I took 0.25g sample. I determined, with reference to a standard curve, 20µg proline/mL. How do I calculate μg proline/mL?
So that's 20µg proline per mL of 3% sulfosalicylic acid extract, and I had 5mL of this 3% sulfosalicylic acid extract, so a total of 100µg proline.
This 100µg proline was from an initial 0.25g homogenized plant material, so 100µg/0.25g = 400µg/g.
Is this the final value, or is there another calculation? I have taken 2mL of toluene. What next?
If all is just as you described it, it should be ok (multiplying by 5 is right). But I'm intrigued that you took 2 ml of toluene. If you describe the complete procedure from beginning to the end we could help you.
Another tip for you: It may not always be necessary, but it would be more stringent if you generated your standard curve by treating the standard exactly as the sample, i.e. start with some quantities in the expected range (100 µg of proline since you've a first hint that that's about the proline content in your tissue; also take 12.5, 25, 50, 200 and 500 µg or so) in powder form and then perform the same extraction (dissolve in sulfosalicylic acid etc) in parallel to your actual sample. At the end, you'll have some read, then you can simply interpolate your sample in the middle of your standard curve.
It would even be better if you additionally calculated the recovery rate of your measuring protocol: Start with your homogenized sample, split it in equal parts or take two equal portions; add let's say 50 µg of proline to one but not to the other, proceed to measure, and at the end you'll know how much of the proline your measurement is really capturing and how much is lost in the process.
Hi Ahmad,
Your calculation is ok if standard curve was generated under the same conditions as the sample.
Vesna
Thank you Mr. Patra. But according to Bates et al, should i calculate again this formula.i want to know which calculation is correct, using standard curve or calculation of Bates et al.
This is elementary mathematics! Of course, your result is arithmetically correct. However, if the problem concerns your method of extraction (and not, I hope for it, how to calibrate a standard curve…), it is another history and you must ask your question in these terms.
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