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This book is freely available for research and educational purposes. XML to PDF by RenderX XEP XSL-FO F ormatter, visit us at lesforgesdessalles.info 𝗣𝗗𝗙 | principle of practical biochemistry and experimental part. BIOCHEMISTRY. Book · January with 25, Reads. Publisher: 1st ed. Try this link Practical Textbook of Biochemistry for Medical Students You may also check out this Textbook of Biochemistry for Medical Students.
Hence, nitric acid is added to prevent such interference. Experiment 2. Elsevier, Notify the teacher immediately, if you observe any unsafe conditions. Observations Time Colour 30 sec. Follow all written and verbal instructions carefully. The needle is removed from the syringe and the blood transferred to an appropriate container, using minimum amount of pressure.
Coagulation factors. Carrier proteins e. Contractile element in the muscle actin, myosin 8. Proteins act as intracellular buffer in maintaining the acid-base balance. Precipitation reactions of proteins 2. Color reactions of proteins. Precipitation Reactions of Proteins Proteins are large molecules with variable sizes, shapes and charges. Solubility of a protein depends on the proportion and distribution of polar hydrophilic groups and non-polar hydrophobic groups in the molecule.
Proteins form colloidal systems in aqueous medium. The stability of protein in solution will depend mainly on the charge and the degree of hydration shell of water molecules around the particles.
Types of Colloids 1. Suspensoids 2. Emulsoids Suspensoids: Suspensoids are stabilized by the electrical charges over the surface of the molecule. Emulsoids are stabilized by: Electric charge over the surface of the molecule 2. Hydration shell around the molecule Proteins can be precipitated by: Removing their shell of hydration 2.
Neutralization of electrical charges 3. Denaturation disorganization of native protein, loss of biological activity 4. Bringing them to isoelectric pH Any factor which neutralizes the charge or removes the shell of hydration will cause precipitation of proteins.
Importance of Precipitation of Proteins 1. Precipitation is used to separate proteins from biological fluids like blood, plasma, CSF, etc. Qualitative Analysis of Proteins 51 2.
Precipitation can also be used under well defined conditions to separate a particular protein from a mixture of proteins, e. Amino Acid Amino acids are organic substances, having two functional groups—amino group and carboxyl group. Amino group is basic while carboxyl group is acidic in nature. The proteins to be studied in the lab are: Albumin 2. Casein Precipitation by Salts Principle: Addition of neutral salts like ammonium sulfate leads to adsorption of hydration shell along with neutralization of surface charges leading to protein precipitation.
Half-saturation with ammonium sulfate: White precipitate a. Protein in the solution in a test is formed. Add equal b. No white precipitated. Protein in the ammonium sulfate is formed.
Filter it. Perform a. No violet Biuret test with color is formed. Filterate which contains protein gives violet color with Biuret test. Full saturation with ammonium sulfate: Experiment Observation Inference Take 3 ml of protein a. Add solid b. No white is precipitated. Protein in the in small quantities is formed. Qualitative Analysis of Proteins 53 Contd Experiment Observation Inference Filter it.
No Violet color Biuret test with the is formed. Therefore, the presence of very small quantities of salts will increase the solubility of a protein by diminishing protein interaction. Higher the molecular weight of a protein the salt required for precipitation is lesser. They hold more water molecules around them. Hence, a higher concentration of salt is required for precipitation of albumin.
Thus, albumin is precipitated by full saturation. Isoelectric Precipitation Principle: The solubility of proteins is minimum at their isoelectric pH as the protein molecules become electrically neutral at this pH. Perform the test with only casein. Add 3 drops will be formed. The net charge is zero. No mobility in an electric field. Qualitative Analysis of Proteins 55 3. Least soluble. Buffering capacity and viscosity will be minimum. Precipitation will be maximum.
Casein is precipitated from milk and the supernatant is called whey. At this pH the color of the solution is dark green.
If the color is not brought to light green, i. So while performing the isoelectric precipitation test for casein, bring the pH of the solution to 4. Precipitation by Organic Solvents Principle: Proteins in solution form hydrogen bonds with water. Organic solvents like acetone, ether or ethanol when added to a protein solution in water, reduce the concentration of water molecules available for keeping the proteins in solution and thus decrease the number of hydrogen bonds.
Experiment Observation Inference Take 1 ml of protein A white Protein is solution in a test precipitate precipitated by tube. Add 2 ml of is formed. This may be achieved by dissolving the protein in saline. Precipitation by Alkaloidal Reagents Principle: The negatively charged ions of the alkaloids neutralize the positive charge on the protein causing denaturation which results in precipitation. Experiment Observation Inference a. Take 2 ml of A thick yellow Protein is protein solution precipitate precipitated by in a test tube.
Add picric acid drop by drop. Qualitative Analysis of Proteins 57 Contd Experiment Observation Inference b. Take 2 ml of A white Protein is protein solution precipitate precipitated by in a test tube. Add trichloro- acetic acid drop by drop. Take 2 ml of A white Protein is protein solution precipitate precipitated in a test tube.
These acids lower the pH of the medium, when proteins carry net positive charges. These protein cations are electrostatically complexed with negatively charged ions to form protein- tungstate, protein-picrate, etc. It is also used to identify proteins in CSF.
