Troubleshooting Low Recovery Rates in Chromatographic Analysis
Author: Chromatography Mound (expert chromatographer, Welch's contract writer)
Introduction
Have you ever encountered a situation where, despite following the procedure from national standard methods step by step, the recovery rate remains too low, leading to inaccurate data? Even after adjusting for recovery rates, you still feel a sense of uncertainty, and you cannot pinpoint the cause or find a solution.
In this article, we will share some insights into the reasons behind low recovery rates and approaches to address them.
Reasons and Solutions towards Low Recovery Rates
It should be noted first that, national standard methods often target a limited number of sample types, while in reality, the matrix composition of different samples within one same category can vary significantly. Therefore, variations in recovery rates are not unusual, and not necessarily due to the operator's technique or errors in the national standards.
From a theoretical perspective, low recovery rates can be attributed to several factors:
1. Insufficient Extraction
In laboratories, two extraction methods are commonly used. The first method is to use organic solvents like water, methanol, acetonitrile, ethanol, acetone, or a mixture of the above. The second method is QuEChERS, which operates on similar principles, extracting target analytes based on polarity similarity.
However, direct extraction relies on a solvent with polarity matching that of the target analyte, leading to competition with the sample matrix. This leads to issues that result in the recovery rates being less than that recorded in the national standards.
For example, for samples of low-fat content (e.g fish), using methanol or water may be enough to yield satisfactory results and obtain a higher selectivity. But in cases where the sample has a high fat content, and the target analyte is somewhat lipid-soluble, using only methanol or water may not be effective.
Solution: Adjust the polarity of the solvent based on the target analyte and sample matrix. For the above example, using solvents with lower polarity like ethanol, acetonitrile, or acetone may improve extraction. In QuEChERS methods, reducing the proportion of water can lower the solvent's polarity and enhance extraction. Additionally, increasing the sample-to-solvent ratio, using water bath heating, sonication, or multiple extractions followed by concentration can help increase the extraction yield.
2. Volatility or Degradation of the Target Analyte
Certain compounds, such as vitamin C, vitamin A, and penicillin, are inherently unstable. During the analysis process, they may decomposite due to being exposed to light, oxygen, or heat, resulting in lower recovery rates.
Solution: Adding antioxidants, carrying out the process under light protection, or using nitrogen gas should prevent degradation. For compounds with low boiling points or thermal instability, careful regulation of water bath temperature during nitrogen evaporation is critical. For substances prone to oxidation, metal ions in the sample may act as catalysts, and EDTA can be used to chelate these metals, protecting the analyte's stability.
3. Losses During the Cleanup Process
When using dispersive solid-phase extraction (dSPE), incorrect amounts or excessive extraction times can cause target analytes to be adsorbed onto the sorbent. Similarly, when using solid-phase extraction (SPE) columns, insufficient activation or inadequate elution volume may result in unstable adsorption or incomplete elution, leading to analyte loss and reduced recovery rates.
Solution: For the former case, reduce the amount of dSPE material and extraction time. For the latter case, use a larger volume of water and/or organic solvents to activate the column or switch to a larger column specification, and add more eluent to ensure complete elution of the target analyte.
4. Way of Calculation
This is particularly significant in mass spectrometry, where matrix effects can cause deviations in the target compound's response, leading to over- or under-estimation of recovery rates. Single-point calibration can also introduce errors.
Solution: Use matrix-matched calibration curves to correct results, or employ standard addition or isotope-labeled internal standards for quantification.
Conclusion and Further Insights
Low recovery rates stem from a wide range of factors. Besides incomplete extraction, decomposition, cleanup loss, and calculation methods, other factors such as instrument stability, changes in mobile phase, selection of different fragment types in mass spectrometry, pH effects, solvent effects, and the form of compounds also contributes. It is advisable to analyze the solution after each step to identify where issues arise, enabling more effective troubleshooting.