Liquid purification or separation – With means to add treating material – Chromatography
Patent
1995-06-06
1996-11-26
Therkorn, Ernest G.
Liquid purification or separation
With means to add treating material
Chromatography
21032189, 21050023, 210656, B01D 1508
Patent
active
055782043
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an apparatus for recovery and buffer exchange and/or concentration of dissolved macromolecules from a mixture of macromolecules, comprising a liquid chromatography unit and at least one hollow fibre membrane cartridge provided in the outlet tubing of the liquid chromatography unit.
Among the standard biochemical techniques there are various chromatographic methods, in which differences in mass, form, affinity, charge or polarity of the molecules are utilized for their separation. In ion exchange, gel filtration and affinity chromatography, liquid chromatography units are used to separate mixtures of macromolecules in their individual constituents.
Besides the individual constituents of a mixture of macromolecules the eluate of a liquid chromatography column contains low-molecular substances, e.g. salts, which were used to compensate for the interactions between the macromolecules and the stationary phase of the chromatography column. After the chromatography, these substances must be removed from the eluate for they are to interfere, e.g. in subsequent steps of detection or further treatment, and are for this reason undesired in the preparations.
In preparative biochemistry so-called "discontinuous equilibrium dialysis" is applied to eliminate low molecular weight components from the eluate of the liquid chromatography unit as well as to exchange the buffer solution containing the mixture of macromolecules. In this method the eluate containing the various molecular species is filled in a dialysis tube, which both ends are closed by clamps. The wall of the dialysis tube consist of a semipermeable membrane, which allows low-molecular substances, e.g. water or salts, to pass while macromolecules are retained. In this way electrolytes or other low molecular weight substances contained in the eluate can diffuse through the membrane until osmotic equilibrium is attained. The dialysis tube filled with eluate is immersed in a vessel containing a large amount of water or buffer solution for several hours. By changing the outer liquid frequently enough, the low molecular weight components can be completely extracted and/or the buffer solution can be exchanged.
When macromolecules are separated according to size by means of gel filtration, the contamination of the preparation with undesired low-molecular compounds is no longer the main problem; it is rather the high dilution of the macromolecules during the elution from the chromatography column which gives reason for concern. This problem is encountered frequently in other chromatographic separation methods as well. Concentrated solutions of high molecular weight substances (for example polyethylene glycol) are used as dialysis fluid to concentrate the eluate, as they show a high affinity for water and thus extract water from the macromolecule solution, but cannot diffuse through the dialysis membrane.
The described procedure is troublesome and requires a considerable amount of time and outlay for equipment. In particular, if the eluate is divided among a large number of individual containers (fractionation) to separate the individual macromolecular species, the method becomes almost unfeasible. Delays, unavoidable when using this method, are particularly disadvantageous, when the sensitive macromolecules remain for too long in an unsuitable buffer solution. This problem appears frequently in the case of enzymatic preparations: the enzyme is often so labile that the troublesome steps of exchanging buffer and/or concentrating by dialysis lead to a considerable loss of enzymatic activity in the preparation.
In the EP-B1-00 58 168, an apparatus has been described, wherein a hollow fibre membrane reactor is used in connection with a liquid chromatography unit. In this hollow fibre membrane reactor the eluate of the liquid chromatography unit is treated with reagents, which react with constituent groups of the samples and/or the eluent to provide a better and faster analysis.
This apparatus is not suited for recovery of biochemical preparations.
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Bartholmes Peter
Kaufmann Michael
Schwarz Thomas R.
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