Analysis of Carbohydrates (Sugars)

Objective:

The purpose of this experiment was to use a spectroscopic method to determine the concentration of carbohydrate solutions. The methods used included a measurement of the carbohydrate’s intrinsic UV absorbance. This experiment allowed for the opportunity to experiment with the various assays available to determine carbohydrate concentration.

Methods:

 The first step was to calculate the standards I would be using. I started this by obtaining seven micro-centrifuge tubes, four of which were for the standard and three of which were the disaccharide samples. I labeled the standards blank, 0.5, 1, and 2, and the samples Fructose, Sucrose, and Lactose. For the standards tubes, I added the following volumes of glucose enzyme reagent, the appropriate standard, and water:

	Reagent	Standard	H20
0.5	1000 ml	20 ml	50 ml
1	1000 ml	20 ml	50 ml
2	1000 ml	20 ml	50 ml
Blank	1000 ml	0 ml	70 ml

For the samples, I added the following volumes of enzyme reagent, the appropriate sample, and their respective enzyme:

	Reagent	Sample	Enzyme
Fructose	1000 ml	20 ml	50 ml
Sucrose	1000 ml	20 ml	50 ml
Lactose	1000 ml	20 ml	50 ml

I started doing the standards first, and then started the sugar samples. I added the volumes into the tubes from smallest volumes to the greatest, making sure to replace the pipette tips when switching between the different solutions. I then capped all of the tubes and put them into the incubator at 37C for 15 minutes.
After the incubation period, I took all of the tubes out of the incubator and transferred the content of each tube to a 1 mL cuvette. I then set up the absorbance meter to read at 500 nm for each of the standards and the samples. I took the blank sample and put it into the absorbance meter to zero the meter out. Afterwards, we put the rest of the  standard and samples into the absorbance meter one at a time and recorded their absorbances. I had to do a double dilution for the sucrose by transferring the solution back into a micro-centrifuge tube from the cuvette and then putting 500 ml of the solution and 500 ml of water into a different cuvette and taking the absorbance again.

Data and Calculations: 

Calculations of Standards

Figure 1: A standard curve plotting the absorbance at 500 nm vs. the carbohydrate concentration in each of our standard.

The known concentrations of Sucrose:

My concentrations of Sucrose:

The known concentrations of Lactose:

My concentrations of Lactose:

Results: 

Overall results for this lab seemed to be acceptable. I had to do a double dilution for the lactose because my first absorbance value for lactose was 1.384, which was outside of my absorbance range. After the double dilution my lactose absorbance went down to 0.640 making both my sucrose and lactose in the range of my standards. The fructose was not within standards due to unknown lab error and I will repeat this process at a later time.