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Purification of organic compounds by surfactant mediated preparative HPLC

A technology for surfactants and organic compounds, applied in the field of purification of organic compounds, can solve the problems of increased purified products, expensive and affordable column hardware, laborious development, etc., to achieve reduced waste treatment, low equipment scale, and easy operation Effect

Inactive Publication Date: 2016-05-04
拉马莫罕·拉奥·达瓦鲁利
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, these large-scale HPLC instruments and associated column hardware are very expensive and limit the affordability of the method
In addition, none of these improvements both address a given column loading capacity, nor do they yield a significant increase in the amount of purified product (output of packed column / mL)
[0006] Despite these above-mentioned advances, there are only two ways to increase the amount of sample that can be purified by Prep-RP-HPLC in a single run when being eluted earlier: (1) The traditional method is Use larger columns (larger amount of stationary phase); and (2) use displacement chromatography, which (while laborious for development) uses stationary phase more efficiently

Method used

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  • Purification of organic compounds by surfactant mediated preparative HPLC
  • Purification of organic compounds by surfactant mediated preparative HPLC
  • Purification of organic compounds by surfactant mediated preparative HPLC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment -1

[0151] Example-1: Prep-RP-HPLC of Leuprolide Acetate Using Triton X-100 as Additional Stationary Phase and Aqueous Phosphate Buffer:

[0152] C-18 reverse phase column (RevelerisC-18, 12g, 40μ, Pore ​​size) was saturated with Triton X-100 (12 g dissolved in 500 mL water). Excess unbound surfactant was washed with 90% aqueous acetonitrile containing 0.1% trifluoroacetic acid to remove unbound surfactant. Next, the column was equilibrated with 5 column volumes (CV) of 0.1% phosphoric acid in water (buffer A). Crude leuprolide (800 mg, net weight by Edelhoch method) dissolved in buffer A was loaded onto the column. The column was washed with 5 CV's of buffer A. Analytical RP-HPLC analysis of the "flow through" eluate revealed the absence of leuprolide. When this proceeding step was omitted, premature elution of crude API was observed because excess surfactant was present at a concentration above its critical micelle concentration. Next, start the gradient elution process. ...

Embodiment 2

[0154] Example 2: Prep-RP-HPLC of Leuprolide Acetate Using Triton X-100 as Additional Stationary Phase and 0.1 mM Cetyltrimethylammonium Bromide Buffer:

[0155] A Reveleris silica derivatized C-18 column (12 g of stationary phase, 40 micron diameter particles, and 60 Angstrom pore size) was chosen and saturated with 12 g of Triton X-100 dissolved in water.

[0156] Excess unbound surfactant was removed by washing with 90% aqueous acetonitrile containing 0.1% trifluoroacetic acid. When this step was omitted, premature elution of crude API was observed because excess surfactant was present at a concentration above its critical micelle concentration.

[0157] Next, the crude API was loaded (81.7% leuprolide at 800 mg, corrected weight of leuprolide was 653.3 mg) and washed with 5 CVs of buffer A (0.1 mM cetyl bromide in water trimethylammonium and 0.1 mM sodium bicarbonate) to wash the column. Analytical RP-HPLC analysis of the "flow through" eluate revealed the absence of leu...

Embodiment 3

[0160] Example 3: Prep-RP-HPLC of Leuprolide Acetate Using Tween 80 as Additional Stationary Phase and 0.1 mM Cetyltrimethylammonium Bromide Buffer:

[0161] A Reveleris silica derivatized C-18 column (12 g of stationary phase, 40 micron diameter particles, and 60 Angstrom pore size) was selected and saturated with 12 g of Tween-80 dissolved in water.

[0162] Excess unbound surfactant was removed by washing with 90% aqueous acetonitrile containing 0.1% trifluoroacetic acid. When this step was omitted, premature elution of crude API was observed because excess surfactant was present at a concentration above its critical micelle concentration.

[0163] Next, the crude API was loaded (81.7% leuprolide at 800 mg, corrected weight of leuprolide was 653.3 mg) and washed with 5 CVs of buffer A (0.1 mM cetyl bromide in water trimethylammonium and 0.1 mM sodium bicarbonate) to wash the column. Analytical RP-HPLC analysis of the "flow through" eluate revealed the absence of leuprolid...

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Abstract

There are only two ways to increase the amount of sample 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 invention 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. The surfactant is bound to the C-18 / C-8 chains of the stationary phase via Van der Waals forces (hydrophobic interactions) and ionic interactions with the residual silanols of the stationary phase.

Description

field of invention [0001] The present invention relates to the purification of organic compounds. More specifically, the present invention relates to a novel method for the purification of organic compounds comprising peptides using preparative reversed-phase high-performance liquid chromatography (Prep-RP-HPLC), which is compatible with the use of surfactants as an alternative to immobilization Surrogate stationary phase (SSP) / additional stationary phase (APS) purification of peptide-containing organic compounds has 7- to 10-fold higher sample loading capacity and output than conventional Prep-RP-HPLC. The increased sample loading capacity is due to the adsorbed surfactant on the C-18 / C8 chain used as the added stationary phase (ASP). Background of the invention [0002] Reverse-phase high-performance liquid chromatography (RP-HPLC) is widely used in academic institutions, forensic laboratories, fine chemistry, and pharmaceutical industries, etc. for small organic molecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/08B01D15/32G01N1/18
CPCB01D15/325B01D15/42C07K1/20C07K7/23
Inventor 穆罕默德·哈利德·安瓦尔
Owner 拉马莫罕·拉奥·达瓦鲁利
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