2

Sampling and
Proximate Analysis

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 19
Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20
Moisture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21
Crude Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Crude Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 28
Crude Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31
Ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Selected References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

D INTRODUCTION

Considerable information about a food sample can be gained through

a general analysis of its main components-moisture, crude fat, crude
protein, ash, and crude fiber. The determination of the percentages of
these components is termed a proximate analysis. In some cases, a
proximate analysis may be all that is required and the more sophis-
ticated instrumental methods discussed in Chapter 3 may be unnec-
essary. For example, a proximate analysis is usually sufficient to es-
tablish the general category of foodstuff to which a particular sample
belongs and the similarity of a particular food sample to materials
previously reported in the literature.
Whether a proximate analysis or more sophisticated analyses are to
be performed, careful sampling is required to obtain accurate and re-
producible values. The chemical and physical properties of foodstuffs
exhibit a certain inherent variability among different samples; vari-
ability within a given sample, however, can be minimized by proper
sampling. Analyses usually are performed on small, discrete samples
rather than on the entire amount of a foodstuff (a so-called perfect
sample). Various techniques (grinding, mixing, etc.) are used to insure
that such small samples are representative of the entire material and
provide a true measure of its overall content.

19
L. W. Aurand et al., Food Composition and Analysis
© Springer Science+Business Media New York 1987
20 2. Sampling and Proximate Analysis

The analyst should consult standard texts for the statistics of sam-
pling. Usually a particular food product has peculiarities that must be
recognized by the sampler. These include heterogeneity of the sample
per se and heterogeneity of the dissolving or suspending media, (e.g.,
inconsistent moisture content). Other factors may be relevant in a par-
ticular food.

D SAMPLING

Two basic types of sampling procedures are used: manual and con-
tinuous. Manual sampling is accomplished with instruments such as
triers, probes, or sampling tubes, which are available in different de-
signs and sizes. Usually a sampling tube varies from 12 to 60 in. in
length. The sample can enter through the end of the tube or through
slots or openings in the side of the tube; then the end or slots are closed,
thus trapping the sample. There are slots or openings at intervals in
the tube to allow simultaneous sampling at various depths of the prod-
uct being sampled. Auger- or drill-type samplers can be used to remove
a sample from solid materials such as cheese, butter, or frozen food
products. To obtain liquid samples, the food product is thoroughly
mixed and then the sample may be removed with a syringe-type sam-
pler or simply by submerging a vessel under the surface (a so-called
"grab" sample.) Commercially available liquid samplers may also be
used.
In continuous sampling procedures, now used by many industries,
samplers or sample boxes mechanically divert a fraction ofthe material
being sampled. For solid materials, a riffle cutter often is used. A liquid
sample may be obtained by "bleeding off" a fraction of the mainstream
line through a smaller diversion line. The rate of the sampling can be
controlled by adjusting flow rates via valves.
An excellent description of sampling devices and their use is given
by Johnson (1963), who discusses the problems of sampling and factors
that must be considered. Generally, the analyst should follow the
AOAC method (or other published method) for sampling if one is avail-
able. Specific directions for sampling various types of foods are given
in later chapters.

Types of Sampling Devices

A riffle cutter is a box-like device with equally spaced dividers which
divide the stream of sample equally. The sample can be proportionally
reduced by passing through successive riffles or recycled through the
Moisture 21

same riffle. This "cutting and quartering" is used in laboratories to

reduce larger samples to convenient laboratory size.
Vezin samplers can be used for intermittent or continuous sampling
of either wet or dry materials. A Vezin sampler is a truncated wedge
of a circle that passes through the stream of material once each rev-
olution. If the cutter wedge is 5% of the volume of the circle, then 5%
of the sample stream is removed. The peripheral speed of the cutter is
usually approximately 30 in.!sec. The formula for determining the
pounds of sample per cut is
S = 0.0925FA
N
where S = pounds of sample per revolution of cutter; N = revolutions
per minute; A = total angle of the cutter(s) expressed in degrees of
the entire circle; and F = feed rate of sample in tons per hour.
Straight-line samplers move in a straight line and at a uniform speed
across and completely through the stream of sample. A detailed dis-
cussion of the geometry of variations of this type is given by Johnson
(1963).

