Internal Standards
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
The goal of many chemical analyses is a quantitative analysis, where the amount of a…
1. Proper Sample Handling: Making a Solution
- 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.
- 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.
- Once the solid has dissolved, pour the solution into the volumetric flask.
- 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
- 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.
- 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.
- 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.
- Make 3 more standards in a similar manner with 0.2, 0.5, and 2 mg/mL caffeine.
- Put 1 mL of each caffeine standard into a sample vial.
- Add 0.2 mL of 2 mg/mL adenine internal standard to each sample vial.
- Run the gas chromatography experiment with each caffeine standard. For each chromatogram, calculate the ratio of peak areas for the caffeine vs the standard.
- 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
- Prepare the desired sample for gas chromatography analysis. In this example, caffeine is extracted from coffee using acetrontrile.
- For the coffee sample, mass out 2 g of coffee into a 100-mL beaker. Record the exact weight of the coffee.
- Add 20 mL of acetonitrile to the beaker.
- Allow it to sit for 20 min, stirring frequently.
- Filter the coffee grounds out using a filter paper in a funnel.
- Rinse the filter paper 3x with small amounts of acetonitrile (5 mL).
- Measure the final volume of the filtrate. It should be about 35 mL.
4. Run the Sample and Calculate the Concentration
- 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.
- 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.
- 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
- 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).
- 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.
Sample loss can occur every time a sample is handled or transferred, thereby making accurate calculations of concentration difficult.
To ensure accuracy, the effects of sample loss must be minimized using careful sample preparation and by limiting the number of sample handling and transfer steps. However, sample loss can also occur due to systematic errors, such as incomplete sample manipulation, matrix effects, and variations in analytic procedure.
These sources of loss can be accounted for by adding a known concentration of a species similar, but not identical, to the compound of interest. This is called an internal standard. Any sample losses that occur to the internal standard should be similar for the analyte, allowing for the concentration to be accurately calculated.
This video will illustrate the use of an internal standard and proper lab technique to account for sample loss when determining the concentration of an unknown.
An internal standard is a substance added in a known amount to standards, samples, and blanks during an analysis.
In chromatography and spectroscopy, the ratio of the signal for the internal standard and the analyte is calculated. This ratio, called the response factor, is proportional to the ratio of the analyte and standard concentrations.
Response factor, R, can be expressed by the following equation, where A represents the analytical signals of the sample and internal standard and C represents the concentrations of the sample and internal standard.
An internal standard can compensate for both systematic and random errors. For example, random errors?such as inconsistencies when measuring a sample?will be the same for both the internal standard and the analyte. Therefore, the ratio of their signals will not change.
For systematic errors, such as matrix effects in solution, the ratio will be unaffected as long as the matrix effect is equal for both the standard and the analyte.
While internal standards provide great benefit, it can be difficult to choose one that is suitable. An internal standard must have a signal that is similar, but not identical, to the analyte. It also cannot affect the measurement of the analyte in any way.
Finally, the concentration must be well known. This is achieved by ensuring that the internal standard is not natively present in the sample; thus, the only source of it in solution is the known concentration added.
In the following experiment, the concentration of caffeine in an unknown sample will be determined by gas chromatography.
This is achieved by creating a calibration curve using known caffeine solutions, with adenine as the internal standard. The slope of the calibration curve is equal to the response factor.
Once the response factor is known, the concentration of the unknown can be calculated from its measured chromatogram area ratio.
Now that you understand the basics of internal standards, let's take a look at the procedure.
To begin the procedure, accurately weigh 100 mg of the internal standard, adenine, into a clean beaker.
Next, dissolve it in roughly 20 mL of dimethyl sulfoxide, and mix the solution.
Once the adenine has dissolved, pour the solution into a 50-mL volumetric flask.
Rinse the beaker and stir bar with 10 mL of DMSO, and pour the rinse into the flask. Repeat this rinse twice, to ensure proper solution transfer. Fill to the calibration mark, resulting in an internal standard with a concentration of 2 mg/mL.
Next, weigh 100 mg of caffeine into a beaker to prepare a stock solution. Dissolve the caffeine with a small amount of methanol. Then, use 3 rinses to transfer this solution to a fresh 25 mL volumetric flask. This is the 4 mg/mL stock solution. Use it to create 3 caffeine standards.
Next, add 0.2 mL of the internal standard, adenine, to each flask. Fill each to the final volume with methanol. Transfer each solution to a sample vial.
Run each caffeine standard through a gas chromatograph. Calculate the ratio of peak areas for the caffeine versus the adenine standard.
First, weigh 2 g of coffee into a 100-mL beaker, and record the weight.
Next, add 20 mL of methanol to extract the caffeine from the coffee. Allow the solution to stir for 20 min.
