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Determination of Water Moisture & Dry Matter in Animal Feed, Grain, Forage by Karl Fischer Titration
Determination of Water Moisture & Dry Matter in Animal Feed, Grain, Forage by Karl Fischer Titration
2, 2002
AGRICULTURAL MATERIALS
Collaborators: D. Amundson; J. Boucher; A. Clark; D. Clark; P. Coco; D. Dow; P. Fahey; H. Hoffmann; D. Mertens;
H. Morris; T. Rittenhouse; M. Wohl
Submitted for publication November 2001. CH3OH + SO2 +RN = [RNH] SO3CH3
The recommendation was approved by the Methods Committee on
Feeds, Fertilizer, and Related Agricultural Topics. Method I was approved
for Official First Action at their meeting in Kansas City, MO, on where RN = imidazole base.
September 8, 2001 and Method II was approved for Official First Action on
February 6, 2002. See “Official Methods Program Actions,” (2001) Inside
Laboratory Management, November/December issue. H2O + I2 + [RNH]SO3CH3 + 2 RN
Corresponding author’s e-mail: nancy_thiex@sdstate.edu. = [RNH]SO4CH3 + 2[RNH]I
THIEX & VAN EREM: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 319
The sulfur dioxide reacts with alcohol to form an ester, Collaborative Study
which is neutralized by the base. The anion of alkyl sulfurous
acid is the reactive component and is present in the KF re- The Study Directors with the cooperation of 8 additional
agent. The titration of water constitutes oxidation of participating laboratories conducted the collaborative study.
alkyl-sulfate by iodine. This reaction consumes water. It These laboratories represented a variety of laboratory types, in-
cluding research, commercial laboratories, manufacturers and
therefore follows that 2 significant prerequisites must be ful-
industry laboratories, and state regulatory laboratories. Two
filled to ensure a stoichiometric course of the KF reaction. The
laboratories were from outside the United States (one Canadian
first of these is the presence of a suitable alcohol to esterify the and one German). Participants received no compensation. The
sulfur dioxide completely. Methanol is the preferred choice, collaborating laboratories provided the Karl Fischer titrator, ho-
because the course of the KF reaction is stoichiometric and mogenizer, circulating water bath, and formamide. Karl Fischer
rapid in methanol. In Method I, methanol is mixed with reagent, methanol, standard reference material, and certified
formamide, which dissolves sugar and helps to distribute pro- water were provided to collaborators.
Collaborating laboratories were asked to analyze 10 mate-
cate in the same solvent as used for the test sample immedi- Alfalfa silage X X
ately after the test sample, thereby retaining interfering Cereal grain mix X X
(birdseed, hog and
compounds in the titration flask. In most cases, if interferences
poultry ration, barley)
are present, they will affect the value for the water standard in
Oats X X
the same manner as the test portion was affected. Additives or
Soybeans X X (Oilseed)
alternative solvents exist to deal with many potential interfer-
Beef feed containing urea, X X X
ences (e.g., 8-hydroquinone can be added to systems that are af-
medicated
fected by FeCl3). The user of the method should be alert to the
Dry cat food X X
possibility of potential interferences, even if unlikely, and run
Milk replacer X
water standards frequently. Extracting replicates of a test sam-
ple in the same solvent may also magnify interference effects.
320 THIEX & VAN EREM: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002
Table 2. Coefficients of variation (CV) for collaborative dictions were made on triplicate test portions. Coefficients of
test samples variation (CVs) and number of values (n) are reported in Ta-
Karl Fischer NIR ble 2. In cases where one value appeared to be an outlying
Material CV (n) CV (n) value, the test was repeated. The CVs for both n = 6 (with the
questionable value removed) and for n = 7 are provided in Ta-
Alfalfa hay 1.1 (6) 0.3 (9) ble 2. An acceptance criterion of a relative standard deviation
Corn silage 2.7 (6) 1.1 (9)
(RSD) of ca 2.0% was considered acceptable for the study ma-
terials. Only the oat sample suitability was questioned. When
Grass hay 0.7 (6) 2.2 (9)
tests were repeated on a second day, the results were improved
Alfalfa silage 0.9 (6), 1.7 (7) 0.1 (9) and the test sample was retained for the collaborative study.