Preparing Samples
The composite sample usually must be reduced to laboratory size.
Mechanical grinding, mixing, rolling, agitation, stirring, or any logical
means of making the sample more homogeneous is desired.
Grinding, which reduces particle size, helps reduce variability in the
weight and size of particles. Usually, a particle diameter of 0.5-1.0
mm is recommended. Good results are obtained if the material is
ground to pass through a 35-mesh sieve. None of the sample should
be discarded because this could remove components that concentrate
in the discarded particles, and lead to erroneous analyses. The Wiley
mill, ball mill, mortar and pestle, mechanical high-speed beaters or
blenders, and meat grinders are commonly used for sample
disintegration.
Liquid samples can be mixed by magnetic stirrers or sonic oscillators,
similar to the devices used to disintegrate cells.

o MOISTURE

Moisture is the measure of the water content of a material. Com-

pounds that volatilize under the same physical conditions as water also
would be included; however, these are usually negligible. Determi-
nation of moisture content is necessary for the analyst in order to
22 2. Sampling and Proximate Analysis

calculate the nutritive value (e.g., vitamin content) of a food product

and to express the results of analytical determinations on a uniform
basis.
Moisture is an important factor in food quality, preservation, and
resistance to deterioration. Grain that contains too much water tends
to deteriorate rapidly due to mold growth, heating, insect damage, and
sprouting. The browning rate of dehydrated fruits and oxygen absorp-
tion by powdered eggs also increase with an increase in moisture
content.
Water exists in three major forms: (1) solvent and dispersing media;
(2) adsorbed on the internal or external surfaces or as fine capillaries
by capillary condensation; and (3) water of hydration. Solvent water
is the solvent for soluble compounds such as sugars, amino acids, and
carboxylic acids. Compounds that do not dissolve are dispersed in
water; these include proteins, gums, and polysaccharides. Adsorbed
water is a very thin film or a fine capillary and is commonly not re-
moved in normal moisture determinations. Hydrate water is a chem-
ical component of sugars such as glucose, maltose, and lactose, of salts
such as potassium tartrate, and of proteins and polysaccharides which
form gels with water firmly bound.
Solvent water (free water) is the most easily removed, but special
precautions are necessary for all samples. Drying temperature, particle
size, vacuum, crust formations on the surface, and surface area of the
sample all affect the rate at which moisture is removed from foods.
Vacuum ovens significantly reduce the deterioration of samples during
heating. At elevated temperatures, chemical reactions such as hy-
drolysis can occur and cause significant errors in the moisture deter-
mination, since the water of hydrolysis is not released from the sample.
Sugar solutions (e.g., honey and fruit syrups and fructose solutions)
decompose at elevated temperatures; thus, the use of an air oven is
not recommended for such materials. Glucose solutions are relatively
stable at 98°C. Residual (bound) water of most foods (1%) is quite dif-
ficult to remove without vacuum drying. Pressures ofless than 25 mm
Hg are most desirable. Most foods require long drying times (up to 16
hr).
Various methods for determining moisture content are described in
detail in the following sections. In some cases, the analysis of a specific
food product is used to illustrate a method.

Vacuum Oven Method

Determination of moisture in wheat flour (AOAC 14.002 and 14.003)
is a good example of the vacuum oven method.
Moisture 23

Apparatus
1. Metal dish approximately 15 mm high by 55 mm in diameter
with cover (according to AACC 44-15A: Sargent-Welch S-25705).
2. Desiccator with drying agent in bottom (dry CaO).
3. Vacuum oven with a pump capable of maintaining 25 mm Hg.
A temperature measuring device should be in place near the
sample. Only dry air should be admitted to the oven after drying.
An H 2 S04 gas-drying bottle will accomplish this.
Procedure
Dry the metal dish and tare. Weigh the flour to nearest 0.1 mg with
cover loose. Dry at 100°C to constant weight and 25 mm Hg vacuum.
Break vacuum with dry air and place lid on dish. Transfer immediately
to desiccator and weigh after the sample and dish reach room tem-
perature. Calculate the percentage moisture and percentage solids.

