Dilutions in an assay

Question

Hello

I'm confused on why we have to dilute our samples? like what is the purpose other than making sure its in the range of a spectrometer? How do dilutions help us find the concentration of something in a solution?

Thank you

Answer 1

Typically, the main reason for diluting a sample being used for assay, is exactly what you suggested regarding having it in the range of linearity for the particular assay. If too concentrated, aberrant results will obtained. Sometimes, the sample is diluted in order to reduce the amount of interfering substance, especially if being used in a bio-assay. Maybe others will chime in with other reasons for diluting an assay sample.

Answer 2

Hi there, I hope this explanation helps add to the previous statement!

Dilution is a common technique used in analytical chemistry for various purposes, including making solutions suitable for analysis with instruments like spectrometers. While adjusting the concentration for spectrometer compatibility is one reason, there are several other important purposes and benefits of dilution:

1. Measurement Range: Diluting a sample allows us to bring its concentration within the linear range of detection for the instrument being used. Spectrometers and other analytical instruments have a limited dynamic range within which accurate measurements can be made. Dilution ensures that the concentration of the analyte falls within this range, avoiding saturation or overwhelming the instrument's detector.

2. Sensitivity: Dilution can enhance the sensitivity of the analysis. For instance, if the concentration of the analyte in the original sample is too high, it may exceed the instrument's detection limit or cause interference with other substances present in the sample. Diluting the sample reduces the concentration of interfering substances and allows for more accurate measurements of the analyte.

3. Matrix Effects: Dilution can help mitigate matrix effects. Samples often contain various matrix components (other substances present in the solution), which can interfere with the analysis by affecting the instrument's response. Dilution reduces the concentration of these matrix components, minimizing their impact on the measurement of the analyte.

4. Precision and Accuracy: Dilution can improve the precision and accuracy of measurements. By diluting a sample, we can decrease the impact of random errors associated with pipetting or sample handling. Additionally, diluting a sample to a known volume allows for more accurate calculations of concentration.

5. Sample Volume: Diluting a sample allows us to work with smaller volumes, which can be beneficial when the original sample volume is limited or when large volumes are impractical for analysis.

Regarding how dilutions help us find the concentration of something in a solution, dilution is often used in conjunction with other analytical techniques, such as titration or spectrophotometry. In these methods, we prepare solutions of known concentrations (standards) and use them to generate calibration curves or standard curves. By comparing the response of the instrument to the analyte in the sample with the responses obtained from the standards, we can determine the concentration of the analyte in the sample through interpolation or regression analysis.