SIMULTANEOUS DETERMINATION OF FREE FATTY ACIDS, PARTIAL ACYLGLYCEROLS AND TOCOLS IN PALM OIL PRODUCTS USING HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY
M.H. MOH'.3, T.S. TANG' and G.H. TAN2

'Advanced Oleochemical Technology Center Malaysian Palm Oil Board No. 6 Persiaran Institusi Bandar Baru Bangi 43650 Kajang, Selangor, Malaysia 'Department o Chemistry f f University o Malaya Lembah Pantai 50603 Kuala Lumpur, Malaysia
Received for Publication May 2,2001 Accepted for Publication May 23,2001

ABSTRACT
A high-peformance liquid chromatographic (HPLC) method is described for the simultaneous separation of @id classes and vitamin E isomers. The detection of both groups was made possible by using an evaporation light scattering detector and a fluorescence detector connected in series. Separation of partial acylglycerols and freefatty acids was satisfactoy . In addition, five major vitamin E isomers in palm oil, namely a-tocopherol, a-, p, y- and Gtocotrienols were also resolved. The accuracy of the method, in terms ofpartial acylglycerols andfiee fatty acid, was in agreement with the result obtainedfrom the gas chromatographic method. The mean recovey of a-tocopherol on spiked tripalmitin was 95.2%.

Torresponding author: TEL: +603-8925661; FAX: +603-89256197; E-mail: felixmoh@porim.gov.my

Journal of Food Lipids 8 (2001) 179-190. AN Righrs Reserved "Copyright 2001 by Food & Nutrition Press, he., Trumbull, Connecticut.

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M.H. MOH, T.S. TANG and G.H. TAN

INTRODUCTION
The separation of lipid classes is commonly carried out by normal-phase highperformance liquid chromatography (HPLC). Under such circumstances, there is very little separation by chain length or degree of unsaturation, allowing each class of lipid to be eluted as a single peak (Hammond and Irwin 1988). By coupling with different types of detectors, many HPLC procedures have been reported. Refractive index (RI) detection was used by Greenspan and Schroeder (1982) for the separation of tri-, di-, monoacylglycerols and free fatty acids. Though baseline resolution of the classes was achieved using a solvent mixture of heptane/tetrahydrofuradformic acid (90: 10: 0.5, v/v/v), each analysis took about 1 h. By using a solvent mixture of heptane/tetrahydrofadformic acid (80: 20: 0.5, v/v/v), Ritchie and Jee (1985) managed to shorten the analysis time by 50%. Riison and Hoffmeyer (1978) described the separation of mono- and diacylglycerols in an emulsifier using isocratic isooctane/iso-propanolmixture (95: 5, v/v) and ultraviolet (W)detection at 213 nm. The major advantage of W detection over R detection I is that the former permits gradient programming. However, according to Greenspan and Schroeder (1982), saturated free fatty acids are not suitable for shortwavelength UV detection as they exhibit almost no absorbance in this region. HPLC with an evaporative light scattering detector (ELSD) has found wide applications in the analyses of lipid classes (Chnstie 1985; Liu et al. 1993; Moreau et al. 1996). The ELSD can be considered as universal because of its versatility, in that it responds to any solute that does not evaporate before passing through the light beam. In addition, it gives excellent results under gradient elution with very little baseline drift during continuous operation even with abrupt changes in solvent composition (Moreau and Christie 1999). Normal-phase HPLC (van Niekerk 1973; Weber 1984; Diack and Saska 1994) is also a common technique for separating vitamin E isomers besides the reversed-phase technique (Strohschein et al. 1999). Usually a mobile phase consisting a mixture of two or three solvents (Such as hexane, heptane or diethyl ether) as a major component, and a more polar solvent as modifier can separate all eight tocopherols or tocotrienols (Rathjen and Steinhart 1999). A number of publications describing the use of W and ELSD detectors for determining vitamin E have been reported (Weber 1987; Warner and Mounts 1990). However, due to its superior sensitivity, fluorescence detector is preferred (Thompson and Hatina 1979; Bourgeios 1992; Balz and Their 1993). In this study we report an HPLC technique for the complete separation of free fatty acid, di- and monoacylglycerols and their detection by ELSD. By connecting to a fluorescence detector in series to the ELSD, simultaneous separation and quantification of the vitamin E isomers typically present in palm oil products under the same HPLC conditions is also achieved.