This is avoided by an initial protein precipitation by alkaloidal reagents. Precipitation by Heavy Metal Ions Principle: Proteins exist as negatively charged ions anions in pH higher than their isoelectric pH generally in an alkaline medium. To such a solution if salt of heavy metals are added, positively charged metal ions can complex with protein anion and metal proteinates are formed which get precipitated.
Take 2 ml of White precipitate Protein is protein solution is seen. Qualitative Analysis of Proteins 59 Points to Remember: On heating the protein loses its structure and becomes denatured to form a coagulum.
It is precipitated after the addition of acetic acid, which provides the suitable pH to get the maximum precipitate. Hold the portion, which gets coagulable test tube over a flame intensified after the protein in a slanting position addition of acetic acid. The lower portion serves as control. This process is known as denaturation.
Some proteins when heated, though denatured, are still soluble. They may be precipitated by bringing to isoelectric pH. Denatured proteins are less soluble than the native proteins. Acetic acid is added: If it disappears it indicates the presence of phosphates. All together there are 20 types of amino acids found in proteins. Due to the presence of the polypeptide bonds and different amino acids residues in their molecules, they react with a variety of reagents to form colored products. These are known as color reactions of proteins.
These reactions are of importance in qualitative detection and quantitative estimation of proteins and their constituent amino acids. So it is known as a first class protein, a protein of high biological value.
Functions of Albumin 1. Maintenance of colloidal osmotic pressure in both vascular and extravascular space. Albumin acts as a transport protein for free fatty acids, bilirubin, calcium and most of the drugs. Physical Properties of Albumin 1. Pale white 2. Cloudy 3. Physical Properties of Casein 1. Characteristic odor 4. Chemical Properties Biuret Test Principle: Cupric ions in alkaline medium form a violet colored complex with peptide bond nitrogen. Copper sulfate is converted to cupric hydroxide which chelates with peptide linkage in proteins to give the purple color.
Biuret Reagent: Contains sodium potassium tartarate and copper sulfate. Qualitative Analysis of Proteins 63 Experiment Observation Inference Take 2 ml of protein Violet color Indicates the solution in a test is formed presence of tube.
Add 2 ml of peptide linkage. Hence non- proteins, e. Therefore, the test should not be carried out with solutions containing these salts. Ninhydrin Test Principle: The reduced Ninhydrin hydrindantin then reacts with ammonia and another molecule of Ninhydrin and produces bluish purple colored complex.
Experiment Observation Inference Take 1ml of protein Bluish purple Indicates the solution in a test color is formed. Heat to boiling. Qualitative Analysis of Proteins 65 Points to Remember: Hence, they give a yellow color with Ninhydrin. Hence, it is positive for proteins, peptones, peptides. Xanthoproteic Test Principle: The benzene ring system in tyrosine and tryptophan undergo nitration on treatment with strong nitric acid at elevated temperature forming a yellow precipitate.
The yellow precipitate turns orange due to ionization, in alkaline medium. Experiment Observation Inference Take 3 ml of protein In acid medium Indicates the solution in a test tube. Nitric is formed. Heat the acids. Tyrosine solution for one and tryptophan. Observe the color. Divide the contents into two parts. Mix well and observe. Sodium nitrite reacts with Sulfuric acid to form nitrous acid reacting acid. The protein gets precipitated by the mercuric sulfate.
The reacting groups phenol group of tyrosine which get exposed on boiling, reacts with nitrous acid to form mercury phenolate. This gives red color precipitate.
Boil gently for 30 seconds. Cool it under tap water. Mix and observe. Mercuric sulfate causes mild oxidation of indole group of tryptophan, which condenses with an aldehyde to give the colored complex.
Add carefully 3 ml of conc. Sulfuric acid along the sides of the test tube. Sakaguchis Test for Guanidine Group Principle: Experiment Observation Inference Take 3 ml of protein Bright red color Indicates the solution in a test is formed. Mix and add 10 drops of bromine water. Qualitative Analysis of Proteins 69 Points to Remember: Sulfur Test for Cystine and Cysteine Principle: On boiling with sodium hydroxide the sulfur present in the protein is converted to inorganic sodium sulfide.
This reacts with lead acetate to form a black precipitate of lead sulfide. Boil residues. Then add 5 drops of lead acetate. Diazobenzene sulfonic acid reacts with imidazole ring of histidine to form a cherry red colored diazotized product under alkaline condition. With the hydroxyphenyl group of tyrosine an orange-red colored product is obtained. Experiment Observation Inference Take 1 ml of 0. After standing for 1 minute, add 2 ml of protein solution.
Qualitative Analysis of Proteins 71 Points to Remember: Carbohydrates, when treated with Conc. Molisch reagent: Experiment Observation Inference Take 2 ml of protein Violet ring at the Indicates the solution in a clean junction of the presence of bound dry test tube; add two liquids carbohydrate. Molisch reagent.
Incline the test tube slightly and add 2 ml of conc. Sulfuric acid along the sides of the test tube so as to form two layers. Point to Remember: On boiling with strong sodium hydroxide, the organic phosphate present in phosphoproteins is released as inorganic phosphate.
