RP 4 - Investigating Membrane Permeability

• Cell-surface membranes are made up of a phospholipid bilayer which makes them selectively permeable.
• The permeability of a membrane can be measured by using beetroot cells, which contain a purple pigment called betalain.
• When the cell-surface membrane has a higher permeability, more pigment leaks out of cells.
• The permeability can therefore be measured by the amount of pigment leaked from beetroot cells into an aqueous solution using a colorimeter.
• To investigate the effect of a named variable on the permeability of cell-surface membranes, equipment required includes beetroot, a scalpel, forceps, a cutting board, a ruler, tongs, distilled water, boiling tubes, a boiling tube rack, a colorimeter, cuvettes, filter paper, a timer, a water bath, a thermometer, and ethanol.
• Cut beetroot into 6-10 identical cubes using a scalpel.
• If investigating temperature, place each of the cubes of beetroot in an equal volume of distilled water (5-15ml) and place each test tube in a water bath at a range of temperatures (30-80 ° C).
• If investigating concentration of solvents, create a dilution series of ethanol using distilled water with ethanol concentrations ranging from 0-100%.
• Leave the samples for 20 minutes - the pigment will leak out of the beetroot.
• Set the colorimeter to a blue filter and zero using a cuvette with distilled water.
• Filter each sample into a cuvette using filter paper.
• Measure the absorbance for each solution - a higher absorbance indicates higher pigment concentration, and hence a more permeable membrane.
• In emergency, cuts from sharp objects can be treated by cutting away from fingers, using forceps to hold the sample, elevating cuts, applying pressure, and seeking medical assistance.
• Cuts from sharp objects can be prevented by taking care when handling glassware, keeping glassware away from the edge of the desk, elevating cuts, applying pressure, and seeking medical assistance.
• Ethanol is an irritant/flammable substance, so it should be handled with care, kept away from naked flames, wash eyes and skin with cold water, and if necessary, seek medical assistance.
• Hot liquids can cause scalding, so they should be handled with care, used with tongs, and if necessary, run under cold water and seek medical assistance.
• Plot a graph of absorbance against ethanol concentration/temperature.
• As the temperature increases, the permeability of the cell-surface membrane increases because proteins in the membrane denature as heat damages bonds in their tertiary structure, creating gaps in the membrane, so it is easier for molecules to pass through it.
• At low temperatures, phospholipids have little energy and are packed closely together to make the membrane rigid, causing a decrease in permeability and restricting molecules from crossing the membrane.
• At very low temperatures, ice crystals can form which pierce the cell membrane and increase the permeability.
• Ethanol causes the cell-surface membrane to rupture, releasing the betalain pigment from the cell.
• Higher concentrations of ethanol will cause more disruption to the membrane as it dissolves phospholipids, thus, as concentration of ethanol increases, the permeability of the cell-surface membrane increases.