Determine the amount of hemoglobin in the blood

Determine the amount of hemoglobin in the blood

Hemoglobin (Hb or Hgb) is the iron-containing oxygen-transport metallo-protein in the red blood cells of vertebrates and the tissues of some invertebrates.

Different types of hemoglobin

1. Hemoglobin A (95%)
2. Hemoglobin A2 (1.5-3.5%)
3. Hemoglobin F
4. Hemoglobin H
5. Hemoglobin S
6. Hemoglobin C
7. Hemoglobin AS
8. Hemoglobin SC

Methodology for estimation of Hb

• Sahlis method
• Cyanmethaemoglobin method (spectrophotometric method)
• Haldanes method
• Talliquist method
• Alkaline haematin method
• Oxyhaemoglobin method
• Determination of specific gravity
• Estimation of iron content of blood (van slyke and stadie method)
• Gowershaemoglobinometer
• Von fleischlshaemometer
• Colorimetric method
  

Hemoglobin concentration measurement is among the most commonly performed blood tests, usually as part of a complete blood count. Results are reported in g/L, g/dL or mol/L (1 g/dL equals about 0.6206 mmol/L). 

Normal values of hemoglobin are

Men: 13.5 to 16.5 g/dl
Women: 12.1 to 15.1 g/dl
Children: 11 to 16 g/dl
Pregnant women: 11 to 12 g/dl .

Sahlis method

1. This method is commonly used to estimate Hb at laboratory level.
2. Dil-HCl (N/10) is used to convert Hb to acid hematite causing brown colorization of test sample which is matched with standard. Reading is recorded as gm/dl of blood.

Procedure

1. Fill graduated tube with N/10 HCl upto 20 mark
2. Take a sprited cotton and wash ring finger ball then prick the finger with strile needle
3. Discard first drop of blood that oozes out after pricking
4. Take Hb pipette with one end touching oozed blood and fill it upto 20 mark
5. Transfer blood to graduated tube containing dilHCl and suck small amount and expel it. Repeat process for 3-4 times
6. Stir the solution and leave for 10 min undisturbed
7. Now add distilled water drop by drop with gentle stirring
8. Continue dilution till it matched exactly with standard plate. Note the result.

Observation

1. Three parameters can be assessed by this method.
2. Hb concentration in gm/dl in blood.
3. Percentage of Hb (14.5 gm/dl Hb is considered 100%).
4. O2 carrying capacity (1.34 ml of O2 is carried by 1 gmHb).

Error limit: - 8-10 %

Precaution

1. Pricking should be appropriate so that no extra bleeding may occur.
2. After blood is drawn using pipette, tip of   pipette should be wiped properly to prevent wrong results and clotting.
3. Wait for exact 10 min after mixing blood with Dilute HCL else it will result in wrong interpretation.
4. Matching with standard should be done against well lightened area otherwise manual error may occur.
5. Read lower meniscus to get a proper interpretation.

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Composition of Saliva

Saliva

Requirement:

Funnel, test tube, TT holder, beaker (100 ml), test tube stand, glass rod, tripod stand, wash bottle.

Saliva

Saliva is a mixture of secretion from three pairs of salivary gland-parotid, submaxillary and sublingual, and numerous small glands in the buccal mucosa. The Composition of the saliva produced from any one gland varies with the rate of flow which itself varies with the type and intensity of stimulus used for obtaining the sample. Saliva varies greatly in different individuals and in the same individual under different circumstances. 

Composition of saliva

Composition of saliva is quite variable. On an average, its water content is 99.5% and solid content 0.5% of the solid, organic constituents comprise 60% and inorganic constituents 40% .The chief organic constituents of saliva are mucin and enzymes .Mucin is a mucoprotein .The lubricating property of saliva is mainly due to its mucin content .The main enzymes in human saliva are amylase and lysozyme, amylase is an amylolytic enzyme whereas lysozyme is anti bacterial.

Procedure for Examination composition of saliva

Collection of saliva: Rinse the mouth with water 3 times then put the water in mouth & hold for 5 minutes after that take out the mouth contents and filter with cotton plug then dilute the  filtrate content with distilled water for 10 times.