Inorganic phosphate reacts with ammonium molybdate in the presence of nitric acid acidic media to form a canary yellow precipitate of ammonium phosphomolybdate. Experiment Observation Inference Take 5 ml protein Canary yellow Indicates the solution in a test precipitate presence of tube. Add 0. Heat strongly and cool under tap water.
Nitric acid. To the filtrate add a pinch of solid ammonium molybdate and warm gently. Most important NPN substances present in urine are uric acid, urea, creatinine and ammonia. Tests for Urea Urea tests are as follows: Colorless 2. Clear 3. Chemical Tests Chemical tests are as follows: Biuret Formation Principle: When heated above its melting point, two molecules of Urea condense to form biuret and ammonia.
Nonprotein Nitrogenous Substances 75 Biuret reacts with alkaline copper sulfate to form a violet color. Urea melts with the liberation of ammonia. On further heating it solidifies. Cool the test tube. This is sometimes mistaken for a positive biuret test.
Sodium Hypobromite Test Principle: When urea is treated with Sodium hypobromite, it decomposes to give nitrogen, carbon dioxide and water. Liberation of nitrogen gas produces brisk effervescence.
Add 5 drops urea. Specific Urease Test Principle: Under optimum pH and temperature, the enzyme urease decomposes urea into ammonia and carbon dioxide which together form ammonium carbonate alkaline substance which changes the solution to pink color in the presence of the indicator. Indicator phenolphthalein changes to pink color in alkaline medium. Experiment Observation Inference Take 3 ml of urea Pink color is formed Urea is solution in a test confirmed.
Add 3 ml of urease suspension and add drops of phenolphthalein indicator. Warm the tube for a few seconds with the hands and keep for 10 minutes. Otherwise the enzyme will be destroyed. Chemical Tests Chemical tests are as follows. Uric acid in alkaline condition reduces phosphotungstic acid to tungsten blue. Composed of sodium tungstate, orthophosphoric acid, concentrated sulfuric acid and solid sodium carbonate.
Experiment Observation Inference Take 3 ml uric Deep blue Indicates the acid solution in a color is formed.
Uric acid reduces salts of silver nitrate to metallic silver. Experiment Observation Inference Moisten a piece of Black spots Indicates the filter paper with on the filter presence of few drops of paper are seen.
Uric acid. Add drops of uric acid on the silver nitrate drops. Murexide Test Principle: When uric acid is treated with conc. The imidazole ring is cleaved and the derivatives condense to give reddish yellow purpuric acid.
Nonprotein Nitrogenous Substances 79 This combines with ammonia to form ammonium purpurate or murexide which is purple red in color. Experiment Observation Inference Take 5 ml of uric A purplish red Indicates the acid in a china dish color is formed.
Warm gently over a low flame. A reddish yellow residue is obtained. Allow the dish to cool. Add two drops of dilute ammonia solution. Creatinine reacts with picric acid in the presence of alkaline medium to form reddish orange colored creatinine picrate. Experiment Observation Inference Take 3 ml of Reddish orange Indicates the creatinine solution color is formed.
The given NPN substance is —————. The composition of urine is a mirror not only of renal function but also of many physiological and metabolic processes occurring in the body. Thus, examination of urine may lead to the diagnosis of many metabolic and systemic diseases. Physical examination 2. Chemical examination 3. Microscopic examination.
Fresh mid-stream specimen of 20 ml is collected in a clean dry container. For most of the qualitative tests, a random urine sample is satisfactory. Morning specimen is desirable for normal analysis. Repeated urine samples are necessary for orthostatic proteinuria. Several changes like urinary decomposition, precipita- tion of phosphates, crystallization of uric acid and bacterial action may alter the urinary composition if it is kept for long periods, especially in the collection of 24 hours urine samples.
Also urine may become alkaline, due to precipitation of uric acid and urates. This requires addition of preservatives to prevent the growth of bacteria and moulds such as 2N hydrochloric acid, conc. Before carrying out any estimation in urine, the urinary deposits must be well mixed. Volume to ml Normal volume. Color Amber yellow Given sample of urine is normal. Odor Aromatic smell Given sample of urine is normal.
Reaction to Blue litmus Normal urine is acidic. Determination of Specific Gravity Specific gravity of urine is measured by an apparatus known as Urinometer. Urinometer consists of a thin stem graduated from to corresponding to Specific Gravity of 1.
Take sufficient urine in a urine Jar. Allow the urinometer to float in it without touching the sides. Observe the reading at the meniscus. This gives the observed specific gravity at the temperature at which the urinometer is calibrated. Note the urine temperature room temperature. Qualitative Analysis of Normal Urine 87 Calculation: Since the room temperature is higher, a temperature correction has to be applied. This when divided by 3 gives 7. The solid content of ml of urine is calculated by multiplying last two digits of specific gravity by 2.
Specific gravity increases with increase in solid content. As the volume increases the specific gravity decreases. Chemical Tests Inorganic Constituents Tests of inorganic constituents are as follows.
