Amino-phase packing solvent

The data of Fig. 11 thus indicate that restricted-access delocalization does not exist for amino-phase packings. Furthermore, the importance of the variation of eo with 6n, shown in Fig. 11, is insignificant, so far as calculations of e" are concerned. We will therefore assume that cb is constant for a given solvent B and amino-phase packings, in the calculation of ° for multicomponent mobile phases (Appendix). 

Since amino acids and nucleotides are all polar and hydrophilic, they will be eluted quickly by the column. The mobile phase (see below) is also selected on the basis of polarity, with a medium- to high-polarity solvent required. The opposite of reverse phase chromatography is normal phase, where the column packing is medium to high polarity and the mobile phase is nonpolar. This technology is generally not applied to the analysis of polar molecules such as amino acids or nucleotides. Some peptides are more hydrophobic, making this method potentially more useful for peptides than for amino acids or nucleotides. 

The retention mechanism in the normal phase is often referred to as adsorption chromatography. It is described as the competition between analyte molecules and mobile-phase molecules on the surface of the stationary phase. It is assumed that the adsorbing analyte displaces an approximate equivalent amount of the adsorbed solvent molecules from the monolayer on the surface of the packing throughout the retention process [18]. The solvent molecules that cover the surface of the adsorbent may or may not interact with the adsorption sites, depending on the properties of the solvent. This retention model, proposed by Snyder, was originally used to describe retention with silica and alumnina adsorbents, but several other studies have shown that this model may also be used for polar bonded phases, such as diol, cyano, and amino bonded silica [10,19]. 

NP phase systems are used for the separation of non-ionic, apolar to medium-polar samples. If the sample is soluble in organic solvents an NP phase system should be applied as first test system (e.g. silica and organic mobile phase). However, if the sample shows best solubility in aqueous solvents the sample is quite polar. In this case RP phase systems with semi-polar (cyano, amino or diol) or apolar packings such as alkylsilica should be used. 

You ako need to consider the chemistry of the packing and its compatibility with the solvent. For example, since primary amines react easily with ketones to form imines, you should not use acetone or methyl ethyl ketone as a slurry solvent for bonded phases containing primary amino groups. 

Amino acids have been separated using PolySulfoethyl Aspartamide and PolyHydroxyethyl Aspartamide columns (PolyLC, USA). The mobile phase was 80% acetonitrile/20% 5-25 mM triethylammonium phosphate buffer, pH 2.8., mall differences in elution order were observed between both packings electrostatic effects combined with the hydrophilic interaction for the PolySulfoethyl Aspartamide column. Retention decreases with increasing buffer concentration. At neutral pH, the PolySulfoethyl Aspartamide retains basic amino acids even in the absence of organic solvent, and acidic amino acids are excluded as a result of ion-exclusion effects. Above 50% acetonitrile, hydrophilic-interaction effects dominate retention. 

The balanced final energy values between the complexes with (J )- and (S)-derivatized amino acids did not correlate well with their separation factors. The final energy values, however, indicated the chromatographic elution order, and van der Waals energy values are useful in the search for the best complex conformation. The precision of prediction of chromatographic separation factors may be improved by using a powerful computer that can handle both a brush-type chiral phase as a model surface for packing, and the solvent effects. 

Figure 4. SFC separation of five FMOC-amino acids 1, acetone (solvent) 2, valine 3, alanine 4, phenylalanine 5, lysine 6, serine. Conditions 15 cm x 4.6 mm ID packed column silica stationary phase CO2 mobile phase with methanol/ water/methylamine (98.99 1.00 0.01 v/v) modifier gradient indicated on chromatogram 42 °C UV detection at 269 nm. (reprinted with permission from Ref, 16).

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