Anionic displacer molecules for hydrophobic displacement chromatography

A technique of displacement chromatography, hydrophobicity, applied in the field of anion displacer molecules for hydrophobic displacement chromatography, which can solve the problem that the displacer compound does not work well

Inactive Publication Date: 2014-07-30
SACHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Anionic displacer molecules for hydrophobic displacement chromatography
  • Anionic displacer molecules for hydrophobic displacement chromatography
  • Anionic displacer molecules for hydrophobic displacement chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0198] 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 actual loading concentration and actual column loading capacity were easily calculated, and these values ​​were then used to adjust the sample concentration and loading for the second DC experiment.

[0199] Sample Preparation - Prepare loading sample solutions at the concentrations and amounts described above. A sufficient excess of solution is required to overfill the loop or to fill the dead volume of the sample loading pump and delivery line. The pH, the amount of pH buffer and the amount of organic solvent are the same as the carrier and displacer buffers. Dissolving the sample in the carrier changes its pH, so the pH of the sample solution must be adjusted again after dissolution. However, the amount of ion-pairing reagent can v...

Embodiment 2

[0252] Example 2: Displacement Protocol for Purification of Crude Synthetic Oligonucleotides

[0253] Instrument configuration : Main pump (1) with 4 buffer lines, sample loading pump (2) with 1 solvent line, pump selector valve, column bypass valve

[0254] 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)

[0255] 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)

[0256] 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.

[0257] 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 = 0 - flow o...

Embodiment 3

[0263] Example 3: Displacement Chromatographic Purification of Crude Oligonucleotides (20-mer) Using Displacer 607b (5-n-Hexyl-2-Hydroxybenzenesulfonate) - Anionic Displacer at Near Neutral pH (See Figure Show 2A)

[0264] Operating conditions:

[0265] Starting peptide: Crude synthetic oligonucleotide (20-mer, A 2 G 6 T 8 C 4 , ammonium salt, monothiophosphate backbone), 82.2% purity, FW=6.7397 mg / μmole, charge=-19.

[0266] Column: Waters Xbridge BEH130, 5 μm, 4.6x250mm SS, -C on silicone 18 .

[0267] Flow Rate: Load = 208 μL / min; Displacement = 208 μL / min

[0268] Ion-pairing reagent: n-butylammonium ( n NH 3 + )

[0269] Temperature = 23°C

[0270] pH=7.0

[0271] Displacer buffer: 15.0 mM Displacer 607b+20 mM H 3 PO 4 (HPLC grade) + 50 mM purified n-butylamine in DI water containing 5% (v / v) MeOH, pH = 7.0 containing 50% HCO 2 H (HPLC grade).

[0272] Loading buffer: 20mM H 3 PO 4 (HPLC grade) + 50 mM purified n-butylamine in water containing 5% (v...

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Abstract

A process for separating organic compounds from a mixture by reverse-phase displacement chromatography, including providing a hydrophobic stationary phase; applying to the hydrophobic stationary phase a mixture comprising organic compounds to be separated; displacing the organic compounds from the hydrophobic stationary phase by applying thereto an aqueous composition comprising a non-surface active hydrophobic anionic displacer molecule and about 10 wt% or less of an organic solvent; and collecting a plurality of fractions eluted from the hydrophobic stationary phase containing the separated organic compounds; in which the non-surface active hydrophobic anionic displacer molecule comprises a hydrophobic anion and a counterion, CI, having the general formula A or B, as defined in the disclosure: [CM][Cl]d[CM-R*-CM'][Cl]d A B.

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 Applications(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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