Anion Displacer Molecules for Hydrophobic Displacement Chromatography

一种置换色谱、疏水性的技术，应用在用于疏水性置换色谱法的阴离子置换剂分子领域，能够解决置换剂化合物不能很好地起作用等问题

Inactive Publication Date: 2016-01-13
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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  • Anion Displacer Molecules for Hydrophobic Displacement Chromatography
  • Anion Displacer Molecules for Hydrophobic Displacement Chromatography
  • Anion 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 of...

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: WatersXbridge BEH130, 5 μm, 4.6x250mmSS, -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: 15.0mM Displacer 607b+20mM 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: 20mMH 3 PO 4 (HPLC grade) + 50 mM purified n-butylamine in water containing 5% (v / v) MeO...

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PUM

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Abstract

A method of separating organic compounds 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 anion displacer molecules and less than about 10% by weight of an organic solvent to displace an organic compound from a hydrophobic stationary phase; and collecting a plurality of fractions containing the separated organic compound eluting from the hydrophobic stationary phase; wherein the non The surface active hydrophobic anion displacer molecule comprises a hydrophobic anion and a counter ion CI, which has the general formula A or B as defined in the description: [CM][CI]d[CM-R*-CM'][CI] d? AB. [CM][CI]d? ? ? ? ? ? A, [CM-R*-CM'][CI]d, 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 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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