Investigation of the effects of concentration and acidity on the rate of absorption of glucose and proteins through osmosis.

Does the rate of osmosis of glucose and amino acids depends on the concentration (g/L) of the solution and pH, determined using colourimetry?

Digestion is a complex biological process which includes both chemical and physical processes. The process begins inside the mouth and continues till the large intestine. Enzymes, the biological catalyst plays an important role in converting the complex molecules – carbohydrates, proteins and lipids into simple molecules like glucose, amino acids, fatty acids and glycerol. These complex molecules are absorbed in the large intestine by the villi. The process through which absorption occurs is osmosis. In this process, the solvent molecules travels from a region of lower concentration to a region of higher concentration across a semi-permeable membrane. The rate at which osmosis occurs depends on various factors like concentration of the solution, temperature and even pH.  Through osmosis, the monomeric nutrient molecules glucose, amino acids, fatty acids and glycerol enters from the large intestine to blood and thus an exploration of the factors which affects the rate of this process is worth of investigation. The concepts, I have studied in Biomolecules and the details of digestion process I have studied in Human physiology has enticed me towards this topic.

Osmosis is the movement of solvent molecules from a region of more concentrated solution to a region of less concentrated solution across a semi permeable membrane. It is a process where movement happens against the concentration gradient and is thus considered as an active transport mechanism.

Osmosis occurs in both plant and animal cells. Based on the osmotic pressure, solutions are classified into three categories -

Isotonic solution - The osmotic pressure of such solutions is equal to that inside the cells. Thus, no movement of solvent molecules (water) occurs from the surroundings to cell or vice versa.

Hypotonic solution - These solutions have a concentration lower than that inside the cells. Thus, movement of water molecules occurs from the solution to the cells causing the cells to swell up.

Hypertonic solutions - These solutions have concentration higher than the cells. Thus, movement of water molecules occurs from the cell to the solution outside. This causes the cells to shrink.

Plant cells have cell wall which enables them to withstand the turgor pressure when the cells are swelled up or becomes shrunk (flaccid). Animal cells do not have that facility. Hence, animal cells demands to be surrounded by isotonic solution. Animal cells are provided with contracting vacuoles which can pump out water from the cell when the water level in the plasma is high. Kidneys performs the role to control the water content of blood and this occurs through a process called osmoregulation.

A colorimeter is an apparatus that is used to measure the absorbance of a coloured solution. The absorbance of a coloured solution is a measure of the concentration of the solution. It is given according to the equation,

A = €cl

A = absorbance

€ = molar absorptivity constant

L = path length

As € and l are empirical constants, the absorbance of a solution can be considered as a measure of the concentration of the solution.

Benedict’s reagent is an alkaline solution of Copper (II) ions. It is used for the quantitative estimation of reducing sugars. Reducing sugars have free aldehyde or ketone group that reacts with Copper (II) ions and reduces it to Copper (I) ions. The Copper (I) ions precipitates out as Copper oxides and are red to orange in color depending on the amount of it. As the mass of reducing sugar tested is increased, the color of Copper oxide deepens. As the compound Copper oxide exhibits maximum absorbance at a wavelength of 565nm, the absorbance of a solution of sample with Benedict’s reagent can be a measure of the quantity of reducing sugar within it.

Reducing sugars + Benedict’s reagent \(\rightarrow\) Copper(I)  oxide (red or orange precipitate)

Proteins are polymers of amino acids connected to each other through peptide linkages. Peptide linkages reacts with Copper (II) ions in alkaline medium to form a coloured chelate complex. The complex formed is usually pink or violet in colour. As the chain length of polymer increases, the number of peptide linkages increases, the colour of the complex also becomes deeper. The complex formed exhibits maximum absorbance at 540nm. Thus, as the absorbance at 540 nm increases, the quantity of protein in the sample increases.

Peptides + Cu(II) ions \(\rightarrow\) Violet coloured complex

There is no correlation between rate of osmosis and the concentration of the solution or the acidity of the medium during the osmosis of glucose and proteins.

