Discover the importance of internal standards in analytical chemistry and how they improve the accuracy of your experiments. This video provides a step-by-step guide to using internal standards, their benefits, and best practices for implementation. Perfect for students, researchers and laboratory technicians who want to improve their quantitative analysis skills. Subscribe for more insights into laboratory techniques and scientific methods.

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Internal standards are crucial in the construction and interpretation of standard curves for dose-response experiments, especially in areas such as pharmacology, toxicology, and biochemistry. Here's how they're used:

Purpose of internal standards
Internal standards are compounds that are added in a constant amount to all samples, standards, and blanks in an analytical experiment. They serve several purposes:
1. **Normalization**: Compensate for variability in sample preparation, instrument response, and experimental conditions.
2. **Accuracy and Precision**: Improve the accuracy and precision of quantitative measurements by correcting for losses during sample preparation and analysis.

### Steps involved in using internal standards for dose-response curves

1. **Selection of internal standard**:
– Choose an internal standard that is chemically similar to the analyte of interest, but does not interfere with its detection.
– It should have similar physical and chemical properties to the analyte to mimic its behavior during analysis.

2. **Preparation of standard solutions**:
– Prepare a series of standard solutions with known concentrations of the analyte.
– Add a constant amount of the internal standard to each of these solutions.

3. **Sample Preparation**:
– Prepare the samples under investigation and add the same amount of internal standard to each sample as to the standards.

4. **Analytical measurement**:
– Analyze both the standard solutions and the samples using an appropriate analytical method (e.g. HPLC, GC-MS, LC-MS).
– Measure the response (e.g. peak area or height) for both the analyte and internal standard in each solution.

5. **Calculation of response rates**:
– Calculate the ratio of the analyte response to the internal standard response for each standard solution and sample.
– This ratio helps minimize variations due to experimental inconsistencies.

6. **Construction of standard curve**:
– Plot the response ratios (analyte/internal standard) of the standard solutions against the known concentrations of the analyte.
– Place a regression line or curve on these points to generate the standard curve.

7. **Determination of unknowns**:
– Use the standard curve to determine the concentration of the analyte in the unknown samples by comparing their response ratios to the standard curve.

### Example in practice
1. **Pharmacokinetics**: When studying drug metabolism, an internal standard structurally similar to the drug can be added to all calibration standards and plasma samples. This corrects for variations in extraction efficiency and instrument response.
2. **Environmental analysis**: For the analysis of pesticide residues in food, an internal standard can take into account matrix effects and recovery rates during sample preparation and analysis.

### References
1. **Analytical Chemistry by Gary D. Christian** – This textbook covers the principles of internal standards in analytical measurements, including their application in constructing standard curves.
2. **Journal of Chromatography A** – Contains numerous studies and articles on the use of internal standards in chromatography for dose-response and other analytical applications.
3. **/"Validation of Analytical Procedures: Methodology/" (ICH Q2(R1))** – Provides guidance for the validation of analytical methods, including the use of internal standards to improve accuracy and precision.

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