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MATERIALS AND METHODS High Performance Liquid Chromatography (HPLC)

The HFLC system (Jasco International Co. Ltd., Tokyo, Japan) consisted of a PU-980 pump, a DG-980-50 3-inline degasser, a LG-980-02s ternary gradient unit, an AS-85 1 autosampler, and a CO-965 column oven. The data was recorded and analyzed using Borwin Version 1.21 chromatographic software (JMBS Developpements, Le Fontanil, France). A Sedex 55 evaporative light scattering detector (ELSD, SEDERE, Alfortville, France) was connected serially to a Jasco FP-970 fluorescence detector. The drift tube temperature of the ELSD was set at 90C, and the flow of the carrier gas (purified air) was set at 2.3 kg/cm3. The fluorescence detector was set at 290 nm (excitation) and 330 nm (emission). The separation was performed on an APEX DIOL I1 (250 mm x 4.6 mm id., 5 pm particle size, Jones Chromatography Ltd., Mid Glamorgan, England), and maintained at 30C in the column oven. HPLC-grade n-heptane and 2-propanol were purchased from Merck (Darmstadt, Germany). The mobile phase gradient program is described in Table 1. The flow of the mobile phase was 1.0 mL/min throughout the analysis with total run of 18 min.

TABLE I . THE MOBILE PHASE GRADlENT PROGRAM

Channel (YO)

Time (rnin)
0 2

R
2 2 15 2

10
11 18

98 98 85 98 98

Channel A, heptane; Channel B, 2-propanol Flow rate, I mL/rnin

Samples were dissolved in dichloromethane (Merck), and 20 pL aliquots were injected for HPLC analysis in duplicates. Calibration curves were constructed by measuring peak areas against concentrations, and the correlation coefficient (12) was used to assess the calibration curve.

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M.H. MOH, T.S. TANG and G.H. TAN

Gas Chromatography (GC) A Hewlett Packard (HP-5890) GC equipped with a 12 m Polyimide-clad HT5 fused silica capillary column (SGE, Austin, TX) of 0.53 mm i.d. and a coating thickness of 0.15 pm was used to separate lipids. Helium carrier-gas column flow rate was 0.80 mL/min. The injector and detector temperature was maintained at 350C. A temperature program with a total running time of 40 min was used. The column temperature, after an initial isothermal period of 0.5 min at 150 C, was increased to 350C at a rate of 6C/min and maintained at this temperature for another 6 min. Samples (1 pL) were introduced by an automatic sampler HP-7673. Lipid standards, purchased from Sigma (St. Louis, MO), were separated according to different carbon numbers. The integration of chromatograms was performed using a HP-3365 I1 ChemStation. Sample Preparation The weights of standards and samples were accurate to 0.1 mg throughout this study. All solutions were filtered through 0.45 pm Whatman filter paper prior to LC analysis, and the injection volume was 20 pL, 1-Monopalmitin, 1,2- and 1,3dipalmitin, trilaurin, tripalmitin, tristearin, triolein, cholesterol, and a-tocopherol were purchased from Sigma Chemical Co. (St. Louis, MO) with 99%+ purity. Palm-based vitamin E concentrate (trade name "Tocomin") and palmitic acid (99% + purity) were supplied by local industries. Lipid standard solutions were prepared by weighing 0.1 g of each standard into a 100 mL volumetric flask and dissolved and made to 100 mL. mark with dichloromethane to provide a stock solution with 1 mg/mL concentration. The stock solution was further diluted to approximately 0.5, 0.2, and 0.1 mg/mL working standard solutions. These four solutions were used for establishing the calibration curves. According to the literature (AOCS 1997), the fluorescence intensity of tocotrienol is the same as tocopherol. Thus, in this study, only a-tocopherol standard solutions were used to construct the calibration graph for the quantitative analysis of vitamin E contents in palm oil. The a-tocopherol stock solution was prepared by dissolving 1.0 g a-tocopherol in 100 mL volumetric flask with dichloromethane. The range of the calibration solutions was 2000, 1000, and 500 I%/*. Statistical Analysis Data are presented as means f SD. The level of significance for differences between groups was tested using the Student t-Test.

