Biological Molecules Practical Endorsement Flashcards
Test for reducing sugars
Reducing sugar ~ can donate an electron to another molecule.
- include all monosaccharides
- some disaccharides (maltose + lactose)
BENEDICT SOLUTION ~ contains the copper ion Cu2+ which makes the solution BLUE.
- Grind up the food with distilled water
- filter away the solid food particles.
- add 3cm cubed of our food solution to a boiling tube.
- Add 3cm cubed of benedict solution.
- Place boiling tube into a beaker of boiling water and leave for 5 minutes.
Results for Reducing sugars
Remains blue ~ no reducing sugar present.
Red precipitate ~ reducing sugars are present and an electron is added to the Cu2+ ion forming the Cu+ ion.
Different amounts of reducing sugar:
Vey small ~ GREEN
More ~ YELLOW
High level ~ ORANGE
Lots ~ BRICK RED
Semi quantitative:
- only gives an APPROXIMATE idea of the amount of reducing sugar.
- It only shows a narrow range of colours changes ~ humans perceive colours slightly differently.
Test for NON - reducing sugars
Non - reducing sugar :
- some disaccharides
- example ~ SUCROSE which contains the monosaccharide glucose and fructose joined by a glycosidic bond.
Cannot test directly ~ we have to break the glycosidic bond releasing the monosaccharide.
- Carry out benedict test and record colour change to see if any reducing sugar is present.
- Use a fresh boiling tube and add 3cm cubed of our unknown solution and 3cm cubed of HDROCHLORIC ACID.
- Place boiling tube in water bath for 5 minutes.
If a non - reducing sugar is present , then the acid HYDROLYSES the glycosidic bond releasing the monosaccharide.
- Add 3 cm cubed of dilute alkali and use pH paper to check that our solution is alkali. ( this is because the benedict test does not work under ACIDIC conditions)
- Add 3 cm cubed of benedict solution , heat and record colour change.
RESULTS for NON - reducing sugars
First benedict test ~ BLUE - No reducing sugar is present.
Second benedict tests ~ ORANGE - contains a non - reducing sugar.
First benedict test ~ GREEN - very small amount of reducing sugar present.
Second benedict test ~ RED - Non - reducing sugar present.
First benedict test ~ RED - Large amount of reducing sugar present.
Second benedict test ~ not possible as even if a non-reducing sugar was present we wouldnt be able to see a colour change beyond red.
Benedict’s solution
- BLUE colour caused by the presence of the copper 2+ ion
- Reducing sugars donate an electron REDUCING the copper 2+ ion to the copper + , forming a red precipitate.
- Leaving the test tube to SETTLE would create a red precipitate at the bottom and a solution above it that will be LESS blue than before as some of the copper 2+ ions have reacted and are no longer in the solution.
The GREATER the concentration of GLUCOSE , the LESS blue the Benedict’s solution will be at the end.
We can QUANTIFY this blueness using a COLORIMETER.
How does a COLORIMETER work ?
- Place a RED filter in front of a lamp which only allows red to pass through.
- We use red as Benedict’s solution absorbs red the most as red is the opposite end of the spectrum to blue.
- Benedict’s solution reacting with glucose makes it LESS blue.
Therefore, the LESS red light absorbed , the GREATER the amount of glucose that must have reacted with our Benedict’s solution
Creating a CALIBRATION CURVE
- Take a series of KNOWN concentrations of reducing sugar.
Concentration of solution:
volume of stock x concentration
volume of stock + water - Carry out the BENEDICT’S test on each of our solutions.
- Use a CENTRIFUGE to separate the solutions from the precipitate.
- Transfer the solutions into a CURVETTE:
- small , plastic container
- 2 transparent and 2 translucent sides.
- always place it into the colorimeter
facing the same way each time.
- Set the colorimeter onto the red filter and place the curvette containing just distilled water into the colorimeter and set it to zero.
