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The Purest Reagents for Separation Optimisation in HPLC
Although reversed
phase HPLC is one of the most important techniques in chromatography, by
itself it is limited to separations of non-ionic (neutral) and non-polar
compounds.
Polar or ionic
substances tend to separate badly on reversed phase systems. So, bearing
in mind that many compounds of biological interest are either ionic or
highly polar, what can the HPLC analyst do to improve such separations?
Either give up or reach for ROMIL-SpR™ Super Purity Ion-Pair Reagents. The
addition to the mobile phase of one of these high purity substances, as a
counter-ion of opposite charge, enables the sample species to form a
neutral ‘ion pair’ that will now readily interact with the reversed phase
packing and thereby permit a clean separation.
Indeed, even though
the chromatographer will be aware of the type of electrical charge on the
species to be separated, we have found that ion-pair reagents are not used
as frequently as they could be because of the time and expense in
selecting the right one for the purpose. Whilst this subject is covered in
detail in many textbooks the following is a brief introduction to
selection of an appropriate ion-pair reagent.
| Basic compounds
R-NH3+ +
R'SO3- >
RNH3SO3--R' |
Such samples (also known
as cationic) are traditionally separated by addition of a
straight-chain alkyl sulphonic acid to the mobile phase. For ease of
solubility this is normally provided in the form of the sodium salt.
The pH of the mobile phase is controlled to around 3-4. It is the
hydrophobic portion of the combined ion-pair that controls the
separation. The greater the hydrophobicity, the greater the
interaction with the reversed phase column packing. So,
heptane-1-sulphonic acid sodium salt (C7) will produce a longer
retention time than, say, pentane-1-sulphonic acid sodium salt (C5).
However, alkyl
sulphonic acids do not work well with HPLC-MS which requires that
the ion-pair reagent should be volatile. For this technique, the
volatile perfluorofatty acids such as trifluoroacetic acid and
heptafluorobutyric acid are recommended. |
| Acidic
compounds
R-COO- +
R4'-N+ >
R-COO-N+-R4' |
These analytes (also
known as anionic) can be separated with straight-chain alkyl
quaternary ammonium salts or alkyl amines, controlling the pH of the
mobile phase to around 7.5. Again, the longer the alkyl chain, the
longer the retention time of the ion-pair species. |
| Ion-Pair
Kits |
ROMIL offers two kits
which each contain nine popular reagents that can be used to
determine optimum separation conditions without the expense of
buying the larger packs. One kit comprises a selection of reagents
for chromatography of anionic or basic compounds whilst the other
covers complementary reagents for separation of cationic or acidic
compounds. Both kits make ideal accessories for a busy development
laboratory. |
| General
points |
Concentration of the
ion-pair reagent should be around 0.005M. A slight change of
concentration by 10-20% can alter the retention time and thereby
resolve overlapping peaks. Where detection is by UV, good UV
transparency is important. Also, the ion-pair reagent if supplied in
a powdered form should be easy to dissolve in small amounts. Equally
the powder should be free from non-soluble particulate matter which
can affect sensitive HPLC pumps and frits. All these are standard
features of ROMIL-SpR™ Super Purity Ion-Pair Reagents. Available in
bulk and laboratory quantities these reagents permit cost-effective
separations of ionic species in either high-volume routine quality
control or specialist separations in research and
development. |
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