Test for Chloride Principle: A white precipitate of silver chloride is formed when acidified urine reacts with silver nitrate. Hence, nitric acid is added to prevent such interference. Urine being acidified with hydrochloric acid forms a white precipitate of barium sulfate by the reaction with barium chloride solution. Test for Phosphates and Calcium Procedure: Take 10 ml of urine in a test tube.
Add 3 ml of ammonium hydroxide boil and cool. A flaky precipitate of calcium phosphate is formed. Filter and discard the filtrate. Collect the filtrate and divide into two parts. Test for phosphates Principle: Phosphates of calcium and magnesium are precipitated by ammonium hydroxide on boiling and these phosphates are dissolved in hot dilute acetic acid.
Experiment Observation Inference To one part of the Canary yellow Indicates the filtrate, add a few precipitate presence of drops of conc. These are crystallized out in alkaline urine. Calcium is precipitated as calcium oxalate with potassium oxalate in acidic condition.
Test for Ammonia Principle: Ammonia present in urine is liberated by heat. The evolution of alkaline ammonium vapors changes the color of red litmus to blue. Add changes to blue. Hold a piece of red litmus paper at the mouth of the test tube. Test for Urea Sodium Hypobromite Test: Experiment Observation Inference Take 3 ml of urine Brisk effervescence Indicates the in a test tube.
Add 5 drops of is seen. Specific Urease Test: The enzyme urease under optimum pH and temperature decomposes urea into ammonia and carbon dioxide which together form ammonium carbonate alkaline substance which changes the solution to pink color in the presence of the indicator. Experiment Observation Inference Take 3 ml of urine in a Pink color is formed.
Urea is confirmed. Add 3 ml of Urease suspension and add drops of phenolphthalein indi- cator. Uric acid in alkaline condition reduces phos- photungstic acid to tungsten blue. Composed of sodium tungstate, orthophosphoric acid, concentrated Sulfuric acid and solid sodium carbonate. Experiment Observation Inference Take 3 ml of urine Deep blue color Indicates the in a test tube; add is formed. Experiment Observation Inference Moisten a piece of Black spots are Indicates the filter paper with seen on the presence of few drops of filter paper.
Add drops of urine on the silver nitrate drops. Creatinine reacts with picric acid in alkaline medium to form reddish orange colored creatinine picrate. This test is done after removing the inorganic sulfate.
Hot hydrochloric acid hydrolyses ethereal sulfate to inorganic sulfate, which then gives precipitate with barium chloride. Mix and filter. Divide the filtrate into two portions. Boil one and compare with the control. Organic constituents present in the given sample of normal urine are urea, uric acid, creatinine and ethereal sulfates. Inorganic constituents present in the given sample of normal urine are chloride, calcium, phosphorus, inorganic sulfates, ammonia, sodium, potassium and magnesium.
Analysis of these abnormal constituents aids in the diagnosis of many diseases. Many of these pathological constituents are present in trace amounts in normal urine but they escape detection due to the low sensitivity of the methods employed. The concentrations of these constituents in urine are increased markedly in different pathological conditions. Usually the analysis is carried out in properly preserved 24 hours urine specimens.
When this is not possible the early morning specimens can be used. On standing, urine undergoes bacterial fermentation and degradation of some compounds. It can be preserved under refrigeration or using chemicals such as toluene or chloroform. The nature of the preservative will depend on the compound to be tested.
An increase in urinary output. Occurs in: A total suppression of urine formation. Analysis of Abnormal Constituents in Urine Contd Heat and Acetic Acid Test Principle: Hold seen at the upper presence of heat the tube over a flame heated portion, which coagulable in a slanting position gets intensified after protein like and boil the upper the addition of albumin.
The lower half serves as control. The plasma proteins of molecular weight of more than 70 kD, hence are absent in normal urine. Acetic acid is to be added to make it acidic. Nitric acid causes precipitation of proteins.
Experiment Observation Inference Take 3 ml of nitric A white ring is formed Indicates the acid in a test tube. Sulfosalicylic acid is an alkaloidal reagent and so it neutralizes the positively charged protein to produce precipitation. Add is formed. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives different shades of color precipitate depending upon the concentration of the sugar.
Cool and observe the contents. Blue Nil Green 0. It is a benign condition seen in anger, fear, etc. It is a benign condition which is seen after excessive intake of carbohydrate or patient is on glucose infusion. Acetone and acetoacetic acid form permanganate colored complex with sodium nitroprusside in presence of ammonia. Add solid colored ring presence of ammonium sulfate a is formed.
Mix well and add 1 ml of strong ammonium hydroxide drop-wise along the side of the test tube. Acetoacetic acid gives a red color with ferric chloride. If the urine is boiled, acetoacetic acid is converted into acetone; but the other substances remain unchanged. Now, if the urine gives negative test, it indicates the presence of acetoacetic acid. Ketone bodies are formed in excess when the glucose metabolism is impaired as in diabetes mellitus or when fat is used exclusively to give energy as in starvation starvation ketosis.
This condition is called as ketosis. It is answered even by small amounts of acetone and acetoacetic acid. It gives positive when converted to acetoacetic acid and then to acetone by oxidation. This occurs in ketosis where there will be ketonemia and ketonuria. In addition to metabolites, drugs excreted can be detected by this test. The editor is to be congratulate d on his efforts As well as practical details nearly all the procedures ar e accompanied by background information and references for further study.
