Lecture 3 - Chromatography Flashcards
What is Ion supression?
Separation of analyte and salt/buffer
What is the advantage of combining MS with chromatography?
Gives MS ability to analyse each component one at a time.
What are the different types of chromatography combined with MS?
Gas chromatography (GC)
High pressure liquid chromatography (HPLC)
Separations are done under atmospheric pressure and must enter the MS (a vacuum)
How does gas chromatography work?
Analyte (gas) is injected into a carrier gas.
The mixture is passed through a colum heated by a colum oven. The gas mixture reaches the detector.
Analyte must be volatile or derivatised
e.g. R-OH > R-O-Si-(CH3)3
What is the advantage of coupling MS with GC?
GC utilizes a long colum, allowing different pressures to be used without disrupting the column.
The column is at atmospheric pressure at one end and at the MS vacuum pressure at the other
What is HPLC?
It has more problems than GC.
Analyte must be in the liquid phase, not gas. Hence there is a lot of liquid to get rid of.
This is fixed through elution one at a time.
State the major parameters of HPLC
Retention volume (Vr): depends on the column type, size and the instrument parameters
Dead volume (Vo): Volume of liquid phase inside the column
Retention factor (k’):
Vr - Vo
______ = k’
Vo
These are independant of the column size and instrument setup.
Optimize retention factor between 1-10
sensitivity should be >1.2
What is the reversed phase separation principle?
Nonpolar interactions of analyte with hydrophobic adsorbent surface (C18, C8, Phenyl, C4) called eluents.
e.g eluents: MeOH, MeCN
Difference in analyte sorption affinities results in their separation.
More polar analytes are retained less
Analytes with higher hydrophobic part are retained longer
Almost no separation of structural isomers
What are the HPLC separation types?
Adsorption - non polar & non ionic / water insoluble
Partition - non ionic polar / Water soluble + insoluble
Ion exchange - ionic & non ionic polar / Water soluble
Exclusion - All of the above
What is HPLC miniaturisation?
Traditional HPLC
4.6mm ID column run at 1ml/min
Nano-HPLC
75uM ID run at 200nl/min
Why? : ESI is a concentration sensitive detector
Lower column ID = greater sensitivity as flow rate is decreased.
What are the features of HPLC-FAB MS?
1-5ul/min flows
Eluent mixed with glycerol
Solvent evaporates leaving glycerol and sample
Continuous analysis
What is the maximum flow rate of ESI?
0.2ml/min
High salt conc. are not tolerated
Source removes the solvent
Large compounds - proteins/large peptides are detected as multiply charged ions
What are the features of HPLC-ESI-MS?
Nano-HPLC at 200nl/min
Injection vol. should be 3nl
What is the equation for Selectivity (a)
k’2
_____ = a
k’1
What is the equation for Efficiency (N)
Vr^2
______ x16 = N
Wb^2
