How to Solve Poor Reproducibility in Laboratory Experiments
Author: Chromatography Mound (expert chromatographer, Welch's contract writer)
Introduction
In laboratory experiments, researchers may notice that, while most measurement results are relatively stable with minimal differences between parallel samples, deviations in some cases can be however significant—quantitative results may vary by a factor of ten or more, and in extreme cases, a compound may not even be detected.
Often, the causes of such inconsistencies are unclear, making it difficult to determine the appropriate corrective measures.
This article provides an overview of potential causes and solutions to poor reproducibility issues based on the general steps of an experimental workflow, with the hope of assisting researchers in improving reproducibility.
1. Issues with Sample Preparation and Storage
Take the determination of additives in preserved fruits as an example. The sampling process significantly affects the results, particularly for solid samples that require cutting or crushing. The manufacturing process determines that additive concentrations vary across different pieces of fruit, potentially influenced by their specific surface area (SSA).
To obtain reliable results for such samples, the sample size must be sufficiently large. Additionally, different sample types should be stored in designated sample bags to prevent cross-contamination. Storage conditions should also be tailored to the sample’s characteristics, including appropriate temperature control.
Solution: Increase the sample size during preparation. Organize sample storage racks or refrigerators to prevent cross-contamination by allocating separate storage spaces for different sample types. Regular cleaning of storage areas is also necessary to prevent mold growth.
2. Issues Related to the Nature of the Compound
Certain compounds are light-sensitive, heat-sensitive, or prone to oxidative degradation, such as penicillin, vitamin A, and lutein. If not handled properly, these compounds may decompose, resulting in lower measured concentrations.
Moreover, some compounds are highly sensitive to pH changes. For example, salts and amines may carry different charges depending on the pH of the solution, leading to variations in chromatographic retention times. If only one form of the compound is accounted for, the final quantification may be underestimated. Similarly, anthocyanins exhibit structural changes depending on pH, which can affect not only retention times in liquid chromatography but also UV absorption spectra.
Solution: Minimize exposure to light and heat based on the compound’s properties. Antioxidants such as vitamin C, sodium sulfite, and potassium iodide can be added to stabilize the sample. Additionally, adjusting the pH after extraction or using a buffer-based mobile phase can help maintain compound stability.
3. Issues with Extraction and Cleanup
Insufficient sample dispersion during extraction can lead to incomplete recovery. For example, in the determination of fatty acids in powdered milk, the sample must be evenly dispersed and heated in a water bath for methylation to occur. If the sample aggregates at the bottom of the container, the measured concentrations will be lower than expected. Extraction time and temperature must also be carefully controlled to ensure optimal recovery.
Target compound loss during cleanup is another factor affecting reproducibility. The extent of loss depends on the specific cleanup method, such as the duration and amount of dispersive solid-phase extraction (dSPE), the polarity and pH of elution solvents in solid-phase extraction (SPE), and potential sample overload.
Solution: Ensure thorough sample dispersion after adding extraction solvent. Agitate the sample multiple times during ultrasonication or use shaking/vortex mixing to promote uniform dispersion, allowing the concentration to reach equilibrium more quickly. If post-cleanup results show lower recoveries or poor reproducibility, analyze the concentration of the target compound in each step to identify the critical point of loss and make necessary adjustments.
4. Contaminants
In some cases, contamination may originate from the target compound itself. Common contaminants include phthalates, bisphenol A, nonylphenol, and perchlorates. These compounds are susceptible to environmental contamination, leading to significant result deviations.
Solution: Perform a background screening of the solvents used. If a solvent is confirmed to be uncontaminated, designate it as a dedicated solvent for that specific analysis. Next, soak and test laboratory instruments with this solvent to identify contamination-prone equipment. Replace contaminated instruments with clean ones to ensure accurate results.
