GLC ANALYSIS OF FREE FATrY ACIDS OF COW AND

Indian !.Dairy Sci., 40, 2, 1987
GLC ANALYSIS OF FREE FATrY ACIDS OF COW AND BUFFALO
GHEE WITHOUT THEIR PRIOR ISOLATION*
DEEPAK SHARMA'"
and M.P. BINDAL
National Dairy Research Institute, Kamal 132001
(Received on 20th December, 1985)
(0.1 ml) of 19:0 acid as an internal standard
followed by methyl urea (300mg) and 14%
Ghee. (clarified butter fat) is an important
BFs-methanol (1 ml). The contents of vial
indigeJl()us milk pwduct. Its consumer appeal were shaken vigorously until methyl urea was
is due to its pleasant flavour.
dissolved and then incubated in a boiling water
Besides the components hke lactones and bath for 5 min after which these were rapidly
carbonyls, the presence of free fatty acids cooled to room temperature. The methyl
(FFA) ar~. known to play an important role in esters thus prepared were thoroughly mixed
the flavour of ghee. The occurrence of high with saturated solution of NaC I (4 m1) and then
levels QfPPA is known to cause rancid flavour. extracted with petroleum ether fraction (4 xl
Therefore analysis of FFA is important. In ml) freshly distilled at 62'C. The ethereal
this paper R method for preparation of methyl layer dried over anhydrous Na.SO, was filtered
esters ofFFA without their prior isolation from through filter paper Whatman No. 4 and the
volume was adjusted to 0.2 ml under a slow
ghee for OLe analysis is described.
stream of dry nitrogen.
MATERIALS AND METHODS
GLe analysis: The methyl esters of FFA
GheesaR/ples: Composite milk samples (six) (0.5 ,.1) were injected into Pye-Unicarn series
collected separately from cow and buffalo herds 304 dual column gas chromatograph equipped
of the mstitute were pwcessed for preparation with FID detector and separated over glass
of ghee ·samples by three different methods, column (1m x 4 mm id) filled with 10% DEGS
name1y,direct cream (DC), creamery butter (Diethyl glycol SUccinate) coated on Diatomite(CB) andindigenou8 (Desi). Butter was clari- CA W (JOO-)20mesh) run at 68°C for 4:0 acid and
fied at lIS-C.
then isothermally at 170"C. Injector and detePreparatitJn oj methyl esters oj FFA of ghee: ctor temperatures were 210"C and 22S·C resMelted ghee (0.1-0.15 ml) was weighed accura- pectively. The flow rates of nitrogen, hydrotely in a Teflon coated screw cap vial to which gen and air were adjusted to 30, 33 and 343
was added: 0.2 % dichloromethane solution m1fmin respectively. The performance of gas
chromatograph was checked with mixture of
standard fatty acid esters and peaks were iden• A part otthe work was presented at IV convention of
tified
by comparing their retention times. The
Association of Food Scientists and Technologists
peak area was measured with CDPI integrator
(I ndi.} Mysore.
fitted
with GLC. The quantification of indi"A part "fthc work was submitted by one of the authors
(DS) to th<,: Kurukshetra University in partial fulfillment vidual FFA was done by the method of Chapman
of the ,requirement for M.Sc. Dairyillg.
(1979).
INTRODUCTION
238
Free fatty acids of ghee
239
RESULTS AND DISCUSSION
Prior to the GLC analysis ofFFA, their isolation from fat was almost a pre-requisite for preparation of their methyl esters (Singhal and lain,
1973: Stark et al; 1976; Woo and Lindsay 1980).
The isolation of FFA from fat over silicic acid
column (lyer et af; 1967; McCarthy and Duthi,
1962;. Bills et 01; 1963) or anion exchange resin
(HUIl1bert and Lindsay, 1969; Singhal and Jain,
1973) !llLve a very low recovery particularly of
short chain fatty acids. The isolation of FFA
as their potassium salts (Singhal and Jain, 1973; : )-+:::::::::;:::....,r--.::::=:::;=---r--~'"l[
Stark;et al., 1976) has also been strongly c r i t i c i s e d
'
~
Rl 'E.~'IQN tiNt
due t~ induced hydrolysis of glycerides and
inconsistence performance of the colwnn. Such Fig. 1 OLC Pattern of GlycerideS (Triolein and Trip.I-.
a sit]L8.tion led us to find out a suitable method
mitin and Acid. (C,.:. And Cwo) in the presence
of Methyl urea.
for preparation of methyl esters of intact FFA
without their prior isolation.
