PHOSPHATE IN COLA BY ION HPLC
AC5 Last Revised:
May 2015
1. PURPOSE This exercise determines the concentration of phosphate in cola drinks by ion chromatography. 2. REAGENTS & EQUIPMENT 2.1 Ion chromatograph with anion column and suppressor unit 2.2 NaHCO3/Na2CO3 mobile phase (10% methanol) 2.3 Coca Cola or similar 2.4 1000 mg/L phosphate standard 3. PROCEDURE 3A. Prework 3.1 Find out the typical level of phosphate in cola drinks (see reference), and determine a suitable dilution. 3.2 Work out how to prepare 50 mL of 2, 5 and 10 mg/L phosphate standards from 1000 mg/L via a suitable intermediate. 3.3 Determine a suitable recovery check. 3B. Solution preparation 3.4 Degas the sample if necessary. 3.5 Prepare the standards as calculated in Prework. 3.6 Prepare duplicate diluted samples, and a recovery check as determined in Prework. 3.7 Filter these solutions and the samples by Millepore. 3C. Analysis 3.8 Inject each solution, running the 10 mg/L standard first. 3.9 Identify the phosphate peak in the samples on the basis of retention time. 3.10 Record the peak area of each in each solution. This may need to be done manually, if the automated process does not measure properly. 3.11 If time allows, identify by spiking, any other peaks of significant size in the sample chromatogram. 4. REPORT Calculations 
prepare a calibration graph plotting peak height against concentration for the standards 
determine the average concentration of the analyte in the samples 
calculate the % recovery 
calculate the number of theoretical plates in the column (use the standards; it is suggested you measure both retention time and peak width with a ruler) Discussion 
compare your result with the expected value 
comment on the %recovery value 
explain the purpose of the methanol in the mobile phase 
comment on the number of theoretical plates 
describe another method (non‐chromatographic) suitable for this analysis 
compare with this method Questions 1.
Outline how an anion exchange resin separates a mixture of anions. 2.
What factors affect the retention order of ions? 3.
What is post‐column ion suppression? Why does it improve sensitivity? 4.
Give TWO ways that the retention times could be increased in this system to allow better separation of early peaks. 5.
Suggest a suitable internal standard for this analysis and explain your choice. 6.
Explain why internal standards are less important in HPLC than in GLC. Reference: Rapid determination of phosphate and citrate in carbonated soft drinks, Dionex Application Note 169 (download from subject webpage) AC5 p2