I have followed Claussen's (2005) extraction procedure and colorimetric determination with acidic ninhydrin reagent (2.5 g ninhydrin/100 ml of a solution containing glacial acetic acid, distill water and orthophosphoric acid 85% at a ratio of 6:3:1) were carried out as follows: Sample of .2 g (fresh weight) were grounded in a mortar after addition of 10 ml of a 3% sulfosalicyclic acid (w/v). The homogenate was filtered and glacial acetic acid and acid ninhydrin reagent (1 ml each) were added to 1 ml filtrate. The closed test tubes were kept in a boiling water bath for 1 hour and reaction was terminated in a water bath of 21 degree centigrade for 5 minutes. Readings were immediately taken at wave length of 546 nm. I too prepared standard calibration curve. Could you please, show me a way to solve this puzzle. Thank you in advance
Typically, the answer is assuming y=mx + b;
(Abs. of sample)/(Abs. of standard) x Conc. of standard = Conc. of sample
[Conc. of sample (like ug/mL) x Volume (mL)]/0.2 g = ug/g Sample
Bruce Neagle,
I have 5 standard solution ( 0, 25, 50, 75, 100 micro M). It is confusing to take the conc and absorbance of standard for the calculation.
Choki,
Your concentrations are too low for a spectrophotometer. Thus, you may have to use an HPLC for analysis (inject 100 uL). A spectrophotometer is useful for concentrations around 1 ppm or mg/L. Therefore, follow my calculations;
MW of Proline is 115.13 g/L (or 1000 mL)
Therefore, 100 mM is (115.13 g/1000)/L or 0.11513 g/L or 115.13 mg/L or ppm
Therefore, 100 uM is (115.13 mg/L)/1000 or 0.11513 mg/L or ppm which is a factor of 10 too low!
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