EXP.

NO: 1 SAFEETY MEASURES IN BIOCHEMISTRY LAB General Safety measure

DATE:

1. Lab can be dangerous places in to work all uses to be aware of the potential hazards and to know what to do process of emergency when starting work in a new lab is important to become familiar with the layout of the room and location of the safety equipments. 2. The portion of the emergency exists, fire alarm and extension should be known, so that the appropriate action can be taken in the current of fire. 3. The main labs for gas, water and switch for electricity should also be located so that the serious can be turned off for on emergency. Safety measures: 1. Lab workers must know the meaning of safety signs. Some of these are in plain English while others are in the form of pictograms. The signs have been standardized in any terms, lettering, diagram and colours so that they can be rapidly identified. Personal protection: 1. Gloves: Heavy gloves must be used when handling corrosive substances such as strong acids and Alkalies. The hazardous nature of there substances is obvious but the danger inherent in skin contact with other chemicals are not always clear. Light weight disposable gloves must be used during weighing and handling of chemicals to avoid the risk of absorption through the skin. 2. Protective clothing: Lab coats are not status symbols but are meant to protect from chemical and infection material. Cotton Is a better material for a lab coat and nylon has a greater absorbing capacity and is generally most resistant to chemicals. The standard open neck coat may be appropriate for most chemical works. But high necked coats are more suitable for work with animals and potentially dangerous microbes. 3. Face masks: These are not always necessary but need to be work. There is a risk of dust from chemicals or from aerosols of microbes. Dangers to avoid: Poisoning often arises from the accidental transfer of a compound through the mouth and their misle can be greatly reduced by always keeping three simple rules in the lab. 1. No smoking 2. No eating or drinking 3. No mouth pipetting First aid First aid, the name implies the help given immediately to on injured person. It is not a substitute for a medical or hospital attention. The following short notes

are not a substitute for a proper course in first aid but should be of same help if you find yourself dealing the subject of the lab accident. Immediate assistance: Artificial respiration: a. The brain is unable to function for more than a few minutes, without oxygen and brain damage or death quickly follows. b. If the casualty has stopped breathing artificial respiration must be started at once. c. It should be really applied by someone trained for this technique. Unconscious: a. If breathing has stopped, apply artificial respiration immediately. b. If the victim is breathing their lay him face down with his head on one side and his arms and legs on that side in a bent position. c. This position makes it easier for the patient and proudest better circulation of blood to all part of the body. Burns: 1. If the causality is on fire, immediately to down the flame, wrap hern in a fire blanket. 2. Then cool the affected area by running water. 3. When acid burns occur in the lab, the suitable alkali must be applied gently with a solution of sodium bi carbonate. 4. When electric burns occur in the lab, switch off the current or if it is not possible free the person using anything non conductive.

QUALITATIVE ANALYSIS OF CARBOHYDRATES AIM: To identify different carbohydrates in the given sample. PRINCIPLE: Qualitative test for carbohydrate classified into three category (i)Those which are base reducing property aldehyde and keto group. (ii)Those which are base are dehydration of hydroxyl group by strong acids and treating the resultant furfural with various compounds to form colour compound. (iii)Preparation of derivatives. (i)Based on reducing properties: Carbohydrate possessing a free or potentially free aldehyde or ketone groups of the properties of readily reducing ion of certain metals such as copper ion Bismuth, silver, mercury etc., Bismuth many test for sugar based on this properties. In the presence of reducing sugar the blue cupric hydroxide is reducing to insoluble yellow (or) red (or) green cuprous oxide. Large particles formed by slow reduction reaction and formed brick red whereas very small particles formed by fast reduction may be formed greenish color. The amount of precipitate is not there color of primary consideration. Fehlings test, Benedicts test and Barfoeds test based on this property. Benedicts is more stable than Fehlings. Barfoeds test is carried in the slightly acidic state. (ii)Test based on dehydration: Stable in dilute acid but treat in strong acids resulting in the loss of water and the formation of furfural (or) furfural derivatives. CHO -3H2O (CHOH) 4 CH2OH Hexose CHO Conc. acid (CHOH) 3 CH2OH Pentose C O (Furfural) Aromatic compound Colored substances C OHCH2 C O (Furfural) Aromatic compound Colored substances C-CHO C OHCH2 C-CHO

The furfural is a reactive compound and it reacts with a number of phenolic and other compounds to produce complete coloured products they are used for the identification and determination. Test which are based on the above reaction (i)Molischs test (ii)Anthrone test (iii)Bials test (iv)Seliwanoffs test

