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