J Sci Food Agric 1989,48,495-505

Fractionation of Hydrogenated Milk Fat using Thin Layer Chromatography

William Banks, John L Clapperton, Anne K Girdler and William Steele

Hannah Research Institute, Ayr KA6 SHL, UK

(Received 1 July 1988; revised version received 2 November 1988; accepted 16 November 1988)

ABSTRACT

Thin layer chromatographic separation of various hydrogenated milk fats on activated silica gel yielded three distinct bands in each case. Fatty acid and triglyceride analyses showed that ,fraction I was composed of high molecular weight material whereas fraction I11 was of low molecular weight and virtually all the triglycerides contained 4:O or 6:O fatty acids. The molecular weight range of fraction I I overlapped those of fractions I and I l l , and the reasons for its forming a distinct band remain obscure. However, fraction I1 had neither the fatty acid composition nor the triglyceride distribution that would allow it to be merely a mixture of fractions I and III. Thermal analysis by means of differential scanning calorimetry showed the three fractions to have different melting characteristics, fraction I melting at the highest temperature and fraction III at the lowest. Hydrogenation markedly increased the heat of melting (AH) of the milk fat . However, values of AH were approximately constant across the three fractions.

Key words: Milk fat, hydrogenated milk fat, fractionation, melting fingerprints, fatty acid composition, triglyceride composition.

INTRODUCTION

Whilst carrying out a study of the effect of silver nitrate content of silica gels on the efficiency of separation of milk fat triglycerides by thin layer chromatography (TLC), it was noted that silica gel plates without silver nitrate resolved milk fat into three distinct fractions. Silica gel has been used in column chromatography to

495

J Sci Food Agric 0022-5142/89/$03.50 0 1989 Society of Chemical Industry. Printed in Great Britain

496 W Batiks. J L Clapperton, A K Girdler, W Steelr

produce from milk fat a range of fractions of different molecular weight (Taylor et at 1978; Parodi 1980), hence some spreading of the fat on the TLC plate was anticipated. However, the fact that three separate fractions were obtained was somewhat surprising. Using unmodified milk fat, Blank and Privett (1964) obtained two fractions on silica gel plates, and Breckenridge and Kuksis (1968) and Arumughan and Narayanan (1982) observed three fractions.

This paper reports the nature of the fractions obtained by TLC on silica gel using hydrogenated milk fats as the starting material. The saturated form of milk fat was chosen to eliminate the possibility that the number and type of double bonds present in the parent fats contributed to the separation.

EXPERIMENTAL

Milk fats

Milks were obtained from two groups of grazing cows (control) both of which subsequently received supplements of either soya oil or palm oil (500 g cow-' d a y - ' ) exactly as detailed previously (Banks et at 1987). Collection of milk and the preparation and storage of butter followed the scheme used in that work.

Hydrogenation of the milk fat

Hydrogenation was carried out using the technique described by Christie (1982) scaled up for I-g quantities of fat. The progress of the reaction was monitored by fatty acid analysis. Stable trans 18: 1 isomers were detected in all samples, but only in the soya oil milk fat did they achieve significant levels (57 mmol mol-' total fatty acids) and were included in the 18:Ogroup. Similarly 15:O and 17:O were ascribed to the 14:O and 16: 0 groups, respectively. Correspondingly, triglycerides containing an odd number of carbon atoms were considered as belonging to their lower neighbour when calculating molar proportions of triglycerides.

Fractionation of hydrogenated milk fat

Anhydrous milk fat (80mg) was separated on thin layers (1.25 mm) of silica gel (Kieselgel 60G; Merck) on glass plates (20 cm x 20 cm). The solvent system was hexane/diethyl ether/formic acid (40:lO:l v).

Triglyceride bands were visualised by spraying with 2,7-dichlorofluorescein and examination under UV light. Three bands of triglycerides were obtained and the fractions were designated I , I1 and 111 according to R, value (0.62, 0.55 and 049, respectively).

Triglycerides were recovered from the silica gel by extraction with chloroform, the solution was evaportated to dryness using a rotary evaporator, and the residue was weighed.

Fatty acid analysis

Milk fat and milk fat fractions were analysed as detailed previously (Banks et at 1976).

