There are many literature that enlightened regarding this but I have some doubts as follows:
1. Introduction of phenol to the reagent, can intensify the color. What it means actually it will give false results or any ?? and if the color development is the indicator of reducing sugar concentration then is this right to add phenol in DNSA reagent preparation??
2. Potassium sodium tartrate can be used initially with the reagents or after the boiling. What is the wise stage of using potassium sodium tartrate in cellulase assay for reducing sugar determination?
3. How to store the reagent and it can be stored for how many days without any loss of action? As GAIL LORENZ MILLER said due to oxidation of sulfite, DNSA reagent can be deteriorate which can be rejuvenated by adding fresh sulfite. My question is for rejuvenation of the reagent how much sulfite can be added?
To understand the Question no. 3, I added the paper of GAIL LORENZ MILLER below and highlighted the point I want to discuss.
WAITING FOR THE SUGGESTIONS AND WELCOME ALL TO DISCUSS REGARDING.
THANKING YOU IN ADVANCE.
-AKM
Dear Abhas,
Normally for cellulase assay people follow IUPAC protocol that is attached herewith for your kind reference. The recipe of DNS reagent is :
DNS Reagent
Mix: Distilled Water 1416 ml
3,5-Dinitrosalicylic acid 10.6 g
NaOH 19.8 g
Dissolve above, then add:
Rochelle salts (Na-K tartarate) 306 g
Phenol (melt at 50°C) 7.6 ml
Na metabisulfite 8.3 g
This reagent can be stored for atleast 2 months when stored in dark without any losses in its activity. Phenol not only intensifies color but increases the stability of the color developed. Rochelle's sallt increase the shelf life of the said reagent and sulfite prevents its from oxidation (photo and air both).
Dear Abhas,
Normally for cellulase assay people follow IUPAC protocol that is attached herewith for your kind reference. The recipe of DNS reagent is :
DNS Reagent
Mix: Distilled Water 1416 ml
3,5-Dinitrosalicylic acid 10.6 g
NaOH 19.8 g
Dissolve above, then add:
Rochelle salts (Na-K tartarate) 306 g
Phenol (melt at 50°C) 7.6 ml
Na metabisulfite 8.3 g
This reagent can be stored for atleast 2 months when stored in dark without any losses in its activity. Phenol not only intensifies color but increases the stability of the color developed. Rochelle's sallt increase the shelf life of the said reagent and sulfite prevents its from oxidation (photo and air both).
Dear Abhas,
I use DNS regent for reducing sugars determination and completely agree with Deepti Agrawal answer. Just prepare reagent according to procedure and everything will work fine.
I understand you concern about its stability and my advice to you is to prepare smaller volume of reagent that you could utilize in some reasonable time (month or two) OR if you have some amount that is ,,older``, just make new calibration curve and thus check if it is still usable.
Thank you all for valuable suggestions. But what would be the differences if I use sodium sulfite anhydrous in-spite of Na metabisulfite ? There will be any variations in this? @ Dr Deepti Agrawal
There is a lot of difference between Sodium metabisulfite and sodium sulphite.... Sodium sulphate will form water of crystallization whereas the function of sodium metabisulphite is to present the oxidation of DNS by first auto-oxidising and keeping the properties of the said reagent intact.
Dear Abhas,
In addition to the above answers related to Q3, you could store your DNS for few months. But while preparing DNS reagent, don't add sodium sulphite. While doing assay, take the required amount of DNS reagent and add sodium sulphite appropriately. For eg. for 100 mL of DNS you would need 50 mg of sodium sulphite. Your DNS reagent is now ready to use !!!
Hi,
What if I don't use sodium metabisulfite and phenol in preparing DNS reagent and I'll use the DNS reagent as soon as prepared.
Thanks
Excuse me what is the color of DNS reagent?
When i make it i get crystal clear color. Is that right?
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