DNS (metabolic biochemistry lab)

Hi, are you referring to dinitrosalicylic acid?

yes

Okay, I got some excellent information for you:

Dinitrosalicylic acid (D.N.S.A. or 3:5-dinitrosalicylic acid) is a reagent used to determine sugar content especially glucose. The DNS technique is employed in order to estimate sugar present in the blood, in the cerebrospinal fluid and in other human bodily fluids. This is also effectively used in the handling of requirements for diabetic clinics in hospital laboratories, considering that only 10 minutes is enough for the process to take place and the reagents are stable, cheap and easily prepared. The amount of blood sugar in the blood has metabolic implications and is used to determine the presence of blood sugar-related disorders such as hyperglycemia. One good way to assess blood sugar level is through the use of dinitrosalicylic acid.

Instructions

1. Place 17.5 g of 3:5-dinitrosalicylic acid in 1,000 ml of hot distilled water (around 60C) and stir to dissolve. Filter the solution through a filter paper and funnel and store at 25C. This is referred to later as Lee's Reagent A.

2. Prepare Glucose-D.N.S.A. solution by adding 1 percent glucose in saturated benzoic acid solution to 100 ml of 1.75 percent Lee's Reagent A and mix thoroughly by inverting the stoppered container repeatedly. Store at 25C.

3. Dissolve 13.8 g of phenol and 13.8 g of sodium bisulfite in 350 ml of 10 percent sodium hydroxide. Add this solution to 510 g of potassium-sodium-tartrate in 180 ml of water. Filter the final solution through filter paper. Store at room temperature. This will serve as Lee's Reagent B.

4. Obtain blood from a capillary puncture using ordinary 0.1 or 0.2 ml pipettes. The 0.1 ml pipette is more qualitatively accurate than the latter when measuring 0.1 ml of blood. However the reverse is true when measuring 0.2 ml of blood.

5. Deliver the blood in the pipette into the glucose-D.N.S.A. solution in a test tube. Mix it in the proportion 1:20. Draw the pipette up and down until you get an even distribution of the mixture between the test tube and the pipette. Leave the pipette in the solution and remove the precipitate from the pipette after it has settled an hour or two later.

6. Add an equal volume of Lee's Reagent B to the clear supernatant. After which, heat in a boiling water-bath for not less than three minutes.

7. Compare the red color developed with one produced by a standard glucose solution.

Things You'll Need:

3:5 dinitrosalicylic acid (17.5 g)
60C hot distilled water (1000 ml)
Filter paper
Funnel
1 percent standard glucose solution in saturated benzoic acid
Stoppered container (greater than 100 ml)
Phenol (13.8 g)
Sodium bisulfite (13.8 g)
10 percent sodium hydroxide solution (350 ml)
Potassium-sodium-tartrate (510 ml)
Water with normal temperature (180 ml)
Pipettes (0.1 or 0.2 ml)
Boiling water-bath
Laboratory equipment for heating
Laboratory equipment for measuring small solids and liquids

Tell me there is anything else you need to know.

thx but i didn't mean to ask about how to prepare a DNS solution.. im assuming the solution is already prepared, and we are using it to study the rate of hydrolysis of glycogen... while doing the experiment, we add at a certain point 2ml of the DNS solution to the tube and we place the tube in boiling water for 10 min, after that we cool the tube and then we measure the absorbing capacity of the solution using a spectrophotometer to compare between different tubes.... the professor told us that when we add DNS the reaction (hydrolysis of glycogen) stops, i didn't understand why it stops ! im also curious about why we placed the tubes in boiling water for 10 min after we added DNS.. also what is the chemical explanation for the change in color occurring while the tubes are being heated ( why is it that higher concentration of glucose causes the solution to become darker upon adding DNS solution (after heating) ). 

