Analysis of Soap and Related Materials

Thomas E. Wood
Consultant,305 Coweto Court, Loudon, Tennessee37774, USA

Introduction
The analytical methods available to the soap chemist for evaluating raw material and soaps include both
the classical physical and chemical analytical procedures that are found in standard reference manuals
of analytical methods, and the modern instrumental methods including gas chromatography and high-
performance liquid chromatography. The two most important reference sources for analytical methods
for soap and soap raw materials include the OJicial Method and Recommended Practices of the American
Oil Chemists’Society (AOCS, 2009) and the Annual Book of ASTM Standard of the American Society
for Testing and Materials, Volume 15.04 (ASTM, 2008). The instrumental methods are generally found
in publications like the Journal of the American Oil Chemists’Society,the Journal of Chromatography, the
JournalofLiquid Chromatography,and other similar publications. Please refer to Table 14.1 for a concise
list of references to selected AOCS Official Test Methods for soap and soap raw materials.

Chemical and Physical Characteristics of Soap Raw Materials

Among the chemical properties of fats, oils, and fatty acids that are important to the soap chemist, three
of the most important are acid value, saponification value, and iodine value. Additionally, the trans-
isomer content can be an important consideration whenever hydrogenated stocks are involved. Other
important characteristics include the titer, free fatty acid, raw color, bleached color, moisture, insoluble
impurities, and unsaponifiable matter.

Acid Value
Acid value is defined as the number of milligrams of potassium hydroxide (KOH) required to neutralize
the free acids in 1 gram of sample.
For samples of fats and oils, the determination of the acid value involves the simple titration, in
alcohol, of an appropriately sized sample to the phenolphthalein end point with essentially no sample
preparation required. In this case, the acid value is virtually a measure of the percentage of free fatty
acid present, and can be expressed as such by simple mathematical conversion. Typically, in the case of
fits and oils, the level of free fatty acid should be very low, ranging from near zero to several tenths of
a unit for higher grade material to a few whole units for lower grade oils. Please refer to AOCS Official
Method Cd 3663 for more information on acid-value determination for fats and oils.
For samples of htty acid stock, the acid-value determination is also a direct titration of the sample
in alcohol to the phenolphthalein end point without any special sample preparation. Since fatty acid
stocks, by their nature, are essentially all free fatty acid, the acid value will range above two hundred
units for most of the common fatty acid blends used in soap making. For fatty acid stocks, the acid value
will approach the saponification value. Please refer to AOCS Official Method Te la-64 for the details
for acid-value determination of fatty acid blends.
For soap products, the acid value is determined on the total fatty acids present including the free
acids, which may range from none to several percent, plus the major portion that is combined with
the cation as soap. Consequently, the acid-value determination for soap requires an initial sample
’399
400 0 T. Wood
Table 14.1, Selected AOCS OfficialTest Methods

TesVAOCS Method for: Fats 81Oils Fatty Acids

Acid value Cd 3d-63 Te la-64
Free fattv acid Ca 5a-40
~~~

Insoluble impurities Ca 3a-46

Iodine value Cd ld-92 Tg la-64
Moisture/volatiles, 130°C Ca 2c-25
Colorimetric color, Wesson method Cc 13b-45
Colorimetric color, Lovibond method Cc 13e-92
Photometric color CC 13~-50 Td 2a-64
Alcoholic samnification color
~
cc 134-94'
Refined 8I bleached color CC 8d-55
R&B; alcoholic saponificationcolor cc 13f-94'
Saponification value Cd 3-25
Titer CC 14-59 Tr la-64
UnsaDonifiable matter Ca 6a-40 Tk l a 4
~~ ~ ~~ ~

Water. Karl Fischer method Ca 2e-84 Tb 2-64

TesVAOCS Method for: Soap Soap with Detergent
Acid value Da 14-48
Anhydrous soap content Da 8-48 Db 6-48
Chlorides Da 9-48 Db 7-48
~~

Free fatty acid/free alkali Da 4a-48 Db 3-48

Glycerin Da 23-56
Iodine value Da 15-48
Moisture 81volatiles Da 2a-48 Db 1-48
SaDonification value Da 16-48 Db 8-48
~~~

Titer Da 13-48
"AOCS Recommended Practice

preparation where the combined fatty acids are liberated by acidulation with an excess of sulfuric acid,
recovered, and then dried. Following this sample preparation, the acid value is determined on the fatty
acids as described above for fatty acid blends. Again, an acid value more than two hundred units would
be expected for the fatty acids associated with most ordinary soaps. Please see AOCS Official Method
Da 14-48 or ASTM Standard Method D 460, Sections 48 and 49 for more details on acid-value
determination of soap.
The acid value is a key indicator of the fatty acid composition of soap since it is inversely and linearly
related to the average molecular weight and chain length of the fatty acids in the blend. For example,
tallow fatty acid has an approximate acid value of 204, while coconut fatty acid has an approximate acid
value of 268. The usefulness of the acid-value determination lies in helping to establish the composition
soap fatty acid blends. In the case of a fatty acid blend that is derived from an 80:20 tallow/coconut oil
blend, for example, the expected acid value should be about 216 based on the weighted average of the
acid values for the blend. If the ratio were to shift toward higher tallow content and lower coconut oil
content, a lower acid value would result. For example, a 90: 10 tallowlcoconut oil blend would result
 Analysis of Soap and Related Materials 0 401

in an approximate acid value of 210. Conversely, a lower tallow-to-coconut oil ratio would result in a
higher acid value. A 70:30 tallow/coconut oil blend would have an acid value near 223.
The acid value is frequently included in specifications for soap with only a lower limit indicated.
This is done for reasons of both quality and economics since tallow, with the lower acid value, is the
lower cost commodity with the poorer performance characteristics.

Saponification Value
Saponification value is defined as the number of milligrams of KOH required to saponify 1 gram of
sample.
The procedure is carried out directly on a sample of the stock for both commercial whole oils and
fatty acid blends. In the case ofsoap samples, the fatty acids must first be prepared by acidulating a sample
of the soap, recovering the liberated fatty acids, and drying the recovered fatty acids. The saponification-
value determination is carried out by refluxing the sample of oil or fatty acid with an excess of KOH in
an alcoholic solution for 30 to 60 minutes. Along with each sample, or set of samples, a blank is also
run. After the reaction is completed and cooled, the excess KOH is titrated with standardized 0.5 N of
hydrochloric acid. With an appropriate calculation, the difference between the titrations of the blank
and the sample is then reported as the saponification value. For more information, see AOCS Official
Methods Cd 3-25 for fits and oils, TI la-64 for fatty acids, and Da 16-48 for soap and soap products.
Note that any difference between the results of the saponification value and the acid value on a given
sample is called the ester value. Not unusually, the saponification value can be one or two units higher
than the acid value on a given sample of soap fatty acid. This results from the reaction of trace amounts
of naturally occurring ester-like compounds that are reactive under the conditions of the saponification-
value determination, but not under the milder conditions of the acid- value determination.

