CHAPTER

Introduction-General methods for

proximate and mineral analysis

Proximate analysis is defined by H. Bennett chemists use the word "crude" before these
in the Concise Chemical and Technical three terms.
Dictionary as the "determination of a group Since aB of these determinations are
of closely related components together, e.g. empirieal, conditions of analysis must be
total protein, fat." It conventionally includes precisely stated and foBowed. Results ob-
determinations of the amount of water, tained in ash and moisture determinations are
protein, fat (ether extract), ash and fiber, with governed primarily by the temperature and
nitrogen-free extract (sometimes termed Ni- the time of subjection to heat. Protein, fat
fext) being estimated by subtracting the sum and fiber figures do not represent single con-
of these five percentages from 100. In order stituents. Any errors made in these five
to emphasize the group nature of the per- determinations are arithmetically cumulative
centage of protein, fat and fiber, many in the figure obtained for nitrogen-free extract.

Water

All foods, no matter what the method of molecules, or adsorbed on the surface of
processing, contain more or less water or colloidal partic1es. These forms of water
moisture. It comprises from 60 to 95 percent require different degrees of heat to remove-
of all natural foods. In plant or animal tissues, some water remaining at temperatures up to
it may be said to exist in two general forms: charring. This means that the phrase "percent
"free water" or "bound water". Free or moisture" is meaningless unless the method of
absorbed water, the most prevalent form, is determination is stated.
readily liberated and may be determined by
most of the methods used for the determina-
Determination of Water
tion of moisture.
Bound water is combined or adsorbed A. Oven-Drying to Constant Weight
water. This may be present as water of While there are a few methods, notably the
crystallization in hydrates or as water even Kar! Fischer titration, that determine water
more firmly bound with protein or saccharide content by a stoichiometric reaction, most

1
F. L. Hart et al., Modern Food Analysis
© Springer-Verlag New York Inc. 1971
 2] Introduction-General methods for proximate and mineral analysis
methods commonly used involve the direct lid, redry 1 hour. Repeat process untilloss between
application of heat by drying the sam pie to successive dryings does not exceed 2-3 mg.
constant weight at specified temperatures and Calculate the loss of weight as moisture.
atmospheric press ures. During such pro-
cedures, hydrates may form through concen- NOTES ON OVEN MOISTURE DETERMINA-
tration rendering the water in such com- TIONS

pounds more difficult to remove by l. Products with high sugar content and meats
with high fat content should be dried in the
volatilization. Other methods involve distilla-
vacuum oven at a temperature not exceeding 70°.
tion, either directly or by azeotropic distilla-
Some sugars, e.g. levulose, decompose about 70°,
tion with an immiscible solvent. An apparatus liberating water.
house summarized the methods for moisture 2. Oven methods are not suitable for sub-
adopted by the AOAC in their 1950 (7th stances, e.g. spices, containing appreciable
Edition) Official Methods of Analysis of the amounts ofvolatile constituents other than water.
Association of Official Agricultural Chemists. 1 3. The removal ofwater from a sampIe requires
that the partial pressure ofwater in the vapor phase
Method 1-1. Moisture, Air Oven Method. be lower than that of moisture in the sampIe.
Hence some air movement is necessary. This is
DETERMINATION accomplished in an air oven by partially opening
Weigh to 1 mg accuracy 2 to 10 g, depending the vents and in vacuum ovens by a slow stream
on solids content, into a weighed metallic flat- of dried air.
bottomed dish equipped with a tight fitting cover, 4. The oven temperature varies at different sites
previously dried at 90°-100°. Loosen cover and on the oven plate, hence the precaution of placing
dry in an air oven, with vents open, for 2-3 hours the sampIe near the thermometer. The variation
at 98°-100°. Remove dish, cover and cool in a may be as much as 3° or even more in older types
desiccator; weigh so on after it reaches room of air or gravity convection ovens. The more
temperature. Return dish to oven, redry one hour modern ovens of this type are equipped with
and weigh. Repeat process until change in weight efficient thermostats and sensors and claim a
between successive dryings does not exceed 2 mg. variant within 1°.
The loss ofweight is calculated as percent moisture. 5. After drying, many substances are quite
hygroscopic; the cover should be placed tightly
Method 1-2. Moisture, Vacuum Oven Method. on the drying dish immediately upon opening the
oven, and the dish should be weighed as soon as
DETERMINA TION it reaches room temperature. This may require as
Weigh to 1 mg accuracy 2 to 10 g, depending long as an hour if a glass desiccator is used.
on solids content, into a weighed, metallic flat- Smith and Mitchell 2 have pointed out that
bottomed dish equipped with a tight-fitting cover, certain dried products are better desiccants than
previously dried at 98°-100°. Loosen cover and those used in the desiccator itself and will pick up
dry in a vacuum oven connected to a pump moisture even in a closed dish. Some desiccants
capable of maintaining a partial vacuum equiva- can be regenerated by following directions on the
lent to 100 mm or less of mercury in the oven label or in the catalog. The choice of desiccant is
chamber and equipped with a thermometer important. Concentrated sulfuric acid, anhydrous
extending into the oven chamber and near the copper sulfate, and granular calcium chloride,
sampIe dishes. Crack the stopcock of the vacuum which are commonly used, are relatively inefficient.
oven to admit a current of air (about two bubbles a Anhydrous calcium sulfate, anhydrous mag-
second) dried by passing through a sulfuric acid nesium perchlorate, anhydrous barium per-
drying tower. Dry 4 to 6 hours at 60°-70°. Stop chlorate, freshly ignited calcium oxide, anhydrous
the suction and carefully admit dried air into phosphorus pentoxide are all more efficient.
the chamber. Insert cover, remove the dish and Boiled, concentrated sulfuric acid (10th ed.
cool in a desiccator. Weigh soon after dish reaches AOAC 22.006) is one of the best, but its corrosive
room temperature. Return dish to oven, loosen properties add an element of danger.
 Water [3
6. The familiar browning reaction between the temperature of distillation by decom-
amino acids and reducing sugars liberates position of any constituents of the sam pie
moisture during drying, accelerated at higher will be included, and measured in the receiver
temperatures. Thus, foods with high protein and as water.
accompanying high reducing sugars must be dried
Xylene was first used as the solvent but it
with caution, preferably in a vacuum oven at 60°.
was found that its boiling point (137°-140°)
7. Forced draft ovens are occasionally used to
lessen the time required for drying to constant was high enough to decompose several
weight. The AOAC uses this as a screening normal constituents of foods and feeds.
method for moisture in cheese (10th ed. Method Fetzer and Kirst 5 compared toluene and
15.158), marine products (10th ed. Method benzene distillations of various food stuffs
18.006) and meat (10th ed. Method 23.003). and recommended use of benzene for he at-
8. The dried residue from the moisture deter- sensitive products.
mination may be used for fat (ether extract The AOAC and ASTA have adopted this
determination) and, if the original sampie were method for the determination of moisture in
2 g, for the crude fiber determination. spices, using toluene (ASTA uses benzene for
spices high in sugars). In spite of its !imitations,
B. Drying in a Desiccator at Room the method has several advantages, particu-
Temperature larly if good judgment is used in selection of
Drying in a vacuum desiccator over the solvent: (I) A constant temperature, that
sulfuric acid at ambient temperature has been of the boiling point of the solvent, is
used at times, particularly on heat-Iabile maintained. (2) The rate of distillation can be
materials. It is quite time consuming. followed visually; when the upper layer of
Windham 3 found that a one week drying solvent in the receiver is cIear the distillation
period was necessary for meats and that the is complete. (3) It is faster than most drying
method did not always give reproducible techniques. (4) No complicated apparatus is
results. The AOAC recommends it for deter- required.
mination of moisture in plant materials and
in grain and stock feeds. Method 1-4. Moisture: Solvent Distillation
(Adapted from official ASTA Method 2).
Method 1-3. Moisture: Vacuum Desiccator
Method (AOAC Method 22.006-22.007). ApPARATUS
AII-glass distilling apparatus.
This method, drying over H2S04 without heat a. Short-necked flask or Erlenmeyer flask, 250
for 24 hours or more, is seldomly used. See or 500 ml, with standard taper joints 24/40.
AOAC method for details. b. Bidwell-Sterling trap, with standard taper
joint 24/40 or its equivalent.
C. Distillation with an Immiscible Solvent c. West condenser, 400 mm long, with stan-
The most frequently used distillation dard taper joint 24/40. Hot plate equipped with a
method (the Bidwell-Sterling method)4 mea- magnetic stirrer and teflon-coated stirring bar.
sures the volume of water released from the
DETERMINATION
sam pie by continuous distillation with an
Transfer 10 to 40 g of sampie (depending on
immiscible solvent. The water is received into
amount of moisture expected and the capacity in
a specially designed receiver which allows it
ml of the receiving trap) into the distilling flask.
to settle into a graduated section where it will Add sufficient toluene, about 75-100 ml, to com-
be measured, while the solvent overflows into pletely cover the sampIe. Insert the magnetic
the distilling flask. It has one disadvantage stirrer, and connect the receiving trap and
common to all moisture methods involving condenser. Fill the trap with toluene by pouring
heat treatment in that any water formed at it through the top of the condenser. Place the
 4] Introduction-General methods for proximate and mineral analysis

