Chapter 4 - Fundamentals of LC in LC-MS - part 1 Flashcards
GC separation involves a
mobile phase and a stationary phase.
In GC, the stationary phase is usually
an immobilized liquid of varying hydrophobicity coated onto the inner walls of fused silica capillary tubing.
GC has ________ options for improving selectivity when compared to LC.
less
In LC, the mobile phase is usually
a liquid mixture of water and an organic solvent plus other additives.
In LC, the stationary phase is usually
materials of varying hydrophobicity chemically bonded to particles of silica as a solid support.
With reverse phase LC, the amount of water in the mobile phase will
determine how strongly a hydrophobic analyte is repelled into the stationary phase.
An LC system consists of the following components:
- mobile phase reservoir
- vacuum degasser
- pump
- injector
- column
- detector
- data system
LC grade solvents are required in order to
prevent column degradation and minimize background signals.
A filter in LC excludes _____ micro meter matter and in HPLC it excludes ______ micro meter matter.
> 0.5 (LC)
>0.2 (HPLC)
Outlet degassing occurs when
the mobile phase moves from the small volume of the capillary tubing to the large volume of the flow cell.
Most effective degassing technique
Offline refluxing (arduous and has risks, so rarely used)
Least favored degassing technique
Offline ultrasonic degassing (least efficient, and risks loss of volatile mobile phase components).
Degassing technique that removes 80% of dissolved gases.
Helium sparging
Most popular degassing technique
Vacuum degassing (low pressure of vacuum chamber draws dissolved gases out through a gas permeable tubing)
Isocratic elution
uses a constant mobile phase composition.
RPLC uses a _________ stationary phase and a ________ mobile phase.
nonpolar, polar
As the organic solvent content increases,
mobile phase polarity decreases, resulting in increase of affinity of the analytes for the mobile phase and a decreased retention time.
General problems of isocratic elution
Early eluting analytes may be poorly resolved.
Late eluting analytes may have broad peak shape and low sensitivity.
Not always suitable to separate complex mixtures with a wide range of chemistry and polarity.
3 fundamentals of an organic solvent that affect chromatographic selectivity.
Polarity (dipole moment)
Acidity (proton donor)
Basicity (proton acceptor)
3 organic solvents commonly used in RPLC whose properties differ significantly.
Methanol
Acetonitrile
Tetrahydrofuran (THF)
Nomograms
can be used to alter the separation such that the overall retention time remains similar, but the selectivity is altered.
THF is the ________ while methanol is the __________ of the three organic solvents commonly used in LC.
strongest, weakest
Gradient elution
gradually increases the content of organic solvent to increase eluting strength.
Advantages of gradient elution:
Improved resolution Increased detection Ability to separate complex samples Shorter analysis times Decrease in column deterioration
Disadvantages of gradient elution:
More expensive instrumentation
Re-equilibration of column after each analysis adds time to analysis time
Most common length of LC column
15 cm
Inner diameter of LC column is between
1-5 mm
Most common LC column inner diameter
2.1 mm
Most common support of LC stationary phase is
highly pure, spherical, microporous particles of silica that have a surface area of several hundred square meters per gram.
Irregular particles provide poorer column efficiency than spherical particles due to
much poorer packing homogeneity.
The smaller the packing material particle in LC column,
the lower the plate height, the better column efficiency, the higher the optimum linear velocity of the mobile phase.
The presence of metal ions within silica can cause
peak tailing.
The composition of silica used for LC is
SiO2 xH2O
Most silica cannot be used above pH
8 because it dissolves in base.
Silanol groups are protonated at pH
~ 2-3
All silanol groups interact with
polar and ionic analytes to different extents.
Acidic silanol groups give the strongest reaction and the
worst peak tailing.
The siloxane bond (Si-O-SiR) hydrolyzes below pH
2 (generally limited to use between pH 2-8)
Monomeric stationary phases
are highly efficient, but not as chemically robust as those produced from di- or trichlorosilanes (which are known as polymeric bonded phases).
Manufacturers may choose to end-cap the column surface, which involves
reacting the silanol groups with a sterically small but highly reactive reagent that ‘caps’ the polar surface silanol with a non-polar trimethylsilyl group.
Longer chain silica provide more steric hinderance and achieve
a lower surface coverage.
RPLC is characterized by having a stationary phase that is
less polar than the mobile phase.
In RPLC, ____ is the most popular stationary phase.
C18 (highly robust hydrophobic phase which produces good retention with non-polar analytes).
Shortening the alkyl chain of RPLC stationary phase will
shorten the retention time.
The use of more polar RPLC stationary phases show
altered selectivity compared to alkyl phases, and can interact with polar analyte functional groups via dipole-dipole interactions.
Traditional reverse phase columns are not suitable for use with aqueous mobile phases above _____% water due to phase collapse (or self-association)
95
Stability in highly aqueous mobile phases is achieved by
the adsorption of a layer of water at the silica surface.
The method of introducing stability in 100% aqueous mobile phases is to use
polar embedded ligands.
Polar embedded ligands contain a modification to the alkyl chain which is usually
an amide, carbamate, or other suitable polar functional group.
Water wettable phase advantages
polar group that gives an alternative selectivity
ability to separate polar, ionizable and highly basic compounds
enhanced robustness over shorter chain and polar bonded phases
