Internal Standards

1. Proper Sample Handling: Making a Solution

1. Take a clean beaker and mass the correct amount of sample into it. Record the actual mass used. In this example, a solution of adenine is made in a volumetric flask for use as an internal standard for the next analysis. The mass of adenine is 100 mg. Do not directly mass into a volumetric flask because it has a long neck and the adenine cannot be easily added or removed.
2. Add roughly 25 mL of solvent (in this case dimethyl sulfoxide (DMSO)) to the beaker and let it stir to dissolve. In this example, the final solution is made in a 50-mL volumetric flask, so only add about 25 mL so the beaker can be rinsed and the solution made up to the final volume.
3. Once the solid has dissolved, pour the solution into the volumetric flask.
4. Rinse the beaker and the stir bar with small amounts of solvent, about 10 mL, and pour the rinse into the volumetric flask. Repeat twice more. This helps ensure proper solution transfer.

2. Preparation of an Internal Standard Calibration Curve

1. Prepare the desired standard samples for gas chromatography analysis. In this example, caffeine is extracted from coffee using acetonitrile and then adenine is used as an internal standard for measurement.
2. For the caffeine samples, weigh out the amount of sample needed to make 1 mg/mL sample. If using a 10-mL volumetric flask, that is 10 mg.
3. Weigh the analyte into a beaker, add a few mL of solvent (here methanol) to dissolve, then quantitatively transfer to the volumetric flask using 3 rinses.
4. Make 3 more standards in a similar manner with 0.2, 0.5, and 2 mg/mL caffeine.
5. Put 1 mL of each caffeine standard into a sample vial.
6. Add 0.2 mL of 2 mg/mL adenine internal standard to each sample vial.
7. Run the gas chromatography experiment with each caffeine standard. For each chromatogram, calculate the ratio of peak areas for the caffeine vs the standard.
8. Make a plot of the area ratio vs the concentration ratio. The slope of that plot is the response factor.

3. Preparation of a Real Sample with Internal Standard for Gas Chromatography

1. Prepare the desired sample for gas chromatography analysis. In this example, caffeine is extracted from coffee using acetrontrile.
2. For the coffee sample, mass out 2 g of coffee into a 100-mL beaker. Record the exact weight of the coffee.
3. Add 20 mL of acetonitrile to the beaker.
4. Allow it to sit for 20 min, stirring frequently.
5. Filter the coffee grounds out using a filter paper in a funnel.
6. Rinse the filter paper 3x with small amounts of acetonitrile (5 mL).
7. Measure the final volume of the filtrate. It should be about 35 mL.

4. Run the Sample and Calculate the Concentration

1. Take 1 mL of the coffee extract sample and add 0.2 mL of the internal standard in a vial. Place the vial into auto-sampler rack.
2. Run a GC analysis of the sample. Make sure that the GC conditions are such that the caffeine and adenine separate. In this example, isothermal separations are performed at 200 °C.
3. After the analysis, compute the peak area for both the internal standard peak and the analyte peak. Using their ratios, calculate the amount of caffeine in the sample.

5. Results: GC Analysis of Caffeine with Internal Standard

1. GC analysis of caffeine is shown in Figure 1. Adenine is used as an internal standard. The ratio of peak areas can be measured and plotted vs the ratio of the concentrations. The slope of the plot is the response factor (in this case 1.8).
2. Figure 2 shows a chromatogram of a coffee sample with adenine internal standard. The ratio of the peak area is 1.78. Using the response factor and the known concentration of adenine (0.33 mg/mL), the concentration of caffeine in the unknown sample is calculated to be 0.33 mg/mL.

Figure 1. Calibration plot using an internal standard. A plot of the area ratios vs concentration ratios for 3 standard samples of caffeine (1, 0.5, and 0.2 mg/mL) with 0.33 mg/mL adenine internal standard added to each. The slope of the line is 1.8, which is the response factor.

Figure 2. Chromatogram of coffee with adenine internal standard. A plot of the response of the FID detector to samples. The three main peaks are adenine (IS), caffeine, and palmitic acid.