Liquid purification or separation – Processes – Chromatography
Patent
1988-06-14
1989-08-22
Therkorn, Ernest G.
Liquid purification or separation
Processes
Chromatography
210635, 210659, B01D 1508
Patent
active
048593427
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to industrial separation by ion-exchange type column chromatography and, more particularly, to a process for separating industrially a large amount of a mixed sample containing biopolymers by column chromatography, an apparatus used for the process and a controlling process thereof.
BACKGROUND ART
Recently, there have been frequently used an ion-exchange type column chromatographical method for the separation of biopolymers to a high purification level in the fields of chemistry, biology, agricultural chemistry, medicine, etc. The ion-exchange type column chromatography is one of the chromatographical methods which employ a filler as a stationary phase. When a sample of a mixture is placed on the top of an ionexchange resin column and developed with an appropriate electrolyte, adsorbed bands of components are caused to flow down separately from each other due to the differences of adsorptivities of respective component ions with respect to the filler. The mobile phase as a developing agent is an eluent. If developing operation, that is, elution, is continued, components are eluted in sequence depending on their adsorptivities, so that they can be collected in certain fractions. When biopolymers such as protein or the like are intended to be purified by separation, an eluent is fed onto the column in which a sample is placed with continuously changing of the salt concentration and/or pH of the eluent with the passage of the time to form a gradient of salt concentration and/or pH along the direction of the column length, and proteins are separately eluted by utilizing the differences of isoelectric points of proteins (gradient elution). Thus, it is necessary to perform gradient elution for the separation and purification of biopolymers such as proteins or the like by means of ion-exchange type column chromatography.
Conventional apparatuses for separating the sample of biopolymers on industrial scale by means of column chromatography are for the most part of a stepwise type comprising a column filled with a filler having ionexchange ability, at least 2 mobile phase baths provided at the upper stream side of the column through passages, a sample bath which will communicate with a sample injecting valve on injecting the sample, fraction collectors provided at the lower stream side of the column, a first detector for concentration and/or pH and a second detector for sample components provided on the passage between the column and the fraction collector, in which as used in bioprocess 3 to 5 baths of mobile phase liquids of which concentration and pH are preliminarily adjusted are provided as gradientors, and the valves of the baths are changed sequentially at a certain interval to perform gradient elution.
The processes of separating and purifying biopolymers such as protein or the like, in which the concentration and/or pH had linear gradients with high precision (within .+-.1%), were not of industrial scale. The conventional apparatuses for separating a biopolymer sample from the gradient column chromatographical system with high precision were proposed only for analytical use, and none of the apparatuses had such a large capacity as to be used on an industrial scale. However, due to the remarkable development of biotechnology in recent years, development of a process and apparatus of high performance and high reliability for separating and purifying a biopolymer has been desired.
The present invention has been achieved on the basis of the above described background. An object of the present invention is to provide a process for separating a biopolymer such as protein or the like from a high precision gradient system, an apparatus therefor and a controlling process thereof.
DISCLOSURE OF THE INVENTION
The present inventors have conducted researches for the processes for separating biopolymer samples from a high precision gradient of column chromatography and have built a variety of apparatuses therefor on a trial basis. As a result, they have found that the
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Eto Shigeo
Saito Hiroshi
Shirasawa Honai
Sugeno Hiroto
Soken Kagaku Kabushiki Kaisha
Suntory Limited
Therkorn Ernest G.
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