Special Properties of Fats, AACC International Method 58-45.

01
Oils, and Shortenings Page 1 of 5

Monoglycerides and Free Glycerol

Final approval April 13, 1961; Reapproval November 3, 1999

Objective
This method determines α-monoglycerides from periodic acid (HIO4)
consumed in oxidation of adjacent hydroxyl groups. β-Monoglycerides are not
oxidized by periodic acid because hydroxyl groups are not adjacent. This
method is applicable to fats, oils, monoglycerides, and blends. It is not
applicable when sample contains, in addition to monoglycerides, chloroform-
soluble polyhydric substances with two or more adjacent hydroxyl groups. This
method is equivalent to Official Methods Ca 11-57 and Cd 14-56 of the
American Oil Chemists’ Society (AOCS).

Apparatus
1. Burets, 50-ml and 100-ml, accurately calibrated.
2. Meniscus magnifier such as to permit reading the buret to 0.1 ml.
3. Volumetric flasks, 1000-ml.
4. Volumetric flasks, 100-ml; glass-stoppered are preferred, but regular volu-
metric flasks and glass stoppers may be used. See Note 3.
5. Volumetric pipets, 10-, 25-, and 50-ml pipets must conform to standards of
. National Institute of Standards and Technology (NIST) and be accurately cali-
brated to deliver 25 and 50 ml, respectively.
6. Pipet, 2-hole rubber stopper, fitted with small-diameter glass tubing.
7. Beakers, 250-, 400-, and 600-ml; watch-glasses to serve as covers.
8. Variable-speed magnetic stirrer with glass stir bar.
9. Graduated cylinders, 100- and 1000-ml.
10. Separatory funnel, 500-ml.
11. Soxhlet flasks, 250-ml.
12. Büchner funnel, about 75-mm diameter.
13. Filter paper, quantitative grade, Whatman no. 40 or equivalent.

Reagents
1. Periodic acid (H5IO6) solution. Dissolve 5.4 g periodic acid reagent grade,
in 100 ml water, add 1.9 liter glacial acetic acid, and mix thoroughly. Store solu-
tion in dark glass-stoppered bottle, or store in dark in clear glass-stoppered bot-
tle. Caution: Cork stoppers must never be used where periodic acid can come in
contact with them.
Test for quality. To 0.5–0.6 g of chemically pure glycerine in 50 ml distilled
water, add 50 ml periodic acid reagent with pipet. Prepare blank using 50 ml
water instead of 50 ml periodic reagent. Let stand 30 min. Add 20 ml KI solu-
tion, mix by gently shaking, and let stand at least 1 min but never more than 5
min before titrating; do not let stand in strong sunlight. Add 100 ml water and
titrate with 0.1N Na2S2O3 solution. (Use variable-speed magnetic stirrer to keep

http://dx.doi.org/10.1094/AACCIntMethod-58-45.01
Special Properties of Fats, AACC International Method 58-45.01
Oils, and Shortenings Page 2 of 5

Monoglycerides and Free Glycerol (continued)

solution thoroughly mixed.) Continue titration to disappearance of brown iodine

color. Add 2 ml starch indicator solution (see reagent 5) and continue titration to
disappearance of blue iodo-starch color. Vigorous agitation is essential.
Titration of solution containing glycerol divided by titration of blank will be
between 0.75 and 0.76 when periodic acid is satisfactory.
2. Sodium thiosulfate solution, 0.1N (Method 70-75.01).
3. KI solution. Dissolve 150 g KI (ACS grade) in water and dilute to 1 liter.
4. Glacial acetic acid, reagent grade, 99.5%. Caution. See Note 1.
5. Starch indicator solution. Make homogeneous paste of 10 g soluble starch
in cold water. Add to mixture 1 liter boiling water, stir rapidly, and cool.
Salicylic acid (1.25 g/liter) may be added to preserve indicator. If long storage is
required, solution must be kept in refrigerator at 4–10°. Fresh indicator must be
prepared when end point of titration from blue to colorless fails to be sharp or
when starch fails to meet sensitivity test.
Test for sensitivity. Place 2 ml starch solution in 100 ml water and add 0.05
ml 0.1N iodine solution. The deep blue color produced must be discharged by
0.05 ml 0.1N sodium thiosulfate.
6. Chloroform, U.S. Pharmacopeia or reagent grade. Caution. See Note 1.
Blank tests run on periodic acid with and without 50 ml chloroform must agree
within 0.5 ml. If they do not, get new supply of chloroform.

