Week 2 biochem - chromatography techniques

Question 1

what is the definition of chromatography

Answer 1

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.

Question 2

why do we separate compounds

Answer 2

we need to isolate things to identify them, we want to see how much of a compound is in our mixture.

Question 3

what is the point of separating things?

Answer 3

• 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.

Question 4

what can chromatography be

Answer 4

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).

Question 5

what are chromatography phases

Answer 5

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.

Question 6

what is the scouting experiment

Answer 6

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.

Question 7

what is chromatography techniques classified by

Answer 7

• 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

Question 8

what are the different types of chromatography

Answer 8

• Planar, which includes:
  > Paper chromatography
  > Thin layer chromatography (TLC)

-column, which includes:
> Gas chromatography (GS)
> Liquid chromatography (LC)

Question 9

what is paper and thin layer chromatography and which one is better

Answer 9

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.

Question 10

what is gas and liquid chromatography

Answer 10

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

Question 11

how does an analyte attain equilibrium

Answer 11

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.

Question 12

what is the equation for the equilibrium constant ( partition coefficient,K) of an analyte

Answer 12

( concentration of analyte in the stationary phase)/ (concentration of analyte in the mobile phase)

Question 13

what is the K value of an analyte

Answer 13

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

Question 14

what is the retention time

Answer 14

the time it takes from entering the column to leaving the column

Question 15

what is the retention time of an analyte

Answer 15

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.

Question 16

what happens if 2 or more analytes have similar K values or retention time

Answer 16

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.

Question 17

what is a chromatogram and what is the point of the detector

Answer 17

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.

Question 18

what happens when analytes travel down the column

Answer 18

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.

Question 19

what is ion-exchange chromatography (liquid chromatography)

Answer 19

it separates analytes based on their electrical charge.
stationary phase resin with their positively charged (anion-exchange) or negatively charged (cation exchange) functional groups.

Question 20

why does the stationary phase resin with their positively charged (anion-exchange) or negatively charged (cation exchange) functional groups in ion-exchange chromatography

Answer 20

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.

Question 21

how are bound analytes removed from the stationary phase in ion-exchange chromatography

Answer 21

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.

Question 22

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

Answer 22

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.

Question 23

what does ion exchange chromatography separate

Answer 23

ion exchange chromatography separates:
- amino acids
-peptides
-proteins
small nucleotides

Question 24

what is size-exclusion chromatography and how is it carried out

Answer 24

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).

Question 25

why are there little holes in the small beads in the stationary phase in size-exclusion chromatography

Answer 25

there are little holes in the small beads in the stationary phase because the small analytes spend a lot of time going in and out of the stationary phase beads as it goes down the column but the big analytes can’t get in the hole and thus travel straight down and out the column and thus this technique separates small analytes and big analytes.
in stationary phase has to be inert as you don’t want any reaction to take place between the analyte and stationary phase.
The mobile phase is aqueous or organic-solvent based.

Question 26

what are other names for size-exclusion chromatography

Answer 26

other names are:
- gel permeation chromatography
- gel filtration chromatography
- molecular sieve chromatography

Question 27

what is affinity chromatography

Answer 27

it separates:
- nucleic acids
- proteins (e.g. antibodies)

it separates analytes based on the interaction between:
- antibody and antigen
- enzyme and substrate

it is very specific/selective as you need to send a column to a manufacturer to make it specific to 1 analyte thus this method can be quite expensive.

Question 28

how is the target analyte obtained in affinity chromatography

Answer 28

the stationary phase is a gel matrix (e.g. agarose) to which the ligand is covalently bound. the ligand binds selectively to the target analyte, other unwanted analytes pass through the column.
the target analyte is then obtained from the column by altering the mobile phase compositions.

Question 29

what does HPLC stand for

Answer 29

high-performance liquid chromatography or high-pressure liquid chromatography (HPLC)

Question 30

why does HPLC get good separation

Answer 30

in HPLC the column is filled with porous inert beads with stationary phase compounds chemically bound to them.
if you have a column with very small beads, as small as possible, you get really good separation as you get sharper peaks and you get analytes separating out very efficiently.

Question 31

why does HPLC have high-pressure pumps attached to it

Answer 31

HPLC is a system that has a high-pressure pump attached to it and this pump pushes the mobile phase at a high pressure thus the analytes get pushed at high pressure however one problem caused is back pressure.

