PRACTICAL 5 AMINO ACIDS AND PROTEINS

Transcription

1 PRACTICAL 5 AMINO ACIDS AND PROTEINS Amino Acids and Proteins Structure 5.1 Introduction 5.2 Amino Acids - The Building Blocks of Proteins Properties of Amino Acids Separation of Amino Acids using Paper Chromatography 5.3 Proteins Proteins - Basic Concepts Methods of Protein Estimation Experiment 1: Studying the Properties of Amino Acids Experiment 2: Separation of Amino Acids By Paper Chromatography Experiment 3: Estimation of Nitrogen and Protein Content Using Microkjeldahl s Method Experiment 4: Determination of Protein Content Using Biuret Method 5.1 INTRODUCTION The last practical focused on studying the properties and estimation of carbohydrates. Moving further, we shall now study the properties of amino acids which we know are the building blocks of proteins. In this practical, we shall carry out specific tests in the laboratory and differentiate between the different amino acids. Further, the process for the separation of amino acids using paper chromatography shall be conducted. The second part of this practical focuses on proteins. We know that all proteins contain nitrogen in addition to carbon, hydrogen, oxygen and some other elements like sulphur. Thus, determination of nitrogen is commonly used to determine the protein content of a sample. In fact, several methods for protein estimation are available. We shall study about two such important and practical methods we can use in the laboratory for protein estimation. These include: kjeldahl s method and the biuret method. Using these methods we shall actually estimate the protein content of the given sample in the laboratory. Objectives After studying this practical and undertaking the activities given herewith, you will be able to: describe the properties of amino acids like tyrosine, tryptophan, arginine, cystine, phenylalanine etc., identify the amino acids on the basis of their properties, carry out the paper chromatography technique for seperation of amino acids, estimate the amount of nitrogen and protein present in a given sample, and determine the protein content in a given sample by biuret method. 5.2 AMINO ACIDS IN THE BUILDING BLOCKS OF PROTEINS You have studied that amino acids are the building blocks of proteins. Can you recall the 20 odd amino acids required by our body? Look up the Nutritional Biochemistry Theory Course, Unit 2. List a few of them in the space provided. Can you also now illustrate the structure of an amino acid? Draw the structure in the space provided. 131

2 Nutritional Biochemistry Amino acids, as you have already illustrated, have a carboxyl group and an amino group bonded to a common carbon atom and differ from each other in their side chains or R groups. The R groups vary in structure, size and electric charge which influence the solubility of amino acids in water. The amino acids are joined together by specific covalent bonds called peptide bonds. All amino acids except glycine have a chiral centre and are optically active and have two possible stereoisomers called enantiomers. You have already studied this term. Can you define it? Define the term enantiomer in the space provided herewith. Also graphically represent the L and D isomers. Enantiomer definition: L-amino acid D-amino acid Can you define the difference between D and L isomerism? You may recall studying in the theory course that all amino acids in proteins belong to the L series. Further, we have also studied that amino acids are classified according to their R groups. In general, they are classified as: 1. Non-polar aliphatic R groups 2. Polar uncharged R groups 3. Positively charge (basic) R groups 4. Negatively charged (acidic) R groups Give 2 examples of amino acids that belong to each of the above mentioned groups: Amino acids with non-polar aliphatic R group Amino acids with polar charged R group Amino acids with positively charged R group Amino acids with negatively charged R group You may also recall studying that amino acid may also be classified as aromatic or nonaromatic. Amino acids, which have a phenyl (benzene) ring in their structure, are called aromatic amino acids. Can you list a few aromatic amino acids? List them in the space provided: 132