PREPARATION OF REAGENTS (i)Molischs reagent: 5gms of alpha napthol dissolved in 100ml of alcohol (ii)Iodine solution: 5gms of potassium iodine and 1.2gms of iodine (or) grounted in mortor pestle and dissolved in water and made up to 500ml. (iii)Fehlings reagent: It is a mixture of copper sulphate and alkaline tartarate solution prepared as follows. Fehlings A: 36.65gms of copper sulphate is dissolved in water and made up in 500ml Fehlings B: 125gms of potassium hydroxide 123gms of sodium potassium tartarate dissolved in water and made up to 500ml. These two solutions are prepared separately in to different bottles whenever needed mix equal volume of solution A and solution B. (iv)Benedicts reagent: 85gms of sodium citrate 50gms of sodium carbonate are dissolved in about 400ml of water by heating 17.3gms of copper sulphate is dissolved separately in about 100ml of water carefully pour the copper solution in the carbonate solution with stirring finally the solution is made up to 1 litre. (v)Barfoeds reagent: 13.5gms of crystalise copper acetate in 200ml of boiling water filtrate is necessary and 1.8ml of glycial acetate. (vi)Bials reagent: 1.5gms of orcinal is dissolved in 500ml of concentrated hydrochloric acid containing 20-30 drops of ferric chloride solution.

(vii)Seliwanoffs reagent: 50mgms of resorcinol is dissolved in 100ml of 0.4N hydrochloric acid. (viii)Tollens reagent: A pinch of glucinol is dissolved in concentrated hydrochloric acid and used for the test. (ix)Phenylhydrazine reagent: 10gms of phenylhydrazine is added to 60ml of water and warmed it and dissolved 50gms sodium acetate is added to 10ml of glycol acetate and stored in brown bottle. (x)picric acid reagent: water 0.2 _____ .2gms of picric acid dissolved in 100ml of distilled

EXPT. NO. GENERAL PROCEDURE OF CARBOHYDRATE

DATE:

Sl.No

EXPERIMENT

OBSERVATION

INFERENCE

Solubility test:

1. Soluble.

A small amount of substance is taken in the test tube and a few drops of water is added. 2.Insoluble 2 Molischs test: To 1ml of sample solution, added 2 drops of Molischs reagent and mixed well. To this mixture 2ml of conc. H2SO4 is added along the side of the test tube. Iodine test: 3.

1.Presence monosaccharide (Or) disaccharides.

of

2. Presence of starch.

A violet colour ring This indicates presence was formed at the of carbohydrate. junction of two liquids.

To 1ml of solution add a few drops of iodine 1. Blue colour was Presence of starch. solution. obtained. 2. Reddish brown colour was obtained. Presence of glycogen. Fehlings test: To 1ml of test solution A red colour solution add a few drops (or) red precipitate fehlings mixture and was obtained. heat in a boiling water bath. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool.

It indicates the presence of reducing sugar. The red precipitate is cuprous oxide.

1. Green and then green precipitate is 1. Due to the reducing obtained. action of sugar. 2. No characteristic change. 2. Presence of non reducing sugars.

6.

Picric acid test:

7.

A red colored solution is obtained. To 2 ml of test solution add 2 ml of picric acid. Due to the formation of picramic acid. It indicates the presence of the reducing sugar. Barfords test: 1. A brick red is To 5ml of Barfords precipitate obtained within 5 reagent, add 5ml of test solution and heat in a minutes. It indicates presence of boiling water bath for 5 2. No characteristic monosaccharide. minutes. change. Presence of maltose and sucrose. This shows Disaccharides.

8.

Seliwanoffs test:

1. Cherry red color To 5drops of test solution is obtained. solution, 3 ml of 2. No characteristic seliwanoffs reagent is change. added and boiled for few minutes in direct flame. Bials test: 1. Green color solution is obtained.

It indicates presence of sugars. Presence sugars. of

the keto aldo

9.

To 2ml of Bials solution and heat in direct 2. No characteristic change. It is due to the flame. presence of pentose. 10. Presence sugar. of aldose

Phenyl hydrazine test: To 5ml of sugar solution, add on equal amount of phenyl hydrazine hydrochloride and sodium acetate. Then add 1ml of glacial acetic acid and shake the mixture well, Filter the above solution, now the filtrate is boiled in a water bath until yellow

1. A white precipitate at room temperature, after heating yellow Presence of mannose. precipitate is formed.

2.Yellow

color

precipitate is formed. The precipitate is observed under the microscope to note the crystals.

precipitate obtained after 5 minutes, the crystals are view 2. Needle shaped under microscope. crystals called as ructosome. It indicates presence of fructose. 3. Yellow color precipitate obtained after 10-20 minutes, needle shape crystals were 3. The needle shape of observed under the crystals is Glucozone. microscope. This indicates presence of glucose.

4. Yellow color precipitate obtained after 30 minutes, flower shape crystals were observed under 4. The flower shape of the microscope. crystals is glactazone. This indicates presence of galactose.

5. Yellow color precipitate obtained after 30 minutes, puff shape or cotton ballshape crystals were observed under 5. The puff shape or the microscope. cotton ball shape of crystals is lactazone. This indicates presence of lactose.

