Assistant Chemist of the Department of Agriculture, Washington, D.C.
Mr. Philip E. Chazal, State Chemist of South Carolina.
Dr. Chas. W. Dabney, Jr., State Chemist of North Carolina.
Dr. W. J. Gascoyne, State Chemist of Virginia.
Dr. E. H. Jenkins, Connecticut Experiment Station.
Prof. John A. Meyers, State Chemist of Mississippi.
Prof. H. C. White, State Chemist of Georgia.
Mr. C. DeGhequier, Secretary National Fertilizer Association.
Dr. Schumann, Dr. Lehmann, Mr. Gaines and others."
Collaborative Study Procedures of the Association of
Official Analytical Chemists
The Association of Official Analytical Chemists (AOAC) is a
unique, nonprofit scientific organization whose primary purpose
is to serve the needs of government regulatory and research
agencies for analytical methods. The goal of the Association is
to provide methods which will perform with the necessary
accuracy and precision under usual laboratory conditions (1).
Since its formation in 1884 the AOAC has provided a mechanism
to select methods of analysis from published literature or
develop new methods, collaboratively test them through inter-
laboratory studies, approve them, and publish the approved
methods for a wide variety of materials relating to foods, drugs,
cosmetics, agriculture, forensic science, and products affecting
the public health and welfare. Its membership is composed of
scientists from Federal, State, Provincial, and other regulatory
bodies who work within the AOAC's established procedures as
researchers, methods collaborators, and committee members.
Although most of the members are from North America, many
nations throughout the world are represented.
The AOAC has almost a century of experience in utilizing the
collaborative study as a means of determining the reliability of
analytical methods for general purposes and, especially, for
regulatory purposes. In fact, the AOAC's major contribution to
analytical science has been to bring the collaborative study
technique for the validation of analytical methods to a high
degree of perfection. In such a study, laboratories analyze
identical sample sets which cover the range of applicability of
a method previously selected as being useful and practical. The
purpose of the study is to establish the characteristics of the
methods with respect to accuracy, precision, sensitivity, range,
specificity, limit of detection, limit of reliable measurement,
selectivity, practicality, and similar attributes, as required.
ORGANIZATION AND PROCEDURES FOR AOAC
The collaborative study is organized and directed by an analyst
designated as the Associate Referee for the specific subject
under investigation. Currently, some 600 Associate Referees
appointed by the Association are responsible for as many topics.
An Associate Referee is selected for his knowledge, interest,
and experience in the subject matter field. He operates under
the scientific guidance, support, and administrative supervision
of a General Referee, who is in turn responsible for a product
area. The Associate Referee reviews the literature and selects
one or two of the better analytical methods available, modifying
them as needed. Alternatively, he may develop or adapt a
method used in his laboratory for the analyte and matrix under
study, testing it thoroughly in his laboratory before designing
a collaborative study. The General Referee is kept informed of
such preliminary studies.
The samples analyzed in a collaborative study are normally
prepared and distributed to the participants by the Associate
Referee. The Association follows the recommendations of You-
den (2) that not fewer than five laboratories participate and that
a minimum of six sample materials be sent to each. These are
minima and, in practice, both are usually exceeded. In addition,
a reference or practice sample is included, where possible.
Laboratories with at least some experience in the general
subject matter are selected as collaborators. Because the objec-
tive of the study is to standardize the method, as contrasted to
standardizing the analyst (3). all analysts are instructed to follow
the method exactly as written even though they may not concur
with the Associate Referee's selection among possible alterna-
tives. The level ofthe analyte in the samples is usually unknown
to the participants.
All individual results obtained by the collaborators are re-
ported to the Associate Referee, who compiles and evaluates
them. Since statistical treatment of the data is considered
essential in a rigorous evaluation of the method for accuracy,
precision, sensitivity, and specificity, it is now required for all
studies. The Association considers this of such importance that
it provides statistical assistance in all cases where it is otherwise
unavailable to the Associate Referee. A statistical manual (4) is
The Associate Referee makes the initial judgment on the
performance of the method. If he recommends approval, it
passes to the General Referee and then to a committee of
experts. If both recommend approval, the method is presented
at the Association's annual business meeting for vote by the
Approved methods and supporting data are published in the
Journal of the Association of Official Analytical Chemists. They
are subject to scrutiny and general testing by other analysts for
at least a year before final adoption. They may be modified and
restudied collaboratively as needed, should feedback from gen-
eral use reveal flaws in the method or in its written set of
directions. Approved methods are included in the Association's
"Official Methods of Analysis", a book of some 1000 pages
which is updated every 4-5 years.
The preceding summary of AOAC's modus operandi recog-
nizes the need for healthy skepticism toward results obtained
by analytical methods which have not undergone such rigorous
scrutiny and interlaboratory testing of their accuracy, precision,
dependability, specificity, and practicality.