There is a positive correlation between the rate of osmosis and the concentration of the solution for glucose and proteins as well as with the acidity of the medium.

The current investigation deals with two independent variables-

Concentration of the solution

Aqueous solutions of glucose and proteins are used. Kidney beans are used for proteins and sugar is used as a source of glucose to mimic the biological processes. All concentrations are measured in g/L. The solutions are prepared by dissolving the required mass of sugar or kidney beans in water. A beaker, a digital mass balance has been used for this process.

Acidity of the medium

The acidity of the medium is controlled by the use of HCl. The concentration of HCl used was kept constant while the volume of HCl added kept on changing. The pH of the solutions added were calculated using the formula pH = -log [concentration of HCl].

The rate of osmosis is the dependent variable. The absorbance of the solution inside the dialysis tube and outside the dialysis tube has been measured initially and after 30 minutes. The difference of absorbance in the solution outside the dialysis tube has been taken as a measure of the amount of the solvent flow across the membrane. The rate of absorbance is calculated as a ratio of the difference in absorbance of the solution outside the dialysis tube and the time taken.

Rate of osmosis = \(\frac{A_2\ -A_1}{time}\)

A1 = absorbance of the solution outside the dialysis tubing after 30 minutes.

A2 = absorbance of the solution outside the dialysis tube initially.

• The time interval given for the osmosis to occur has been kept constant throughout all the trails. In all trials, the dialysis tube was kept inside the solution for 30 minutes.
• The concentration of the solution inside the dialysis tube was always kept constant. A constant value of  0.5 g^L-1 was used for both glucose and kidney beans.
• The same dialysis tube was used in all trials.
• The height of the dialysis tube was kept fixed in all trials. A fixed length of 5 cm has been used.
• The volume of solution inside the dialysis tube was kept same in all cases. 10 cm³ of solution was taken inside the dialysis tube in all cases.

Safety precautions

• No eatables were ingested or exposed to skin.
• Safety masks, safety goggles and lab coats were used.
• The work station was always kept clean.
• All solutions were handled carefully to avoid spillage.

Environmental considerations

No toxic waste products were produced. All unused materials were disposed safely into the waste bin.

Ethical considerations

No ethical contradictions as all IB guidelines and safety protocols were followed.

Preparation of glucose solutions

The required mass of sugar was weighed on a watch glass using a spatula and a digital mass balance. The weighed mass of sugar was then transferred to a glass beaker. 100 cm³ of distilled water was then added to it using a graduated measuring cylinder. The concentrations of the solutions prepared are – 0.5 g^L-1, 1.0 g^L-1, 1.5 g^L-1, 2.0 g^L-1, 2.5 g^L-1 and 3.0 g^L-1. For example, 0.05 g of sugar was dissolved in 100 cm³ of distilled water to prepare the 0.5 g^L-1 solution.

Preparation of protein solutions

Kidney beans was used as a source of protein. The solutions of kidney beans was also prepared in the same process and the strength of the solutions prepared were same as that of glucose.

Preparation of alkaline Copper (II) solution

1.59 ± 0.01 g of Copper (II) sulphate penta hydrate was weighed on a watch glass using a digital mass balance and the weighed solid was transferred to a 100 cm³ volumetric flask. 0.04 ± 0.01 g of solid NaOH was added to it. Distilled water was then added to it using a graduated measuring cylinder.

Determination of absorbance

A dialysis tube was taken and a tube of length 5 cm was cut out from it using a ruler and a scissor. The cut out of the dialysis tube was then kept inside a beaker containing distilled water. One end of the dialysis tube was tied with a rubber band and the other end was kept open. A 250 cm³ glass beaker was taken and filled with 0.5 g^L-1 of glucose solution. The dialysis tube was taken and filled with 10 cm³ of 0.5 g^L-1 of glucose solution using a graduated pipette. The s