Fractionation of hydrogenated milk fat using TLC 491

Triglyceride analysis

Separation of the triglycerides and the fractions by capillary GC has been described previously (Banks et al 1985).

Melting profiles

Melting profiles of the parent milk fats, their hydrogenated derivatives and the fractions of the saturated fats were obtained using differential scanning calorimetry (Banks et al 1980, 1985).

RESULTS AND DISCUSSION

Hydrogenated milk fats

The fatty acid compositions of the parent hydrogenated milk fats are shown in Table 1; the corresponding triglyceride distributions are shown in Table 2 . The differences between the two groups on the control diet were, in terms of both fatty acid composition and triglyceride distribution, within experimental error. Therefore, only a mean value is recorded for the control treatment.

The fatty acid compositions, in terms of the carbon skeleton, are similar to those produced by the same type of dietary treatment used previously (Banks et al1987). Supplementation ofthe basal diet with soya or palm oil causes the expected decrease in the proportion offatty acids containing 6:@16:Ocarbon atoms (soya oil) or 6:O- 14:O (palm oil). Consequently, the mean carbon atom chain length (p,,) of the control fat is less than the corresponding values for the oil-rich treatments.

The triglyceride distributions are also similar to those published previously (Banks et al 1987), the control having a bimodal distribution with peaks at carbon numbers 38-40 and at 50. The high molecular weight component is emphasised in both milk fats derived from oil-rich diets. Reasonable agreement is observed

TABLE 1 The fatty acid compositions (mmol mol- ') of the parent hydrogenated milk

fats

Fatty acid Control, no oil Soya oil Palm oil

4: 0 6:O 8:O

lo:o 12:o 14:O 16:O 18:O -

P"

90 49 26 48 48

152 255 332

14.1

100 38 16 26 29

112 206 472

14.7

98 33 14 28 30

102 326 369

14.6 ~~~~~ ~ ~ ~ ~

Pn is the number-average carbon atom content of the fatty acids in each hydrogenated milk fat.

498 W Banks, J L Clapperton, A K Girdler, W Sreele

TABLE 2 Relative proportions (mmol mol- ') of triglycerides of different carbon number present in the parent, hydrogenated milk fats and the number-average value (z,) for each fat compared with the theoretical value (3 x P.) derived from fatty acid

composition

Carbon number Control, no oil Soya oil Palm oil

2 28 5 30 10 14 5 32 22 12 11 34 45 36 37 36 86 78 107 38 111 127 157 40 117 138 106 42 91 61 53 44 84 41 42 46 85 55 51 48 92 74 80 50 112 116 I40 52 91 133 149 54 52 126 58

3 x P, 42.3 44.1 43.8

-

~

C N , 43.0 44.7 44.1

between the calculated and experimental values for the number-average value of carbon number, indicating that there were no serious discrepancies in the analyses.

Fractionation of the hydrogenated milk fats

The fatty acid compositions of the three fractions obtained from each of the thre, hydrogenated milk fats are shown in Table 3; the corresponding values for .Ile distribution of molecular species in the triglycerides are shown in Table 4.

In each case, fraction I was composed virtually completely of 14:0,16:0and 18:0, the small amounts (14 mmol mol-' on average) of 12:O and shorter acids could be regarded merely as contaminants. Fraction I1 contained the highest proportions of the medium chain length acids, ie 8:0, 10:0, 12:O and 14:0, and fraction 111 contained the highest proportions of 4:O and 6:O. The proportions of 16:O in fractions I and I1 were broadly similar for each pair; the lowest proportion of 16:O was on each occasion found in fraction 111. However, for each fat the variation in 16:O across the three fractions was not very marked. By contrast, the proportion of 18:O varied by a factor of c 2.4 between fraction I (highest) and fraction I11 (lowest) with fraction I1 occupying an intermediate position.

In terms ofmolecular size, fraction I always had the highest P, and fraction I11 the lowest; the P, of fraction I1 was intermediate between these values, but somewhat closer to that of fraction I. The ratio pn/c was 1.1 for each of the three fats, and p"/F!," was similarly constant at 1.3.

