Lab 1

Title: Laboratory 1 Quantification of Sodium Benzoate in Food
Objective:
i.
To detect the quantities of sodium benzoate found in food.
Introduction:
Sodium benzoate is a type of salt that may occur naturally in some foods but it is
more likely to be chemically produced and added as a preservative to foods. When used as
a preservative, sodium benzoate is typically added to foods in small amounts only. If too
much is added, food may have a very bitter taste. (http://www.wisegeek.com/what-is-sodiumbenzoate.html)
Sodium benzoate used as the ingredients of foods as it works very well at killing
bacteria, yeast and fungi. It commonly used as a preservative in foods with a high acid
content, since sodium benzoate will only work when the pH balance of foods is less than 3.6.
It is therefore effective in most sodas, vinegar, fruit juice, and in mixed ingredients like salad
dressing.
It
is
additionally
used
to
stop
the
fermentation
process
in
wines.
(http://www.wisegeek.com/what-is-sodium-benzoate.html)
Sodium benzoate is widely used in carbonated and still beverages, syrups, cider,
salted margarine, olives, sauces, relishes, jellies, jams, preserves, pastry and pie fillings, low
fat salad dressing, fruit salads, prepared salads, and in storage of vegetables. It has been
incorporated in those foods as an antimicrobial agent at concentrations of 0.05 to 0.1%
(Sivasankar, 2008).
There
have
been
some
health
concerns
about
the
combination
of sodium benzoate and ascorbic acid or vitamin C. When the two are mixed, they can form
the chemical benzene, which is carcinogenic. However, sodium benzoate on its own is not
considered a carcinogen and consumption of a huge amount of it in order to have toxic
levels in body. (http://www.wisegeek.com/what-is-sodium-benzoate.html)
Materials:
Deionized water, micropipette, nylon syringe filter (0.45m), C18 column, sodium acetate,
acetic acid glacial, HPLC grade methanol, micropipette tips, small beaker, HPLC grade
Sodium benzoate and sample (Sprite soft drink).
Methods:
a) Sample Preparation
i. 5mL of sample (Sprite soft drink) was
transferred into a small beaker by using
a syringe.
ii. Approximately 20mL of deionized

was then water added into the beaker.
iii. The mixture then was mixed with

35mL of methanol.
iv. The mixture then transferred into

volumetric flask. The volume was
topped up to 100mL with deionized
water.
v. Samples then were filtered through

0.45m syringe filter prior to injection.
b) HPLC conditions
i. The chromatographic
separation was achieved with a
C18 column.
iii. 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 15 minutes at
wavelength of 235nm.
c) Preparation of standard curve

2
i. The external standard plot method

was used.
ii. Duplicate injections of 20L sodium

benzoate standard solutions were used
to construct linear regression lines
(peak area versus concentration).
iii.The peaks were identified based on

the retention time.
iv.The standard curves were obtained

with five points (5, 10, 20, 40 and
80mg/L) for sodium benzoate.
Result:
From the instrumental result:
Instrument Conditions
Column Temp. (left)
Column Temp. (right)
Pressure
Flow
At start
40.0
40.0
91.3
0.800
At stop
40.0o C
40.0o C
92.0 bar
0.800 ml/ min
Using Beers Law:

A=ebc
A = absorbance of the sodium benzoate (proportional to the peak height of the sodium
benzoate on the chromatogram.)
e = constant particular to sodium benzoate
b = path length of the cell in the instrument (which remains unchanged)
c = concentration of sodium benzoate
Since eb is a constant, it can be represented as k, and the equation can be simplified to:
A = kc
Rearranging this equation gives us:
k=A
c
k = 7.7cm
7.66337 mg/L
k = 1.0478
So, the concentration of sodium benzoate, c = A
k
Expanding c, we get = Mass of sodium benzoate (mL)
Volume of solution (mL)
Concentration of sodium benzoate, c [Mass of sodium benzoate (mL)] = A
Volume of solution (mL)
c (5mL) = 7.7 cm
100mL 1.0478 cm per mg/L
c = 7.3487mg/L
0.05 mL
c = 146.97mg/L
c =0.14697 mg/ mL
Discussion
Buffer solution is an aqueous solution consisting of a mixture of a weak acid and its
conjugate base or a weak base and its a conjugate acid. Buffer solutions are functioning to
keep pH at a nearly constant value in a wide variety of chemical applications. Plus, buffer
solutions are necessary to keep the correct pH for enzymes in many organisms to work.
Industrially, buffer solutions are used in fermentation processes and in setting the correct
conditions for dyes used in colouring fabrics. They are also used in chemical analysis and
calibration of pH meters.
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 CH 3COO. The
esters contain another radical, such as ethyl, attached to the acetic acid radical. 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, HC2H3O2, and 0.10 M sodium acetate, NaC2H3O2.
Typically the pH can be set to one pH unit above or one pH unit below the pK a for the acid.
The pH used is 4.74 because the pKa of acetic acid is 4.7, which can be figured out using
Henderson-Hasselbalch equation, pH = pKa + log [A1-]/ [HA]. 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.
According to the result obtained from the HPLC instrumentation, the pressure used is
91.3 bar at start and 92.0 bar at stop. While the flow is 0.800 mL/ min both at start and stop.
The amount of sodium benzoate can be determined by using the equation below:
A=ebc
where A is the measured absorbance. The absorbance will be proportional to the peak
height of the sodium benzoate on the chromatogram; e is the constant particular to the
sample; b is the path length of the cell in the instrument, which remains unchanged and last
but not least, c is the concentration of the sample. The Beer-Lambert Law is useful for
characterizing many compounds but does not hold as a universal relationship for the
concentration and absorption of all substances. The Beer-Lambert law has implicit
assumptions that must be met experimentally for it to apply. For instance, the chemical
makeup and physical environment of the sample can alter its extinction coefficient. The
chemical and physical conditions of a test sample therefore must match reference
measurements for conclusions to be valid.
Conclusion:
At the end of the experiments, the quantity of sodium benzoate in food (Sprite soft
drink) was able to be detected. The amount of sodium benzoate obtained from the
instrument is 7.66 mg/L. The exact amount of sodium benzoate calculated is 0.14697
mg/mL.
References:
Sivasankar, B. (2008). Food Processing and Preservation ,5th ed., Prentice Hall of India
5
Private Limited, New Delhi.

Ellis-Christensen. T., 2011. What Is Sodium Benzoate? Date of retrieved 8th November 2011
http://www.wisegeek.com/what-is-sodium-benzoate.html.

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Title: Laboratory 1 Quantification of Sodium Benzoate in Food

To detect the quantities of sodium benzoate found in food.

ii. Approximately 20mL of deionized

iii. The mixture then was mixed with

iv. The mixture then transferred into

v. Samples then were filtered through

iii. The injection volume was

c) Preparation of standard curve

i. The external standard plot method

ii. Duplicate injections of 20L sodium

iii.The peaks were identified based on

iv.The standard curves were obtained

Using Beers Law:

Private Limited, New Delhi.

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