assembled apparatus on an electric hot plate, and

slowly bring the toluene to a boil. Distill at the D. Chemical Methods
rate of 1 or 2 drops per second until most of the The only chemical method commonly used
water passes into the trap, then increase distillation for foods is that which employs the Kar!
rate to about 4 drops per second. Fischer reagent. This reagent (discovered in
When all moisture is apparently over, as shown
1936 by Karl Fischer 7) is composed of
by a layer of c1ear toluene in the upper section of
iodine, sulfur dioxide, pyridine and a solvent
the trap, wash down the condenser by pouring
toluene in the top. Dislodge any water droplets in that may be methanol or methyl cellosolve.
the condenser tube by brushing with a long The reactions involved are:
handled nylon burette brush or a copper wire
spiral. Rinse the brush or spiral with a few ml of a. CsHsN.I2 +CsHsN.S02 +CsHsN +H20-+
toluene poured through the condenser. Continue 2CsHsN.HI + CsHsN.S03.
the distillation about five minutes longer, cool the
trap to room temperature, and read the volume of
water, estimating to nearest 0.01 ml.
Normally an excess of sulfur dioxide,
N OTES ON SOLVENT DISTILLA TION PRO- pyridine and methanol (or other hydroxyl-
CEDURE containing compound) is used so the effective
1. Other solvents recommended by various strength of the reagent is established by the
workers include: iodine concentration. The most widely used
Carbon Tetrachloride B.P. 77° reagent is a methanolic solution containing
Benzene B.P.80° the other components in the ratio:
Methyl Cyc10hexane B.P. 100°
12 : 3 S02 : 10 CsHsN.
Toluene B.P. 111 °
Tetrachloroethylene B.P.121° Because the Kar! Fischer reagent is in
Xylene B.P. 137°-140° effect a powerful desiccant, the sampIe and
2. The American Spiee Trade Association, in reagent must be protected from atmospheric
their Official Analytical Methods, recommends moisture in all procedures. Both visual and
the use of benzene instead of toluene for spices electrometric titration procedures are possible
.such as red peppers (this inc1udes cayenne, chili and in the simplest form of the method the
pepper, chili powder, and paprika), dehydrated reagent acts as its own indicator. The sampIe
on ion and dehydrated garlic, which contain large
solution remains canary yellow as long as
proportions of sugars and other materials which
water is present, changing to chromate
may decompose, liberating water at the tempera-
ture of boiling toluene. For example, levulose yellow and finally to the brown color of
begins to decompose at about 70°.6 unused iodine at the end point. The visual
3. The entire apparatus should be c1eaned each titration is, however, less precise than pro-
time before use with sulfuric acid-dichromate cedures employing electrometric means of
solution, rinsed thoroughly with distilled water, determining the end point. For this reason
then with alcohol, and dried. most Kar! Fischer determinations are now
4. The receiving trap should be calibrated by made using various forms of market appara-
distilling successive accurately measured quanti- tus based on electrometric principles. Most
ties ofwater into the calibrated portion ofthe trap of these instruments use the "dead stop"
with toluene. Readings should be estimated to the
technique 8 employing platinum electrodes.
nearest 0.01 milliliter.
In the simplest form, (Fig. 1-1), apparatus
5. Various modifications of the original receiv-
ing trap are available. Choice depends on the requirements are a battery, variable resistor,
expected volume ofwater to be distilled, degree of galvanometer or microammeter and platinum
calibration required, ease of flow-back and other electrodes. A potential is applied across the
factors. electrodes to just balance the system, that is,
 Water [5

to the point where the galvanometer is not cathode polarized. At the end point, the
deflected. During titration, as long as water sm all excess of iodine depolarizes the
is present, the anode is depolarized and the cathode, resulting in a surge of current.

A B

Electrode
leads

s
Read

Fig. 1-1. Electrical circuit for direct dead-stop end point: C, dry cell, 1.5 v; G, galvanometer (Leeds &
Northrup No. 2330-C) or microammeter; RI, resistor, wire-wound, 9000 ohms; R2, resistor, wire-wound, 1500
ohms; R3, potentiometer, 1000 ohms; R4 ,resistor, 400 ohms, variable; S, switch, single pole, double throw.

It is obvious that, like any titration method, formamide was substituted for methanol as
the Kar! Fischer method will not work unless the extracting medium, all of the moisture
there is contact between the reagent and the was removed from ground dehydrated pota-
ingredient being titrated-in this case, water. toes, sweet potatoes, carrots and peas in 15
If the food is one that is alm ost completely minutes.
soluble in anhydrous methanol or methyl
In spite of the fact that the Kar! Fischer
cellosolve there is no problem. Most solid
method is widely used for the rapid deter-
foods, however, are only very partially soluble
mination of moisture in foods and many other
in these solvents and in such cases techniques
such as Soxhlet extraction with methanol materials, few collaborative comparisons with
followed by titration of the extract have been results by oven drying have been made. For
resorted to. Some foods do not yield all of this reason and because this chapter is a
their moisture rapidly under such extraction general one, only the following two modifica-
procedures, and Johnson 9 has shown that for tions of the method are described:
sweet and white potatoes soaking for 4 to 6
hours in methanol is required to give results Method 1-5. Moisture in Molasses, Kar! Fischer
comparable to those of vacuum oven drying. Reagent. 11 (This is an AOAC first action official
McComb and McCready 1 0 found that if method).
 6] Introduction-General methods for proximate and mineral analysis
ApPARATUS sam pies. If time lapse occurs between titration of
a. Buret with automatie zero reservoir for sampies, adjust liquid in titration vessel to end
reagent. point by titration with reagent before adding next
b. Magnetic stirring device. sampie.
c. Titration vessel (300 ml Berzelius beaker with
stopcock attached to side at bottom for with- Method 1-6. Moisture in Dehydrated Vegetabfes,
drawing excess solution is recommended. Karf Fischer Reagent. 12
d. Electrodes.
e. Circuitry for deadstop end point detection. ApPARATUS. See Method 1-5.
All openings must be tightly cIosed or protected
with drying tubes to prevent contamination from REAGENTS
atmospheric H20. Various titration assemblies a. Formamide-Practical grade.
may be obtained from laboratory supply houses, b. Karf Fischer Reagent-Obtain ready made
or may be assembled. from a laboratory supply house or prepare as
Assemble titration apparatus and follow manu- folIows: Dissolve 84.7 g resublimed 12 in 269 ml
facturer's instructions; set for direct titration. Set reagent grade pyridine «0.1 % H 20) in I-liter
timer for 30 second end point. Add enough dry Pyrex glass-stoppered bottle. Add 667 ml of dried
methanol to cover electrodes on electrode probes absolute methanol (preferably <0.05% H20).
and turn on stirrer. Adjust speed to obtain good To this add 64 g of sulfur dioxide gas. To avoid
stirring without splashing. Do not let stirrer bar appreciable heating add the S02 slowly at rate
contact electrodes. Titrate until satisfactory end of about 40 g/hour. Stopper solution tightly and
point is reached. Newly assembled or not recently store 2-3 days be fore use.
used apparatus may require repetition of this Some commercial methanol is suitably dry for
step to dry out system. use in the reagent. If it is found necessary to dry
the available supply, add 5 g of Mg turnings to
REAGENTS each liter and after the initial vigorous reaction
a. Karl Fischer Reagent-Available from lab- subsides, distill off the alcohol. Take care to keep
oratory supply houses or prepare as folIows: the distillation system free from contamination by
Dissolve 133 g h in 425 ml dry pyridine in dry atmospheric moisture.
glass-stoppered bottle. Add 425 ml dry methanol
or ethylene glycol monomethyl ether. Cool to 4° DETERMIN AnON
in ice bath and bubble in 102-105 g S02. Mix weIl Determine the water equivalent of the Kar!
and let stand 12 hours. (There is less trouble with Fischer reagent and the blank titer of the fOfma-
stopcock leakage when ethylene glycol mono- mide daily, or each time aseries of determinations
methyl ether is used.) The reagent is reasonably is made, because parasitic reactions decrease the
stable, but restandardize for each series of deter- reagent's effective strength. To determine the
minations. blank, titrate 10 ml of formamide in a 250 ml
b. Anhydrous Methanol - Reagent grade Erlenmeyer fiasko To the same fiask add by means
CH30H containing <0.1 % H20. Prepare by of a weight buret 70-100 mg of water and titrate.
distilling over Mg. Calculate the water equivalent of the reagent-
mg H20/ml reagent. (H is advisable to average at
DETERMIN AnON least three blanks and water equivalent values
Add about 120 mg H20 from weighing pipet or for each standardization. The net titer of the
other suitable device and titrate with Kar! fiasks should check within 0.1 ml and the range of
Fischer reagent. the water equivalent values should not exceed 6
Calculate C = mg H20/ml reagent. parts per thousand). Carry out all titrations drop
For titration of molasses, C = about 5 mg/mI. by drop near the end point until an end point
Weigh quantity of molasses estimated to give constant for 30 seconds is obtained. Grind the
20-40 ml titer into titration apparatus and titrate. sam pies of dehydrated vegetables to pass a 40
Percent H20 = (C x ml reagent)/(g sampie x 10). mesh sieve and store in small tightly sealed con-
Drain excess liquid and repeat with succeeding tainers. Weigh a sampie of approximately 500 mg
 Water [7

of the ground material into a dry, glass-stoppered Brown-Duvel Moisture Tester devised in 1907
250 ml Erlenmeyer ftask which contains astirrer. by V.S. Department of Agriculture chemists
Add 10 ml of formamide with slight agitation to for the determination of moisture in grains.
disperse the sampie and prevent dumping. Heat Water in the sampIe is distilled along with a
40 seconds on a hot plate held at ISO ± 10°. hydrocarbon oil and the volume of water
Release the stopper slightly and gently rotate the
distilled is measured.
ftask during the heating; cool to room temperature
and titrate. (Because of the viscous nature of some The Brabender Moisture Tester is a
ofthe sample-formamide mixtures, it is sometimes combination drying oven, desiccator, and
expedient to rotate or agitate the flask slightly analytical balance. The Ohaus Moisture
during the titration in addition to using the Balance is similarly designed. The percentage
magnetic stirrer.) of moisture is read directly on a scale.
Calculate the percent water as folIows: Another instrument, the Speedy Moisture
Tester, depends on the reaction of calcium
%H20 = carbide with the water of the sampIe. The
100 (sampie titer-blank titer) (H20 equivalent) pressure of the evolved acetylene is read on a
(sampie weight in milligrams) dial directly calibrated in percent of moisture.
There are other instruments on the market
NOTES
wh ich measure the conductivity ofthe sampIe.
1. Besides methanol and formamide listed
One is the Tag-Heppenstahl Moisture Tester
above, pyridine, dioxane and dirnethyl formamide
have been employed as sampie solvents. used for the determination of moisture in
2. Direct titration usually gives total water, grains. Others, such as the Tag Dielectric
that is, free plus hydrated water. When a suitable instrument, measure the dielectric properties
water-miscible liquid can be found in which the of the sampie. All of these are speedy and
sampie is insoluble, free water can be determined moderately precise if instructions are followed
by extraction with this liquid and titration of the exactly. Many of them must be calibrated
extract. for each particular food product tested. They
3. The method is obviously not applicable are thus suitable for routine in-process or
without modification to materials containing sub- finished product testing of a particular food.
stances that react with iodine. For other inter- During recent years, instruments have been
ferences refer to Selected Reference Q.
devised using nuclear magnetic resonance
4. Instead ofweighing out portions ofwater for
standardization of the Kar! Fischer reagent, finely principles, radio frequency power absorption,
ground sodium tartrate dihydrate (1 - 1.5 g) microwaves as sensors, and other properties
dispersed in 50 ml of pretitrated methanol may be of matter. The use of all of these specialized
used as a standard. instruments is considered to be beyond the
5. Some dehydrated plant material, e.g. spices, scope of this text. Their manufacturers issue
contain active aldehydes and ketones that react detailed instructions for these instruments.
with the methanol in Kar! Fischer reagent to form In 1966 the AOAC adopted a near-infrared
water. Rader l2 has proposed a method to the spectrophotometric method for moisture in
AOAC that substitutes methyl cellosolve (ethy- dehydrated vegetables and spices. See Method
lene glycol monomethyl ether) for methanol in 17-10.
the Kar! Fischer reagent and formamide as the
sampie solvent.
Reference Method for
E. Special Instrumentation Methods Moisture Determination
Many specialized instruments have been Makower 13 of the USDA Western
developed for the direct and rapid determina- Regional Research Laboratory has pointed
tion of moisture. One of the earliest was the out the need for a "reference method" for the
8] Introduction-General methods for proximate and mineral analysis

determination of moisture. It would be a Iyophilization apparatus and dried in vaCIIO

means of comparing or calibrating other while still in the frozen state to a moisture
methods that are more or less empirical but are level of 2 to 3 %. The Iyophilized sam pIe is
used extensively because of their convenience. dried in a vacuum oven at 60° or 70° and a
He and his co-workers have developed a pressure of 0.05 to 0.15 mm of mercury, or
procedure based on Iyophilization. Briefly at room temperature in an evacuated desic-
stated, the ground sam pIe along with an cator over an efficient desiccant such as
eight-to-ten-fold addition of water is held magnesium perchlorate. The original papers
over night at about 5° and then frozen by should be consulted for a description of the
immersion in asolid carbon dioxide-ethanol lyophilization apparatus and the details of
slurry. The frozen mixt ure is transferred to a the method.