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183

RESULTS AND DISCUSSION

The study investigated the possibility of separating free fatty acid, partial acylglycerols and vitamin E isomers of palm oil in a single HPLC run. The identification of the free fatty acid and partial acylglycerol peaks using ELSD were straightforward using primary standards. For the identification of vitamin E isomers, Tocomin was fractionated into tocopherols and tocorienols according to the HPLC procedure described by AOCS (1997). Individual fractions of these isomers were collected via stream-splitter installed between the column and the fluorescence detector. These fractions were therefore used for the identification of tocopherols and tocotrienols in this study.
1 2

00

5.0

L
10 0

I5 0

Tme ( ) A

FIG. 1. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC (HPLC) ANALYSIS OF VARIOUS LIPID STANDARDS USING EVAPORATIVE LIGHT SCATTERING DETECTION (ELSD) Peak Identification; 1, Tripalrnitin; 2, Palrnitic Acid; 3, 1,3-Dipalrnitin; 4, 1,2-Diplamitin and 5,2-Monopalrnitin.

Figure 1 illustrates the ELSD chromatogram of various lipid standards. The least polar tripahtin eluted first followed by p a h t i c acid (5.6 min) while the most polar 1-monopalmitin (13.1 min) came last. For partial acylglycerols, 1,3dipalmitin eluted at approximately 6.1 min and its positional isomer 1,2-dipalrnitin at 7.8 min. The cholesterol was found to elute between the two diacylglycerols. The

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M.H. MOH, T.S. TANG and G.H. TAN

calibration curves of free fatty acid, mono- and diacyl-glycerols are shown in Fig. 2. The nonlinearity pattern between sample concentration and ELSD response was also reported elsewhere (Liu et al. 1993;Moreau et al. 1996). However, by plotting a log against log calibration curve, linearity could be achieved with correlation coefficient (2)close to unity. The accuracy of the HPLC-ELSD detection of free fatty acid and partial acylglycerols was also compared with those obtained from the GC-FID method (Table 2). The co-efficients of variation obtained for the analysis of free fatty acid, mono- and diacyl-glycerols by HPLC-ELSD detection are 0.241.05%, 0.01-0.22% and 1.47-5.88%, respectively.

1000000

al

600000

e I? W
n

400000

200000

0
0
02

04

0.6

0.8

12

Concentration (mglmL)

FIG. 2. NONLINEAR CALIBRATION CURVES OF FREE FATTY ACID AND PARTIAL GLYCERIDES OBTAINED FROM ELSD

The total monoacylglycerols content found in two separate lots of crude palm oil (CPO) (Fig. 3a) and crude palm olein (CPOoL) was very low (<1%), but high in free fatty acids (3.80-5.30%) and diacylglycerols (4.48-6.36%). As the oil undergoes steam distillation under vacuum, most of the free fatty acids are removed.

5
L] I

TAE3LE 2. COMPARISON RESULTS OF FREE FATTY ACID AND PARTIAL ACYLGLYCEROLS OBTAINED FROM HPLC AND GC

HPLC, %

GC, % MG
0.68 (0.01) 0.34 (0.02)

Samples

Code

FFA

DG
FFA
5.57 (0.04) 3.84 (0.02) 4.20 (0.02) 4.18 (0.01) 4.48 (0.01) 5.79 (0.00) 5.67 (0.01) 6.36 (0.01) 0.22 (0.01) 0.28 (0.01)

DG
4.62 (0.01) 5.98 (0.01) 5.82 (0.01) 6.67 (0.01)

MG
0.41 (0.01) 0.21 (0.01) 0.14 (0.01) 0.16 (0.01)

E w ! 2

CPO

A0039 A004 1

5.30 (0.02) 3.80 (0.04)

8
z

CPOOL

B0912 B0914

4.12 (0.02) 4.1 1 (0.01

5?

CPO = crude palm oil CPOoL = crude palm olein Values in paratheses are standard deviations of two readings.

<

2
b
B

186

M . H . MOH, T.S. TANG and G.H. TAN

00

50

10.0

15 0

Tmc (I&)

FIG. 3 . HPLC CHROMATOGRAMS OF CRUDE PALM OIL USING (A) ELSD AND (B) FLUORESCENCE DETECTION Peak Identification: 1, Triacylglycerol;2, Free Fatty Acid, 3, 1,3-Diacylglycerol; 4, 1,2Diacylglycerol; 5. I -Monoacylglycerol; 6, or-tocopherol; 7, a-tocotrienol; 8,P-tocotrienol;9, ytocotrienol and IO,&tocotrienol.

