PAG - membrane practicals

Question 1

what is the DV of the experiment

Answer 1

absorbance (measures quantity of pigment that leaked into solution)

Question 2

what is the IV of the experiment

Answer 2

pH or temperature

Question 3

method

Answer 3

using a cork borer to cut a section of the beetroot
using a scalpel and a ruler, a cut 7 equal-sized (5 millimetres) cubes of beetroot and weigh the pieces
rinse the beetroot pieces under running water for 5 minutes until no more excess stain appears in the wash water
add the beetroot pieces to 7 different test tubes: 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C , add a beetroot piece in each.
make a water bath using a large beak and water from a kettle. when the water bath has reached 30°c place the test tube labelled 30°c into you water bath for 10 minutes
after 10 minutes stir the contents of the test tube and observe the colour and compare to the colour of the remaining test tubes
repeat the process three times for each temperature to allow anomalous data to be identified (with greater certainty)

Question 4

why must the beetroot pieces have the same dimensions / use a cork borer

Answer 4

gives uniform disc sizes as the SA control variable will be controlled and will make the test MORE valid
other control variables - source to ensure the same concentration of pigment, same variety and age: this ensure the same concentration / volume of pigment in the beetroot slices

Question 5

why must you rinse the beetroot pieces

Answer 5

the vacuoles and cells were cut open and the discs needed to be washed to remove all the stain BECAUSE colour would be more intense than it should be if not rinsed or results would not be representative / overestimate / more intense

Question 6

why do you need a thermostatically controlled water bath

Answer 6

to ensure uniform temperature throughout experiment, same kinetic energy and the same rate of diffusion
more valid results

Question 7

why do you stir the contents of the test tube

Answer 7

to distribute the pigment throughout the whole test tube - ensure the measurement of absorption is precise

Question 8

why do you repeat the process three times per temperature

Answer 8

to produce a mean value or statistic test e.g. standard deviation
this is to increase reliability

Question 9

how could the data be made more precise

Answer 9

pour the solution into a cuvette and put in into a colorimeter
add the water in a cuvette to ensure the absorbance is zero
measure the absorbance of the pigment, this provides a quantitative measurement

Question 10

why do you ensure that the absorbance of water is zero

Answer 10

it is a control - ensures all values are measured to the same standard and comparable which increases its validity

Question 11

what colour wavelength should the colorimeter be set on

Answer 11

green

Question 12

why do you wash the pieces of apparatus and get new apparatus each time

Answer 12

this prevents contamination

Question 13

conclusions of experiments and why

Answer 13

at higher temperatures the membranes are damaged or broken down
phospholipids gain kinetic energy - move round so are not as packed increasing the membrane permeability
further increases in temperature - proteins in the membrane denature due to the hydrogen bonds breaking / tertiary structure destroyed or changed
phospholipid bilayer breaks down, phospholipids melt
phospholipids within membrane become more permeable / membrane breaks down which means that more pigment is able to leave the cell by diffusion
pigment has more kinetic energy at higher temperatures therefore, diffusion happens faster and thus more/darker pigment
once the bilayer has been broken down, osmosis (the net movement of water into or out of a cell) can not occur down a diffusion gradient as the partially permeable membrane is non existent

Question 14

when plotting the graph what does the S-shape / steep slope suggest

Answer 14

suggests pigment leaks out through denatures membrane proteins
sharp increase in absorbance corresponds to temperatures causing proteins to denature

Question 15

what is the aim of the practical

Answer 15

to investigate the effect of temperature on the selectively permeable membrane of beetroot

Question 16

what does betalain diffuse outwards

Answer 16

concentration inside vacuole may be higher than outside and diffusion takes place down a concentration gradient
vacuole has a membrane (tonoplast)
the pigment i too large to fit in between the tails of fatty acids (or phospholipid molecules)
when damaged the pigment will diffuse out through the membrane and then the cell surface membrane of the cell

Question 17

What is the effect of pH on membrane permeability

Answer 17

Above / below optimum
E.g. too acidic (too many H+) or too below (too many OH-)