Lyophilization Method
Commercial lyophilizers (freeze dryers) are quite convenient for
moisture measurements. A vacuum of approximately 5 /-lm of Hg and
a cold trap (finger) temperature of - 50°C are possible. National Bu-
reau of Standard Reference Material 1577-Bovine Liver is prepared by
removing fat, major blood vessels, and skin, then grinding. The ground
liver is then transferred to polyethylene trays and lyophilized, at a
pressure not greater than 30 Pa (0.2 mm Hg) with a cold trap tem-
perature of - 50°C, for 24 hr. If analyses are contemplated beyond
moisture measurement, the lyophilized liver is ground in a Toronado
mill then relyophilized for at least 24 hr using a cold trap at or below
- 50°C and at a pressure not greater than 30 Pa (0.2 mm Hg). This
method is particularly good for preparing tissue for metals analysis or
for nonvolatiles. A sample of at least 250 mg should be used for metals
analysis.

Distillation Method
Foods that contain only small amounts of water or may contain sig-
nificant amounts of substances other than water volatile at 100°C re-
quire special methods for determining moisture. One of these is the
distillation method in which a solvent immiscible with water is cod-
istilled from a weighed sample. The solvent usually is toluene, xylene,
or a mixture of these with other solvents. The solvent itself or the
solvent mixture has a boiling point slightly higher than that of water.
Upon boiling, the solvent and water distill over, are condensed, and
24 2. Sampling and Proximate Analysis

drop into a graduated collection tube (the Bidwell-Sterling tube is most

commonly used).
The determination of moisture in blue and similar cheeses illustrates
the distillation method.
Apparatus and Solvent
1. Receiver-Bidwell & Sterling, or modified Bidwell & Sterling,
with 5-ml volumetric tube with $" or upper 24/40, lower 24/40
(Sargent-Welch No. S-28317-A).
2. Condenser-Cold-finger type with $ 24/40 joint.
3. Boiling flask-250 ml, round bottom, short neck; $" 24/40 joint
with receivers with lower joint $" 24/40.
4. Heating mantle connected to voltage controller.
5. Distillation solvent-Toluene (boiling point = 111°C).
Standardization of Apparatus
Support the apparatus. Lubricate lower joints with silicone stopcock
grease. Clean and dry the interior of the apparatus. Rinse with toluene,
and fill volumetric tube of receiver with solvent. Also rinse the interior
of the condenser, dry, and immediately insert condenser into appa-
ratus. Remove all moisture from exterior of apparatus. Add glass beads
to boiling flask to prevent bumping, and heat flask to redry.
Add 5 ±0.0001 g of water and 75 ml of toluene to the sample flask
and connect to the receiver. Heat until refluxing starts and adjust heat
to distill at a rate of 0.25-0.5 ml of water per min. Increase the rate
of refluxing gradually to maximum. When no more water distills, add
a few milliliters of toluene to the sample flask and bring to boil. Repeat
this procedure until no more water is distilled over.
Cool the Bidwell-Sterling tube and read the volume of water col-
lected (repeat whole procedure at least four times). From these data,
a distillation recovery factor is calculated: f = g H 2 0 added/g H 2 0
recovered. The reproducibility of this factor is determined by the op-
erator and the acceptable variability is determined by the accuracy
required in the analysis.
Procedure
Set up apparatus as outlined under Standardization. Weigh into the
250-ml sample flask a sample that will give 2-5 ml of water on dis-
tillation. Add boiling beads. Add sufficient toluene to cover the sample
completely (approximately 75 mI). Connect the flask to the side arm
of the Bidwell-Sterling tube. Pour toluene through the condenser until
the collecting tube is filled. Heat the flask to boiling and distill slowly
(2 drops/sec) until most of the water is in the Bidwell-Sterling tube.
Moisture 25

Then increase the distillation rate to 4 drops/sec until no more water

comes over. Wash down the condenser with toluene. If water droplets
are evident in the condenser, pour more toluene through the condenser.
Continue the distillation to determine if additional water is present
in the sample or apparatus. Remove the heating mantle and cool the
Bidwell-Sterling collector to approximately 25°C. Be sure that all
water droplets are in the tube. Read the volume of water collected and
calculate the percentage of water in the sample:
% H 20 = f x g H20 distilled from sample x 100
g of sample

