I am getting the dark blue colour even before adding phosphate to my reagents when estimating phosphate with ascorbate method. Could someone troubleshoot this for me?
I dont know which methodology following u right now Vikas, but one of the best methods which I know for phosphate (Inorganic) estimation is Fiske- Subba Rao method; which was invented by our Indian Scientist Dr Y Subba Rao; Just go through any basic practical biochemistry books for detailed procedure and method in detail. My answer may serve ur purpose or not; if so it will be appreciated, thank you.
Thank you very much Krishna.....Fiske Subbarao method is very good but is not stable (colour). You have to take readings with in 10-15 min. The Method I am following is Molybdenom ascorbate method by Ames. But Unfortunately I am getting the blue colour even without adding the phosphate in it. I changed the reagents etc but no luck. Could sulfuric acid be a problem...or something else.....I have no idea.....I am also using MilliQ water as well as normal deionised water.........The other method which aslo uses the same reagent as well as Zn acetate is also giving me the same result....
What is the problem- the sample with blue colour without adding of phosphate is your control?
Sorry for the late reply.... I have changed reagents 3 times....using MQ and RO water.....but about the maintenance of these I am not sure......washed my glassware in chromic acid.........but still i am getting the intense blue colour.......i am checking the reagents and mixing them in eppendorfs and falcon tubes.......the blue colouration appears within 5 min and the colour is very dark....like lots of phosphate.The only difference is during my PhD in india where i never faced this problem was distilled water which was made through boiling method....
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