Nitrogen
Nitrogen, expressed either as total nitrogen catalyst. Later he suggested addition of ben-
or "protein" (N x 6.25), is almost always zoic acid and sugar to digest aromatic
determined by a form of wet combustion in substances more difficult to analyze, and
which the nitrogen present is converted to advocated the combined use of copper and
ammonium sulfate and finally to ammonia. mercury. It might be noted here that A. E.
The ammonia formed is distilled and titrated Paul and E. H. Berry showed that the use of
with a standard acid solution. This method, the two catalysts gave no faster digestion
originally devised by J. Kjeldahl in 1883 14 , has than did mercury alone. Arnold's main con-
been modified by many investigators. The tribution appears to be the accumulation of
modifications that have become most widely much analytical data on the method and use
accepted are in what is now known as the of improved apparatus, particularly the trap
Kjeldahl-Gunning-Arnold (KGA) Method. bulb. Vickery maintains, with considerable
H. B. Vickery has written an interesting justification, that credit is due to Wilfarth for
historical review of this method, which is his fundamental contribution of a metallic
briefly abstracted here (Selected Reference catalyst, and that the method known since
DD). Kjeldahl originally digested the sam pIe 1912 as the KGA method should be called
in fuming sulfuric acid fortified with phos- the Kjeldahl-Wilfarth-Gunning method. In
phorus pentoxide; then potassium perman- 1955, a joint Association of Official Agri-
ganate was added to complete the oxidation cultural Chemists-American Oil Chemists'
to ammonium sulfate. He diluted the oxidized Society Committee 15 recommended that use
mixture, added excess of sodium hydroxide of copper as a catalyst in official methods be
and zinc granules and distilled the ammonia dropped. This was approved by the AOAC,
thus formed. and the official method given in Methods of
In 1885, H. Wilfarth used a metallic Analysis, 10th ed. (I965) was revised accord-
catalyst to shorten the time of oxidation ingly. This is given as Method 1-7 in this text:
(mercury or copper as oxides were found to
be most effective). A few years later, J. W. Method 1-7. Total Nitrogen in Nitrate-Free
Gunning suggested the use of potassium Products.
sulfate to hasten the removal of water in
order to increase the rate of digestion. About REAGENTS
this same time, C. Arnold confirmed a. Sulfuric acid, 93-98 % H2S04, N-free.
KjeldahI's and Wilfarth's techniques and b. Mercuric oxide, HgO, or metallic mercury,
recommended mercury as the most efficient reagent grade, N-free.
 Nitrogen [9

c. Potassium sulfate, powdered or anhydrous gently until frothing ceases. (If necessary, add a
sodium sulfate reagent grade, N-free. small amount of paraffin to reduce frothing.) Boil
d. Sulfide or thioslilfate solution, Dissolve 40 g briskly until solution c1ears and then for at least
commercial potassium sulfide, K2S, in I L water 30 minutes longer (2 hours for sampies containing
(a solution of 40 g sodium sulfide, Na2S, or 80 g organic material).
sodium thiosulfate, Na2S203.5H20, in I L water Cool, add about 200 ml water, cool below 25°,
may be used instead). add 25 ml of the sulfide or thiosulfate solution,
e. Sodium hydroxide pellets or solution, N- and mix to precipitate mercury. Add a few zinc
free; for solution dissolve about 450 g solid granules to prevent bumping, tilt fiask, and gently
NaOH in water and dilute to I L (sp g should be add along the side of the neck, without agitation,
1.36 or higher). 25 g sodium hydroxide pellets, or equivalent water
f. Zine granules, reagent grade. solution, to make contents strongly alkaline. (lf
g. Methyl red indicator, Dissolve 1 g methyl desired, the sulfide or thiosulfate solution may be
red in 200 ml a1coho\. mixed with the sodium hydroxide solution before
h. Standard 0.5 N or 0.1 N hydrochloric or addition to the fiask.) Immediately connect the
sulfllrie acid. (Use 0.1 N standard acid solution fiask to the distilling bulb on the container. Place
when amount of N is smal\.) under the condenser a 500 ml Erlenmeyer fiask
i. Standard 0.5 N or 0.1 N sodill/1/ hydroxide containing 25 to 50 ml of the standard acid solu-
solution. tion, reagent h, accurately measured by a pipette
or burette. The tip of the condenser should extend
NOTE: Reagents hand i should each be standard- beneath the surface of the acid in the Erlenmeyer
ized against a primary standard, and then checked fiasko
one against the other. A blank determination Ignite the burner under the distilling fiask and
should be made on all reagents by substituting rotate fiask to mix contents thoroughly. Heat until
2 g of sucrose for the weighed sampie, and pro- all the ammonia has been distilled (at least 150 ml
ceeding according to Method 1-7. of distillate). After distillation has been com-
pleted, lower the receiving fiask until the con-
ApPARATUS denser tip is above the liquid in the fiask and wash
a. 500 mlor 800 ml Kjeldahl flask. off the condenser tip with distilled water. Titrate
b. Kjeldahl distilling bulb. the excess standard acid in distillate with the
c. Straight-tube condenser. standard alkali solution, reagent i, using methyl
d. Heater (gas or electric) for digestion, ad- red as indicator. Correct the titration for the
justed to bring 250 ml water at 25° to a rolling blank determination on reagents used and ca1cu-
boil in about 5 minutes if electric. To test heaters, late percent of nitrogen, as N, in the sampie. If
preheat 10 minutes if gas, or 30 minutes if electric. the percent of crude protein is desired, multiply
Add 3 or 4 boiling chips to prevent superheating. the percent of N by 6.25. This is the factor used
The Kjeldahl digestion and distillation racks conventionally for all proteins. Occasionally other
iIIustrated in most apparatus supply catalogs are factors are used: 5.7 for wheat, 6.38 for milk,
suitable and convenient for the purpose. These 5.55 for gelatin, 5.95 for rice, 5.77 for soybeans.
may be obtained for either gas or electricity as a
source of heat.
Total Nitrogen in Sampies
DETERMINATION Containing Nitrates
Place weighed sampie (0.7-2.2 g, depending
The unmodified Kjeldahl digestion of
upon amount of N) in a Kjeldahl digestion fiasko
Method 1-7 cannot be relied on to convert
Add 0.7 g of mercuric oxide or 0.65 g metallic
mercury, 15 g of powdered potassium sulfate or a11 of the nitrate nitrogen in a sam pie to
anhydrous sodium sulfate, and 25 ml sulfuric acid. ammonium sulphate, so this method is not
(If larger sampie than 2.2 g is used, increase satisfactory for determining total nitrogen in
amount of sulfuric acid 10 ml for each gram of sam pies containing nitrates. Because Method
sampie.) Place fiask in inc1ined position and heat 1-7 does transform an uncertain proportion
10] Introduction-General methods for proximate and mineral analysis

of the nitrate to ammonium salts, it must be Method 1-8. Total Nitrogen in Sampies Contain-
emphasized on the other hand that it is not a ing Nitrates and/or Difficultly-Digestible Com-
measure of the non-nitrate nitrogen in pounds.
mixtures containing nitrate.
REAGENTS
Since nearly all human foods are essentially
a. Chromium metal powder, JOO mesh-Fisher
nitrate-free, Method 1-7 can be used with
C-318 or Sargent SC-11432.
little chance of error to determine total b. Norton alundum 14x.
nitrogen in foods. However, cured meats c. Silicone anti/oam-General Electric No. 66
contain small proportions of nitrate (and or Dow Corning Antifoam Q is suitable.
nitrite), and tobacco (if this can be classified d. Sodium thiosul/ate or potassium sulfide
as a food) is high in nitrate. When it is solution-Respectively 200 g NaZSZ03.5HzO or
necessary to take nitrate nitrogen into con- 100 g KzS per liter.
sideration in determining total nitrogen, there
are several modifications of the Kjeldahl DETERMINATION
Transfer a sampie containing not more than
method that may be used. The best known of
80 mg of nitrate N to a 500 ml Kjeldahl flask; add
these are those employing salicylic acid to fix
1.2 g of Cr powder (a) and 35 ml of water, and let
the nitrate, and sodium thiosulfate or zinc
stand 10 minutes with occasional swirling. Then
dust to reduce it l6 • However, in sampies add 7 ml of HCI and two drops of silicone anti-
containing high proportions of chloride (ratio foam (c) and let stand until visible reaction takes
of chloride ion to nitrate ion I: 3 or greater), place. Then place on an electric burner adjusted to
these methods yield low results because the cause boiling in 7-7.5 minutes, and let stay there
nitric and hydrochloric acids in the digestion until the contents of the flask come to a roIIing
mixture react to form nitrosyl chloride boi I. Remove from the heat and aIIow to cool.
(NOCI), wh ich is volatilized and lost. Several Add 22 g of K ZS04, 1.0 g of HgO, 25 ml of
methods have been devised to eliminate this HZS04 and 1.5 g of the alundum (b). (If the sampie
contains considerable organic matter add 0.5 ml
error, among wh ich are those employing
of silicone antifoam.) Place on burner regulated to
reduced iron 17 and a special Raney nickel
cause boiling in 5.0-5.5 minutes, aIIow about 15-
alloy (containing 40% Ni, 10% Co and 50% 20 minutes for the copious white fumes to cIear
AI)18 as reducing agents. Both of these out of the bulb of the flask, swirl gently, and
methods have yielded excellent results in our digest an additional 30 minutes. If material con-
laboratory, but the reduced iron method is taining refractory nitrogen is present, continue the
slightly shorter. digestion for a total of 60 minutes after the copious
However, a chromium reduction method white fumes cIear out of the bulb of the flask.
recently introduced by Gehrke et al. '9 is Remove from the heat and cool.
claimed not only to be even shorter than the Add 20 ml of the NaZSZ03.5HzO or KzS
iron method but to be a truly universal solution (d) and complete the analysis as directed
in the second paragraph of Method 1-7.
method in that it gives accurate results with
sampies containing high chloride/nitrate ratios
NOTE: Instead of employing a separate solution
and with difficultly digestible materials like
of NaZSZ03.5HzO or KzS, 200 g of NaZSZ03.
nicotinic acid and other pyridine derivatives 5HzO or 100 g of KzS may be added to the 450 g
as weil as with the more tractable substances of NaOH in preparing the sodium hydroxide
that can be handled satisfactorily by Method solution, Method 1-7, (e). This eliminates the
1-7. The method is: addition of one solution to the digestion mixture.
 Ash [11