3 The distinctive physical, chemical and biological properties associated with an amino acid are the result of the R-group. There are 20 major amino acids that differ in their R-group. The R-group can be hydrophobic or polar, aromatic or aliphatic, charged or uncharged as we have seen above. The different R-groups are responsible for amino acids having different polarities, solubilities and chromatographic behavior. Amino Acids and Proteins Amino acids, you may also recall studying in the theory course, when dissolved in water, exist as zwitterions. A zwitterion can act as an acid (proton donor) or base (proton acceptor). They exhibit chemical properties due to COOH and NH 2 groups. Let us study some of the properties of amino acids in the laboratory Properties of Amino Acids Some of the important properties of amino acids are tabulated herewith: Properties 1. Solubility (a) In Cold water (b) In Hot water (c) In Dilute acid like HCl (d) In Strong alkali like NaOH solution (e) In Weak alkali like NH 4 OH solution (f) In Alcohol 2. Ninhydrin Test 3. Folin s Test 4. Hofmann s Test 5. Sulphuric acid Test 6. Morner s Test 7. Hopkins-Cole test Amino acids are soluble in various solvents due to presence of polar groups. Tyrosine is the least soluble in water. It is also insoluble in organic solvents but readily soluble in dilute alkalies and acids. Tryptophan and arginine are soluble in all solvents except alcohol. Cystine is poorly soluble. It is only slightly soluble in water but soluble in solutions of dilute acids and alkalies. While phenylalanine is insoluble in cold water, it is easily soluble in hot water. It is insoluble in alcohol, but soluble in acids and alkalies. Amino acids on heating with ninhydrin reagent are quantitatively deaminated to give a blue colour. When some of the ninhydrin reacts with its reduction product hydrindantin and ammonia, a blue coloured substance (Ruhemann s purple) is formed. This test is given due to the aromatic ring in the aromatic amino acids. It is given by tyrosine as well as tryptophan. The reaction is due to formation of a coloured mercury compound with the hydroxy phenyl group and hence is given positive by all phenols. Tyrosine is the only phenolic amino acid, thus this test confirms tyrosine. The purple colour that is seen is due to the formation of ferric salt of tyrosine-sulphuric acid. This reaction is due to the presence of phenolic hydroxyl group in tyrosine. The reaction is due to the presence of indole ring in tryptophan. It reacts with glyoxylic acid to form a coloured complex. The copper is required for the test since pure tryptophan gives the test in presence of copper or ferric ions. 133

4 Nutritional Biochemistry 8. Sakaguchi Test 9. Xanthoproteic Test 10. Lead Acetate Test This test is given by any substance having a guanidine group. Arginine has a guanidine group and thus gives a positive Sakaguchi test. In an alkaline medium, α-naphthol combines with the guanidino group of arginine to form a complex which is oxidized by sodium hypochlorite to produce a red colour. This test is due to nitration of phenyl ring. However, this test is generally given by phenylalanine when combined in peptide linkage and not in pure form. On boiling with NaOH, the sulfur present in cystine (the R group contains S) is released as Na 2 S. This reacts with lead acetate to form a brown or black ppt of lead sulfide. While cystine (and cysteine) responds to this test, the sulfur present in methionine is not released by the above treatment. We shall carry out these tests in the laboratory and differentiate between the different amino acids. Activity 1 later in this practical focuses on studying the properties of amino acids on the basis of their properties like solubility and colour reaction due to their structure and the side chain (R). Before we move on to the activity, let us also quickly review another process which we can use in the laboratory for the separation of amino acids i.e. separation of amino acids using paper chromatography Separation of Amino Acids using Paper Chromatography One of the important techniques used in the laboratory for seperating amino acids is paper chromatography. We have already described the technique earlier in practical 2. Look up Practical 2, once again and get an overview on chromatography. The theory/ principle behind the chromatography process is once again described herewith for your reference. Theory/Principle Chromatography is a collective term used for those processes which allow resolution of mixture by affecting the separation of all or some of their components in zones or phases different from those in which they are originally present. It is a technique used for the separation of a mixture brought about by dynamic partition or continuous distribution of dissolved material between two immiscible phases, of which one is mobile and the other is stationary. The mobile phase flows through the stationary phase. During the process of movement of mobile phase, small differences in adsorption-desorption, or partitioning or ion exchange behaviour of different components of the mixture are multiplied many-fold thus enabling their separation. The ability of chromatography to separate different solutes depends on the selectivity of the process and the degree to which the system can distinguish between different solutes. Paper chromatography is a very useful technique for separating mixtures of metal ions, anions, amino acids, sugars, dyes, drugs etc. It is based on the principle of partition of a substance between two immiscible solvents in which one of the solvents is static while the other is mobile. In paper chromatography, the water held in cellulose fibers acts as a static phase whereas water or any other solvent acts as a mobile phase. 134 Paper chromatography can separate different amino acids based on their varying solubilities in two different solvents. In this method, a sample of an amino acid (or mixture of amino acids) is applied as a small spot near one edge of a piece of

