1. Experiment 4-4: Determination of Glucose in Blood Serum
Procedure

2. Preparation of Glucose Samples
Stock solution: Dissolve 1.00g of glucose in water in 100mL volumetric flask
Standard 1: dilute 5mL of stock solution to 100mL in volumetric flask
Standard 2: dilute 10mL of stock solution to 100mL in volumetric flask
Standard 3: dilute 20mL of stock solution to 100mL in volumetric flask
Standard 4: dilute 30mL of stock solution to 100mL in volumetric flask

3. Test Tubes
#1: 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of Standard 1
#2: 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of Standard 2
#3: 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of Standard 3
#4: 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of Standard 4

4. Test Tubes …continued
#5-7: 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of blood serum sample
#8 (blank): 10mL of o-toluidine reagent (in glacial acetic acid), 0.1mL of water
Cover test tubes with Parafilm and mix well to increase probability of collision between reactants.

5. Reagents and Reaction
Reaction between glucose (an aldehyde) and o-toluidine (a primary aryl amine) in glacial acetic acid
Imine formation mechanism
A carbinolamine is an intermediate formed

6. Heating and Cooling
Put all test tubes in boiling water AT THE SAME TIME for exactly 7.5 minutes
To make reaction go forward: this reaction is reversible
To expose all test tubes to the same exact environment in order to have accurate absorbance readings
Cool test tubes on ice for 2 minutes and wait 5 minutes after taking them out of ice bath before taking absorbance
To make solutions suitable for taking absorbance measurements in spectrophotometer

7. Absorbance Take all absorbencies at 630nm against blank (Test Tube #8)
630nm is the wavelength of red light, the complimentary color of green, which is the color of the imine complex product

8. Quiz Question
What is the purpose of taking the absorbance of the first four test tubes?
They are used to make the calibration curve, from which the concentration of glucose in the serum sample can be calculated using the absorbance of the sample.