Iodine Value
Iodine value is defined as the number of centigrams of iodine absorbed by 1 gram of sample.
For samples of fats, oils, and fatty acids, the iodine-value determination is performed directly on an
appropriately sized sample of the material that was liquefied by melting and filtered to remove any trace
impurities including moisture. For samples of soap, the fatty acids must first be liberated with sulfuric
acid, recovered, and then dried. The iodine value is then determined on the prepared fatty acids.
The procedure is carried out both by reacting the sample of oil or fatty acid with an excess of an
iodine monochloride solution (Wij’s solution), and by titrating the excess iodine with a standardized
sodium-thiosulfate solution by using a starch indicator for the end-point determination. Each sample,
or set of samples, is done with a blank to determine the quantity of iodine consumed by the sample.
The difference between the blank and the sample is attributable to the iodine absorption by the sample.
With appropriate calculation, the difference is reported as the iodine value for the sample. See AOCS
Official Methods Cd ld-92 for fats and oils, T g la-64 for fatty acids, and Da 15-48 for soap and soap
products for detailed information on the procedures. Also, for soap and soap products, refer to ASTM
Standard Method D 460, Sections 50 to 52, for detailed procedures.
The iodine value is directly related to the degree of unsaturation present in the fat, oil, or fatty
acid. The iodine value also serves as an indicator of the relative hardness of fats and of the derived fatty
acids and soap product for otherwise comparable fat stocks. In general, higher levels of unsaturation, as
indicated by higher iodine values, indicate a tendency toward softer fat stocks and softer soap product.
This relationship is clearly evident when comparing natural animal fats such as tallow and grease that
have roughly comparable average chain-length distributions and average molecular weights, as indicated
by their saponification values, but which have markedly different amounts of unsaturated fatty acids.
Tallow, with its lower iodine value indicating less unsaturation, will melt at a higher temperature and
will result in firmer soap compared with grease.
402 0 T. Wood
The underlying basis for the physical effects of lower melting point and softer material associated
with natural animal fats that have a higher degree of unsaturation is found in the predominance of cis-
unsaturated fatty acids in naturally occurring fats. Due to the geometry around the cis double bonds,
the molecules with the cis configuration have a distinctive bend in the carbon chain at the site of the
carbon-to-carbon double bond. This results in the looser packing of molecules in the solid, causing
reduced intermolecular forces, and consequently lower, melting point. The effects on the properties
of fatty acids and soap resulting from varying the cis- and trans-isomer content are discussed in a later
section.

Titer
Titer is defined as the temperature, expressed in degrees centigrade, at which fatty acids solidify.
Generally, titers of fatty acids will vary inversely with iodine values. Titer is frequently used as a
quality-control and process-control measure in soap making since it is a good indicator of the processing
characteristics of the resultant soap at the bar-finishing stage of manufacturing. Soap made from higher
titer fatty acid blends tends to be firmer, and vice versa. The effects of trans-isomer content on this
relationship are discussed in a later section.
The typical inverse relationship between titer and iodine value for otherwise comparable fats is
illustrated in Table 14.2. Both lard and tallow are similar in average molecular weights, but lard, with
the significantly higher iodine value, has a much lower titer and will form a softer soap
For tallow, Grompone ( 1 784) reported that titer is fundamentally dependent on the stearic/oleic
acid ratio. The relationship between titer of tallow and stearic-acid content is direct and linear. The
relationship between titer and oleic-acid concentration is inverse and linear. Apparently, the titer is not
influenced significantly by the varying levels of palmitic acid.
The titer test is always performed on the sample in the form of free fatty acids. For fatty acid blends,
the procedure requires no special sample preparation. For fats and oils, a sample of the stock to be
evaluated must first be saponified, followed by acidulation, and then the recovery and drying of the
fatty acids. For soap samples, the sample needs to be acidulated with subsequent recovery and drying
of the fatty acids. Please refer to AOCS Official Methods Cc 14-57 for fats and oils, Tr la-64 for fatty
acids, and Da 13-48 for soap and soap products. Also, for soap and soap products, see ASTM Standard
Method D 460, Sections 46 and 47.
Table 14.2. Comparative Data for Lard and Tallow
Property Lard Tallow
SaponificationValue 190-202 190-200
~ ~~~~~~

IodineValue 53-77 35-48

liter (“C) 32-43 40-46

Effects of Trans Isomers on Fatty Acid and Soap Properties

The variation in the tram-isomer content of fatty acid blends impacts the titer, soap characteristics, and
the relationship between titer and iodine value for otherwise similar fatty acid stocks. While the iodine
value can serve as a useful indicator of hardness for naturally occurring fats where cis unsaturation
is prevalent and tram unsaturation is low and consistent, this reliability can significantly diminish
whenever hydrogenation is involved. If hydrogenation is carried out under conditions of higher-than-
normal temperature, lower-than-normal hydrogen feed, or with a poor catalyst, a significant amount of
rearrangement from cis to tram isomers can occur rather than the reduction of the double bonds. During
the hydrogenation of fatty acids, the reduction of cis-unsaturated fatty acids (with their characteristically
nonlinear structure) to saturated fatty acids (that are essentially linear molecules) will predictably result
 Analysis of Soap and Related Materials 0 403

in the formation of harder fatty acid with a lower iodine value. However, the rearrangement of cis to
tram isomers, insofar as it occurs, will not contribute to the lowering of the iodine value, but will result
in harder fatty acid stock with a higher melting point.
The relative amount of cis and trans isomers not only has an impact on the melting point and
hardness of the fatty acid, but also affects the hardness and plasticity of the resulting soap. The trans-
isomer content will influence titer independent of the degree of saturation. Again, this phenomenon
can become important when fatty acids used in soap making are subjected to hydrogenation. The
conversion of a significant amount of cis to tram isomers will result in harder-than-expected soap for a
given iodine value. Under these conditions, titer may be a more useful process-control measure for the
soap maker than iodine value.
Some interesting relationships between melting points and iodine values are seen in Table
14.3 (Unichema Chemicals, 1987), where analogous sets of Fatty acids that have varying degrees of
unsaturation and &trans ratios are listed. For the four groups of isomers listed, both the iodine values
and melting points are given. In the first pair, both isomers have a double bond at the number-6 carbon
atom: the first is a cis structure, and the second is a tram structure. Both have an iodine value of 90.
Note, however, the difference in melting points, with the trans isomer having a melting point of 21°C
higher than the cis isomer. The other two pairs of isomers listed here show the same kind of relationship
between isomeric structure and melting point. In the last set, each of the three Fatty acid isomers
contains three double bonds. Note that in the first structure shown, all three double bonds are cis; in
the second case, one is cis and the other two are trans; and in the third case, all three are trans. All three
of these isomers have the same iodine value of 274. However, a dramatic increase occurs in the melting
point with increasing trans-isomer content.
Table 14.3. Comparative Iodine Values and Melting Points
~~ ~ ~~ ~~ ~~~