The ions break down hydrogen and ionic bonds in the protein channels/carrier in the phospholipid bilayer - as a result the tertiary structure changes, the proteins denature, the cytoskeleton is no longer held together the membrane break down

Question 18

What is denaturation and what does it ALWAYS cause

Answer 18

Process of disrupting protein structure by breaking bonds

ALWAYS cause a loss of function and is irreversible

Question 19

How to increase the validity of your findings

Answer 19

Repeat to find the mean to identify anomalies

Control variables e.g. with temperature vs permeability, control the SA of beetroot so that there is the same concentration of pigment in each temperature - control so that the validity of results increases

Have narrow intervals and consistent intervals - e.g. every 10 degrees

Question 20

Describe an experiment by which you could test to see whether alcohol concentration affect membrane permeability

Answer 20

Same volume discs of beetroot - use a cork borer, scalpel and ruler

Same volume of alcohol - use a measuring cylinder

Same temperature - use a water bath and a thermometer = thermostatically controlled

Same time in alcohol: leave from mim 20 minutes; max 60 minutes - using a stopwatch

Range of alcohol concentrations - 0%(control), 10-100 increasing 10% intervals

Place into a cuvette and use a calorimeter - same green wavelength - to read amount of pigment in solution

Repeat experiment 3x - repeatability to find mean

Graph of colour intensity (% absorbance) over alcohol concentration

Question 21

Conclusion of ethanol conc vs absorbance

Answer 21

As the percentage of alcohol increased the less the transmission percentage was

Ethanol is nonpolar and is a fat solvent

The phospholipids dissolves in the ethanol and the membrane becomes disrupted and is no longer held in a tight gel state

The higher the alcohol the more the solution becomes less polar so more betalyin and diffusion out

Once the membrane has been dissolves, osmosis (the net movement of water into or out of the cell) can not occur down the diffusion gradient

Question 22

What are limitations to membrane permeability investigations and what are their solutions

Answer 22

Limitation: colorimeter can only detect a range of absorbances - there is a definite maximum absorbance that can be detected and solutions darker than this will give the same reading

Solution: dilute each sample before placing in cuvette or use a smaller number of beetroot discs per boiling tube

Question 23

What are factors that affect rate of diffusion and how can they be controlled

Answer 23

SA:V ratio / diffusion distance
Use the same cork borer to cut cylinders of exactly same diameter and rule to cut cylinders of the same length

Time
Leave discs in each solution / temperature for the same lengths of time

Temperature
Place all boiling tubes in the water bath of the same temperature
Use a thermometer to control / stabilise the temperature so the temperature does not fluctuate

Question 24

how to use a calorimeter

Answer 24

use a green filter - purple pigment absorbs green light and set to read absorbance
zero colorimeter using cuvettes filled with distilled water
measure absorbance of each solution for each boiling tube
plot graph: x-axis independent variables (temperature/concentration) / y-axis dependent variable (absorbance)

Question 25

membrane permeable investigations: TEMP

Answer 25

cut cylinders from the same beetroot using cork borer
place cylinder next a ruler and cut discs of the same thickness using a scalpel and ruler
set up thermostatically controlled water baths over a suitable range of temperatures (0 - 50 degrees)
place one piece of beetroot in each boiling tube and place one boiling tube in each water bath
leave the boiling tubes in the water bath for the same time: min 20 mins / max 60 mins (use a timer / stopwatch)
remove same volume of water from each boiling tube, place into cuvette and use a calorimeter to measure absorbance

Question 26

membrane permeable investigations: ETHANOL / DETERGENT CONCENTRATION

Answer 26

cut cylinders from the same beetroot using cork borer
place cylinders next to a ruler and cut discs of the same thickness using a scalpel
make suitable ranges of concentrations using serial dilution (0% - 40% increasing in 10% for ethanol) and (0% - 2% increasing in 0.5% increments for detergent)
pour the same volume in each concentration into each boiling tube - use a measuring cylinder
place one beetroot disc into each boiling tube
leave in for the same time: min 20 mins / max 60 mins (use a timer / stopwatch)
remove the same volume of solution from each boiling tube, place into a cuvette and use a calorimeter to measure absorbance