Fischer Method
The Fischer method is particularly applicable to foods that give er-
ratic results when heated or under a vacuum. Low-moisture foods such
as dried fruits and vegetables, candies, chocolate, roasted coffee, oils,
and fats are commonly analyzed for moisture by the Fischer titration
method; high-moisture foods are not analyzed by this method.
The method is based on the reduction of 12 by S02 in water according
to the equation
2H20 + S02 + 12 ~ H2S04 + 2HI
Karl Fischer modified and quantitized the procedure to include Iz,
S02, pyridine (C 5H5N), and methanol in a four-component system. Eth-
ylene glycol monomethyl ether has been substituted for anhydrous
methanol. The overall equation for the reaction has been given as
C5H 5N·1z + C5H 5N·S02 + C5H5N + H20
~ 2 C5H 5N·HI + C5H 5N·SO g C5H5·SOg + CHgOH
~ C5H 5N·S0 4 CHg
The titration is carried out using a commercial titrimeter equipped
with platinum electrodes. The water extraction is achieved with sol-
vents such as methanol, formamide, pyridine, dioxane, and
dimethy Iformamide.
Apparatus and Reagents

1. Any commercial titrimeter equipped with two platinum

electrodes.
2. Karl Fischer reagent (Sargent-Welch SC 12960) with water
equivalent of approximately 5 mg H 20/ml (for preparation see
AOAC method 32.048).
26 2. Sampling and Proximate Analysis

3. Water in methanol standard (Sargent-Welch SC 12966) with 1

ml = 1 ± 0.01 mg H 2 0 at 25°C. (This may be used to recheck
the water concentration of the Karl Fischer reagent.)
Procedure
Weigh a 3.0-g sample of dried beans ground to powder into a 50- to
100-ml glass-stoppered flask. Add 20 ml N,N-dimethylformamide.
Close top and seal. Place in 90°C oven for 1 hr, then mechanically shake
flask for 10 min. Cool to 25°C. Place supernatant in a glass-stoppered
centrifuge tube and centrifuge to remove debris.
Place 100 ml of formamide into a 250-ml flask and titrate to Karl
Fischer reagent end point. Add 15 ml of the supernatant from the
sample into the same 250-ml flask and titrate to the same end point.
Determine a dimethylformamide blank (15 mI). Calculate percentage
water in the sample as follows:
% H2 0 = {[mg H 2 0 in 15-ml sample supernatant - mg H 2 0 in 15-ml
blank (20/15)] -;- mg sample} x 100

D CRUDE FAT

Crude fat is the term used to refer to the crude mixture of fat-soluble
materials present in a sample. The lipid materials may include tri-
glycerides, diglycerides, monoglycerides, phospholipids, steroids, free
fatty acids, fat-soluble vitamins, carotene pigments, chlorophyll, etc.
The two methods most commonly used to determine crude fat are
wet extraction and dry extraction. Wet extraction is performed with
the water remaining in the sample. The Babcock method and the Mo-
jonnier method both are wet extraction methods used for crude fat
determinations in milk and milk products. These methods have also
been applied to other food products such as raw, canned, and frozen
fish (AOAC method 18.045). A common dry extraction method is de-
scribed in AOAC 7.055. Soxhlet extraction is performed with anhy-
drous ether. This technique extracts the crude fat into the ether which
is finally evaporated. Details of the procedure are given in Woods and
Aurand (1977). Dry extraction is preferred when it is inconvenient to
remove most of the water from a food.