Preparation of Phosphate buffer pH 6.8: Weigh out 2.8g of Disodium hydrogen phosphate and dissolve dissolved in 25 mL of water. Dissolve 1.45g dihydrogen potassium phosphate in 25 mL of water then mix the both solution and make up the volume up to 100 mL.

Preparation of 10% starch Solution: Weigh out 10 g Starch and transfer in beaker; dissolve in sufficient amount of distilled water and make up the volume up to 100 mL.

Test for protein (Biuret test): Take 2 mL sample solution in test tube and add 2 mL sodium hydroxide and mix well then add copper sulphate drop wise. Appearance of violet colour confirms protein. 

Test for carbohydrate (Molish test): Take 2 mL sample solution in test tube and add 2 drops of molish reagent. Then incline the test tube and add sulphuric acid along with wall of test tube. Appearance of violet colour confirms carbohydrates.

Test for mucine: Take 2 mL sample solution in test tube and add acetic acid (1%) drop by drop. Appearance of precipitate confirms presence of mucine.

Result: In own saliva protein, carbohydrates, and mucine are present.

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Detect various adulterants present in milk

Requirements: Milk, test tube, beaker, and test tube stand etc.

Theory: Quality control tests for milk are very important to assure adulterant free milk for consumption. Adulteration of milk reduces the quality of milk and can even make it heazardous. Adulteration like soap, acid, starch, table sugar and chemical like formalin may be added to milk. Most of the chemicals used for as adulterates are poisonous and cause health hazards. 

Adulterats are mainly added to increase the shelf life of milk. Some of the preservatives like acid and formalin are added to the milk as adulterants, thereby increasing the storage period of milk. Generally water is added to the milk to increase the volume content of the milk. Some of them common adulterants found in the milk are as micro-organism, table sugar, starch, acid, soap, formalin, ammonium sulphate, urea etc.

Procedure: 

Detection of miroorganism in milk

Measure out 5 mL milk into test tube and 2 drops of methylene blue indicator is added into it. Mix the content of test tube well. A blue colour is observed. The test tube now kept in an incubator at 37.c for 30 minutes. After incubation, if the colour changes from blue to white in a short period of time, indicates the presence of bacteria in milk.

Detection of table sugar in Milk

Measure out 5 mL milk into test tube and 2 mL of conc. HCl is added to the test tube. The content is mixed well and then 0.1 g of resorcinol powder is added to the test tube. Mix the content in the test tube. Now the test tube is placed in a boiling water bath for 5 minutes. After the incubation if red colour is observed,indicateds the presence of table sugar in milk.

Detection of starch in Milk

Measure 2 mL of milk in test tube. The test tube is then kept for incubation in boiling water bath for5 minutes. After incubation, the test is cooled and few drop 1% iodine solution and mix thr content well. Appearance of blue colour indicates presence of starch in milk.

Detection of acid in Milk

Measure out 5 mL milk into test tube and. Add few drops of Conc. Sulphuric acid and gently shake the test tube. Add 0.5% ferric chloride solution dropwise into the test tube. Mix the content well. Development of buff colour indicates the presence of benzoic acid and if violet colour is observed shows the presence of salicylic acid.

Detection of soap in Milk

Measure out 5 mL milk into test tube and. Add 5 mL of hot water into the test tube containing milk. Now adds 1-2drops of phenolphthalein indicator solution into test tube. Mix the content of test tube. Development of pink colour confirms the presence of soap in milk.

Detection of formalin in Milk

Measure out 2 mL milk into test tube and. Add 2 mL of 90% sulphuric acid and ferric chloride mixture into the test tube. Formation of purple colour ring at theinterface of two layers indicates the presence of formalin in milk.

Detection of ammonium sulphate in Milk

Measure out 2 mL milk into test tube. Add 0.1 mL of 2% sodium hydroxide into the test tube. Mix the contents well. Add 0.1 mL of 2% sodium hypochloride solution into the test tube. Finally add 0.1mL of 5% phenol into the test tube. Mix the content well. A light blue colour will be observed on mixing. Keep the test tube in a boliling water bath for 20 secconds. Formation of deep blue colour indicates the presence of ammonium sulphate in milk.

Test for detection of urea in Milk

Measure out 5 mL milk into test tube and mixed with 5 mL of paradimethyl amino benzaldehyde (16%). If the solution turns yellow in colour, confirm the presence of urea in milk.