Cereal grain mix 0.9 (6), 2.5 (7) 0.3 (9) Collaborators were asked to report results on an “as is” ba-
(birdseed, hog and sis, to determine triplicate analysis for each test sample, and to
poultry ration, barley)
report data to 4 significant figures. In addition to data on test
Oats 4.3 (6) on 1st day 1.4 (9) on 1st day
Table 2001.12A. Statistical results for interlaboratory study on determination of water in animal feed, cereal, and
forage (blind duplicate design)
Table 2001.12B. Statistical results for collaborative study on determination of dry matter in animal feed, cereal, and
forage (blind duplicate design)
Feeds Mean Laba sr RSDr, % sR RSDR, % HORRAT
a
Number of laboratories retained after eliminating outliers. Number of laboratories removed as outliers shown in parentheses.
the Karl Fischer titration vessel by high-speed homogeniza- H2O + I2+ [RNH] SO3CH3 + 2RN
tion. The formamide content must not exceed 50% to maintain = [RNH] SO4CH3 + 2[RNH] I
reaction conditions. A subsequent titration of the water is per-
formed at 50°C with one-component Karl Fischer reagent
Imidazole is used as the base to neutralize the acids and
based on imidazole.
buffer the titration system. (Note: The pH of the system could
In Method II, boiling methanol is used instead of the meth- be upset if a feed, such as a mineral mix, which was either a
anol–formamide mixture at a higher temperature of 66°C strong acid or base were to be analyzed.)
without high-speed homogenization. A totally tight titration One source of interference is from aldehydes and ketones,
vessel is critical. which react with methanol to form acetals and ketals, respec-
These methods provide for the dry, ground test sample to tively, and water. Do not use acetone to rinse glassware or
be weighed and added directly to the titration cell to minimize equipment. The acetone residue reacts with methanol to form
contamination by atmospheric moisture. The reactions are: acetone dimethyl acetal and water, and thus bias water results
high. Additional potential interferences include metal oxides,
such as CaO, or other compounds that can form water when
CH3OH + SO2 + RN = [RNH] SO3CH3
neutralized; or thiols, such as cysteine hydrochloride, or other
compounds that can be oxidized by iodine. If interferences are
where RN = imidazole base. suspected, verify by running the water standard in triplicate in
Table 2001.12C. Statistical results for interlaboratory study on determination of water in animal feed, cereal, and
forage, including data from laboratory using alternative extraction (blind duplicate design)
ID Mean No. labsa(b) sr RSDr, % sR RSDR, % HORRAT
Soybeans 7.01 10 0.10 1.41 0.42 6.03 2.02
Urea feed 4.57 10 0.18 3.97 0.47 10.20 3.20
Grass hay 6.34 10 0.28 4.44 0.48 7.59 2.51
Cereal grains 8.60 10 0.37 4.26 0.44 5.08 1.76
Alfalfa silage 7.40 9(1) 0.15 2.05 0.68 9.17 3.10
Corn silage 6.43 10 0.21 3.23 0.45 7.02 2.32
Cat food 6.94 10 0.23 3.33 0.44 6.27 2.10
Milk replacer 5.18 10 0.30 5.85 0.40 7.76 2.49
Alfalfa hay 7.40 10 0.49 6.64 0.64 8.59 2.90
Oats 7.51 10 0.15 1.99 0.57 7.54 2.55
a(b)
a = Number of laboratories retained after eliminating outliers, (b) = number of laboratories removed as outliers.
322 THIEX & VAN EREM: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002
the same solvent as used for the test sample immediately after HYDRANAL®–Composite 5 (available from Riedel-de Haën
the test sample, thereby retaining interfering compounds in 34805, Sigma Aldrich, St. Louis, MO), or equivalent.
the titration flask. In most cases, if interferences are present, (b) Methanol.—Anhydrous, for moisture determinations,
they will affect the value for the water standard in the same water content not to exceed 0.05% (HYDRANAL–Methanol
manner as the test portion. Be alert to the possibility of potential Dry; Riedel-de Haën 34741, Sigma Aldrich, St. Louis, MO), or
interferences and run water standards frequently. Extracting equivalent.
replicates of a test sample in the same solvent may also magnify (c) Formamide.
interference effects. If interfering compounds are found, addi- (d) Solvent.—Methanol–formamide (1 + 1). Mix fresh
tives or alternative solvents exist to deal with many. each day.
(e) Sodium tartrate dihydrate.—Primary standard (water
Method I
Extraction into Methanol–Formamide (1 + 1)
content, 15.66 ± 0.05%), HYDRANAL®–standard sodium
Using High-Speed Homogenization tartrate–2-hydrate (Riedel-de Haën 34803, Aldrich Chemical
Co., Milwaukee, WI), or equivalent.