Babcock Method
The Babcock method is a rapid method for fat determination in var-
ious food products. The test depends upon the fact that when milk or
Crude Fat 27

other foods are treated with concentrated sulfuric acid, the proteins
are first precipitated and then dissolved, permitting the fat to rise in
a layer at the top. The fat layer is measured in a calibrated tube; from
this value, the percentage of fat present may be calculated.
Reagents and Apparatus

1. Concentrated sulfuric acid with a specific gravity of 1.82 at 20°C.

2. Centrifuge capable of being electrically or otherwise heated to
55°-60°C. The proper speed of the centrifuge depends upon the
size of the head. The following rpms should be used for the cor-
responding head diameters: 10 in., 1074; 12 in., 980; 14 in., 909;
16 in., 848; 18 in., 800; 20 in., 759; 22 in., 724; and 24 in., 693.
3. Divider or calipers for measuring the height of the fat column.
4. Graduated cylinder or pipette graduated to deliver 17.5 ml of
sulfuric acid.
5. Standard Babcock test milk bottle approximately 6 in. in height,
with a neck not less than 63.5 mm long and graduated from 0
to 8 in percent units with intermediate graduations between the
units representing tenths of a percent.
6. Standard milk pipette graduated to contain 17.6 ml of water at
20°C, with a delivery time of 5-8 sec and a maximum error in
graduation not to exceed 0.05 ml. Check the pipette by measur-
ing from a burette the volume of 20°C water that it holds up to
the graduation mark.
7. Water bath held at 55°-60°C.
Procedure
Secure with the aid of the milk pipette, a 17.6-ml sample of well-
mixed milk. Transfer the sample to the standard test milk bottle, blow-
ing out the milk in the tip after it has ceased flowing. Hold the test
bottle at an angle and pour the sulfuric acid (15°-20°C) into it slowly,
in small portions, while rotating the bottle to wash all milk into the
bulb of the bottle. Thoroughly mix the acid and milk with a rotary
motion (so that no liquid gets into the neck of the bottle) until all traces
of curd have disappeared. When the acid and milk are properly mixed,
the mixture becomes hot and turns uniformly dark colored.
Transfer the bottle to a centrifuge and counterbalance with another
sample bottle or bottle containing water. When the centrifuge has at-
tained the proper speed (depending upon the diameter of the head),
centrifuge the sample for 5 min. Remove the bottle and add hot distilled
water (60°C or higher) until the bulb of the bottle is filled. Centrifuge
for 2 min at the proper speed. Again add hot water until the fat column
28 2. Sampling and Proximate Analysis

is forced into the neck of the bottle, and the liquid approaches the top
graduation on the bottle. Centrifuge 1 min at 55°-60°C.
Place the bottle into the water bath, immersing it to the level of the
top of the fat column. When equilibrium is attained, as evidenced by
no change in the lower fat surface, remove the bottle from the bath,
dry, and measure the height of the fat column with dividers or calipers.
The fat column is measured from its lower surface to the highest point
of the upper meniscus and is read directly as percentage by weight of
fat in the milk.
Remarks
When measured, the fat column should be translucent golden yellow
in color and free of visible suspended particles. Light-colored fat with
white particles beneath it indicates that the acid was too weak, the
milk was too cold when the acid was added, or insufficient acid was
used. Dark-colored fat containing dark specks indicates that too much
acid or too strong an acid was used.

Dry Extraction of Peanuts

Determine the moisture content of the sample by an appropriate
method. Weigh a 2- to 3-g sample into an extraction thimble and place
into a Soxhlet extraction apparatus. Attach the apparatus to a weighed
flask. Half-fill the flask with anhydrous ether. Using a heating mantle,
bring the ether to boil and extract for 16 hr. Rearrange the apparatus
for distillation and evaporate the ether to near dryness. Evaporate the
remaining ether under a hood and with gentle heating in the mantle.
(Care should be taken to remove peroxides from the ether prior to use.)
Dry the flask containing the crude fat extract for 2 hr at 90°C. Weigh
and calculate percentage of crude fat.

% Crude fat = Wt. of crude fat x 100

Wt. of dry sample

o CRUDE PROTEIN

For routine analysis, the determination of protein per se is not per-

formed due to the difficulty of extracting protein from a sample. Gen-
erally, uncomplicated samples that do not contain unusually high con-
centrations of nonprotein nitrogen-containing compounds (e.g., NH4 +,
free amino acids, urea, and other more complex nitrogenous com-
Crude Protein 29

pounds) may be analyzed by simply determining the percentage of ni-

trogen (as NH 3 ) and making the assumption that this nitrogen was
released from protein during digestion. The standard method for de-
termining nitrogen, the Kjeldahl procedure, can be adapted for appli-
cation to a wide variety of foodstuffs.