Systematic Name Formula IV MP("C)

6c-octadecenoic acid C..H,.O, 90 33
6t-octadecenoic acid
~
C..H..O. 90 ~~ ~~
54
9c-octadecenoic acid C18H340, 90 16.3
9t-octadecenoic acid C..H,.O, 90 45
9~,12~-octadecadienoic
acid C18H3202 181 -5.0
9t,l2t-octadecadienoic acid Cl*H,,O, 181 28-29
9~,12~,15c-octadecatrienoic
acid C..H.,O. 274 -1 1
~ ~~

921 1 t,l3t-octadecatrienoic acid C18H,02 2 74 49

9t,ll t,l3t-octadecatrienoic acid C,.H,,O, 274 71 .S

Trans Isomer Measurement

The classical method of analysis for iodine value will establish the relative amounts of saturated and
unsaturated fatty acids present, but will not differentiate between the cis and trans isomers. Infrared
spectroscopy can quantify the tram-acid content of fitty acid blends. The method is based on the
absorption at 966 cm-' by the trans double bonds in the sample. The absorption is directly proportional
to the concentration of trans double bonds present in the sample. The absorption is thought to be
independent of the position and number of trans double bonds present. Conjugated tram double bonds
in excess of 5% may interfere with this method. The presence of such interference is indicated by
absorption at 987 cm-I. The details of this method are found in AOCS Official Method Cd 14-95. One
may achieve a complete analysis of cis/trans isomers by capillary gas chromatography by using AOCS
Official Method Ce lh-05.
404 0 T. Wood
Color Evaluation of Soap Raw Materials
In the following sections, we review the various methods for evaluating the color of soap raw materials
and of the resulting neat-soap product. The color of raw fats and oils stock is an indicator of the degree
of abuse and degradation to which the material was subjected. To that extent, it is a measure of the
degree of additional processing that is required to produce white soap and of the potential for the loss
of fat in the soap-making process. In the case of fatty acid stocks, the color is also an indicator of the
quality of the material, and will have a direct bearing on the color of the neat soap made from the fatty
acid stock. If the color is high, it may also be an indicator of degradation that will affect the odor of the
resulting soap.

Raw Color of Fats and Oils by Colorimeter

Probably the most widely used method of color measurement for fats and oils in the United States is the
Wesson method that uses the Lovibond AOCS Tintometer, Model AF710, that is manufactured by The
Tintometer, Ltd., Amesbury, Wiltshire, United Kingdom (www.tintometer.corn).
This method determines the color of fats, oils, and fatty acids by the comparison of a column of the
oil with standard glass slides under specific controlled conditions. The procedure is also commonly used
for commercial fatty acid blends where, in the absence of any turbidity, the material can be evaluated
while molten.
In the Lovibond method, a 5.25-inch column of material is placed in a standard color tube that
is specially designed for use with the AOCS Tintometer. ‘The tube is inserted into the port of the lit
cabinet where the color of the material in the sample tube can be compared with the standard red- and
yellow-glass slides that have numerical scale values. Usually, both the red and yellow values are specified.
Frequently, only the red value is specified and reported, particularly for tallow and coconut oil. The
details of this method are found in AOCS Official Method Cc 13b-45.
The accepted international standard based on BS 684 is I S 0 15305, the Lovibond method. The
Model F (BS 684) Lovibond Tintometer that is also made by The Tintometer, Ltd. is suitable for this
method. Please refer to the AOCS Official Method C c 13e-92 for details of this method.

Color of Fats, Oils, and Fatty Acids by Photometric Measurement

Another common method of measuring the color of fats and oils utilizes visible spectrophotometry. The
photometric measurement of the color of fats and oils measures the absorbance at 460, 550, 620, and
670 nm. The photometric color is expressed as follows:

Photometric color = 1.29A4, + 69.7A550t 41 .2A620- 56.4A6,,

(Eq.14.1)

Please refer to AOCS Official Method C c 13c-50 for the details of this method.
Frequently, the color of fatty acids is also determined by photometric measurement. Either the
absorbance or transmission is measured at 440 nm and 550 nm. The results are reported either as “%
transmission at 440/550 nm” or in terms of the “photometric index” that is expressed as: 100 x A440and
100 x AZso. Please refer to AOCS Official Method T d 2a-64 for details of this method.

Refined and Bleached Color of Fats and Oils

O n e can use the refined and bleached color test to predict the color after the processing of intermediate
and lower grades of tallow that will require pretreatment before use in soap making.
The tallow sample is refined by neutralizing the free fatty acids with caustic soda. The neutral fat is
then separated by filtration through cheesecloth or filter paper to remove any soap formed during the
 Analysis of Soap and Related Materials 0 405

refining step. The neutral fat is then treated with activated bleaching earth. The mixture is then filtered,
followed by the reading of the Lovibond color of the clear filtered fat. Please refer to AOCS Official
Method Cc 8d-55 for the details of this procedure.

Direct Bleach Test forFats and Oils

The direct bleach test for tallow is useful when working with higher grades of inedible tallow that are
low in free-fatty acid content. The method uses 300 grams of fat that is heated to 1 10°C, followed by the
addition of 15 grams of activated bleaching earth. The mixture is then agitated for 10 minutes at 80°C
or higher, followed by filtration through a heated funnel at 70°C. After the bleaching and filtration, the
Lovibond color is determined. All supplies used for this procedure are the same as those specified for the
refined and bleach test above. The results of this test indicate the potential for making white soap from
fats that will be pretreated by bleaching without refining.

Alcoholic Saponification Color for Fats and Oils

In AOCS Recommended Practice Cc 13g-94, a method is presented for saponification color
determination of high-quality tallow and coconut oil intended for use in soap making. In this method,
a sample of the untreated oil is reacted with an excess of alcoholic KOH solution. Upon completion of
the saponification reaction, a sample of the soap solution is placed in a 5.25-inch tube color evaluation
using the Wesson method for Lovibond-color measurement found in AOCS Official Method Cc
13b-45.
In AOCS Recommended Practice Cc 13894, methods are presented for refined and bleached color
and for saponification color of lower grade tallows and greases intended for use in soap making. First, the
sample is subjected to a caustic refining and bleaching operation. Following the refining and bleaching,
a portion of the sample is reacted with an excess of an alcoholic KOH solution. After saponification is
completed, a sample of the soap solution is transferred to a 5.25-inch tube for color reading using the
procedure in AOCS Official Method Cc 13b-45.

