ppm concentration of caffeine and decaffeine

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procedures.docx

HPLC Analysis of Caffeine in Regular and Decaffeinated Coffee

You will (1) use the method of standard addition to determine the concentration of caffeine in both regular and decaffeinated coffees, and (2) observe the effect of varying the mobile phase composition on the retention time of caffeine. This water-soluble alkaloid is found in many plants and is a stimulant. Below is a procedure, but bear in mind that different coffee samples will have different concentrations of caffeine. So the procedure may need to be modified to fit your sample. You will be using an autosampler. So be sure to label your vials well.

Supplies: HPLC grade water – 18 MΩ water passed through a 0.46 μm filter, HPLC grade methanol, 0.46 μm syringe filter, caffeine, regular coffee, and decaffeinated coffee

Procedure

Prepare the column by ramping up the solvent to 90 MeOH / 10 water at 0.5 mL/min and washing the column until a stable baseline is seen. Then ramp the column down to 47 MeOH / 53 water and run until a stable baseline is seen. At the end of all runs, clean the column again with the 90 MeOH / 10 water solvent until a stable baseline is seen.

Prepare 100 mL of a 100 ppm caffeine standard in HPLC grade water. Set the HPLC pump to 47% MeOH/53% water at 0.8 mL/min and set the HPLC detector to 254 nm. Record the column information.

Run and save the chromatogram both in pdf form and as a xy-data table. Be sure to get the time and areas for the peaks.

Filter 40 mL of coffee using a syringe filter. The filter may be re-used, just rinse it with HPLC grade water. Transfer via pipet 5 mL of filtered coffee to 50 mL volumetric flask and dilute to the mark (*save this soln for later). Run this sample as you did the standard and save the data.

For the standard addition runs, you will need to mix 5 mL of your coffee with four different volumes of the standard and dilute to 50 mL. I will leave these four different volumes for you to decide. The only requirement is that the ratio of the largest signal to the smallest signal is . Save all chromatograms. Prepare a standard addition plot and calculate the concentration of caffeine in your coffee sample. Look online for the typical range of caffeine in coffee.

Repeat with decaffeinated coffee. Compare/contrast values and comment on how coffee is decaffeinated.

Using the solution previously prepared (labeled above with *), perform a study where the MeOH/water ratio is varied. You have 47/53 data, so perform two runs where the %MeOH is greater than 47% and two runs where it is less than 47%. Note the retention times and separation of the caffeine. Plot tr vs. %MeOH and explain the trend based on the idea of eluent strength and column polarity.

All water-based solutions may be disposed of in the sinks. Methanol solutions must go in waste containers.

Regular Coffee

Solution#

Stock soln (mL)

Regular Coffee (mL)

total volume(mL)

1

0

5

50

2

2

5

50

3

4

5

50

4

6

5

50

5

8

5

50

Decaffeinated Coffee

Solution#

Stock soln (mL)

Decaffeinated Coffee (mL)

Total volume (mL)

1

0

5

50

2

2

5

50

3

4

5

50

4

6

5

50