POLYHYDROXYETHYL ASPARTAMIDE

II. INTRODUCTION OF PolyHYDROXYETHYL ASPARTAMIDE AND THE EFFECT OF CHAOTROPES ON THE FRACTIONATION RANGE... 

A SEC material should be hydrophilic if it is to be used for biological applications. One such material, introduced by PolyLC in 1990 (8), is silica with a covalently attached coating of poly(2-hydroxyethyl aspartamide) the trade name is PolyHYDROXYETHYL Aspartamide (PolyHEA). This material was evaluated for SEC of polypeptides by P.C. Andrews (University of Michigan) and worked well for the purpose (Fig. 8.1). Because formic acid is a good solvent for polypeptides, Dr. Andrews tried a mobile phase of 50 mM formic acid. The result was a dramatic shift to a lower fractionation range for both Vq and V, (Fig. 8.2) to the point that V, was defined by the elution position of water. 

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. 

Dipeptides can be separated also using the PolyHydroxyethyl Aspartamide column. Alpert used a mobile phase of 80% acetonitrile with 10-40 mM triethylammonium phosphate buffer, pH 2.8 or 5.0. The elution pattern can be changed by variation of the pH. 

As an example of longer peptides. Substance P fragments with amino acids sequentially removed from the N-terminus were separated on both using PolySulfoethyl Aspartamide and PolyHydroxyethyl Aspartamide columns (Fig. 11.2). Tbe separation on both columns was carried out in 80% acetonitrile, 10 mM triethylammonium-methyl phosphonate buffer, pH 3.0 with an increasing salt gradient from 0 to 125 mM sodium perchlorate. 

Oligonucleotides were separated on the PolyHydroxyethyl Aspartamide column using a shallow dual gradient of increasing salt concentration with concomitant decrease in acetonitrile. Mobile phase A was 40 mM triethylammonium phosphate buffer, pH 5.0, in 70% acetonitrile mobile phase B was 75 mM triethylammonium phosphate buffer, pH S.0, in 80% acetonitrile. 

Aminolysis of the intact rings with taurine leads to the formation of poly(2-sulfoethyl aspartamide) silica and the reaction with ethanolamine to the formation of poly(2-hydroxyethyl aspartamide) silica. Poly(succinimide)-based silica phases are manufactured by PolyLC (Columbia, MD, USA) under the trade names of PolyCAT A for poly(aspartic acid) silica, PolySulfoethyl A for poly(2-sulfoethyl aspartamide) silica, and PolyHydroxyethyl A for poly(2-hydroxyethyl aspartamide) silica. All three poly(succinimide)-based columns have a pore size of 200 A and a surface area of 188 m /g. Various poly(succinimide)-based columns have been used for the separation of carbohydrates, phosphorylated and nonphosphorylated amino acids, petides and glycopeptides, oligonucleotides, and various other polar analytes under HILIC conditions, but lately lost some of their momentum due to a lower chromatographic efficiency in comparison to more modern HILIC phases and column bleed [44]. 

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