The book will have a ready appeal to teachers who are looking for new ideas at all levels of expertise in practical biochemistry. Endeavour qu: Teachers of biochemistry will turn to this book again and again to find their inspiration This book can be recommended to all teachers as a valuabe collection of practical biochemistry experiments. We are always looking for ways to improve customer experience on Elsevier.
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When you read an eBook on VitalSource Bookshelf, enjoy such features as: In a modest attempt, additions have been made about the methods of expressing concentrations, instrumentations, collection and preparation of blood specimen, SGOT, SGPT, etc. The inquiring mind will certainly benefit from such exposures to manage the clinical situation in a more creative and challenging manner.
Some modifications became necessary in various chapters and thus the matters have been updated in a befitting manner to serve the demanding needs of the consumers. Lastly, I thank many people like Prof B Misra and Publishers for their guidance and assistance respectively.
This book in a concise but equally satisfactory form will help the user, students and practitioners either to a great extent. Besides being handy it has been kept up to its spirit of recent approaches by virtue of which one has the best utility in a busy time. For every medical practitioner and enlighted patients, Biochemistry has been playing a significant role.
This book will certainly be of significant importances to the practitioners as well as laboratories. I thank all my colleagues and friends in contributing to have brought out this book in its near perfect shape.
My extreme heartful thanks are due to the incessant guidance from Prof B Misra, Department of Physiology, MAM College in bringing out every chapter in an excellent way. The contributions that I have received by the constant cooperation of my parents and wife cannot be ignored. I hope the book will bring out greater number of readers keeping in view the worth of this book.
The author always keeps an open eye for suggestions. Methods of Expressing Concentration Physical Chemistry Achromic Point Isoelectric Point Saponification Number Iodine Number Formal Titration Gastric Analysis Urine Analysis Food Analysis Collection and Preparation of Blood Specimen Urinary Reducing Sugars Urinary Chlorides Urinary Creatinine Ascorbic Acid in Urine Serum Uric Acid Blood Sugar Blood Urea Urea Clearance Blood Cholesterol Serum Calcium Inorganic Phosphorus Serum Total Proteins and Albumin: Globulin Ratio Serum Bilirubin Prothrombin Time Liver Function Tests The most common expressions are: Percent 2.
Molarity 3. Normality 4. Molality 5. Percent According to Caraway there are three ways of expressing percentage of solution, i. Molarity A molar solution contains 1 gm mol. Molarity of a solution of 2 litres containing NaCl dissolved. Normality A normal solution contains 1 gm equivalent Wt. Normality of a solution of 2 litres containing gm of glucose. Molality Molality is defined as number of moles of solute dissolved in gm of solvent Not in solution.
Formality Formality parameter is not used nowadays because all of the above parameters are better than it and are able to express concentration sufficently. Formality is same as molarity if molecular weight in the formula is replaced by formula weight.
The most convenient way of expressing hydrogen ion concentration is by the term pH. Electrometric method is the most accurate one and is done by using a pH meter whereas colorimetric determination of pH can be simply done by the following methods: Indicator papers also called narrow range pH papers.
Universal indicators. Indicators are substances which change in colour with change in the pH of the solution to which they are added. Indicators are weak organic acids or bases. Their unionized forms show a colour while their ionized forms, i. The colour of the solution in presence of an indicator depends upon the relative proportions of ionized and unionized forms of the indicator which in turn depend upon the hydrogen ion concentration.
For each indicator there is a definite pH range in which it is present as a mixture of its ionized and unionized forms. In this specific range, variations in the pH of the solution will bring visible change in the colour of the indicator. It is necessary that the effective pH range of the indicator includes the pH of the unknown sample.
Add 2 drops of the indicator in each tubes. Mix and observe the colour in all the three test tubes. Test tube containing acetic acid will display acid colour of the indicator whereas test tube containing sodium carbonate will display alkali colour of the indicator. If the colour obtained with the unknown solution lies in between i. Repeat the same procedures with the other indicators till a suitable indicator is selected.
Some common indicators useful for biological pH range are: Indicator pK pH range Acid colour Alkali colour 1. Thymol blue acid range 1. Methyl orange 3. Methyl red 5. Phenol red 7. Thymol blue alkaline range 9. Phenolphthalein 9.
Determining the end point in acid-base titration. Determining the pH of the unknown solutions. Indicator paper Indicator paper consists of a strip of a sensitized paper and is accompanied by a colour chart which shows different colour which the indicator exhibits at different pH values.
Take a strip of indicator paper and moisten it or dip it in the solution whose pH is to be determined. Remove the excess of the fluid adhering to the indicator paper strip by means of pressing between the folds of filter papers. Compare the colour of the pH paper with the colour chart on the indicator paper and thus determine the pH of the solution. Universal indicator Universal indicator is a wide range indicator solution having pH between 0 to Take 5 ml of the unknown solution. Add to it 0.
