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Introduction
Experimental
a) Standard Preparation
- The sorbic acid was weigh about 0.1 g and transferred into 50 mL beaker that contain
25 mL of deionised water, it was stir until sorbic acid was dissolved.
- Sorbic acid solution was transferred into 100 mL volumetric flask and added deionised water
until calibration mark and it was shake to homogenized the mixture. It then was transferred
into 50 mL beaker to pipette 10 mL and transferred into 100 mL volumetric flask and was
diluted with deionised water until calibration mark.
- The solution was homogenized and filter it with millipore filter.
b) Sample preparation
- Sausages was grounded into small pieces and was weigh about 1 g and added with 25 mL
methanol. Then, it was stir to ensure all part being contact with methanol.
- The mixture was placed in sonicator about 50 C in 30 minutes.
- The sample was filtered by using 0.45 m nylon membrane and it was transferred into vial.
Data/Result
Standard preparation,
Stock solution = 1000 ppm =
X = 100 mg = 0.1 g of sorbic acid
Diluted to 100 ppm (working solution),
M1V1 = M2V2
(1000 ppm) (V1) = (100 ppm) (100 mL)
V1 = 10 mL of stock solution
Compound
2.04
8774230.31 V.s
Sausages sample
2.03
590397.32 V.s.
Discussion
This experiment is to determine the amount of sorbic acid in sausages by using HPLC. Chicken
Frankfurter, Farms Gold brand was used in this experiment. Sausage was grounded into small
pieces and was weighed about 1.005 g and added with 25 mL methanol. Then, it was stir to
ensure all part being contact with methanol. The mixture was placed in sonicator about 50 C in
30 minutes to make sure that all the sausages completely go to pieces. The sample was filtered
by using 0.45 m nylon membranes and it was transferred into vial. This is because nylon
membranes are suitable for filtering aqueous solutions and most organic solvents. Solvents
must be degassed to eliminate formation of bubbles because it will give the best result and
peak. If has bubble, it will effects the result and the peak that will come out. The pumps (Perkin
Elemer) provide a steady high pressure with no pulsating, and can be programmed to vary the
composition of the solvent during the course of the separation.
HPLC depends on the relative polarity of the solvent and the stationary phase. Chromatography
has two types of partition which is normal phase and reversed phase. Reversed phase HPLC is
the most commonly used form of HPLC. For this experiment, it stationary phase is non-polar
which is 18 carbon atoms in them and the mobile phase is polar which is methanol (mixture of
water and ACN) and interacts with solute. Flow rate of mobile phase was set to 1.0 mL/min and
was run about 5 minutes. Flame ionization detector (FID) was used as a detector. From the
experiment, we also can get the retention time of a standard solution (sorbic acid) which is 2.04
min and peak area of sorbic acid, 8774230.31 V.s. Some precautions should be taken which is
before injected the solvent we need to make sure that there is no air bubble in the syringe.
Other than that, we need to weigh approximately the standard (0.1 g) and sample (1 g) and
need to wash the apparatus before use it to avoid any residual solution inside the apparatus.
Conclusion
As a conclusion, we can determine the retention time of a standard solution (sorbic acid) which
is 2.04 min and the peak area is 8774230.31 V.s , for sausages sample, the retention time is
2.03 and the peak area of sorbic acid in sausages sample is 590397.32 V.s. We also can
determine the amount of sorbic acid in sausages by using HPLC and also can calculate the
amount of sorbic acid in sausages by using response factor method which is 6.73 ppm.
References
Datasheet