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Reversed-Phase is the Most Common Type of HPLC
- Columns are rugged, efficient, reproducible, and stable.
- Aqueous mobile phases are less expensive then organic phases.
- Less time is required for equilibration of the column between runs.
- Detection is often easier.
Retention Times in Reversed Phase
Polar (hydrophylic) molecules are eluted first.
Non-polar molecules are are eluted last.
Medium polarity molecules are eluted in the middle.
Stationary Phases
Alkane chains of 8 (C-8) or 18 (C-18) carbon atoms
bonded to silica are the most commonly used stationary phases
For analyses conducted with a high pH mobile phase,
alkanes bonded to a polymer support are used. Polymer
supports are also preferred over silica for the analysis of
basic compounds because the residual silanol groups on
silica can cause severe band tailing.
Polar stationary phases such as amino, cyano, diol, can
also be used in reversed phase chromatography
Retention Time and pH in Reversed Phase
When an acid or a base is ionized it becomes much less
hydrophobic and will elute much earlier. Acids lose a
proton and become ionized (negative charge) as pH increases.
Bases on the other hand, gain a proton and acquire a positive
charge as pH decreases.
not charged
Relative Retention Time
partially charged
fully charged
1
2
pKa
3
4
pH
5
6
7
Buffers and pH Control
When the mobile phase pH is near the pKa of an analyte,
the retention of the analyte varies considerably with small
changes in pH (as small as 0.1 unit) and peak shapes can
become skewed. It is therefore important that the mobile
phase is well buffered, and the pH is accurately measured.
Buffers and pH Control
It is also important to note that the analyte itself can change
the pH of the mobile phase within its concentration band
so concentrations of the analytes should not be too large
and the sample pH should not be too different from the buffer pH.
Buffers are limited in the pH range that they can control so it is
best if the pKa of the analyte is similar to the pKa of the buffer.
Buffers and pH Control
Citrate buffer has the advantage of being able to stabilize pH
over a wide range (2.1-6.4). This is because the citrate molecule
has 3 ionizable groups with different pKa’s. However, because
of its UV absorbance, it can only be used with UV detectors
at wavelengths above 230 nm. Citrate has also been claimed
to be corrosive to stainless steel, so if it is used, extra care is
needed to flush the system after a series of runs.
Many other buffers are also available.
Amphoteric Molecules
Some molecules, amino acids are good examples,
have both acidic and basic groups on the same molecule.
This property is called amphoteric. Amphoteric
molecules have positive charges at low pH and negative
charges at high pH. At intermediate pH values, both the
acidic and basic groups are partially charged.
Because both groups are charged, the molecule has
its highest ionization and therefore its lowest retention
at intermediate pH.
Water and C-18 Stationary Phases
Mobile phases used should not contain less than 10% organic
solvent when using stationary phases such as C-18.
When the mobile phase contains more than 10% organic solvent
the alkane chains are more or less expanded out straight from
the surface.
With less than 10% organic solvent, the C-18 chains
collapse and it is difficult to bring them back to the straight
conformation.
Organic Solvents
Three water-miscible solvents, methanol, acetonitrile
and tetrahydrofuran differ considerably in their selectivity
properties and are also acceptable in terms of UV absorbance
and viscosity and therefore are the most commonly used
organic solvents for reversed-phase separations.
Reversed Phase and Basic Compounds
Basic compounds interact with the silanols of silica-based
columns resulting in increased retention, band tailing
and column-to-column variability.
- Use Type B columns which have a minimum of acidic silanols
- Use a low pH mobile phase to minimize ionization of silanols
- Use buffer cations that are strongly held by the silanols and will
not readily exchange with the analyte ions.
- Use high pH where basic analytes are not charged and will
not be affected by the silanols. At higher pH, silica columns
may degrade, so polymer based stationary phases should be used.
Non-Aqueous Reversed-Phase HPLC
For very hydrophobic samples that are retained so strongly
that they are not eluted with 100% acetonitrile, a non-aqueous
mobile phase can be used.
Typically methanol or acetonitrile is used as the more polar
solvent, while chloroform, methylene chloride,
methyl-t-butyl ether, or tetrahydrofuran are commonly used
as the less polar solvent (stronger solvent).
The separation of carotenoids such as a-carotene, b-carotene
and lycopene is an example of a food science use of
non-aqueous reversed-phase chromatography.
Effect of Temperature
Increasing temperature has the effect of decreasing k
so that compounds elute earlier.
As temperature is increased the retention of planar compounds
decreases faster than non-planar compounds resulting in
a change in selectivity.
Temperature can have a large effect on selectivity
for ionizable compounds.