Saponification Color of Fatty Acids

The color of fatty acids for soap making can be evaluated by measuring the alcoholic saponification
color that determines the color of a potassium soap solution. This method is applicable to normal-soap
fatty acid blends with a saponification value in the range of 214 to 220.
In this procedure, a sample of the fatty acid is dissolved in ethanol, followed by neutralization with
an aqueous 39% of a KOH solution. The percentage of transmission at 440 and 550 nm is determined
for the potassium soap solution. This method serves as a good indicator for the expected color of
finished soap to be made from the fatty acid blend. Due to the relatively short period of time required
to run this procedure, it can be useful as a receiving quality-control method for bulk fatty acids.

Approximate Conversionsfor Various Color Scales:

Figure 14.1 gives the approximate equivalence of various visual color scales commonly used for fats and
oils, including Gardner, APHA, F.A.C, Lovibond, and percent transmission. Additional correlations for
several methods of measuring the color of Fats and oils are given in AOCS Official Method Cc 13b-45.
The American Oil Chemists' Society (AOCS) does not encourage the use of conversion charts for color.
They should be used with caution for an estimation or an approximation purpose only.
406 0 T. Wood

TRANOMIWION H Ilkn,440/860

Fig. 14.1, Approximate equivalents of various visual color scales.

Free Fatty Acid Content of Fats and Oils

For determining the free fatty acid content of fats and oils, a weighed sample is dissolved in ethanol,
followed by titration with a standardized NaOH solution to the phenolphthalein end point. The result
is calculated and conventionally reported as percentage of oleic acid for most samples. Please refer to
AOCS Official Method Ca 5a-40 for details of this method.
The presence of elevated levels of free htty acid can result from the storage of fats and oils at
elevated temperatures in the presence of water, causing hydrolysis of a portion of the triglycerides. Also,
prolonged enzymatic activity in the case of animal fats can result in undesirable levels of free Fdtty acid.
The presence of excessive free fatty acid will indicate a decreased level of available glycerine for recovery
and an increased potential for color and odor degradation of the stock.

Moisture Content of Fats, Oils, and Fatty Acids

The moisture content of tallow and other nonlauric oils is generally determined by the 130°C air-oven
method found in the AOCS Method Ca 2c-25. The air-oven method is not recommended for coconut
and palm kernel oils. For the standard Karl Fischer titration method that is recommended for lauric oils
and other fat stocks, please see AOCS Method Ca 2e-84. For the Karl Fischer method for fatty acids,
see AOCS Method T b 2-64.

Insoluble Impurities in Fats and Oils

?his procedure determines dirt, bone meal, and other insoluble impurities present, under the conditions
of the test, in all commercial fats and oils.
A properly sized sample of the fat is dried to constant weight in an oven; one can use the residue
from the standard air-oven moisture determination if available. The dried sample is dissolved in a
warm 50-mL portion of kerosene, and filtered through a dried, tared Gooch crucible with a glass-fiber
filter. The filter is washed with five 10-mL portions of hot kerosene, followed by a thorough washing
with petroleum ether. The crucible and contents are dried to constant weight in a 101°C air oven,
cooled under desiccation, and weighed. The weight of residue is reported as the percentage of insoluble
impurities. See AOCS Official Method Ca 3a-46 for more information.

Unsaponifiable Matter in Fats and Oils

Unsaponifiable matter can be composed of such things as cholesterol, fatty alcohols, and denaturants
in fats. These materials do not react with NaOH to form soap and are extractable with fat solvents. The
level of unsaponifiable material in fats and oils can range as high as a few tenths of a percentage for
better grade materials. Apart from the obvious economic consideration associated with the purchasing
of fats for soap making, the unsaponifiable matter can also have a negative impact on soap performance.
One unit of unsaponifiable matter in soap can reduce the detergency action of at least three times its
weight in soap (The Procter & Gamble Co., 1967).
 Analysis of Soap and Related Materials 0 407

The unsaponifiable-matter content of fats is determined by vigorously reacting a sample with

KOH, followed by petroleum-ether extraction of the unreactive organic matter. Several petroleum-
ether extractions are collected and dried, with the amount of residue determined gravimetrically.
Unsaponifiable-matter content can be determined by AOCS Method Ca 6a-40 in fats and oils, T k
la-64 in fatty acids, and Da 11-42 in soap. Also, for soap and soap products, see ASTM Standard
Method D 460, Sections 36 to 38.

MIU Content of Fats and Oils

The sum of the moisture, insoluble matter, and unsaponifiable matter present in commercial fats and
oils is typically part of the material specification, the MIU, and may serve as the basis for a price
adjustment if the total exceeds specified limits.

Analysis of Soap and Minor Ingredients

In the following sections, we review the principal test methods used for analyzing soap and soap
products. Included here are methods for anhydrous soap, moisture, glycerine, chlorides, free fatty acid
or alkalinity, and certain hnctional ingredients.

Anhydrous-Soap Content
In the anhydrous-soap determination, a precisely weighed soap sample is treated with mineral acid to
liberate the fatty acids. The fatty acids are recovered and reacted with a NaOH solution to form soap.
The resulting soap is dried and weighed to establish the anhydrous-soap content of the original sample.
Please refer to either AOCS Official Method Da 8-48 or ASTM Method D 460, Sections 24 to 25, for
details of this procedure.

Moisture Content of Soap

The moisture content ofordinary sodium soap can be reliably determined by the 105°C air-oven method
as described in the AOCS Method Da 2a-48 and by the ASTM Method D 460. This method is not
reliable in the presence of elevated glycerine and free-fatty acid levels.
Another use611 method that is specific for water is the Karl Fischer titration. This method provides
for a rapid determination of water content, and is especially useful when water is present in small
quantities. It may be used on glycerine, fats and oils, fatty acids, soap, and soap product. Among
the automated systems available for a Karl Fischer moisture analysis is the Photovolt Aquatest 2010,
manufactured by Photovolt Instruments Inc. (www.photovolt.com), that generates a Karl Fischer reagent
on demand based on the sample. Since a Karl Fischer reagent is generated electrolytically, no volumetric
measurement of reagent is required, and no standardization of the solution is needed. Instead, the water
content of the sample is determined and computed by the instrument based on the equivalence of one
coulomb of electricity to 186.53 micrograms of water.

