WO2003055862A1 - Procede d'isolation d'ectoine par chromatographie - Google Patents

Procede d'isolation d'ectoine par chromatographie Download PDF

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Publication number
WO2003055862A1
WO2003055862A1 PCT/EP2002/012512 EP0212512W WO03055862A1 WO 2003055862 A1 WO2003055862 A1 WO 2003055862A1 EP 0212512 W EP0212512 W EP 0212512W WO 03055862 A1 WO03055862 A1 WO 03055862A1
Authority
WO
WIPO (PCT)
Prior art keywords
column
ectoin
ectoine
hydroxyectoin
usually
Prior art date
Application number
PCT/EP2002/012512
Other languages
German (de)
English (en)
Inventor
Klaus FÜLBERTH
Harald Müssig
Bernd Wesp
Original Assignee
Merck Patent Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent Gmbh filed Critical Merck Patent Gmbh
Priority to AU2002351926A priority Critical patent/AU2002351926A1/en
Priority to EP02787611A priority patent/EP1461322A1/fr
Publication of WO2003055862A1 publication Critical patent/WO2003055862A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/06—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member

Definitions

  • the present invention relates to a chromatographic process for separating ectoin from a mixture containing ectoin and hydroxyectoin.
  • ectoin When ectoin is obtained as a natural product from cells by biotechnical processes, as described, for example, in EP-A-0 677 044, DE-A-19925615, EP-A-0 999 277, DE-A-4427617 or T. Sauer et al. GIT FACHZ. Lab. 10/95, the desired product, ectoin, is usually obtained together with by-products, especially hydroxyectoin. It is therefore necessary to separate ectoin not only from contamination by cell material, but also from other cell substances that are also obtained from these cells. Unwanted cell material is usually first removed by filtration. For the separation of the ectoin / hydroxyectoin mixture there are HPLC methods, but also extraction methods.
  • This object is achieved by a process for the chromatographic separation of ectoin from a mixture containing ectoin and hydroxyectoin, characterized in that the separation is carried out using an ion exchange column which has a ratio of diameter to height of 1: 8 to 1:20.
  • the mixture to be purified, containing ectoin and hydroxyectoin, is usually filtered before the process according to the invention is carried out in order to rule out the presence of possible cell components which occur in biotechnical processes.
  • Any column which has the dimensions mentioned above is suitable as a chromatography column.
  • the material from which this column is made is not critical. Metal, plastic or glass is usually used as the material.
  • the chromatographic column is packed with an ion exchange material. It is usually a cation exchange resin. A strong acid cation exchange resin is particularly preferred.
  • the resin preferably has a small and uniform particle size. The average particle size is 450 to 700 ⁇ m, more preferably 500 to 650 ⁇ m.
  • Examples of the ion exchange resin include DOWEX MARATHON C,
  • DOWEX HCR S DOWEX 50 WX 8-100, AMBERLITE IR 122, FRACTOGEL EMD SE,
  • the process according to the invention is preferably carried out without pressure, in contrast to conventional HPLC columns in which pressure is used.
  • pressure in contrast to conventional HPLC columns in which pressure is used.
  • the slim column used with the pressure-free This way of working clogging is avoided and at the same time an excellent separation of ectoin is obtained.
  • the ion exchange resin is usually in a loosely washed-in state.
  • the ion exchange column used has a ratio of diameter to height of 1: 8 to 1:20, preferably 1: 9 to 1:15, more preferably 1: 9 to 1:12, most preferably 1:10.
  • the column usually has a diameter of 20 to 60 cm and a height of 160 to 500 cm. Columns in which the diameter is 25 to 40 cm and the height is 200 to 450 cm are particularly preferred.
  • the column particularly preferably has a diameter of 30 cm and a height of 300 cm.
  • the term “height” means the filling height of the column. The column itself can of course be much higher than the fill level.
  • the column is packed with the ion exchange resin.
  • the ion exchange resin is usually slurried in an aqueous solution and this slurry is filled into the column.
  • the aqueous solution is usually a weak salt solution.
  • This salt solution preferably contains sodium, potassium or magnesium ions as cations.
  • a sodium chloride solution, potassium chloride solution or magnesium chloride solution is preferred, and a sodium chloride solution is particularly preferred.
  • This solution is preferably 0.1 to 2 M, more preferably 0.5 to 1.5 M, most preferably 1 M.
  • the column is then washed. This is preferably done until the conductivity drops to ⁇ 100 ⁇ S. Water, especially demineralized water, is suitable for washing the column.
  • the column is then loaded with the ectoin solution.
  • the ectoin solution usually has a pH of 1.0 to 2.5, preferably 1.5 to 2.0. It is preferred that the flow rate when loading the column with the ectoin solution is 200 to 800 l / h, preferably 400 to 600 l / h, more preferably 500 l / h.
  • the column After loading the column with the crude octoin solution to be separated and purified, which also contains hydroxyectoin in addition to ectoin, the column is washed from below with water, preferably demineralized water.
  • the amount of water is preferably 500-1000 l / h, preferably 700 l / h. This washing step elutes Hydroxyectoin, while Ectoin remains bound and thus the purification is achieved.
  • the product can then be eluted.
  • Weakly basic solutions are suitable as eluents, for example aqueous solutions of sodium hydroxide solution, potassium hydroxide solution or an aqueous ammonical solution.
  • the weakly basic solution is usually 0.01 to 0.5 M, preferably 0.05 to 0.25 M, particularly preferably 0.08 to 0.2 M.
  • Elution with weakly basic solutions preferably sodium hydroxide solution, removes the ectoin and possibly the remaining hydroxyectoin evenly from the gel.
  • the residual amount of hydroxyectoin, if any, is usually ⁇ 4%, more preferably ⁇ 1%.
  • the elution rate depends on the height of the column and the ion exchange resin. Usual speeds are in the range of 500 to 1000 l / h, preferably 600 to 900 l / h, more preferably 700 to 800 l / h.
  • the elution can be carried out in several fractions.
  • the elution is usually monitored by known detection methods. This is preferably done by means of UV detection and / or monitoring of the pH.
  • ectoine is obtained with a UV signal of 210 to 230 nm, preferably 220 nm.
  • the fractions can be divided so that they contain the product which was eluted at different pH values.
  • a first fraction may contain eluted material up to a pH of 7 and a second fraction may contain the eluted material at a pH greater than 7.
  • the column is usually washed. It is common for the column to have a conductivity of ⁇ 100 ⁇ S after washing. Water is suitable for washing, especially demineralized water.
  • the column was loaded with an ectoin solution to be purified, containing ectoin and hydroxyectoin, at 500 l / h from below.
  • the solution had a pH of 1.5 to 2.0.
  • the product was then eluted from below with 0.1 N NaOH at 750 l / h. The following fractions were collected:
  • Ectoin was predominantly in the first fraction and to a lesser extent in the second fraction in a ratio of 4: 1.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