SELECTION OF METHODS FOR STUDY
A certain degree of variability is associated with all measure-
ments. Much of the research on analytical chemistry is an
attempt to minimize that variability. But there are many different
types of variability in analytical work. We often find that when
we attempt to minimize one kind, we must necessarily permit
expansion in another kind. In practical analytical chemistry, the
problem often comes down to which variability is to be mini-
Some examples ofthis point may be helpful. In atomic weight
determination, everything-especially practicality-is sacrificed
for accuracy. A high degree of accuracy and practicality is
required in the assay of precious metals, but the fire assay used
is generally applicable to little else besides metals and minerals.
In clinical chemistry, within-laboratory precision (repeatability)
is critical, and often is of greater interest to clinical laboratories
than absolute accuracy or agreement with the values of other
laboratories (reproducibility). In drug analysis, a high degree of
accuracy is required in the therapeutic range because the
analytical values determining the identity, strength, quality, and
purity of pharmaceutical preparations, as laid down in phar-
macopoeial specifications, are directly related to clinical value.
With polynuclear hydrocarbons, specificity is important, since
some of these compounds are carcinogenic while others are
not. In applying the famous Delaney clause of the United States
Federal Food, Drug, and Cosmetic Act, all attributes of the
analytical methods are secondary to the detection of extremely
small concentrations (detectability), or to exhibiting a high
degree of response for small changes in concentration (sensi-
There is a very special case involving accuracy, where the
"true value" is determined by the method of analysis. Many
legal specifications and standards for food and agricultural
products define ill-defined components such as moisture, fat,
protein, and crude fiber in terms of reference methods. There-
fore, the precision of these methods becomes the limiting factor
for their performance. In fact, most analyses involved in com-
mercial transactions require primarily that the buyer and seller
agree on the same value (analytically and economically), re-
gardless of where it stands on an absolute scale.
The point of these examples is that although methods of
analysis are characterized by a number of attributes-accuracy,
precision, specificity, sensitivity, detectability, dependability,
and practicality-no method is so flawless that all these qualities
can be maximized simultaneously. For any particular analysis,
the analyst must determine, on the basis of the purpose of the
analysis, which attributes are essential and which may be
Unfortunately, the literature is replete with examples indicat-
ing that an individual analyst, and especially the originator of
a method of analysis, is not an unbiased judge of the relative
merits of the methods of analysis which he develops and uses.
In our experience, the collaborative study provides impartial
data on the suitability of the method. The data, in many cases,
speak for themselves.
The collaborative study, or ring test or round robin test, as it
is called in other organizations, provides the basic information
on the performance of analytical methods. The extent of the
information will depend on the number of samples provided,
the number of analyses performed, and the number of labora-
tories participating. The data should be unbiased because the
composition of the samples is known only to the administrator
of the study. Some of the requirements of the study and their
relationship to the characteristics and attributes of the method
are as follows:
(1) Accuracy. Samples must be of defined composition (by
spiking, by formulation, or by analytical consensus).
(2) Specificity. Samples should contain related analytes.
(3) Sensitivity. Samples should differ from each other or from
negative samples by a known amount.
(4) Applicability. Samples should include the concentration
range and matrix components of interest.
(5) Blanks. Samples should include different matrices with
"none" of the component of interest.
(6) Precision. Instructions should request replicate analyses
by the same or different analysts in the same laboratory,
preferably on different days. By far a better procedure is to
include "blind" (unknown to the analyst) replicate samples in
(7) Practicality. Instructions should request information as to
the actual and elapsed time required for the analyses; the
availability of reagents, equipment, and standards; and any
necessary substitutions. When practice samples are included,
the number of analyses required to achieve the stated recovery
and repeatability, should be reported.
PROCEDURAL DETAILS OF COLLABORATIVE STUDY
As numerous beginners in this field have discovered, much
preliminary work must be done before sending out samples:
(1) The method must be chosen and demonstrated to apply
to the matrices and concentrations of interest.
(2) The critical variables in the method should have been
determined and the need for their control emphasized [a rug-
gedness test (5) is useful for this purpose].
(3) The method should be written in detail by the Associate
Referee and tested by an analyst not previously connected with
(4) Unusual standards, reagents, and equipment must be
available from usual commercial sources of supply, or sufficient
quantities must be prepared or obtained to furnish to the
(5) The samples must be identical and homogeneous so that
the analytical sample error is only a negligible fraction of the
expected analytical error.
(6) A sufficient number of samples must be prepared to cover
typical matrices and the concentration range of interest (toler-
ance, maximum or minimum specifications, likely levels of
(7) Samples must be stable and capable of surviving the
rigors of commercial transportation.
(8) Reserve samples should be prepared and preserved to
replace lost samples and to permit reanalysis of samples con-
sidered as outliers to attempt to discover the cause of abnormal
(9) The instructions must be clear. They should be reviewed
by someone not connected with the study to uncover potential
misunderstandings and ambiguities.
