Spectrophotometric Determination of Cholesterol in Food Biochemistry 2 Lab Section D

Introduction
Thirty percent of the cholesterol used in the body comes from the diet. It can be found in animal products such as cheese, eggs, meats and seafood. Cholesterol is the cause of so much anxiety in our American society due to our high intake of these foods. The following experiments were performed for the purpose of measuring the amount of cholesterol extracted from a food sample. A spectrophotometer was used to measure the concentration of cholesterol from a given food extract via percent transmittance.

Materials and Methods

Part A: Lipid Extraction Five grams of cheese and equal amounts of acid-washed sand were weighed and transferred to a mortar. While grinding the contents with a pestle, 10 mL of hexaneisopropanol (3:2) was used to extract the lipids. All contents were centrifuged at 10,000 g for 5 minutes. The supernatant fluid was then decanted and stored separately in a clean centrifuge tube. A re-extraction was performed with the ground food sample and sand mixture, using another 10 mL of hexane-isopropanol (3:2). After centrifuging again at 10,000 g for 5 minutes, the supernatant fluid was decanted and combined with the first supernatant fluid. Five mL of Na2SO4 were added to the combined supernatants, vortexed, and centrifuged for another 5 minutes. Using a pipette, the organic layer was removed and transferred to a new tube. Using a micropipet, 10 L of this was transferred to cuvet 1; using a 1 mL pipet, 0.05 mL was transferred to cuvet 2; using a 1 mL pipet, 0.1 mL was transferred to cuvet 3. These solutions were evaporated using a boiling water bath. When dry, 2mL of ethanol were pipeted into each tube and mixed with the vortex mixer. These solutions were saved for use in part B. Part B: Determination of Cholesterol Standard solutions were prepared by pipeting the volumes, shown in the table below, into cuvets to achieve a final volume of 2 mL.

Table 1: Standard Solutions Standard solution Concentration g/mL (Blank) 0 10 20 40 80 100 Volume of Stock Cholesterol (mL) 0 0.2 0.4 0.8 1.6 2 Volume of Ethanol (mL) 2 1.8 1.6 1.2 0.4 0

Two mL of acid-ferric chloride was added to the blank and to each of the standard solutions and to the samples from part A. After vortexing thoroughly, these solutions were incubated for 30 minutes at room temperature. The % Transmittance was for each solution was read using a spectrophotometer (Spec 20) and recorded in the Results section below.

Results
In Table 2, see below, are the transmission results from the spectrophotometer readings. Also in the table are the calculated absorbance for each concentration of the stock cholesterol. The calculations to convert the % transmission to the calculated absorbance are found below the table. Table 2: Cholesterol Standard Cholesterol Concentration of Standards, g/mL 0 10 20 40 % Transmission (%T) Calculated Absorbance (A) 0.00 0.119 0.252 0.480

00.0 76.1 56.0 33.1

80 100

12.0 11.5

0.921 0.939

Calculations:

The graph seen below in Figure 1: Spectrophotometric Determination of Cholesterol in Food shows the absorbances from the stock cholesterol solution as noted above in Figure 1. The last concentration of 100 g was not used because it caused too much of a deviation in the R2 value.

For the cholesterol sample (cheese), the measured % transmission from the Spec 20 was used to calculate the absorbance. These values are found in Table 3, which is below. The calculations are found below the table. Then the equation generated from the trendline found in Figure 1 above, a = 0.011c + 0.009, was used to calculate the cholesterol concentration for the cholesterol sample. These total are also found in the

table below. The calculations for these concentrations are found below the absorbance calculations.

Table 3: Cholesterol Sample Cuvet # 1 2 3 % Transmission (%T) 91.0 58.0 00.0 Calculated Absorbance (A) 0.0410 0.237 0.00 Cholesterol Concentration, g 2.91 20.7 -0.818

Absorbance Calculations:

Cholesterol Concentration Calculations: #1 0.0410 = 0.011c + 0.009 0.0410 - 0.009 = 0.032 0.032/0.011 = 2.909 = 2.91 g g #2 0.237 = 0.011c + 0.009 0.237 - 0.009 = 0.228 0.228/0.011 = 20.727 = 20.7 g

#3

0 = 0.011c + 0.009 0 - 0.009 = -0.009 -0.009/0.011 = -0.818

Discussion and Conclusions

Part A: Lipid Extraction The preparation of the cholesterol extraction from the cheese, the lipid used in Part A, went as specified.

Part B: Determination of Cholesterol The cholesterol concentration in cuvet #1 was determined to be 2.91 g. The % transmission was outside the range of the stock cholesterol solution, at 91.0. The solution in cuvet #3 was determined to have a % transmittance of 0.00 and did not contain any cholesterol. Cuvet #2, however, had a % transmission within range of the stock cholesterol solution, of 58.0, and it was determined it had 20.7 g of cholesterol. In conclusion, the purpose of the lab, to use a spectrophotometer to test extracts from specific foods to determine their concentration of cholesterol, was successfully implemented. Post Lab Questions: 1. Why is cholesterol called a non-saponifiable liquid? Cholesterol, although a lipid, is not a fatty acid. The process of saponification requires a fatty acid and a salt resulting a soap. 2. What was the purpose of preparing three different dilutions of the sample extracts? Because a spectrophotometer requires a minimum volume of a solution for to accurate test, three different dilutions of the sample extract were needed so that at least one of the three would be within readable range for the Spec-20 at 550 nm.