High-Performance Liquid Chromatography (HPLC)

1. Making the Mobile Phase

1. Prepare the mobile phase by adding 400 mL of acetonitrile to approximately 1.5 L of purified DI water.
2. Carefully add 2.4 mL of glacial acetic acid to this solution.
3. Dilute the solution to a total volume of 2.0 L in a volumetric flask with purified DI water. The resulting solution should have a pH between 2.8 to 3.2.
4. Adjust the pH to 4.2 by adding 40% sodium hydroxide, drop-wise with the use of a calibrated digital pH meter. Add very slowly once the pH reaches 4.0. This should take around 50 drops to accomplish.
5. Filter the mobile phase through a 0.47-µm Nylon 66 membrane filter under vacuum to degas the solution and to remove solids that could plug the chromatographic column. It is important to degas the mobile phase to avoid having a bubble, which could either cause a void in the stationary phase at the inlet of the column or work its way into the detector cell, causing instability with the UV absorbance.

2. Creating the Component Solutions

The three components that need to be made are caffeine (0.8 mg/mL), potassium benzoate (1.4 mg/mL), and aspartame (L-aspartyl-L-phenylalanine methyl ester) (6.0 mg/mL). These concentrations, once diluted in the same fashion, put the standards at the levels found in the soda samples.

1. Add 0.40 g of caffeine to a 500-mL volumetric flask, then dilute to the 500-mL mark with DI water.
2. Add 0.70 g of benzoate to a 500-mL volumetric flask, then dilute to the 500-mL mark with DI water.
3. Add 0.60 g of aspartame to a 100-mL volumetric flask, then dilute to the 100-mL mark with DI water. Place this solution in a refrigerator to avoid decomposition during storage.

3. Making the 7 Standard Solutions

The three components all have differing distribution coefficients, which affects how each interacts with both of the phases. The larger the distribution coefficient, the more time the component spends in the stationary phase, resulting in longer retention times in reaching the detector.

1. Following the chart in Table 1, pipet the proper amount of each component into a 50-mL volumetric flask.
2. Dilute each of the stock solutions to the 50-mL mark on the volumetric flasks with mobile phase.
3. Pour each standard solution into labeled small vials in a sample rack.
4. Store the racks of samples in a refrigerator, along with the remaining solutions in the 50-mL volumetric flasks.

4. Checking the Initial Settings of the HPLC System

1. Confirm that the waste line is in a waste container and is not recycling back into the mobile phase.
2. Verify that the flow rate of the mobile phase is set to 0.5 mL/min. This is high enough to allow all peaks to elute within 5 min and slow enough to allow for nice resolution.
3. Verify that the minimum and maximum pressure and the flow rate are set to the correct values on the front panel of the solvent delivery system (the pump).
   1. Minimum pressure setting: 250 psi (this is to shut off the pump, if a leak occurs).
   2. Maximum pressure setting: 4,000 psi (this is to protect the pump from breaking, if a clog forms).
4. Press "zero" on the detector's front panel in order to set the blank (the blank is the pure mobile phase).
5. Rinse a 100-µL syringe with deionized water, then with several volumes of one of the working standards to be analyzed, and fill the syringe with that solution. Start with the 3 single-component samples, which allows for identifying the peak of each component of interest.

5. Manually Injecting the Sample and Data Collection

1. With the injector handle in the load position, slowly inject 100 µL of solution through the septum port.
2. Verify that the data collection program is set to collect data for 300 s, which allows enough time for all 3 peaks to elute through the detector.
3. When ready to start the trial, rotate the injector handle to the inject position (which injects the sample into the mobile phase) and click "Start Trial" on the computer data collection program immediately. For standards 1-3, only one of the three sequential peaks appear on the screen during the run (Figure 1).
4. Once 300 s have passed, the data collection sends a prompt to save the data file. Save the data under a suitable file name (e.g., STD#1).
5. Note the time in seconds for the peak of each trial, which is used in identifying that component.
6. Remove the syringe from the septum and repeat the process for each of the remaining working standards, using the same time per chromatogram as determined from the first run.

Figure 1. The chromatogram of the 3 components. From left to right, they are caffeine, aspartame, and benzoate.

6. The Samples of Diet Sodas

Diet Coke, Diet Pepsi, and Coke Zero are the "unknowns." They have been left out in open containers overnight to get rid of the carbonation, as bubbles are not good for the HPLC system. This sufficiently gets rid of any gases in the samples.

1. Draw around 2 mL of the diet soda into a plastic syringe.
2. Attach the filter tip to the syringe via Luer-Lok by twisting it in place.
3. Push the liquid in the syringe through the filter and into a small glass vial. This gets rid of unwanted particulates that could potentially clog the separation column.
4. Dilute each sample with an equal amount of DI water, so they are at 50% purity.
5. Inject 100 µL of the sample into the sample loop, and run trials with the same parameters as for the standards.

7. Calculations

1. From the concentrations of the component solutions, calculate the concentration of all of the components in the standards, based upon the dilutions that were made for the 7 samples.
2. Determine peak areas on the chromatograms for each standard and the unknown samples by the triangular method, which equals peak height times the width at ½ height (Figure 2). After determining which peak corresponds to each component based upon the time it takes for each component to show their respective peak, enter these peak areas into a computer spreadsheet.
3. Create calibration curves of peak area vs. concentration (mg/L) in the standards for all three components.
4. Determine the least-squares fit for each calibration curve.
5. Calculate the concentration of each component in the diet sodas from the peak areas shown from the HPLC trials for the samples. Remember that the diet soda was diluted by a factor of 2 prior to injecting into the HPLC system.
6. Calculate the amount, in mg/L, of each component in the diet sodas.
7. Based upon the results, calculate the milligrams of each component found in a 12-oz can of soda. Assume 12 oz = 354.9 mL.

Figure 2. A basic example of a curve's peak height and width, which are to be multiplied (peak height times width at ½ height).