Right, I see what you mean. I recall that when you add 2 ml of DNS reagent, it stops the reaction. According to my notes, 3,5-dinitrosalicylic acid reacts with the sugar to form 3-amino,5-nitrosalicylic acid in a reduction reaction. In other words, one mole of sugar will react with 1 mole of DNS to form 3-amino,5-nitrosalicylic acid. The chemical that is formed is able to absorb light strongly at 540 nm and depending on the sugars, the darker the reaction will be (I'm assuming it is copper-colour red). So the more sugar there is, the more of carbonyl groups there are, and the darker it will be. This method tests for the presence of free carbonyl group (C=O) - the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in
fructose.

The reaction is shown below:

                      oxidation
aldehyde group carboxyl group
                                   reduction
3,5-dinitrosalicylic acid 3-amino,5-nitrosalicylic acid

Heating to 100C caused the reaction to stop. However, I'm not sure if you used any enzymes in your lab, but if you had, it would have denatured them at 100C after 10 minutes. I think these enzymes work best at 50 C and beyond that, they are nonfunctional. Regardless, heating the solution liberates the reducing sugars which allows the 3,5-dinitrosalicylic acid to react with the reducing sugar and in turn, producing the colour. Did you guys then rapidly cool the test tubes in running tap water?

yes we did cool the tubes right after heating them for 10 minutes by running tap water, we also added 15 ml of distilled water to each tube to dilute the solution so the color of tubes would still be in the " reading range " of the spectrophotometer, im not sure why we had to cool down the tubes, i guess it could be to stop the DNS from reacting with reducing sugars to prevent the color from getting darker... even though i dont see why that would be a bad thing if we were going to dilute it later either ways....

We used HCl to hydrolyze the bonds... and i don't think that the heating caused the reaction to stop... because i recall the professor telling us that adding DNS will get the reaction to stop.. i think heating was just to get the reaction going between the DNS and the reducing sugars..

I've seen variations of this lab where the test-tubes are only submerged in the hot bath for 5 minutes or even 7 minutes. I believe the cooling stops the glycogen from breaking down (basically prevents the liberation of glucose). Therefore I think you're right that it stops the DNS from reacting with reducing sugars - not because DNS requires heat to react, but because it stops the glycogen molecule from breaking down, thereby releasing reducing sugars that DNS requires to change colors. Essentially, it is the heat that allowed the glucoso units to be released from the glycogen.

yea that would make sense.. i also posted something which i found out to be wrong... before adding DNS we added NaOH, this is what made the hydrolysis stop, not the DNS. thx 

I'm guessing the NaOH outcompetes the DNS for the reducing ends of the sugar molecules.

im not sure about that.. but i know that we added it to suppress the hydrolysis of glycogen by HCl ( to be honest i never knew that glycogen can be broken down by HCl before this lab.. and i still have doubts )

That's interesting. I researched this a little further and I found out that the acid hydrolysis of glycogen by HCl does not take place immediately; rather it takes place over a period of time. Initially, little or no glucose is evident, but as the reaction progresses, with the addition of heat, increasingly more glucose is released until all of the glycogen is degraded. Thus, the addition of heat speeds up the process. The addition of NaOH, however, causes the following reaction: HCl + NaOH H2O + NaCl... Therefore it neutralizes HCl, which is why it stops the reaction I initially thought it outcompetes DNS, but that's not the case.

Hi, I had the same lab. So from what I understood the reason why we added water following boiling with DNS was to dilute the color so that the spectrophotometer could read it? Also I have 2 more questions if anyone please can help me: 1. List the components of a positive control that can be used in this experiment and 2. What use should be put of the controls values before plotting a graph? Thank you.

Hi Lili and welcome to the forum, wish I could help, but can you give us a quick run down of the experiment, perhaps even post the protocol? It was a while since this one was looked at

Recall that a positive control is a procedure that is very similar to the actual experimental test, but which is known from previous experience to give a result that is hypothesized to occur in the treatment group (positive result). This is opposed to a negative control where it is known to give a negative result. The positive control confirms that the basic conditions of the experiment were able to produce a positive result, even if none of the actual experimental samples produce a positive result.