Quantitative Determination of Caffeine by External and Internal Calibration Methods by
Partner’s Name: ____________________________
Unknown Sample Code:___5___________________
1) The purpose of this experiment is to investigate the HPLC determination of caffeine by
2) Prepare a calibration curve for the analysis of caffeine by HPLC.
3) Investigate the method of internal standard using theophylline
4) Distinguish between an Internal and External standards.
5) Calculate the concentration of caffeine in an unknown using the calibration curve
(external standard) and by the internal standard method.
6) Investigate the absorbance spectra of each compound in the ultraviolet. What does the
HPLC detector really detect?
7) Calculate the absorptivity and molar absorptivity of each compound at 254 nm.
8) Calculate the resolution and capacity factor for the HPLC chromatograms.
Prelab: Complete the following calculations before coming to the lab.
Calculate the concentration of caffeine prepared if 1000. ppm caffeine if the following aliquots
are diluted into a 10.00 mL volumetric flask. The solvent is 50:50 methanol:water for all dilutions.
Table 1. Dilutions Calculations: Dilute the stock caffeine sol’n using the following
volumes to a total volume of 10.00 mL. The solvent is 50:50 methanol:water for all
Aliquot Vol. of Stock
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If a 500. L aliquot of your sample is diluted to 10.00 mL, the dilution factor is……
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Caffeine Unknown #=_5_______________
Calibrate the micro syringe using the analytical balance. Assume that 1.000 g of
water has a volume of 1.000 mL
1) Record the mass for 100, 250, 350, and 500 micro litres volumes delivered by the
2) Use these volumes as V1 in C1V1=C2V2 for calculation of the caffeine concentrations.
Table 1. Calibration of the micro syringe
Average Vol. V1
3) Inject the following 9 samples (Milli Q water and flasks 1-8) into the HPLC. Complete data
tables 6a and 6b. Calculate the concentration of caffeine or theophylline and record the
retention time and peak areas for each sample/solution.
4) Record the UV spectrum for samples in flasks 1-3 over the wavelength of of 200-300 nm.
Table 6a. Summary table for HPLC data
Milli Q Water
Note: 1) the peak for milli Q water will be very small and around 2-2.26 min. 2) Please don’t
panic if there are no peaks for flask 3 (adipic acid) or very small.
Table 6b. Summary table for HPLC data. Peak area or area ratio plotted in the y-axis
and concentration on the x-axis.
Theophylline Area Acaffeine/Atheophylline
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1. Tabulate and Plot the calibration using external standards for the HPLC data from Figure 6a.
Plot the HPLC Peak Area of caffeine as a function of caffeine concentration in ppm. Apply a
trend line to the data and report the trend line equation and R 2 value. Please remember that
this is not a Beer’s law plot as such and the intercept may not go through 0,0 and, therefore,
please do not force it through 0,0. Calculate the concentration of the caffeine unknown.
Insert Calibration Curve – External Standard
2. Calculate the peak area ratio of Acaffeine/Atheophylline for each internal calibration standards
and the unknown. area ratio of Acaffeine/Atheophylline for each internal calibration standards
and the unknown. Apply a trend line to the data and report the trend line equation and R 2
value. Again, please remember that this is not a Beer’s law plot as such and the intercept may
not go through 0,0 and, therefore, please do not force it through 0,0. Calculate the
concentration of the caffeine unknown.
Insert Internal Calibration Curve – Internal Standard
3. Attach the UV spectra from the UV2600 for the caffeine, theophylline and adipic acid. Examine
the spectra and absorbance data at ~254 nm.
If the HPLC detector only measured the absorbance at 254 nm, discuss why adipic
acid was not a good choice as an internal standard?
Are there any other reasons why adipic acid may not have been an ideal choice as
an internal standard?
Is 254 nm the best wavelength to analyze caffeine and theophylline and if not
suggest an alternative wavelength?
4. From the UV2600 data, calculate the absorptivity “a” of caffeine, theophylline, and, if possible,
and adipic acid at 254 nm. Use dimensional analysis to calculate the molar absorptivity “” of
each. If this sounds completely foreign, check the lab 1T1 or the lecture notes on
spectroscopy, or ask.
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Where A is the absorbance, b is the path length and c is the concentration.
Calculate the absorptivity (a) and molar absorptivity (ɛ) of each reagent.
Show a sample calculation.
5. Calculate the resolution Rs of caffeine and theophylline. Convert all times to minutes or
seconds, but not both, before starting the calculation. For these data assume that all the peak
widths for both theophylline and caffeine =0.20 min.
2 (t R )
− (t R )
WCaffeine + WTheophylline
2 tCaffeine − tTheophylline
WCaffeine + WTheophylline
t or t R is the retention time in min.
W is the peak width in min.
Note: the resolution should be >0.5 and < 10 and unit less. If they are not you have made Page 5 of 6 INST507/CY601/10054 an error. 6. Calculate the capacity factor for theophylline. t −t Capacity factor= k A = R M tM Where tRA = retention time for compound peak A tM = retention time for solvent peak (~2.66 min) Table. Calculate the capacity factor K for theophylline and caffeine Compound Capacity Factor K Theophylline Caffeine Are the capacity factors within the acceptable range? Briefly explain. 7. Calculate the %Relative Error between the molar absorptivity of caffeine at ~271273 nm. The molar absorptivity of caffeine at 273 nm =9.74x10 3 L/mol·cm. (https://www.rjb.ro/wp-content/uploads/art04-master-1-2.pdf) Page 6 of 6 Hplc Injection 1: 1 mL /min 60 water 40 methanol – 10 ppm theophylline Hplc Injection 2: 1 mL /min 60 water 40 methanol -10 ppm caffeine Hplc Injection 3: 1 mL /min 60 water 40 methanol - 10 ppm adipic acid Hplc Injection 4: 1 mL /min 60 water 40 methanol – 10 ppm caffeine Hplc Injection 5: 1 mL /min 60 water 40 methanol - 25 ppm caffeine Hplc Injection 6: 1 mL /min 60 water 40 methanol – 35 ppm caffeine Hplc Injection 7: 1 mL /min 60 water 40 methanol – 50 ppm Caffeine Hplc Injection 5: 1 mL /min 60 water 40 methanol - Unknown