Separation and Quantitation by HPLC
Introduction
HPLC is a speedy and efficient way to analyze the components of unknowns. This is especially useful in its application to the pharmaceutical field. The concentrations and percentages of compounds in a drug can be precisely and accurately determined by this method. Using HPLC a sample drug containing unknown quantities of acetaminophen and caffeine was analyzed and compared to a set of standard dilutions containing known quantities of these components. A calibration curve using the HPLC given values was designed to determine the concentrations and calculate percentages of each compound in the unknown.
Experimental Methods
A 0.6065 g sample of solid unknown # 2220 was dissolved in approximately 80 mL of 0.0100 M HPLC grade methanol. This solution was transferred quantitatively into a 100 mL volumetric flask which was filled to volume with methanol. A 10x dilution was made by pipetting a 10 mL aliquot of the unknown solution into a clean 50 mL volumetric flask that was brought to volume with methanol. A sample of this unknown solution was transferred to a labelled autosampler vial for HPLC analysis.
A series of four standard solutions were designed to increase in acetaminophen concentration and decrease in caffeine concentration. Four 50 mL volumetric flasks were conditioned with methanol and labelled 1-4. Using calibrated volumetric pipets 1 mL of 0.0100 M liquid acetaminophen and 25 mL of 0.0100 M liquid Caffeine were pipetted into flask one. 5 mL of acetaminophen and 10 mL of Caffeine were pipetted into flask two. 10 mL of acetaminophen and 5 mL of Caffeine were pipetted into flask three. 25 mL of acetaminophen and 1 mL of Caffeine were pipetted into flask four. All four flasks were brought to volume with methanol. A sample from each flask was placed in a separate labelled and corresponding autosampler vial for HPLC analysis. 1µl aliquots from each sample were analyzed in an HP Agilent 1100 series with a C-18 column. They were processed in order of vials 1-4, then the unknown. The parameters of the HPLC machine were preset to a mobile phase that was 60% water and 40% methanol with a flow rate of 1.500ml/min.
Results and Discussion
Standard sample 1 was designed to be 50 mL and have a maximum concentration of 0.005 M Caffeine. So the volume of caffeine needed was calculated as follows:
In contrast, the smallest volume of acetaminophen that could be measured quantitatively by pipet was 1 mL. So the concentration was calculated as follows:
These equations were used to calculate the necessary volumes and concentrations of the compounds for each standard solution.
The HPLC analysis gave retention times and peak areas for each compound in each sample. The results were as follows:
Peak 1 increases in peak area corresponding to an increase in acetaminophen concentration. Peak 2 decreases in area corresponding to the decrease in caffeine concentration. Calibration curves were made for each compound by plotting Concentration vs. Peak Area.
The “best fit” trendline of each curve gave an equation that shows the linear relationship between concentration and peak area for the compounds. These equations were used to calculate the concentrations of each compound in the unknown. The peak areas given by the HPLC analysis of the unknown were plugged into the y-values of their corresponding equations to give the following:
Acetaminophen
Caffeine
Conclusion
Unknown # 2220 was found to be 29% acetaminophen, 54% caffeine, and 17% fillers. One fundamental error that was made in this experiment was a failure to carry exact calibrated pipet measurements throughout every calculation. This was caused by a loss of labels on all pre-calibrated pipets. Therefore the concentrations used to plot the calibration curves were not exact causing a possible higher level of uncertainty in the final percentages calculated for acetaminophen and caffeine.