As a result, the refmed, bleached and deodorized (RBD) palm olein contains very low free fatty acid content (<O.1%). However, its diacylglycerol content remains relatively high (2.8-3.4%) (Fig. 4a). In terms of vitamin E analysis, the separation of major tocopherol and tocotrienol peaks commonly found in palm oil was satisfactory. These peaks identified as a-tocopherol (4.8 min), a- (5.1 min), p- (5.8 min), y- (6.8 min) and 6tocotrienols (8.1 min) are illustrated in Fig. 5 . The calibration graph of the fluorescence intensity against the concentration of a-tocopherol is linear (9 = 0.9997), producing a regression equation of y = 3828x - 927893 (Table 3). The mean recovery of a-tocopherol was 95.2%, as determined from spiked tripalmitin (n=3).

SIMULTANEOUS DETERMINATION OF FREE FATTY ACID

187

(a)

I
00
5.0 Tune (min)
10.0

15 0

FIG. 4. HPLC CHROMATOGRAMS OF REFINED, BLEACHED AND DEODORIZED PALM OLEIN USING (A) ELSD AND (B) FLUORESCENCE DETECTION Peak Identification: 1, Triacylglycerol; 2,1,3-diacylglycerol; 3, 1,2-diacylglycerol; 4, a-tocopherol; 5, a-tocotrienol, 6, P-tocotrienol; 7, y-tocotrienol and 8.6-tocotrienol.

A typical vitamin E profile of CPO is illustrated in Fig. 3b. The mean ratio of individual isomer was found to be 14% of a-tocopherol, 12% of a-tocotrienol, 1% of p-tocotrienol, 55% of y-tocotrienol and 18% of 6-tocotrienol. One lot of CPO analyzed gave high vitamin E contents of 1449 pg/g, while the other four lots ranged from 890 to 978 pg/g. The total vitamin E contents in RBD palm olein (654-754 pg/g) was slightly reduced after the deodorization process (Fig. 4b).

188

M.H. MOH, T.S. TANG and G.H. TAN

00

50
T c (&) m

10 0

I5 0

FIG. 5. H P I L ANALYSIS OF PALM-BASED COMMERCIAL VITAMIN E CONCENTRATE USING FLUORESCENCE DETECTION Peak Identification: I , a-tocopherol; 2. a-tocotrienol; 3, p-tocotrienol;4, y-tocotrienol and 5.6-tocotrienol.

TABLE 3. CALIBRATION DATA OF a-TOCOPH1:ROL

~~ ~

Conc IidrnL

a
6744237 2809522 1026421

Mean
6748437 3 2839917 7 10263570

SD

2000
1000

500
r
= 0.9997

6706424 2583355 102655R

679465 I 2826036 1026092

44263 2 38595 5 239 5

y - 3821 x -927893

CONCLUSION

The HPLC procedure described provides a simple and convenient method for n monitoring the changes in the content of partial acylglycerols and vitamin E i a palm oil processing plant.

SIMULTANEOUS DETERMINATION OF FREE FATTY ACID

189

ACKNOWLEDGMENTS The authors thank the Director-General of Malaysian Palm Oil Board for his permission to publish this paper, and Khosatum Telepok for her technical assistance.

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RIISOM, T. and HOFFMEYER, L. 1978. High performance liquid chromatography analyses of emulsifier: 1. Quantitative determinations of monoand diglycerols of saturated fatty acids. J. Am. Oil Chem. SOC.55,649-652. RITCHIE, AS. and JEE, M.H. 1985. High-performance liquid chromatographic technique for the separation of lipid classes. J. Chromatogr. 329,273-280. STROHSCHEIN, S., RENTEL, C., LACKER, T., BAYER, T. and ALBERT, K. 1999. Separation and identification of tocotrienol isomers by HPLC-MS and HPLC-NMR coupling. Anal. Chem. 71,1780-1785. THOMPSON, J.N. and HATINA, G . 1979. Quantitative determination of tocopherols and tocotrienols in foods and tissues by HPLC. J. Liq. Chromatogr. 2, 327-344. VAN NIEKERK, P.J. 1973. The direct determination of free tocopherols in plant oils by liquid-solid chromatography. Anal. Biochem. 52, 533-537. WARNER, K. and MOUNTS, T.L. 1990. Analysis of tocopherols and phytosterols in vegetable oils by HPLC with evaporative light-scattering detection. J. Am. Oil Chem. SOC. 827-83 1. 67, WEBER, E.J. 1984. High performance liquid chromatography of the tocols in corn 61, grain. J. Am. OilChem. SOC. 1231-1234. WEBER, E.J. 1987. Carotenoids and tocols of corn grain determined by HPLC. J. Am. Oil Chem. SOC.64,1129-1 134.