TITLE

Quantification of Sodium Benzoate in Food

OBJECTIVE
To measure the quantity of sodium benzoate in food.
INTRODUCTION
Food and beverage spoilage has been a problem throughout history. Most
food spoilage is due to enzyme action upon food. Enzymes are complex organic
compounds that may act as catalysts and cause a chemical change to occur. Most
enzymes that cause food to spoil are produced by living organisms, e.g., bacteria,
molds and yeast. Fresh foodstuffs may have some of these microorganisms present
and others may be encountered by exposure to the air or in processing.

The

enzymes that cause spoilage may be generally described as two types;

endoenzymes which exist within the microorganism and exoenzymes which are
released by the microorganism.

Consequently, the quality of microorganism

enzymes of both types which cause food to spoil is directly related to the
concentrations of the microorganism present, its species, and its general activity.
The addition of chemical preservatives to food is not new and been practiced
for centuries. Some of the most familiar preservation methods, those of brining,
pickling with vinegar, smoking, and preserving with sugar solutions, depend upon
chemical preservatives. These methods inhibit microorganism activity and retard
microorganism growth and multiplication.

These methods act in one of two

generalized way is by physically increasing the density of the microorganisms

environment or chemically, by a direct inhibiting action on the microorganism
themselves. Consequently, chemical preservatives which perform by a direct
inhibiting action on the microorganisms themselves are not new.
Sodium benzoate is a chemical preservative which in very low concentration
inhibits

the

activity

of

the

microorganisms

themselves.

It

is bacteriostatic and fungistatic under acidic conditions. It is most widely used in

acidic foods such as salad dressings (vinegar), carbonated drinks (carbonic acid),

jams and fruit juices (citric acid), pickles (vinegar), and condiments. It is also used
as a preservative in medicines and cosmetics.
Sodium benzoate is produced by the neutralization of benzoic acid with
sodium

hydroxide. Benzoic

acid

is

detectable

at

low

levels

in

cranberries, prunes, greengage plums, cinnamon, ripe cloves, and apples. Though
benzoic acid is a more effective preservative, sodium benzoate is more commonly
used as a food additive because benzoic acid does not dissolve well in water.

MATERIALS
Deionized water, Stomacher bag, Whatman No.1 filter paper, micropipette, Nylon
syringe filter (0.45m), C18 column, sodium acetate, acetic acid glacial, HPLC grade
methanol, micropipette tips, HPLC grade Sodium benzoate.

METHODS
Sample Preparation
1) 5ml of each samples is added to approximately 20 mL of water and then
homogenized on Stomacher for 10 minutes.
2) 35ml of methanol is mixed thoroughly with the mixture.
3) 100mL of water is used to brought up the volume of mixture and filtered through
Whatman paper No.1.
4) The 0.45m syringe filter is used to filter the samples prior to injection.
HPLC Condition
1) The chromatographic separation was achieved with a C18 column
2) The injection volume was 20L, mobile phase was 65:35, acetate buffer (pH 4.74)
and methanol with flow rate of 0.8mL/min for 20 minutes at wavelength of 235nm.

Preparation of Standard Curve

1) The external standard plot method was used.
2) Duplicate injections of 20L sodium benzoate standard solutions were used to
construct linear regression lines (peek area versus concentration).
3) The peaks were identified based on the retention time.
4) The standard curves were obtained with five points (5,10,20,40 and 80mg/L) for
sodium benzoate.

RESULT
A = kc
k=A
c
k = 7.7cm
7.66337 mg/L
k = 1.0478
So, the concentration of sodium benzoate, c = A
k
c = 7.3487mg/L
0.05 mL
c = 146.97mg/L

c =0.14697 mg/ mL

DISCUSSION
In this experiment, the quantity of sodium benzoate in food (soft drink Sprite)
was measured. According the result that has been obtained the concentration of
Sodium Benzoate is 0.14697 mg/ mL. Theoretically the concentration of the sodium
benzoate should be 0.1 mg/ mL. Thus, the result is accepted. The k value is 1.0478.
The value of k is calculated from the formula A = kc. The formula is derived from
the Beers Law that is A=ebc, Since eb is a constant, it can be represented as k, and
the equation can be simplified to A = kc. According to the Beers Law A is the
absorbance of the sodium benzoate (proportional to the peak height of the sodium
benzoate on the chromatogram.), e is the constant particular to sodium benzoate, b
is the path length of the cell in the instrument (which remains unchanged), c is the
concentration of sodium benzoate. Beer's law states that the absorbance is directly
proportional to the concentration of a solution. If you plot absorbance versus
concentration, the resulting graph yields a straight line.
HPLC is a chromatographic technique that can separate a mixture of
compounds

and

is

used

in biochemistry and analytical

chemistry to

identify,

quantify and purify the individual components of the mixture. HPLC typically utilizes
different types of stationary phases, a pump that moves the mobile phase(s) and
analyte through the column, and a detector to provide a characteristic retention
time for the analyte. The detector may also provide additional information related to
the analyte. Analyte retention time varies depending on the strength of its
interactions with the stationary phase, the ratio/composition of solvent(s) used, and
the flow rate of the mobile phase. It is a form of liquid chromatography that utilizes
smaller column size, smaller media inside the column, and higher mobile phase
pressures.
A buffer is

an aqueous

solution that

has

highly

stable pH.

If

you

add acid or base to a buffered solution, its pH will not change significantly. Similarly,
adding water to a buffer or allowing water to evaporate will not change the pH of a
buffer. A buffer is made by mixing a large volume of a weak acid or weak
base together with its conjugate. A weak acid and its conjugate base can remain in

solution without neutralizing each other. The same is true for a weak base and its
conjugate acid.
The buffer used in this experiment is acetate buffer. Acetate is a salt or ester
of acetic acid. Salts of acetic acid contain a metal attached to the acetic acid radical
CH3COO. In this experiment, the pH of the acetate buffer used is 4.74. This buffer
can be made using an equal volume of 0.10 M acetic acid, HC 2H3O2, and 0.10 M
sodium acetate, NaC2H3O2. Typically the pH can be set to one pH unit above or one
pH unit below the pKa for the acid. The pH used is 4.74 because the pKa of acetic
acid is 4.7. Despite, a buffer solution resists change of pH, water does not. That is
the reason why acetate buffer solution is used instead of using water.

CONCLUSION
At the end of the experiments, the quantity of sodium benzoate in food (Sprite soft drink) was
able to be detected. The quantity of sodium benzoate calculated from the formula is 0.14697
mg/mL.

REFERENCE
Saad, B., 2004. Simultaneous determination of preservatives (benzoic acid, sorbic
acid,methylparaben and propylparaben) in foodstuffs usinghigh-performance
liquid
chromatography. Journal of Chromatography A, 393397.
Sivasankar, B. (2008). Food Processing and Preservation ,5th ed., Prentice Hall of India
Private Limited, New Delhi.

UNIVERSITI MALAYSIA SABAH

SCHOOL OF FOOD SCIENCE AND
NUTRITION
SEMESTER 1, SESION 2011/2012

NT 30703
FOOD SAFETY AND QUALITY CONTROL

PRACTICAL 1
QUANTIFICATION OF SODIUM BENZOATE IN FOOD
LECTURER
DR. CHYE FOOK YEE

DEMONSTRATOR
MR TIN

NAME: SHATESH KUMAR A/L CHANDRAHASAN

MATRIX NOMBOR: BN09110073