(10) If the analyte is subject to change (e.g., bacterial levels,
nitroglycerin tablets), provision must be made for all participants
to begin the analysis at the same time.
(11) Practice samples of a known and declared composition
should be furnished with instructions not to analyze the un-
knowns until a specified degree of recovery and repeatability
(or other attribute) has been achieved.
(12) Provision should be made when necessary for submis-
sion of standard curves, tracings of recorder charts, or photo-
graphs of thin-layer plates in order to assist in determining
possible causes of error.
OTHER TYPES OF INTERLABORATORY STUDIES
This type of collaborative study, which is designed to deter-
mine the characteristics of a method, must be carefully distin-
guished from other types of interlaboratory studies which by
design or through ignorance provide other kinds of information.
The most important types of other studies are:
(1) Those studies which require the collaborators to investi-
gate the variability of parts of methods or applicability to
different types of samples. (An interlaboratory study is usually
an inefficient way of obtaining this type of information.)
(2) Those studies which permit an analyst to use any method
he desires. Such studies invariably produce such a wide scatter
of results that the data are of little value for evaluation of
methods. They may be useful in selecting a method from a
number of apparently equivalent methods, provided the purpose
is emphasized beforehand and the participants provide a de-
scription of the method used in order to permit a correlation of
the details of the methods with apparent biases and variabilities.
(3) Those studies which are used for quality control purposes,
whose participants are not permitted sufficient time to gain
familiarity with the method, or who permit deviations to enter
into the performance of the analyses on the grounds that the
deviation is obviously an improvement which could not possibly
affect the results of the analysis, or who claim to have a superior
With this background information, it is now appropriate to
introduce the following definitions which were agreed upon as
part of the guidelines for collaboration between the AOAC and
the Collaborative International Pesticide Analytical Council L~d.
Collaborative study. An analytical study involving a number
of laboratories analyzing the same sample(s) by the same
method(s) for the purpose of validating the performance of the
Preliminary interlaboratory study. An analytical study in which
two or more laboratories evaluate a method to determine if it
is ready for a collaborative study.
Laboratory performance check. The analysis of very carefully
prepared and homogeneous samples, normally of known active
ingredient content, to establish or verify the performance of a
laboratory or analyst.
The collaborative study i's an experiment designed to evaluate
the performance of a method of analysis through the analysis
of a number of identical samples by a number of different
laboratories. With proper design, it provides an unbiased eval-
uation of the performance of a method in the hands of those
analysts who will use it. A collaborative study must be distin-
guished from those studies designed to choose a method or to
determine laboratory or analyst performance.
(1) AOAC, "Handbook of the AOAC", 4th ed., AOAC, Box 540,
Benjamin Franklin Station, Washington, D.C. 20044, 1977.
(2) W. J. Youden, "Accuracy of Analytical Procedures", J. Assoc.
Off. Anal. Chern., 45, 169-73 (1962).
(3) Harold Egan, "Methods of Analysis; An Analysis of Meth-
ods", ibid., 60,260-7 (1977).
(4) W. J. Youden and E. H. Steiner, "Statistical Manual of the
AOAC: Statistical Techniques for Collaborative Tests. Plan-
ning and Analysis of Results of Collaborative Tests", AOAC,
Box 540, Benjamin Franklin Station, Washington, D.C. 20044,
(5) W. J. Youden, "The Collaborative Test", J. Assoc. Off. Anal.
Chern., 46, 55-62 (1963).
(6) "Guidelines for Collaboration Between the Association of
Official Analytical Chemists (AOAC) and the Collaborative
International Pesticide Analytical Council Ltd. (CIPAC)", ibid.,
Daniel Banes, "The Collaborative Study as a Scientific Concept",
J. Assoc. Off. Anal. Chern., 52,203-06 (1969).
William Horwitz, "Problems of Sampling and Analytical Meth-
ods", ibid., 59, 1197-203 (1976).
Reprinted with permission from: Analytical Chemistry (March 1978) 50, 337A-340A.
Published 1978 American Chemical Society
Definitions of Terms and Explanatory Notes
(1) Term "H20" means distilled water, except where otherwise
specified, and except where the water does not mix with the
detn, as in "H20 bath."
(2) Term "alcohol" means 95% ethanol by vol. Alcohol of
strength x% may be prepd by dilg x mL 95% alcohol to 95 mL
with H20. Absolute alcohol is 99.5% by vol. Formulae of specially
denatured alcohols (SDA) used as reagents are as follows:
SDA No. 100 parts alcohol plus
1 5 wood alcohol
benzene or rubber
"Reagent" alcohol is 95 parts SDA 3-A plus 5 parts isopropanol.