Mix well and find out the pH by matching the colour of the solution with the colour chart on the universal indicator bottle. The one with which it coincides or matches, is the pH of the unknown solution. When superimposed, then the pH can be calculated by using Handerson-Hasselbatch equation. Now distribute the same number of drops in acid solution because any drop of indicator solution going into the solution, will give its acid form and alkali solution because any drop of indicator solution going into alkali will give its alkali form.
Mix the contents of each tube and examine the colours by white light. If the colour viewed through tube no. Now again observe colour. If the colour viewed through the tube nos 1 and 2 matches with the colour viewed through the tube nos 3 and 4, then count the number of drops added to acid tube and alkali tube.
The manner in which matching has been done it can be argued that the pH of the solution which contains ionized and unionised indicator in the ratio they are present in tube nos 1 and 2. Water passes through the membrane until the concentration on both sides becomes same. Such a movement of solvent molecules from a pure solvent or dilute solution, through a semipermeable membrane is called osmosis.
Osmotic pressure is the pressure that must be applied on a solution to keep it in equilibrium with the pure solvent when the two are separated by semipermeable membrane or osmotic pressure is the force required to oppose the osmotic flow. Since osmotic pressure is proportional to the total number of solute particles in solution so the substances which ionize, will have the higher osmotic pressure as compared to those which do not ionize.
If the solution containing crystalloids and colloids is placed in a cellophane sac and this is immersed in a jar of distilled water, the crystalloids diffuse across the membrane while the large colloidal molecules are retained.
By repeatedly changing the distilled water outside the sac, it is possible to free the colloidal material virtually completely from salts and other crystalloids. This process is known as dialysis. Cover the top of the thistle funnel by tying a piece of cellophane over it. Clamp the funnel on a stand and suspend this into a breaker of distilled water.
Physical Chemistry 7 After half an hour, take out a portion of solution from the beaker and perform the iodine test and barium chloride BaCl2 test. No change in colour is observed.
A white precipitate of barium sulphate is obtained. The starch test will be negative and sulphate test positive. To Study the Phenomenon of Adsorption and to Compare Two Eluters—Acetone and Water Adsorption is a phenomenon in which a substance is adsorped on the surface of a substance.
It takes place due to the presence of free valencies on the surface of the adsorbate which attracts and takes up the adsorbant. Procedure Take two test tubes.
Add 10 ml of 0. Shake the tube vigorously and filter the contents into the separate test tubes. Both dyes are completely adsorbed by the charcoal and are retained on the filter paper.
Wash the charcoal on the filter paper in one funnel with acetone and the other funnel with water. The colour of the filtrate with acetone washings will be blue while with water it will be colourless. Interpretation The solution looses its blue colour due to the adsorption of methylene blue particles on the activated charcoal.
This proves that the charcoal is a good adsorbant. On adding water to it, water cannot elute methylene blue and hence colourless filtrate is obtained. But acetone dissolves the methylene blue particles and thus the solution regains its original blue colour. This acetone is a better eluter than water and a strong adsorbant than the charcoal. To Study and Compare Surface Tension of Two Liquids—Water and Soap Solution Surface tension is a phenomenon concerned with the force acting at the surface of a liquid giving an appearance of a stretched membrane.
A liquid or a fluid is made up of molecules. But molecules on the surface are unequally attracted because of the absence of forces from above. This leads to certain unbalanced forces on the surface. Due to these forces, the surface acts as a membrane. Surface tension is defined as the workdone in ergs in streching the membrane by 1 square centimeter.
Add 3 ml of water in one and 3 ml of soap solution in the other. Sprinkle sulphur powder in both the test tubes. Interpretation Due to lower surface tension of soap solution, the surface could not keep the sulphur powder floating. Therefore, the surface tension of water is more than that of soap solution. Molisch Test This is a general test for carbohydrates. If oligosaccharides or polysaccharides are present, they are first hydrolysed to the constituent monosaccharides which are then dehydrated.
Pentoses yield furfural and hexoses yield 5-hydroxymethyl furfural. Mix and incline the test tube and cautiously add 2 ml of concen- trated sulphuric acid by the side of the test tube so that the acid forms a layer under the carbohydrate solution. Gently rotate the test tube between the palms of the hands to bring about slight mixing at the interface.
An appearance of violet or purple ring at the interface junction of two solutions indicate the presence of carbohydrates. Reducing sugars mono or disaccharides by virtue of free aldehydic or ketonic group in their structure reduce cupric ions in alkaline solutions at high temperature. Sodium carbonate Makes medium alkaline. Sodium citrate Prevents the precipitation of cupric ions as cupric hydroxide by forming a loosely bound cupric-sodium citrate complex which on dissociation gives a continuous supply of cupric ions.
Slightly heat the contents to dissolve. Filter the solution and make the volume to ml. Dissolve separately Add this solution slowly and with stirring to the above solution—the mixed solution is ready for use.
Add to it 8 drops of given carbo- hydrate solution. Boil over a flame or in a boiling water bath for 2 minutes. Cool the solution. An appearance of green, yellow or red precipitate indicates the presence of reducing sugars.
The colour of the solution or precipitate gives an approximate amount of reducing sugars present in the solution.