B. Apparatus
(mL reagent) in mL to the nearest 0.001 mL. Repeat 4 times. mogenize momentarily to rinse down moisture that may have
Calculate titer, then average the 5 values. The relative stan- been under the cap. This ensures that the moisture of the parti-
dard deviation should be <2%. cles under the cap or hanging on the glass walls above the liq-
uid surface is titrated. The end point is reached when no
mg H2 O mg Na 2 C4 H4 O6 ⋅ 2H2 O× 0.1566
Titer = = change in potential is observed for 10 s (stop criterion: time;
mL reagent mL reagent delay: 10 s). Record the volume of titrant (V). Repeat determi-
nation in triplicate. The relative standard deviation of repli-
where mg Na2C4H4O6⋅2H2O is S – T, in mg. cates should be <5%. The cell need not be emptied between
each titration. Usually about 3 titrations can be performed be-
G. System Suitability
fore the cell requires emptying and replenishing.
Heat cell to 50 ± 1°C. Dry the cell as in E. Check drift in the
I. Calculations
titration cell. A dried titration cell should have a maximum
drift consumption of 5–10 µL Karl Fischer reagent/min. Ana- mg H2O = V × titer
(c) Glass weighing spoon.—With opening for dispensing tip, reweigh the syringe and record the weight (S1). Obtain the
test portion into the titration flask through the septum stopper weight of standard solution (Ss – S1) by subtracting the weight
(Brinkmann 6.2412.000 or equivalent). (S1) from the weight of the syringe plus test portion (SS). Re-
(d) Magnetic stirrer. cord the water standard weight to the nearest 0.1 mg, and enter
the weight into instrument. Turn on the heating system, start
K. Reagents the stirrer and begin the titration as soon as the methanol re-
See C. turns to an active boil. Record the volume of titrant (V1).
Perform the titration procedure 2 additional times, recording
L. Preparation of Test Sample weights of the syringe after each subsequent addition (S2, S3)
See D. and the respective volume of titrant (V2, V3). Calculate the
percent recovery as in G.
M. Drying or Conditioning the Cell Average % recovery should be 100 ± 1%. If system is not
Dispense ~50 mL methanol into the titration vessel. Close within specifications, correct before continuing with determina-
7.03a
7.46b
iarization samples were reported to them, and attempts were
6.86
7.37
8.11
6.67
7.87
8.52
7.55
7.38
B19
made to correct problems at that point. However, in some
Oats
cases many months elapsed between the time some laborato-
7.23a
8.00b
6.84
7.20
8.24
6.73
7.82
8.39
7.41
7.55
A17
ries analyzed the familiarization samples and the test samples.
Unfortunately, since the concentration of water can be af-
fected by atmospheric conditions in the laboratory after the
7.63a
7.29
7.21
8.19
6.38
7.57
7.88
6.83
7.09
6.37
B20
Alfalfa hay
6.87
7.89
6.28
7.40
8.09
7.02
8.38
7.98
A16
and time over which data were submitted, results of the collab-
orative study are encouraging. Eight laboratories used
Metrohm/Brinkmann (Westbury, NY) equipment and one
4.93a
4.76
4.87
5.69
4.91
Milk replacer
5.29
5.61
4.84
5.08
4.69
B18
4.85
5.68
4.93
5.52
5.67
4.81
5.86
5.74
A14
6.53
6.72
7.06
7.17
6.69
6.87
7.36
5.96
B15
tem suitability.
Cat food
6.56
6.77
7.04
7.06
7.46
6.63
6.90
6.34
A11
6.28
6.81
5.81
6.66
6.66
6.22
6.81
5.44
B13
Corn silage
Table 3. Collaborative study results for determination of water in animal feed, cereal, and forage
6.15
6.97
5.66
6.21
6.94
6.50
6.98
6.05
A9
7.81b 7.72b
8.35
7.14
7.47
7.39
6.78
7.55
5.87
Alfalfa silage
B12
7.08
7.52
7.35
6.91
7.43
6.34
A8
Data excluded by Cochran’s test when excluding 100% boiling methanol extraction data.
study statistics.