Free-Glycerine Content of Soap

This method determines the free-glycerine content of soap by way of the oxidation of the glycerine with
periodic acid.
The sample of soap containing glycerine is mixed with chloroform and glacial acetic acid, and then
quantitatively transferred to a volumetric flask. Distilled water is added, and the sample is dissolved
with heating, if necessary, to completely dissolve the sample. The flask is filled to volume with distilled
water and stoppered, followed by agitation to effect thorough mixing. The water and chloroform layers
are then allowed to separate. An aliquot of the aqueous layer containing the glycerine is withdrawn and
added to a beaker containing an excess of periodic-acid reagent where the oxidation-reduction reaction
is allowed to proceed for 30 minutes. Two blanks are prepared for each batch of samples being analyzed.
408 0 T. Wood
Afier the reaction period has passed, potassium iodide is added to each beaker to liberate the excess
iodine. Each sample and blank are then diluted with deionized water and titrated with standardized
0.1 N of a sodium-thiosulfate solution. The titration difference between the blank and the sample is
converted with appropriate calculation and reported as a percentage of free glycerine in the soap sample.
See AOCS Official Method Da 23-56 and ASTM Standard Method D 460, Sections 82 to 84.
Another useful procedure for the determination of glycerine content of soap is a modification of
the sodium metaperiodate method in the AOCS Official Method Ea 6-5 1. In this procedure, a 40.0g f
0.001 g sample (or other sample size based on the expected results found in the table in AOCS Method
Ea 6-51) is weighed into a 250-mL Erlenmeyer flask. Add 100 mL of deionized water and heat on a
steam bath to dissolve. Once dissolved, the solution is acidified to a slight excess with 1:4 sulfuric acid
using a methyl-orange indicator. The flask is covered with a watch glass and heated to clarify the fatty
acid layer. The solution is filtered through a qualitative filter paper into a 400-mL beaker, retaining the
fatty acids on the filter paper. The fatty acids are washed with several small portions of hot deionized
water until the washings are neutral to methyl orange. Cool and neutralize to methyl orange with 50%
of NaOH very carefully. Adjust the pH of the solution so that it is definitely acid to methyl orange.
Add a few glass beads and boil for 5 minutes to expel any CO,. Boil long enough to reduce the volume
to 75 mL. Some glycerine may be volatilized if the solution volume goes below 50 mL. Cool to room
temperature. Prepare a blank of distilled water and process with the sample solutions in an identical
manner. Buffer the pH meter using pH 7.0 and pH 10.0 buffer solutions. Neutralize the sample and
blank to a pH of 8.1 f 0.1 with NaOH. (The strength of NaOH used depends on the acidity of the
sample.) Final adjustment should be made with 0.125 N or weaker of NaOH or with 0.1 N or weaker
of H,SO,. The volume of the pH-adjusted solution should not be more than 100 mL. Pipet 50 mL
of sodium metaperiodate solution to the pH-adjusted samples. Swirl the beaker to ensure thorough
mixing, cover with a watch glass, and immediately place in a dark cupboard at room temperature for 30
minutes. Remove from the cupboard, add 10 mL of 50% of an ethylene-glycol solution by graduated
cylinder, swirl gently, and allow to stand for 20 minutes. Titrate by using 0.125 N of NaOH to a pH of
8.1 f 0.1. A dropwise addition of NaOH should be made as the 8.1 pH is approached. The results are
calculated as follows, where A = mLs of NaOH used for sample titration and B = mLs of NaOH used
for blank titration.
(A - BXNX9.209)
%Glycerin =
Sample weight in grams
(Eq. 14.2)

Chlorides in Soap
?he soap sample, usually 5 grams, is dissolved in about 300 mL of chloride-free deionized water, with
boiling as needed. The soap is then reacted with an excess of magnesium nitrate to form insoluble
magnesium soaps, filtered, and washed with chloride-free deionized water. The filtrate is then titrated
with a standardized 0.1 N of a silver-nitrate solution with a potassium-chromate indicator. The result
is calculated and usually reported as a percentage of sodium chloride. See AOCS Official Method Da
9-48 and ASTM Standard Method D 460, Sections 53 to 55.

Free Fatty Acid and Free Alkalinity in Soap

The free fatty acid or free alkalinity in soap is determined by titration, with standard alkali or acid as
appropriate, to the phenolphthalein endpoint. For most soaps that contain no significant amount of
alkaline salts, the procedure involves the titration of the sample, usually 10 or 20 grams dissolved in
neutralized ethanol, with either a standardized NaOH solution (0.1 N or 0.25 N) for acidic samples
or sulfuric acid (0.1 N or 0.25 N) for alkaline samples to the phenolphthalein end point. The results
 Analysis of Soap and Related Materials 0 409

for alkaline soaps are usually reported as either a percentage of NaOH or Na,O for sodium soaps and a
percentage of KOH or K,O for potassium soaps. For acidic soaps, the results are typically reported as a
percentage of oleic acid, coconut acid, or lauric acid.
Note that the titration is always performed in neutralized ethanol, rather than water, due to the
hydrolysis ofsoap in water that would buffer the solution and interfere with the endpoint determination.
See AOCS Official Method Da 4a-48 and ASTM Standard Method D 460, Section 21 for more
detai I.

Triclocarban and Triclosan in Soap by Ultraviolet Absorbance

Two important and widely used additives found as the active ingredients in many antibacterial soaps
and as the functional ingredients in many deodorant soaps are triclocarban and triclosan. The chemical
structures of these two compounds make them amenable to quantitation by ultraviolet (vv) absorbance
spectroscopy methods. Triclocarban absorbs at 265 nm, and triclosan at 282 nm.
Note that interference may be encountered due to absorbance by fragrance components or other
soap ingredients at these same wavelengths. This interference can be nullified by preparing a calibration
curve for each formulation of soap product. By preparing stock solutions of the product matrix without
the active ingredient and a separate stock solution of the active ingredient, various concentrations
of the active ingredient over the concentration range of interest can be prepared for W-absorbance
measuremen t.
Listed in Table 14.4 are the W-absorbance values obtained for triclocarban concentrations over
a range from 0.0 to 1.0 mg/l00 mL in a typical soap formulation. Since the W absorbance is a
direct linear function of the analyte's concentration, the relationship between U V absorbance and
concentration can be expressed by an equation in the form of a straight line:

A,,, = mC+ b
(Eq. 14.3)

where ACoRR is the U V absorbance and Cis the triclocarban (or triclosan) concentration in mg/l00 mL.
The slope of the line, m,and theyintercept, 6, can be derived by the treatment of the concentration and
absorbance data using linear least-squares equations (Arnold & Ford, 1972) as follows:

(Eq. 14.4)

(Eq. 14.5)

Thus, the linear equation for the data in this example would be:
A,, = (1.4 I)@)+ 0.0624

(Eq. 14.6)
Upon rearrangement, the equation becomes:

C=
- 0.0624
ACoRR
1.41
(Eq. 14.7)
410OT.Wood
The equation obtained in this manner can be applied at any time in the future during a routine
analysis of the product of this same formulation. By preparing a test solution of the product and
determining its W absorbance at the specified wavelength, the triclocarban (or triclosan) concentration
in the test solution can readily be determined. By dividing the triclocarban (or triclosan) concentration
obtained from the soap sample by the soap-sample concentration in the test solution, the analyte’s
concentration in the product can then be expressed. ?he y-intercept value, 0.0624 in this example,
represents the correction for interference from other formula components such as fragrance in the
product matrix. In Fig. 14.2, the equation that was derived from the data in Table 14.4 is represented
graphically.

