General Chromatography Flashcards
What is goal of separation
1.Resolution of peaks (Ability to separate 2peaks)
2.retention time
3. Zone width- efficiency
What is a good resolution value
Resolution have to be 1.5 means that we don’t have missing and our peaks are well separated
If Rs is low than 1,5 means that there is mixing of 2 peaks
What affect resolution
Different in peak size can cause inseparable problems of peak/ not be able to distinguish them
How to improve resolution
-increase retention time of analytes
- improve selectivity by choosing other phases
-improve efficiency
What does retention and selectivity depends on
Intermolecular interactions the analytes experience with M and S phase molecules
Why does analytes interact with M phase in LC only
Because in GC M phase is gas (N) and they have idle behaviour means they will not interact with analytes.
Gas purposes in GC is to transport analytes away from column where is not dissolved in S phase
What are different chromatographic mode
Reverse phase chromatography: when S phase is non polar and M phase is polar.
Normal phase chromatography: when S phase is polar and M phase is less polar
What is retention factor
Is how long time analytes stays in stationary phase compared to mobile phase
What affect retention factor and retention time
It’s can be affected by flow rate, if we reduce flow rate retention time will increase
Ex: 1 ml/min flow rate give 1 retention factor but if we reduce to 0,5 ml/min flow rate retention factor will be twice
Separation factor
Is time two analytes stays in stationary phase
What is the characteristic of separation factor
It has always to be great than 1, if less or equal to 1 means that 2 cpds has same retention(co-elute) time and no separation occurs
What does retention factor depends on
It depends on distribution constant
Separation factor depends on what
Depends on distributions constant ratio of 2 compounds
Can pressure drop affect retention factor
In LC no effect- If you have liquid, the compressibility of liquid is insignificant that will not give any effect on the retentativity of a compound Cz the thermodynamic properties will not change by pressure.
In GC pressure have effect- But when mobile phase is compressible, the it can affect retentativity due to change in solubility
Can temperature affect retention factor and. Separation factor
In GC, k is affected by temperature and separation factor
- so the higher temperature the higher vapour pressure and faster analyte will come out.
- the more volatile the compound is the less time it will spend in the system.
What else does temperature affect
It affect kinetic properties of systems and linked to the zone width (broadening of a peaks) .
-increase of temperature lead to increase of diffusivity leading to wider zone
How is temperature affect in LC
- retention factor is reduced at low %(no po)
- affects kinetic properties increase mass transfer(moving of analytes from M.phase to S.phase ) lead to fast equilbria led to narrow peaks and less mixing of the zones
What does pressure drop
In LC increase in temperature reduce viscosity lead to increase of flow rate
What determines efficiency
Is determined by the width of zone, better control of zone width better efficiency
Effect on N and H
The bigger plate number the narrow plate height is and a small plate height the small width of peaks
What does plate height depend on
Zone broadening causes large plate heights
- the higher the resistance the wider peak becomes
Zone broadening
The faster flow rate the more zone broadening cause equilibrium resistance increases with the flow rate
Mass transfer resistance
The analytes close to the boundary will have short time to diffuse into M.phase from S.phase
What happened at high pressure in GC
At high pressure gas are more compressible lead to the effect on linear velocity and diffusion rate.
So because is compressible it become more viscous which affect it’s flow ability
Explain bidding’s equation
Ko= measure of the porosity, so the higher the porosity the bigger the contribution of the pores we have to the zone broadening
O = show geometry of pores. so straight pores O=1, pores are tortuous O>1 so the zone broadening become more
Small particles size
You can increase flow rate without reducing efficiency
- by reducing particle size the minimum of plate height is reduced
Big particles size
If you increase flow rate you will reduce efficiency
Fact that affect zone broadening in extra- column
- Injected volume : the bigger volume injected the wider zone width resulting in zone broadening
- Detection: use cell that are less in % than peak volume
- Tubing design: if you use wider tubing peak will be dispose in higher volume lead to zone broadening
- Void volume If you have narrow zone of molecules and enter (dissolved ) in bigger volume will lead to wide peak at low flow rate same as high flow rate
Cause of mixing chamber
It happens when tubil didn’t connect well with cavity so they will creation of mixing chamber that cause wide zone lead to wide peak and zone broadening
How can solvent cause zone broadening
If we dissolved the analytes in the stronger solvent that eluent this can cause zone broadening
Explain concentration effect
When 100% of water of solvent is used distribution ratio will be increased and analytes will stick to the stationary phase, as water doesn’t have the same composition as the mobile phase.
But when we use 100% methanol will give opposite effect, distribution ratio will be reduced. So the analytes will follow sample solvent until is mixed with mobile phase as they have the same composition.
Zone broadening due to the isothermal effects
When we increase the concentration and column is overloaded there will be analyte-analyte interactions and they will contribute to the distribution constant by increasing it lead to the increase in retention time resulting in peak broadening
Zone broadening due to the isothermal effect (adsorption chromatography )
Analytes interact with s.phase by diffusion but when we overload analyte concentration, analyte will not have more space to stick to s.phase surface leading to increases of analytes concentration in M.phase which reduce distribution ratio And reduce retention time resulting in peak broadening
What is the difference between fronting and tailing
Fronting: occurs in liquid s.phase, when we overload columns with analyte concentration this will cause analyte analyte interaction which contributes to the increase of distribution ratio lead to increase of retention time resulting in peak broadening
Tailing: occurs in solid s.phase, so when analyte interact with s.phase by diffusion and we overload it there will be no more space for analyte to stick on s.phase surface so this will increase the analytes concentration in M.phase causing the reduction of distribution ratio resulting in peak broadening and as the overload increases we increase the tailing
How to solve the fronting and tailing
We can solve the problem by diluting our sample
Relationships between resolution and efficiency(N), selectivity(&), retentivity(K)
Retention time: by increasing retention time to 5 this will increase the resolution but after 5 no need of increasing k cause Rs will be constant (no effect), so the ideally 1<k<5, if k is less than one means that compound spend less time in s.phase than in M.phase leading to bad separation.
Separation factor: small change in & can cause a big change in Rs, Rs increase with &. And & >1 because If is equal to 1 means that there is co-elution means both cpds have same retention time. & is the ratio of retention factor.
Plate number: Rs can be increased by reducing plate height
Which factor cause flow rate going from normal to micro column
Cross sectional area
How can flow rate be obtained in micro columns, by which equipment
It can be obtained by splitting flow with micro pumps
What happened when we have same velocity in M.phase
H,N and Rs will be the same
What happens in micro column whe flow rate is 1
H will increase leading to decrease of Rs and decrease of N
What happened to the pressure drop when flow rate is keeper to 1
Pressure drop will be higher
What happens to k when columns diameter reduces
Nothing, k Depends on nature of 2 phase M and S phase and their relative amount
What does k depend on
M and S phase
What happens to retention time in micro column
If linear velocity is small no effect. but, when we increase linear velocity with flow rate equals to 1 retention time reduce
What happens to the peak height in micro column
It goes there with higher concentration lead to height peak
