Making sure the drugs you get from the pharmacy function as they are intended to is important. The drugs you buy go through rigorous testing to make sure they have the right chemical properties. Gas chromatography is one method scientists use to ensure this. Measuring how materials respond to light helps scientists separate and prepare these compounds.
When chemists perform quantitative analyses, they measure how much of a particular substance is in a sample. They need accurate measurements to retain as much of the compounds they need as possible. If an instrument for measuring how much a sample weighs isn't properly calibrated, it might not display the correct weight of a substance. Gas chromatography (GC) is one way scientists can minimize these errors.
GC experiments involving mixing a liquid sample with a solvent that scientists insert in the gas chromatograph apparatus. The liquid sample then vaporizes into a gas in the chromatograph near an inert gas such as argon or helium which doesn't react with the sample.
The two gases are heated and enter a long tube so that the components of the sample separate. The detector at the end of the tube records the presence of various components of the sample and produces a graph based on how much of each component is present.
For general chromatography and spectroscopy experiments, scientists and engineers use response factors to measure quantitative responses that can determine how much of a sample is present. This lets scientists use a response factor to correct for how much of a sample may be lost in certain parts of experiments. Measuring the difference in response of different samples lets them account for these errors.
RRF Calculation for Impurities
The general formula for a response factor for GC is peak area divided by its concentration for a chemical component. In some cases, the height of the peak is used instead of the area. The relative response factor (RRF) is, then, one response factor divided by another. By comparing these factors to standards of response factors from known compounds, chemists can determine the composition of a particular sample to figure out if there are any impurities.
RRF is generally used to compare the peaks of impurities to the primary peak or peaks of the substance you are analyzing. The separation of components of the substance also depends on vapor pressure, polarity of the components, temperature of the gas chamber and the amount of material you initially insert into the apparatus.
Preparing an Internal Standard Calibration
The advantages of internal standard method calculations include setting the fraction of a component's peak area to its concentration equal to the peak area and concentration of a known standard. Plotting a graph of the response against concentration, you can calculate RRF by dividing the slopes of two different substances. For an internal standard calculation in gas chromatography, you can calibrate your gas chromatograph apparatus to ensure you have the correct amounts of chemical compounds present.
You can calibrate your apparatus through a series of steps.
- Make sure your sample material is ready to be analyzed through GC. Weigh them and measure them to check the mass, volume or other properties of interest.
- Place the substance in a beaker or graduated cylinder and add solvent to dissolve it. Transfer it to a volumetric flask by rinsing the beaker or cylinder.
- Create more standards of your sample for comparison.
- Add 1 mL of each dissolved sample into a separate vial.
- Add a small amount of internal standard to each vial. Make sure you keep track of how much you add and that you add the same amount to each vial.
- Perform the GC experiment for each vial.
- With the resulting chromatogram graph and data, calculate the ratio of the peak areas for the sample of interest and the internal standard.
- Plot these ratios, and find the slope of the plot. This should be the RRF.
- The response factor for the same analyte will be different for different detectors.
- The response factor may change over time so the GC should be recalibrated and new response factors determined on a regular basis.
About the Author
S. Hussain Ather is a Master's student in Science Communications the University of California, Santa Cruz. After studying physics and philosophy as an undergraduate at Indiana University-Bloomington, he worked as a scientist at the National Institutes of Health for two years. He primarily performs research in and write about neuroscience and philosophy, however, his interests span ethics, policy, and other areas relevant to science.