Ash

Mineral elements are present in foods as temperature, and ashing time for over 40
both organic and inorganic compounds. Their classes of food products, and proposed
exact composition as they exist in the food is "general directions" for the determination of
often difficult to determine. Ashing the food total ash. The method given below is that
to destroy all organic matter changes its proposed by Klemm (Selected Reference FF):
nature; metallic salts of organic acids are con-
Method 1-9. Total Ash, General Method.
verted to oxides or carbonates or may react
during ashing to form phosphates, sulfates or Accurately weigh an amount equivalent to about
halides. Some elements, such as sulfur and the 2-5 g of solids from the weIl-mixed sampie (or
halogens, may not remain completely in the measure by means of a pipet, if a free-flowing
ash, but may be volatilized. liquid) into a tared, shaIlow, rather wide, flat-
The determination of total ash in foods is bottomed ashing dish that has been previously
empirical, as are the other determinations dis- ignited. Use a Pt dish unless otherwise directed.
cussed in this chapter. It is therefore essential If considerable moisture is present, dry on a
steam bath to apparent dryness, then ignite to
that the detailed instructions in a method of
constant weight in an electric muffle equipped
analysis be followed exact1y, and that the
with an indicating pyrometer and a thermostatic
pertinent factors, such as time, temperature and
heat contro!. The control should be set at the
method of ashing, be recorded by the analyst. temperature recommended in the method given
Ash determinations were originally made for the food product concerned. If no temperature
over an alcohol burner, agas burner, or a gas- is recommended, set the control at 525°-550°.
fired muffle. Directions varied from "low red Ignite to a white or gray ash, transfer directly to a
heat" on up. The advent of the electric muffle desiccator (single desiccator for each dish is pre-
with indicating pyrometer and thermostatic- ferable), cool to room temperature and weigh
ally controlled heat made control of ashing immediately. Caleulate as percent ash.
temperature possible for the first time. NOTE: Bring the muffle to the stated temperature
Wichmann, in his classic series of papers on slowly, without flamingo Too active combustion
ashing technique, made decomposition curves maycause lossofash,orthe ashmay fuseandcause
of the ash of typical agricultural products by encIosed carbon to escape ignition. Take care to
plotting weight loss against temperatures. avoid loss oflight, fluffy ash. Cover the dish with a
(Selected Reference HH). Potassium com- small watch glass even while in the desieeator.
pounds predominate in plant ash, and sodium If a carbon-free ash cannot be obtained by
following this procedure, remove the dish from
compounds in animal ash. Potassium car-
the muffle, cool and leaeh the mass with hot water.
bonate volatilizes appreciably at 700° and
Filter through an ashless paper, evaporate the
alm ost entirely at 900°. Sodium carbonate is filtrate to dryness and dry the residue and paper at
unchanged at 700°, but suffers considerable 150°-200°. Then ignite at the stated temperature
loss at 900°. Reactions between carbonates to a white or gray ash. If a small amount of carbon
and phosphates also occur (HH). still remains, moisten the ash in the dish with
Klemm, in his survey of approximately 50 several drops of water, dry at 150°-200° and re-
ashingprocedures listed in Methods 0/ Analysis, ignite.
AOAC, 9th ed. (1960), remarked that it is
Method 1-10. Acid-Insoluble Ash.
virtually impossible to incorporate every
applicable factor governing ashing of different This determination is of value in detecting such
types offood into one general method. He has adulterants as sand or soil in spices, tale in con-
devised a table listing sam pie size, ashing fectionery, ete.
12] Introduction-General methods for proximate and mineral analysis

DETERMINATION NOTES ON ASH DETERMINATION

To the ashed sampie obtained from Method 1-9, 1. Sinee temperatures may vary in different
add 25-30 ml of diluted hydrochloric acid (1 + 2.5 parts of the muffle, the sampie dishes should be
by volume). Cover the platinum dish with a watch plaeed as e10se to the pyrometer terminals as
glass to prevent spattering and boi I for 5 minutes. possible. Many analysts prefer to plaee the dishes
Filter through an ashless filter paper and wash on a siliea plate elevated 1 to 2 em above the
with hot water until washings are acid free. muffle floor on 4 small poreelain crueibles. This
Transfer the filter paper and contents to the origi- pro vi des a more even temperature distribution.
nal dish, dry and ignite at a temperature of 625- 2. See Note 5 in Notes on Oven-drying Methods,
650 to a white ash. Cool in a desieeator and
0
moisture, for preeautions on choice and use of
weigh. desiceants.

Crude fiber
The term "erude fiber" is a measure of the b. Sodium hydroxide solution-O.313 N. 1.25 g
material in a food of vegetable origin that has ofNaOH/loo ml,free or, nearly sO,from Na2C03.
no appreciable food value other than rough- (Concentrations of these solutions must be
age. It consists largely of cellulose, lignin and checked by titration.)
c. Prepared asbestos-Spread a thin layer of
pentosans, which comprise the cellular struc-
aeid-washed, medium or 10ng fiber asbestos in an
ture of the plant along with small amounts of
evaporating dish and heat 16 hours at 600° in a
nitrogenous matter. By definition of a com- muffle. Boil 30 minutes with 1.25 % H2S04, filter,
bined liaison committee of the AOCS and wash thoroughly with water, and boil 30 minutes
the AOAC 20 , "crude fiber is loss on ignition with 1.25 % NaOH. Filter, wash onee with 1.25 %
of dried residue remaining after digestion of H2S04, wash thoroughly with water, dry and
sampIe with 1.25 %H 2 S0 4 and 1.25 %NaOH ignite 2 hours at 600°.
solutions under specific conditions." The Determine the blank by treating 1.0 g of pre-
principle of the method of determination has pared asbest os with acid and alkali as in Deter-
suffered no essential change since its introduc- mination. Correet crude fiber results for any blank,
tion by German agricultural chemists in whieh should be negligible (about 1 mg). Asbestos
reeovered from the determination may be used in
1864 21 •
subsequent deterrninations.
The AOCS-AOAC Crude Fiber Liaison
d. Alcohol-Methanol, isopropyl alcohol, or
Committee of 15 members was established in 95 % ethyl alcohol.
1958. After 4 years of intensive study, the e. Anti/oam-Dow Corning Antifoam A Com-
Committee recommended a completely revised pound diluted I + 4 with mineral spirits or
method based on the original acid-alkali petroleum ether, or Antifoam A Emulsion diluted
digestion but defining more precisely the 1 + 4 with water.
equipment and reagents used and the con- f. Bumping chips or granules-Broken Alundum
ditions of the test. They also devised new erueibles or equivalent. Granules of RR Alundum
fiItering equipment since filtering seemed to 90 Mesh, manufactured by Norton Co., Wor-
be the greatest potential source of error. cester, Mass., are satisfaetory.
Method 1-11 is the method recommended ApPARATUS
by the combined liaison committee and a. Digestion apparatus-With a condenser to
adopted by the two societies: fit a 600 ml beaker and a temperature-adjustable
hot plate that will bring 200 ml of water at 25°
Method 1-11. Crude Fiber.
to a rolling boil in 15 ± 2 minutes (may be
REAGENTS obtained from Laboratory Construction Co.,
a. Suljuric acid solution-O.255 N. 1.25 g of 8811 Prospect Avenue, Kansas City, Mo.).
H2S04!100 ml. b. Ashing dishes-Silica (Vitreosil) 70 x 15 mm;
 Crude fiber [13

or porcelain, Coors No. 450, size 1; or the equiva- with an aspirator or some other source of vacuum
lent. with a valve to break the vacuum.
c. Desiccator-With efficient desiccant such as f. Liquid preheater-(See Figure 1-4.) A sheet
4-8 mesh Drierite. (Calcium chloride is not copper tank with 3 coils of inCu tubing 12.5 ft
satisfactory.) long. Solder inlets and outIets where the tubing
d. Filtering device-With No. 200 Type 304 or passes through the tank walls. Connect to a
316 stainless steel screen (W. S. Taylor Co., 3165 reflux condenser and fill with water. Keep the
Superior Ave., Cleveland, Ohio), easily washed or water boiling with two 750 watt thermostatically
digested residue. Either the Oklahoma State filter controlled hot plates. Use Tygon for inlet leads
screen (see Figure 1-2; available from Laboratory to reservoirs of water, acid and alkali; use gum
rubber outlet tubing. The capacity of the pre-
heater is adequate for 60 analyses in 8 hours.

PREPARATION OF SAMPLE
Reduce the sampIe (a riffle is suitable) to 100 g
and place a portion in a sealed container for the
moisture determination. Determine moisture
immediately. Grind the remainder to uniform
fineness (a Webber mill with a 0.033-0.040"
screen, a Mikro mill with a 1/25-1/16" screen, and
I' 2f .[ a Wiley mill with a 1 mm screen, give comparable
fineness.) Since most materials lose moisture
Ir--------------71 during grinding, determine moisture on the

A--.---------------
B-I I I I I I I I I
ground sampIe at the same time a sampIe is taken
for the crude fiber determination.

