LC Flashcards
What is the component of the column support
-Have to be solid particle ex: silica
- spherical and porous
Hplc and Hplc
Hplc: The small particles you use the high performance you get and the high pressure drop. Particle bigger than 3 and can tolerate small pressure of 400 bar
Uhplc: particle less than 3 microns can tolerate pressure up to 1500 bar
Advantage of particle to be fully porous
Means that analyte can enter the pore and diffuse to the center of particle out again
Characteristics of Superficially porous material
Contains solid core that are not accessible for the analytes
Characteristics of Perfusion particles
Have bigger pore structures.
Can permit flow through the particles.
Diffusive pores for analyte
Separation characteristics of particles in Hplc
-Total porous with 5/3.5 commonly used
- superficially porous give us better efficiency Cz distance of analytes to move in and out is reduced
How does fully porous particles affect resistance to mass transfer
It’s become bigger when we use fully porous particles
How does fully superficially/ porous-shell particles affect resistance to mass transfer
It’ll reduced by using superficially particles lead to better efficiency
How can molecule size relate to pores size during separation
When we use large molecules size use larger pores btn 15-100. Small molecule use small pores btn 7-10.
What is xerogel / sil- gel
-Is the silica particles that are made by soluble silicate.
- produced in water free solvent
-big surface where molecules can interact and 70% porosity
What is the sol-gel
- small spherical silicate
- are boiled in water and get hydroxylate silica
-when dissolve they make ikiraro and aggregate together - low porosity and surface area
- better pH
Characteristics of silica
Purity: the more metal it contains the poor performance of separation will become Cz molecule can interact with metal due to the ionic interaction which cause secondary mechanisms(bad)
Type of silica
Silica A is acidic means it can loss H and form positive charge which can interact with positive analyte to get ion exchange pka of 5, less purity
Silica B is less acidic
Effect of choose of silica have on total separation
- by comparing silica A and B, both contain C18
- compounds are basic drugs pka larger than 6 so around pH 5/6 they will be positively charged we can know it too by looking on eluent
- as silica A have acidic back bone so it can interact with the analytes which give us secondary interaction mechanisms.which will have big effect on retention time by increasing it
- by using silica B which is less acidic the interaction is low so this will not have big effect on retention
How can acidity of silica affect retention time and tailing
-Buy reducing acidity of silica means from silica A to silica B we reduce retention time
- due to the strong secondary interaction of silica A with analyte it cause tailing
What does coverage of silica surface depends on
It’s depends on length of ligand so, the longer ligand the less coverage of material will have
Characteristics of silica ligand
Horizontal polymerisation ligand the better coverage
The bulky the branches the better coverage/ better protection of silica back bone
Why do we need to protect phases
-At low pH below 2 where there is higher concentrations of hydroxynium ion so bond attaches ligand to the S.phase surface will be blocken lead to bleeding
- base ph a
> 8 can dissolve silica
How can we protect the phase
1.Use of bulky side chain can protect phase for low ph
2. Use column that are end-capped means the second or third rx are done on material by adding s.phase ligand and attach small molecules (TMS) and less bulky
Silica columns selectivity
- Length of the column
- when the retentivity of analyte is high we choose the short chain means that when you want to reduce Kd or retention time you use short chain so where the surface area where analyte will interact will be reduced - Interactions
Selectivity based on interaction, how s.phase interactions interact with analyte
Cation exchange interaction / mixed retention mechanism
If Analyte are positively charged for basic compounds at low M.phase pH , pH have to be higher enough to be higher than pka value of the silica so that is charged and low enough for the analytes to be positively charged
What does column life time depends on
Silica type
If is protected
Temperature
Comparing non Capping and endo capping
The stronger the retention of the basic compounds is, it indicate that more silica surface will be exposed to retain analyte based on h bonds. So end- capping will not bleed as much as column which is not protected.
Effect of pH buffer
Better to use phosphate buffer and carbonate buffer to set higher ph=10
Effect of temperature on silica dissolution
With higher temperatures amounts of silica dissolved will increase
How can we measure column efficiency
We have to consider the injected volume, how tubing narrow are, type of sample diluent,
What plate number for well packed Hplc
We need packed column with small particles size reduce column length without any loss in efficiency,
why?
Cz when we have long column will increase retention time (analysis time)
How to know the wellness of peak by peak asymmetry
If we have the excellent performance then the distance between an and b will be the same, so the peak asymmetry will be 1 but 1.2 is okay too not 4
When tailing peak the distance will be bigger than 1
Why other materials than silica
-Chemical stability (pH range)
-Temperature stability
How to suppress ionisation
By increasing pH so that we have more basic conditions to get neutral form
Characteristics of detector
High selectivity; sensitivity and have no effect on temperature and flow rate
How do wavelength affect selectivity and sensitivity
- the higher wavelength, the more selective measurement you can do
Why do we choose other detector systems
- when analyte absorbance is low means molar absorbativity is low we use UV-Vis
- when we need low control then is good to use selective detector
Cause of long time noise
Temperature cause noise and can cause drifting
Noise give limitation in trace analysis (detection and quantify very small amounts of substance)
Consequences of noise
Cause limit in detection and can produce peak and we won’t be able to differentiate it from normal one
What cause drifting base line in LC
Temperature or dying lamp
How to reduce noise
You reduce data collection interval by lowering frequency to 1Hz
But if you measure at high frequency of 15Hz will collect high noise
What indicat detector performance
By signal to noise ratio
How can you increase separation of peak in LC
When you increase the selectivity of detectors we decrease interfering effect
Uv-Cut off
This give us info about mobile phase transparency (means M.phase doesn’t absorb too much light)
so we need absorbance which are less than 0.5).
Impact of wavelength choice
It can affect the selectivity and sensitivity of the analytes
What are the polar interactions in LC
H bonds
How does temperature affects retention in LC
By increasing temperatures you reduce the h bonding interaction which reduce water polarity by increasing acetonitrile % which increase elutition strength then reduce k
Effect of changing column
If you change columns from C18 to cyano column retention reduce Cz cyano column is less retentivy in reserve phase column
How to improve resolution in terms of retention
When you decrease the concentration of eluent (acetonitrile) you reduce elution strength which increases retention and peak are separated well
How does organic solvent affect separation
When you decrease percentage or concentration of organic solvent can cause good separation but not really
How does elution strength depends on organic solvent
Acetonitrile have good elution strength .so, the lower the organic solvent the better elution strength
What does the reversed phase mechanism depend upon
Reverse phase mechanism depends on the hydrophobic interaction. So the stronger hydrophobic interaction the more retention time
How does analyte elution in the LC
The one which is more acidic elute last, strong interaction between acidic analyte and s.phase (hydrophobic )
How does reducing particle size affect efficiency
Affect efficiency by reducing mass transfer resistance and zone broadening by multiple flow rate