Tables 2001.12A and B show a summary of results of sta-
8.80a
Cereal grains
8.74
8.22
8.84
8.62
8.95
8.01
8.59
9.04
7.67
B10
8.05
8.19
8.59
9.56
9.08
8.36
8.79
8.43
6.01
6.97
6.31
6.13
6.05
6.01
7.23
6.72
B7
Grass hay
6.03
6.92
6.28
6.68
5.92
6.03
7.07
6.32
A3
4.29
4.92
4.57
5.63
4.33
4.12
5.23
4.26
B4
4.33
5.00
4.54
5.07
3.78
4.14
5.16
4.37
A2
6.70
6.99
6.91
7.76
6.54
6.62
7.18
6.72
B5
Soybeans
6.63
7.14
6.97
7.86
6.54
6.49
7.47
6.63
A1
c
326 THIEX & VAN EREM: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002
Table 4. Comparison of laboratory ranking scores including and excluding alternative extraction procedure data
Laboratory ranking score
Titration
Lab equipment used Excluding data using alternative extraction Including data using alternative extraction
a
Boiling methanol extraction.
b,c
Showed statistical bias compared to other laboratories based on laboratory ranking score.
Steiner (4) showed that the alternative-extraction procedure water to collaborators and for coming to our rescue in the ship-
had no bias compared to the original extraction procedure. ment of these materials. We thank Helga Hoffmann for
The boiling methanol extraction procedure had a laboratory persisting with the alternative extraction and for generating
ranking score of 44. Results of that test are shown in Table 4. the comparability data. We thank Brian Steinlicht (South Da-
Comparability data for the study including the boiling kota State University, Olson Biochemistry Laboratories,
methanol extraction procedure are provided in Table 3. The Brookings, SD) for assistance with preparing, splitting, and
within-laboratory relative standard deviation (RSDr, repeat- sealing familiarization and test samples. We also thank the
ability) ranged from 1.41 to 6.64% when including the 100% following collaborators for their participation in this study:
boiling methanol data compared to 1.14 to 6.99% when ex- Jacques A. Boucher and Andy Clark, Canadian Food In-
cluding it. Among-laboratory (including within-) relative spection Agency, Laboratory Services Division, Ottawa, On-
standard deviation (RSDR, reproducibility) ranged from 5.08 tario, Canada
to 10.20% when including the 100% boiling methanol data Doug Clark, Riedel-de Haen, St. Louis, MO
compared to 5.35 to 10.73% when excluding it. Including David Dow and Pam Fahey, 3M, Maplewood, MN
100% boiling methanol data did not affect collaborative study Helga Hoffmann, Rdh Laborchemikalien Gmbh & Co.
results (see Table 2001.12C). The values obtained with the KG, HYDRANAL-Labor, Seelze, Germany
boiling methanol extraction were not detected as outliers for Dave Mertens and Diane Amundson, U.S. Dairy Forage
laboratory variance by Cochran’s test (4). Research Center, Madison, WI
Herschel Morris and Paul Coco, Louisiana Department of
Recommendations Agriculture and Forestry, Division of Agricultural Chemistry,
Baton Rouge, LA
On the basis of this study, the Study Directors recommend Tina Rittenhouse, Hershey Foods, Hershey, PA
that the described method for Determination of Water (Mois- Myriam Wohl, Brinkmann Instruments, Inc., Westbury, NY
ture) in Animal Feed, Grain, and Forage (Plant Tissue) by
Karl Fischer Titration method be adopted as Official First Ac- References
tion. Due to the high bias associated with the method modifi-
cation for the Orion Turbo 2 equipment, this modification (1) Van Erem, T., Thiex, N., Pohmer, J., Poffenbarger, M.,
should be further evaluated before being adopted as Official Smith, V., & Patel, E. (1998) J. AOAC Int. 81, 25–32
First Action. It is recommended that the alternative boiling
(2) Riedel-de Haën HYDRANAL®-Manual, available through
methanol extraction procedure be adopted Official First Ac- Allied Signal, HYDRANAL® Technical Center, Morristown, NJ
tion in addition to the original method extraction procedure.
(3) Scholz, E. (1984) Karl Fischer Titration: Determination of
Water-Chemical Laboratory Practice, Springer-Verlag, New
Acknowledgments York, NY
(4) Youden, W.J. & Steiner, E.H. (1975) Statistical Manual of
We thank Riedel-de Haen for providing Karl Fischer re- the AOAC, Association of Official Analytical Chemists,
agent, methanol, standard reference material, and certified Arlington, VA
THIEX & VAN EREM: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 2, 2002 327