ESTIMATION OF REDUCING SUGAR BY BENEDICT’S QUANTITATIVE REAGENT AIM: To estimate the amount of reducing sugar present in the given unknown sample by Benedict’s method. PRINCIPLE: Benedict’s quantitative reagent is the modified form of qualitative reagent. It consists of cupric sulphate, sodium carbonate and sodium citrate, potassium thiocyanate, and potassium ferrocyanide. The alkali present in the Benedict’s reagent enolises the sugar, thereby causing them to be a strong reducing agent. Ferrocyanide serves to dissolve the copper hydroxide while thiocyanate helps to convert the red cuprous oxide to whit crystals of cuprous thiocyanate, which gives the clear end point. REAGENTS REQUIRED (i)Benedict’s quantitative reagent (BQR) (a)Cupric sulphate, (b) Sodium carbonate, (c) Sodium citrate, (d) Potassium thiocyanate, (e) Potassium ferrocyanate. (ii)Anhydrous sodium carbonate (iii)Working standard glucose solution (100mg of glucose in 100 ml of distilled water) (iv) Porcelain beads. PROCEDURE: Titration I: Standardisation of Benedict’s Qualitative Reagent Accurately pipette out 10 ml of Benedict’s quantitative reagent into a clean conical flask. One spatula full of 1 g of sodium carbonate was added into the conical flask. Few pieces of porcelain beads were also added in order to avoid bumping. The contents were brought to temperature approximately 60-700C. The contents were titrated against the standard glucose solution with regular shaking until the blue colour disappeared. The end point is the appearance of chalky white precipitate. The titrations were repeated for concordant values. Titration II: Estimation of Glucose The given unknown sample solution was made up to 100ml with distilled water in a standard flask. It was shaken well for uniform concentration. The burette
was filled with this unknown solution and the titrations were performed as mentioned above till the appearance of chalky white precipitate. The titrations were repeated for concordant values. Tabulation: Titration I: Standardization of Benedict’s Quantitative reagent Std Glucose Vs Benedict’s Quantitative reagent Indicator: Self S.No.
Contents in conical
Burette
ValueFlask
reading
Concordant
(ml) Initial
Final (ml)
1. 2. 3. Titration II: Estimation of Glucose Standard Benedict’s Quantitative reagent Vs Unknown Glucose Indicator: Self Unknown Glucose solution Vs Benedict’s Quantitative reagent Indicator: Self S.No.
Contents in conical
Burette
ValueFlask
reading
Concordant
(ml) Initial
Final (ml)
1. 2. 3. Calculation Standardization of glucose: ---- ml of standard glucose is needed for reducing 10 ml of Benedict’s quantitative reagent. But 100mg of glucose is present in 100 ml of solution. Hence 1 ml contains 1mg of glucose. But X ml of standard glucose is used up for the reduction of 10 ml of Benedicts reagent, so X ml of standard glucose solution corresponds to X mg of glucose. But,
During estimation of glucose present in unknown solution, Y ml of unknown glucose is used up for the reduction of 10 ml of Benedict’s quantitative reagent. Hence X mg of glucose is present in Y ml of unknown sugar sample. Hence, 100 of the unknown glucose solution contains =
X mg x 100 ml Y ml
Result: The amount of glucose present in 100ml of the given solution =__________mg
ASSAY OF AMYLASE ACTIVITY AIM: to assay the activity of the given salivary α – amylase enzyme sample. Principle: In any enzyme assay the rate of the reaction can be known by measuring the amount of substrate that is utilized or amount of product that is formed in unit time. Amylase is a hydrolytic enzyme which breaks down starch (polysaccharides) into smaller units called as maltose. However, they can be converted to a colored product by specific chemical reactions. Here maltose reacts with alkaline dinitrosalicylic acid to give a reddish orange or orange color.
The amount of maltose thus produced in unit time can be
measured by using a colorimeter at 520nm, which gives an idea about the enzyme activity. Reagents required: 1. 0.1N phosphate buffer (pH 6.7) 2. Dinitro salicylic acid reagent (DNS reagent): it was prepared by dissolving 1 gram of 3.5 – dinitro salicylic acid, 30 grams of sodium potassium tartarate and 1.6 grams of sodium hydroxide in distilled water and made up to 100 ml with the same. 3. Starch solution: it acts as a substrate. 0.5% starch solution was prepared by weighing 1 gram of soluble starch in 200 ml of phosphate buffer. 4. 1% sodium chloride solution: 1 gram of sodium chloride in 100 ml of distilled water.
5. Enzyme solution: Dilute 1 ml of saliva with distilled water and make up the volume to 20 ml. This gives 1:20 dilution of enzyme. 6. Standard maltose solution (1mg/ml): 100 mg or 0.1 gram of maltose was weighed and dissolved in distilled water and final volume was made up to 100 ml by using standard volumetric flask. 7. 2N Sodium hydroxide solution: act as a denaturing agent for enzyme. It was prepared by dissolving 8 grams of sodium hydroxide pellets in 100 ml of distilled water. Procedure: A. Prepartion of standard graph for maltose: Pipette out different aliquots (0.2 to 1.0ml) of standard maltose solution and make up the volume to 2 ml by using distilled water. To this solution 2 ml of DNS reagent was added. The cocktail or mixtures in the test tubes were kept in boiling water bath for about 10 minutes. The tubes were cooled and the mixture was diluted by addition of 10 ml of distilled water. The resultant reddish orange or orange color was read at 520 nm by using a digital colorimeter. The blank was prepared in just the same way but by addition of distilled water instead of maltose solution. Prepare the standard graph by plotting the concentration of maltose on x – axis and the optical density values on y – axis. Tabular column 1: preparation of standard graph Sl.
Name of
Vol. of std,
Conc of
Vol. of
Vol. of DNS
No.
the test
maltose soln
standard
dist.
(ml)
(μg)
Water
0.0 0.2 0.4 0.6 0.8 1.0
0000 0200 0400 0600 0800 1000
(ml) 2.0 1.8 1.6 1.4 1.2 1.0
01 02 03 04 05 06
Blank Std 1 Std 2 Std 3 Std 4 Std 5
Condition
Vol. of
OD at
reagent
distilled
520nm
(ml)
water
2.0
10 ml
Keep the test tuves in boiling water bath for 10 minutes , cool
B. Enzyme Assay: For the enzyme assay 0.5 ml of 1: 20 diluted enzyme sample was taken. The enzyme assay can be done by estimating the maltose produced by the enzyme. The amylase enzymatic assay was performed as shown in tabular column 2. Tabular column 2: Enzyme assay: Sl. No. 01 02 03 04 05 06 07 08 09 Calculation:
Substrates to be Blank tube Control tube added (ml) (ml) Phosphate buffer 2.5 2.5 Starch solution 2.5 2.5 1% NaCl 1.0 1.0 Enzyme solution 0.0 0.5 2N NaOH 0.5 0.5 Leave the tubes at 370C for 15 minutes 2N NaOH ----DNS reagent 2.0 2.0 Keep the tubes in boiling water bath for 10 mins, cool Distilled water 5.5 5.0 OD at 520 nm
