Introduction to Practical Biochemistry. György Hegyi István Venekei. XML to PDF by RenderX XEP XSL-FO F ormatter, visit us at caite.info —daily tech on the manga guide to calculus. “The art is fantastic, and the teaching method is both A Practical Introduction to Python Programming. Try this link Practical Textbook of Biochemistry for Medical Students You may also check out this Textbook of Biochemistry for Medical Students.
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𝗣𝗗𝗙 | principle of practical biochemistry and experimental part. PDF | On Jul 10, , Anand Tiwari, PhD and others published Practical Biochemistry: A Student Companion. We are hopeful that this practical biochemistry book will help the medical students to envisage about the various facts encountered in the reactions in the body.
These are used for performing titration, and for boiling the solutions. On hydrolysis the linkage is cleaved and it becomes a reducing sugar. Updating Results. Note the bright crimson red color of the oxyhemoglobin is changed to purple due to reduced hemoglobin. Principles of Colorimetry
Different pipettes are used to pipette different fixed volumes. Variable volume adjustable type: The volume of fluid to be dispensed can be adjusted with the adjusting screw as required.
Variable volumes, e. Test Tubes Test tubes are of uniform thickness which can withstand mechanical and thermal shocks. Tubes with rim are preferred when reagent in the test tube is directly heated on the flame using a test tube holder.
Test tubes are available in capacities of various volumes. Folin-Wu Tubes Folin-Wu tubes have markings at They are used for determination of blood sugar by Folin- Wu method. Dispensers Dispensers are used to dispense large fixed volumes of reagents. They are usually used to dispense strong acids and alkalies. Desiccators Desiccators are used to keep solid or liquid materials dry. They usually have an area at the bottom where a desiccant water absorbing material is placed, which removes the water of hydration from compounds.
Finally, it should be rinsed with tap water followed by distilled water. The apparatus can be dried quickly by rinsing with alcohol followed by ether. The cleaned glassware except the graduated glassware is dried in a hot air oven. Dichromate-Sulfuric acid mixture chromic acid is used for cleaning glasswares which removes even the last traces of grease.
Monosaccharides Monosaccharides are also called as simple sugars. They have only one potential sugar group. They consist of a single polyhydroxy aldehyde or ketone unit, and thus cannot be hydrolyzed into simpler form. They may be subdivided into different groups as follows: Depending upon the number of carbon atoms they possess, e. Depending upon the functional groups: Oligosaccharides Oligosaccharides consist of a short chain of monosaccharide units 2 to 10 units , joined together by a characteristic bond called glycosidic bond.
Oligosaccharides are subdivided into different groups based on the number of monosaccharide units present. Type of No. In reducing disaccharides one of the functional groups is free.
Non-reducing disaccharides do not have free functional group. The potential functional groups are involved in glycosidic linkage.
They are also called glycans or complex carbohydrates. They are classified into two types according to the type of monosaccharide units present. Made up of repeated units of same type of monosaccharide units. Starch, glycogen, cellulose, inulin, dextrins, dextrans 2.
Made up of different types of monosaccharide units and their derivatives. Agar, gum, pectins, glycosaminoglycans such as hyaluronic acid, heparin sulfate, keratin sulfate, chondroitin sulfate. Carbohydrates are the main source of energy in the body. Storage form of energy starch and glycogen 3. Excess carbohydrate is converted to fat. Glycoproteins and glycolipids are components of cell membranes and receptors. They form structural basis of many organisms. The functional group of the sugar molecule takes part in most of the chemical reactions.
Carbohydrates such as sucrose, which do not contain free functional groups are not enolized by alkali and relatively stable in alkali solution. Strong Alkalies On boiling with strong alkali, the aldehyde polymerizes to form resin which is called caramelization.
Thus, glucose loses its reducing property. Reaction with Acids Weak Acids These have no appreciable action on sugars. Strong Acids With strong acids, sugar undergoes dehydration forming furfural. The pentoses yield furfural and hexoses give hydroxy- methyl furfural. Keto- hexoses like fructose yield greater amount of furfural derivative than aldo- hexoses like glucose. Their responses to individual carbohydrates are given later.
Learning this way will also aid in the examination, when an unknown solution is given for qualitative analysis.
Qualitative Analysis of Carbohydrates 33 The various tests for carbohydrates are given below: Molisch test: Iodine test: Osazone test: Physical Properties 1. Colorless except Starch which is pale white 2. Clear except Starch which is cloudy 3. Odorless 4. Reaction to litmus: Experiment Observation Inference Take 2 ml of given Violet ring at the junction Given solution in a clean of the two liquids is solution is a dry test tube; add formed.
Incline the test tube slightly and overlay 2 ml of conc. Points to Remember: Therefore, acid should be layered very slowly and carefully to minimize this interaction. This occurs as a result of the action of concentrated sulfuric acid on it. This occurs if these substances have a bound carbohydrate moiety attached to them, e. Iodine Test Principle: The test depends upon the property of adsorption possessed by the large polysaccharide molecules which adsorb the smaller iodine molecules on their surface to form the blue colored complex of ill-defined chemical nature.
The property of adsorption decreases on heating, the complex dissociates and, therefore, the color disappears. Qualitative Analysis of Carbohydrates 35 Iodine reagent: Experiment Observation Inference Take 2 ml of given Blue color is formed. Given solution is a solution in a test polysaccharide.
Add drops of Iodine solution. When it is treated with iodine solution, Iodine is trapped inside the coil and the complex has an intense blue color. When the amylose solution is heated the helical conformation is disrupted and loses its capacity to bind iodine. On cooling the original conformation is regained and the capacity to bind iodine is also recovered.