DETERMINATION
Extract 2 g of the ground material with ether or
petroleum ether (Method 1-13). If the fat content
r-, is less than 1 %, extraction may be omitted.
I I
I
I
I
I
Transfer the residue to a 600 ml beaker, avoiding
I
I
I
I fiber contamination from paper or a brush. Add
about 1 g of prepared asbestos, 200 ml of boiling
1.25 %H2S04, and one drop of diluted antifoam.
(An excess of antifoam may give high results; use
only if necessary to control foaming.) Bumping
chips or granules mayaiso be added. Place the
beaker on the digestion apparatus with pre-
Fig. 1-2. Oklahoma State Filter Screen. adjusted hot plate, and boil exactly 30 minutes,
rotating the beaker periodically to keep the solids
Construction Co.) or California modified poly- from adhering to the sides. Remove the beaker
ethylene Büchner funnel (see Figure 1-3; consists and filter as in a or b.
of two-piece polyethylene funnel manufactured a. Using the Oklahoma filter screen-Turn on
by Nalge Co., Inc., 75 Panorama Creek Drive, the suction and insert the screen (precoated with
Rochester, New York, as their Catalogue No. asbestos if extremely fine materials are being
J1060, item 4280, 70 mm, without No. 200 screen, analyzed) into the beaker, keeping the face of the
or an equivalent. Seal the screen to the filtering screen just under the surface of the liquid until all
surface of the funnel, using a small-tip soldering liquid is removed. Without breaking the suction
iron.) or raising the filter add 50-75 ml of boiling water.
e. Suctionfilter-To accommodate the filtering After this wash is removed, repeat with three 50
devices. Attach a suction flask to a trap in line ml washings. Work rapidly to keep the mat from
14] Introduction-General methods for proximate and mineral analysis

Büchner funnel (precoated with asbestos if ex-

tremely fine materials are being analyzed), rinse
the beaker with 50-75 ml ofboiling water and wash
through the Büchner funnel. Repeat with three
50 ml portions ofwater and suck dry. Remove the
mat and residue by snapping the bottom of the
Büchner funnel against the top of the beaker
while covering the stern with the thumb or fore-
finger and replace in the beaker. Add 200 ml of
boiling 1.25 %NaOH and boil exactly 30 minutes.
Remove the beaker and filter as above. Wash with
25 ml of boiling 1.25 % H2S04, three 50 ml
portions ofwater and 25 ml of alcohol. Remove the
mat and residue, and transfer to an ashing dish.
Dry the mat and residue 2 hours at 130 ± 2°.
Cool in a desiccator and weigh. Ignite 30 minutes
at 600 ± 15°. Cool in the desiccator and reweigh.
I"
~------38------~'1
Percent crude fiber in ground sampIe = C = (Loss
in wt on ignition -Ioss in wt of asbestos blank x
loojwt sampIe. Percent crude fiber on desired
moisture basis = Cx (100- % moisture desired)j
100- % moisture in ground sampIe). Report to
0.1 %.

NOTES ON CRUDE FIBER DETERMINATION

1. Chemists using this method should study the
four reports of the Crude Fiber Liaison Co m-
mittee listed under R. E. Holt, in Selected Refer-
ence LL. The reports discuss in detail the possible
sources of error, choice of filtering equipment
and the necessity for following directions exactly
Fig. 1-3. Modified California State Büchner Funnel,
2-piece, polyethylene. Covered with 200-mesh screen, as written.
A, heat-sealed to edge of filtering surface. 2. Provided a 2 g sampIe was taken, the residue
from ether extraction may be used as the sampIe
becoming dry. Remove the filter from the beaker for crude fiber. Some laboratories routinely
and drain all water from the line by raising above determine moisture by oven drying, ether extract,
the trap level. Return the mat and residue to the and crude fiber, successively on the same 2 g
beaker by breaking the suction and blowing back. sampIe.
Add 200 ml of boiling 1.25 % NaOH and boi!
exactly 30 minutes. Remove the beaker and filter Van Soest and Wine 22 of the Agricultural
as above. Without breaking suction, wash with Research Service of USDA have introduced a
25 ml of boiling 1.25 % H ZS04 and three 50 ml new concept of the meaning of the term
portions of boiling water. Drain free of excess "crude fiber". They define fiber on a nutri-
water by raising the filter. Lower the filter into the tional basis as "insoluble vegetable matter
beaker and wash with 25 ml of alcohol. Drain the which is indigestible by proteolytic and
line, break the suction and remove the mat by
diastatic enzymes and wh ich cannot be utilized
blowing back through the filter screen into the
ashing dish. Proceed as in the final paragraph except by microbial fermentation in the
below. digestive tract of animals". These authors
b. Using the Cali/ornia Büchner /unnel- point out that the classic acid-alkali digestion
Filter the contents of the beaker through the to obtain the fibrous portion of plant material
 Crude fiber [15

9"

Fig. 1-4. Continuous heater for distilled water, 1.25% alkali, and 1.25% acid.

reported as "crude fiber" gives a figure that Van Soest obtained fiber residues of low
has an uncertain and variable relationship to nitrogen content by the use of neutral deter-
the nutritive value of the "fiber" so obtained. gents 23 in 1963. Further studies led to the
The ideal method is one that will separate development of a method that yields a residue
lignin, cellulose and hemicellulose with a representing the cellulose and lignin portions
minimum ofnitrogenous material. The residue of the cell-wall, incl uding insoluble ash
obtained by acid-alkali digestion retains a (mainly silica). lt is included in this text as
considerable portion of plant protein, and part Method 1-12.
of the lignin is gelatinized or dissolved and is This method cannot be considered a re-
lost. placement for the acid-alkali digestion Method
16] Introduction-General methods ror proximate and mineral analysis

1-11, especially when it is desired to compare detergent solution, 2 ml decahydronaphthalene,

the results with the many recorded analyses of and 0.5 g sodium sulfite with a scoop. Heat to
authentic sam pies of plant material in the boiling in 5-10 minutes. Reduce heat as boiling
literature which were obtained by the estab- begins, to avoid foaming. Adjust boiling to an
lished "crude fiber" technique. It does, how- even level and reflux 60 minutes, timed from
onset of boiling. Swirl beaker and fill previously
ever, give a truer picture of the nutritional
tared crucible. Admit vacuum only after crucible
availability for evaluation of foods and feeds
has been filled. Use low vacuum at first, increasing
of plant origin, particularly for non-ruminant it only as more force is needed. Rinse sam pIe into
animals. In this connection see a critique of crucible with minimum of hot (80-90°) water.
this method by Kimm, Gillingharn and Remove vacuum, break up mat, and fill crucible
Loadholt: J. Assoe. Offtc. Agr. Chemists 50; with hot water. Filter liquid and repeat washing
340 (1967). procedure. Wash twice with acetone in same
manner and suck dry. Dry crucibles at 100° 8 hours
Method 1-12. Plant Cell-Wall Constituents. or overnight. Cool in efficient desiccator and
Fiber Insoluble in Neutral Detergents. weigh. Report yield ofrecovered neutral-detergent
fiber as cell-wall constituents. Estimate noncell-
REAGENTS wall material by subtracting this value from 100.
a. Neutral-detergent solution. To 1 liter distilled Ash for 3 hours at 500-550°. Cool in desiccator
water add 30 g of sodium lauryl sulfate, USP, and weigh. Report ash content of neutral-deter-
18.61 g disodium dihydrogen ethylenediamine- gent fiber.
tetraäcetate dihydrate, reagent grade, 6.81 g
sodium borate decahydrate, reagent grade; 4.56 g
NOTES
disodium hydrogen phosphate, anhydrous, reagent
I. With concentrated plant material much
grade and 10 ml 2-ethoxyethanol (ethylene glycol
gelatinous material may form which may tend to
monoethyl ether), purified grade. Agitate to dis-
clog the filter. As an alternative the Oklahoma
solve. Adjust pH to a range of 6.9-7.1.
State Filter Screen described in Method I-lI
b. Decahydronaphthalene. Technical grade.
(Figure 1-2) may be used for the initial filtration
c. Acetone. Use a grade that is free from color
before the sampIe is washed into the crucible.
and leaves no residue upon evaporation.
d. Sodium sulfite. Anhydrous, reagent grade. 2. Crucibles must be clean for efficient filtra-
tion. Used crucibles may be cleaned by ashing at
ApPARATUS 500° and then forcing water in reverse flow through
a. Refluxing apparatus. Use any conventional the filter plate. When crucibles become clogged
apparatus suitable for crude fiber determinations. with ash particles after repeated use, they may be
Berzelius beakers (600 ml) and condensers made cleaned by forcing in reverse flow a hot solution
from 500 ml round bottomed flasks are preferred. of 20% KOH, 5% Na3P04 and 0.5% disodium
b. Sintered glass crucibles. Tall form, coarse EDTA. Overuse of this cleaning solution should
porosity with plate 40 mm in diameter and large be avoided as it tends to erode the glass.
enough to hold 40-50 ml liquid. 3. For many purposes, it will be important to
know the quantity of cell wall organic matter;
DETERMINATION therefore an ashing procedure is included in the
Weigh 0.5-1.0 g air-dried sampIe (ground to method. For non-ruminants with little fiber-
pass 40 mesh) into refluxing apparatus. Add, in utilizing capacity, the neutral detergent fiber will
order, 100 ml cold (room temperature) neutral- be the only fiber value required.

Ether extract (crude fat)

The term "ether extract" embraces all sub- sterols, waxes, fatty acids, carotenoids, chloro-
stances extracted by ethyl ether. In addition phyll and other pigments. This determination,
to fats, it includes phospholipides, lecithins, like that of crude fiber, goes back to work done
 Nitrogen-free extract [17

by early researchers in the German agri- DETERMINATION

cultural experiment stations, and later at Dry a 2 g sampIe, preferably in a vacuum oven
experiment stations in this country and in at 70°, to remove moisture (or use residue from
England. These workers used anhydrous ethyl the oven determination of moisture). Transfer
ether, and many of the results in literature on dried material to an extraction thimble with
porosity permitting a rapid flow of ether. Extract
the fat content of foods and feeds were
in a Soxhlet extractor at a rate of 5 or 6 drops per
obtained by use of this solvent.
second condensation for about 4 hours, or over-
Solvents other than ethyl ether have been night at a rate of 2 or 3 drops per second. Remove
used but the yield and composition of their ether by cautious evaporation of the contents of
resultant extracts differ somewhat from that the Soxhlet flask, dry in air oven at 100° for 30
obtained by ethyl ether. In order to avoid minutes, cool and weigh.
confusion, it is essential that the solvent be
named. The determination is made on a sam pie
N OTES ON ETHER EXTRACT DETERMINATION
that has previously been dried in an oven to I. Some methods for specific foods call for use
remove moisture. of some other solvent, for example, the AOAC
Two types of extractors are in general use: method for meats permits use of either ether or
the continuolls extractor, exemplified by the petroleum ether, whereas certain federal specifica-
Underwriters, Knorr, Goldfisch or Bailey- tions, such as that for pork sausage PP-S-91,
Walker, and the intermittent extractor, such require use of petroleum ether.
as the Soxhlet and its many modifications. 2. For powdery substances, such as flour, the
The latter is more efficient. It has the one weighed sampIes may be mixed with an equal
disadvantage of using a relatively large amount weight of clean, dry sand before drying.
of solvent. 3. The residue remaining after extraction may
be dried, and quantitatively transferred to a 600 ml
Method 1-13. Ether Extract (Crude Fat). (This beaker for use for crude fiber determination,
is the official AOAC method for grains, feeds, provided a 2 g sampIe was used.
flour, meats, etc.). 4. The approximate fat content of various
cIasses of basic foods is given below as a guide to
REAGENT
sampIe size:
Anhydrous ethyl ether. Wash commercial ether
two or three times with water, add several pieces fresh fruit (other than olives or
or pellets of sodium or potassium hydroxide, and avocados), leaf or root vege-
let stand overnight or longer until most of the tables, immature corn, beans
water has been absorbed. Decant into a dry and peas up to 1%
bottle, add small pieces of cIeaned, freshly cut mature or dried beans or peas
metallic sodium and let stand until evolution of (other than soy) 1 to 5%
gas ceases. Keep over metallic sodium in a bottle, cereals and grains 1 to 5%
protected by a calcium chloride tube. nuts (other than coconut) 45 to 75%