Result: In the given urine sample no abnormal constituents (protein, sugar, mucus, ketone body etc.) are present.

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Definition of pH and measurement

Prepare acetate buffer of pH 4.6 to 6.0 and measure their pH using pH meter

Requirements: Wash bottle, volumetric flask, beaker, Sodium acetate, glacial acetic acid.

Definition of pH

The hydrogen ion concentration of most solution is extremely low. pH is strictly defined as the negative logarithm of the hydrogen ion activity, but in practice the hydrogen ions concentration is usually taken and this is virtually the same as the activity except in strongly acid solution.

Measurement of pH

pH indicator:  An approximate idea of the pH of a solution can be obtained using indicators. These are organic compounds of natural or synthetic origin whose colour is dependent upon the pH of the solution. Indicators are usually weak acid which dissociate in solution. 

Accurate measurement of pH: The most convenient and reliable method for measuring pH is by the use of a pH meter which measure the e.m.f of a concentration cell formed form a refrence electrode, the test solution and a glass electrode sensitive to hydrogen ions.

Glass electrode: The glass electrode consists of a very thin bulb about 0.1mm thick blown on to a hard glass tube of high resistance. Inside the bulb is a solution of hydrochloric acid (0.1 mL/litre) connected to a platinum wire via a silver chloride electrode, which is reversible to hydrogen ion. A buffer solution is one that resists pH. Change on the addition of acid or alkali such solution are used in many biochemical experiment where the pH needs to be accurately controlled.

From the Henderson-Hasselbalch equation the pH of a buffer solution depends on two factors. One is the pka value and other the ratio salt and acid.

Procedure for Measurement of pH

Preparation of acetate buffer pH 4.6: 5.4 gram of sodium acetate was dissolved in sufficient amount of distilled water then 2.4 mL of glacial acetic acid was added and volume was made up to hundred mL and pH was noted down using pH meter.

Preparation of acetate buffer pH 4.7: 0.84 gram of sodium acetate was dissolved in sufficient amount of distilled water then 0.33 mL of glacial acetic acid was added and volume was made upto 100mL and pH was noted down using pH meter.

Preparation of acetate buffer pH 5.0: 1.36 gram of sodium acetate was dissolved in sufficient amount of distilled water then 0.6 mL of glacial acetic acid was added and volume wad made up to 100 mL and pH was noted down using pH meter.

Preparation of acetate buffer pH 5.5: 27.2 gram of sodium acetate was dissolved in sufficient amount of distilled water then 50 mL of glacial acetic acid was added and volume was made upto 100mL and pH was noted down using pH meter.

Result: Acetate buffer solutions are prepared and submitted.

Prepare phosphate buffer of pH 5 and pH 7and measure their pH using pH meter

Requirement: Wash bottle, volumetric flask, and beaker, potassium dihydrogen phosphate, potassium hydroxide, Disodium hydrogen phosphate.

Phosphate

This is probably most popular buffer but phosphate readily forms complexes with heavy metal. Phosphate is also inconvenient as it plays an active part in a no. of biochemical reaction where it can act as an activator, inhibitor or metabolite. The buffering capacity above pH 7.5 is poor.

Procedure for Measurement of pH for Phosphate

Preparation of phosphate buffer (pH 5): 0.68 gram of potassium dihydrogen phosphate was dissolved in sufficient amount of distilled water and volume was made up to 100 mL then adjusts the pH to 5 with 10 M potassium hydroxide.

Preparation of phosphate buffer (pH 7): 0.050 gram of anhydrous disodium hydrogen phosphate and 0.030 gram of potassium dihydrogen phosphate was dissolved in sufficient amount of distilled water and volume was made up to 100 mL.

Result: Phosphate buffer solutions are prepared and submitted

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Examination of abnormal urine

Requirements:

 
Glassware: Test tube, test tube stand, beaker, burner, funnel, filter paper.

Chemicals: Acetic acid, Ammonium sulphate, Sodium nitro prosside, concentrate ammonia, Benedic reagent

Theory:

Protein

The term albumin is applied to the heat coagulable proteins in the urine. Such proteins (albumin and globulin both) are found in urine under pathological condition known as albuminuria. Normal urine contains 3-10 mg% of albumin protein is not normally found in the urine and proteinuria may indicate disease although occasionally healthy persons may excrete small amount of protein at intervals.

