Neutral Zwitterionic Displacer Molecules for Hydrophobic Displacement Chromatography

A displacement chromatography and zwitterion technology, which is applied in the field of neutral zwitterion displacer molecules for hydrophobic displacement chromatography, and can solve problems such as the inability of displacer compounds to work well

Inactive Publication Date: 2015-12-23
SACHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the strongly surface-active displacer compounds disclosed in U.S. Patent No. 6,239,262 do not work well, resulting in relatively poor to moderate quality displacement trains where significant levels of impurities can be present in the "purified" product

Method used

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  • Neutral Zwitterionic Displacer Molecules for Hydrophobic Displacement Chromatography
  • Neutral Zwitterionic Displacer Molecules for Hydrophobic Displacement Chromatography
  • Neutral Zwitterionic Displacer Molecules for Hydrophobic Displacement Chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0224] (2) Select the initial concentration of the sample by one of two methods:

[0225] (a) Initial sample concentration (mg / mL) = 0.25x replacement agent concentration (mM) x formula weight (mg / μmole)

[0226] = 0.25 x 10 mM x 1.7466 mg / μmole = 4.37 mg / mL (for Example 2 below)

[0227] (b) Select an estimated column binding capacity for the sample, eg 50 mg sample / mL matrix. Assuming the displacement flow rate and the sample loading flow rate are the same:

[0228] Initial sample concentration (mg / mL) = (column binding capacity (mg / mL) m ) x column volume (mL m ) / ((T 2 -T 1 ) x sample flow rate (mL / min))

[0229] =(50mg / mL m x4.155mL m ) / ((434min-220min)x0.208mL / min)

[0230] =4.67mg / mL

[0231] (for Example 2 below)

[0232]If the first DC experiment with loaded samples resulted in an overloaded condition (>100% loading), the experiment was rerun at half the sample concentration. From the results of the first successful DC experiment using the sample, the actua...

Embodiment 1

[0260] Example 1: Displacement Protocol for Purification of Crude Synthetic α-Endorphin

[0261] Instrument configuration : Main pump (1) with 4 buffer lines, sample loading pump (2) with 1 solvent line, pump selector valve, column bypass valve (ColumnBypassValve)

[0262] Pump selector valve: 6-way valve controlled by single-channel toggle logic (S3=0, pump 1 to column-pump 2 to waste; S3=1, pump 1 to waste-pump 2 to column)

[0263] Column valve: 6-way valve, which is controlled by a single channel switching logic (S6 = 0, liquid flows through the column; S6 = 1, liquid flow bypasses the column)

[0264] A UV photodiode array detector (flow cell: 0.5 mm flow, 9 μL volume) after the column, followed by a conductivity detector (flow cell: 170 μL volume); the conductivity flow cell was removed when fractions were collected for analysis.

[0265] Loading buffer = A-line on pump 1 (S1 = 1 - flow on, S1 = 0 - flow off); Displacer buffer = B-line on pump 1 (S2 = 1 - flow on, S2 =...

Embodiment 3

[0295] Embodiment 3: HPLC analysis-

[0296] Method 3a, 3b - reversed phase for neutral zwitterions: using Waters996PDA detector and WatersXbridge BEH130 equipped in series with Dionex / ESABiosciences (Chelmsford, MA) CoronaPlusCAD detector and WatersXbridge BEH130, 5 μm, 4.6x250mmSS, -C on Silicone 18 , gradient HPLC on a reversed-phase column (Chelmsford, MA) from Waters (Milford, MA).

[0297] Sample injection: 25 μL of ~1 mM sample solution in A buffer

[0298] UV detection: 208-220nm, depending on the compound to be analyzed

[0299] Flow rate: 1.0 mL / min.

[0300] A buffer: 5% CH with 0.1% (v / v) trifluoroacetic acid 3 CN (v / v) in HPLC-grade distilled water.

[0301] B buffer: 5% H with 0.1% (v / v) trifluoroacetic acid 2 O(v / v) in HPLC-grade CH 3 CN.

[0302] Survey gradient method: 100% A0-2min

[0303] 100%A to 100%B2-62min

[0304] 100% B62-70min

[0305] Analytical gradient method:

[0306] Method 3c – reverse phase for long chain alkyl halides:

[0307]...

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Abstract

A method of separating an organic compound from a mixture by reversed-phase displacement chromatography comprising providing a hydrophobic stationary phase; applying to the hydrophobic stationary phase a mixture comprising the organic compound to be separated; by applying to the hydrophobic stationary phase a non-surface active hydrophobic an aqueous composition of a neutral zwitterion displacer molecule and optionally an organic solvent to displace the organic compound from the hydrophobic stationary phase; and collecting a plurality of fractions containing the separated organic compound eluting from the hydrophobic stationary phase; wherein The non-surface active hydrophobic neutral zwitterion displacer molecule comprises a hydrophobic zwitterion having the general formula [CM-R*-CM'] as defined in the specification.

Description

Background technique [0001] Displacement chromatography (DC) is one of three known forms of column chromatography—elution chromatography, displacement chromatography, and front chromatography. DC is primarily a preparative method, but there are also analytical applications using "micropreparative" DC with packed "narrow bore" or capillary columns. [0002] Displacement chromatography can be performed using any of four conventional chromatographic methods when a suitable high-purity displacer molecule is available. DC is used for (a) ion exchange chromatography (cation exchange, anion exchange), (b) hydrophobicity chromatography (reverse phase, hydrophobic interaction, hydrophobic charge induction, thiophile), (c) normal phase chromatography, including hydrophilic interaction interaction chromatography (HILIC), and (d) immobilized metal ion affinity chromatography (IMAC). [0003] With an optimized DC, high purity (high resolution), high recovery (high yield) and high column ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/32B01D15/42
CPCB01D15/325B01D15/422C07K1/20
Inventor 百瑞·L·海莫尔
Owner SACHEM INC
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