PAG - membrane practicals Flashcards
what is the DV of the experiment
absorbance (measures quantity of pigment that leaked into solution)
what is the IV of the experiment
pH or temperature
method
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)
why must the beetroot pieces have the same dimensions / use a cork borer
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
why must you rinse the beetroot pieces
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
why do you need a thermostatically controlled water bath
to ensure uniform temperature throughout experiment, same kinetic energy and the same rate of diffusion
more valid results
why do you stir the contents of the test tube
to distribute the pigment throughout the whole test tube - ensure the measurement of absorption is precise
why do you repeat the process three times per temperature
to produce a mean value or statistic test e.g. standard deviation
this is to increase reliability
how could the data be made more precise
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
why do you ensure that the absorbance of water is zero
it is a control - ensures all values are measured to the same standard and comparable which increases its validity
what colour wavelength should the colorimeter be set on
green
why do you wash the pieces of apparatus and get new apparatus each time
this prevents contamination
conclusions of experiments and why
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
when plotting the graph what does the S-shape / steep slope suggest
suggests pigment leaks out through denatures membrane proteins
sharp increase in absorbance corresponds to temperatures causing proteins to denature
what is the aim of the practical
to investigate the effect of temperature on the selectively permeable membrane of beetroot
what does betalain diffuse outwards
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
What is the effect of pH on membrane permeability
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
What is denaturation and what does it ALWAYS cause
Process of disrupting protein structure by breaking bonds
ALWAYS cause a loss of function and is irreversible
How to increase the validity of your findings
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
Describe an experiment by which you could test to see whether alcohol concentration affect membrane permeability
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
Conclusion of ethanol conc vs absorbance
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
What are limitations to membrane permeability investigations and what are their solutions
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
What are factors that affect rate of diffusion and how can they be controlled
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
how to use a calorimeter
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)
membrane permeable investigations: TEMP
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
membrane permeable investigations: ETHANOL / DETERGENT CONCENTRATION
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
