SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OILS

AOCS Recommended Practice Cd 11c-93

Reapproved 2009

Quantitative Separation of Monoglycerides,

Diglycerides, and Triglycerides by Silica Gel
Column Chromatography
DEFINITION
This method determines glyceride mixtures by solid-liquid adsorption chromatography (SLAC), using
silica gel as the adsorbent. Triglycerides are eluted with 10% diethyl ether in petroleum ether, diglycerides
with 25% diethyl ether in petroleum ether, and monoglycerides with 100% diethyl ether. Free fatty acids
present are found predominantly in the triglyceride fraction; the remainder in the diglyceride fraction.
Thin-layer chromatography (TLC) is used to verify the purity of the fractions and/or the effectiveness of
the separation.
SCOPE
This method is applicable to emulsifiers and shortenings.

APPARATUS
1. Chromatographic column assembly—25 × 2 cm column with Teflontm stopcock and solvent reservoir, 250 mL, with glass
joint, fitting the top of the column.
2. Receiving flasks—four 250 mL.
3. Variable-thickness thin-layer chromatographic spreader (see Notes, 1).
4. Glass plates—Pyrextm, 5 × 20.5 × 0.32 cm (see Notes, 1).
5. Cylindrical thin-layer developing chamber—6.5 cm diameter × 24 cm high, with cap (see Notes, 1).

REAGENTS
1. Silica gel—Davison grade 923, adjusted to 5% moisture.
2. Silica gel—GF-254 for thin-layer chromatography, E. Merck (Brinkmann Instruments, Inc., distributor).
3. Chloroform—reagent grade (see Notes, Caution).
4. Petroleum ether—reagent grade (see Notes, Caution).
5. Diethyl ether—anhydrous, reagent grade (see Notes, Caution).
6. Ethyl alcohol—SDA Formula 3A (see Notes, Caution).
7. Acetone—reagent grade (see Notes, Caution).
8. Sulfuric acid—concentrated (sp. gr. 1.84), reagent grade, diluted to give a 70:30, v/v, sulfuric acid/water solution (see Notes,
Caution).
9. Chromium trioxide—technical grade.

PROCEDURE
1. Prepare the column with 30 g silica gel (Reagents, 1) slurried in 50–60 mL petroleum ether (Reagents, 4).
2. Accurately weigh 0.7–0.9 g of test sample into a 30 mL beaker. Add 3 mL chloroform (Reagents, 3) to dissolve the test
sample and quantitatively transfer it to the top of the column. Rinse beaker with up to 3 mL of chloroform, adding the rins-
ings to the column.
3. Elute the test sample at a flow rate of 2 mL/min, using 250 mL of solvent for each fraction, as noted below in this section
for Fractions I through IV. If the separation of methyl esters from triglycerides is desired, 200 mL of a solvent system of 5%
diethyl ether (Reagents, 5) in petroleum ether (Reagents, 4), v/v, should be used prior to the elution of the triglycerides in
Fraction I.
Use the following solvent systems to elute the indicated fraction:
(a) Fraction I (triglycerides)—250 mL 10% diethyl ether (Reagents, 5) in petroleum ether (Reagents, 4).
(b) Fraction II (diglycerides)—250 mL 25% diethyl ether (Reagents, 5) in petroleum ether (Reagents, 4).
(c) Fraction III (monoglycerides)—250 mL 100% diethyl ether (Reagents, 5).
Note—If the elution of glycerol and/or other polar material is desired, a fourth fraction is collected:
(d) Fraction IV (glycerol and/or polar material)—200 mL 100% ethyl alcohol (Reagents, 6).
4. Collect the effluents separately for each fraction and evaporate to dryness in tared 250 mL flasks on a steam bath under a
stream of nitrogen. A boiling chip should be placed into each flask before the tare weight is determined in order to prevent
bumping.
5. Weigh the fraction and repeat drying until a constant weight is obtained.

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