Cationic displacer molecules for hydrophobic displacement chromatography

A technique of hydrophobic cation, displacement chromatography, applied in the field of cation 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

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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

Method used

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  • Cationic displacer molecules for hydrophobic displacement chromatography
  • Cationic displacer molecules for hydrophobic displacement chromatography
  • Cationic displacer molecules for hydrophobic displacement chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 6b

[0196] (for Example 6b(a))

[0197] (2) Select the initial concentration of the sample by one of two methods:

[0198] (a) Initial sample concentration (mg / mL) = 0.25x replacement agent concentration (mM) x formula weight (mg / μmole)

[0199] = 0.12 x 10 mM x 1.7466 mg / μmole = 2.10 mg / mL (for Example 6b(a))

[0200] (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:

[0201] 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))

[0202] =(50mg / mL m x4.155mL m ) / ((586min-270min)x0.208mL / min)

[0203] =3.16mg / mL

[0204] (for Example 6b(a))

[0205] 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...

Embodiment 1a

[0233] Example 1a: Exemplary protocol. Displacement chromatography purification of crude synthetic angiotensin I

[0234] Instrument configuration : Single main pump with 4 solvent lines, sample injection valve with 40mL loop, column bypass valve

[0235] Sample injection valve: 6-port valve controlled by single-channel toggle logic (S3=0, bypass loop, S3=1, loop inline)

[0236] Column Bypass Valve: 6-way valve controlled by single channel switching logic (S6=0, column inline, S6=1, bypass column)

[0237] A post-column UV photodiode array detector (flow cell: 0.5 mm flow, 10 μL volume) followed by a conductivity detector (flow cell: 170 μL volume). Bypass the conductivity cell when collecting fractions for analysis.

[0238] Loading buffer = A-buffer (S1 = 1, flow on, S1 = 0 flow off); Displacer buffer = B-buffer (S2 = 1, flow on, S2 = 0 flow off);

[0239] Displacer removal buffer = C-buffer (S4 = 1, flow on, S4 = 0 flow off); column storage buffer = D-buffer (S5 = 1,...

Embodiment 1b

[0246] Example 1b: Displacement Chromatographic Purification of Crude Angiotensin I Using Displacer 14 - Higher Loading at Lower Concentrations (see Figure 1b -analyze)

[0247] Operating conditions:

[0248] Starting peptide: desalted crude synthetic angiotensin I, 82.7% pure, FW ~ 1.296 mg / μmole, charge = +4

[0249] Column: Waters Xbridge BEH130, 5 μm, 4.6x250mm SS, -C on silicone 18

[0250] Flow Rate: Load = 208 μL / min; Displacement = 208 μL / min

[0251] Ion-pairing reagent: trifluoroacetate (CF 3 CO 2 - )

[0252] Temperature: 23°C

[0253] pH: 2.0

[0254] Displacer Buffer: 10.0 mM Displacer 14 + 12 mM CF 3 CO 2 H contains 3% (v / v) MeCN in DI water, pH = 2.0 with NH 4 Oh

[0255] Loading buffer: 12 mM TFA with 3% (v / v) MeCN in water, pH=2.0 with NH 4 Oh

[0256] Sample solution: 4.38mg / mL peptide in water containing 3% (v / v) MeCN and 27mM CF 3 CO 2 - ;pH=2.0 contains NH 4 Oh

[0257] Loading: 155.0mg, 35.4mL, from 40mL loop;

[0258] Loading ti...

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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 cationic 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 cationic displacer molecule comprises a hydrophobic cation and a counterion, CI, having the general formula A or B, as defined in the disclosure.

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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IPC IPC(8): B01D15/32B01D15/42
CPCB01D15/325B01D15/422B01D15/426
Inventor 百瑞·L·海莫尔
Owner SACHEM INC
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