-
Chapman (1979) reported that methyl urea
form$ some unknown inclusion complexes with
glycerides whereas the associated FFA do not
form these complexes and thus easily can fonn
methyl esters under suitable conditions of
reacii':>n, This principle has been made use in
this method also. It is evident from the analysis
of model mixture including triolein, tripalmitin
and 14:0and 18:0 acids (Fig I) that the glycerides
in the presence of methyl urea remained inert
,
,
towards BFI-methanol reagent and did not fonn
Rlllit'(Uoi T"'E: _. • _
......_
esterS whereas 14:0 and 18:0 acids could be
easily converted into their respective methyl Flg. 2 OLC Pattern of Gly""rides (Triolein and Tripiil_ .
mitin and Acids (Cwo and Cwo) in the abstnce
e~ter.s. In the absence of methyl urea (Fig 2),
of Methyl Urta.
all the four peaks of 14:0 16:0, 18 :0 and 18:1
were· nbserved showing that both acids and presented in Table 1. The data was also analysglycerides formed esters.
ed statistically (Table 2) according to 2x2 way
analysis of variance with interaction.
The method requires very small amount of
fat (7()"100 rug), is much less time consuming
The total concentrations of FFA (mgJg fat)
and a[so very simple.
in cow ghee (5.9 to 12.3) was 0.97, L24and
1 .70 times that of buffalo (5.8 to 7.6) in DC;
GLC analysis of FFA: The average fatty acid CD and 'desi' ghee respectively. It is aisoevi,
composition (mg/g) of FFA in cow and buffalo dent that the total concentration of FIlA was
ghee prepared by three different methods is maximum in 'desi' ghee of both tIte species.
Deepa~
Sharma and M,P. Bindal
TABLE 1
Average composftlon of FFA mgjg present in cow and buft'a1o IIhce SIIpl. prepared by different methods"
cow
Ack1
GHEE
Direct Cream Creamery blllter
BUFFALO GHEE
'De,i'
Direct Cream Creamery butter
'Des;'
4:()
0 .01
ti:Q
8:0
10 :0
0.04
0 . 11
o. r1
0 .08
10 :1
0 . 20
0 .23 .
0. 14
0.06
0.02
0.03
0.24
0.27
0.25
0 .32
0.01
0 .01
12 :0
0 .26
0 .37
0 .$9
0 .31
0.43
0.43
12: I
0.01
0 .02
0 .02
.02
0.02
0.02
14 :0
0.74
0 .91
1.00
1.27
1.12
14:1
0. 03
0 .03
1.33
0.10
0.10
0.03
0.03
14 :2
0.03
0. 04
0. 01
16;0
2. 82
2 . 16
4.14
2.39
2.92
2.87
16 :1
0 .03
16 :2
0.02
IS :0
0 .73
0 . 24
0. 52
0 . 16
0. 21
0.24
18:1
2.31
I. 74
1.25
UO
18:2
0 .07
0. 20
4.27
0.37
0 .25
O. f9
1.11
0.35
Unidentified
0.13
0.07
O. [5
0.05
0.18
0.19
Total
7.32
5. 99
12.29
5. 83
7 . 25
7. 58
0.04
.- -~.
"Tnese are the mean values of six samples analysed in duplicate.
Shor( clIaiJl(4:0 ti) 8:0) FFA: The analysis
of model mixtures of these fatty acids and rancid
ghee samples gave three peaks showing the
recovery of 4:0 6:Q and 8:0 acids by this method
. whereas fresh ghee samples did not give any
peak. This showed that the short chain fatty
acids did not escape esterification nor were washed away during washing of methyl esters. Tl1u~
it is eyideilt that fresh ghee was practically devoid
of shott chain fatty acids. Singhal and Jain
(I973) also reported the absence of these acids
in fresh ghee.
although the tota.! concentration of FFA was
higher (1.32-1.65 times) in cow ghee, The
concentration of major individual fatty acids
i.e. 10:0, 12 :0 and 14 :0 was more in bumlio
!han cow ghee.
