MARDI Res.Bull.

, (1985)13, 2: (190-193)

DETERMINATION OF BENZOIC ACID IN CHILLI SAUCE BY HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY
J.S.CHIA* andZ. BADRIAH*

Chilli sauce.
Keywords:Benzoicacid, High performanceliquid chromatography,

RINGKASAN

Satu kaedah untuk penentuan asid bcnzoik dalam sos cili dengan kromatografi cccair tckanan
tinggi (HPLC) menggunakan turus Hewlett Packard RP-8 dan 57. mctanol dan metanol (50:50)
s e b a g a i f a s a b e r g e r a k d i t e r a n g k a n . I a d i k e s a n p a d a 2 5 4 n m d e n g a n m c n g g u n a k a n p e n g e s a nj a r a k
g e l o m b a n g p e m b o l e h - u b a h . P e m u l i h a n 8 1. 5 c / , , + 2 . l l c / r ' t e l a h d i d a p a t i a p a b i l a - 5 p g a s i d b e n z o i k
s t a n d a r d d i g u n a k a n d a l a m k a j i a n i n i . P e r s a m a a ny a n g b a i k d i d a p a t i a n t a r a h a s i l d a r i p a d a p e n g g u n a a n
HPLC dan kaedah penyulingan wap. kecuali sedikit gangguan yang mungkin discbabkan oleh asid
v o l a t i l d a l a m h a s i l k e p u t u s a n y a n g l e b i h t i n g g i y a n g d r p e r o l e h i d e n g a n k a e d a h p e n y u l i n g a nw a p .

INTRODUCTION w i t h t i t r i m e t r i c a n a l v s i s( P e n n s o N , 1 9 7 0 ) .

Advancements in methodology and MATERIALS AND METHODS

instrumentation have made high perfor-
mance liquid chromatography (HPLC) a Instrument
very powerful tool for the analysis of food
components and additives. High perfor- Analyses were performed with a
mance liquid chromatography methods for Hewlett Packard 10848 Liquid Chroma-
the determination of benzoic acid and sorbic tography equipped with a variable wave-
acid in fruit juices and other food products length detector (190-6(X) nm). a variable
h a v e b e e n r e p o r t e d ( N e l s o N , 1 9 7 3 ;S n v l v , v o l u m e i n j e c t o r i n j e c t i o n s y s t e m ,a b u i l t - i n
WoonwRnn and CoNpno. 1916: printer/plotter and a digital integrator. A
E r s E N s e r s sW, e n E n a n d E H l e n o r N c . 1 9 7 7 1 reversed phase RP-8. l0 pm Hewlett
CnnruEvnlE, 1980). Advantages of this Packard Column (200 x 46 mm i.d.) was
w i t h t h e s p e e d .t h e
t e c h n i q u ea r e a s s o c i a t e d used with 57r' methanol:methanol(Merck,
specificity of the analysis and most HPLC grade) as the mobile phase (A:B).
important of all, minimal sampleprepara- The solvent was filtered through a 0.i3 p
tion was required. Titrimetric and spectro- Millipore filter with the aid of gentle
photometric methods are the official suction. The flow rate of the mobile phasc
methods recommended (Pennsoru, 1970: was set at 1.5 ml/minute. The variable
A . O . A . C . , 1 9 7 5 ) . H o w e v e r , t h e s em e t h o d s wavelength detector and the reference
are tedious and at times results are not w a v e l e n g t hw e r e s e t a t 2 5 4 n m a n d 4 ( X )n m
reproducible. r e s p e c t i v e l yT. h e o v e n a n d s o l v e n tt e m p e r a -
tureswere set at 35' Centisrade.
Chilli sauceis a product prepared from
c h i l l i , s u g a r .v i n e g a r ,s a l t a n d s p i c e sw i t h p H Sample Preparation
v a l u e sa r o u n d . 3 . 5 .S o d i u m b e n z o a t e ,w h i c h
i s a p r e s e r v a t i v ew i t h a p H r a n g eo f 2 . 5 - 4 . 0 Seven bottles of chilli sauce were
for optimum microbial inhibition (Funrn, randomly collectedfrom the market. The
1 9 6 8 ) ,i s a c o m m o n p r e s e r v a t i v eu s e d i n t h i s p H o f 4 g o f h o m o g e n i z e ds a u c es a m p l ew a s
product. The present paper described a a d j u s t e dt o 2 w i t h d i l u t e s u l p h u r i ca c i d a n d
simple and rapid estimation by reverse e x t r a c t e dt w i c e w i t h l 0 m l o f d i e t h y l e t h e r
phase HPLC of benzoic acid in chilli sauce, (BDH, Anala grade). The ether solution
and the percentage recovery as compared was extracted twice with l0 ml of 0.1 N
*Food Technology Division. MARDI. S e r d a n g , S e l a n g o r ,W c s t M a l a v s i a