1.60

1.40

1.20

I .oo

0.80

0.60

0.40

0.20

0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 I .oo

Trirlocarban Cone, C (mg/lOO mL)

Fig. 14.2. UV Absorbance vs. Triclocarban Conc. for a Typcial Antibacterial Bar Soap

Table 14.4. UV Absorbance vs.Triclocarban Conc. for a Typical Bar Soap

Conc (mg/lOO ml) A265 A320 ACORR
0.0 0.17 0.1 1 0.06
0.4 0.73 0.1 2 0.61
0.6 1.03 0.10 0.93
0.8 1.33 0.11 1.22
1.o 1.49 0.14 1.45
 Analysis of Soap and Related Materials 0 4 1 1

Chromatographic Methods
Triglyceride Analysis by HPLC
Some work is reported in the literature for the separation of triglycerides using high-performance liquid
chromatography (HPLC). Plattner et al. (1977) performed triglyceride separation by chain length
and degree of unsaturation by using a C18 p-Bondapak column with an acetonitrile-acetone mobile
phase and a differential refractometer detector. Waters Associates has a specialty column for triglyceride
analysis (Waters, #84346) which is used with a 50:50 acetonc-tetrahydrohran mobile phase Waters
Division of Millipore Corp., 1986). Supelco’s reversed-phase “Supelcosil” LC-8 and LC-18 columns,
using acetone-acetonitrile (63.6:36.4) mobile phase, are also reported to effect triglyceride separations
(Supelco, 1980). Triglyceride composition may be determined using AOCS Official Methods Ce 5b-89
and Cc 5c-93.

Derivatized Fatty Acid Analysis by HPLC

Scholfield (1975) reported the use of a C18/Corasil column and an aqueous acetonitrile mobile phase
for the separation of fatty acid methyl esters (FAMEs) by unsaturation and chain length. Work by
Warthen ( I 975) achieved the analytical separation of FAMEs, including cis and tram isomers, by using
a p-Bondapak C18 column and methanol-water mobile phase.
Work was reported by Jordi (1978) for the separation of the phenacyl and naphthacyl derivatives
of Fatty acids by using a p-Bondapak column and the Fatty Acid Analysis column Waters) with an
acetonitrile-water gradient. The HPLC of geometric isomers of the fatty acids of coconut oil and
other seed oils was also reported by Wood & Lee (1983). This method also utilized the phenacyl and
naphthacyl derivatives chromatographed on a C18 reversed-phase column by using an acetonitrile-
water gradient.

Free Fatty Acid Analysis by HPLC

Analysis of free fatty acid, without derivatization, by HPLC was described by King et a]. (1982). In
this method, a Free Fatty Acid column (Waters) was used with varying combinations of a ternary
mobile phase consisting of tetrahydrofuran, acetonitrile, and water at a reduced pH, and employing
a refractive-index detector. Using this system, the identification and semi-quantification of fatty acid
mixtures derived from industrial oils and alkyd resins are accomplished in about 15 minutes.
Additional work was reported by George (1994)- quantitation of fatty acids from triglycerides
and soap without derivatization. The triglycerides are first saponified, and then acidulated to free the
fatty acids. The fatty acids are then dissolved in a methanol or a tetrahydrofuran-methanol solution for
injection. The mobile phase consists of 45% of acetonitrile, 20% of tetrahydrohran, 34.5% of water,
and 0.5% of glacial acetic acid at a flow rate of 1.1 mL/minute on a Waters fatty acid stainless-steel
column. Work was also done by using radial-compressed stainless-steel columns. Soaps are dissolved in
methanol, and then injected directly into the HPLC where they are acidulated on the column by the
acidic mobile phase. A refractive-index detector is used in conjunction with the isocratic reverse-phase
chromatography.

Derivatized Fatty Acid Analysis by Capillary GC

Capillary gas chromatography (GC) on wall-coated open-tubular fused silica columns has greatly
enhanced the accuracy and speed of analysis due to the development of technology to attach very polar
stationary phases to highly deactivated fused silica. Slover & Lanza (1979) used capillary glass columns
coated with SP2340 for quantitation of FAMEs. The columns were used for extended periods, and, with
up to 1900 samples, analyzed on a single column during an 1l-month period with little deterioration
412 0T.Wood
of the column. The separation included the resolution of cis and trans isomers. Capillary G C analysis of
cis and trans FAMEs is also described in Supelco’s literature (1985) in which most of the C,, isomers are
resolved. In this method, the FAMEs are injected into a GC equipped with a glass split-injection port
(split ratio 100:1), SP2560 column (Supelco, #2-4056) with 100 m x 0.25 m m i.d., 0.20 pm film, and
a flame-ionization detector (FID). By using an oven temperature of 175°C and helium as the carrier
gas, an excellent resolution of all the C,, through C,, FAMEs is obtained including partial separation of
most of the C,,:, cis- and trans-positional isomers, but run times can exceed 60 minutes.
Lanza et al. (1980) reported the use of short glass capillary columns coated with SP2340 for the
rapid analysis of fatty acids that provides some resolution of geometric isomers. Sampugna et al. (1982)
also reported the rapid analysis of trans fatty acids by using an SP2340-coated glass capillary column.
The official AOCS method for FAME preparation (AOCS, 1994a) uses a boron trifluoride-
methanol reagent. The official AOCS method for FAME analysis by G C (AOCS, 1994b) also utilizes
packed-column technology. A complete analysis of cishrans isomers may be achieved by capillary GC
by using AOCS Official Method C e 1h-05.

Glycerine Content of Soap by HPLC

Glycerine determination in soap base was accomplished by using HPLC as described by George and
Acquaro (1 982). A Carbohydrate Analysis column (Waters) is used with an acetonitrile-water (92.5:7.5)
mobile phase, a 1.O mL/minute flow rate, with a differential refractometer (Waters, Model 401). Sample
analysis time is about 30 minutes, including sample preparation time under the above conditions.