(3) Term "ether" means ethyl ether, peroxide-free by follow-
ing test: To 420 mL ether in separator add 9.0 mL 1% NH4V03
in H2S04 (1+16). Shake 3 min and let sep. Drain lower layer into
25 mL g-s graduate, dil. to 10 mL with H2S04 (1 +16), and mix.
Any orange color should not exceed that produced by 0.30 mg
H20 2(1 mL of soln prepd by dilg 1 mL 30% H20 2to 100 mL with
H20) and 9.0 mL 1% NH4V03in H2S04 (1 + 16). Peroxides may be
eliminated by passing .;;700 mL ether thru 10 cm column of
Woelm basic alumina in 22 mm id tube.
(4) Reagents listed below, unless otherwise specified, have
approx. strength stated and conform in purity with Recom-
mended Specifications for Analytical Reagent Chemicals of
American Chemical Society:
Sulfuric Acid . . . . . . . . . . . . . . . . . . . . . . . .. 95.0-98.0% H2S04
Hydrochloric acid .................... 36.5-38.0% HCI
Nitric acid ........................... 69.0-71.0% HN03
Fuming nitric acid. . . . . . . . . . . .. . . . . . .. ;,,90% HN03
Acetic acid . . . . . . . . . . . . . . . . . . . . . . . . . .. ;,,99.7% HC2H30 2
Hydrobromic acid ......... " ..... . ... 47.0-49.0% HBr
Ammonium hydroxide. . . . . . . . . . . . . . .. 28-30% NH3
Phosphoric acid. . . . . . . . . . . . . . . . . . . . .. ;,,85% H3P04
Where no indication of diln is given, reagent is of concn given
(5) All other reagents and test solns, unless otherwise de-
scribed in text, conform to requirements of American Chemical
Society. Where such specifications have not been prepd, use
highest grade reagent. When anhyd. salt is intended, it is so
stated; otherwise the crystd product is meant.
(6) Unless otherwise specified, phenolphthalein (phthln) used
as indicator is 1% alc. soln; Me orange is 0.1 % aq. soln; Me red
is 0.1 % alc. soln.
(7) Directions for stdzg reagents are given in Chapter 50.
(8) Unusual reagents not mentioned in reagent sections or
cross referenced, other than common reagents normally found
in laboratory, are italicized first time they occur in method.
(9) Com. prepd reagentsolns must be checked for applicability
to specific method. They may contain undeclared buffers, pre-
servatives, chelating agents, etc.
(10) In expressions (1 +2), (5+4), etc., used in connection with
name of reagent, first numeral indicates vol. reagent used, and
second numeral indicates vol. H20. For example, HCI (1 +2)
means reagent prepd by mixing 1 vol. HCI with 2 vols H20.
When one of reagents is solid, expression means parts by wt,
first numeral representing solid reagent and second numeral
H20. Solns for which the solv. is not specified are aq. solns.
(11) In making up solns of definite percentage, it is understood
thatx g substance is dissolved in H20 and dild to 100 mL. Altho
not theoretically correct, this convention will not result in any
appreciable error in any of methods given in this book.
(12) Chromic acid cleaning soln is prepd by (1) adding 1 L
H2SO. to ca 35 mL satd aq. Na2Cr207 soln; or (2) adding 2220 mL
(9 Ib) H2S04 to ca 25 mL satd aq. Cr03 soln (170 g/100 mL).
Reagents may be tech. grade. Use only after first cleaning by
other means (e.g., detergent) and draining. Mixt. is expensive
and hazardous. Use repeatedly until it is dild Of has a greenish
tinge. Discard carefully with copious amts of H20.
(13) All calcns are based on table of international atomic
(14) Burets, vol. flasks, and pipets conform to following
Federal specifications (available from General Services Admin.,
Specification Activity 3F1, Washington Navy Yard, Bldg. 197,
Washington, DC 20407):
Buret NNN-B-00789a May 19, 1965
Flask, vol. NNN-F-00289d Feb 7, 1977
Pipet, vol. NNN-P-395c March 13, 1970
Pipet, measuring NNN-P-350c July 16,1973
See also NBS Circular 602, "Testing of Glass Volumetric Ap-
paratus" (available as Com 73-10504 from NTIS, Springfield, VA
(15) Standard taper ($) glass joints may be used instead of
stoppers where the latter are specified or implied for connecting
(16) Sieve designations, unless otherwise specified, are those
described in Federal Specification RR-S-366e, Nov 9,1973 (avail-
able from General Services Admin.). Designation" '100-mesh'
(or other number) powder (material, etc.)" means material
ground to pass thru std sieve No. 100 (or other number).
