Week 2 biochem - chromatography techniques Flashcards
what is the definition of chromatography
A physical process that separates compounds in a mixture (solute) as a result of their different affinity for a mobile phase and stationary phase.
this is used because in order to analyse a compound properly, we need to get it away from everything else.
why do we separate compounds
we need to isolate things to identify them, we want to see how much of a compound is in our mixture.
what is the point of separating things?
- we may want to isolate, identify, and or quantify the compound of interest we want to analyse.
- we may want to remove other compounds that could interfere with our compound of interest.
what can chromatography be
chromatography can be analytical (mostly done in research labs) or preparative ( industries use chromatography to prepare analysts, they take a massive amount of mixture, put it through a chromatography column and separate out grams/kg of analyst of interest).
what are chromatography phases
the phases are mobile phase (MP) and stationary phase (SP)
the mobile phase is always liquid or gas (cause it has to move)
the stationary phase is liquid or solid.
what is the scouting experiment
in chromatography a lot of parameters are involved thus there are a lot of things we can change through scouting experiments. when you do an experiment , you have a set of parameters, you see what happens in the experiment and then you can change 1 of the parameters, in this case separation. either in order to improve the experiment or see what is interfering in the experiment.
what is chromatography techniques classified by
- the type of chromatographic bed
> planar (flat) or column - the type of mobile phase
> gas or liquid - the type of separation mechanism ( by which the chromatography system operates)
> size, polarity, ect
what are the different types of chromatography
- Planar, which includes:
> Paper chromatography
> Thin layer chromatography (TLC)
-column, which includes:
> Gas chromatography (GS)
> Liquid chromatography (LC)
what is paper and thin layer chromatography and which one is better
paper:
- you have a chromatography paper and you put your sample at the bottom
- you then stand the paper in a tank of organic solvents
- the solvents travel up the paper through your sample
- the analytes will start separating out, this separation depends on their attachment to the mobile phase or stationary phase.
TLC:
this is similar to paper chromatography but it uses a microscope slide.
the microscope slide is coated with acidic gel, alumina or cellulose,
environmental scientists use this method because it doesn’t require electricity.
TLC is better than paper chromatography because it works faster and you get better separations and better quantitative analysis.
what is gas and liquid chromatography
gas chromatography
it requires the sample to be heated for the sample to work, thus this is not suitable for proteins.
the gas usually used is helium or nitrogen because they are inert.
liquid chromatography
the mobile phase is liquid
the stationary phase is solid
how does an analyte attain equilibrium
an analyte in a sample mixture will attain equilibrium as it passes through the chromatography column and move between the mobile phase or stationary phase.
how fast and slow the analyte moves through the chromatography column depends on its attachment to the mobile phase or stationary phase.
what is the equation for the equilibrium constant ( partition coefficient,K) of an analyte
( concentration of analyte in the stationary phase)/ (concentration of analyte in the mobile phase)
what is the K value of an analyte
K = [A]SP/ [A]MP
each analyte has its own K value for any chromatographic system, chromatography systems have a lot of parameters, you need to set the parameters, set the system, and then there is the own K value for each system.
alter 1 parameter, e.g. temperature, you will change the K value of the analyte
what is the retention time
the time it takes from entering the column to leaving the column
what is the retention time of an analyte
the retention time of an analyte = time from sample injection to elution from the column.
each analyte has its own retention time for any chromatography system. as some analytes will spend longer in the column and other analytes won’t.
what happens if 2 or more analytes have similar K values or retention time
it would make it difficult to separate out the analytes.
to make difference in the values, the chromatography system can be modified for example a different mixture of the mobile phase can be used.
what is a chromatogram and what is the point of the detector
chromatogram gives an idea of how much of an analyte there is. the bigger the peak the more analyte/ dye is present, the smaller the peak the less of the analyte/dye is present.
there is a detector that is plugged in at the end of the column (e.g. vv detctor). the detector detects response against time and the bigger the detector response, the more dye/analyte is present.
what happens when analytes travel down the column
when analytes travel down the column, analytes start separating out but not all analytes will separate out completely.
when the analytes are not separated out completely you get fused peaks as they exit the column. to solve this problem the easiest way is to change the constituents of the mobile phase.
example
in the graph, fronting can be caused because there is too much analyte in the column.
tailing can be caused because the analyte is hanging onto the stationary phase.
to solve these problems, you need to alter the components of the mobile phase.
what is ion-exchange chromatography (liquid chromatography)
it separates analytes based on their electrical charge.
stationary phase resin with their positively charged (anion-exchange) or negatively charged (cation exchange) functional groups.
why does the stationary phase resin with their positively charged (anion-exchange) or negatively charged (cation exchange) functional groups in ion-exchange chromatography
if the stationary phase is negatively charged then the analytes that are negatively charged will repel and want to leave the stationary phase quickly however if the analytes are positively charged then the analyte with be attracted to the stationary phase and want to remain in the stationary phase.
different analytes have different affinities for the stationary phase, depending on their charge.
we can use this technique for proteins because have different electrical charges depending on what type of amino acid is in them.
how are bound analytes removed from the stationary phase in ion-exchange chromatography
bound analytes are removed from the stationary phase by altering the pH of the stationary phase.
we can separate the positively charged amino acid from the negatively charged ones by first letting the negatively charged amino acids leave the stationary phase then we can change the pH of the stationary phase and thus make the stationary phase positively charged and this causes the positively charged amino acids to leave the stationary phase.
what is another way, other than changing the charge of the stationary phase, to get the analytes out of the stationary phase in ion exchange chromatography
you can alter the salt concentration because if you put salt in (salt has anions and cations in it) it will compete with the proteins/analytes for the binding sites in the stationary phase. thus you can get your proteins/analytes out if they are being stubborn by altering either the pH or salt concentration.
what does ion exchange chromatography separate
ion exchange chromatography separates:
- amino acids
-peptides
-proteins
small nucleotides
what is size-exclusion chromatography and how is it carried out
it separates analytes based on physical size.
what happens is you have your analyte mixture at the top and you put the column upright and the analyte mixture runs down due to gravity (it would take a long time for gravity to work itself so liquid-handling pumps are used to speed things up).