Triclocarban and Triclosan in Soap by HPLC

George et al. (1 980) also developed an HPLC method for the determination of triclocarban and triclosan.
This method uses a radially compressed C18 column (Waters, Radial Pak A). The sample preparation
involves using C18 solid-phase-extraction cartridges to remove most of the soap and fragrance
components from the sample. The mobile phase for the separation consists of a tetrahydrofuran/water
mixture (58:42) at a flow rate of 2 mL/rninute with a W detector at 280 nm. Sample preparation time
is reported to be 15 minutes with a 15-minute analysis time.

Chelating Agents in Soap by HPLC

Identification and quantification of aminocarboxylate chelating agents used in bar soaps- such as
ethylenediaminetetraacetic acid (EDTA), N-hydroxyethylenediaminetriacetic acid (HEDTA), and
diethylenetriamine pentaacetic acid (DTPA) by HPLC-are described by Goldstein and Lok (1988).
This method uses cupric sulfate to precipitate the soap and to form a water-soluble aminocarboxylate-
copper complex. The copper complex is then isolated and chromatographed on an anion- exchange
column (Wescan, Anion R Analytical) with a mobile phase consisting of 0.003 M of sulfuric acid at
a flow rate of 1.5 mL/minute. A W detector was used at a wavelength of 254 nm. Under the above
conditions, the retention times for the copper complexes were 3.5 minutes for HEDTA, 6.2 minutes
for DTPA, and 6.9 minutes for EDTA.

BUT in Soap by Capillary GC

A method for the determination of 2,6-di-tert-butyl-methylphenol (BHT) in soap products is offered
by Goldstein et al. (1982). The authors present a method that can quantitate B H T in fragranced bar
soap without employing the cumbersome standard addition method that was previously reported
(Sedea & Toninelli, 1981). After blending the sample with dimethylformamide and adding 2,4-di-
tert-butylphenol (DTBP) as an internal standard, bis-trimethylsilyltrifluoroacetamide (BSTFA) is
added to a filtered aliquot to convert the BHT and DTBP to their silyl derivatives. The sample is then
introduced into a GC equipped with a glass split-injection port (split ratio 200:1), methyl silicone
 Analysis of Soap and Related Materials 0 4 13

column (Hewlett-Packard, #17071-60010) with a 12 m x 0.2 mm i.d., and an FID. By using helium
as the carrier gas, the column temperature is held at 100°C for 2 minutes and then programmed up to
144°C at 2”C/minute. The program rate is then changed to 3 0 W m i n u t e up to 240°C and held for 5
minutes. Under the above conditions, the reported retention times of the silylated derivatives of DTBP
and BHT were 15.5 minutes and 22.4 minutes, respectively. O n e can also apply this method to neat
soaps, pellets, and fatty acids as well.

Evaluation of Soap Color and Translucency

Visual Color Comparisons
Bar-soap color evaluation was traditionally performed by a visual comparison of fresh product against
various standards such as paper or plastic color chips and retained standard product samples. Visual color
comparisons are prone to be subjective, and individual differences in color perception and background-
lighting conditions can affect them. All physical standards, including both color chips and retained
product standards, must be carehlly handled and protected to avoid fading. All such standards will
change with time and must be periodically updated.
For visual color comparisons, a controlled lighting environment is a necessity. The use of a
standardized light source and a working environment, such as that which a Pantone Color Viewing
Light can provide, can be helpful in providing such standardized conditions. The Pantone Color Viewing
Light is available from Pantone, Inc. (www.pantone.com). The Pantone Color Viewing Light provides
three different light sources-including artificial daylight, fluorescent store light, and incandescent
home light-that one can use independently or in combination. The interior of the cabinet has a matte
neutral-gray finish that minimizes reflection and glare.

Soap Color by Hunter Reflectance Color Measurement

The instrumental approach to color evaluation can provide many advantages over visual comparison
techniques. The instrument in common use by many soap manufacturers is the Hunter D25LT
Colorimeter that is manufactured, sold, and serviced by Hunter Associates Laboratory, Inc. (www.
hunterlab.com). Among the advantages of the instrumental approach are the objective basis for color
comparison to standard, a defined target that is not subject to deterioration, and quantifiable data that
can be retained indefinitely. Variables such as light source, background lighting, and differences in
individual color perception are eliminated. Also, the nature of the instrumental approach easily lends
itself to interfactory and intercompany color control.
The Hunter Opponent Color Scale (Hunter & Harold, 1987a) expresses color in terms of three
values: “L” expresses “lightness,” and ranges from 0 to 100 units; “a”expresses “redness” when positive,
“gray” when zero, and “greenness” when negative; and “6” expresses “yellowness” when positive, “gray”
when zero, and “blueness” when negative. The Hunter L, a, and 6 Color Solid is illustrated in Fig.
14.3.
The L, a, and 6 values are determined from fresh-product samples that were cut to a planar surface.
The values obtained for each sample are then compared with previously established target values. The
differences between sample and target L, a, and 6 values can then be interpreted individually, or they
can be resolved into the total color difference (Hunter & Harold, 1787b) or delta-E ( AEj value that
serves as a very usehl tool for color control at the production level. The total color difference or A E is
expressed by the following equation:
AE = JAL2 + Aa2 + Ab2
(Eq. 14.8)

This equation resolves the three component differences, A L, A a and A 6, into the direct difference, in
Hunter units, between the target and the sample. The A Eserves as a very usehl tool for routine quality
4 14 0 T. Wood

control of bar-soap color. For example, typical A E limits might allow:

Product with a A E below 2.0 units to be packed without qualification,
Product with a A E between 2.0 and 4.0 units to be packed with an ongoing effort to bring the
product back below 2.0 units, and
Product with A E above 4.0 units not to be packed.
The Hunter colorimeter can also measure neat-soap whiteness. The color can be reported in terms of
L, r2 and b values, or can be expressed in terms of the Whiteness Index (Hunter & Harold, 1 9 8 7 ~ )The
.
perception of whiteness, in terms of the Hunter Opponent Color Scale values, is favored by a higher L
value (lightness) and a more negative b value (higher degree of blueness). The Hunter Whiteness Index
is expressed as:

WI = L - 36.
(Eq. 14.9)

‘The human eye can consistently perceive the differences of 1.5 units of Hunter Whiteness Index
(Appleby & Halloran, 1990).
In performing a color evaluation on neat soap, one must wait until the neat soap has thoroughly
cooled to room temperature before measuring the whiteness. The sample needs to be cut to a flat surface
before performing the Hunter readings. As recommended, one must read the sample six times, turning
the sample about 60 degrees for each reading. One should then report the average of the six readings.
In a similar manner, the color of soap to be commercially made from fatty acid blends can be
reliably estimated by quantitatively preparing a sample of sodium soap in the laboratory. For an 80:20
tallowxoconut oil blend, for example, 130 grams of fatty acid would be melted and weighed. A solution
of NaOH would be prepared in a 600-mL beaker. The fatty acid would be added slowly with constant
mixing to the caustic solution until the soap is formed. Soap would then be packed into a 125-mm
Petri dish, and let stand for about an hour to cool before covering. The soap would then set until the
following day when the color would be evaluated. If the color is to be measured with the Hunter
colorimeter, six readings should be taken and averaged. This procedure is not very practical as a routine
quality-control procedure, but can be very useful in qua1if)ing a vendor‘s material.