- We can use this to record the percentage of absorbance of light through each supernatant.
- Plot a graph with glucose concentration on the X -axis and absorbance of light on the Y - axis.
This is known as a calibration curve , which can be used to determine the concentration of sugar in an UNKNOWN solution.
Test for PROTEINS
- Add 3cm^3 of food solution into a test tube.
- Add 3cm^3 of BIURET reagent ( premixed sodium hydroxide and copper II sulfate)
- In the PRESENCE of protein , the solution turns PURPLE or LILAC.
- In the ABSENCE of protein, the solution remains BLUE.
This test actually detects PEPTIDE BONDS.
Therefore you get a POSITIVE result with proteins as they contain peptide bonds.
However, a solution of amino acids would give a NEGATIVE result as these do not contain peptide bonds.
Cell fractionation
CELL FRACTIONATION ~ used to isolate different organelles so they can be studied.
COLD ~ reduce enzyme activity, release of enzymes could damage the organelles.
ISOTONIC ~ same water potential to stop osmosis from occurring , preventing the organelles from shrivelling or bursting.
BUFFERED ~ to keep a constant PH (neutral) to prevent damage of organelles.
MOST DENSE
nucleus
chloroplast
mitochondria
lysosomes
ribosomes
LEAST DENSE
Test tube consists of :
Supernatant liquid & the pellet ( the organelle)
What is chromatography?
- Thin layer chromatography is commonly used to monitor the progress of reactions.
- This is because it works relatively QUICKLY.
- The aim of chromatography is to separate a mixture into its individual components.
The two PHASES in chromatography
STATIONARY PHASE:
- the chromatography paper or the TLC plate.
- made of cellulose
- the TLC plate is often a sheet of plastic , coated in a thin layer of silica gel or aluminium hydroxide.
-there are free -OH groups pointing outwards ,in contact with the mobile phase.
MOBILE PHASE:
-the solvent for the biological molecules
- WATER is used for polar molecules
- ETHANOL is used for non-polar molecules
The MOBILE phase flows through and across the STATIONARY phase , carrying the biological molecules with it.
Rf value
RETENTION FACTOR:
Distance moved by solute / Distance moved by solvent
Why do some spots move faster than others in chromatography?
- The speed at which molecules move along the paper or TLC plate depends on their POLARITY and SOLUBILITY in the solvent.
- Exposed -OH groups make the surface of the paper or plate very polar, and allow it to form hydrogen bonds with the molecules , alongside other dipole interactions.
POLAR ~ will be ADsorbed , hence move more slowly.
NON-POLAR ~ travel very quickly up the plate.
If two molecules travel at exactly the same speed , we can use a different SOLVENT or change the pH.
METHOD for separating a mixture of AMINO ACIDS
1) A spot of the UNKNOWN amino acid sample mixture is placed on the PENCIL LINE of the TLC plate using CAPILLARY TUBING.
2) Spots of KNOWN standard solutions of different amino acids are also placed on the pencil line EQUALLY SPACED.
3) The TLC plate is lowered into the solvent, ensuring the level of the solvent at the start is BELOW the pencil line.
4) Cover the beaker with a GLASS PLATE.
5) Let the apparatus ‘run’ until the solvent has reached a point JUST BELOW the top of the TLC plate.
6) The amino acid can then be identified by comparing and matching them with the CHROMATOGRAMS of the known standard solutions of the different amino acids.
How to make COLOURLESS molecules visible.
NINHYDRIN ~
- sprayed onto the TLC plate after it had dried.
- This binds to AMINO ACIDS which are then visible as brown or purple spots.
UV LIGHT ~
- TLC plates have a chemical which fluoresces under UV light making it glow.
- Those places where the spots have travelled to , DO NOT glow , making them easily identified.
IODINE ~
- The dried TLC plate is placed in an enclosed container with a few iodine CRYSTALS.
- The iodine forms a GAS , which BINDS to the molecules in each of the spots.