Kjeldahl Nitrogen Determination

In the Kjeldahl procedure, the nitrogen in an organic compound is
converted to ammonium salts, from which the ammonia is liberated
by adding a nonvolatile alkali. After distillation, the ammonia is de-
termined by titration. The initial decomposition of the organic com-
pound is usually accomplished by acid digestion, often in the presence
of catalysts.
Apparatus and Reagents
1. 500-ml Kjeldahl flasks.
2. Digestion apparatus with fume exhaust and distillation appa-
ratus. A combination unit specifically designed for Kjeldahl de-
terminations is convenient (Sargent-Welch S-63215).
3. Catalyst mixture (96% Na2S04, 3.5% CUS04, and 0.5% Se02).
4. 0.10 N Sulfuric acid.
5. 2% Boric acid solution.
6. Methyl red-bromcresol green indicator (0.016% and 0.083%, re-
spectively, in ethanol).
Procedure
Weigh a sample that is known to contain 0.03-0.04 g N into a 500-
ml Kjeldahl digestion flask. Add 8-10 g of catalyst mixture and 20 ml
of concentrated H 2S0 4. Heat gently in the digestion apparatus, then
vigorously until boiling begins. Continue heating at least 1 hr after
the mixture has cleared.
Add approximately 400 ml of deionized distilled water to the diges-
tion flask and a large piece of metallic zinc. The receiving flask should
contain 50 ml of 2% boric acid solution and a few drops of methyl red-
bromcresol green indicator. The delivery tube of the distillation ap-
paratus should be below the surface of the boric acid solution.
Add approximately 75 ml of 50% NaOH to make the mixture basic
and distill the ammonia into the boric acid solution; collect at least
300 ml of distillate. Wash the walls of the receiver and the condenser.
Titrate the distillate with 0.10 N sulfuric acid.
30 2. Sampling and Proximate Analysis

Calculations
The milliequivalents (meq) of NH3 released from the protein sample
equals the meq of acid required in the titration step; thus, meq NH3
= ml acid x normality acid. Since the meq of N in the protein sample
equals the meq of NH3, the grams of N can be obtained as follows: g
N = meq N x (0.014077 g N/meq N). The percentage nitrogen in the
sample is then calculated from the expression: % N = (g N/g sample)
x 100. These formulas can be combined to give
%N = (ml acid x normality acid) x 1.4
g sample
The weight of nitrogen in a sample can be converted to protein by
using the appropriate factor based on the percentage of nitrogen in the
protein. Protein in most foods ranges from approximately 15% to 20%
N; the average is near 16%. Thus, to convert g ofN to g of protein, the
common factor is 6.25 (100 -:- 16). However, the nitrogen-to-protein
conversion factor does vary among different food products, as shown
in Table 2.1. It is advisable to check appropriate sources and to use
the most accurate factor known for converting g of N to g of protein.
Remarks
Potassium or sodium sulfate is added to the digestion mixture to
increase the boiling point; this permits a shorter digestion time. Me-

Table 2.1. Factors for Converting Nitrogen to Protein in

Various Food Products

Food product Conversion factor

Milk 6.38
Rye 6.25
Oats 6.25
Corn 6.25
Buckwheat 6.25
Rice 6.25
Barley 6.25
Wheat 5.70
Wine 6.25
Peanuts 5.46
Brazil nuts 5.46
Almonds 5.18
Other tree nuts 5.30
Coconut 5.30

Source: AOAC 1980.

Crude Fiber 31

tallic zinc granules are added to prevent bumping. The zinc slowly
reacts with sodium hydroxide to produce hydrogen bubbles, which stir
and prevent superheating of the digestion mixture.
AOAC method 2.057 uses sulfuric acid as an oxidizing-digestion
agent and mercury or HgO as the metal catalyst. This alternative
method, in which ammonia is distilled into a known volume of standard
acid and the excess acid back-titrated with standard alkali, is described
in Chapter 6. Other metals (e.g., copper and selenium) have been used
as catalysts; mercury appears to be superior but suffers from its slow-
ness to catalyze the digestion. Selenium promotes a faster digestion,
but losses of nitrogen may occur.