Nitrogen-free extract

This term is applied to the figure obtained fiber. For example, the authors of USOA
by subtracting the sum of the percentages of Agriculture Handbook No. 8, Composition of
moisture, crude protein (N x 6.25), ash, ether Foods (revised 1963) define "total carbo-
extraet and erude fiber from 100. It is some- hydrates" as "the remainder after the sum of
times called "carbohydrates by difference", or the fat, protein, ash, and moisture have been
"total carbohydrates", although this latter deducted from 100".
term is used by most authors to include crude It must again be pointed out that any errors
18 1 Introduction-General methods for proximate and mineral analysis

made in laboratory determinations of fat, methods, such as the c1assical combustion

protein, ash, moisture, and crude fiber are method for carbon and hydrogen, the Dumas
reflected in the figure reported for nitrogen- combustion method for nitrogen, nor some
free extract. determinations of physical characteristics, for
Ihis conc1udes our discussion on ~neral example, meIting points and boiling points.
methods for proximate analysis of foods. We These are given in all books on general chem-
are not inc1uding certain long established ical analysis and need not be repeated here.

Mineral elements

The term "mineral elements" is, of course,

inexact because organic elements such as Silicon
carbon, hydrogen, nitrogen, oxygen, phos-
phorus and sulfur do occur in minerals, Method 1-14. Silicon (AOAC Method 6.005).
Nevertheless, it serves as a convenient group
c1assification for those elements, mostly Ignite a 10-50 g sampie in a flat-bottomed Pt
metallic, that occur in relatively minor propor- dish in a muffle at 500-550° until the residue is
tions in foods and are usually determined as white or nearly so. (Pt dishes must be used with
caution in ashing plant materials high in Fe; for
elements rat her than as specific compounds or
such materials it may be advisable to use a well-
groups of compounds.
glazed porcelain or a Vycor dish and run a blank
The number of elements that may occur in determination.) Moisten with 5-10 ml of HCl,
traces in foods is very large, inc1uding besides boil about 2 minutes, evaporate to dryness, and
the relatively major group composed of Si, heat on a steam-bath 3 hours to render the Si02
Ca, Mg, Na, K, P, Sand Cl, such elements insoluble. Moisten the residue with 5 ml of HCl,
as Fe, Al, Mn, F, As, B, Co, Cu, Hg, Mo, boil 2 minutes, add about 50 ml of water, heat a
Pb, Se, Sn, Zn, and I. While As, Hg, Pb and few minutes on a water-bath, filter through a
Sn probably always represent contamination hardened paper, and wash thoroughly. To this
(with the possible exception of As in shellfish), filtrate add the filtrate and washings from the
Fe, Mn, Cu, Mo, Zn and I occur as traces as alkali-soluble Si0 2 determination b, and dilute
integral parts of enzymes in animal tissues, to 200 ml. Designate as Solution A. Save far Ca
determination.
and these elements and F, Band Se are
a. Sand-Wash the residue from the filter into
natural ingredients of plant foods grown on a Pt dish, and boil about 5 minutes with about
certain soils. It was originally intended to 20 ml of saturated Na2C03 solution; add a few
inc1ude in the present text methods for all of drops of 10% NaOH solution, let the mixture
these elements, but it was impossible to do settle, and decant through an ignited and weighed
this without sacrifice of space needed for dis- Gooch crucible. Boil the residue in the dish with
cussion of the various types of foods, if the another 20 ml portion of the Na2C03 solution,
book were to be kept of reasonable size. We and decant as before. Repeat the process. Transfer
are therefore listing in this chapter methods the residue to the crucible and wash thoroughly,
for only those elements we have c1assed as first with hot water, then with a !ittle HCl (l + 4),
and finally with hot water until Cl-free. Dry the
"relatively major". Methods for calcium,
filter and contents, ignite at 500-550°, and weigh
copper, iron and iodine specially adapted for
as sand. Confirm by microscopic examination.
dairy products are given in Chapter 6. b. Alkali-soluble Si02-Combine the alkali ne
Analysts interested in determining the minor filtrates and washings, acidify with HCl, again
elements are referred to the texts listed under evaporate, and dehydrate by heating 2 hours at
"Mineral Elements" in the Selected References. 110-120°. Moisten the residue with 5-10 ml of
 Mineral elements [19

HCI, boil about 2 minutes, add about 50 ml of 250 ml portions of H Cl (3 +22), mixing thoroughly
water, heat on a water-bath 10-15 minutes, filter after each treatment. Rinse with water until color
through an ashless filter or an ignited and weighed is removed, and then transfer to the column with
Gooch crucible, wash with hot water, ignite at water. The column is ready for use after the water
500-550°, and weigh as Si0 2. Add the filtrate to has drained to the top of the resin. [The exchange
Solution A. capacity for phosphate is about 1500 mg, so a
number of aliquots can be passed through the
column before regeneration is necessary. Rinse
NOTES
the column until the eluate is colorless (about 250
1. The above separation of sand from alkali- ml of water) before each use.]
soluble Si02 is necessary when information on the
constitutional ("normal") Si content of vegetable
or other plant material (as distinguished from the REAGENTS
Si coming from adhering sand) is desired-or, a. Buffer solution-pH 10. Dissolve 67.5 g of
conversely, when contamination of a food with NH4C1 in 200 ml ofwater, add 570 ml ofNH40H,
excessive sand must be proved. In those foods and dilute to I liter with water.
where the "normal" Si content is low enough to b. Potassium hydroxide-potassium cyanide so-
be ignored, this separation may be omitted. lution-Dissolve 280 g of KOH and 66 g of KCN
2. When the filtrate from the above procedure in I liter of water.
is not being used in other determinations, or the c. Potassium cyanide solution-2 %. Dissolve 2 g
presence of perchloric acid will not interfere in of KCN in 100 ml of water.
such determinations, HCI04 may be substituted d. Calcium carbonate-Primary standard grade,
for HCl with some saving in time, because with dried 2 hours at 285°.
this acid evaporation to fumes is sufficient to e. Hydroxy naphthol blue-Calcium indicator.
dehydrate the Si02, and evaporating to dryness Obtainable from Mallinckrodt Chemical Works,
and heating the residue on the steam bath may be 2nd & Mallinckrodt Sts., St. Louis, Mo. 63160, in
omitted. a dispenser bottle as their No. 5630.
3. If the quantity of the Si02 appears to be f. Calmagite [l-(J -hydroxy-4-methyl-3-phenyl-
excessive, or it is dark-colored, its percentage can azo )-2-naphtI101-4-sulJonic acid]-Calcium and
be checked by evaporation with H2F2 and a magnesium indicator. Obtainable from Mallinc-
few drops of H2S04, ignition, and determination krodt as their No. 4283, or from G. Frederick
of the loss in weight, provided the Si02 was Smith Chemical Co., Station D, Box 5906,
originally collected on an ashless paper and Columbus, Ohio, as their No. 278.
ashed in a Pt dish. g. Disodium dihydrogen ethylenediamine tetra-
äcetate (EDTA) standard solution--O.Ol M. Dis-
Calcium solve 3.72 g of EDTA (99+ % purity) in water in
a liter volumetrie flask, and dilute to volume.
Method 1-15. Calcium by EDTA Titration. 24 Accurately weigh enough CaC03 to give a titra-
tion of about 40 ml with the 0.01 M EDTA
ApPARATUS solution, and transfer to a 400 ml beaker. Add 50
a. Titration stand-Fluorescent illuminated, ml ofwater and enough 10% HCl to dissolve the
such as the "Titra Lite" of the Precision Scientific CaC03. Dilute with water to about 150 ml and
Co., Chicago, Ill. add 15 ml of 1 NNaOH (disregard any precipitate
b. Ion-exchange colllllln-Approximately 20 x or turbidity). Add about 200 mg of the hydroxy
600mm, fitted with a coarse porosity sintered glass naphthol blue indicator, and titrate the pink
disk and a Teflon stopcock. Place 30--40 g from solution to a deep blue end point, using a magnetic
a fresh bottle of Amberlite lR-4B resin (obtainable stirrer. Add the last few ml of the EDTA solution
from Mallinckrodt as their No. 3327) in a 600 ml dropwise. Molarity of the EDTA solution equals
beaker, and exhaust with three 250 ml portions
of 5 % Na2C03 or NaOH. Wash with water until mgCaC0 3
excess base is removed. Treat the resin with three ml EDTA x 100.09
20] Introduction-General methods for proximate and mineral analysis

PREPARATION OF SAMPLE %Ca = Titer B x 0.4008 x 10 x 100/mg sampie.