Corresponding international std and US std sieves are given in
(17) Term "paper" means filter paper, unless otherwise spec-
(18) Term "high-speed blender" designates mixer with 4
canted, sharp-edge, stainless steel blades rotating at the bottom
of 4-lobe jar at 10,000-12,000 rpm, or with equiv. shearing
action. Suspended solids are reduced to fine pulp by action of
blades and by lobular container, which swirls suspended solids
into blades. Waring Blendor, or equiv., meets these require-
(19) "Flat-end rod" is glass rod with one end flattened by
heating to softening in flame and pressing vertically on flat
surface to form circular disk with flat bottom at end.
(20) Designation and pore diam. range of fritted glassware
are: extra coarse, 170-220 /-Lm; coarse, 40-60; medium, 10-15;
fine, 4-5.5; Jena designations and pore diam. are: 1, 110 /-Lm;
2, 45; 3, 25; 4, 8.
(21) Unless otherwise indicated, temps are expressed as
Table 1. Nominal Dimensions of Standard Test Sieves (U.S.A.
International Sieve Wire
Standard' U.S.A. Opening, Diameter,
(ISO) Standard inches mm
12.5 mmb '/2 in.b 0.500 2.67
11.2 mm 7/'6 in. 0.438 2.45
9.5 mm 3/8 in. 0.375 2.27
8.0 mm 5/,6 in. 0.312 2.07
6.7 mm 0.265 in. 0.265 1.87
'/. in." 0.250 1.82
5.6 mm No. 3'/2 0.223 1.68
4.75 mm No.4 0.187 1.54
4.00 mm No.5 0.157 1.37
3.35 mm No.6 0.132 1.23
2.80 mm No.7 0.111 1.10
2.38 mm No.8 0.0937 1.00
2.00 mm No. 10 0.0787 0.900
1.70 mm No. 12 0.0661 0.810
1.40 mm No. 14 0.0555 0.725
1.18 mm No. 16 0.0469 0.650
1.00 mm No. 18 0.0394 0.580
850 "me No. 20 0.0331 0.510
710 "m No. 25 0.0278 0.450
600 "m No. 30 0.0234 0.390
500 "m No. 35 0.0197 0.340
425 "m No. 40 0.0165 0.290
355 "m No. 45 0.0139 0.247
300 "m No. 50 0.Q117 0.215
250 "m No. 60 0.0098 0.180
212 "m No. 70 0.0083 0.152
180 "m No. 80 0.0070 0.131
150 "m No. 100 0.0059 0.110
125 "m No. 120 0.0049 0.091
106 "m No. 140 0.0041 0.076
90 "m No. 170 0.0035 0.064
75 "m No. 200 0.0029 0.053
63 "m No. 230 0.0025 0.044
53 "m No. 270 0.0021 0.037
a These standard designations correspond to the values for test sieve
apertures recommended by the International Organization for Standard-
ization, Geneva, Switzerland.
b These sieves are not in the standard series but they have been
included because they are in common usage.
c 1000 I-'m = 1 mm.
(22) Operations specified as "wash (rinse, ext. etc.) with two
(three, four, etc.) 10 mL (or other voL) portions H20 (or other
solv.)" mean that the operation is to be performed with indicated
vol. of solv. and repeated with same vol. of solv. until number
of portions required have been used.
(23) Definitions of terms used in methods involving spectro-
photometry are those given in JAOAC 37, 54(1954). Most
important principles and definitions are:
(a) More accurate instrument may be substituted for less
accurate instrument (e.g., spectrophtr may replace colorimeter)
where latter is specified in method. Wavelength specified in
method is understood to be that of max. absorbance (A). unless
no peak is present.
(b) Absorbance(s) (A).-Neg. logarithm to base 10 of ratio of
transmittance (T) of sample to that of ref. or std material. Other
names that have been used for quantity represented by this
term are optical density, extinction, and absorbancy.
(e) Absorptivity(ies) (a).-Absorbance per unit concn and cell
length. a = A/be, where b is in cm and c in gil, or a = (A/be)
x 1000, if c is in mg/L. Other names that have been used for
this or related quantities are extinction coefficient, specific
absorption, absorbance index, and £16';'".
(d) Transmittance(s) (T).-Ratio of radiant power transmitted
by sample to radiant power incident on sample, when both are
measured at same spectral position and with same slit width.
Beam is understood to be parallel radiation and incident at right
angles to plane parallel surface of sample. If sample is soln,
solute transmittance is quantity usually desired and is detd
directly as ratio of transmittance of soln in cell to transmittance
of solv. in an equal cell. Other names that have been used for
this quantity are transmittancy and transmission.
(e) Standardization.-Spectrophtr may be checked for accu-
racy of wavelength scale by ref. to Hg lines: 239.95, 248.3,
253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 404.66,
435.83, 546.07, 578.0, and 1014.0 nm. To check consistency of
absorbance scale, prep. soln of 0.0400 g K2CrO./L 0.05N KOH
and det. absorbance at following wavelengths in 1 cm cell: 230
nm, 0.171; 275,0.757; 313.2,0.043; 375, 0.991; 400, 0.396. See
"Standards for Checking the Calibration of Spectrophotome-
ters," Letter Circular LC-l017, reissued Jan 1967, NBS.