Procedure
Preparation of samples
1. Solid samples in flake form. Mix without melting and take portion for test.
2. Solid samples not in flake form. Melt at not more than 10° above melting
point, mix thoroughly, and take portion for analysis. Do not attempt to test sam-
ple that contains so much free glycerol that it separates when sample solidifies.
3. Semisolid and liquid samples. Liquefy by heating at not more than 10°
above melting point, mix thoroughly, and take portion for analysis. Do not
attempt to test sample that contains so much free glycerol that it separates from
sample when cooled to room temperature. Caution: Sample must not be sub-
jected to temperatures in excess of that required to melt them, since monoglyc-
eride content may be reduced if any soap is present.

Procedure for monoglyceride

1. Weigh duplicate samples accurately into 100-ml glass-stoppered
volumetric flasks. Proper size of sample is indicated in Table I.
2. Add 50 ml chloroform with graduated cylinder. Dissolve sample in chloro-
form and mix thoroughly by shaking. Warm flask on steam bath if necessary to
effect complete solution and then cool to room temperature.
Special Properties of Fats, AACC International Method 58-45.01
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Monoglycerides and Free Glycerol (continued)

TABLE I
Sample Size for Monoglycerides
Monoglyceride Approximate Size of Sample Weighing Accuracy
(%) (g) (g)
100 0.30 ±0.0002
75 0.40 ±0.0002
50 0.60 ±0.0003
40 0.70 ±0.0005
30 1.00 ±0.001
20 1.50 ±0.001
10 3.00 ±0.002
5 6.00 ±0.004
3.0 or less 10.00 ±0.010

3. Add 25 ml water, stopper flask tightly, and shake vigorously for 30–60 sec
so that contact is good between aqueous and chloroform phases. Flask must be
tightly stoppered and adequate precaution taken so that none of aqueous extrac-
tion solution or chloroform solution is lost.
4. Set flask aside until aqueous and chloroform layers separate. Transfer aque-
ous layer to glass-stoppered 100-ml volumetric flask, using glass siphon. Aque-
ous layer must be transferred as completely as possible without including any of
chloroform layer. (Note: When emulsion forms because of presence of soap in
sample, add 3 or 4 ml glacial acetic acid to break emulsion.)
5. Extract three more times with 25, 25, and 20 ml water as directed in para-
graphs 3 and 4.
6. Add chloroform to original flask until level of chloroform coincides with
100-ml mark on flask. Transfer as much as possible of aqueous layer above
chloroform layer to flask containing aqueous extracts. (Save aqueous extracts in
volumetric flask for determination of free glycerol.) Stopper flask tightly and
mix thoroughly by inverting.
7. Pipet 50 ml periodic acid solution (reagent 1) into series of 400-ml beakers.
Prepare three for blanks, adding 50 ml chloroform to two and 50 ml water to
third. Titrations of water and chloroform blanks are used as a check on chloro-
form (reagent 6).
8. Pipet 50 ml chloroform-sample solution into 400-ml beaker containing 50
ml periodic acid solution, and shake gently to effect thorough mixing. Cover
with watch-glass and let stand 30 min. (Note: Samples may be allowed to stand
1.5 hr at room temperature before titration, but never longer.)
9. Add 20 ml KI solution (reagent 3), mix by gently shaking, and let stand at
least 1 min but never more than 5 min before titrating. Do not let stand in strong
sunlight. Add 100 ml water and titrate with 0.1N Na2S2O3 solution (reagent 2).
Use variable-speed magnetic stirrer to keep solution thoroughly mixed.
Special Properties of Fats, AACC International Method 58-45.01
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Monoglycerides and Free Glycerol (continued)

Continue titration to disappearance of brown iodine color from aqueous layer.

