My aim is to estimate proline content from plant tissues using Ninhydrin method with a spectrophotometer(colorimetric method). Briefly, Proline when in contact with Ninhydrin yield a colured solution and the absorbance can be measured at 520nm. I found a simple method for the proline quantification in this book at Section 3.2 (also some other protocols have similar steps). It's pretty clear until blanking the spectrophotometer using toluene and quantifying the chromophore containing toluene. However, I am a bit confused about making the standard curve at Step 10.
My question is 'since the readings are done based on chromophore in the above step, Do I need to add ninhydrin to the serial dilution with known proline solutions? OR do I need to make a serial dilution with a range for instance, 0-100 micromol with only proline stock as described here(under Standard Curve Preparation?