[turbosof]

please, I need your help, I am working on exopolysaccharides produced by lactic acid bacteria, I use the  total carbonhydrate quantification by the method of Dubois, I would like to know if it is possible to dilute the mix reaction (sample + phenol+ sulfuric acid) when the absorbance value is outside linear range instead of diluting sample before the add of reagents. can you give me a reference, please it is very important for me.
best regards

[Arkcon]

I don't have specific experience with this method, so someone else may have to fill you in better.   Offhand, I wouldn't deviate in such a manner, on general principals.  Its very plausible too much analyte will saturate the reaction, and misrepresent the results.  It would even give me pause if you kept the sample, but altered optical parameters (narrowed slit, shortened scan, or reduced detector sensitivity)

You could Google for the technique, and see if you find a reference that says just what you propose, but I'd doubt someone would submit for peer-review something like that.  You could get lucky 'tho, just check them all and see if someone has deviated in just such a way.

Of course, you can always test it for yourself -- prepare a bunch of standards at double strength, diluting before and after assay, and see if the results are comparable to your satisfaction.

This really is all up to the rigor your application demands.

[turbosof]

thank you very much for your answer, therefore I will test both and see what happens

[Furanone]

I have used this method very often with a Pharmacia agarose-packed size exclusion column separating starch fractions (amylose & amylopectin) and collecting in test tubes using a fraction collector. I would split up the test tubes half for total carbohydrates with phenol-sulfuric acid method and other half for iodine-binding analysis (amylose turns blue due to linear helices and polyiodide inclusion & amylopectin turns red due to highly branched structure allowing shorter iodide complexes within helices).

It was found by myself as well as many other grad students that dilution with water could only be done succesfully before addition of phenol solution then concentrated sulfuric acid. After addition of phenol & sulfuric acid, if dilution was attempted, results came out a bit wonky and standard deviation increased dramatically.

Therefore, keep excess of your sample. If you measure absorbance at 490 nm once and it is outside limits (phenol-sulfuric acid method is so incredibly sensitive!), you have some remaining sample to dilute to appropriate levels to get it in range before adding phenol & sulfuric acid.