Mucus 

Although a small amount of mucus may be found in normal urine, large amounts may indicate an inflammatory disorder.

Reducing substances (sugar)

The condition characterised by the excretion of readily detectable amounts of glucose in urine is known as glucosuria. Pathological glucosurias occur in diabetes mellitus and other disorders, in which there is a marked elevation of the blood sugar and usually an increased volume of urine (polyuria). The glucose content of the urine in diabetes mellitus may reach 10 percent or more but is usually around 3 to 5 percent. The urine may be light in colour and has a high specific gravity.

Blood

Hematuria and hemoglobinuria. The conditions in which blood occurs in the urine may be classified as hematuria and hemoglobinuria. In hematuria there are present not only hemoglobin but unruptured corpuscles as well, whereas in hemoglobinuria the pigment alone is present.

Test for Protein

Boiling test

 
Take five mL urine in the test tube and heat the content its turns cloudy then add few drop of acetic acid. Disappearance of cloudiness indicates the absence of protein.

Nitric acid test

 
Take two mL urine take in the test tube followed by addition of concentrate nitric acid with the wall of test tube, no ring form at the junction of two indicates the absence of protein.

Test for Mucus

Take two mL urine in the test tube and dilute with equal volume of water, then add few drop of acetic acid if no precipitates forms confirm the absence of mucus.

Test for sugar

Benedict test

Take three mL benedict’s reagent in test tube and add 8 drop sample solution. Heat the content for 3 minutes. No Formation of yellowish green precipitate indicates the absence of reducing sugar.

Test for Ketone body

Take three mL of Ammonium sulphate in the test tube then add three mL of urine followed by addition of few crystal of nitro prusside. Shake well the content for dissolution of crystal. Then add two drop concentrate ammonia, mix well and allowed to stand. No formation of purple color confirms the absence of ketone body.

Result: In the given sample of urine abnormal constituents………………are present.

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Examination of normal urine

Requirements:

Glassware: Test tube, test tube stand, beaker, burner, funnel, filter paper.

Chemicals: Ammonium hydroxide, dilute acetic acid, Concentrate Nitric acid, Ammonium molybedate, Potassium oxalate, Silver Nitrate solution, Hydrochloric acid, Barium chloride, Picric acid, Sodium hydroxide.

Theory: Urine is the major route for water loss from the body. In a healthy individual the urine output is about 1-2 l/day. Water loss through kidney although highly variable is well regulated to meet the body demands to get rid of water or to retain. It should however, be remembered that man cannot completely shut down urine production, despite there being no water intake.

Composition of urine

Water: It is the largest component of urine. It makes about 95% of total urine.

Inorganic components: This contains two types of ions i.e. cation and anion. Major cations are sodium (Na+), potassium (K+), calcium (Ca+2), magnesium (Mg+2), ammonium (NH4+), iron (Fe+2), copper (Cu+2), zinc. Chief anions are chloride (Cl-), phosphate (PO4-3), sulphate (SO4-3), bicarbonate (HNO3-), Nitrate (NO3-) etc.

Organic compounds: The principle nitrogenous constituents are urea acid, creatinine, hippuric acid, purine base and amino acid.

Procedure Physical examination

(a) Appearance: Normal urine is clear and transparent when freshly collected.
(b) Color: Normal urine is pale yellow colour. This colour is due to pigment urochrome.
(c) Odor: Freshly collected urine has characteristic aromatic odor due to the presence of some volatile organic acid.

Chemical examination

Their are different test for urine examination. They are given below-

Test for Ammonia

Take three mL fresh urine in the test tube and heat then put red litmus paper over the mouth of test tube. Litmus paper turn blue confirm the presence of ammonia.

Test for Calcium and Phosphate

Take 5 mL urine in test tube and add little amount of ammonium hydroxide  and boil and white flanky precipitate form, filter the content and dissolve the residue in 3 mL of diulte acetic acid them collect the filterate and divide in two part. (

a) In first part add few drop of concentrate nitric acid followed by ammonium molybedate then boil. Formation of lemon yellow color precipitate indicates the presence of phosphate. 

(b) In second part add one mL potassium oxalate solution. Formation of white precipitate indicates the presence of calcium.