D CRUDE FIBER

Crude fiber is a measure of the quantity of indigestible cellulose,

pentosans, lignins, and other components of this type present in foods.
These components have little food value but provide the bulk necessary
for proper peristaltic action in the intestinal tract.
Recent research into the roles of dietary fiber components have
caused reevaluation of some of the traditional concepts concerning
fiber. Actually, dietary fiber may be an unfortunate misnomer; a term
such as nondigestible portion may be more correct since this dietary
component mayor may not have a fibrous structure.
The nondigestible or dietary fiber fraction is a complex mixture of
different substances. The major ones are cellulose, the glucose polymer
that is the predominant material of plant cells; hemicellulose, a shorter
version of cellulose; pectin, the glue that binds plant cells together;
and lignins, amorphous, aromatic polymers that together with cellu-
lose form the woody cell walls of plants.
Dietary fiber acts to lower the concentration of low-density lipopro-
tein cholesterol in the blood, possibly by binding with bile acids. The
lignin fraction has been identified as the possible binding agent.
The most common technique for measuring a food's fiber content is
the crude fiber method, which dates from the 1800s and does not dif-
ferentiate one polysaccharide from another. AOAC method 7.073 de-
scribes the details ofthis frequently used method for determining crude
fiber in grains and similar products.

Crude Fiber Determination in Grains

The sample is digested with boiling dilute acid to hydrolyze the car-
bohydrate and protein materials contained in it. Further digestion with
32 2. Sampling and Proximate Analysis

boiling dilute alkali causes the saponification of the fatty materials

not extracted by ether. Both treatments contribute to the solution of
most of the mineral matter. The residue after digestion-consisting
mainly of fiber and a little mineral matter-is filtered off, dried, and
weighed. It is then ignited to constant weight and again weighed. The
difference in the two weights represents the weight of crude fiber
present in the sample.
Reagents and Equipment
1. Sulfuric acid solution containing exactly 1.25 g ofH2 SO,J100 ml
of solution.
2. Sodium hydroxide solution containing exactly 1.25 g of carbon-
ate-free NaOH/100 ml of solution.
3. Filtering asbestos-prepare by digesting on a steam bath for 8
hr or longer with a 5% NaOH solution and then thoroughly wash
with hot water. Again digest on a steam bath for 8 hr or longer
with a dilute HCI solution (1 part acid + 3 parts H 2 0) and thor-
oughly wash with hot water. Dry and ignite at a bright red heat.
4. Erlenmeyer flasks-'lOO to 750 ml in capacity.
Procedure
Carefully transfer the dried residue remaining in the extraction
thimbles after dry extraction of the crude fat to a 750-ml Erlenmeyer
flask; add about 0.5 g of the ignited asbestos along with the sample.
Add 200 ml of boiling sulfuric acid solution, connect the flask with a
reflux condenser, and digest the sample at boiling temperature for 30
min. (The contents of the flask should come to boiling within 1 min
and boil for exactly 30 min.) Rotate the flask at 5-min intervals to keep
the contents of the flask thoroughly mixed. The solid matter has a
tendency to stick to the sides of the flask, out of contact with the so-
lution, and this must be avoided as much as possible. A blast of air
directed into the flask will help to reduce frothing, which may carry
the sample up the walls of the flask and even into the condenser at
times.
At the completion of the digestion period, filter the contents of the
flask through a linen filtering cloth, using an appropriate filtering
device, and wash free from acid with boiling water. Bring a quantity
of the sodium hydroxide solution to boiling and keep it at this tem-
perature under a reflux condenser until used. Transfer as much as
possible of the washed residue on the filter back to the Erlenmeyer
flask by means of a spatula; the remainder of the residue is washed
into the flask with 200 ml of the boiling sodium hydroxide solution. A
Ash 33

