Preparing the Standard Caffeine Samples

**DO NOT dispose of these samples; they will be used in Lab 6-Capillary Electrophoresis**

1. Accurately weigh out 10.0 mg of caffeine. The caffeine can be found on the shelf near the weigh station area.

2. Transfer the caffeine into a clean 100 mL volumetric flask.

3. Dilute to the mark with HPLC/CE grade water. The stock solution will have a final concentration of 0.1 g/L.

4. Carry out a series of dilutions to obtain standard solutions of 0.01 g/L, 0.025 g/L, 0.05 g/L, and 0.075 g/L. Make 10 mL of each solution and use HPLC/CE grade water to make the dilutions.

5. Shake each of the mixtures to ensure adequate mixing.

6. Filter the solutions using the provided filter. Do not do this until you are ready to run your samples.

a. The filter and syringe can be reused if the solutions are filtered from low concentration to high concentration.

b. The syringes can be found in the plastic drawers near the door of the lab.

c. Filter approximately 1 mL and dispose of it into a waste beaker to wash the filter.

d. Filter the solution into the appropriate vial. (Figure 1.1) DO NOT put tape on the vials; ask your TA for a sharpie to write directly on the vials.

Figure 1.1: Appearance of HPLC/CE vials. The HPLC can also use plastic vials that look quite similar. Image is taken from www.thermofisher.com

Preparing the Beverage Samples

**DO NOT dispose of these samples; they will be used in Lab 6-Capillary Electrophoresis**

1. The beverages can be found in the refrigerator in the lab.

2. Prepare the Coffee Sample.

a. Pipette 5 mL of coffee into a clean and Dry 50 mL volumetric flask and dilute to the mark with HPLC/CE grade water.

b. Filter the sample using the provided filter.

c. Rinse the filter by filtering the first 1-2mL of the sample into the waste beaker.

d. Fill a vial with the appropriate volume and label the vial.

3. Prepare the Tea Sample.

a. Pipette 10 mL of tea into a clean and Dry 50 mL volumetric flask and dilute to the mark with HPLC/CE grade water.

b. Filter the sample using the provided filter.

c. Rinse the filter by filtering the first 1-2mL of the sample into the waste beaker.

d. Fill a vial with the appropriate volume and label the vial.

4. Prepare the Soft Drink Sample

a. If the soft drink you selected is carbonated, decarbonate the soft drink by pouring it back and forth between two beakers until the bubbles cease.

b. Pipette 25 mL of the soft drink into a clean and Dry 50 mL volumetric flask and dilute to the mark with HPLC/CE grade water.

c. Filter the sample using the provided filter.

d. Rinse the filter by filtering the first 1-2mL of the sample into the waste beaker.

e. Fill a vial with the appropriate volume and label the vial.

Preparing a Sequence for the Caffeine Standards and Samples

1. Set the method for this experiment. All of the samples will use the same method. **Your TA will be assisting you while you set up your sequence.**

2. Put standard caffeine solutions in slots 1-5 with the least concentrated in slot 1 and the most concentrated in slot 5. Place the beverage samples in slots 6-8.

3. Select “CHE 115 CAFFEINE.M” in the “method” menu bar.

4. Click “method” in the selection menu and select “edit entire method.”

5. Confirm that the solvent mixture is 47% Methanol and 53% Water.

6. Go to “sequence” menu bar and select “CAFFEINE_LC.S.”

7. Click “sequence” followed by “sequence template” and check to see that your samples are in the correct sample order/slots. Click “run sequence.”

8. While the samples are running, make sure all of your bulk samples and caffeine standards are in containers that can be stored and are properly labeled if you are performing this lab before Lab 6 (CE experiment).

9. Confirm with your TA that your data looks appropriate before disposing of any solutions. You can dispose of the small vials for this experiment once you have collected all of your data. If you have already performed Lab 6, then dispose of your samples.