Objective

To determine benzoic acid in chili sauce by using High Performance Liquid Chromotography.

Introduction

The purpose of the experiment is to determine benzoic acid in food. Benzoic acid C7H6O2 or
C6H5COOH, is a crystalline solid and a simple aromatic carboxylic acid. Benzoic acid occur
naturally in many plants and it serves as a food preservatives and benzoic acid is an important
precursor for the industrial synthesis of other organic substances. Benzoic acid are determine by
High Performance Liquid Chromatography (HPLC) because this instrument can separate liquid
sample and solvent-soluble solid sample. The separation occur when the component have higher
affinity towards mobile phase. The retention time is determine and calculate the concentration of
benzoic acid. The HPLC instrument commonly include a sampler, pump, and detector. The
sampler brings the sample mixture into the mobile phase stream which carries it into the column.
The pump deliver the desired flow and composition of the mobile phase through the column. The
detector generates a signal proportional to the amount of sample component emerging from the
column, then allowing for quantitative analysis of the sample component.
Apparatus

1. Vacuum
2. Conical flask
3. Beaker
4. Glass rod
5. Pipette
6. Volumetric flask

Chemical

1. Benzoic acid
2. Methanol
3. NaCl
4. chloroform
Procedures:

Preparation of HPLC Mobile Phase (Buffer solution)

I. 200-300 ml of benzoic water was added into a 1L in the volumetric flask

II. 15 ml of concentrated acetic acid has been added into the flask
III. 15g of ammonium acetate was weight into a 250 ml beaker and dissolved in 100ml
deionized water.
IV. The ammonium acetate was added with acetic acid into 1L flask and dilute to mark with
deionized water. The solution has been mixed and filtered.

Preparation of Methanol HPLC Grade

i) The HPLC methanol grade was sparged and degassed if necessary.

Preparation of 70% ethanol

i) 700 ml of 95% ethanol has been added into 1L beaker and dilute up to mark with
deionized water. It was transfer into a storage container
Preparation of standard
I. The benzoic acid has been prepared by dissolve 0.4g of solid benzoic acid with 50
ml 70% of ethanol.
II. It was transfer into 100 ml volumetric flask and dilute to the mark with the 70%
ethanol
III. 20g/ml was prepared by pipette the 0.25 ml of stock solution and transferred into
50 ml volumetric flask and dilute with 70% ethanol up to the mark.

Preparation of sample
I. Solid sodium chloride has been add into beaker that containing 25cm3 of deionized water
until cannot dissolved. The solution was filtered
II. The 2.5g chilies source was add into 25cm3 of saturated chloride and filtered the mixture
and transfer it to separating funnel.
III. It has been extracted and was washed 4 time by using 5cm3 of chloroform
IV. The solution collected was dilute with 25cm3 of the HPLC methanol grade and it was
centrifuged before run the analysis.
Data and Results:

Students Concentration of benzoic acid in sample

(ppm)
1(asma) 1.9327

2(latif) 1.7934

3(rahimi) 1.1822

4(iffa) 1.4725

Calculation:

Concentration of standard = 20g/ml

Peak area standard = 214621

Peak area of benzoic acid in sample = 19245

RF = Peak area standard / Concentration of standard

= 214621/ 20

= 10731.05

Concentration of benzoic acid in sample = Peak area of benzoic acid / RF

= 19245 / 10731.05

= 1.7934 ppm
Discussion

Based on the experiment the result obtain by following the procedure using instrument in lab.
Based on the experiment the concentration of benzoic acid 1.7934 ppm. Compare the other
student the result obtain near each other its means the successful when doing the experiment. The
benzoic acid was prepared weight 2.5023g of it. Condition of HPLC was set depends the
procedure in lab. The retention time of the peak sample has been obtain and it was compare to
the standard it means to ensure the benzoic acid contain present or not in the sample. When
doing the experiment that have several precaution should avoid to obtain exactly result such as
prevent the parallax error and zero error. Parallax error is an error in reading an instrument due to
the eye of the observer and pointer are not in line perpendicular to the plane of the scale, and
zero error is error arises when the measuring instrument does not start from exactly zero. In other
the sample, mobile phase and benzoic acid standard should be sonnicate to remove air bubble
because it may affect the flow rate and immediately disturb the result. The sample and standard
must be filter by 20m pourous filter to remove any impurities that will damage the instrument
during run the instrument. We should nfilter because it will prevent column from clogging. The
statinory phase is refer to the column in this experiment benzoic acid is polar so the mobile phase
should polar mobile phase and the column must non-polar column. The result it will read which
detector.

Conclusion

As a conclusion, the average concentration of benzoic acid contained in the chili sauce sample is
1.7934 ppm.
References
(2014, august 14). Retrieved from http://www.hplc2014.org/

(2014, august 14). Retrieved from http://www.chemguide.co.uk/analysis/chromatography/hplc.html