The triglyceride distributions (Table 4) showed that in fraction I most of the material (> 800 mmol mol-' total triglycerides) was present in the three highest

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500 W Banks, J L Clapperton, A K Girdler, W Steele

TABLE 4 Relative proportions (mmol mol- I ) of triglycerides of different carbon number present in the three fractions obtained from each of the parent hydrogenated milk fats; the number-average value (En) for each fraction compared with the theoretical value (3 x Pn) derived from fatty

acid composition

number I II I I I I II III I II

Carbon Control, no oil Soya oil Palm oil -

-

- - 28 30 32 34 36 5 7 38 6 34 40 5 97 42 4 147 44 7 158 46 40 155 48 126 146 50 291 145 52 345 84 54 170 21

- -

- -

- -

__ 50.7 45.1 49.8 45.3

C N , 3 x P,

- - 1 24 61

- -

- -

3 I27 -

12 260 -

274 - 40 90 199 -

41 1 102 6 2 98 4 20 130 - 73 151 - 206 181 - 340 130

351 64 36.9 51.9 46.7 36.0 51.9 46.8

-

~ - ~

- - 12 33 - ~

95 202 - ~

29 8 - 11 53 287 -

53 - 103 10 1 135 6 15 159 2 98 200 1 305 207 - 399 111 - 181 19 37.8 51.3 47.0 37.2 51.0 47.1

- -

III

-

-

23 81

245 356 216 50

8 4 2 2 1

31.7 36.3

-

triglycerides, ie those with carbon numbers 50, 52 and 54; only very small proportions were found in triglycerides with carbon numbers < 44. The distribution for fraction I1 (control fat) showed a broad plateau region extending from carbon numbers 42 to 50; fraction I1 derived from both oil-rich diets did not possess this plateau, but rather a peak at carbon number 50 (soya oil) or at carbon numbers 48- 50 (palm oil). Fraction I11 was characterised in each case by (i) carbon number 38 being the dominant triglyceride and (ii) very little material (only 15 mmol mol- on average) with carbon numbers > 42.

Again, the measured values of the number-average triglyceride were in very reasonable agreement with the values calculated from the fatty acid compositions.

From the results recorded in Tables 3 and 4, the natures of fraction I and fraction I11 are easily discerned. Fraction I is the high molecular weight component, whereas fraction I11 represents that component (or at least a very large proportion of it) of the parent milk fat in which 4: 0 and 6: 0 are found at position sn 3. One would expect that such a fraction would contain 333 mmol mol-' of 4:0+6:0 (experimental values lie in the range 319-326 mmol mol-') and that no triglyceride with carbon number > 42 should be present. To an excellent approximation, both expectations are fulfilled.

Fraction 11, however, presents an enigma. Its molecular weight range encompasses those of both fractions I and 111, and yet it is obviously not merely a simple mixture of the two. It is possible that partitioning into fractions I and I1

Fractionation of hydrogenated milk fat using TLC 501

depends on the steric placement of the individual acids in the triglyceride moiety, eg that 18:0, 16:0, 18:0, and l8:0, 18:0, 16:0, go into different fractions. However, positional isomerism should have no part to play in the partition of triglyceride with carbon number 54 in hydrogenated fat. The possibility remains that the material with carbon number 54 in fraction I1 contains 18:l trans: certainly, the amounts of this isomer present in the three samples of fraction I1 are approximately proportional to the amounts of carbon number 54. Nevertheless, much more work is required to define the underlying causes for the partition between fraction I and fraction I1 of any particular triglyceride.

Using silicic acid in column chromatography, Morrison and Hawke (1979) divided milk fat into three fractions of high, medium and low molecular weight. However, in comparison with the separations reported here, their high molecular weight fraction contained rather more acids in the size range 6:O-12:0 (94 mmol mol-' vs 13 mmol mol-I). Also, the4:0+6:0 content oftheir low molecular weight fraction was on average 307 mmol mol-' as opposed to the value of 322 mmol mol- '. However, the greatest difference lies in the middle molecular weight fraction in which Morrison and Hawke (1979) recorded 4:0+6:0 values of 181 mmol mol - ; fraction I1 in the present work yielded a mean value for 4: 0 + 6:O of 32 mmol mol-'. The separation by Parodi (1980), again using a silica gel column, was comparable to that reported by Morrison and Hawke (1979) except that the low molecular weight fraction contained 4:O or 6:O in all the triglycerides. However, only - 70% of the total 4:0+ 6:O was found in this fraction, the remainder being in the medium molecular weight fraction.