Question 32

how do the analytes bind to the stationary phase in HPLC

Answer 32

analytes bind to the stationary phase through:
- polar-polar interaction or
- non-polar-non-polar interactions

Question 33

how do the analytes interact with the stationary phase

Answer 33

analytes interact with the stationary phase through:
- ion-exchange
- size-exclusion
- affinity chromatography

Question 34

what are the advantages of HPLC

Answer 34

• faster elution times due to high pressure
• better resolution due to smaller stationary phase particles

Question 35

what is HPLC normal phase

Answer 35

it separates analytes based on polar interactions (e.g. hydrogen bonding or dipole-dipole interactions(electrical interactions)) with the polar stationary phase (e.g. silica)
the stationary phase is polar
the mobile phase is non-polar, non-aqueous (e.g. chloroform)

Question 36

what type of mobile phase is usually used for HPLC normal phase

Answer 36

the type of mobile phase solvent used can be hexane and dichloromethane. Hexane is very non-polar, it got almost no electrical charge to it, and dichloromethane has a couple of chlorine atoms in it which gives it a little bit of polarity. thus what can be done is start of with the mobile phase of 90% hexane and 10% dichloromethane, the 90% hexane will attract very non-polar lipids so they will pass through the column first with the help of the HPLC pump as well, then change the amount of the organic solvents in the mobile phase (this is called gradient elution), change the amount very slowly and over time is will swap around so at the end you have a mobile phase flowing through the column with 10% hexane and 90% dichloromethane and at this point, the polar lipids will be attracted to the mobile phase and leave the column.
thus the more polar molecules will elute last from the column.

Question 37

in HPLC normal phase, what time of analytes are usually used

Answer 37

the analytes used are usually a mixture of lipids, this is because lipids are generally hydrophobic.
you usually dissolve the lipid mixture into the non-polar, non-aqueous mobile phase, however, lipids have a slight electrical charge as they travel through the column thus they will be attracted to the stationary phase by the electrical charge through polar-polar interactions.

Question 38

how is the strength of the analyte bond and stationary phase linked in HPLC normal phase and why is it called HPLC normal phase

Answer 38

It’s called HPLC normal phase cause it was developed first.
the strength of the analyte bond to the stationary phase is proportional to analyte polarity.

Question 39

what is HPLC reverse phase

Answer 39

it is most frequently used in HPLC
it separates analytes based on non-polar, hydrophobic interaction with the non-polar stationary phase (e.g. RMe2SiCl (R group is a straight chain alkyl group [e.g. C18H37]))
it used non-polar-non-polar interactions between the mobile phase and stationary phase.

Question 40

what are mobile phase and stationary phase polarity in HPLC reverse phase

Answer 40

the mobile phase is moderately polar
it is usually a mixture of water and organic solvent (e.g. water and acetonitrile, methanol).
the stationary phase is non-polar
you are using the hydrophobicity of the stationary phase and the hydrophobicity of your analytes (your proteins) to make the analytes bind to the stationary phase.

Question 41

how is the strength of the analyte bond and stationary phase linked in HPLC reverse phase

Answer 41

the strength of the analyte bound to the stationary phase is proportional to analyte hydrophobicity.
this means the more hydrophobic protein will favour the stationary phase and thus hang on to the stationary phase beads in the column and the less hydrophobic proteins will leave the column first.

Question 42

how are the non-polar analytes eluted from the stationary phase in HPLC reverse phase

Answer 42

you change the constituents of the mobile phase and the non-polar proteins will elute from the column.

Question 43

what is the definition of isocratic elution

Answer 43

it means if you have a mobile phase that stays the same all the way through meaning it remains constant in composition.

Question 44

in HPLC, what are the ancillary equipment

Answer 44

ancillary equipment in HPLC are:
- Liquid handling pump
it must provide a constant flow of mobile phase at constant (high) pressure
-mixing system
its got a mixing system so you can alter the compositions of the mobile phase
-detector
the top end of the column is the mobile phase, at the other phase is the detector
- UV-VIS spectrophotometer (non-destructive)
- Mass spectrometer (destructive) - LC-MS (Liquid chromatography detector mass spectrometry) (this method destroys the same)
- fraction collectors
you use it if you want to collect the analytes.
the fraction collectors can be automated or you can spend 8-10 hours collecting the analytes manually.

Question 45

what is the uses for HPLC

Answer 45

• manufacturing
  > pharmaceuticals for analytical or preparative)
• Legal/ forensic
  > illegal drugs/ doping in urine
• research
  > very wide range of applications in chemistry, biochemistry and pharmacology
• medical
  > nutrients e.g. vitamins in blood serum

Question 46

what are the negatives of HPLC

Answer 46

• an expensive technique
  analytical HPLC columns cost £400-£2000
• eventually “wear out”
• can easily become irreversibly blocked

make sure you don’t get gas in the mobile phase because the bubbles will interfere with separation and affect the results

Question 47

what are the main HPLC equipment

Answer 47

• fluorescence detector
• column chamber
• chromatogram on computer
• sample manager
• solvent manager