Add 2 ml starch indicator solution (reagent 5) and continue titration to
disappearance of iodine from chloroform layer and disappearance of blue iodo-
starch color from aqueous layer. Vigorous agitation is essential for complete
removal of iodine from chloroform layer.
10. Read buret to hundredths of ml.
11. Blanks are handled exactly like samples, as in paragraph 9.
12. If titration of sample (paragraph 10) is less than 0.8 of titration of blank
(paragraph 11), there is not sufficient excess of periodic acid to ensure complete
reaction. When this occurs, repeat analysis with smaller portions—25, 10, or 5
ml in paragraph 8—until titration of sample is more than 0.8 of that of blank.
When 10 ml (or less) is required, repeat analysis by starting at beginning with
smaller sample, referring to paragraph 1 to find proper amount of sample to
weigh.
13. For best results, difference between titration of blank (paragraph 11) and
titration of sample should be more than 4 ml. When <4 ml, it is advisable to
repeat analysis beginning with paragraph 1 and using twice as much sample. If
sample size doubled exceeds 10 g, use only 10 g.

Procedure for free glycerol

Add water to combined aqueous extracts from monoglyceride test (paragraph
6 above) until volume is 100 ml, and mix thoroughly. Analyze in same manner
as prescribed for monoglycerides, paragraphs 7–13 inclusive.

Calculations
For monoglycerides
The following calculations assume monoglyceride to be monostearin.
( B − S ) × N × 17.927
Monoglyceride, % =
W
where B = titration of blank containing 50 ml chloroform, S = titration of
sample, N = normality of Na2S2O3 solution, W = weight of sample, represented
by aliquot pipetted for test (paragraph 8), 17.927 = molecular weight of
monostearin ÷ 20.

For glycerol
( B − S ) × N × 2.30
Free glycerol, % =
W
where B = titration of blank containing 50 ml water, S = titration of sample, N =
normality of Na2S2O3 solution, W = weight of sample, represented by aliquot
taken for test, 2.30 = molecular weight of glycerol ÷ 40.
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Monoglycerides and Free Glycerol (continued)

Notes
1. Acetic acid in the pure state is moderately toxic by ingestion and
inhalation. It is a strong irritant to skin and tissue. The threshold limit value
(TLV) in air is 10 ppm.
Chloroform is a known carcinogen. It is toxic by inhalation and has anesthetic
properties. Avoid contact with the skin. Prolonged inhalation or ingestion can
lead to liver and kidney damage and may be fatal. It is nonflammable, but will
burn on prolonged exposure to flame or high temperatures. The TLV is 10 ppm
in air. A fume hood should be used at all times when using chloroform.
Hydrochloric acid is a strong acid and will cause severe burns. Protective
clothing should be worn when working with this acid. It is toxic by ingestion
and inhalation and a strong irritant to eyes and skin. The use of a properly
operating fume hood is recommended. When diluting the acid, always add the
acid to the water, never the reverse.
2. The AOCS Commercial Fats and Oils Analysis Committee has considered
alternate methods, including titration, column chromatography (AOCS Official
Method Cd11b-93), GC (AOCS Official Method Cd11b-91), HPLC, and NIR.
3. Rubber or cork stoppers must never be used where periodic acid can come
in contact with them.
4. “Potato Starch for Iodometry” is recommended, because this starch pro-
duces a deep blue color in the presence of the iodonium ion. “Soluble Starch” is
not recommended because a consistent deep blue color may not be developed
when some soluble starches interact with the iodonium ion.
5. Pipet chloroform sample solution with care and in such manner that none
of the aqueous layer is included.

Reference
American Oil Chemists’ Society. 1998. Official Methods and Recommended Practices, 5th ed.
Methods Ca 14-56 and Cd 11-57. The Society, Champaign, IL.