5 chromatography paper. The edge of the paper is then placed in a shallow layer of solvent mixture in a chromatography tank. Amino Acids and Proteins The solvent mixture contains several components, one of which is usually water and another of which is a more non-polar solvent. As the solvent mixture moves up the paper by capillary action, the water in the mixture binds to the hydrophilic paper (cellulose) and creates a liquid stationary phase of many small water droplets. The non-polar solvent continues to move up the paper forming a liquid mobile phase. Since amino acids have different R-groups, they also have different degrees of solubility in water vs. the non-polar solvent. An amino acid with a polar R-group will be more soluble in water than in the non-polar solvent, so it will dissolve more in the stationary water phase and will move up the paper only slightly. An amino acid with a hydrophobic R-group will be more soluble in the mobile non-polar solvent than in water, so it will continue to move up the paper. Different amino acids will move different distances up the paper depending upon their relative solubilities in the two solvents, allowing for separation of amino acid mixtures. The movement of amino acids can be defined by a quantity known as R f value, which measures the movement of an amino acid compared to the movement of the solvent. At the start of the chromatography, the amino acid is spotted at what is called the origin. The chromatography is then performed, and the procedure is stopped before the solvent runs all the way up the paper. The level to which the solvent has risen is called the solvent front. The R f value of an amino acid is the ratio of the distance travelled by the amino acid from the origin to the distance travelled by the solvent from the origin. R f = distance travelled by the substance distance travelled by the solvent front Since R f value for an amino acid is constant for a given chromatography system, an unknown amino acid can be identified by comparing its R f value to those of known amino acids. Certain technical aspects are important when performing paper chromatography. First, it is necessary to keep the applied amino acid spot very small. The spot tends to spread out as it moves up the paper, so starting with a big spot will produce a large smear by the end of the procedure, making it difficult to measure an accurate R f value. Second, the chromatogram paper must be kept very clean. Fingerprints or other types of contamination will interfere with the chromatography and give poor results. Finally, since amino acids are colourless, something must be done to detect the amino acids at the completion of the chromatography. One of the simplest methods for this involves spraying the paper with ninhydrin. When heated, ninhydrin reacts with amino acids to produce a bluepurple colour (yellow in the case of proline), making the amino acids spots visible for analysis. With this basic understanding, we are now equipped to carry out the exercise relating to separation of amino acid using paper chromatography. The activity 2 in this practical will give you good practice and experience with paper chromatography. But before we move on to the activities, let us quickly also review the literature for proteins. 5.3 PROTEINS The discussion below focuses on the basic concepts of proteins with regards to structure and properties and also describes the methods commonly used for protein estimation in the laboratory. We begin our study with the basic concepts. 135

6 Nutritional Biochemistry Proteins _ Basic Concept Proteins, as we already knows by now are products of amino acids. Each molecule of protein is composed of many molecules of amino acids joined by peptide bond as can be seen in Figure 5.1. Figure 5.1: Peptide bond formation Proteins contain nitrogen in addition to carbon, hydrogen, oxygen and some other elements like sulphur. Thus determination of nitrogen is commonly used to determine the protein content of a sample. Proteins contain about 16% nitrogen and multiplying the nitrogen content by a factor of 6.25 gives us the amount of crude protein present in the given sample. Several methods for protein estimation are available. Let us get to know about these methods Methods of Protein Estimation Several methods for protein estimation are available. These methods are based on several analytical characteristics of the protein. Laboratory estimation of protein is based on any of the following principles: i) Nitrogen estimation based on the principle of Kjeldahl s method, ii) Estimation of tyrosine in protein (as in Lowry s method) or both phenyl alanine and tyrosine in protein (Folin s method). iii) Biuret method - most commonly used method in clinical practice and teaching laboratories. We have studied some of the tests in qualitative methods and the principle is the same. The Biuret method for example can be used for both qualitative and quantitative estimation of proteins. Let us learn about these methods in details. We shall, however, focus on the kjeldahl s method and the biuret method here in this section. A. Nitrogen Estimation-Kjeldahl s Method Nitrogen in the protein is estimated by a method referred to as Kjeldahl s method. Although over a period of time many other methods have emerged for determination of organic nitrogen, this method still remains an old favourite because it is reliable and has very well standardized procedures. 136 The method involves the digestion of the weighed sample with concentrated sulphuric acid, selenium oxide, potassuim sulphate and copper sulphate in a long necked special digestion flask known as kjeldahl s flask. Potassium sulphate increases the boiling point of the mixture. Since the digestion results in fuming of the sulphuric acid, the procedure has to be carried out in a fume hood and the procedure takes several hours. The sample is digested until the solution in the digestion flask becomes clear after which it is transferred to an appropriate volumetric flask (usually 50 or 100 ml flask) and the volume is made up to the mark with distilled water. Aliquots of this sample are used for distillation in the kjeldahl s apparatus and nitrogen in the protein is estimated. Most proteins contains 16% nitrogen. Thus, percent nitrogen content is multiplied by 6.25 to get crude protein content.

7 Amino Acids and Proteins Figure 5.2: The kjeldahl s apparatus The experiment involves decomposition of ammonium sulphate by a fixed volume of alkali (NaOH). The reaction is highlighted in Figure 5.3. Ammonium sulphate on decomposition releases ammonia which is collected in a flask of boric acid solution Tashiro s indicator. With boric acid ammonia forms a complex ammoium borate. As ammonia is distilled into the boric acid, the Tashiro indicator slowly changes colour from pink to green. This is because the colour of the indicator is ph dependent - it is pink in acidic medium and green in alkaline medium. When the green coloured solution containing the boric-acid-ammonia complex is retitrated against H 2, the solution slowly becomes acidic again and the indicator becomes pink. This point when colour changes from green to pink is taken as the end point. The Reaction involved includes: Protein + H 2 (NH 4 ) 2 + CO 2 + H 2 O (conc. sulphuric acid) (ammonium sulphate) (NH 4 ) 2 + 2NaOH 2NH 3 + Na 2 + 2H 2 O (ammonium sulphate) (sodium hydroxide) (Ammonia) (Sodium sulphate) Figure 5.3: Reaction involving decomposition of amonium sulfate The amount of nitrogen can be calculated for this value using the equation: 1000 ml of 1N H 2 = 1000 ml of 1N NH 3 = 1000 ml of 1N (NH 4 ) 2 S0 4 = 1000 ml of 1 N Nitrogen = 14 g of Nitrogen (based on molecular weight calculations) The Kjeldahl s method is commonly used in estimation of protein content of foods and has been extensively used for protein estimation of various foodstuff. We shall look at the method in details in activity 3 in this practical and try to find the amount of ammonium sulphate in the sample from which protein can be calculated. Next, let us learn the oldest colorimetric method for protein estimation i.e. the biuret method. B. Biuret Method The Biuret reaction was one of first colorimetric assays developed for protein estimation. It is most often used in applications requiring a fast but not a highly accurate value. The principle of biuret method is described next. Principle/Theory If a strongly alkaline solution of Biuret is heated with very dilute copper sulphate, a violet colour is obtained. The substances which contain two carbamyl (-CONH 2 )groups joined together either directly or through a single carbon or nitrogen atom and those which contain 137

8 Nutritional Biochemistry two or more peptide links give a blue or purple coloured complex with alkaline copper solution. Proteins respond positively since there are pairs of -CONH 2 groups in the molecule. The reaction is due to coordination of cupric anion with the unshared electron pair of peptide nitrogen and the oxygen of the water. A coordination complex is formed which is purplish coloured and can be colorimetrically measured. When protein solution is made strongly alkaline with NaOH or KOH and very dilute Cu is added, a purplish to pink violet colour is obtained, the intensity of the colour depending upon the concentration of protein or the number of peptide bonds undergoing the reaction. The biuret reaction is shown in Figure 5.4. NH 2 O O NH 2 C NH 2 O C NH 2 C O O NH C C O NH NH Cu ++ NH NH C C O NH Urea NH 2 Biuret Coloured coordination complex Figure 5.4: The Biuret reaction The detailed procedure involved in biuret method is described in activity 4. So with this knowledge, we can start with the experiments given in this practical. There are 4 experiments in this practical. Organize your time in such a manner that you can carry out these experiments one by one. 138

9 Amino Acids and Proteins EXPERIMENT STUDYING THE PROPERTIES OF AMINO ACIDS 1 Date:... Aim: To study the properties of amino acids like tyrosine, tryptophan, arginine, cystine, phenylalanine. Now conduct the various tests indicated in the format herewith and note down your observations and inference in the space provided: Tyrosine 1. Solubility Test Observation Inference Test the solubility of the amino acid in the following solvents (a) Cold water (b) Hot water (c) Dilute acid like HCI (d) Strong alkali like NaOH solution (e) Weak alkali like NH 4 OH solution (f) Alcohol 2. Ninhydrin Test Take 5 ml of tyrosine solution add 0.5 ml of 0.1% ninhydrin solution. Boiling for 1-2 minutes and allow to cool. 3. Folin s Test To 1-2 ml of tyrosine solution add equal amounts of phenol reagent (folin-ciocalteau reagent-25 g sodium tungstate g sodium molybdate ml phosphoric acid + 25 ml conc. HCI + 37 g Li + a few drops of bromine) and 10 ml of a saturated solution of sodium carbonate. 4. Hofmann s Test (Millon s test for tyrosine) To 3 ml of tyrosine add a few drops of Million s reagent. Heat. 139

10 Nutritional Biochemistry 5. Sulfuric Acid Test Take a small amount of tyrosine in a small 10 ml beaker and add 3-5 drops of conc H 2 ' Keep on a boiling water bath for 20 minutes. Cool. Wash the contents into another small beaker with distilled water. Slowly add solid CaCO 3 with stirring until the reaction of the solution is no longer acid. Filter. Concentrate the filtrate, and add it to a few drops (avoid an excess) of very dilute neutral ferric chloride. 6. Morner s Test (formaldehyde -sulphuric acid test) To 3 ml of Morner s reagent (1 ml of formalin + a 100 ml solution of equal amounts of distilled water and conc H 2 ) add a little tyrosine solution. Heat gently. Tryptophan 1. Solubility Test the solubility of the amino acid in the following solvents (a) Cold water (b) Hot water (c) Dilute acid like HCI (d) Strong alkali like NaOH solution (e) Weak alkali like NH 4 OH solution (f) Alcohol 2. Ninhydrin test (write the procedure here in the space provided) 140

11 3. Folin s test (write the procedure here in the space provided) Amino Acids and Proteins 4. Hopkins- Cole test To about 3-4 ml of tryptophan solution add 3 ml of glyoxylic acid solution. Mix. Carefully add 5 ml of conc. sulphuric acid down the side of the tube. Add a drop of copper sulphate solution. Arginine 1. Solubility Test the solubility of the amino acid in the following solvents (a) Cold water (b) Hot water (c) Dilute acid like HCI (d) Strong alkali like NaOH solution (e) Weak alkali like NH 4 OH solution (f) Alcohol 2. Ninhydrin test (Same as before. Write the procedure in the space provided) 3. Sakaguchi test Take 2-3 ml of arginine solution. To this add 1-2 drops of α-naphthol solution and 2 ml of sodium 141

12 Nutritional Biochemistry Cystine 1. Solubility Test the solubility in the following solvents: (a) Cold water (b) Hot water (c) Dilute HCI (d) KOH solution (e) NH 4 OH solution (f) Alcohol 2. Ninhydrin test Same as before. 3. Lead Acetate test To 5 ml of cystine solution add a few drops of 10% solution of lead acetate. Mix well. Make the solution strongly alkaline by adding 40% KOH or NaOH. Boil for a few minutes and let it stand for some time. Phenylalanine 1. Solubility Test the solubility in the following solvents: (a) Cold water (b) Hot water (c) Dilute HCI (d) KOH solution (e) NH 4 OH solution (f) Alcohol 2. Ninhydrin test Same as before 3. Xanthoproteic test 142 To 2-3 ml of phenylalanine solution, add 1 ml of conc. HNO 3. Cool. Make it alkaline with strong NH 3

13 Now conduct the various tests indicated in the format herewith and note down your observations and inference in the space provided: Amino Acids and Proteins Results The given solution contains amino acids Give the structure of these amino acids. Write the reaction involved in the tests used to determine these. Submit your experiment for evaluation.... Counsellor signature 143

14 Nutritional Biochemistry EXPERIMENT 2 Date:... SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY In Experiment 1 you learnt methods of identification of amino acids on basis of their properties like solubility and colour reactions due to their structure and R groups. Let us also study the separation of amino acids by using chromatography. Aim: To separate and identify the unknown amino acids in a given mixture by paper chromatography. Theory/Principle You have already studied the principle of chromatography in Section 5.2 above. Based on your understanding of the topic, write the principle here in the space provided. Materials: Petridishes (200 mm diameter) with lid, Whatman Chromatographic paper, brown paper, 500 ml separating funnel, capillary tubes, small beaker, durham tubes, a pair of scissors stoppered glass tubes, cotton for making wick. Reagents The following reagents will be required for carrying out the experiment: 1. Amino acid solution- Weigh abour 3-4 mg of each amino acid and dissolve it in dil HCl (1-2 drops). Make the volume upto 10 ml. 2. Chromatographic solvent Take a separating funnel. To this add 500 ml of freshly shaken mixture of equal volumes of water and butanol. Add 60 ml of glacial acetic acid. Mix. Shake. Discard the lower layer. Take the upper layer. 3. Ninhydrin solution 1% ninhydrin in acetone. Procedure 144 Now carry out the procedure following the steps given herewith: 1. Take a circular Whatman No 1 filter paper. 2. Take a Petri dish the diameter of which is slightly smaller than the diameter of the paper. 3. From the centre of the paper draw a circle of 2 cm diameter. 4. Mark the spots with a pencil for spotting on the circle. 5. With the help of a fine capillary tube spot the amino acid standards and unknown mixture on the marked spots. This process is called spotting. Allow the spot on the paper to dry completely. 6. Insert a cotton wick in the central hole of the filter paper. 7. Put some solvent in the Petri plate and place the paper on top of it ensuring that the wick dips into the solvent in the centre of the petridish. Cover the petridish. 8. Leave this arrangement for 3-3½ hours so that the solvent reaches nearly to the end of the circular paper.

15 9. Remove the lid and mark the solvent front with a lead pencil. 10. Dry the paper in air by clipping it on to a thread line. 11. For visualization of amino acid, spray ninhydrin over the paper and dry the paper again (for 10 minutes). 12. Areas where the amino acid has moved will get a yellow, pink or purple colour. Mark the areas with a pencil. 13. For identification of amino acids, estimate and measure the distance between the point of application of amino acid and the centre of the amino zone acid, as well as, the distance between the point of application and the solvent front. 14. Ratios of the two measurements give the R f value of standard amino acid and that of an unknown amino acid to identify the amino acids. Amino Acids and Proteins Precautions 1. Hold the paper with the extra strip kept at one side. Handling of whatman paper with hand should be avoided. 2. The spotting of amino acid should not be more than 1 mm in diameter since large spots lead to diffused and large amino acid patterns with no sharp outline. 3. The petridish should be saturated with solvent vapours. 4. Solvent front should be marked immediately after removing teh chromatogram, since it disappears rapidly. 5. The outline of each amino acid should be immediately marked with pencil, Do not mark with ball point or ink pen. Observations and Result Attach the chromatograph prepared in the space provided on next page. Observation: Circular paper chromatography Calculations Calculate the R f value as indicated: R f value = distance travelled by the substance distance travelled by the solvent front R f value of amino acid 1 Distance travelled by the solute =... Distance travelled by the solvent front =... Therefore R f value =... R f value of amino acid 2 Distance travelled by the solute =... Distance travelled by the solvent front =... Therefore R f value =... R f value of amino acid 3 Distance travelled by the solute =... Distance travelled by the solvent front =... Therefore R f value =... Result Record your results here as indicated: The R f of the amino acids in the mixture are

16 Nutritional Biochemistry Thus, Amino acid 1 is... Amino acid 2 is... Amino acid 3 is... Circular paper Chromatography Submit your experiment for evaluation Counsellor signature

17 ESTIMATION OF NITROGEN AND PROTEIN CONTENT USING MICRO KJELDAHL S METHOD Aim: To estimate the amount of ammonium sulphate in the given solution by Micro Kjeldahl method. Amino Acids and Proteins EXPERIMENT 3 Date:... Reagents Collect the following reagents to conduct the experiment: 40% NaOH Tashiro s indicator - 2% boric acid + methyl red + methylene blue Phenolphthalein Standard sulphuric acid (0.1N) Anhydrous sodium carbonate Methyl orange Principle/Theory Write the principle behind the kjeldahl s method in the space provided herewith. You can look up section 5.3 for information on this methodology. Look at the Kjeldahl s apparatus and draw the diagram. This will enable you to remember the distillation process. 147

18 Nutritional Biochemistry The Kjeldahl s apparatus Procedure You would realize that this proceedure is carried out in two steps. In the first step the standardization is done followed by the sample titration in the second step. Carry out the experiment following the proceedure enumerated herewith:- 1. Standardization of H 2 - Prepare a solution of 0.1 N H 2 by diluting 2.8 ml of the concentrated acid to 1000 ml in a volumetric flask. This is approximately 0.1N. Standardize the acid against 0.1 N Na 2 CO 3 solution. Accurately weigh g of anhydrous Na 2 CO 3. Transfer to a 100 ml volumetric flask and make up the volume to the mark with distilled water. Pipette 10 ml of this solution in a conical flask and titrate against the diluted H 2 prepared using 1% solution of methyl orange as an indicator. Calculate the exact normality of H 2 and dilute it accordingly to obtain exactly N H Sample titration- Take the given ammonium sulfate solution into a 100 ml volumetric flask and dilute to the mark with distilled water. Mix well. Set up the steam distillation apparatus and pass steam through the apparatus for cleaning it. After the apparatus is cleaned, pipet 10 ml Tashiro s mixture into a 100 ml conical flask and place it at the delivery end of the apparatus making sure that the delivery tube is dipping in the solution. Add 10 ml of the dilute sample from the funnel in the apparatus and few drops of phenolphthalein. After this wash the funnel with about 5 ml distilled water to ensure that all the ammonium sulphate has reached the distillation flask. Then add 10 ml of 40% NaOH and immediately close the stopper. Now heat the flask to pass the steam through this mixture. The liberated NH 3 from ammonium sulphate is collected in the Tashiro s mixture in the receiving flask. Continue distillation until the level of the solution in the receiving flask becomes approximately double. Remove the receiving flask and rinse the tip of the delivery tube with distilled water. Titrate the solution in the flask against standard H 2 till the color changes from green to pink. Calculate the amount of ammonium sulfate in the given solution. Precautions 1. The apparatus should be airtight to prevent leakage of NH The tip of the delivery tube should be immersed in Tashiro s mixture in the receiving flask. 3. After completion of distillation, remove the receiving flask first to prevent back suction. Results and Observations Record your observations in the format given in this section. Follow the reactions and the quations spelled out for your convenience herewith and fill up the gaps as indicated.the equations in this section require a lot of concentration and systematic study. So please be very attentive and alert while writing your observations and results Standardization of H 2 Normality of Na 2 CO 3 solution = 0.1 N

19 Volume of 0.1 N Na 2 CO 3 solution = 10 ml Volume of 0.1 N H 2 required Amino Acids and Proteins S.No. Pilot Initial Buret reading (ml) Final Difference Titer value =..(a) ml Calculations Now carry out the calculations as indicated: N1V1 = N2V2 Where N1 = Normality of Na 2 CO 3 solution =.. V1 = Volume of 0.1 N Na 2 CO 3 solution =.. ml N2 = ( to be calculated) V2 = (a) ml (i.e. the titre value) Now write the values for V1, N1 and V2 and calculate N2. N2 = N1 V1 =.... =.. (b) N =...N H SO 2 4 V2 2. Sample titration: Given Unknown sample taken in the volumetric flask and volume made to 100 ml with distilled water Volume of dilute (NH 4 ) 2 solution for distillation = 10 ml Volume of.. (b) N H 2 required S.No. Pilot Initial Buret reading (ml) Final Difference Titer value =.(c) ml 1000 ml of 1 N H 2 = 1000 ml of 1N NH 3 = 1000 ml of 1N (NH 4 ) 2 = 1000 ml of 1 N Nitrogen = 14 g of Nitrogen (based on molecular weight calculations) therefore, 1000 ml of 1N H 2 = 14 g Nitrogen 0. 1 ml of 1N H 2 = 14 mg Nitrogen 0. 1 ml of 0.1 N H 2 = 1.4 mg Nitrogen 0. 1 ml of (b) N H 2 =.(b) 1.4 =..(d) mg Nitrogen (c) ml of (b) N H 2 = c d =..(e) mg Nitrogen 10 ml of dilute solution contains.. e mg nitrogen 149

20 Nutritional Biochemistry 100 ml of dilute solution contains = 100 (e) = (f) mg of nitrogen 10 Results The unknown sample contained. mg nitrogen. Submit your experiment for evaluation.... Counsellor signature 150

21 DETERMINATION OF PROTEIN CONTENT USING BIURET METHOD The Biuret reaction was one of first colorimetric assays developed for protein estimation. It is most often used in applications requiring a fast but not a highly accurate value. Amino Acids and Proteins EXPERIMENT 4 Date:... Let us carry out this experiment and learn the oldest colorimetric method for protein estimation. Aim: To plot a standard curve for albumin and determine the protein content in the given solution. Apparatus We would require the following appratus to carry out the experiment: Test tubes Borosil glass (6x3/4 inch) Funnel Volumetric flasks 100 ml Glass marker/permanent marker Conical flasks Labels Pipettes 1 ml, 5 ml, 10 ml Spectrophotometer or colorimeter Beakers Single pan balance Measuring cylinder Butter paper for weighing Reagents Collect the following reagents to conduct the experiment: N NaOH solution - Dissolve 8 g of NaOH pellets in 1 litre of distilled water. 2. Biuret reagent - Dissolve 90 g of Rochelle salt (sodium potassium tartarate) in 500 ml of 0.2 N sodium hydroxide solution. Add 10 g of copper sulphate and 10 g KI and volume is made to 2 L with 0.2 N alkali. 3. Working standard - Dissolve 200 mg albumin in 100 ml distilled water (2 mg/ml). Principle We have already studied about the principle of biuret method earlier in section 2.3. Look up the section now and write down the principle here in the space provided based on your understaning about the method. 151

22 Nutritional Biochemistry Procedure Now carry out the proceedure as enumerated herewith: 1. Preparation of standard albumin: A standard solution of 2 mg albumin/ml is prepared in distilled water. For this weigh 200 mg of albumin and transfer This amount into a 100 ml volumetric flask. Make up the volume to the mark with distilled water. 2. Colour development for standard solution: Take different volumes of standard albumin solution (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 ml) in different test tubes In each case, make up the volume to 3 ml with distilled water. Add 3 ml of biuret reagent to each test tube and keep the tubes at room temperature for 30 minutes for colour development after shaking. 3. Preparation of unknown solution: Take the unknown solution and dilute the given solution to 100 ml mark and take one volume (e.g. 2.0 ml) in duplicate similar to that prepared for the standard. Add all other reagents just as you did for the standard solution. 4. Preparation of blank: Pipet 3 ml of distilled water. (Do not add the standard solution in the blank). Now repeat the above procedure for colour development. 5. Measurement of O.D: Read the absorbance of all solutions at 540 nm after setting the colorimeter to 100% transmittance with the blank solution. 6. Standard Curve: Plot a graph between concentration albumin in standard solution and O.D. 7. Calculate the amount of protein with the help of the standard curve. Plotting and Calculations I. Preparation of Standard Albumin 200 mg of albumin was dissolved in distilled water and the volume made to 100 ml. therefore, the strenght of the standard albumin solution = 2 mg/ml Since 1.0 ml of the standard albumin solution = 2.0mg albumin ml of the the standard albumin solution contains = mg albumin ml of the the standard albumin solution contains = mg albumin ml of the the standard albumin solution contains = mg albumin ml of the the standard albumin solution contains = mg albumin ml of the the standard albumin solution contains = mg albumin II. Colorimetric Reading for Standard Solution Record the colorimetric reading for standard solution here in the format provided. For concentration of albumin write the amount calculate above in point I. Note the O.D reading in column V. I Volume of Std. Sol. II Conc. of albumin (mg) III Distilled Water (ml) IV Biuret Reagent (ml) V Optical Density at 540 nm VI Optical Density for 2.0 mg albumin

23 Mean OD for 2.0 mg albumin = = (A) = mg. 6 Now prepare the standard curve for albumin (on a graph paper) with concentration of albumin (figure included in item II above) on x-axis and the optical density (figures in item V above) on y-axis. Stick the graph in the space provided on page. Amino Acids and Proteins III. Colorimetric Reading for Unknown Solution a) Optical density of unknown albumin sample, 1 = 2 =... mean optical density of unknown sample = (B). IV. Determination of Albumin Content a) From Standard Curve On the standard curve prepared earlier, plot the OD of the unknown solution on the Y- axis. Now check the corresponding concentration of albumin (in the unknown solution) for this OD on the X-axis. Say the value obtained is E. So 2.0 ml of unknown solution contains E ml of albumin ml of unknown solution will contain = E 100 = F =..mg. 2 Write the calculations in the space provided: Observed Value = F =. Expected value = D = (Take it from the counsellor) Calculate the % error (based on optical density) using the formula given herewith: = D F 100 D 153

24 Nutritional Biochemistry 154 Standard curve between concentration of standard albumin solution and OD

25 b) From Optical Density Mean O.D for 2.0 mg albumin = A mg... If OD of albumin is A.., then concentration of albumin is = 2.0 if OD of the unknown solution is B, then concentration of unknown albumin solution = 2.0 B = C =.mg A So C.. mg is present in 1ml of unknown ml of unknown contains = C x 100 = = F mg. Amino Acids and Proteins Observed Value = F =. Expected Value = D = (Take it from the counsellor) Calculate the % error (based on optical density) using the formula given herewith: = D F 100 D Result The given solution contains... mg of protein. Now, also answer the review questions given next. These will help you consolidate your understanding on this topic. Review questions 1. Why is the nitrogen content multiplied with 6.25 to obtain the protein value? 155

26 Nutritional Biochemistry 2. Why do proteins give biuret test? 3. Why do you add NaOH during distillation of ammonium sulphate? Submit your work for evaluation.... Counsellor Signature 156