La présente invention concerne un procédé pour isoler de l'ectoïne par chromatographie à partir d'un mélange contenant de l'ectoïne et de l'hydroxyectoïne, se caractérisant en ce que la séparation s'effectue au moyen d'une colonne échangeuse d'ions qui a un rapport diamètre : hauteur de 1 : 8 à 1 : 20. Ce procédé permet une excellente isolation d'ectoïne, sans produire d'impuretés indésirables avec des quantités élevées d'hydroxyectoïne.
PCT/EP2002/012512 2002-01-03 2002-11-08 Procede d'isolation d'ectoine par chromatographie WO2003055862A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002351926A AU2002351926A1 (en) 2002-01-03 2002-11-08 Method for the chromatographic isolation of ectoine
EP02787611A EP1461322A1 (fr) 2002-01-03 2002-11-08 Procede d'isolation d'ectoine par chromatographie

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10200068.9 2002-01-03
DE2002100068 DE10200068A1 (de) 2002-01-03 2002-01-03 Verfahren zur chromatographischen Trennung von Ectoin

Publications (1)

Publication Number Publication Date
WO2003055862A1 true WO2003055862A1 (fr) 2003-07-10

Family

ID=7711466

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/012512 WO2003055862A1 (fr) 2002-01-03 2002-11-08 Procede d'isolation d'ectoine par chromatographie

Country Status (4)

Country Link
EP (1) EP1461322A1 (fr)
AU (1) AU2002351926A1 (fr)
DE (1) DE10200068A1 (fr)
WO (1) WO2003055862A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111965281A (zh) * 2020-08-17 2020-11-20 华熙生物科技股份有限公司 一种依克多因的检测方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0866061A1 (fr) * 1997-03-18 1998-09-23 Degussa Aktiengesellschaft Procédé de séparation des dérivés de pyrimidine à partir de solutions aqueuses
EP0957174A1 (fr) * 1998-05-13 1999-11-17 Degussa-Hüls Aktiengesellschaft Procédé pour la séparation de dérivés de tétrahydropyrimidine

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0866061A1 (fr) * 1997-03-18 1998-09-23 Degussa Aktiengesellschaft Procédé de séparation des dérivés de pyrimidine à partir de solutions aqueuses
EP0957174A1 (fr) * 1998-05-13 1999-11-17 Degussa-Hüls Aktiengesellschaft Procédé pour la séparation de dérivés de tétrahydropyrimidine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
T. SAUER: "BACTERIAL MILKING", BIOTECHNOLOGY AND BIOENGINEERING. INCLUDING: SYMPOSIUM BIOTECHNOLOGY IN ENERGY PRODUCTION AND CONSERVATION., vol. 57, no. 3, 1998, JOHN WILEY & SONS. NEW YORK., US, pages 306 - 313, XP000919323, ISSN: 0006-3592 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111965281A (zh) * 2020-08-17 2020-11-20 华熙生物科技股份有限公司 一种依克多因的检测方法

Also Published As

Publication number Publication date
DE10200068A1 (de) 2003-07-17
EP1461322A1 (fr) 2004-09-29
AU2002351926A1 (en) 2003-07-15

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