AC4 SUGARS IN HONEY BY HPLC Last Revised:
May 2015
1. PURPOSE This exercise determines the concentration of glucose & fructose in honey by HPLC. 2. REAGENTS & EQUIPMENT 2.1 Agilent HPLC with column 2.2 75% acetonitrile/25% water mobile phase 2.3 Glucose & fructose (solid) 3. PROCEDURE 3.1 Prepare 100 mL of 1, 2 & 5 g/L glucose and fructose mixed standards. 3.2
In triplicate, weigh accurately about 2.5 g of honey into a 400 mL beaker (labelled S1, S2 & RC). 3.3
To the RC beaker, weigh accurately about 0.5 g of fructose. 3.4
Add 200 mL of distilled water to each beaker, and stir until the honey has dissolved. 3.5
Transfer to 250 mL volumetric flasks and make up to the mark with water. 3.6
Filter the three samples by Millepore. 3.7
Run all solutions on the HPLC, using the provided operating procedure. 4. REPORT Calculations For each analyte, 1.
Plot a calibration graph of peak area vs concentration. 2.
Determine the line of best fit values. 3.
Calculate the concentration of the analyte in each sample (in g/L). 4.
Calculate the mass of analyte in each sample. 5.
For S1 & S2, calculate the %w/w in the sample. 6.
For fructose, calculate the %recovery, using the equation below. %
100 4
1& 2
3.3
4
Discussion 
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compare your results to those in Reference article 1 comment on the recovery check value comment on the order of elution of the two sugars, in terms of their attraction to the stationary phase Questions 1.
What other sugars are typically found in sugars? 2.
The manufacturers of the column in this exercise recommend gradient elution as the optimum method for the analysis (see Reference article 2). a. What other instrumental component would be required for this? b. What is the specified gradient condition? c. What detector is specified for their method? How would this deal better with gradient elution than the one in this exercise? Acknowledgement This exercise is based on the project conducted by Linda Stubbs in 2009. AC4 p2 AIT23. SUGARS IN HONEY BY HPLC RESULTS SHEET Standard masses Fructose Glucose 1 g/L 2 g/L 5 g/L Sample masses S1 S2 RC Retention times Fructose Glucose Peak areas Fructose Glucose 1 g/L 2 g/L 5 g/L S1 S2 RC