METHOD USED:
Gas Chromatography ( GC)
A common analytical method in analysis for Benzoic acid in food samples. For example, the determination is done in soft drink. The reason why we would not chose this method is because of general elution problem. To confirm the identity of Benzoic acid, spiking or peak enhancement should be done. GC would be a better method when it is coupled with Mass Spectrometry.
REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
We have decided to
use Reversed Phase HPLC ( High Performance Liquid Chromatography) because the sample that we would be working on is a
polar substance: Benzoic Acid.We could not use GLC (Gas liquid chromatography) as our mobile phase is liquid not gas.
Materials and apparatus:
1) Orange Juice
2) Agilent 1100 Series HPLC instrument equipped
quaternary pump, a degasser, an autosampler, a diodearray detector
3) Agilent Chemstation for data acquisition and analysis
4) Kromasil 100-5C18 column (250 mm×4.6 mm, 5µm)
Reagents:
1)
Solvents: ammonium acetate/acetic acid and methanol ( both HPLC
Grade)
2)
Mobile phase: ammonium acetate/acetic acid buffer solution and methanol (60:40)
Things to take note of:
1)
Injection technique must be fast and accurate.
2)
Mobile Phase must undergo degassing to removes
gas particles.
3)
They must pass through sintered glass filters to
remove particulate matters.
Chromatographic conditions:
1)
Injection volume : 20 Microliters
2)
Detection wavelength: 235nm
3)
Column temperature: 25 degree celcius
4)
Flow rate: 1ml/min
Steps:
Preparation of solvents:
1)
To prepare 500ml of Mobile phase consisting of ammonium acetate/acetic acid buffer solution and methanol
2)
Measure 200ml of Methanol and transfer into
the Pyrex bottle.
3)
Measure 300ml of ammonium acetate/acetic acid buffer solution, and transfer
into the same Pyrex bottle.
4)
This will be our mobile phase solution.
5) Prepare 100ml of solvent solution with ammonium acetate/acetic acid and methanol ( 50:40).
Preparing sample:
1) Measure 10ml of orange juice an add it into a 100 ml flask.
2) 75 ml of mobile phase is added into the 100ml flask.
3) Place solution in ultrasonic bath for 10 mins to ensure that everything dissolves.
4)Dilute to the mark with mobile phase at 20°C.
5) Add 1 ml of Carrez solution I and 1ml od Carrez solution II fo clarification.
6) Filter solution with nylon filter cartridge into
a sample vial and label it.
Sample analysis
1)Place all solutions at the rack and take note of their number.
2)Program autosampler to inject 20 microliters of solution in seperate runs in HPLC.
3)Detemine Peak area of our sample solution with standard solution.
4)Plot calibration graph of concentration (ppm) against peak area.
5)Calculate amount of benzoic acid in orange juice.
Results:
1) Precision of the analytical method was evaluated
by measuring the peak chromatographic area of benzoic
acid (six times) on the same sample. We have found that RSD % was 0.33%. Which means the method is very precise.
2) Calibration graph was plotted and the correlation coefficient for standard curve was 0.9997.
( we could not get the picture as it did no allow us to copy and paste,)
3) Limit of detection (LOD) = 0.97 mg·L-1
4) Limit of quantification (LOQ)= 2.94 mg·L-1
In conclusion,the highest concentration of benzoic acid that was found in orange juice was within the range of 71 mg·L-1 being in accordance with the maximum limits allowed. Revered phase HPLC method is good for identify and quantifying the amount of benzoic acid in orange juice as this method provided good sensitivity.
http://www.angelo.edu/faculty/kboudrea/molecule_gallery/04_aromatics/benzoic_acid_01.gif