Drain the liquid from eanned whole tomatoes,
eentrifuge and filter through a fast filter paper. NOTES
Weigh 100 g of the filtrate into a Pt or poree- 1. Never pipet any KCN solution by l11outh.
lain dish, and evaporate to dryness using a forced- 2. The direetions for preparation of the sampie
draft oven, infrared radiation or other eonvenient are specifieally direeted to eanned whole tomatoes,
means. Ash at not more than 525 0 until apparently and eall for sampling only the drained liquid,
earbon-free (ash gray to brown). Cool, add 20 ml beeause the author of Method 1-15 was interested
of water and then eautiously add 10 ml of HCl in the question of whether market eanned toma-
under a wateh-glass while stirring with a gl ass rod. toes eontained more than the 0.026 % Ca per-
Rinse the wateh-glass off into the dish and evap- mitted by Federal regulations. He found by
orate to dryness on a steam bath. Add 50 ml of experiment that analysis of the liquid was suffi-
HCl (l +9), heat 15 minutes on a steam bath and cient sinee its Ca eontent was identieal with
filter through a quantitative paper into a 200 ml that of the solid portion. The direetions should
volumetrie fiasko Thoroughly wash the paper and apply without alteration to eanned lima beans and
dish with hot water. Cool the filtrate, dilute to the potatoes for whieh the regulations set Ca limits
mark and mix. For other foods, see Note 2. ofO.026 and 0.051 % respeetively. For other foods
it would be advisable to ash an appropriate
DETERMINATION quantity of the whole material and proeeed as
a. Removal oJphosphate-Transfer a 500r 100 direeted above. Solution A, Method 1-14, may
ml aliquot of the prepared sampie to a 250 ml be used.
beaker, and adjust the pH to 3.5 with 10% KOH
solution (added drop by drop), using a pH meter
and magnetie stirrer. Pass the adjusted solution Magnesium
through the resin eolumn and eolleet the emuent
in a 250 ml volumetrie fiask-adjusting the fiow Method 1-16. Magnesium by EDTA Titration. 24
rate to 2-3 mI/minute. Wash the eolumn
thoroughly with two 50 ml portions of water, Apparatus, reagents, preparation of sampie and
passing the first 50 ml through the eolumn at the removal of phosphate are the same as in Method
same rate as the sampie solution and the seeond 1-15.
50 ml at a rate of 6-7 mI/minute. Finally freely Pipet a 100 ml aliquot of the 250 ml of emuent
pass enough water through the column to make into a 400 ml beaker and adjust its pH to 10 with
to the mark. Mix thoroughly. the buffer solution, using a pH meter and mag-
b. Titration-Pipet a 100 ml aliquot into a 400 netie stirrer (about 5 ml are required). Add 2 ml
ml beaker and adjust the pH to 12.5-13.0 with the of the 2 % KCN solution and 200 mg of Cal-
KOH-KCN solution (about 10 ml) using a pH magite indieator; titrate immediately with the
meter and magnetic stirrer. Add 0.100 g of 0.01 MEDTA solution, through red to deep blue,
aseorbie acid and 200-300 mg of the hydroxy- using a magnetie stirrer. MI EDTA solution
naphthol blue indicator. Titrate immediately with consumed = Titer A
the 0.01 M EDTA solution through a pink to a
deep blue end point, using a magnetie stirrer. %Mg=
MI EDTA solution eonsumed = Titer B. (Titer A - TiterB) x 0.2432 x 10 x l00/mg sampie

Potassium and sodium

The classical way to determine potassium solvents. 26 With careful handling the chloro-
is by the Lindo-Gladding ehloroplatinate platinate method can give very precise results,
method. 25 There are also several variations but lately it has tended to be superseded by
of a method depending on the insolubility of methods based on the recent discovery of the
the perchlorate in alcohol or other organic insolubility of potassium tetraphenylborate 2 7,
 Potassium and sodium [21

or by the use of flame photometry. Details of solution increases. Prepare separate extracts for
a flame photometrie method applieable to Na and K when their concentrations in the sampIe
both potassium and sodium are given below: greatly differ. For K, use a sampIe weight not
exceeding 0.1 g/50 ml extracting solution; for low
Method 1-17. Flame Photometrie Met/IOd [01' Na concentrations, use at least I g sample/50 ml
K and Na. (ADAC Met/IOd 6.016-6.019). extracting solution. For higher concentrations,
prepare weaker extracts by reducing the ratio of
REAGENTS
sampIe to extracting solution rat her than by
a. Potassitllll stock solution-l,ooO ppm K.
diluting stronger extracts.
Dissolve 1.907 g of dried KCI in water and dilute b. Fruit juices and [mit f/avored beverages-
to I liter.
Evaporate an aliquot of the sampIe, ash at low
b. Sodiufll stock solutioll-l,ooO ppm Na.
temperature and take the residue up in a measured
Dissolve 2.542 g of dried NaCI in water and dilute
volume of the extracting solution.
to I liter.
c. Dther [oods 28-Weigh I-50 g (according to
c. Lithium stock solution-I,ooo ppm Li.
the expected Na and K contents) ofthe sampIe into
Dissolve 6.109 g of dry LiCl in water and dilute
a shaHow Vycor dish. If damp, dry in an oven
to I liter.
just below 100°. Then place in a cold muffle
d. Ammonium oxalate solution--O.24 N. Dis-
furnaceand heat to 550°,maintainingthistempera-
solve 17.0 g of (NH4h C204.H20 in water and
ture until a gray ash is formed. A110w the furnace
dilute to I liter.
to cool and remove the dish. Moisten the ash with
e. Extracting solutioll-To 250 ml of the NH 4
redistilled, constant-boiling HCI, evaporate on a
oxalate solution add enough of the Li stock
steam bath and again heat the residue in the fur-
solution to bring the resulting intensity of the Li
nace to 550°, maintaining this temperature until
line within the range of the instrument being used
the ash is white (genera11y 1-2 hours). If the ash
(see Preparation o[ Sampie), and dilute to I liter
fails to become white after several hours at 550°,
with water.
cool the dish and contents, moisten the residue
PREPARATION OF STANDARD SOLUTIONS with a 25 %solution of redistilled HN03, dry and
Dilute mixtures ofappropriate aliquots ofthe K heat again to 550°. (Any color still remaining is
and Na stock solutions in order to prepare aseries usuaHy due to Fe, and cannot be eliminated.)
of standards containing K and Na in stepped Fina11y take up the residue in a measured volume
amounts (including 0) covering the instrument of the extracting solution.
range, together with Li and NH4 oxalate in the DETERMINATION
same concentrations that will be employed in the Rinse a11 glassware with diluted HN03 fo11owed
extracting solutions. by several portions of water. Protect solutions
from airborne Na (or K) contamination. Operate
PREPARATION OF SAMPLE
the instrument according to the instructions of
a. Dried plant materials (including vegetables)-
the manufacturer. A110w the instrument to reach
Transfer a weighed portion of the finely ground
operating equilibrium before using. Atomize por-
and weH mixed sampIe to an Erlenmeyer ftask
tions of the standard solutions toward the end of
large enough to contain twice the volume of
the warm-up period until reproducible readings
extracting solution to be used. Add a measured
for the series are obtained.
volume of extracting solution, stopper the ftask
Run standards covering the concentration
and during 15 minutes shake vigorously at fre-
range ofthe sampIes involved at frequent intervals
quent intervals. Filter through dry, fast paper. If
during atomization of aseries of sampie solutions.
the paper clogs, pour the contents onto additional
Repeat this operation with both standard and
fresh paper and combine the filtrates. Use the
sam pIe solutions enough times to result in a
filtrates for the determination.
reliable average reading for each solution. Plot
NOTE: Do not make extracts more concentrated the analysis curves (K/Li and Na/Li ratios) from
than required for the instrument because there is a readings of the standards and caJculate the per-
tendency toward incomplete extraction as the centages of K and Na in the sampies from these
ratio of sampIe weight to volume of extracting curves.
22] Introduction-General methods for proximate and mineral analysis

Phosphorus

Most ofthe methods for phosphorus depend on a hot plate in a weil ventilated hood and boil
at some point on conversion of this element to one minute. Cool to room temperature, swirling
the phosphomolybdate. After the ammonium carefully 3-4 times during the cooling. Filter onto
phosphomolybdate is filtered off, it may be a Gooch crucible, lined with a glass fiber filter
dissolved in an excess of standard alkali paper that has been dried at 250 0 and weighed.
Wash five times with 25 ml portions of water. Dry
wh ich is back-titrated with acid, or it may be
the crucible and contents for 30 minutes at 250°,
dissolved in an excess of ammonia and the
cool in a desiccator to constant weight and
phosphorus precipitated as magnesium am- weigh as (C9H7N))[P04.12Mo03]. Subtract a re-
monium phosphate, which is ignited and agent blank. Multiply by 0.01400 to obtain the
weighed as magnesium pyrophosphate. For weight of P.
traces of phosphorus, the phosphomolybdate
can be reduced to a blue molybdenum com- NOTE: Drying the precipitate at the high tem-
pound which is estimated colorimetrically. perature specified is necessary to remove variable
Two relatively recent variations are a water of crystallization and obtain stoichio-
colorimetric method depending on formation metric results.
of the yellow phosphomolybdovanadateZ 9 ,
and a gravimetrie method involving precipi-
Method 1-19. Molybdenum Blue Micro Method.
tation of quinoline phosphomolybdate. It is (AOAC Method 6.062-6.064).
this latter method and one of the Mo blue
colorimetric methods that we have chosen to
REAGENTS
present:
a. Phosphorus standard solution-Dissolve
0.4394 g of pure dry KHzP04 in water and dilute
Method 1-18. Quinoline Phosphomolybdate to 1 liter. Dilute 50 ml of this solution to 200 ml
Method. (AOAC Met/IOd 2.023-2.025). (2 ml = 0.05 mg P).
b. Ammonium molybdate solution-Dissolve 25
REAGENTS g of NH 4 molybdate in 300 ml of water. Dilute
Quimociac reagent-Dissolve 70 g of Naz 75 ml of H ZS0 4 to 200 ml and add this to the NH4
M004.2HzO in 150 ml of water. Dissolve 60 g molybdate solution.
of citric acid in a mixture of 85 ml of HN03 and c. Hydroquinone solution-Dissolve 0.5 g of
150 ml of water and cool. Gradually add the hydroquinone in 100 ml of water and add 1 drop
molybdate solution to the citric-nitric acid of HZS04 to retard oxidation.
mixture with stirring. Dissolve 5 ml of synthetic d. Sodium sulfite solution-Dissolve 200 g of
quinoline in a mixture of 35 ml of HN03 and NaZS03 in water, dilute to 1 liter and filter. Either
100 ml of water. Gradually add this solution to keep the solution weil stoppered or prepare
the molybdate-citric acid-HN03 solution, mix fresh each time.
and let stand for 24 hours. Filter, add 280 ml of e. Magnesium nitrate solution-Dissolve 950 g
acetone, dilute to I liter with water and mix. of P-free Mg(N03)z.6HzO in water and dilute to
Store in a polyethylene bottle. 1 liter.