Test for chloride

Take 2 mL urine in the test tube and add few drop of concentrate nitric acid followed by two mL of silver nitrate solution. Formation of white precipitate confirms the presence of chloride.

Test for sulphate

Take 3 mL urine in test tube and add few drop of concentrate hydrochloric acid then one mL of barium chloride solution. Formation of white precipitate indicates the presence of sulphate.

Test for creatinine or Jaff’s test

Take 3 mL of picric acid solution in the test tube and add 0.5 mL of sodium hydroxide solution and divide into two parts. 

(a) In first test tube add 2 mL urine. Deep reddish orange colour confirms the presence of creatinine. 

(b) In second part add 2 mL water; no colour change indicates the absence of creatinine.

Result: In the urine Ammonia, Chloride, Phosphate, Calcium, Sulphate, and creatinin urea present. They are confirmed by ammonia test, calcium, phosphate test, chloride test, sulphate test, and creatinin test respectively.

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Chemical tests for lipids

Requirements:

Glassware: Test tube, test tube stand, beaker, wash bottle, burner,    filter paper, Chemicals: chloroform, benzene, concentrate sulphuric acid, formaldehyde sulphuric acid solution, acetic anhydride, sodium carbonate, sodium chloride, calcium chloride.

Theory for lipids

Lipids are a large group of naturally occurring compound, characterized only by their water repellant property hence called hydrophobic. Their solubility in organic solvent is variable. Some are soluble in polar organic solvent like alcohol and other in non-polar solvents. Like hexane, benzene etc although many proteins and polysaccharides are also not soluble in water, they do not repel water. They are easily wetted by water. by water may absorb and swell with water hence called hydrophilic unlike the protein or carbohydrate the lipid have no specific functional group or linkage characterizing all of them although a majority belong to the group of oils and fats. Lipids like carbohydrates provide energy for work and life processes. They also form part of some structure in the cell membrane, wall of tissue and organ. The lipids also provide a non-aqueous medium in the body which helps in regulation of transport of many chemicals, due to their partition between lipid and aqueous layers. Thus lipids play an important role in living organism. Lipids also occur in conjugation with protein and are termed lipoproteins. They may be classified into (1) simple lipid (2) phospholipids (3) complex lipid.
 

Procedure for Chemical tests for lipid

Solubility test

Take two mL sample solution in different three test tubes, followed by addition two mL chloroform, water, and benzene is added in respective test tube. Mix and allow to stand. Oil or lipid soluble in organic solvent and insoluble in inorganic solvent.

Spot test

Take three mL chloroform in test tube and add five drop of sample. Mix and take three drops on filter paper and allow drying till chloroform evaporated.

Salkowaski test (Sulphuric acid test)

Take few drop of sample solution in the test tube and dissolve in chloroform followed by addition of equal amount concentrated sulphuric acid. In chloroform bluish red to purple color observe and in acid green fluorosence indicates the presence of cholesterol.

Formaldehyde sulphuric acid test

Take two mL chloroform solution in test tube followed by addition of two mL formaldehyde sulphuric acid solution. Pour the chloroform layer into other test tube and add two-three drop acetic anhydride. Appearance of blue colour confirms the presence of cholesterol.

Acetic anhydride Sulphuric acid test or (Liebermann–Burchard test)

Take small amount of sample solution in test tube and dissolve in chloroform followed by addition of  ten drops of acetic anhydride and two- three drop of concentrate sulphuric acid. Solution become red finally bluish green indicate the presence of cholesterol.

Saponification test

Take four mL of 2 % sodium carbonate solution in test tube and add four drops sample solution. Shake well and boil, and cool and divided into three parts. 

(a) In first part add few drop of concentrate hydrochloric acid. Fat separate and float, confirms the presence of fat. 

(b) In second part add sufficient amount of sodium chloride. White precipitate appears and floats on surface that indicates the presence of fat. 

(c) In third part add few drop of calcium chloride solution. In soluble calcium precipitate form, confirms the presence of fat.

Result: In the given sample lipid is present. It is confirm by the different tests such as solubility test, spot test, salkowaski test, formaldehyde sulphuric acid test, acetic anhydride. sulphuric acid test, saponification test.

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Tests for Carbohydrate

Requirements:
 

Glasswares: Test tube, test tube stand, beaker, wash bottle, burner, glass rod, funnel, filter paper, and spatula.

