Experiment 4 - Effect of pH Changes on Detection of Macromolecules Experimental

Question

Experiment 4 - Effect of pH Changes on Detection of Macromolecules Experimental Procedure thesis that connects low pH and the detection of macromolecules Determine an experimental procedure. Include all variables and the control and experimental groups. 2. a. Materials to choose from: i. Any or all of the solutions in Table 1. ii, 1% Hydrochloric Acid IMAC ii. pH Paper (Broad and Narrow) iv. Any or all of the test solutions (Benedict's, Iodine, Lipid Paper, Biuret) 3. Perform the experiment. 4. Record data 5. Analyze data and evaluate if it supports the hypothesis.

Explanation / Answer

1. Hypothesis: Low pH affects detection of macromolecules

Similar to acid-base systems, macromolecules also accept and donate protons acting as acids and bases. But instead of one such group that participates in this proton exchange and owing to the size of the macromolecule, there are multiple groups on the macromolecule that accept and donate protons. These groups, acidic or basic, act as weak acids and bases with Ka values that determine the dissociation extent depending on the pH of the solvent in which the macromolecule is placed. Therefore, changes in the pH of the system decide the number of groups on the macromolecule being protonated and deprotonated, and hence the properties of the macromolecule. For example, the solubility of protein is affected by changes in pH. In highly acidic system, the groups on the protein macromolecule will be positively charged, and subsequently will be attracted to the acid anions causing them to precipitate.  

Likewise, the reagents used for detection of macromolecules can also be affected by changes in pH. Few possibilities are listed below.

1) In Biuret test, a pink-purple or violet blue coloured complex is formed when the proteins are made to react with an alkaline solution of copper sulphate. The alkaline solution is essential to initiate the reaction and form the biuret complex. This reaction will be aborted in presence of acid, i.e. low pH.

2) In Benedict's test, the copper (II) ions in the blue colored Benedict's solution are reduced to Copper (I) ions, which causes the solution to change its colour to orange red/ brick red. This happens due to the reducing property of monosaccharides (reducing sugars), when they are heated in presence of Benedict's solution. Complex sugars such as disccharides and polysaccharides fail to repond to this test. Barfoed's test is essentially the same reaction with Benedict's reagent except in lower pH (~ pH 4.5) and reduced heating time. While no color change will indicate di- and polysaccharides, rusty or brownish-red color will indicate monosaccharides.

3) In iodine test, the triiodide ion is formed first that forms a coordinate complex with the helically coiled starch macromolecule. The iodine is centrally placed within the coil due to adsorption. The color obtained depends on the length of linear chain available for adsorption. At low pH, when the system is more acidic, the color changes to dark blue, while in presence of NaOH, the complex breaks and the color disappears.

2. Experiment

Hypothesis: Low pH affects detection of macromolecules

Aim: To check the effect of low pH on detection of macromolecules.

Materials Required: Iodine solution, Benedict's reagent, Biuret reagent, Lipid paper,1% HCl, test tubes, test tube holder, burner, dropper, macromolecule samples (Starch, monosaccharides, lipids, proteins), 1 M sodium hydroxide (NaOH).

Experimental procedure: The methods for each test is given below, which should be applied for each macromolecule sample. Positive results will indicate the nature of the macromolecule, while the negative results will serve as controls. Ideally each test serves a as a control for the other.

1. Iodine test:

a) The iodine solution is added dropwise, using a dropper, to each test tube containing the sample. b) Blue black color change in slution indicates presence of Starch. c) 1% HCl is added dropwise to this; the color darkens further if the sample is starch. d) 1M NaOH is added to this and the color slowly reverts back to original (colorless), again indicating that the sample is starch e) No color change since the first step indicates that the sample is not starch.

2. Benedict's test / Barfoed's test:

a) The Benedict's reagent is added dropwise using a dropper, into the test tube containing the sample. b)holding the test tube firmly using a test tube holder, boil the solution for more than 2 minutes till a brick read precipitate appears. c) Brick red precipitate indicates the presence of glucose or similar reducing monosaccharides. No color change will indicate the absence of monosaccharides. d) The method can be repeated but this time HCl is added dropwise and the boiling time is reduced to 2 minutes. Brownish red coloration of the solution indicates presence of monosaccharides.

3. Biuret's test: Biuret's reagent is essentially 40% NaOH + 1% CuSO4

a) A small volume of NaOH solution is added to the test tube containing sample, followed by addition of CuSO4 solution dopwise. b) The mixture has to be shaken well c) Appearance of a violet color indicates presence of protein. d) Addition of acid will lead to no color change

4. Acid precipitation of proteins:

a) In a test tube , about 3 ml of conc. HCl is added. b) To this the sample is added using a dropper, down the inner walls of the test tube to form a layer above the acid. c) Appearance of a curdy white precipitate indicates presence of proteins.

5. Lipid paper test:

a) On the lipid paper, add a drop of each sample and allow them to dry up. b) Appearance of a translucent spot indicates lipids. c) Mix a drop of acid with the lipid sample, and again spot on the lipid paper. the translucent spot still appears.

5.

blue black coloration, turns darker on addition of acid, becomes colorless on addition of base

Violet coloration upon addition of biuret's reagent. No color change when reaction done in presence of acid

A translucent spot seen even after drying.

Addition of acid mixed sample still retains the spot

Test Starch Monosaccharide Lipids Proteins Interpretation Iodine test

blue black coloration, turns darker on addition of acid, becomes colorless on addition of base

no color change, even after addition of acid or base no color change, even after addition of acid or base no color change, even after addition of acid or base lower pH positively confirms detection of starch sample Benedict's Test No color change Brick red precipitate upon heating No color change No color change - Barfoed's test no color change Rusty or brick red coloration upon heating with acid no color change no color change Lower pH accelerates the Benedict's reaction and positively confirms monosaccharides Biuret's test no color change no color change no color change

Violet coloration upon addition of biuret's reagent. No color change when reaction done in presence of acid

Lower pH or addition of acid disrupts the alkaline environment required to form the violet colored biuret complex Acid precipitation no change no change no change A curdy white precipitate is formed Lower pH hydrolyses starch or the glycosidic bonds in the monosaccharides , but do not affect their solubility. It has no effect on lipids. Only for proteins, lower pH affects their solubility, denaturing them and precipitating them out of solution. Lipid paper test No translucent spot seen even after drying No translucent spot seen even after drying

A translucent spot seen even after drying.

Addition of acid mixed sample still retains the spot

No translucent spot seen even after drying Lower pH has no effect on lipids. Therefore the spot is still visible.

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