Hello!

What is the absorbance reading you're looking at 10 and 60 mg / L?

What wavelength are you detecting?

Probably your curve it is outside the range of detection why you look the same reading, would recommend you to do a curve from 1-10 mg / L of proline and see how it behaves

Hello

i have prepared standard curve of proline in the range of 20 - 100 micro g/ml, and absorbance was taken at 520 nm wavelength.

Hai!

I suggest you to do the range from 0 to 50 micro g/ml at 520 nm where the stock solution of L-proline is 50 micro g/100 ml. All the best.

Hi Philisiwe!

I usually work with a standard curve from 0.007 to 1.790 micro mol/mL, and absorbance is taken at 520 nm.

Hi there!

There is an improtant point about standard curves. You do not do standard curve for concentration but rather you do it for a quantity of a substance. For proline, the range is 20-100 micrograms. Thia means you prepare your stock solution of proline at a concentration that allows you to handle 20-100 micrograms accuratley, preferably the stock solution should be 1 microgram/microlitre. If you are doing a standard of 20, 40, 60, 80 and 100 micrograms of proline, you will take 20, 40, 60, 80 and 100 ul respectively of the stock and react them with reagents to produce colour. You will then extract the colour with toluene and read at 520.

Like I said, you are not dealing with a concentration of proline but rather with an absolute amount in each measurment.

Good luck

Hi

Thanks you all for the suggestions. I will try them.

Cheers.

You might like to see simplest and eco-friendly protocol for proline quantification that appeared online in Protoplasma (you may download the same from my ResearchGate site).