Laboratory Techniques: Liquids & Solutions, and Separation Techniques Flashcards
What is a hazard and a risk?
- A hazard is a potential source of harm which can be chemical (e.g. acid), physical (e.g. glass), biological (e.g. parasites) etc.
- A risk is how likely the hazard is to cause harm.
What is a risk assessment?
Risk assessment is the process of identifying all hazards, determining the risk, and taking measures to reduce the risk of harm occuring.
What are some control measures that can be used?
1) Elimination - Redisgn the job so the hazard is eliminated.
2) Substitution - Use alternatives that don’t produce the same level of risk.
3) Isolation - Carry out the experiment in a contained environment.
4) Education - Train people to follow standard methods of practice.
5) PPE(personal protective equipment) - A last line of defence which includes wearing gloves, goggles, lab coat etc.
What are dilutions used for?
Dilutions are used in experiments to control confounding variables, generate a suitable range in an independent variable or modify the dependent variable so that a measurable value can be obtained. They can either be linear or log/serial.
Describe linear dilution series.
Consists of a range of dilutions that differ by an equal interval (e.g. 0.1, 0.2, 0.3ml…). Different volumes of the stock solution should be added to different volumes of solvent, meaning each concentration is made individually. Any measurement errors will only effect one concentration.
Describe log dilution series.
Consists of a range of different dilutions that differ by a constant proportion (e.g. 10^-1, 10^-2..). Each dilution series should act as the stock for the next dilution. This means that each concentration depends on earlier measurements so errors are compounded in later dilutions.
What is a colorimeter and how is it used?
A colorimeter is used to measure the concentration of a pigment in a solution, the turbidity (cloudiness) of a liquid or the density of cells in a culture. A small sample of the test substance is held in a cuvette and illuminated by a coloured light source. The amount of light absorbed is recorded electronically. The machine is calibrated using a blank cuvette containing solvent only which acts as control value.
What is a standard curve/calibration graph?
A standard curve is made by plotting the absorbance readings of known concentrations of a substance. This curve can then be used to determine the concentration of a substance using its absorbance. This is called interpolation.
What are buffers?
Buffers are aqueus solutions that show little difference in their pH when an acid/alkali is added. Since most biological processes are pH dependent, most body substances and cells contain buffers, e.g. blood.
What is centrifugation and how does it work?
Centrifugation is the method for separating materials in suspension according to their density. It is often used in biology to separate cell constituents. The material is rotated in a centrifuge tube and the resultant g-force causes constituents to separate. The most dense items form a pellet at the bottom of the tube and the the less dense items/liquids stay above and is called the supernatant.
Describe paper and thin-layer chromatography.
Substances such as amino acids are separated according to their solubility. The materials are spotted on the base of the chromatography paper and a solvent mix pulls up the different constituents through the chromatogram. The different affinities of the constituents results in different rates of movement so the constituents travel to different heights and can then be identified.
Describe affinity chromatography.
This is used to separate a specific protein from a mixture. An antibody/ligand specific for binding with the protein of interest is immobolised in agarose gel and packed into a column. The mixture of proteins is then poured through the column and the protein of interest will bind to the antibody/ligand within the column. The other proteins cannot bind and are washed away. The column can then be washed with a buffer to lower the pH as this will lower the affinity of the protein to the ligand and the target protein can be recovered.
Describe protein electrophoresis.
This separation technique uses current flowing through a buffer to separate the proteins. The proteins are passed through the buffer gel and will move through the gel at different rates due to differences in size and charge.
Describe what isoelectric points are.
The isoelectric point (IEP) of a protein is the pH at which it has an overall neutral charge. Above and below this point, there will be a positive or negative charge allowing the protein to be soluble in water. Therefore, at its isoelectric point, a protein will form and solid and precipitate out of solution. This can be used to separate different proteins.