Green color — upto 0. Fehling solution consists of: Fehling solution A It contains copper sulphate solution. It is prepared by dissolving It is prepared by dissolving gm of KOH and gm of Rochelle salt in ml of distilled water. Mix, equal volume of Fehling A and Fehling B before use. It is semiquantitative and more sensitive.
These complexes dissociate sufficiently to provide a continuous supply of ready available cupric ions for oxidation. Mix and boil. Appearance of yellow or red precipitate of cupric oxide indicates the presence of reducing sugars. The acidity makes it a weaker oxidising reagent. Therefore only monosaccharides, will reduce cupric ions. However if heating is prolonged, disaccharides may be hydrolysed by the acid and the resulting monosaccharide will give the test positive. Reagent Cupric acetate in lactic acid.
To this add 25 ml of 8. Stir cool the solution and dilute to ml. Place the test tube in boiling water bath for 3 minutes. An appearance of brick red precipitate of cuprous oxide indicates the presence of monosaccharides.
Precautions The solution should be boiled for 3 minutes only. Overheating should be avoi- ded because on prolonged heating disaccharides will also give this test positive. Ketohexoses, i. Overheating of the solution is avoided because on continuous boiling, aldoses will also give this test positive because of their conversion to ketoses by hydrochloric acid.
Heat over a flame for 30 seconds only. An appearance of cherry red colour indicates the presence of fructose. Phenylhydrazine Test Osazone Formation Test Reducing sugar can be distinguished by phenylhydrazine test when characteristic osazone crystals are formed. These osazones have definite crystal structure, precipitation time and melting point and hence help in the identification of reducing sugars.
Examine the shape of crystals under low power of microscope. The shape of the osazones are as follow: Needle shape osazone. It is prepared only at the time of reaction. Test In a clear and dry test tube, take approximately 0. Add 5 ml of carbohydrate solution and drops of glacial acetic acid. Mix and place the test tube in boiling water bath for 30 minutes. Pentoses on heating with strong acid are converted to furfural which reacts with the coloured compound produced when orcinol and ferric chloride react with each other.
Appearance of green colour. To Detect Galactose Mucic acid test Galactose on oxidation with strong acid gives mucic acid which crystallises out and can be observed microscopically. Test In a test tube take 1 ml of galactose solution followed by 1 ml of concentrated nitric acid. Keep it overnight. Examine a drop of the crystals under low power of microscope. Maltose and lactose are reducing disaccharides where as sucrose is a non-reducing disaccharide.
One percent solution of each maltose, lactose and sucrose are provided. Molisch test Dissaccharides are first hydrolysed to constituent monosaccharides which are then dehydrated. Test In a clean and dry test tube, take 2 ml of the carbohydrate solution.
Incline the test tube and cautiously add 2 ml of concentrated H2SO4 by the side of the test tube. An appearance of violet or purple ring at the junction of two solutions indicate the presence of dicarbohydrates. Add 8 drops of given dicarbohydrate solution. Boil for 2 minutes. An appearance of green, yellow or red precipitate indicates the presence of disaccharides. No change in colour indicates the presence of disaccharides in the solution.
Negative for disaccharides. Osazone formation i. Test In a clean and dry test tube, take roughly 0. Add 5 ml of disaccharide solution and 2 drops of glacial acetic acid. Place the test tube in boiling water bath for 30 minutes.
After 30 minutes, take out the test tube from the boiling water bath and allow it to cool by itself in a test tube rack Do not disturb the test tube in between as the osazones of disac- charides separates out on slow cooling.
Appearance of yellow crystals takes place. Observe the shape of crystals under low power microscope. Sunflower shape Lactose: Add to it drops of concentrated hydrochloric acid. Boil the contents for few minutes minutes. Divide it in two parts. The test will be positive. It will be positive now. Osazone Formation Carry out the osazone test with the hydrolysate solution of sucrose.
Appearance of needle shaped crystals. Molisch test 2. Iodine test. This test is used for polysaccharides detection and differentiation. Iodine forms a coordination complex between the helically coiled polysaccharides chain and the iodine centrally located with in the helix due to adsorption. Amylose a linear chain component of starch gives a deep blue colour. Amylopectin, a branched chain component of starch gives a purple colour. Glycogen gives a reddish brown colour. Dextrins, formed from the partial hydrolysis of starch gives colours ranging from brown red to colourless depending on the size of the molecule.
Cellulose, inulin, disaccharides or monosaccharides gives no colour with iodine. Mix and observe the colour. No change in colour indicates the absence of polysaccharides.
Hydrolysis i. Divide the solution in five equal parts i. Remove the tube from the boiling water bath at an intervals of 1, 5, 8, 12 and 20 minutes. Now divide the solution in each tubes in two parts: Second part, perform iodine test. Enzymatic hydrolysis Take a clean test tube and collect some saliva in it. Take two dry test tubes and label them as blank and experimental. Mix well. Now add 1 ml of distilled water only in the blank and 1 ml of saliva in the test.
Keep the test tubes for 30 minutes at room remperature. In Blank a. Iodine test: Blue colour b. In Test a.
Negative b. Red precipitate. Where as starch without saliva is not broken up in to smaller molecules because there is no hydrolysis. No need to perform Molisch test, as the unknown solution is carbohydrate in nature. Iodine test Positive for polysaccharides.