Soap Translucencyby Hunter Opacity Measurement

This technique has proven to be useful in quantifying the translucency of bar soap using the Hunter
D25LT Colorimeter. The method is an adaptation of the “contrast ratio method” for measuring opacity
(Hunter & Harold, 1987d). The procedure for performing the opacity reading is outlined in the Hunter
instruction manual that accompanies those instrument models, such as the D25LT, that are designed
to perform this measurement.
The application of this procedure for measuring the translucency of bar soap involves preparing
the soap sample for reading by cutting a flat slice to a uniform thickness of .25 inch. Then the standard
routine for opacity measurement, as outlined in the Hunter manual, is performed. This procedure
involves calibrating the instrument, followed by two simple operations with the meter. In the first step,
the translucent slab is backed by the uncalibrated white tile to achieve maximal reflectance through
the sample, followed by the second step where the sample is backed up by the black tile for minimal
reflectance through the sample. The processor in the instrument then converts this data into an opacity
value. The opacity value provides an inverse indicator of translucency. A reasonable scale of values for
interpreting these results might rate an opacity value below 10% as “excellent,” between 10 and 25%
 Analysis of Soap and Related Materials 0 4 1 5

as "good," benveen 25 and 40% as "fair," between 40 and 50% as "poor," and greater than 50% as
essentially a nontranslucent product. Action steps in the plant can then be set for these various grades.

L= 1 0 0 q

a = 175Cl.OZX-Y)
fl
b =70CY- 0.8472)
7 T
Fig. 14.3. Hunter L, a, 6 Color solid.

Foreign Particulate Matter in Soap

A convenient method for quantifying the presence of any particulate foreign matter in the soap base or
finished bars utilizes a Model J BulkTank Sediment Tester (used in the dairy industry) which is available
from the Clark Dairy Equipment Company of Greenwood, Indiana.
The procedure involves finely dividing 100 grams of sample and dissolving them in 1 to 2 L of hot
deionized water on a steam bath with agitation. The resulting soap solution is then filtered through
a specially designed 3.5-cm filter disk that is designed to be used with the sediment-tester device.
The sediment tester is mounted on a large filter flask that is connected to an aspirator for suction.
The particulate matter trapped on the filter disk can then be visually compared with a standard set of
photographs to provide a grade for the sample.

Acknowledgments
The author was formerly Vice President/Director of Technical Services at Valley Products Company,
Memphis, T N , USA, and The Hewitt Soap Company, Inc., Dayton, O H , USA.

References
American Oil Chemists' Society (AOCS). Ojicial Methodc and Recommended Practices of the American Oil Chemists'
Society, 6th ed.; R.C. Walker, Ed.; Champaign, IL, 2009a; Method Ce 2-66.
Ibid., 2009b; Method Ce 1-62.
Ibid., 6" ed.; 2009~.
4 16 OT. Wood
American Society for Testing and Materials (ASTM). Annual Book ofASTM Standad; : West Conshohocken, PA,
2008; Vol. 15.04.
Appleby, D.B.; K.A. Halloran. Soap Technologyfor the 1990s; L. Spitz, Ed.; American Oil Chemists' Society: Cham-
paign, IL, 1990; p. 104.
Arnold, J.G.; R.A. Ford. The Chemist: Companion: A Handbook OfPractical Data, Gcbniques, and References;John
Wiley & Sons: New York, 1972; p. 188.
George, E.D. J. Am. Oil Cbem. Soc. 1994, 71, 789.
George, E.D.; J.A. Acquaro. J. Liq. Cbromatogr. 1982,5, 927.
George, E.D.; E.J. Hillier; S. Krishnan.J. Am. Oil Cbem. Sor. 1980, 57, 131.
Goldstein, M.M.; W.P. Lok. Ibid. 1988,65,1350.
Goldstein, M.M.; K. Molever; W.P Lok. Ibid. 1982,59, 579.
Grornpone, M.A. Ibid. 1984,61, 788.
Hunter, RS.; R.W. Harold. The Memurement ofAppearance, 2nd ed.; John Wiley & Sons: New York, 1987a; pp.
173-1 74.
Ibid., 1987b; pp. 174-175.
Ibid., 1 9 8 7 ~pp.
; 206-207.
Ibid., 1987d; p. 90.
Jordi, H.C.J. Liq. Cbromatogr. 1978, I , 215.
King, J.W.; E.C. Adams; B.A. Bidlingrneyer, Ibid. 1982,5, 275.
Lanza, E.; J. Zyren; H . 1 Slover. J. Agric. Food Cbem. 1980,28, 1182.
Plattner, RD.; G.F. Spencer; R. Kleiman.]. Am. Oil Cbem. Sor. 1977,54,511.
Sampugna, J.; L.A. Pallansch; M.G. Enig; M. Keeney. .] Cbromatogr. 1982,249, 245.
Scholfield, C.R. J. Am. Oil Cbem. Soc. 1975,52, 36.
Sedea, L.; G. Toninel1i.J. Cbromatogr. Sci. 1981, 19,290.
Slover, H.T.; E. Lanza. J. Am. Oil Cbem. Sor. 1979,56, 933.
Supelco, Inc. One-Step Triglyccride Sepuration; HPLC Bulletin 787B, Bellefonte, PA, 1980.
Supelco, Inc. Capilhty AnalyscS OfPositional Cis/Trans Fatty Acid Methyl Ester Isomers;G C Bulletin 822, Bellefonte, PA,
1985.
The Procter & Gamble Company. Better Rendering; Cincinnati, O H , 1967; p. 17.
Unichema Chemicals, Inc. F a q A c i d Data Book, 2nded.; Chicago, IL, 1987; pp. 4-5.
Warthen, J.D. Ibid. 1975,52, 151.
Waters Division of Millipore Corp. Waters Sourcebookfir Cbromatograpb Columns and Supplies; Milford, PA, 1986; p.
43.
Wood, R.; T. Lee. .] Cbromatogr 1983,254, 237.