Chemicals: Molisch reagent, concentrate sulphuric acid, hydrochloric acid,  iodine solution, Benedict reagent, fehling solution, barfoed’s reagent, selivanoff reagent, glacial acetic acid, phenylhydrazine hydrochloride, sodium acetate.

Theory for chemical tests

Carbohydrates are defined chemically as aldehyde or ketone derivatives of polyhydroxy alcohols or compounds which yield these derivatives on hydrolysis.  “Carbohydrate" means hydrated carbon, which indicates the relative proportion of carbon, hydrogen and oxygen atoms in a carbohydrate. For example glucose has a molecular formula C6H12O6 .It also indicates that these are the only three elements in this group of natural compound. However, like the complex protein, some carbohydrate also may have amino group. Carbohydrates also have two major functions in living organisms: (1) they provide energy needed for life processes. (2) Some are parts of structural unit of cell and tissue especially in plant. Simple carbohydrates are glucose, fructose, (both occur in fruits), lactose (occur in milk) and sucrose (cane sugar). Starch (rice, potato, maize) is a high molecular weight carbohydrate. Carbohydrates are classified on the basis of units of sugar present in the molecule as follows (1) monosaccharide (2) disaccharides (3) oligosaccharides (4) polysaccharides.

Procedure for chemical tests for carbohydrate

Different chemical tests for carbohydrate are given below-

Molisch test

Take two mL sample solution in test tube and add two drops of molisch reagent. Mix well and inclined the test tube and add one mL concentrate sulphuric acid by the side of the test tube. Appearance of reddish violet colour confirms the presence of caebohydrate.

Iodine test

Take two mL sample solution in test tube followed by addition of two drops of dilute hydrochloric acid and few drops of iodine solution. Appearance of blue color indicate the presence of starch.

Benedict’s test

Take three mL benedict’s reagent in test tube then add eight drops sample solution. Mix the content and heat for three minutes. Appearance yellowish green precipitate confirms the presence of reducing sugar.

Fehling test

Take two mL fehling solution in test tube, warm the solution and add one mL sample solution. Mix well and heat the solution. Brownish red colour confirms the presence of reducing sugar.

Barfoed’s test

Take two mL barfoed reagent in test tube followed by addition of one mL sample solution, and heat the solution up to boiling. Appearance of reedish orange precipitate indicates presence of monosaccharides.

Selivanoff’s test

Take two mL sample solution in test tube and add three mL selivanoff reagent. Mixed well and gently heat the solution. No red color confirms the presence of aldose sugar.

Osazone test

Take three mL sample solution in test tube and add ten drops glacial acetic acid followed by addition of few amount of phenyl hydrazine hydrochloride and double amount of sodium acetate. Mix and warm the solution and filter the solution and filtrates is keep in boiling water bath. Needle shape crystal confirms the presence of glucose.

Result: In the given sample carbohydrate (glucose) is present; it is confirmed by the different test such as molisch test, iodine test, benedict’s test, fehling test, barfoed test, selivanoff test, osazone test.

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Chemical test for protein

Requirements:

 
Glassware: Test tube, test tube stand, beaker, test tube holder, glass rod, funnel, pipette, wash bottle, wire gauze, burner.

Chemical: sodium hydroxide, copper sulphate, ninhydrine solution, nitric acid, ammonium hydroxide, mercuric sulphate, lead acetate, glacial acetic acid, formaline solution, ammonium molybodate, sulphuric acid, sodium nitrate.

Theory  for Chemical test

Amino acid- Amino acid are the monomer unit or building block of the biopolymer protein amino acid contains both amino and carboxylic acid functional group attach acid are present in nature , in which only 20 are present in protein form all from of life plant, animal or microbial. Essential amino acid- those amino acid which are not synthesized in our body are called essential amino acid like histadine, isoleucine. 

Non essential amino acid- those amino acid which are synthesized in our body are called non essential amino acid like aspargin, aspartic acid, glutamic acid. Proteins are high molecular weight compound made up of number of amino acid. protein are also be defined as high molecular weight polymer of   amino acid joined together with peptide bond .protein is the polymerized form of peptide. Proteins are classified in three groups namely simple, conjugatedand derived protein. 

