The vanillin-sulphuric acid assay mixture appears yellow before incubation and develops a stable red to red-purple color after heating, which is used to quantify saponins like Diosgenin. Inconsistent colors indicate possible issues with reagent freshness, incubation conditions, or solvent interference.

Thank you, @Abdelhak.

May I ask a bit? For the diosgenin solution, I am using absolute methanol and diluting it through serial dilution using the same absolute methanol. Let's say I serially diluted the diosgenin methanol stock solution with water. Is it correct?

It sounds like you have set up your diosgenin standard curve correctly in principle, but the main issue seems to be inconsistency in the vanillin–sulfuric acid color reaction, which explains why your absorbance values are not giving a clean linear relationship. There are a few key areas you can focus on to troubleshoot. First, make sure your diosgenin stock solution is accurately prepared and fully dissolved in methanol, since incomplete solubility or slight weighing errors can affect the concentrations and lead to non-linear plots. Preparing it in a volumetric flask and sonicating if necessary helps ensure accuracy. Fresh reagents are also crucial because vanillin solutions and sulfuric acid can degrade or vary over time; always prepare the vanillin solution fresh and use the same grade of sulfuric acid each time. The reaction itself is highly sensitive to the order of addition, timing, and temperature, so you should always add sample, then vanillin, then sulfuric acid in the same order, vortex immediately, and transfer the tubes quickly to a pre-heated water bath at 60 °C for exactly 10 minutes. Even small deviations in heating time or temperature can shift the color from yellow to pink or purple, so consistency is key. After incubation, cooling should also be standardized, ideally 4 minutes in ice-cold water, and the absorbance should be read promptly at 544 nm, since the color can continue to change over time.

Another common cause of non-linearity is the concentration range itself: if the standards are too concentrated, the absorbance values can saturate and deviate from linearity. A better approach is to prepare a dilution series that yields absorbance values roughly between 0.05 and 1.0, for example 10, 20, 40, 80, and 160 µg/mL. Plotting absorbance against these concentrations and working only in the linear portion of the curve usually gives the best results. Running triplicates and including both a reagent blank and a methanol blank will help you correct for any background signal. It is also useful to scan one standard across 400–700 nm to confirm that 544 nm is indeed the absorbance maximum under your conditions, since sometimes the peak can shift slightly depending on the batch of reagents.

The reason you see different color hues—from yellow before incubation to pink or purple afterwards—is that the vanillin–sulfuric acid reaction produces chromophores whose exact shade depends on the concentration, local pH, and even slight differences in mixing or heating. This variability is normal but can be minimized by maintaining very strict consistency in your protocol. If you still cannot achieve linearity, you may need to narrow your concentration range, verify the purity of your diosgenin standard, or adjust the wavelength slightly based on your scan. In short, the key to a good standard curve in this method is meticulous attention to reagent freshness, concentration range, and especially strict control over timing, temperature, and mixing.

Hi, thank you so much Athira Prameela . And it's so helpful for me to cross-check my error! And really love your detailed explanation, which is easy to understand.