Why am I getting opposite results in my chlorophyll assay using the OptiSciences equipment and the spectrophotometric method?

Valeria Adrianzen Castillo @Valeria_Adrianzen_Castillo

25 September 2023 2 4K Report

Hi! My name is Valeria and my research focuses on species A and B, both shrubs, with obvious differences in the thickness and weight of their leaves. During the trials, I have observed that in both species, the level of chlorophyll increases as the level of salt stress increases. However, what is intriguing is that there is a significant difference in the results obtained using two different methods: with the OptiSciences equipment, species A shows a higher amount of chlorophylls in all treatments, while when using the spectrophotometric method, The results suggest that species B has a higher concentration of chlorophylls. This discrepancy puzzles me and raises questions about the accuracy of the results.

It is important to note that I have performed the spectrophotometric method both with and without fresh weight correction, and the results in this method remain consistent. The procedure for this method consists of cutting leaf discs, immersing them in ethanol and measuring the corresponding absorbances. On the other hand, with the chlorophyll equipment, the amount of chlorophyll is quantified directly in a portion of the leaf using tweezers.

I welcome any suggestions or explanations that may shed light on this discrepancy in results between the two methods and how I might address this issue in my research.

Ricarda Koopmann

For any analytical method used, you must perform an uncertainty calculation. What is the error of measurement ?

This is not a false handling but the normal uncertainty, any measurement has, caused by different parts of handling, dilutions and equipment.

Probably if you do that, you may see, that both results within the ranges of the error bars. For biological samples you have an statistical error, as well

on top of the method error.

For your study, you must discuss pros and cons of the methods, search for literature describíng the method accuracy and performance and find your conclusion. Maybe one method is better suited here, maybe both have positive aspects. If these methods are used in testing labs worldwide, you may find results of ring tests which are performed in different laboratories.

Rahul Prasad Singh

Dear, Dr.Valeria Adrianzen Castillo

Here are some suggestions and explanations that might help you address this issue:

Method Validation: Ensure that both the OptiSciences equipment and the spectrophotometric method are correctly calibrated and validated. Calibration and validation can help confirm the accuracy and reliability of your measurements with each method. Double-check that the methods are being executed correctly and consistently.

Measurement Variability: Understand that different measurement techniques may have inherent variability. The spectrophotometric method and direct chlorophyll quantification with tweezers may have different sensitivities to factors like the thickness and weight of the leaves. Variability can be influenced by factors such as leaf structure, extraction efficiency, and sensitivity to specific chlorophyll types (e.g., chlorophyll-a and chlorophyll-b).

Absorbance Correction: Ensure that you are accounting for background absorbance and correcting for it in your spectrophotometric measurements. Background absorbance can arise from the solvent used (ethanol) or impurities in your samples. Properly correcting for this can help improve the accuracy of your spectrophotometric results.

Fresh Weight Correction: It's excellent that you've considered fresh weight correction in your spectrophotometric method. Continue to use this correction method as it helps normalize the chlorophyll content to the actual leaf tissue mass.

Leaf Structure: Consider the differences in leaf structure between species A and B. Differences in leaf thickness, cellular arrangement, and chloroplast distribution can affect how chlorophyll is measured with different methods.

Species Variability: Recognize that different plant species can respond differently to stressors like salt stress. The variation in chlorophyll content you observe may be a genuine biological difference between species A and B. Carefully document and analyze these differences in the context of your research objectives.

Replication: Ensure that you have a sufficient number of replicates for each treatment and each species. Statistical analysis can help determine if the differences you observe are statistically significant.

Consultation: Consider consulting with colleagues or experts in plant physiology or chlorophyll measurement techniques. They may offer insights or suggestions for refining your methods or interpreting your results.

Additional Techniques: If possible, consider using additional techniques to measure chlorophyll, such as high-performance liquid chromatography (HPLC), which can provide highly accurate quantification of chlorophyll-a and chlorophyll-b.

Data Integration: Rather than relying solely on one method, consider integrating data from both methods into your analysis. This can provide a more comprehensive understanding of chlorophyll dynamics in species A and B under salt stress conditions.

Is There Any Feasible Method To Test Fluorescent Compounds Other Than Fluorescence Spectrometers ??

What are the LAB methodologies (procedures, equipment etc) for CO2 enrichment experiments to study crop responses?

Can I measure absorbance efficiency with wavelength in a spectrophotometer?

CAT assay in microplate?

Are there any differences between setting automatic or manual number of measurements in DLS?

What's support me on Simulink Matlab Model of Regenerative Braking in DC Traction ?

How can I quantify the color of a solution?

Bradford assay for insulin: discrepancies between standard curve and sample concentration during crystallisation?

RNA concentration: Nanodrop vs Agilent 2100 Bioanalyzer?

What dilution do I use for my GNP solution UV-Vis and DLS characterization?