(24) Least square treatment of data and calculation of regres-
sion lines.-This technic finds the best fitting straight line for
set of data such as std curve. It calcs that straight line whose
sum of squares of vertical deviations (usually A) of observations
from the line is smaller than corresponding sum of squares of
deviations from any other line. Equation of straight line is:
Y = a + bX,
where a is intercept at Y axis (X = 0). and b is slope of line.
Least square estimates of constants are:
b = I (X, Y,) - [(IX,I Y,)/n]
IXf - (IX,)2/n
where I = "sum of" the n individual values of indicated
operation, and X and Yare the averages of the X and Y points.
Example: To find "best" straight line relating A (Y) to concn
Observation Concn Absorbance
No. CI X, Y, X~ X,V,
1 80 1.270 6400 101.6
2 60 1.000 3600 60.0
3 40 0.700 1600 28.0
4 30 0.550 900 16.5
5 20 0.250 400 5.0
6 10 0.100 100 1.0
7 0 0.050 0 0.0
n=7 IX, = 240 IY, = 3.92 IXl = 13000 I(X,Y,) = 212.1
x= IX,!n = 240/7 = 34.29
Y = IY,!n = 3.92/7 = 0.56
b = 212.1 - (240)(3.92)/7 77.7 = 0.0163
13000 - (240)2/7 4771
a = 0.56 - 0.0163(34.29) = 0.001
Best equation is then:
Y = 0.00 + 0.0163X
If for sample, A = 0.82, corresponding concn (X) would be:
X = (Y - 0.00)/0.0163 =0.82/0.0163 = 50.3.
Many scientific and statistical calculators are preprogrammed
to perform this calcn.
(25) Common safety precautions are given in Chapter 51.
(26) For sake of simplicity, abbreviations CI and I instead of
CI2 and 12 are used for chlorine and iodine. Similar abbreviations
have been used in other cases (0, N, H). The same abbreviation
may also be used for the ion where no ambiguity will result.
(27) Reagents and app. referenced with only a letter, e.g., (c).
will be found in the reagent or apparatus section of that method.
(28) To conserve space, most of the articles and some prep-
ositions have been eliminated.
(29) Names and addresses of manufacturers and suppliers,
and trade names of frequently mentioned materials, are fur-
nished below solely as a matter of identification and conven-
ience, without implication of approval, endorsement, or certifi-
cation. The same products available from other suppliers or
other brands from other sources may serve equally well if
proper tests indicate their use is satisfactory. These firms when
mentioned in a method are given by name only (without
Manufacturers and Suppliers
Ace Glass, Inc., PO Box 688, 1430 N West Blvd, Vineland, NJ
Aldrich Chemical Co., Inc., 940 W St. Paul Ave, Milwaukee, WI
Allied Chemical Corp., Specialty Chemicals Div., PO Box 1087R,
Morristown, NJ 07960
Aluminum Company of America, 1501 Alcoa Bldg, Pittsburgh,
American Cyanamid Co., Agricultural Div., PO Box 400,
Princeton, NJ 08540
American Instrument Co., Div. of Travenol Laboratories, Inc.,
8030 Georgia Ave, Silver Spring, MD 20910
(ASBC) American Society of Brewing Chemists, 3340 Pilot Knob
Rd, St. Paul, MN 55121
(ATCC) American Type Culture Collection, 12301 Parklawn Dr,
Rockville, MD 20852
Analabs Inc., 80 Republic Dr, North Haven, CT 06473
Applied Science Laboratories, Inc. (Applied Science Division,
Milton Roy Co.). PO Box 440, State College, PA 16801
Baird-Atomic, Inc., 125 Middlesex Tnpk, Bedford, MA 01730
J. T. Baker Chemical Co., 222 Red School Ln, Phillipsburg, NJ
Barber-Colman Co., see Searle Analytic, Inc.