6. Yellow color precipitate obtained

after 45 minutes, flower bud shape crystals were observed under the microscope. 6. The flower bud shape of crystals is maltazone. This indicates presence of maltose. 7. Yellow color precipitate obtained after 20 minutes and within 30 minutes, sharp needle shape crystals were observed under the microscope. 7. The sharp needle shape of crystals is xylazone. This indicates presence of xylose.

HYDROLYSIS OF SUCROSE: To 10ml of sugar solution, add 5-10 drops of hydrochloric acid and heat in a boiling water bath for 20 minutes and then cool it. Then neutralized with sodium carbonate. Then filter the solution, the filtrate used for the further studies. HYDROLYSIS OF POLYSACCHARIDES: 1% of starch solution is taken in 100 ml of conical flask and add 5ml of concentrated HCL. The resulting mixture is divided in to 5 equal parts into different test tubes and placed the test tubes in a boiling water bath. Remove the test tubes from boiling water bath at the interval of 1, 5,8,12 and 20 minutes. Divide the solution of each test tubes in two parts. Then the solution is neutralized by sodium carbonate. One part of the test solution is performing with Benedicts test and second part is performed with Iodine test. This result, hydrolyzed starch is conformed. RESULT:

EXP.NO: REACTIONS OF GLUCOSE S.No EXPERIMENT OBSERVATION

DATE:

INFERENCE

Solubility test: A small amount of substance is taken in the test tube and a few drops of water is added.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs . reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube.

3.

Iodine test: To 1ml of solution add a few drops of iodine solution. Fehlings test: To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool. Picric acid test:

6.

To 2 ml of test solution . add 2 ml of picric acid. Barfords test: To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5

7.

minutes. Seliwanoffs test: 8. To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame. Bials test: 9. 10. To 2ml of Bials solution and heat in direct flame. Phenyl hydrazine test: To 5ml of sugar solution, add on equal amount of phenyl hydrazine hydrochloride and sodium acetate. Then add 1ml of glacial acetic acid and shake the mixture well, Filter the above solution, now the filtrate is boiled in a water bath until yellow precipitate is formed. The precipitate is observed under the microscope to note the crystals.

RESULT: The given sugar sample is 1. 2. 3. 4.

EXP.NO:

DATE:

REACTIONS OF FRUCTOSE S.No EXPERIMENT OBSERVATION INFERENCE

Solubility test: A small amount of substance is taken in the test tube and a few drops of water is added.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs . reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube.

3.

Iodine test: To 1ml of solution add a few drops of iodine solution. Fehlings test: To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool. Picric acid test:

6.

To 2 ml of test solution . add 2 ml of picric acid. Barfords test: To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5 minutes.

7.

Seliwanoffs test: 8. To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame. Bials test: 9. To 2ml of Bials solution and heat in direct flame. Phenyl hydrazine test: 10. To 5ml of sugar solution, add on equal amount of phenyl hydrazine hydrochloride and sodium acetate. Then add 1ml of glacial acetic acid and shake the mixture well, Filter the above solution, now the filtrate is boiled in a water bath until yellow precipitate is formed. The precipitate is observed under the microscope to note the crystals.

RESULT: The given sugar sample is 1. 2. 3. 4.

EXP.NO:

DATE:

REACTIONS OF LACTOSE S.No EXPERIMENT OBSERVATION INFERENCE

Solubility test: A small amount of substance is taken in the test tube and a few drops of water is added.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs . reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube.

3.

Iodine test: To 1ml of solution add a few drops of iodine solution. Fehlings test: To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool. Picric acid test:

6.

To 2 ml of test solution . add 2 ml of picric acid. Barfords test: To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5 minutes.

7.

Seliwanoffs test: 8. To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame. Bials test: 9. To 2ml of Bials solution and heat in direct flame. Phenyl hydrazine test: To 5ml of sugar solution, add on equal amount of phenyl hydrazine hydrochloride and sodium acetate. Then add 1ml of glacial acetic acid and shake the mixture well, Filter the above solution, now the filtrate is boiled in a water bath until yellow precipitate is formed. The precipitate is observed under the microscope to note the crystals.

10.

RESULT: The given sugar sample is 1. 2. 3. 4.

EXP.NO: REACTIONS OF MALTOSE

DATE:

S.No

EXPERIMENT

OBSERVATION

INFERENCE

Solubility test: A small amount of substance is taken in the test tube and a few drops of water is added.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs . reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube. Iodine test: To 1ml of solution add a few drops of iodine solution. Fehlings test: To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool. Picric acid test: 6. To 2 ml of test solution . add 2 ml of picric acid. Barfords test:

3.

7.