DETERMINATION PREPARATION OF SOLUTION

Pipet into a 500 ml Erlenmeyer flask a 50 ml To a 1 or 2 g sampie in a small porcelain
aliquot of Solution A, Method 1-14, and dilute to crucible add 1 ml of the Mg(N03h solution and
about 100 ml with water. Add 50 ml of the place on a steam bath. After a few minutes,
quimociac reagent, cover with a watch-glass, place cautiously add a few drops of HCl, taking care
 Sulfur [23

that gas evolution does not push portions of the volume and filter, discarding the first portion of
sampie over the edge of the crucible. Make 2 or 3 the filtrate.
further additions of a few drops of HCI while the
sampie is on the bath, so that as it approaches DETERMINATION
dryness it tends to char. If the contents of the To a 5 ml aliquot of the filtrate in a 10 ml
crucible become too viscous for further drying volumetrie flask, add 1 ml of the NH4 molybdate
on the bath, complete drying on a hot plate. solution, rotate the flask to mix, and let stand a
Cover the crucible, transfer to a cold muffle and few seconds. Add 1 ml of the hydroquinone solu-
ignite at 500° for 6 hours until an even gray ash is tion, again rotate the flask and add 1 ml of the
obtained. (If necessary, cool the crucible, dissolve Na2S03 solution. (The last three additions may be
the ash in a little water or alcohol-glycerol, made with a Mohr pipet.) Dilute to volume with
evaporate to dryness and return to the muffle water, stopper the flask with a thumb or fore-
uncovered for 4-5 hours longer.) Cool, take up in finger and shake to mix thoroughly. Let stand 30
HCl (l +4), and transfer to a 100 ml beaker. Add minutes. Then in a photoelectric colorimeter
5 ml of HCl and evaporate to dryness on a steam (equipped with a filter, maximum transmittance
bath to dehydrate Si0 2 • Moisten the residue with 625-675 mIL) immediately compare with 2 ml of
2 ml of HCl, add about 50 ml of water and heat a the standard KH2P04 solution that has been
few minutes on the bath. Transfer to a 100 ml treated simultaneously and identically with the
volumetrie flask, cool immediately, dilute to sampie. Report as percent P.

Sulfur

Sulfur occurs in natural foods chiefly as an added to ensure complete oxidation and fixation
ingredient of. proteins or amino acids such as of all S present. For larger sampies and for sampies
cystine and methionine. Trace amounts are with a high S content, proportionally larger
present in the vitamin thiamine, and in spices quantities of this solution must be used.) Heat on
such as mustard and vegetables such as an electric hot plate at 180° until no further action
occurs. Transfer the crucible while hot to an
cabbage and onions, in the form of organic
electric muffle and let it remain at low heat (not
sulfides. Therefore, while so me natural sulfate
over 500°) until the charge is thoroughly oxidized.
may be present, the fact that unoxidized forms (No black particles should remain. If necessary,
of this element predominate means that pro- break up the charge and return to the muffle.)
vision must be made for so me form of oxida- Remove the crucible from the muffle and let it
tion when the sam pie is ashed prior to deter- cool. First add water, then HCl in excess. Bring
mination oftotal sulfur as barium sulfate. The the solution to a boil, filter and wash thoroughly.
usual oxidizing agents are sodium peroxide, If preferred, transfer the solution to a 250 ml
magnesium nitrate or perchloric acid. The volumetrie flask before filtering and dilute to the
following method employs the second ofthese: mark with water.

DETERMINATION
Method 1-20. Total Sulfur. (AOAC Method Either dilute the entire filtrate to 200 ml or take
6.059--6.060). a 100 ml aliquot of the solution in the 250 ml
volumetrie flask (= 0.4 g sampie) and dilute to
PREPARATION OF SOLUTION 200 ml. Neutralize with HCl and add 5 ml in
Weigh a I g sampIe into a large porcelain excess. Heat to boiling and add 10 ml of 10 %
crucible. Add 7.5 ml of Mg(N0 3h solution, BaCI 2 solution drop by drop stirring constantly.
Method 1-1ge, in such a way that all of the Continue boiling for about 5 minutes and let
material comes in contact with the solution. (It is stand 5 hours or longer in a warm place. Decant
important that enough Mg(N03h solution be through an ashless paper or an ignited and weighed
 24] Introduction-Genera1 methods for proximate and minera1 analysis

Gooch crucible. Add 15-20 ml of boiling water to free. Dry the precipitate and filter or crucible,
the precipitate, transfer to the filter or crucible ignite and weigh as BaS04.Wt. BaS04 x 0.1374
and wash with boiling water until the filtrate is Cl = S.

Chlorine

The chlorine content of natural foods is low c. Ferric indicator-A saturated solution of
and, while traces of organic chlorine exist in FeNH4(S04h. 12 H20.
some of them, essentially all of this element is d. Nitric acid-Free from lower oxides of N
present in the form of chloride. The amount of by diluting the usual pure acid with about one-
chloride is high in some manufactured foods fourth its volume ofwater. Boil until colorless.
because salt has been added as a seasoning or
preservative. PREPARATION OF SOLUTION
The following method is simple and should Moisten a 5 g sampIe in a Pt dish with 20 ml of
suffice for sam pies containing more than just 5 % Na2C03 solution, evaporate to dryness and
traces of Cl: ignite as thoroughly as possible at 500°. Extract
with hot water, filter and wash. Return the residue
Method 1-21. Vo/hard Method. (AOAC Methods to the dish and ignite to ash; dissolve in HN03
6.065 and 6.067--6.068). (1 +4), filter, wash thoroughly and add this solu-
tion to the previous aqueous extract.
REAGENTS
a. Silver nitrate standard so!ution-Prepare a
solution slightly stronger than O.1N and stan- DETERMINA TION
dardize by thiocyanate titration against KCI that To the prepared solution add a known volume
has been recrystaIIized three times from water, of the AgN03 solution (a) in slight excess. Stir
dried at 100° and heated to constant weight at weH; filter and wash the AgCI precipitate
500°. Adjust to exactly O.1N. 1 ml = 0.0355 g thoroughly. To the combined filtrate and washings,
Cl. add 5 ml of the ferric indicator and a few ml of the
b. Ammonium or potassium thiocyanate standard HN03. Titrate the excess of Ag with the thio-
solution-O.l N. Prepare a solution slightly stronger cyanate to a permanent light brown. From the
than 0.1N. Standardize against Solution a, and ml of AgN03 used, calculate the quantity of CI
adjust to exactly 0.1N. in the sampIe.

TEXT REFERENCES

MOlSTURE 7. Fischer, K.: Angew. Chem., 48, 394 (1935).

1. Central Scientific Co., Chicago: Pamphlet FM-2 8. Faulk, C. W. and Bawden, A. T.: J. Am. Chem.
(March 19, 1954). Soc., 48, 2045 (1926); (a) Kao and Hsu: Acta
2. Smith, E. R. and MitchelI, L. C.: Ind. Eng. Chem., Chim. Sinica, 24, 1 (1958).
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10. McComb, E. A., McCready, R. M.: J. Assoc.
4. Bidwell, G. L. and Sterling, W. A.: Ind. Eng.
Ojfic. Agr. Chemists, 35, 437 (1952).
Chem., 17, 147 (1925).
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Agr. Chemists, 38, 130 (1955). 49, 551 (1966).
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Trade Association, American Spice Trade Associa- 726 (1966).
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 Selected References [25
(a) Makower, B. and Nielsen, E. : Z. Anal. Chem., CRUDE FIBER
20, 856 (1948). 20. Holt, K. E.: J. Assoc. Offic. Agr. Chemists, 45,
578 (1962).
NITROGEN 21. Henneberg, Landw. Versuch Stat., 6, 497 (1864).
14. Kjeldahl, J.: Z. Anal. Chem., 22, 366 (1883). 22. Van Soest, P. J. and Wine, R. H.: J. Assoc. Offic.
15. Report of the Committee on joint collaborative Agr. Chemists, SO, 50 (1967).
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Society on total Nitrogen in feeds and fertilizers, 46, 829 (1963).
J. Assoc. Offic. Agr. Chemists, 38, 56 (1955).
16. Horwitz, W. (ed.): Official Methods 01 Analysis 01 MINERAL ELEMENTS
the Association 01 Official Agricultural Chemists, 24. Steagall, E. F.: J. Assoc. Offic. Agr. Chemists, 49,
10th ed. Method 2.045. Association of Official 287 (1966); Ibid., SO, 195, 219 (1967).
Agricultural Chemists, Washington (1965). 25. Ref. 16, Method 2.069-2.071.
17. Gehrke, C. W., Beal, B. M., and Johnson, F. J.: 26. Ref. 16, Method 6.021.
J. Assoc. Offic. Agr. Chemists, 44, 239 (1961); 27. Wittig, G. Keicher, G., Ruchert, A. and Raff, P.:
Gehrke, C. W., and Johnson, F. J.: J. Assoc. Ann., 563, 100, 126 (1949); Wittig, G.: Angew.
Offic. Agr. Chemists, 45, 46 (1962). Chem., 62A, 231 (1950); Wittig, G. and Raff, P.:
18. Burch, W. G., Jr. and Brabson, J. A.: J. Assoc. Ann., 573, 195 (1951).
Offic. Agr. Chemists, 48, 1111 (1965). 28. Bills, C. E., McDonald, F. G., Niedermeier, W.
19. Gehrke, C. W., Ussary, J. P., Perrin, C. N., and Schwarts, M. c.: Anal. Chem., 21, 1078
Rexroad, P. R. and Spangier, W.: J. Assoc. Offic. (1949).
Agr. Chemists, SO, 965 (1967). 29. Ref. 16, Methods 2.018-2.022 and 22.074-22.077.

SELECTED REFERENCES

PROXIMATE COMPOSITION OF FOODS Sherman, H. c.: Chemistry 01 Food and Nutrition,

A Bailey, E. M.: Analyses of common foods. Conn. 8th ed., New York: Macmillan Co. (1952).
Agr. Exp. Sta. Bul. 373 (1934). J Watt, B. K. and Merrill, A. L.: Composition of
B Bureau of Human Nutrition and Home Econo- foods-raw, processed, prepared. U.S.D.A.
mics: Tables of food composition in terms of Agriculture Handbook 8 Rev. (1963).
eleven nutrients. U.S.D.A. Misc. pub. 572, Rev. K Winton, A. L. and Winton, K. B.: The Structure
(1949). and Composition 01 Foods. Vols I-IV. New York:
C Canada Dept. of National Health and Welfare, John Wiley and Sons, Inc. (1932-1939).
Nutrition Division: Tables of Food Values L Woodman, A. G.: Food Analysis. 4th ed. New
Recommended for Use in Canada (1951). York: McGraw-Hill Book Co. (1941).
D Chatfield, C. and Adams, G.: Proximate com- M Wooster, H. A., Jr.: Nutritional Data. 2nd ed.
position of American food materials. U.S.D.A. Pittsburgh: H. D. Heinz Co. (1954).
Circular 549 (1940).
E Consumer and Food Economics Research MOISTURE
Division, Agricultural Research Service: Nut- N Moisture Determination by the Karl Fischer
ritive value offoods. U.S.D.A. Home and Garden Reagent, 2nd ed. Dorset: British Drug Houses,
Bulletin 72 (1960). Ltd. (1966).
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