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Objective

To determine amount of sorbic acid in sausages.


To determine the retention time of a standard solution (sorbic acid).
To identify the sorbic acid peak in a sausages sample.

Introduction

High-performance liquid chromatography (HPLC) is used to separate the components in a


mixture, to identify and quantify each component. It has been used in medical, to detect vitamin
D level in blood serum, research, to separate the components of a complex biological sample
and used in manufacturing, during the production process of pharmaceutical products. HPLC
instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column,
and a detector. 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 reversed phase HPLC, it
stationary phase is non-polar which is 8 or 18 carbon atoms in them and the mobile phase is
polar which is methanol (mixture of water and alcohol) and interacts with solute. Solvents must
be miscible because if it in immiscible solvent system such as water and toluene it would create
a mess in the column. In HPLC, the most common detector was used is UV-vis absorbance
detector. Almost any compound can be detected by UV-vis absorbance, either direct or indirect.
Typical limits of detection are 1 to 10 M for compounds that show considerable absorbance
limited to single wavelength.
In this experiment, sample was used is sausages. We used Chicken Frankfurter, Farms Gold
brand. It is made from ground meat with a skin around it. Sausages may be preserved by curing
or drying. Different countries around the world have different techniques to made sausages.
Sausage is most favourite food among teenager and kid. Sausages contain sorbic acid in it
ingredients as a preservative so that it can keep in a long time. Therefore, in this experiment we
need to determine the amount of sorbic acid in sausages by using HPLC whether it has a
suitable amount of sorbic acid or not compared to standard that has been used. Sorbic acid
preservative, and its role is affected by pH, but it is less acidic than benzoic acid and weak, the
optimum pH range than benzoic acid and wide. The preparation of sorbic acid solution first
sorbate was dissolved in ethanol, a solution of sodium bicarbonate or sodium carbonate, then
add in foods such as sausages.

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

Retention time (min)

Peak area of sorbic acid

Sorbic acid Std.

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

Faridah Hanum Hj Badrun, Nor Azira Irma Muhammad, Zailuddin Ariffin.


Universiti Teknologi Mara, Industrial Chemistry Programme, Basic Instrumental Analysis,
Laboratory Manual, CHM 260,
2013, 7

Faridah Hanum Hj Badrun, Basic Analysis (CHM 260),


Note Basic Analysis,
2013, 7

HPLC, 11 July 2013, http://www.files.chem.vt.edu/chem-ed/sep/lc/hplc.html

UV-Vis, 13 July 2013, http://www.files.chem.vt.edu/chem-ed/spec/uv-vis/uv-vis.html

Sorbic acid, 13 July 2013, http://sorbicacid.org/sorbicacid.html

Datasheet