US 2016/0237112 A1 · PURIFICATION OF ORGANIC COMPOUNDS BY SURFACTANT MEDIATED PREPARATIVE HPLC

Abstract

A sample is provided that can be purified by preparative reversed phase high performance liquid chromatography (Prep-RP-HPLC) in a single run in spite of recent advances in the production of reversed phase derivatized silica stationary supports: (1) The traditional approach is to use a bigger column (greater amount of stationary phase); and (2) Use displacement chromatography which (while labor intensive to develop) uses the stationary phase more effectively. This disclosure describes a unique Prep-RP-HPLC technique that uses a C-18/C-8 derivatized silica coated with a surfactant such as Triton X-100 to result in 7 to 10 fold increase in sample loading (of the crude mixture of organic compounds including synthetic crude peptides) in contrast to the conventional Prep-RP-HPLC technique. This increase in sample loading capacity and output is due to the additional surrogate stationary phase characteristic of the C-18/C8 adsorbed (bound) surfactant.

Claims19

Dependent claims

A method of purification of organic compounds including:

peptides with increased sample loading capacity of reverse phase column in Preparative Reversed Phase High Performance Liquid Chromatography (Prep-RP-HPLC) using one of a surrogate stationary phase and an additional stationary phase in conjunction with one of a C-18 and a C-8 derivatized silica stationary phase.

A method of purifying a multi-component sample of organic compounds including peptides by Preparative Reversed Phase High Performance Liquid Chromatography (Prep-RP-HPLC) comprising the steps of:

(a) configuring a chromatographic system having a hydrophobic stationary phase;
(b) saturating the chromatographic stationary phase with one of a surrogate stationary phase and additional stationary phase surfactants selected from alkyl glycoside, bile acids, glucamides and poly-oxyethylenes;
(c) washing the column to remove excess un-bound surfactant employing a mixture of organic solvents and water;
(d) equilibrating the column with the starting mobile phase;
(e) applying a multicomponent sample to one end of the chromatographic bed comprising stationary phase coated with the surfactants;
and
(f) eluting the multicomponent sample using a linear gradient of buffers A & B, wherein the buffer A is 0.1 mM Cetyltrimethylammonium bromide and 0.1 mM sodium bicarbonate in water and buffer B is 0.1 mM Cetyltrimethylammonium bromide and 0.1 mM sodium bicarbonate in 50% aqueous acetonitrile;
(g) eluting the multicomponent sample using a linear gradient of buffers A & B, wherein the buffer A is 0.1% phosphoric acid in water and buffer B is 0.1% phosphoric acid in 50% aqueous acetonitrile;
or
(h) eluting the multicomponent sample using a linear gradient of buffers A & B, wherein the buffer A is 1% phosphoric acid in water and buffer B is 1% phosphoric acid in 50% aqueous acetonitrile;
or
(i) eluting the multicomponent sample using a linear gradient of buffers A & B, wherein the buffer A is 25 mM to 150 mM triethylammonium phosphate (pH 3) in water and buffer B is 25 mM to 150 mM triethylammonium phosphate (pH 3) in 50% aqueous acetonitrile;
and
(j) recovering the desired component of the sample.

11 more elements

Dependencies are inferred from the claim text.

Purification of organic compounds by surfactant mediated preparative hplc

Life of this patent

Purification of organic compounds by surfactant mediated preparative hplc

Life of this patent

Abstract

Claims19