190
sodium hydroxide (BDH, Anala grade) anc Table 1. Retentiontime of standardbenzoic
the ether portion was discarded. The acid at different flow rates and Vomethanol
aqueous phase was acidified to pH 2 and re- Flow rate % B Retention time
extracted first with 20 ml and then with 10 (ml/min) (methanol) (min)

ml of ether. The ether was evaporated off I 70 4.05

and the residueswere dis-solvedin 10 ml of 1.5 50 3.75
1.5 80 ) i 1

methanol (Merck, HPLC grade). 2.0 30 3.94

2.0 40 3.20
The extracted sample was filtered 2.0 50 2.86
through a 0.5 pcMillipore filter with the aic 2.0 75 2.10

of Luer Lock syringe into a sample vial and

10 pl of this extract was injected into the lnj-start

column. 0

Preparation of Standard Benzoic Acid (500

g.g/ml) I
3
o
J.6.2.

Twenty-five milligrammes of benzoic 3.

acid (BDH, Anala grade) was accurately
weighed in a weighing boat and dissolvedin
methanol in a 50-ml volumetric flask. The
standard solution was filtered through a 0.5
ir, Millipore filter with the aid of Luer Lock
syringeinto a sample vial. Ten microlitres of
this standard benzoic acid was used in the
external standard calibration.
RESULTS AND DISCUSSION
External standard calibration method
was adopted in this study. The RP-8
column was equilibrated with the mobile
phase solvent at a maximum flow rate of 4
ml/minute. Very stable baselines were
obtained at slope sensitivity (ss) set at 0.5
and zero. Trial runs at various flow rates of
1, 1.5 and 2.0 ml/min as well as at different
solvent compositions were carried out.
Table 1 shows the retention times (R.T.) of
the standard benzoic acid at different flow
rates and solvent compositions.A flow rate
of 1.5 ml/min and a mobile phase of 5OVo
composition B was chosen. A typical
chromatogram of standard benzoic acid is
shown in Figure l.
Solvent temperature was found effect-
ing the R.T. of the peak. This was observed
when the solvent temperature was set at
Figure L Chromatogram of 10 p"l of
standard benzoic acid (500 p.glml).
ambient temperature (25'C) where fluctua-
tions in the R.T. occurred. When the
solvent temperature was set at 35"C or at a
slightly higher temperature than the room
temperature, reproducibility of the reten-
tion time was within the 5Vo retention time
window.
In the solvent container A. a 5Vo
methanol solution was used instead of the
pure distilled water. This was to avoid
excessive heat produced when methanol
(50%) was mixed with HrO (50%) in the
mixing chamber prior to flowing into the
chromatography column.
Recovery testsof the standardbenzoic
acid (5 g.g) were carried out in order to test
the efficiency of the method of extraction.
Table 2 shows the percentagerecovery and
the reproducibility of the peak. An average
of 8l.5Vo + 2.7IVo of the standard were
recovered if standard benzoic acid was
extracted using the method described for

191
 T a b l e 2 . R e t e n t i o n t i m e s a n d p e r c e n t a g er e c o v e r y o f t h e e x t r a c t e d
benzoic acid (amount injected : 5 pg)

R r - t c n t i t r nt i m c ( m i n ) Standardbenzoic acid recovercd

Standardbcnzoic acid Extractcd bcnzoic acid Amount (pg)
3.lt-s 3.flO 1 .t 3 r.t2.
ri
3.n-s 3.81 :1.I ti ft3.6
3.n5 -3.ul 1.tJ 112.1
3.ri3 3.r33 . +l.2 ri2.1
3.60 3.60 3.93 7rJ.6
3.60 3.60 3.95 79.1)
Avc:fll.5'ri+ 2.ll'r't

s a m p l ep r e p a r a t i o n .T e n m i c r o l i t r e s( 5 + g ; Table 3 shows the results of a com-

o f n o n - e x t r a c t e ds t a n d a r db e n z o i ca c i d w a s parative study of the amounts of benzoic
used as the external standardin the cali- a c i d i n s o m e c h i l l i s a u c es a m p l e so b t a i n e d
bration run. Figure 2 shows the chroma- by the HPLC method describedand the
togram of the extractedstandardbenzc'ric steam distillation method (A.O.A.C.,
acid. 1 9 7 5 ) .I n s a m p l e sC S 3 a n d C S 3 - A a g r e e -
a b l e r e s u l t sw e r e o b t a i n e db y b o t h m e t h o d s .
I n s a m p l e sC S - 1 . C S - 2 a n d C S - 4 b e n z o i c
Dctcctor rcsp()nsc
a c i d w a s n o t d e t e q t e db y t h e H P L C m e t h o d
but 12 to 21 ppm were obtained by the
steam distillation method. The higher
I r e s u l t so b t a i n e d b y t h e l a t t e r m e t h o d m i g h t
=
a b e d u e t o t h e i n t e r f e r e n c eo f o t h e r v o l a t i l e
3 a c i d sp r e s e n ti n t h e p r o d u c t s .
3
a T h e e x t r a c t o f t h e b e n z o i ca c i d f r o m
t h e s a m p i es t a y e dr a t h e r s t a b l ei n m e t h a n o l .
T h i s w a s s h o w n b v r e s u l t so b t a i n e di n T a b l e
Figure 2. Chromatogram oJ':itandord 3 . I n s a m p l e sC S 3 a n d C S 3 - A . o n t h e f i r s t
benzoic ocid extrocted in methanol. day of extraction, the amounts obtained