Depending upon colour, the polysaccharide is identified. If negative, polysaccharides are absent. Reducing sugars can be monosaccharides or disaccharides. Indicates the presence of fructose.
Osazone test For the identification of particular carbohydrates. This is different from acid hydrolysis. Enzymatic hydrolysis gives bigger units and does not break the branched point i. Achromic point is that point at which no colour is obtained with iodine. Chromic period is that time period which is required to obtain achromic point when enzymatic hydrolysis is being performed. Once enzyme activity starts we get first soluble starch.
The first product hereafter formed is amylodextrins. Next we get erythrodextins which gives reddish colour with iodine. No colour with iodine is got when we get achrodextrin. This point is called achromic point. Add 2 ml of buffer pH 6. Take out 5 ml of it. This is prepared buffer-starch solution.
Rinse your mouth with water. Take ml of warm water in mouth and rotate the water with tongue. Take this in a polythene beaker and now taken 5 ml of this. Take a tile having grooves and put iodine in equal amount in each groove. Note the chromic period. Observations Time Colour 30 sec. Biuret Test Biuret test is given by all compounds that contains two or more peptide bonds. Since proteins are polypeptide, hence it is a general tests for proteins.
The name of the reaction is derived from the organic compound, a biuret, obtained by heating urea at high temperature which gives a positive test. Biuret Reagent Stock It is prepared by dissolving Then is added 2. Working biuret reagent is prepared by dissolving 50 gm of stock biuret reagent to ml with 0.
Divide N the solution, i. They give only a yellow colour. Test Take 1 ml of protein solution, add to it drops of freshly prepared ninhydrin solution. Heat the solution. Appearance of blue colour indicates the presence of proteins. To Study the R-Groups of Proteins The most important aspect of R-group of proteins is that of their nutritional significance. These acids which the animal body is unable to synthesise either completely or in amounts sufficient to normal growth maintainence must be supplied in the food.
These so called essential amino acids can be tested for in various way and can in many cases, be detected and determined by means of simple colour reactions based upon the qualitative test. A variety of colour reactions specific to particular functional groups in amino acids are known.
They are useful in both the qualitative and quantitative identification of particular amino acids. Xanthoproteic test For benzenoid radical The reaction is based upon the nitration of the benzene ring with concentrated HNO3 yielding yellow derivatives of nitrobenzene which turns to orange in alkaline medium. Add 1 ml of concentrated HNO3. A white precipitate due to denaturation of protein is formed. Boil the solution. A yellow solution derivative is formed due to nitration of benzene ring.
A pink colour is obtained in this test is due to mercury complex of nitrophenol derivatives. Reagent Mercuric nitrate dissolved in concentrated HNO3. Test Take 1 ml of test solution in a test tube.
Acidify with dilute-H2SO4. Check with litmus. A yellow precipitate adheres to the side of the test tube. Cool the solution under tap water. The precipitate or solution turns red. Hopkins-Cole test For indole group This test is specific for tryptophan and is an indication of the presence of tryptophan in the protein. Test Take 1 ml of test solution and add few drops of 1: Add 2 drops of mercuric sulphate i.
Incline the test tube and add 1 ml of concentrated H2SO4 by the side of the test tube. A purple ring is formed at the junction of the two layers. Sakaguchi test For guanidino group This test is given by all compounds containing guanidino group and thus is an indication of the presence of arginine present either in free or in combined form.
Test Take 3 ml of test solution in a test tube. Wait for 5 minutes. Development of bright red colour takes place. Run a control by taking 3 ml of distilled water instead of protein solution and add all other reagents as in test.
Test for cystine or cysteine —S—S—and—SH radicals Sulphur is present in proteins as cystine, cysteine or methionine. Cool and add lead acetate solution. A black precipitate of PbS insoluble in dilute HCl is formed. Test for free—SH radical Take 1 ml of test solution. Add few crystals of ammonium sulphate. To it add few drops of freshly prepared solution of sodium nitroprusside and 1 ml of liquor ammonia. Development of rose red colour takes place.
Precipitation by heavy metals. Precipitation by alkaloidal reagents. On the alkaline side of the isoelectric point proteins exits as negative ions. Test 1. Take 3 ml of protein solution. Appearance of white precipitate. A light blue precipitate appears. An intense white precipitate appears. Precipitation by alkaloidal reagents Proteins are precipitated from the solution by combination between the acid anions and the positively charged protein molecule by forming all insoluble complex.
The alkaloidal reagents precipitate proteins by combination of the acidic radical of the former with the cationic form of the protein, which predominates when the solution is on the acidic radical of the former with the cationic form of the protein, which predominates when the solution is on the acidic side of the isoelectric point.
To 3 ml of protein solution, add few drops of metaphosphoric acid. A dirty white precipitate appears. White precipitate appears. To 3 ml of protein solution, add 3 ml of Esbach reagent. A precipitate appears. A deep yellow precipitate appears. Precipitation of proteins by heating Proteins which are precipitated when their solutions are heated are termed as heat coaguable proteins.
This property of proteins is made use of in the detection of albumin in urine simply by heating the urine. Solubilities of several proteins in various solvents are shown below: