Chromatographic Separation of Amino Acids, pH Profile of Amino Acids - Lab Report Example

Biochemistry Laboratory Reports: A Practical Portfolio Practical 1. Chromatographic Separation of amino acids Introduction: Chromatographic separation technique employs a stationary phase on which the solute moves accompanied by the mobile phase (solvent). The separation of amino acids depends on the rate at which it moves while in simultaneous contact the two phases. This migration depends upon the molecular weight, shape and structure and polarity of the molecule to be separated. The ratio of the distance covered by the molecule to be separated and by the solvent is called the relative front and is used to identify an unknown amino acid. Since the amino acids are colorless, a ninhydrin solution is used to develop color for identification. Paper chromatography is actually liquid-liquid chromatography, the paper should not be considered as solid phase, but the water molecules trapped in the cellulose of the paper form the ‘stationary’ phase. To saturate the cellulose, most paper chromatographic solvents have some amount of water in it. So the components with very high water solubility will move slower ( Paré and Bélanger, 1997). Method: A 60:40 v/v acetonitrile: ammonium ethonate mobile phase is made, pH 7.2, and placed in a covered tall jar. Aqueous solutions of amino acids are spotted on the specified location (origin) on the stationary phase using a capillary and allowed to dry. The stationary phase is then put into the jar with mobile phase and allowed to run for 40 minutes. Mark the solvent front. Make sure the solvent stays well below the top edge. The stationary phase is then dried and sprayed with ninhydrin solution in the fume hood, and heated to allow the color to develop. Results: Fig 1. Cromatogram of amino acids showing relative migration. Blue arrows show finger prints. From the Rf values (Table 1) it seems neither molecular weight nor the polarity had any significant effect on the migration. Glycine being the smallest did not travel the farthest. Looking at both, Rf and the color developed the sample X is Lysine and Y is Proline. Finger prints are seen on both the lateral sides of the paper, probably at the places used to handle the paper. They appear due to reaction between ninhydrin and the terminal amines of the lysine de-bonded from the amino acid. Also the sweat-gland secretions in the ridges of the fingers has proteins too (Sens,Simmons and Spicer, 1985). Table 1. Relative fronts of respective amino acids. Amino acid Solute distance (mm) Solvent distance (mm) Rf Polarity Approximate Mt gm/mol Proline 45 59 0.76 NP 115 Glutamic acid 41 0.69 P/acidic 147 Leucine 47 0.8 NP 131 Valine 45 0.76 NP 117 Glycine 38 0.64 NP 75 Lysine 37 0.62 P/basic 146 Y 44 0.74 x 38 0.64 Legend: P, polar, NP, non-polar amino acid Conclusion: Paper chromatography can be used to separate amino acid from a mixture of amino acids. The migration of amino acids on the solid phase is a complex interplay between the molecular eight, shape, structure and polarity of the amino acids and their affinity towards the solid and the mobile phase. Use of ninhydrin to identify amino acids can be extrapolated in the forensics to locate finger print. Reference List Paré, J. R. J. and Bélanger, J. M. R., 1997. Instrumental Methods in Food Analysis. Amsterdam:Elsevier. Sens, D.A., Simmons, M.A. and Spicer, S.S., 1985. The analysis of human sweat proteins by isoelectric focusing. I. Sweat collection utilizing the macroduct system demonstrates the presence of previously unrecognized sex-related proteins. Pediatr Res,19(8),pp.873-8. Practical 3: pH profile of Amino Acids Introduction: Amino acids are both amphiprotic (can accept or donate protons) and amphoteric (can react with acids or bases). The carboxylic acid group being acidic tends to lose a proton, and the amine group being basic tends to gain a proton. Amino acids in solution exist as an equilibrium mixture of neutral molecules and dipolar ions called zwitterions. In acid solution the zwitterion converts to a positively charged ion and in alkaline solution the zwitterion converts to a negatively charged ion. For each amino acid there is a particular pH, the isoelectric point, at which the amino acid exists as the neutral zwitter ion. Below this pH the amino acid exists as a positively charged ion (cation). Above this pH the amino acid exists as a negatively charged ion (anion). These studies are important because transport of amino acid across the intestinal walls, reabsorption by the kidney glomeruli or intercellular transport, are all pH dependent. A disturbance in this transport can cause medical problems (Maenz, Chenu, Breton and Berteloot, 1992). Besides, being acids, all amino acids have a buffering capacity. Histidine, with pKa of 6.8, close to the physiological pH, can buffer some pH changes in the blood and can function as one of the blood-buffering systems. Proteins rich in histidine also show this capacity (Szebedinszky and Gilmour, 2002). Method: 10 cm3 of glutamic acid solution was prepared and pH measured. Using a burette, small amounts of 0.1 M HCl was added to the amino acid solution initially with constant stirring and pH measurements. The amount of acid was gradually increased till the pH fell to 1.5. The exercise was repeated with 0.1M NaOH solution. A graph was plotted of pH against the volume of acid/base added. Results: Table 1: pH profile of glutamic acid HCl Volume cm3 pH NaOH Volume cm3 pH 0.5 1.4 3 4.0 1.0 1.6 6 4.2 1.5 1.5 9 4.6 2.0 1.5 12 5.3 2.5 1.5 15 5.8 3.0 1.5 18 6.5 4.5 1.4 21 9.8 5.0 1.4 24 10.5 6.0 1.4 27 11.0 7.0 1.4 30 11.9 8.0 1.4 33 12.2 9.0 1.4 36 12.3 10.0 1.4 39 12.4 12.0 1.4 42 12.5 13.0 1.3 45 12.5 Fig 1: Titration curve of glutamic acid against NaOH Fig 2: Titration curve of glutamic acid against HCl Dissociation of Glutamic acid can be represented as follows: (Picture from http://cti.itc.virginia.edu/~cmg/Demo/analyzeAA/glutamic/glutamic.html) At low pH, below pH = 2.0, glutamic acid carries a net positive charge due to two carboxyl groups. After the addition of one equivalent of base, the lowest pKa group will have been deprotonated, giving no net charge on a glutamic acid. We do not see this in the graph since the readings started at pH 4 (Fig 1). The half-way point of this first wave of the titration will be at pH = pK1 = 3.2 and the pH of the first equivalence point will occur at the average of the first two pka's. This is also the isoelectric point of glutamic acid, the pI. Addition of more OH- begins to remove the second acidic proton, from the COOH of the R-group. Fig. 2 shows the titration of glutamic acid against HCl. It shows no amount of acid has any effect on the glutamic acid ionic species. Conclusion: The graph does not display the actual biphasic graph, peculiar of glutamic acid. This is due the fact that instead of starting to add small amounts of NaOH initially, 3 cm3 was added and that missed the readings which could have shown the pKa1. However from the graph it can be seen that glutamic acid will try to resist any change in pH around 8+1. Reference List Szebedinszky, C. and Gilmour, K.M., 2002. The buffering power of plasma in brown bullhead (Ameiurus nebulosus). Comp Biochem Physiol B Biochem Mol Biol,131(2),pp71-83. Maenz, D.D., Chenu, C., Breton, S. and Berteloot, A., 1992. pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles. J Biol Chem, 267(3),pp1510-6. Prac 4: The Iodine Number of an Oil or Fat Introduction: Iodine number is the measure of degree of unsaturation of a fat or oil and is defined as the number of grams of iodine taken up by 100g of fat. In principle, fatty acids react with iodine, resulting in the addition of iodine at the C=C double bond site.  In this reaction, iodine monochloride (Wij's solution) reacts with the double bonds to produce a di-halogenated single bond. After the reaction is complete, the amount of iodine that has reacted is determined by adding a solution of potassium iodide to the reaction product. This causes the remaining unreacted ICl to form molecular iodine. The liberated I2 is then titrated with a standard solution of 0.1N sodium thiosulfate. Iodine monochloride is used as a catalyst. The process is used to characterize oils, to follow hydrogenation process in refining and an indicator of lipid oxidation (Neilsen, 2010). It is also used to see the relationship between types of dietary fats and the abdominal fat deposition (Wongsuthavas, et al., 2008). Method: 1 cm3 of chloroform is added to about 0.3 gm sample and then using a burette, 25cm3 of Wij's solution is added. The flask is then kept in the dark for 30 - 45 minutes for the addition reaction to occur. A spatula of potassium iodide is then added and mixed well. After dilution with water, the solution is titrated using starch as an indicator against 0.1 M solution of sodium thiosulphate. Wij’s solution is sensitive to temperature, moisture and light. Small changes in temperature affect titer values of the Wij’s solution. So it is a good practice to titrate the blanks and samples at the same time. Also a 50-60% excess of Wij’s solution is preferred and addition of 10 cm3 2.5% mercury (II) acetate in glacial acetic acid (after Wijs solution is added) will shorten the reaction time to 5 minutes (Rotz, 1984). Result: The following formula was used to calculate the iodine number I2 No I2 No = ( B-T) x 0.1 x 127 x 100 1000 x W From the formula, the Iodine number for the given fat is: I2 No = (50.8-37.6) x 0.1 x 127 x 100 1000 x 0.31g = 54.1 I2 No for the given vegetable Oil: I2 No = (49.2-21.2) x 0.1 x 127 x 100 1000 x 0.31g = 114.7 Conclusion: According to Rotz (1984), the fat is most likely to be lard and the vegetable oil could be Rape seed oil. Reference List Rotz, R. Iodine Value of Edible Oils and Fats According to Wijs (AOAC Method). Mettler Toledo. [Online] Available at < http://www.ibchem.com/IB/ibfiles/organic/org_doc/iodine_value.pdf> [Accessed 28 April 2012]. Neilsen, S.S. ed., 2010. Food Analysis. New York: Springer. Wongsuthavas, et al., 2008. Influence of amount and type of dietary fat on deposition, adipocyte count and iodine number of abdominal fat in broiler chickens. J Anim Physiol Anim Nutr (Berl),92(1),pp92-8. Practical 5: Saponification of a Triglyceride Introduction: Triglycerides can be hydrolyzed by several procedures; the most common methods use alkali or enzymes. Triglycerides are composed of three fatty acids linked to glycerol by fatty acyl esters (-O-CO-R). The fatty acids (FAs) may be saturated (no C=C double bonds) or unsaturated. Liquid triglycerides are oils, while solid triglycerides are fats. The “saponification number” is used as an indicator of fatty acid chain length in triglycerides. The value is simply a measurement of the cm3 of KOH required to complete the hydrolysis of one gram of fat or oil. Triglycerides containing long fatty acids will have a lower saponification number than triglycerides with shorter fatty acids. Saponification literally means "soap making". It is important in oil industry to know the amount of free fatty acid present in waste, since this determines the refining loss. Since fatty acids are important components of the cell membrane their studies in biological samples is of importance. Larger chain length FAs are poor substrate of the cyclooxygenase and lipoxygenase enzymes, and so are not incorporated into the cell membranes (Robinson, Johnson, Ferrante and Poulos, 1994). Method: 2.0 gms of TG was weighed and put in a round bottom flask. 50cm3 of ethanolic potassium hydroxide was added using a safety pipette, and the solution refluxed for 30 minutes and then cooled. (KOH is very corrosive, extreme care is needed while handling!). Do not forget to turn on the running water! Upon cooling, a few drops of phenolphthalein were added to the solution and titrated against 0.5 mol dm-3 HCl. Saponification number and average molar mass of fat was calculated using the following formulae. With advance in technology now Fourier transform infrared spectroscopy is available for rapid estimation of the saponification number of triglycerides (van de Voort, Sedman, Emo and Ismail, 1992). Saponification value (S) = (B-T) x 0.5 x 56 mg/g fat W B = Blank value T = Test value W = Weight of fat/oil Average molar mass of fat = 3 x 56 x 1000 S Results: B = 22.0-15.5 = 6.5 ml T = 30.5-14.5 = 16 ml S = {(16.0 -6.5) x 0.5 x 56}/2 = 133 mg/gm fat Average molar mass of fat = 3 x 56 x 1000 133 = 1263.2 gm/mol Subtracting molar mass of glycerol, Average mass of just the 3 FAs = 1263.2 - 92.2 = 1171 gm/mol Average molar mass each FA = 1171/3 = 390 gm/mol No of carbon atoms present = 390/12 (12 is molar mass of each carbon atom) = 32 carbon atoms approximately Adjusting for oxygen and hydrogen atoms, each FA is around 30 long, triacontatetraenoic acid (30:4, n-6) (Robinson, Johnson, Ferrante and Poulos, 1994). Conclusion: The given sample may be having a triglyceride with very long chain fatty acids as indicated by the saponification number. It can be 30:4, n-6 triacontatetraenoic acid. It is consistent with the notion that sap no is inversely proportional to the chain length of the fatty acids (Das, 1978). Reference List Das, D., 1978. Biochemistry. Kolkata: Academic Publishers. Robinson, B.S., Johnson, D.W., Ferrante, A. and Poulos, A., 1994. Differences in the metabolism of eicosatetraenoic (20:4(n - 6)), tetracosatetraenoic (24:4(n - 6)) and triacontatetraenoic (30:4(n - 6)) acids in human neutrophils. Biochim Biophys Acta,1213(3),pp325-34. van de Voort, F. R., Sedman, J., Emo, G. and Ismail, A. A., 1992. Rapid and direct lodine value and saponification number determination of fats and oils by attenuated total reflectance/fourier transform infrared spectroscopy. Journal of the American Oil Chemists' Society, 69(11), pp1118-23. Practical 6: Chromatographic Separation of Carbohydrates Introduction: Chromatography is essentially a physical method of separation in which components of a material are distributed between two phases. One phase is stationary (stationary phase) while the other (the mobile phase) percolates through it in a definite direction .The principle behind this technique is that the solvent mixture travels up the plate by capillary action, the components from the sample travel up at different rates due to their interaction with the coating on the plate (the stationary phase) and the moving solvent system (the mobile phase). Method: A jar containing 40:50:10 butan-1- ol:acetone:phosphate buffer (pH 5) 1cm from the bottom was prepared and covered. On the plate, samples were spotted well away from the edge of the plate using a capillary. Enough sample volume was spotted using repeated spotting and drying. The solvent was allowed to run within 1cm from the top edge. The plate was dried in fume cupboard. The samples were visualized by dipping the plates in a mixture of anisaldehyde with 0.5% concentrated sulphuric acid. The plate was heated until the color developed. The method is rapid and inexpensive and is widely used to check sugars in beet juices, and other factory made juices. It can also be employed, for example, to see the different fermentation products by several species of yeasts (Vrbaški, Jakovljević and Lepojević, 1992). Results: Table 1: Rf values of different carbohydrates Exercise 1 Exercise 2 Sugar (approx. molar mass gm/mol) Solute front mm Solvent front mm Rf Sugar (approx. molar mass gm/mol) Solute front mm Solvent front mm Rf D-Ribose (150) 33 74 0.45 D-Glucose 25 74 0.34 D-Glucose (180) 21 0.28 D-Galactose 18 0.24 D-Fructose (180) 24 0.32 Maltose (342) 20 0.27 D-Galactose(180) 16 0.22 Lactose (342) 10 0.13 The exercise 1 entailed monosaccharaides. As can be seen from the Table 1, Ribose being a pentose, with lower molar mass than the other hexoses, travels the farthest. Amongst the hexoses, the sugars have migrated based on their polarity and solubility. The solubility of fructose>glucose>galactose and so is their Rf values. Exercise 2 involved mono- and disaccharides separation. The results of this exercise reveal that disaccharides migrate slower than monosaccharaides in general, but again this is dependent on their solubility. Amongst the disaccharides, lactose migrates lesser than maltose because it is sparingly soluble in water. It is interesting to note that D- glucose and D-galactose migrate differently on the two plates, practically under the same conditions (exercise 1 and 2). Conclusion: The chromatographic separation of carbohydrates is based on the molar mass of the sugar. Pentoses move faster than hexoses, and monosaccharaides move faster than disaccharides, provided they have same polarity. Hence this separation technique separates carbohydrate molecules based on molar mass and polarity. Reference List Vrbaški, L., Markov, S., Jakovljević, J. and Lepojević, Ž., 1992. Improved thin-layer chromatography separation of carbohydrates in wort and beer. Biotechnology Techniques, 6(5), 413-416. Practical 7: Estimation of Reducing Carbohydrates Introduction: This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Reducing carbohydrates may be estimated by reaction with 3,5-dinitrosalicylic acid (DNS), which is reduced to 3-amino-5-nitro-salicylic acid, the anion of which has a strong red colour. The red colour is directly proportional to the amount of glucose present and is readable at 540. Dissolved oxygen in water can interfere with the assay, so sulphite is added to the assay to absorb the dissolved oxygen. The method is convenient and inexpensive but has low specificity (Miller, 1959). Method: DNS solution is added to 0.8g/ dm3 glucose solution in several dilutions. The mixture is heated in a water bath at 90 degree Celsius. The tubes are then cooled, diluted with deionized water and read on a spectrophotometer at 540 nm. Proper blank is used (without glucose). Result: The obtained graph is almost linear, and can be used to estimate the unknown. Table 1: Absorbance readings for the glucose standard curve Concentration g/L Absorbance 540 nm 0 0.0 2 0.34 4 0.58 6 0.78 8 1.2 Unknown 0.305 Fig 1: Standard curve of glucose From Graph, y = 0.142X + 0.012 or 0.305 = 0.142X + 0.012 or X = (0.305 -0.012)/0.142 = 2.06 g/ dm3 Conclusion: The above method is inexpensive and convenient to assay reducing sugars. The unknown sample has about 2 g/ dm3 of glucose/reducing sugar in it. This method is fast; requiring around 15 min, hence can be used in pathological laboratories to estimate determining blood and cerebrospinal fluid sugar levels (Mohun and Cook, 1962). Reference List Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem,31(3),pp426-428. Mohun, A.F., and Cook, I.J., 1962. An improved dinitrosalicylic acid method for determining blood and cerebrospinal fluid sugar levels. J Clin Pathol, 15,pp169-80. Practical 8: The acid and enzymatic hydrolysis of glycogen Introduction: In vitro, glycogen content can be demonstrated by its total hydrolysis with acid. The acid hydrolysis does not occur right away but occurs gradually with time. One can follow the hydrolysis reaction by sampling the reaction mixture at regular interval of time. Acid breaks the glycosidic linkages of the glycogen. Glycogen is present in all cells of the body, except neurons. Its metabolism is different and important in liver and muscles. Glycogen is not only important for glucose storage in muscles, but is medically important as well. Glycogen storage disease is commonly seen and there are 7 different types of this disease. These are inborn metabolic disorders involving enzymes of glycogen metabolism. Due to this reason, enzymatic kinetics of hydrolysis of glycogen is also important to study (Guinovart, 2007). Method: Acidic Digestion: Seven tubes are prepared with 1.2 M NaOH in each and labeled 0 through 30 minutes in interval of 5 minutes. 0.4 ml of glycogen and 0.6 ml of concentrated HCl are added in a larger tube. 0.1 cm3 reaction mixture is withdrawn into the tube labeled 0 minutes right away, and then at regular interval of 5 minutes, for which the acid digestion is put in a boiling water bath. For colorimetric estimation of the glucose released, 0.5 cm3 of 3,5-dinitrosalicylate reagent is added to the stopped incubation tubes and heated for 5 minutes. It is then diluted with water and read at 540 nm in the spectrophotometer. It is approximated that total hydrolysis occurs after 25 minutes. Enzymatic Hydrolysis: 0.4 cm3 of glycogen was incubated with 0.6 cm3 of the enzyme amylase at room temperature. 1cm3 of 3,5-dinitrosalicylate reagent was pipetted in several tubes labeled with regular time interval required. This reagent even stops the reaction. The reaction mixture was pipetted to these labeled tubes at regular time interval. Upon dilution with water, the color is read at 540 nm. Results: Table 1: Absorbance readings for the glucose standard curve Concentration g/ dm3 Absorbance 540 nm 0 0.0 2 0.34 4 0.58 6 0.78 8 1.2 Figure 1: Standard curve for the glucose estimation. Table 2: % hydrolysis of glycogen as a function of time Acid Hydrolysis Enzyme Digestion Time min Aborbance 540 nm Conc of Glucose g/L % hydrolysis Time min Aborbance 540 nm Conc of Glucose g/L % hydrolysis 0 0.0 0 0 0 0 0 0 1.5 0.778 5.4 34.2 1.5 0.134 0.9 14.1 3.0 0.977 6.8 43.0 3.0 0.257 1.7 26.6 4.5 1.176 8.2 51.9 4.5 0.358 2.4 37.5 6 1.312 9.2 58.2 6 0.555 3.8 59.4 9 1.548 10.8 68.3 9 0.665 4.6 71.9 12 1.692 11.8 74.7 12 0.797 5.5 85.9 15 1.704 11.9 75.3 15 0.841 5.8 90.6 20 2.260 15.8 100 20 0.889 6.2 96.9 25 1.948 13.6 ------- 25 0.92 6.4 100 Figure 2: Concentration of reducing sugar released by acid and enzyme hydrolysis as a function of time. Figure 3: Graph showing % hydrolysis of glycogen by acid and enzyme Figure 2 shows that the concentration of the reducing sugar released by the acid is much more than that released by the enzyme. It can be seen from the graph of Figure 3 that hydrolysis of glycogen by the enzyme, Amylase, follows a smoother path then the acid hydrolysis. Although acid starts the hydrolysis at a faster pace than the enzyme, it slows down a bit by the 6th minute in terms of its activity. Enzyme activity, however, continues at a constant rate. Conclusion: Hydrochloric acid can release more glucose from rat glycogen at any given time, compared to the enzyme amylase. This is because of the well-known fact that amylase cannot cleave the branch point alpha 1,6 glycosidic linkages of the glycogen, while acid can. Use of combination of the glycogen catabolizing enzymes, glycogen phosphorylase and debranching enzyme, could have given better results. Acid starts the glycogen cleavage at a faster pace initially compared to amylase. However, amylase acts at a constant rate on the glycogen but acid shows uneven rate of hydrolysis. Reference List Guinovart, J.J., 2007. Study of the regulatory mechanisms of glycogen metabolism, its alterations in pathologies and characterisation of new therapeutic targets. Scientific Report: Molecular Medicine Programme [Online] Available at: < http://www.irbbarcelona.org/files/File/15-meta-07.pdf > [Accessed 28 April 2012]. Practical 9: The effect of pH on the reaction velocity of salivary amylase Introduction: Salivary amylase starts starch digestion in the mouth itself. It splits the alpha-1,4 glycosidic bonds of glycans. From starch it can produce maltose, glucose and ‘limit dextrins’. One molecule of the enzyme can ‘attack’ a number of linkages on different glucose chains. The activity and stability of amylase depends on calcium and chloride ions. It has a molecular weight of 55 KD (Jacobsen, Melvaer, and Hensten-Pettersenm 1972). Like all enzymes, salivary amylases have optimum temperature, pH etc at which they show maximum activities. This exercise was done to see the optimum pH of this enzyme. When an alkaline solution of maltose is heated with methylamine, a bright violet-carmine colour develops read at 450 nm. The colour results from ring-opening, followed by rearrangement to form an ene-diol, and reaction with the methylamine. Method: Add 1 cm3 of enzyme solution to differently buffered (with different pH) substrate in separate tubes. Incubate at room temperature for 10 minutes and the stop the reaction by adding NaOH. Then add 5% methylamine hydrochloride solution to each test tube, mix, and heat in a boiling water bath for two minutes. Cool, dilute and read at 450 nm. Water can be used as blank. Then a graph of absorbance against pH to obtain the pH profile of the enzyme is plotted. Results: Table 1: Amylase velocity curve: pH vs absorbance pH Absorbance 450 nm 3 0 3.8 0.1 4.2 0.4 5.8 0.7 6.2 0.9 6.8 1.2 7.2 1.18 7.8 0.95 8.2 0.8 Fig 1: pH profile of salivary amylase at room temperature Results: The results show that at acidic pH between 2 and 4, the enzyme has negligible activity. Beyond pH 4, the activity picks up rather exponentially and maximizes at pH 7. It stars falling after pH 7. Conclusion: Salivary amylase has optimum pH of 7, at room temperature and in presence of chloride ions. This is consistent with the literature (Marini, 2005). Reference List Jacobsen, N., Melvaer, K.L., and Hensten-Pettersen, A., 1972. Some properties of salivary amylase: a survey of the literature and some observations. J Dent Res,51(2),pp381-8. Marini, I. 2005.Discovering an accessible enzyme: Salivary α-amylase : Prima digestio fit in ore: A didactic approach for high school students. Biochem Mol Biol Educ,33(2),pp112-6. Practical 10: Chromatography of 2, 4 DNP Derivatives of -keto acids Introduction: The principle of this method is based on the formation and subsequent separation by paper chromatography of 2,4 dinitrophenylhydrazone derivatives of -keto acids. -keto acids measurements in blood and urine are of importance in diabetes, malnutrition and protein deficiency. Taylor and Smith (1955), however are not satisfied with the usage of 2,4 DNP for laboratory purpose. They are of the opinion that 2,4 DNP does not appear to be completely satisfactory reagent for this purpose, it is not specific for -keto acids. In addition, multiple spots may be obtained owing to the separation of geometrical isomers of the -keto acids-hydrazone complexes and because of production of an artifact from the reagent. Hence they propose to use 2,4 diamino-nitrobenzene instead. Method: Mix the given keto acids with 2,4 DNP and incubate at room temperature for 10 minutes. Add 8 cm3of ethyl acetate to each tube and shake for 2 minutes. Discard the lower aqueous layer and add 5 cm3 of 10% sodium carbonate to the ethyl acetate portion and shake for 2 minutes. Discard the upper organic solvent and acidify the content with 1 cm3 of concentrated HCl and then with dilute acid. Extreme care is taken while handling concentrated acids. Add 1 cm3 of ethyl acetate to extract the yellow colour and spot enough at the origin of the chromatogram to leave a yellow spot. Result: As can be seen from the figure below, a keto acid and the sample (spot 2 and 3) have barely moved while one (spot 1) has moved significantly and shows two spots (1 of 1 and 2 of 1). The -keto acids spotted at spot 1 seems to have isoforms, and the two forms have migrated differently. The other two specimens may not be true keto-acid, including the sample, which shows negligible migration. Table 1: Rf for the different -keto acids. Spots Distance moved (cm) Solvent front (cm) Rf Spot1of 1 4.1 13.8 0.30 Spot2of 1 7.2 0.52 Spot 2 0.5 0.04 Spot 3 0.6 0.04 Fig 1: Chromatogram showing the migration of different -keto acids Conclusion: -keto acids can have isoforms, and these isoforms migrate differentially on the solid phase. According to Taylor and Smith (1955) this could be an artifact of the reagent 2,4 DNP also. Reference List Taylor, K.W. and Smith, M.J.H., 1955. 1:2 Diamino-4-nitrobenzene as a reagent for the detection and determination of -keto acids in blood and urine. Analyst, 953,pp 607-13. Practical 2: Changes in pH during acid-base neutralisation Introduction: Acids release protons while bases release hydroxyl ions. A chemical change takes place when an acid reacts with a base. Depending on the pH strength of the acid or base, neutralization may occur. If a base neutralizes an acid, there is no longer an acid or a base in solution and the solution would, therefore, lose its acid or basic properties. A neutral solution has a pH value of 7. pH is actually a measure of the concentration of hydrogen ions in the solution. A buffer is a solution containing a weak acid and one of its salts or a weak base and one of its salts. These studies are important any from waste water treatment to enzymatic reactions. Method: Strong acid vs strong base: A 50 cm3 burette is filled with the given base; the acid is taken in a conical flask. Few drops of the indicator are added. 1 cm3 of base is run into the flask at a time with constant stirring using a magnetic stirrer. Note the pH at each interval. When the neutralization approaches, the base is added in smaller volumes. Weak acid strong base: Repeat the above exercise with acetic acid and NaOH. To study the pH behaviour of carbonate-bicarbonate buffer, 25cm3 of 0.05M sodium carbonate solution is titrated against 0.1 M hydrochloric acid. Result: Table 1: Volumetric titrations of 0.1 M Glycine with HCl or NaOH and pH Volume of HCl added (cm3) pH Volume of HCl added (cm3) pH 0.5 5.5 1.0 5.3 1.5 5 2.0 5 2.5 4.7 3.0 4.5 3.5 4.5 4.0 4.4 4.5 4.4 5.0 4.3 5.5 4.3 6.0 4.3 6.5 4.3 7.0 4.2 7.5 4.2 8.0 4.1 8.5 4.1 9.0 4.1 9.5 4.1 10.0 4 10.5 4 11.0 4 11.5 4 12.0 3.9 12.5 3.9 13.0 3.9 13.5 3.9 14.0 3.9 14.5 3.9 15.0 3.9 15.5 3.9 16.0 3.9 16.5 3.8 17.0 3.8 17.5 3.8 18.0 3.8 18.5 3.8 19.0 3.8 19.5 3.8 20.0 3.8 20.5 3.8 21.0 3.8 21.5 3.8 22.0 3.8 22.5 3.8 23.0 3.8 23.5 3.8 24.0 3.8 24.5 3.8 25.0 3.8 25.5 3.8 26.0 3.8 26.5 3.8 27.0 3.7 0.1 8.4 0.2 9.1 0.4 9.3 0.6 9.4 0.8 9.5 1.2 9.7 1.6 9.9 2 10 2.4 10.1 2.8 10.2 3.4 10.3 4 10.4 4.6 10.5 5.2 10.6 5.8 10.7 6.4 10.8 7 10.9 7.6 11 8.2 11.1 8.8 11.2 Figure 1: Titration of 0.1 m Glycine against HCl Figure 2: Titration of 0.1 M Glycine against NaOH Result: From Fig 1 it is evident that when 0.1 M glycine is titrated against strong acid, there is gradual decrease in pH, while when it is titrated against NaOH, the pH increases gradually. Fig 2 shows unusual curve against NaOH. The curve should be biphasic, but instead it shows gradual increase. Due to the lack of biphasic graph, it is not possible to calculate the pKa. The reaction of sodium carbonate with HCl occurs as follows: Na2CO3 + HCl ------------------ NaCl + Na2HCO3 Na2HCO3 + HCl -------------- NaCl + CO2 +H2O Overall reaction: Na2CO3 + 2HCl ---------------- 2NaCl + CO2 +H2O 2H3O+ + CO32- ------ H2CO3 +2H20 Due to two end points in the titration, 2 pKa values are expected. Conclusion: Acid-base titrations are useful in understanding the buffering capacity of a system. Carbonic acid-bicarbonate buffer system is an important physiological buffer in the body. Kreb Cycle: Set of Experiments: 1. If it gives O consumption of 5 moles it has pure pyruvate in. Batch 1 2. If the sample + Arsenite gives O consumption of 2 moles, sample is contaminated with malate. Batch 6 3. If the sample gives O consumption of only 1 mole, it is pure Malate. Batch 3 4. If the sample + pure Pyruvate + Arsenite give no O consumption, it has mixture of pyruvate and alpha-keto glutarate (KG). Batch 5 5. If the sample gives O consumption of 3 moles, sample is pure Alpha-KG. Batch 2 6. If the sample + malonate gives no O consumption, it is pure succinate. Batch 4 7. If the sample + malonate gives 5 moles of O consumption, it is mixture of pyruvate and succinate. Batch 7 These seven experiments will distinguish between the samples. NADH, FADH2, P:O ratio estimation. Pyruvate: Produces 4 NADH and 1 FADH2 molecules. Hence the P:O ratio is 14:5. Succinate: Produces 1 NADH and 1 FADH2 molecules. Hence the P:O ratio is 14:2. Fumerate and Malate both: Produces 1 NADH and 0 FADH2 molecules. Hence the P:O ratio is 3:1. Read More