Chromatography Flashcards
What are the three applications of chromatography
Quantitation
Identification
Purification
What is extraction chromatography
You have 2 immiscible phases, the solutes move to each diff phase depending on their solubility’s
The strength of their interaction with each other determines where they go
(Ex. If lower layer is water, more polar things go in the lower layer)
What are the 3 ways the extraction can be adjusted to move things into different phases
Ph
Chelators
Surfactants
How does ph affect extractions
Affects the efficiency,
if protonated or deprotonated the solvent goes to less or more polar layer because the charge has changed
How do chelators affect extractions
They change the solubility of metal ions
If the ions are chelated, they can go into the organic (nonpolar) layer by decreasing the charge/polarity of the ion
Makes things neutral/less polar
How do surfactants affect extractions
They interact with the hydrophobic and hydrophilic phases by making micelles and liposomes
These surfactants can force things to go into the nonpolar layer by surrounding the particle and making it hydrophobic (less polar)
What is regular chromatography
Similar to extractions
There is a stationary phase and a mobile (eluent) phase that the solvent can interact with differently depending on the strath of their interactions with thhat phadr
If the eluent is flowing, things that interact with it will flow too
Do things have zero interaction with either mobile or stationary phase
No there’s always some interaction but just less
If something interacts less with stationary phase when does it get seen in a column
First solvent (mobile phase) always elutes first then the thing that interacts least with the stationary phase comes after
If something elites out after adding a diffent solvent than before when it’s wasn’t eluding what does this mean
The new solvent and the thing have stronger interactions with each other then the old solvent
What does the types of chromatography depend on and what are the 5 types of chromatography
Depends on the type of stationary phase used
Adsorption chromatography
Partition
Ion exchange
Size exclusion
Affinity
What is adsorption chromatography
The solutes interact with the surface of a solid stationary phase
What is partition chromatography
The solutes dissolve into a liquid stationary phase
What is ion exchange chromatography
The solute has a chemical reaction with a resin stationary phase instead of just interacting
It exchanges ions
What is size exclusion (gel) chromatography
The solutes get filtered through a mesh gel or interact with the pores of the gel
Larger travel slower smaller travel faster
What is affinity chromatography
The molecules of the solute has specific antibody or antigens or DNA/RNA pairings that bind to the stationary phase via chemical reactions
What are chromatograms
A plot of signal vs time from the detector as the solutes flow down the elution column
What ever elites first shows up on the graph first
The area under the peaks is proportional to their respective amount of solute
What is retention time
The amount of time it took for the sample to elute
TM TR TR PRIME IN TABLET pics
WRITE ON FOLUMA SHEET
What is separation factor
The ratio of how far apart the peaks are in a chromatogram (the adjusted retention time of 2 over the adjusted retention time of 1)
It’s independent of flow rate which means it’s good for comparing across experiments
It’s symbol is alpha
What is retention factor (K)
How fast the peak goes vs the fastest (unretained) compounds
The adjusted retention time over the TM
Bigger K = slower
Measuring peak resolution
Slide 16 in tablet pics
What resolution is best to see peaks and quantify them in a chromatogram
1.50
And the peaks a 6 sigmas apart
What happens to the peaks when there’s a larger retention time
The resolution of the peaks gets worse because the bands widen
They widen because they diffuse inside the columns along with having concentration gradients
