ROMIL-SpR Ion-Pair & Buffer Reagents for HPLC

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.

	ROMIL-SpR^™ Ion-Pair & Buffer Reagents