Bausch & Lomb, Inc., Analytical Systems Div., 820 Linden Ave,
Rochester, NY 14625
BBL, Div. of Bioquest, PO Box 243, Cockeysville, MD 21030
Beckman Instruments, Inc., 2500 Harbor Blvd, Fullerton, CA
Becton, Dickinson, & Co., Rutherford, NJ 07070
Bio-Rad Laboratories, 32nd and Griffin Ave, Richmond, CA 94804
Brinkmann Instruments, Inc., Cantiague Rd, Westbury, NY 11590
Burdick & Jackson Laboratories, Inc., 1953 S Harvey St, Muske-
gon, MI 49442
Burrell Corp., 2223 Fifth Ave, Pittsburgh, PA 15219
Calbiochem, 10933 N Torrey Pines Rd, La Jolla, CA 92037
Carborundum Co., PO Box 423, Niagara Falls, NY 14302
Cenco Inc., 2600 5 Kostner Ave, Chicago, IL 60623
Coleman Instruments Division, Perkin-Elmer Corp., 2000 York
Rd, Oak Brook, IL 60521
Corning Glass Works, Laboratory Products Dept., Corning, NY
Curtin Matheson Scientific, Inc., PO Box 1546, Houston, TX
Ddco Laboratories, PO Box 1058A, Detroit, MI 48232
Dohrmann Div. of Envirotech Corp., 3240 Scott Blvd, Santa
Clara, CA 95050
Dow Chemical Co., Ag-Organics Dept., PO Box 1706, Midland,
Dow Corning Corp., Midland, MI 48640
E. I. du Pont de Nemours & Co., Wilmington, DE 19898
Eastman Kodak Co., Eastman Organic Chemicals, 343 State St,
Rochester, NY 14650
Eaton-Dikeman Co., Mt. Holly Springs, PA 17065
Elanco Products Co., Div. of Eli Lilly Co., Elanco Analytical
Laboratory, Dept. MC757, Indianapolis, IN 46206
Fisher & Porter Co., Lab Crest Scientific Div., County Line Rd,
Warminster, PA 18974
Fisher Scientific Co., 711 Forbes Ave, Pittsburgh, PA 15219
Floridin Co., Berkeley Springs, WV 25411
Foss America Inc., PO Box 504, Route 82, Fishkill, NY 12524
GAF Corp., 140 W 51st St, New York, NY 10020
G.B. Fermentation Industries, Inc., 1 N Broadway, Des Plaines,
Geigy Chemical Corp., Saw Mill River Rd, Ardsley, NY 10502
Hamilton Co., PO Box 17500, Reno NV 89510
Hess & Clark Laboratories, Div. of Rhodia, Inc., 7th and Orange
Sts, Ashland, OH 44805
Hewlett-Packard Co., 1501 Page Mill Rd, Palo Alto, CA 94304
Hoffman-La Roche, Inc., Nutley, NJ 07110
ICI-America, Inc., Chemical Research Dept., Wilmington, DE
ICN-K&K Laboratories, Inc., 121 Express St, Plainview, NY
ICN Pharmaceuticals, Inc., Life Sciences Group, 26201 Miles Rd,
Cleveland, OH 44128
johns-Manville Products Corp., Greenwood Plaza, Denver, CO
Kimble Products, Owens-Illinois, PO Box 1035, Toledo, OH 43666
Kontes Glass Co., Spruce St, Vineland, NJ 08360
Labconco Corp., 8811 Prospect Ave, Kansas City, MO 64132
Eli Lilly & Co., 740 5 Alabama St, Indianapolis, IN 46206
Mallinckrodt Chemicals Works, Science Products Div., 2nd &
Mallinckrodt Sts, St. Louis, MO 63147
MC/B Manufacturing Chemists, 2909 Highland Ave, Norwood,
Matheson Scientific, Inc., see Curtin Matheson Scientific, Inc.
Merck & Co., Inc., 126 E Lincoln Ave, Rahway, NJ 07065
Miles Laboratories, Inc., Elkhart, IN 46514
Monsanto Chemical Co., 800 N Lindberg Blvd, St. Louis, MO
(NBS) National Bureau of Standards, Washington, DC 20234
(NF) National Formulary, see USP
New York Laboratory Supply Co., 510 Hempstead Tnpk, West
Hempstead, NY 11552
Orion Research Inc., 380 Putnam Ave, Cambridge, MA 02139
Perkin-Elmer Corp., 702-G Main Ave, Norwalk, CT 06856
Phillips Chemical Co., Division of Phillips Petroleum Co., Spe-
cialty Chemicals, Drawer '0', Borger, TX 79007.