wash bottle marked to deliver 200 ml of solution is helpful in effecting

this transfer.
Immediately connect the flask to a reflux condenser and boil for 30
min, rotating the flask at 5-min intervals. At the end of this period,
remove the flask and filter the contents through the same linen fil-
tering cloth used at the completion of the acid hydrolysis. Wash thor-
oughly with boiling water and then transfer the contents of the filter,
with the aid of a spatula and wash water, to a Gooch crucible prepared
with a thin asbestos mat. Wash the crucible and contents thoroughly
with water and then with 15 ml of ethanol.
Dry the crucible and contents at lOoo-HO°C until a constant weight
is attained, cooling the crucible in a desiccator before weighing. Then
ignite the crucible and contents in a muffie furnace at a dull red heat
(approximately 600°C) until all organic matter has been destroyed (ap-
proximately 20 min). The loss in weight during incineration represents
the weight of crude fiber in the sample from which the percentage
present in the sample may be calculated.
Calculations
The % crude fiber is calculated by the equation

% Crude fiber
(loss in wt of sample - loss in wt of asbestos blank) x 100
wt of sample

The asbestos mat blank is necessary to eliminate error due to ignitable

materials in the asbestos. The % crude fiber also may be expressed on
a desired moisture basis, as follows:
% Crude fiber on desired moisture basis
=
01.
-10
Cru de fib (100 - % moisture desired)
1 er x -:----------------'----
(100 - % moisture in ground sample)

DASH

When either organic or inorganic compounds are decomposed or re-

leased at high temperatures (500°-600°C), the remaining residue is
the ash. This residue consists of oxides and salts containing anions
such as phosphates, chlorides, sulfates, and other halides and cations
such as sodium, potassium, calcium, magnesium, iron, and manganese.
During the ashing process organic salts decompose, losing the car-
34 2. Sampling and Proximate Analysis

bon-containing moiety. The metal from such salts forms an oxide or

reacts with other anions of the matrix. Some metals (e.g., cadmium
and lead) may be volatilized during ashing; therefore, if the ash is to
be examined for trace elements, care should be exercised to prevent
losses during ashing. For most foods, ashing at 485°C or less for 12 hr
will give acceptable results for trace element analysis. Losses of min-
erals due to carbon (soot) release can be appreciable from carbon-con-
taining samples. This mechanical loss of ash can be avoided by starting
the incineration in the mume furnace at a low temperature (room
temperature) and allowing the temperature to rise slowly. Air currents
may be a problem if the door of the furnace is opened suddenly. Gen-
erally, samples should not be placed in areas of the furnace closer than
1 in. from the rear wall and 1.5 in. from the front.

Ashing Sugar and Sugar Products

Ashing of sugar and sugar products is described in AOAC methods
31.012 and 31.013. AOAC method 31.014 gives the procedure for sul-
fated ash, and 31.015 describes the determination of soluble and in-
soluble ash.
Procedure
Heat a 5- to 10-g sample in a previously tared 100-ml platinum or
Vycor crucible to HO°C until the residual water is lost. Add 5 drops
of olive oil and heat for 30 min under a heat lamp. Place the dish or
crucible in a cold mume furnace and bring slowly to 525°C for 12 hr.
Cool and weigh the crucible and ash residue.

D SELECTED REFERENCES

AACC. 1976. Approved Methods of the American Association of Cereal Chemists. Vols.
I and II. American Assoc. of Cereal Chemists, St. Paul, MN.
AOCS. 1979. Official and Tentative Methods of the American Oil Chemists' Society.
Vols. I and II. American Oil Chemists' Society, Champaign, IL.
AOAC. 1980. Official Methods of Analysis. 13th ed. W. Horowitz (editor). Assoc. of Of-
ficial Analytical Chemists, Arlington, VA.
AOAC. 1984. Official Methods of Analysis. 14th ed. S. Williams (editor). Assoc. of Official
Analytical Chemists, Arlington, VA.
JOHNSON, N. L. 1963. Sampling devices. Food Technool. 17, 1516-1520.
POMERANZ, Y. and MELOAN, C. E. 1978. Food Analysis: Theory and Practice. Rev.
ed. AVI Publishing Co., Westport, CT.
WOODS, A. E. and AURAND, L. W. 1977. Laboratory Manual in Food Chemistry. AVI
Publishing Co., Westport, CT.