Advanced Materials Research Vol 506 (2012) pp 182-185 Online: 2012-04-25

© (2012) Trans Tech Publications, Switzerland

doi:10.4028/www.scientific.net/AMR.506.182

Simplified Qualitative Analysis of Glycerides Derived from Coconut Oil

Using Thin Layer Chromatography

S. Pengon1,2, C. Limmatvapirat3,* and S. Limmatvapirat1,2

1
Department of Pharmaceutical Technology, Faculty of Pharmacy,
Silpakorn University, Nakhon Pathom 73000, Thailand
2
Pharmaceutical Biopolymer Group (PBiG), Faculty of Pharmacy,
Silpakorn University, Nakhon Pathom 73000, Thailand
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Silpakorn University, Nakhon Pathom 73000, Thailand
*Chutima@su.ac.th

Keywords: Coconut oil; Glycerides; Thin layer chromatography

Abstract. Coconut (Cocos nucifera L.) oil is composed predominately of medium-chain

triglycerides which have been reported to be beneficial to human health. It also contains free fatty
acids (FFAs) which can combine with glycerol to form monoglycerides, diglycerides, and
triglycerides. The analysis of FFAs and their glycerides has been proposed to assess the quality of
coconut oil used as raw materials in various industrial fields. The aim of this study was to develop
the qualitative method for investigation of FFA and their glycerides in coconut oil using thin layer
chromatography (TLC). Coconut oil and standards of FFA and their glycerides were
chromatographed separately on Silica gel 60 F254 TLC plates using hexane: ether: acetic acid
(60:40:1) and hexane: ethyl acetate: acetic acid (60:40:0.5) as solvent systems A and B,
respectively. The spots on developing TLC plates were detected and compared using 254-nm UV
light and iodine vapor. The results showed that the resolution of solvent system A was better than
that of solvent system B. However, both solvent systems were used to confirm the results. The
retention factor (Rf) values of the components were in good agreement with their polarity. This
method should provide a guideline for qualitative analysis of coconut oil.

Introduction
Coconut oil is an edible oil extracted from the kernel or meat of matured coconut (Cocos nucifera
L.). It contains medium-chain triglycerides, vitamin E and polyphenols which have been exhibited
various biological activities, including antioxidant, hypolipidemic, antitumor and antithrombotic
activities[1]. It is also an important source of medium-chain triglycerides (MCTs) which are used
as dietary supplements for patients with malabsorption, disorder of lipid metabolism, or glucose
control and also as a component of modified infant milk formulas [2]. The MCT is composed of a
glycerol backbone and three saturated fatty acids with chain length of 6-12 carbons. MCTs and
their free fatty acids (FFAs) have been found to inhibit most Gram-positive bacteria [3], enveloped
viruses [4], yeasts [5] and protozoa [6].
Coconut oil consists of about 92% saturated fatty acids, among which MCTs contribute 70% of
the total fatty acids [7]. The remaining unsaturated fatty acids are composed of monounsaturated
fatty acids (6%) and polyunsaturated fatty acids (2%). Among the saturated fatty acids, coconut oil
contains medium-chain fatty acids (including lauric acid and caprylic acid) and other fatty acids
(such as myristic acid and palmitic acid). Many studies have been carried out to determine FFAs
and their glycerides using a gas chromatography equipped with a flame ionization detector [1,2,7]
that is required an expensive instrument and difficult to prepare samples. So this study was to
develop the more simplified qualitative method for investigation of FFAs and their glycerides in
coconut oil using thin layer chromatography (TLC). This analytical technique was proposed to be
sensitive, fast, simple, and inexpensive.

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 Advanced Materials Research Vol. 506 183

Experimental Procedure
Coconut oil and standards of FFAs (lauric acid, myristic acid, palmitic acid, capric acid, oleic acid
and stearic acid) and their glycerides (each 20 µg) were separately dissolved in 95% methanol (250
µl). All standard abbreviations were shown in Table 1. Sample solutions and standard solutions (2
µl) were separately spotted onto the baseline of a TLC plate and then left until dried. The spotted
TLC plates were developed with the solvent systems A (hexane: ether: acetic acid 60:40:1 v/v/v)
and B (hexane: ethyl acetate: acetic acid 60:40:0.5 v/v/v). The elution distance of the solvent front
was approximately 8 cm from the baseline. After developing, the plates were air dried for 5 min in
a fume hood. The spots on developing TLC plates were detected by 254-nm UV light and iodine
vapor. The retention factor (Rf) was calculated using the ratio of the distance traveled by the
compound to the distance traveled by the solvent. The developing TLC plates and the Rf values
were shown in Fig. 1-2 and Table 2, respectively.

Table 1 Standard abbreviations

Free fatty acids (FFAs) Monoglycerides (MGs) Diglycerides (DGs) Triglycerides (TGs)
Lauric acid LA Monolaurin mL Dilaurin dL Trilaurin tL
Myristic acid MA Monomyristin mM Dimyristin dM Trimyristin tM
Palmitic acid PA Monopalmitin mP Dipalmitin dP Tripalmitin tP
Capric acid CA Monocaprin mC Dicaprin dC Tricaprin tC
Oleic acid OA Monoolein mO Diolein dO Triolein tO
Stearic acid SA Monostearin mS Distearin dS Tristearin tS