Pierce Chemical Co., PO Box 117, Rockford, IL 61105
H. Reeve Angel & Co., Inc., 9 Bridewell PI, Clifton, NJ 07014
Rohm & Haas Co., Independence Mall West, Philadelphia, PA
Salsbury Laboratories, Charles City, IA 50616
Sargent-Welch Scientific Co., 7300 N Linder Ave, Skokie, IL
(S&S) Schleicher & Schuell, Inc., 543 Washington St, Keene, NH
Schoeffellnstrument Corp., 24 Booker St, Westwood, NJ 07675
SGA Scientific, Inc., 735 Broad St, Bloomfield, NJ 07003
Scientific Products, Div. of American Hospital Supply Corp.,
1430 Waukegan Rd, McGaw Park, IL 60085
Searle Analytic, Inc., 2000 Nuclear Dr, Des Plaines, IL 60018
Shell Oil Co., PO Box 2463, Houston, TX 77001
Sigma Chemical Co., PO Box 14508, Sl. Louis, MO 63178
G. Frederick Smith Chemical Co., PO Box 23344, Columbus, OH
Sterwin Chemicals, Inc., 90 Park Ave, New York, NY 10016
Supelco, Bellefonte, PA 16823
Technicon Instruments Corp., 511 Benedict Ave, Tarrytown, NY
Arthur H. Thomas Co., Vine St at 3rd, PO Box 779, Philadelphia,
Ultra-Violet Products, Inc., 5100 Walnut Grove Ave, San Gabriel,
Union Carbide Corp., Chemicals and Plastics, 270 Park Ave, New
York, NY 10017
Union Carbide Corp., Agricultural Products and Services, PO
Box 1906, Salinas, CA 93901
Uniroyal Chemical, Elm St, Naugatuck, CT 06770
The Upjohn Co., Kalamazoo, MI 49001
(USDA) U.S. Department of Agriculture, Office of Information,
Washington, DC 20250
(USP) United States PharmacopeiaI Convention, Inc., 12601
Twinbrook Pkwy, Rockville, MD 20852
Varian Aerograph, 2700 Mitchell Dr, Walnut Creek, CA 94598
Varian Instrument Div., 611 Hansen Way, Palo Alto. CA 94303
Velsicol Chemical Corp., 341 E Ohio St, Chicago, IL 60611
VWR Scientific, PO Box 3200, San Francisco, CA 94119
Wallerstein Co., see G. B. Fermentation Industries, Inc.
Waters Associates, Inc., Maple St, Milford, MA 01757
Winthrop Laboratories, Special Chemicals Dept., 90 Park Ave,
New York, NY 10016
Amberlite. Ion exchange resins. Rohm and Haas Co.
Anakrom. Gas chromatography supports. Analabs, Inc.
Celite. Diatomaceous products. Johns-Manville Products Corp.
Chromosorb. Chromatographic supports and packings. Johns-
Manville Products Corp.
Dowex. Ion exchange resins. Dow Chemical Co.
Florisil. Chromatographic adsorbents. Floridin Co.
Gas-Chrom. Gas chromatography solid supports. Applied Sci-
ence Laboratories, Inc.
Hyf/o Super-Cel. Diatomaceous products. Johns-Manville Prod-
Skellysolve. Hydrocarbon solvents. Getty Refining and Market-
ing Co., PO Box 1650, Tulsa, OK 74102
Tef/on. Chemically resistant polytetrafluoroethylene. E. I. du
Pont de Nemours & Co.
Tygon. Halogenated vinyl plastic. Norton Co., Plastics & Syn-
thetics Div., 12 E Ave, Tallmadge, OH 44278
(30) The foillowing abbreviations, many of which conform
with those of Chemical Abstracts, are used. In general, principle
governing use of periods after abbreviations is that period is
used where final letter of abbreviation is not the same as final
letter of word it represents. Periods are not used with units,
except inch(es) and gallon(s).
absorbance(s) thruout (not restricted to for-
mulas); not absorption. A' is used for std;
Ao for blank; 3 digit subscript numerals
usually denote wavelengths in nm
CH3CO- (acetyl, not acetate)
American Chemical Society
alcoholic (not alcohol)
alkaline (not alkali)
American Oil Chemists' Society
American Society for Testing and Materials
average (except as verb)
degrees Celsius (Centigrade)
Collaborative International Pesticides Ana-
concentrate (as verb or noun)
counts per minute
crystalline (not crystallize)
ethylenedinitrilotetraacetic acid (or -tetra-
ethanol (the chemical entity C2H50H)
degrees Fahrenheit (DC = (5/9) x (OF - 32))
Food and Agriculture Organization
fluid ounce(s) (29.57 mL)
foot (30.48 cm)
gravity (in centrfg)
gallon(s) (3.785 L)
acetic acid (not HAc)
high pressure (or performance) liquid chro-
inner diameter (or dimension)
inch(es) (2.54 cm)
International Organization for Standardiza-
Journal of the Association of Official Analyt-
ical Chemists (after 1965)
Journal of the Association of Official Agri-
cultural Chemists (before 1966)
pound(s) (453.6 g)
meter(s); milli-as prefix
molar (as applied to concn). not molal
milliampere (et amp)
mm); use nanometer (nm)
normal (as applied to concn); in equations.
normality of titrating reagent
Newton (105 dynes)
National Bureau of Standards
National Canners Association (now National
Food Processors Associa.tion)
m); formerly mIL
acetate (cf Ac)
outer diameter (or dimension)
ounce(s) (28.35 g)
) as prefix
Pascal (1 Newton/m2
; 9.87 x 10-6
x 10-3 mm Hg (torr); 1.45 x 10--4 psi)
powdered (as adjective)
parts per billion (1/109
parts per million (1/106
pounds per square inch (absolute)
pounds per square inch gage (atmospheric
pressure = 0)
pint(s) (473 mL)