Using a B?chner funnel, filter out the coffee grounds. Rinse the beaker with a small amount of methanol, and pour this rinse into the funnel. Repeat the rinse twice.
Measure the final volume of the filtrate; it should be approximately 35 mL.
To prepare the sample for analysis, add 1 mL of the coffee extract to a sample vial. Then, add 0.2 mL of the adenine internal standard, and place the vial into the instrument's auto-sampler rack.
Run a gas chromatography analysis of the sample, ensuring that the conditions are such that the caffeine and adenine are?separate.
After completing the analysis, compute the peak area for both the internal standard and the analyte.
Once all the samples have been analyzed, the standard calibration curve can be determined for the caffeine/adenine solutions by plotting the ratios of the peak areas versus the ratios of the concentrations. The slope of this line, which represents the response factor, was 1.8.
Next, the GC data from the extracted coffee sample is analyzed. The ratio of the peak areas was calculated to be 1.78. Using the response factor and the known concentration of the internal standard, adenine, the concentration of caffeine in the unknown sample was calculated to be 0.33 mg/mL.
Many different types of reactions, across various scientific disciples, utilize internal standards to minimize the effects of errors and sample loss.
The effects of sample loss encountered during sample preparation can be minimized using internal standards, keeping their concentration ratio nearly constant.
In this example, bioactive lipids were extracted from lysed cells using a liquid-liquid extraction process. Stable isotope internal standards were added at the beginning of extraction to account for errors during sample preparation.
Internal standards were not only critical for the preparation of the bioactive lipids, but also for the analysis. The lipids were separated using high-performance liquid chromatography, and analyzed via mass spectrometry.
In spectroscopy, internal standards can help correct for random errors due to changes in light source intensity. If a lamp or other light source has variable power, it will affect the absorption and consequently, emission of a sample. However, the ratio of an internal standard to analyte will stay constant, even if the light source does not.
In chromatography, one of the largest sources of error is the injection. Auto-samplers help minimize this, but error can still be 1?2% relative standard deviation.
In this example, vapor standards containing an internal standard were analyzed using gas chromatography to establish a calibration curve. Once this was complete, the unknown sample could then be measured and the losses due to volatility of the sample accounted for.
You've just watched JoVE's introduction to internal standards. You should now understand best practices for minimizing sample loss, internal standards, and response factors.
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Q1: What is an internal standard and why is it used in analytical chemistry?
An internal standard is a substance added in a known amount to standards, samples, and blanks during analysis. It compensates for both systematic and random errors that occur during sample handling and measurement. By calculating the ratio of signals between the internal standard and analyte, called the response factor, accurate concentration calculations are possible even when sample loss occurs.
Q2: How does an internal standard help account for sample loss?
Any sample losses occurring after the internal standard is added should be similar for both the internal standard and the analyte. Since both substances experience proportional losses, their signal ratio remains constant. This allows the response factor to accurately reflect concentration ratios, making it possible to calculate analyte concentration despite sample loss during preparation and transfer steps.
Q3: What are the key requirements for choosing a suitable internal standard?
An internal standard must have a signal similar but not identical to the analyte, allowing the measurement method to distinguish between them. It cannot affect the analyte measurement and must not be natively present in the sample. The concentration must be well known, ensuring the only source in solution is the known amount deliberately added during analysis.
Q4: What is a response factor and how is it calculated?
The response factor is the ratio of analytical signals for the internal standard and analyte, which is proportional to their concentration ratio. It is calculated by plotting the ratios of peak areas versus concentration ratios from known standards. The slope of the resulting calibration curve equals the response factor, which can then be used to determine unknown analyte concentrations.
Q5: How do internal standards correct for systematic errors like matrix effects?
Matrix effects interfere with analyte measurement by affecting the signal in solution. When an internal standard experiences the same matrix effect as the analyte, the ratio of their signals remains unaffected. Performing calibrations in the same matrix as the sample ensures both substances are equally influenced, allowing the response factor ratio to accurately reflect true concentration differences.
Q6: What analytical techniques commonly use internal standards?
Internal standards are widely used in chromatography, spectroscopy, and electrochemistry. In gas chromatography, the ratio of peak areas for the internal standard and analyte is calculated. In spectroscopy, internal standards correct for random errors from variable light source intensity. These techniques rely on internal standards to minimize errors and ensure accurate quantitative analysis across diverse scientific disciplines.
Q7: How is sample preparation optimized to minimize sample loss before using an internal standard?
Sample loss is minimized by reducing the number of handling and transfer steps. Massing solids directly into the final flask eliminates unnecessary transfers. When transfers are necessary, triple rinsing glassware ensures complete sample transfer. For samples that adsorb to glass, using polypropylene tubes prevents losses. These preparation strategies work alongside internal standards to ensure accurate concentration determination.