To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5 minutes. Seliwanoffs test: To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame. Bials test: To 2ml of Bials solution and heat in direct flame. Phenyl hydrazine test: To 5ml of sugar solution, add on equal amount of phenyl hydrazine hydrochloride and sodium acetate. Then add 1ml of glacial acetic acid and shake the mixture well, Filter the above solution, now the filtrate is boiled in a water bath until yellow precipitate is formed. The precipitate is observed under the microscope to note the crystals.

8.

9. 10.

RESULT: The given sugar sample is 1. 2. 3. 4.

EXP.NO: REACTIONS OF SUCROSE AIM:

DATE:

To determine the presence of sucrose in a given sample. HYDROLYSIS OF SUCROSE: To 10ml of sugar solution, add 5-10 drops of hydrochloric acid and heat in a boiling water bath for 20 minutes and then cool it. Then neutralized with sodium carbonate. Then filter the solution, the filtrate used for the further studies.

S.No

EXPERIMENT

OBSERVATION

INFERENCE

1.

Solubility test: A small amount of substance is taken in the test tube and a few drops of water is added.

2.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs . reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube. Benedicts test: To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to cool. Fehlings test: To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath.

3.

4.

Barfords test:

5.

To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5 minutes. Seliwanoffs test: To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame.

6.

RESULT: The given sugar sample is 1. 2. 3. 4.

EXP.NO: REACTIONS OF POLYSACCHARIDES AIM:

DATE:

To determine the presence of polysaccharides in a given sample. HYDROLYSIS OF POLYSACCHARIDES: 1% of starch solution is taken in 100 ml of conical flask and adds 5ml of concentrated HCL. The resulting mixture is divided in to 5 equal parts into different test tubes and placed the test tubes in a boiling water bath. Remove the test tubes from boiling water bath at the interval of 1, 5,8,12 and 20 minutes. Divide the solution of each test tubes in two parts. Then the solution is neutralized by sodium carbonate. One part of the test solution is performing with Benedicts test and second part is performed with Iodine test. This result, hydrolyzed starch is conformed.

S.No

EXPERIMENT

OBSERVATION

INFERENCE

1.

Solubility test: A small amount of substance is taken in the test tube and a few drops of water are added.

2.

Iodine test: To 1ml of solution add a few drops of iodine . solution.

3.

Molischs test: To 1ml of sample solution, added 2 drops of Molischs reagent and mixed well. To this mixture 2ml of conc H2SO4 is added along the side of the test tube. Benedicts test:

4.

To 3ml of sugar solution and few drops of Benedicts reagent is added and heated in boiling water bath for 3 minutes then allowed to

cool. Fehlings test: 5. To 1ml of test solution add a few drops fehlings mixture and heat in a boiling water bath. Barfords test: 6. To 5ml of Barfords reagent, add 5ml of test solution and heat in a boiling water bath for 5 minutes. Seliwanoffs test: 7. To 5drops of test solution, 3 ml of seliwanoffs reagent is added and boiled for few minutes in direct flame. .

RESULT: The given sugar sample is 1. 2. 3. 4.

ESTIMATION OF REDUCING SUGAR BY BENEDICTS QUANTITATIVE REAGENT AIM: To estimate the amount of reducing sugar present in the given unknown sample by Benedicts method. PRINCIPLE: Benedicts 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 Benedicts 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)Benedicts 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 Benedicts Qualitative Reagent Accurately pipette out 10 ml of Benedicts 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 Benedicts Quantitative reagent Std Glucose Vs Benedicts Quantitative reagent Indicator: Self S.No. Contents in conical Initial 1. 2. 3. Titration II: Estimation of Glucose Standard Benedicts Quantitative reagent Vs Unknown Glucose Indicator: Self Unknown Glucose solution Vs Benedicts Quantitative reagent Indicator: Self S.No. Contents in conical Initial 1. 2. 3. Calculation Standardization of glucose: ---- ml of standard glucose is needed for reducing 10 ml of Benedicts 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 Benedicts 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 Burette reading Concordant ValueFlask (ml) Final (ml) Burette reading Concordant ValueFlask (ml) Final (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 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:

water. Procedure:

act as a denaturing agent for enzyme.

It was

prepared by dissolving 8 grams of sodium hydroxide pellets in 100 ml of distilled

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 Condition Vol. of OD at

No.

the test

maltose soln (ml)

standard (g) 0000 0200 0400 0600 0800 1000

dist. Water (ml) 2.0 1.8 1.6 1.4 1.2 1.0

DNS reagent (ml) Keep the test tuves in boiling water bath for 10 minutes , cool

distille d water

520nm

01 02 03 04 05 06

Blank Std 1 Std 2 Std 3 Std 4 Std 5

0.0 0.2 0.4 0.6 0.8 1.0

2.0

10 ml

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 Test (ml) 2.5 2.5 1.0 0.5 --0.5 2.0 5.0