Results and Discussion

Effect of Detection Method. Fig.1 showed the spots of standard compounds including FFAs and
their glycerides on TLC plates which were observed under 254-nm UV light and detected with
iodine vapor. All standards except for MGs couldn’t be seen under UV light. UV-light is good for
visualizing compounds which are UV-active, particularly those with double bonds and extended
conjugation. Nevertheless, all FFAs and their glycerides could be detected by staining with iodine
vapor. It is based upon the observation that iodine has a high affinity for unsaturated compounds.
Due to the hydrophobic character of compounds, they adsorb iodine and readily appear on TLC
plates as brownish spots. Both detection methods can be used for qualitative determination of FFAs
and their glycerides.
Effect of Mobile Phase. Solvent systems A (hexane: ether: acetic acid 60:40:1 v/v/v) and B
(hexane: ethyl acetate: acetic acid 60:40:0.5 v/v/v) were used as mobile phases. The polarity of
solvents arranged from high to low was acetic acid > ethyl acetate > ether > hexane. Therefore, the
elution strength of solvent system B was higher than that of solvent system A. The spots and Rf
values of FFAs and their glycerides on TLC plates developed with solvent systems A and B were
shown in Fig. 2 and Table 2, respectively. Because of different elution strength, the Rf values
calculated from solvent system B were higher than those from solvent system A. The more polar
compound has a stronger interaction with the silanol group on silica gel. Therefore, the less polar
compound moves higher than the more polar compound (resulting in a higher Rf value). In this
experiment, both solvent systems were used to confirm the results.
184 Biomaterials and Applications

FFA MG DG TG FFA MG DG TG FFA MG DG TG FFA MG DG TG

(a) (b) (a) (b)

Fig. 1 Spots of FFAs and their glycerides with
different detection methods: (a) 254-nm UV
light, (b) iodine vapor
Fig. 2 Spots of FFAs and their glycerides with
two solvent systems: (a) system A (hexane:
ether: acetic acid 60:40:1 v/v/v), (b) system B
(hexane: ethyl acetate: acetic acid 60:40:0.5
v/v/v)

Table 2 Rf values of FFAs and their glycerides from solvent systems A and B
Rf values
Solvent system A Solvent system B
LA 0.3125 - 0.4250 0.6000 - 0.6875
mL 0.0125 - 0.0875 0.1375 - 0.2438
dL 0.1875 - 0.3125 0.6625 - 0.7500
tL 0.6000 - 0.7250 0.8500 - 0.9375
MA 0.3250 - 0.4375 0.5000 - 0.6000
mM 0.0000 - 0.0625 0.1250 - 0.2000
dM 0.1875 - 0.2625 0.5750 - 0.6500
tM 0.5628 - 0.6375 0.7250 - 0.8375
PA 0.3625 - 0.4750 0.5250 - 0.6125
mP 0.0125 - 0.0750 0.1313 - 0.2000
dP 0.8750 - 0.2750 0.4500 - 0.5813
tP 0.6000 - 0.6875 0.6688 - 0.7875
CA 0.3125 - 0.4500 0.4750 - 0.5875
mA 0.0125 - 0.0875 0.1125 - 0.1875
dA 0.2063 - 0.3125 0.5500 - 0.6500
tC 0.5875 - 0.6500 0.7625 - 0.8625
OA 0.3125 - 0.4750 0.5750 - 0.6875
mO 0.0000 - 0.4625 0.1375 - 0.3125
dO 0.1875 - 0.3250 0.6500 - 0.7750
tO 0.5875 - 0.6750 0.8500 - 0.9500
SA 0.3625 - 0.4500 0.6250 - 0.7125
mS 0.0125 - 0.0750 0.1688 - 0.2375
dS 0.1500 - 0.2375 0.5625 - 0.6875
tS 0.6500 - 0.7250 0.9000 - 0.9625
 Advanced Materials Research Vol. 506 185

Separation of Glycerides. FFA has a carboxylic group (-COOH) on one end. So, its polarity
depends on the number of C atom in hydrocarbon (HC) chain. Table 2 showed the Rf values of
standard FFAs. The results demonstrated Rf values of CA < LA < MA < PA < OA < SA because
the number of C atom of CA(C10) < LA(C12) < MA(C14) < PA(C16) < OA(C18, double bond) <
SA(C18). In case of glycerides derived from the same FFAs demonstrated Rf values of
momoglycerides (MGs) < diglycerides (DGs) < triglycerides (TGs). MG has two free hydroxyl
group (free –OH) and one HC chain so it has the highest polarity. DG has one free –OH and two HC
chains while TG has three HC chains but without the free –OH. Therefore, DG is more polar than
TG. These reasons were related with the results of Rf values. The Rf values of the compounds
were in good agreement with their polarity.

Conclusions
Iodine vapor was a simple method for detection of FFAs and their glycerides on the Silica gel 60
F254 TLC plates. Solvent systems A (hexane: ether: acetic acid 60:40:1 v/v/v) and B (hexane: ethyl
acetate: acetic acid 60:40:0.5 v/v/v) were appropriate for separation of those compounds. TLC was
the sensitive technique and could provide a guideline for qualitative analysis of coconut oil.

Acknowledgements
This work was supported by the Thailand Research Fund; the Commission on Higher Education; the
Royal Golden Jubilee Ph.D Program (PHD/0343/2551); the Silpakorn University Research and
Development Institute and the Faculty of Pharmacy, Silpakorn University.

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Biomaterials and Applications
10.4028/www.scientific.net/AMR.506

Simplified Qualitative Analysis of Glycerides Derived from Coconut Oil Using Thin Layer
Chromatography
10.4028/www.scientific.net/AMR.506.182