In mild alkaline medium reducing sugars undergo tautomerization to form enediols which reduce cupric ions to cuprous ions. Cuprous hydroxide is formed. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives different shades of colored precipitate depending upon the concentration of the sugar.
Thus prevents the precipitation of cupric ions as cupric hydroxide by forming cupric sodium citrate complex.
It acts as a stabilizing agent. Improves the shelf- life of the reagent by preventing an interaction between sodium carbonate and copper sulfate, which may otherwise get precipitated as cupric carbonate.
Add 8 drops of the given solution. Mix and boil for 2 min. Allow to cool spontaneously. Observation Inference Sign Approx. Green precipitate. Hence, the below given procedure has to be followed. Acid Hydrolysis Principle: Heating in an acidic environment leads to the hydrolysis of the glycosidic bonds present in disaccharides or polysaccharides. To 5 ml sucrose Add drops of Conc. Hydrochloric acid. Boil for 3 min. Divide the solution into two parts. Acid hydrolysis To 5 ml of starch Add drops of Conc.
Boil for 5 min. Boil for 2 min. In mild acidic medium reducing sugars undergo tautomerization to form enediols which reduce cupric ions to cuprous ions. During the process of heating cuprous hydroxide is converted to cuprous oxide which gives red precipitate. Since acidic medium is unfavorable for reduction, stronger reducing agents like monosaccharides give reduction within 30 seconds. Copper acetate in glacial acetic acid. Mix and boil for 30 seconds. Allow it to cool at room temperature.
Furfural derivative of ketosugar condenses with resorcinol to form a chromogen cherry red color. Boil for 30 seconds and allow it to cool at room temperature. In the presence of glucose along with fructose sensitivity decreases. Boil for 30 seconds. Osazone Test Principle: Osazone mixture: To 1 part of Phenyl hydrazine Hydrochloride add 2 parts of sodium acetate and few drops of glacial acetic acid. Sodium acetate acts as a buffer to maintain the pH.
Phenyl hydrazine HCl reacts with C1 and C2 of a reducing sugar first to form phenyl hydrazone and then to form osazones. To 5 ml of given solution add 3 spatula of osazone mixture. Mix well and keep in boiling water bath for 5 min. Take out some crystals on a slide and observe under microscope. In case of lactose and maltose, mix well and keep in boiling water bath for 30 min. Qualitative Analysis of Carbohydrates 43 Points to Remember: So they form crystals only when the test tube is cooled.
During osazone formation phenyl hydrazine reacts with the 1st and 2nd carbon atoms and hence the structural difference between these three sugars is masked. Hence glucose, fructose and mannose form the same needle shaped osazone crystals. Hence, sucrose does not produce osazone crystals.
To differentiate glucose and lactose that is excreted in the urine. The carbohydrates to be studied in the lab are: Lactase is deficient in lactose intolerance.
Since, there is no free functional group sucrose is non-reducing. On hydrolysis the linkage is cleaved and it becomes a reducing sugar. The test results for different carbohydrates are sum- marized below: They are complex organic substances made up of carbon, hydrogen, oxygen and nitrogen.
Some proteins also contain sulfur and phosphorus. Simple proteins: Albumin, globulin and protamines 2. Conjugated proteins: Derived proteins: They are of two types: Proteons, metaproteins. Maintain the structural integrity of bones, tendons, hair and teeth- collagen, elastin, keratin. Acts as enzymes catalytic function. Hormones regulatory function. Antibodies- immunoglobulins defense protein. 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. Some of the compounds detected are listed below. Gently sinks to the bottom. Observe without mixing. Bile pigments are oxidized by nitric acid to various colored products, e.
Various colored rings Indicates the Nitric acid in a will be formed at the presence of bile test tube. Add 5 ml point of contact pigments. Experiment Observation Inference Take 5 ml of urine A white precipitate Indicates the in a test tube.
Add of barium presence of bile few crystals of sulfate is formed. A green color develops Then add 3 ml of on the filter paper. Unfold the filter paper. Bile contains conjugated bilirubin which is excreted into the intestine. It oxidizes bilirubin to biliverdin green or bilicyanin blue.
Test for Blood Principle: Hemoglobin peroxidase of blood decomposes hydrogen peroxide catalytically and liberates nascent oxygen. This oxygen oxidizes benzidine to a blue or green compound. This color changes to brown within a few minutes on exposure to air. Benzidine reagent: It contains benzidine and glacial acetic acid. Experiment Observation Inference Take drops of A blue or green color Indicates the benzidine solution is formed, which is stable presence in a test tube.
Add drops of and changes to brown. Add 1 or 2 drops of this mixture to 2 ml of urine. These cells contain a peroxidase, which is responsible for the positive reaction. The benzidine reaction then becomes negative although sufficient blood is present in urine. The hemoglobins differ depending on the type of polypeptide chains they are composed of. The normal hemoglobins are Hb-A hemoglobin of adult , Hb-F hemoglobin of fetal life and Hb-A2 hemoglobin of postnatal. The following are the derivatives of hemoglobin.
Native hemoglobin: It serves as oxygen carrier in the blood. Hemoglobin in combination with 4 molecules of oxygen. Suggestions are made for further work in more advanced classes. As well as the practical method the experiments are accompanied by background information, discussion of results, references for further study and illustrations. A volume that has been long awaited 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. We would like to ask you for a moment of your time to fill in a short questionnaire, at the end of your visit.
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