Long chain (15:0 and above) FFA: Among
tong chain FFA, 16:0 and 18:0 are the major
constituent acids. Acid 16:Q contributed to the
level of 34- 37% of total fatty acids in cow
ghee (2. 84. I) and 34--48 % in buffalo ghee
(2.4.2.9) whereas 18:1 acid contributed around·
30-34 % in cow ghee (1.7-4 .3) and only 16-22%
Medium chain (10;0 to 14:0) FFA: The level in buffalo ghee (I .2-1.2). The concentration
of medium chain FFA in buffalo ghee (1.6--2 .1) of 16:0 acid was about 2-3 times that of 18;0
was 1 .15to 1.69 times that of cow ghee (! .2-2 .3) . acid. Other acids 15:0, 16;1, 16:2 and 17;0)
Free fatty acids of ghee
TABLE 2
ANOVA table for free fatty adds (IN"enl In row and bmfnlD ghce prepared by three dilrerent metbocb
(Dc, CB and ded)
--
.
SQurces of variation
Between Replicates
Degree
of
freedom
·BetV.'eCII
species and
10:0
14;0
12:0
16;0
18;0
18;/
0.014664
0.0732169
O. 0806319
0.044090
O.OO788S
0.099749"
0.0016233
0.176459
0.885060**
0.676737·. 29.92197·'
2
0.070385"
0.152939
0.231935
3.52852
0.0977954
7.79328·'
2
0.019929
0.049257
0.467021
3.124772
0.1440311
10.656829··
25
0.0126607
0.050501
0.156938
1.30994
0.667155
1.378923
5
'!Jelween species
lle1ween methods
Mean sum of square
0.8S99.lj
.!llethods
"Significant at S % level
··Significant at 1 % ..""I
were either absent or present only in trace
.amounts. The higher level of FFA in 'desi'
. ghee. might be due to more extensive hydrolysis
of glycerides during fermentation process
illvolved. It was noticed that such hydrolysis
was much less in buffalo than in cow milk.
Similar observation was reported by Rama.
.Murthy and Naraya.nan (1974).
The data on statistical analysis (Table 2)
teyealed that the variations in the concentration
of 10;0, 16:0, 18:0 and 18:1 acids were highly
Significant (P";;O .1) behveen cow and buffalo
ihee samples whereas such variations in ghee
,samples prepared by different methods were
significant only for 10:0 and 18 :1 acid.
SUMMARY
A simple method was developed for the :prC'oparation of methyl esters of free fatty Ilotids
(FFA) of milk fat without their prior isolation .
for subseqw:nt analysis by GLC. In this
method, ghee (0.1 ml) is incu.bated with BF;smethanol reagent in the presence of methyl tirea
and an internal standard (19;0 acid) at lOO·C
for 5 min, followed by extra.ction of the resultant
methyl esters with petroleum etller. The methyL
esters were analysed by GLC.
The short chain (4;0 and 6:0) FFA were either
absent or present only in trace amounts in: g~
prepared by different methods. The conc;en~
trations of both medium chain (10;0 to 14:0)
and long chain (15:O and above) FFA Werehigher in cow than in buffalo ghee.
242
Deepak Sharma and M.P. Bindal
REFERENCES
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3, ! 17.
'!jilts'. D.D., Kn.1Iri, L.L and Day. E.A. (l 96}) J. Dairy
S~{.• 46, 1342
Ram. Munhy, M.K. and Narayanan, K,M, (1974) Ind.
Chapm.n, O.w. (1979) JAOeS, 56, 77.
Singhal, 0.1'. (lnd lain, M.K. (1973) Ind. J. All/nt, Sci.,
43. 1026.
JfUJ).pert, ES. and Lindsay, R.C. (J969) J. Dair)' Sci..
~ •. 1962,
iYcr.
M .T., Richardson, 'T., Annmdw", C.H. and
lli>urd"",,,. A. (1967) J. Dairy Sci., 52, 2&5.
J • D Ofr}
' ' .><1..
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Stark, W., Urbach, G. , and Hamilton, J.S. (1976) J. DIliry
Re.<.. 43, 469.
Woo, A.H., and Lin<isay, R.C. (l980) J. Doi,y Sci., 63.
t05S.

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