Simple proteins are also classified into sub group- albumin, glutelins, albuminoids, histones, prolamines, globulin, globin protamine. Conjugated proteins are classified as: nucleoprotein, metalloprotein, lipoprotein, phosphoprotein, glycoprotein, chromoprotein. Structures of protein are classified as: primary structure secondary, tertiary, quaternary structure.

Procedure  for Chemical test for protein

Different different chemical test for protein is given below-

Biuret Test

Take two ml sample solution in test tube. Add two ml sodium hydroxide solution. Mix well then add drop wise copper sulphate solution and mix. Purplish violet color appears that indicates presence of protein.

Ninhydrin test

Take two mL sample solution in test tube then add 0.5 mL ninhydrin solution.  Heat the mixture up to boiling and cool. If blue colour appears that confirm protein is present.

Haller’s test

Take two mL concentrate nitric acid in test tube then inclined the test tube and add two mL sample solution through wall of test tube. White precipitate appeared at junction of two fluids indicates presence of protein.

Xenthoprotic test

Take two mL sample solution in test tube followed by addition of 1 mL concentrate nitric acid. A white precipitate appears then heat the mixture until solution become yellow.  Cool it. Add ammonium hydroxide solution in excess. Appearance of orange colour indicates the presence of aromatic amino acid.

Millan-Nasse’s test

Take three mL of sample solution in test tube then add mercuric sulphate.  Mixed well then boil for two minutes then cool and add sodium nitrite sulphate. Appearance deep red colore confirms the tyrocin amino acid.

Lead sulphide test or Lead blackening test

Take two mL sample solution in a test tube and add two mL sodium hydroxide solution, mix well and boil for one minute then add five drop lead acetate solution to the above mixture. Brown colored precipitate confirms the presence of cysteine.

Lead acetate test

Add two mL of sample solution a test tube containing two mL sodium hydroxide solution. Then heat up to boil and cool it.  Add little glacial acetic acid followed by one mL lead acetate solution. Precipitations of blackish precipitate indicate the presence of sulpher containing amino acid. 

Aldehyde test

Take three mL sample solution in a test tube followed by addition of two mL formaline solution. Then add two drop mercuric sulphate and mix properly. Inclined the test tube and add two mL concentrate sulphuric acid with the side of wall of test tube. Appearances of reddish violet color indicate the presence of tryptophan amino acid.

Neumann’s test

Take three mL of sample solution in test tube; add five drops sulphuric acid followed by three drops concentrate nitric acid. Mix well and heat the mixture until solution becomes colorless then add one mL concentrate nitric acid and one mL ammonium molybodate and heat the content. Cannary yellow colours appear, that confirms presence of casein protein.

Result: In the given sample solution protein (aromatic amino acid, tyrocin, cestine, tryptophan, and casein) is present. It is confirmed by the different test such as Biuret test,Ninhydrin test, Haller’s test, Xenthoprotic test, Millon-Nasse’s test, Lead sulphide test, Lead acetate test, Aldehyde test, Neumann’s test.

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To prepare various chemicals used in biochemistry laboratory

Requirements: Beaker, Funnel, concentrate hydrochloric acid, distilled water, measuring cylinder, copper sulphate, and acetic acid etc.

Theory: According to Arrhenius theory, on dissolution in water acid forms hydrogen ion (H+) and bases form hydroxyl ion (OH-). According to Bronsted Lowry theory acid are the substances which can give proton and bases are substances which can accept proton. According to Lewis theory acid are those molecule which can accept lone pair of electron and base are those molecule which can donate lone pair of electron.

Strong acid: An acid which yields protons easily in high concentration. Ex- HCL, HNO3. A base which yields hydroxyl ion in high concentration called strong base. Ex- NaOH, KOH.

Weak acid- If the degree of ionization of acid is less they are called weak acid. Ex- CH3COOH. 

Weak base- if the degree of ionization of base is less they are called weak base. Ex- Ca(OH)2.

Hydrochloric acid: Category-Pharmaceutical Aid (acidifying agent), Description-clear, colourless, fuming liquid, odor, pungent. Solubility: Miscible with water.

Sodium hydroxide (NAOH): Category- Pharmaceutical aid (alkalizing agent). Description- White crystalline masses supplied as sticks, pellets or slabs, deliquescent, readily absorbs carbon dioxide. Solubility: very soluble in water, freely soluble in ethanol (95%).