Separation of milk fat triglycerides by silica gel TLC yielded only two fractions according to Blank and Privett (1964), namely one including (i) only long chain fatty acids and (ii) at least one short chain (4:O or 6:O) fatty acid. However, both Breckenridge and Kuksis (1968) and Arumughan and Narayanan (1982) reported three fractions. The former also showed that the triglyceride distribution of the middle fraction overlapped those of the other two fractions. Thus the present results obtained with hydrogenated milk fat appear to be comparable with published observations for unmodified milk fat.

Relation of the fractions to the parent fats

It is of course axiomatic that, if a fractionation is carried out properly, the fatty acid composition and triglyceride distribution of the parent fat constructed from the proportions of the fractions should be identical, within experimental error, to those measured for the parent fat. Values calculated in this way for the fatty acid composition are shown in Table 5, and Table 6 contains the corresponding triglyceride distributions.

Considering the errors involved in measuring the relative proportions of fatty acids and triglycerides and in obtaining the weight of each fraction, the agreement between Tables 1 and 5, and between Tables 2 and 6, must be regarded as highly satisfactory.

Thermal properties of the parent milk fats and their hydrogenated derivatives

The melting curves of the parent milk fats and their hydrogenated derivatives are

502 W Bunks, J L Clapperton, A K Girder, W Steele

TABLE 5 Fatty acid composition (mmol mol-') ofthe parent hydrogenated milk fats

calculated from the proportions of the fractions

Fatty acid Control, no oil Soya oil Palm oil

4: 0 6:O 8 : O

1o:o 12:o 14:O 16:O 18:O

82 43 24 52 44

147 252 355

95 104 36 38 15 15 27 29 27 26

119 1 06 203 308 477 373

TABLE 6 Relative proportions (rnmol mol- ') of triglycerides of different carbon number present in the parent hydrogenated milk fats calculated from the proportions of the

fractions

Carbon number Control, no oil Soya oil Palm oil

28 30 32 34 36 38 40 42 44 46 48 50 52 54

Trace 8

21 44 93

111 116 86 79 84 92

122 101 44

- 4

12 35 77

122 137 59 42 58 78

123 137 115

-

4 10 34

103 153 1 06 51 43 52 87

149 149 58

shown in Fig 1. The effect of hydrogenation was to move the melting curve to higher temperatures and dramatically alter the form of the curve. In particular, the melting curve of each hydrogenated fat was resolved into two distinct areas. The saturated milk fat derived from the control diet exhibited major melting peaks at 21°C and 44°C; supplementation of the diet with oil, and subsequent hydrogenation of the milk fat, moved these peaks to higher temperatures, 26°C and 55°C in the case of soya oil, and 27°C and 54-56°C for the palm oil sample. Given the large increase in 16:0+ 18:O in going from the control fat to those derived from oil-rich treatments, the movement of the high melting fraction from 44°C to 5656°C was not unexpected.

Hydrogenation produced the expected increase in the heat of melting (AH) of the

Fractionation of hydrogenated milk f a t using TLC 503

al 1

I

- 4 0 -20 0 20 4 0 60 Temperature (OC)

Fig 1. Heat flow rate as a function of temperature for the melting of the milk fats (-) and their hydrogenated forms [----); fat derived from (a) control diet, (b) diet containing palm oil, and (c) diet

containing soya oil.

milk fats. The parent fats had a mean AH of 70.6 J g- whereas after hydrogenation the average value of A H was 102Jg-'. Using comparable crystallisation and melting conditions, Timms (1980) reported that hydrogenation caused AH to increase from 73.6Jg-' to 1OOJg-'.

Thermal properties of the fractions obtained from the hydrogenated milk fat

The melting curves of the various fractions obtained from the three parent hydrogenated fats are shown in Fig 2.