T a b l e 3 . C o m p a r i s o no f r e s u l t sf o r t h e e s t i m a t i o no f b e n z o i c
a c i d b y H P L C a n d s t e a m d i s t i l l a t i o nm e t h o d s

Samplecodc Estimatedbenzoicacid (pg/g)

HPLC method S t c a m d i s t i l l a t i o nm c t h o d

CS3 I 3llt + 20(n:.{) 2 ( X ) . +7 0 ( n : 2 )

. l -332 + 2 7( n : 3 )
CS3-A I l . l J + 3 0( n : 2 ) 3 ( X+
) 60(n=2)
3 l 2 0 6+ . 1 1( n : 2 )

C S I N . D .( n : 2 ) 2 l + l J( n : 2 )
CS_2 N . D .( n : 2 ) 12 (n: l)
CS_.I N . D .( n : 2 ) 1 7+ . l ( n = 2 )
S C 3 5 2 - s+ 7 ( n = 3 ) N.A.
SC_3,A 5;ll + l3 (n=3) N.A.
n : No. o f d c t e r m i n a t i o n sc a r r i e do u t .
N.D. : Not detcctable.
N.A. : Not analysed.
Thc cxtract was kept firr two davs beforc it was analysqd

192
were 1 318 + 20 ppm and I 144 + 30 ppm overcome by using a U-Bandapak CN
respectively.When tfe extractswere kept in reverse phase column with 27c acetic acid'.
sample vials for two days, the amounts m e t h a n o l ( C n n N e v n l e , 1 9 8 0 )a s t h e m o b i l e
obtained were 1 332 + 27 ppm and | 206 + p h a s e , i f q u a n t i t a t i o ni s r e q u i r e d f o r t h e s e
4 1 p p m , t h u s n o n - s i g n i f i c a n ct h a n g e sw e r e two preservatives.
observed.
ACKNOWLEDGEMENTS
Under the chromatographiconditions
used in this study, benzoic acid was not T h e a u t h o r sw i s h t o t h a n k t h e D i r e c t o r
totally resolvedfrom sorbic acid. a preserva- of Food Technology Division for his
tive which is not permitted to be used in p e r m i s s i o nt o p u b l i s h t h i s p a p e r .
sauce. However; this problem could be

ABSTRACT

A m c t h o d i s d e s c r i b e df o r t h e d et c r m i n a t i o n o f b c n z o i c a c i d i n c h i l l i s a u c eb y h i g h p c r f o r m a n c c
liquid chromatographv (HPLC) using a RP-13 Hewlett Packard column. and 5-ti mcthirnol and
m c t h a n o l ( 5 0 : 5 0 ) a s t h c m o b i l c p h a s c . I t w a s d e t e c t c d a t 2 5 ; 1n m b y u s i n g t h c v a r i a b l c w a v c l c n { t h
d c t c c t o r . R c c o v c r i c so f l t l . 5 l i t 2 . l l ' / ( w a s o b t a i n e d w h e n 5 p g o f s t a n d a r d b c n z o i c a c i d u , a su s c d i n
t h c r c c o v c r v s t u d i c s . G o o d a g r c c m c n t w a s o b t a i n e d b e t w c e n r e s u l t sf r o m H P L C a n d s t c i l m d i s t i l l a t i o n
m c t h o d s . c x c c p t s o m c i n t c r f c r c n c c . p o s s i b l yd u e t o t h c p r c s c n c c o f v o l a t i l c a c i d s w a s o b s c r v c d i n t h c
h r q h c rr c s u l t so b t a i n c d b v t h c s t c a m d i s t i l l a t i o nm c t h o u .

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r s. .sW
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c h r n m a t n g r a p h v . ( ' h r o r n u r o g r a p h i u1 0 . 2 6 2 1 . hcnzoatc and caffcinc in bcvcragcsbl rcrcrsc
phasc high prcssurcliquid chromatographr. "/.
Funte. T.E. (1968). Handhook oJ Food Additive pp. Assot. OlJ. Anul. Charn. -59. l-i 9.

Acceptedfor publicationon ISth Mav, 1985

r93
 Diterbitksnoleh:
InstitiutPeny€li6ik8n
d8n KemajusnPertanian
Mslsysia{MARDI),
PetiSurat202,U.P.M., Serdang,Selsngor,Malsysia.

Dicstskoleh Percstakan
Nadzam,KualsLumpur.