Procedure For To prepare various chemicals used in biochemistry laboratory

Preparation of dilute hydrochloric acid (5N, HCl): Forty two mL of concentrated hydrochloric acid was transferred in beaker previously containing ten mL distilled water and volume was made up to hundred mL by distilled water.

Preparation of dilute sulphuric acid (5N, H2SO4): Fourteen mL of concentrated sulphuric acid was transferred in beaker previously containing ten mL distilled water and volume was made up to hundred mL using distilled water.

Preparation of dilute nitric acid (5N, HNO3)

Thirty one mL of concentrated nitric acid was transferred in beaker previously containing ten mL distilled water and volume was made up to hundred mL using distilled water.

Preparation of 5N sodium hydroxide (NaOH) solution

Twenty grams of sodium hydroxide was weighed out using electrical balance. It was transferred in beaker and dissolved in sixty mL distilled water. After complete dissolution, volume was made up to hundred mL using distilled water. It provided hundred mL sodium hydroxide solutions (5 N).

Preparation of 1% acetic acid (CH3COOH) solution

One mL of glacial acetic acid was pipette out and transferred into beaker previously containing ten mL distilled water. Its volume was made up to hundred mL using distilled water. It provided hundred mL acetic acid solutions (1%).

Preparation of 1% sodium nitrite (NaNO2) solution

One gram of sodium nitrite was weighed out using electrical balance and transferred in beaker. It was dissolved in 50 mL of distilled water. After complete dissolution, volume was made up to hundred mL by distilled water.

Preparation of 5N ammonium hydroxide (NH4OH) solutions

Thirty four mL liquor ammonia was measured out and transferred into beaker previously containing ten mL distilled water. Volume was made up to hundred mL using distilled water.

Preparation of 10% copper sulfate (CuSO4) solution

Ten g copper sulphate was weighed out using electrical balance. It was transferred into beaker and dissolved in eighty mL distilled water. Then 0.3 mL sulphuric acid solution was added. Then volume was made up to hundred mL using distilled water.

Result- We prepared several chemicals such as 5N hydrochloric acid, 5N sulphuric acid, 5N Nitric acid, 5N sodium hydroxide, 1% acetic acid, 1% sodium nitrite, 5N ammonium hydroxide and 10% copper sulfate.   

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Qualitative test for proteins and amino acids by using Biuret test methods

Biuret Test

Chemical requirement: Protein solution, sodium hydroxide, copper sulphate, Millon’s reagent, ninhydrin.

Apparatus 

Burette stand, heating mental, Test tube, Pipette, beaker, burette.

Principle 

Proteins are compounds consisting of amino acids. Proteins exhibit colloidal properties and there for do not diffuse through an intact animal membrane. Hydrolysis, with acids or alkalis, breaks the protein into constituent amino acids.

Procedure Biuret test

To 2-3 ml of protein solution in a test tube add an equal volume of 10% sodium hydroxide solution. Mix thoroughly and add 0.5% copper sulphate solution drop by drop until a purplish- violet color is produced.

(Blue precipitate of copper hydroxide is formed if excess copper sulphate is added).

Biuret reaction is due to the peptide linkage O+C-N-H of due to two carbamyl groups (CONH2). Hence, it is positive with all proteins.

CONH2
|
NH
|
CONH2

|
Biuret

Ninhydrin reaction 

To 5 ml of dilute protein solution (ph between 5 and 7) add 0.5 ml of a 0.1% solution of ninhydrin. Heat to boiling for one or two minutes and allow cooling. A blue colour appears.This test is given by all amino acids except proline and hydroxyproline. Proline and hydroxyproline react with ninhydrin and give yellow colour.

Millon’s reaction

To 5 ml of protein solution add 3-4 drops of Millon’s reagent and mix. Heat to boiling point over a small flame. Proteins like egg albumin. Gives white precipitate which gradually turns red on heating. Proteoses and peptones give red colour. If no colour develops, add 2-3 more drops of Millon’s reagent and heat again. However, large quantity of Millon’s reagent gives yellow colourwhich is not a positive reaction. This reaction is due to the presence of the hydroxyphenyl group in protein molecule e.g. Tyrosine.

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