For each fat, the melting point of the fraction increased with molecular weight, fraction I having the highest melting point and fraction I11 the lowest. In the case of the fats derived from the oil-rich diets, fraction I contained, in addition to the main melting peaks at 55-59"C, small amounts of material giving melting peaks at 17°C (soya oil) and 46°C (palm oil). Fraction I1 exhibited two peaks in all cases, the major one being found in the range 44-50°C and the minor one at 19-23°C. The major melting peak in fraction I11 was composed of two overlapping peaks occurring at 24 and 29°C (control), 30 and 34°C (soya oil) and 29 and 33°C (palm oil); additionally, the sample derived from soya oil exhibited a melting peak at 15°C. This last peak,

504 W Banks, J L Clapperton. A K Girdlcr, W Steele

la)

g-1

g-1

- .

-20 0 20 40 60

Temoerature (OC)

Fig 2. Heat flow rate as a function of temperature for the melting of the fractions (fraction I -; fraction I1 ---; fraction 111 obtained from the hydrogenated milk fats; fat derived from (a) control diet,

(b) diet containing palm oil, and (cj diet containing soya oil:

and the minor component of fraction I melting at 17"C, may result from the presence of residual trans double bonds in the sample obtained from feeding soya oil.

No pattern could be observed between AH and molecular weight (or melting point); the mean value of AH for the nine fractions was 113 J g- ' (SD 10 J g- ' .

CONCLUSIONS

On TLC plates coated with activated silica gel it is possible to obtain three distinct fractions from hydrogenated milk fat. Fraction I11 is composed of molecules

Fructionution of hydrogenated milk Jut using TLC 505

containing either 4:O or 6:O at triglyceride position s n 3 , whereas fraction I is composed of high molecular weight triglycerides. In terms of molecular weight, fraction I1 is intermediate between I and 111 but somewhat closer to the former fraction. Both fatty acid and triglyceride compositions indicate that fraction I1 cannot be merely a mixture of fractions I and 111. Similarly, thermal studies show that the melting properties of fraction I1 are quite distinct from those of fractions I and 111. Nevertheless, no explanation can be given for the mechanism by which the technique produces three distinct fractions which show considerable overlap with respect to molecular weight. Perhaps the most significant point to emerge from this study is that the TLC technique allows a virtually quantitative separation of that fraction of the total triglyceride that contains 4:O or 6:O at position sn 3.

REFERENCES

Arumughan C, Narayanan K M 1982 Triacylglycerol composition of cow milk fat. J Food Sci Technol (India) 19 71-74.

Banks W, Clapperton J L, Ferrie M E 1976 The physical properties of milk fats. J Soc Dairy Tecknol 29(2) 86-90.

Banks W, Clapperton J L, Kelly M E, Wilson A G, Crawford R J M 1980The yield, fatty acid composition and physical properties of milk fat obtained by feeding soya oil to dairy cows. J Sci Food Agric 31 368-374.

Banks W, Clapperton J L, Girdler A K 1985 On the fractional melting of milk fat and the properties of the fractions. J Sci Food Agric 36 421-432.

Banks W, Christie W W, Clapperton J L, Girdler A K 1987 The trisaturated glycerides of bovine milk fat. J Sci Food Agric 39 303-316.

Blank M L, Privett 0 S 1964 Structure of milk fat triglycerides. J Dairy Sci 47 481488. Breckenridge W C, Kuksis A 1968 Structure of bovine milk fat triglycerides. I . Short and

Christie W W 1982 Lipid Analysis (2nd edn). Pergamon Press, Oxford, p 61. Morrison I M, Hawke J C 1979 Triglyceride composition of bovine milk fat with elevated

Parodi P W 1980 Separation of milk fat triglycerides into classes by silver ion adsorption

Taylor M W, Norris G E, Hawke J C 1978 The thermal properties of bovine milk

Timms R E 1980 The phase behaviour and polymorphism of milk fat, milk fat fractions and

medium chain-lengths. Lipids 3 291-300.

levels of linoleic acid. Lipids 12 994-1004.

thin-layer chromatography. Aust J Dairy Technol 35 17-22.

triacylglycerols. N Z J Dairy Sci Technol 13 236-241.

fully hardened milk fat. Aust J Dairy Technol 25 47-53.