INTRODUCTION
TO
HPLC
Modes of HPLC
Adsorption Chromatography is another name for liquid-solid chromatography.
The solid phase is usually silica or alumina, which have highly polar
surfaces. The mobile phases are commonly some of the less polar solvents.
Normal-Phase Chromatography is based on a polar liquid phase coated or
bonded onto a silica support. For historical reasons, this is called normal
phase because the stationary phase is polar and the mobile phase is
typically a nonpolar solvent such as hexane or isopropylether. In normal
phase, the least polar component is eluted first.
Reversed-Phase Chromatography uses a non-polar stationary phase (also
coated or bonded onto silica or another support) and polar solvents such as
water, acetonitrile or methanol. In reversed-phase chromatography, the
most polar component is eluted first.
Ion-Pair Chromatography is one of the methods used to separate ions. It uses
regular reversed phase columns, and can separate acids, bases and neutral
compounds during the same chromatographic run.
Ion-Exchange Chromatography is based on the use of ion-exchange resins as
the stationary phase.
Ion Chromatography was developed to separate the ions of strong acids and
bases. The equipment used is different from that of ion-exchange
chromatography.
Solvent Handling
Degassing
1) Dissolved gasses such as nitrogen and oxygen that can
cause problems by forming bubbles in the column or the
detector.
2) Oxygen from the air is chemically reactive and may react
with the sample.
3) Dissolved oxygen also strongly absorbs at low UV
wavelengths
Methods of Degassing Solvents
1)
2)
3)
4)
Filtration
Sonication under vacuum
Sparging with Helium
Interal Degassing
Solvent Handling
Inlet Filters
Particulate matter may damage the pumping or injection
system by wearing out valves and seals.
Evaporation
The composition of a mixture of solvents can change over
time as evaporation depleted the most volatile solvent from
the mix. Place sparge tube above the liquid to reduce this
problem.
Bacteria Control
- Make up new buffer solutions daily
- Use sodium azide
Corrosion
PTFE(Teflon) - Mostly resistant to HPLC Solvents
PEEK - Strong organic solvents may give problems
Stainless Steel - hydrochloride salts and hydrochloric
salts are the most aggressive towards stainless steel.
Acidic samples may damage injector systems.
Long-Term Solvent Storage
Alcohols, alkanes, toluene, xylenes, acetonitrile
and ethyl acetate are chemically stable for more
than a year.
Ethers form peroxides which are very reactive
compounds that can seriously affect
chromatography. Peroxides can be explosive
when they build up and therefore pose a safety
hazard.
Chlorinated solvent degrade to form reactive
free-radical by-products and hydrochloric acid.
The acid can alter the pH of the mobile phase
and seriously affect the chromatography.
Requirements of HPLC
Pumps
1) should be able to generate pressures up to
5000 psi.
2) should provide uniform “pulse free” flow.
3) should be able to provide accurate flow rates
from 0.1 to 10 mL/min.
4) should have accurate control of flow rate
changes (0.5% or less relative error).
5) should contain corrosion- and leak- resistant
components.
HPLC Pump Design
Chamber Filling:
This valve closed
This valve open
Pumping Stroke:
This valve open
This valve closed
Chamber
HPLC Pump Design
Gradient Elution
One of the most important methods of improving
separation of components of complex samples is
to change the mobile phase composition during
the run. The change of mobile phase
composition is called gradient elution and it can
be accomplished either by high-pressure mixing
or low-pressure mixing.
Solvent Mixing Systems
High-Pressure Injection
HPLC Column Packing
The stationary phase is most commonly made of
porous spherical particles with diameters from 310 mm.
The typical porous particles used in analysis
of small (non-protein) molecules have 7-12 nm
pores. The total surface area of such
particles ranges from 150 – 400 m2/g.
Micropellicular Particles
Micropellicular particles have a stationary phase
coating only on the outside surface are useful for
rapid separation of macromolecules such as
proteins. Because the mobile phase does not
diffuse into the particles, the mass transfer
kinetics allow for very sharp peaks but since the
total surface area of these particles is small
sample volume is very limited.
The Surface of Silica
For adsorption chromatography, the silica is used without
modification, but for most other types of HPLC, the silica is
coated with one of many types of stationary phases. More
commonly, in modern chromatography columns, the
stationary phase is chemically bonded to the silica
Bonding Phases onto the
Silica Support
Silica
+
OH
CH3
|
Silica -0-Si- CH3
|
CH3
+
CH3
|
Cl -Si- CH3
|
CH3
HCl
Could be many different
functional groups here
The Surface of Silica Bonded
with C18
End Capping
Usually half or more of the silanol groups remain
unreacted after bonding with C18. One method
used to reduce the effects of these residual
silanol groups is a process called “endcapping.
End Capping
After bonding with C18. use small silane
molecules such as trimethylchlorosilane to react
some of the remaining –OH groups
Steric Protection
CH3
|
Hydrolysis may
degrade the column by
breaking off the bonded
phase.
CH2
|
Silica -0-Si- CH3
|
CH2
|
CH3
All C18 Columns Are Not Created
Equal
See Handout…..
Differences in:
Particle Size
Pore Size
Surface Area
Carbon Load
End-Capping
Silica Type
*Bonding Density
Comparison of Different C18 Columns
Column: Symmetry C18
Mobile Phase:
60% CH3CN
40% 50mM KH2PO4,
pH 3.2
Hypersil HyPURITY C18
1.
2.
3.
4.
5.
6.
7.
Sample:
Uracil
Pyridine
Phenol
Dimethyl phthalate
N,N-Dimethylaniline
4-Butylbenzoic acid
Toluene
C18 Phases Ranked According to Retention for
Toluene
Other Bonded Phases
Phenyl phases show weak dipole - induced dipole
interactions with polar analytes. Usually this type
of bonded phase is used for separating closely
related groups of molecules.
Other Bonded Phases
Cyano columns are very slightly polar and are
useful for fast separations of mixtures consisting
of very different components that might have a
very broad range of retention times on the
usual columns. Cyano columns can be used with
both normal- and reversed-phase HPLC.
Other Bonded Phases
The amino-phase is the most polar, and it can
also act as weak anion-exchanger, (protonated at
low pH). Amino columns are mainly used in
normal-phase mode, especially for selective
retention of aromatic compounds.
Other Bonded Phases
Diol columns are slightly polar and are used for
normal-phase separations. Diols are useful for
samples containing many compounds with
different polarities , and which usually have
strong retention on unmodified silica.
Non-Silica Supports
The most common polymer support material for
reversed-phase separation is made of
divinylbenzene cross-linked polystyrene
The main advantage of porous polymers is that
they can be used in the pH range from 1 to 13.
(silica supports tend to dissolve at pH greater
than 9).
Because of the surface acidity of silica
supports, polymer supports can be a better
choice for separating basic compounds.
Column Improperly Installed in the Lab……..