I am trying to estimate educing sugars using the method described by Miller (1958). I make solutions of glucose from 100 microgram/ml to 1000 microgram /ml and use for making the standard curve , taking water as blank.However the first reading in the spectrophotometer(OD 540) is always negative. I don't get a line originating from 0.I have repeated the experiment multiple times using different reagents and changing the spectrophotometer. have attached two graphs, one including the blank and other excluding the blank and some other points.I am really interested to know why this is happening. Can somebody sort it out?
I have used reagent blank. It was reagent plus same volume of water as the sample
A +ve y-intercept would indicate that the sample/standard was contaminated. A -ve y-intercept indicates that some of the glucose is not reacting (unlikely) or some of the glucose is being destroyed before analysis (again unlikely) OR the cuvettes are not identical (very likely).
1. Clean all cuvettes overnight with aqua regia and check they are identical
2. Repeat curves with fresh standard.
3. Use a different test.
I don't know the method, so I can't say if that is normal or not. I've seen this behavior in phosphate calibrations where a longer reaction time is needed at low P concentrations.
However, Bruce is right: glucose std solutions are not very stable so that is the next thing to check.
When checking cuvettes you need to do that with water and with the coloured solution at 540 nm.
Thanks Milham and Neagle for the answers.
I made fresh glucose solutions and changed other reagents as well. I think my cuvette is OK, because I have tried using new one. I also took the OD using microtiter plate in plate reader.
Did you try setting the 0 con 0 absorption in the machine
Yes Sir, the blank was set as zero which contained the reagents + water.
But the first reading of standard containing 100 microgram / ml of glucose gave negative readings every time (less than the blank).
It was repeated 7 times in triplicates and gave same results all the time. its against the logic.
we really wonder if any body has ever got, what we are not getting.
are you weighing properly and baseline correction with solvent
Ignore the 100 microgram/ml glucose and plot only the standards higher than that in the standard curve. It indicates lack of sensitivity of the method at lowest concentrations. While estimating unknown samples also see that its concentration falls in the range of concentrations in the standard curve where the method shows sensitivity.
As i am trying to do analysis of Glucose by using glucose oxidase enzyme by DNS method... How much amount of glucose is left over after the reaction is estimated by adding DNS... Absorbance at 575nm...can anybody help me.... M not getting desired results... As i uploaded 2 graphs of my results...
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