Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
2002-11-13
2004-10-12
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S143000, C210S656000, C210S659000
Reexamination Certificate
active
06802969
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a chromatography system and a method for separating and purifying a target material from a mixture using the chromatography system.
2. Description of the Related Art
In the separation and analysis of trace components a variety of chromatographic techniques are regularly widely used. In particular, it can be said that the liquid chromatography technique, which can be applied to any sample as long as it dissolves in a solvent, has been established for almost all compounds. As a result, the application of liquid chromatography has been attempted not only for trace amounts of samples on an analytical level, but also for larger amounts of samples using various extensions of analytical techniques (Journal of Chromatography A, 908 (2001) 243-250, JP, A, 5-204, JP, A, 8-262001, JP, A, 10-282079 and JP, A, 2000-81422).
However, where the object is preparation or manufacture, the development of separation technology is currently still in progress.
Where the object is preparation, conventional methods can be broadly divided into the following three techniques.
One method is that in which the size of an analytical column is increased and a large amount of sample is separated all at once. This is extremely easy to understand, but since the usual amount of sample processed in an analysis is a few tens of micrograms or less, even in the case where, for example, a few grams are separated, this is a 100,000 times increase. Therefore, even when considering an analytical column with a size where the volume is in units of mL (or the weight of a packing material is in units of g), since several hundred liters of volume or several hundred kg of a packing material are required, it is not entirely practical.
Of course, in the case of analysis, if the detector can detect the target sample, a small amount of sample can give a margin for resolution, and as long as the detector's sensitivity allows it, the smallest amount of sample is therefore separated. However, when the whole surface area of the packing material is covered with the sample, it is obvious that sample in excess of this cannot be adsorbed on the surface, and for a low molecular weight molecule this is usually assumed at around 30 mg/100 m
2
. It is assumed that the limit at which a chromatographic separation can be carried out is usually where in the order of one tenth of the surface area of the packing material is covered. Since the surface area of high surface area porous silica gel a packing material is generally around 400 m
2
/g, the limit for the amount of sample is considered to be around 12 mg/g. Even in this case the ratio by weight of the sample and the required packing material is at least about 100 times. Therefore, even when scaled up, it is necessary to maintain the proportion of the sample relative to the packing material at this value or less all the time during the separation operation and in all parts of the column.
Another reason for a large amount of a packing material being required relative to the sample in liquid chromatography is that, in the chromatography column during the separation process, the part where the actual separation takes place is limited to the part where the target component is. That is, when looked at from the point of view of separation, the part of the column where the target component has already passed through, and the part of the column where the target component has not yet passed through, are not only just occupying space, but also have the negative effect of imposing pressure that opposes the flow of the mobile phase to no purpose, without contributing to the separation. However, in order to carry out a separation between components that only have a small difference in distribution coefficient, a column with a large number of theoretical plates, that is, a sufficiently long column is required, and in the case where a high resolution is required the parts of the column before and after the target component that are not involved in the separation effect at a specific point in time cannot evade the drawback of being inevitably relatively large.
Another problem when applying column chromatography to large-scale fractionation is the issue of what form of column should be used for the packing material, which is several hundred times the amount of the target fraction, and as a second point, even if the required amount of a packing material is decided, what kind of particle size should be used for the packing material.
As is well known, since in chromatography separation is carried out along the length of the column, in order to separate the target component at the required purity from a mixture, it is necessary to have a fixed minimum required length. This length corresponds to the number of theoretical plates needed to provide the necessary resolution, which is expressed as the number of theoretical plates. While the necessary resolution corresponds to the length, the amount processed corresponds to the cross-section of the column. If, as when talking about a normal analytical column, it is possible to load in the order of 0.5 mg of sample for 1 g of adsorbent, since about 1.2 g of a packing material can be packed in a representative column normally used for analysis having a diameter of 4.6 mm and a length of 15 cm , it can be estimated that 0.6 mg of sample can be loaded for a diameter of 4.6 mm. Therefore, by calculation, in order to load 1 g of sample, a column with a diameter of 188 mm and a length of 15 cm is necessary.
When calculating scale-up, the following can therefore be considered.
That is, when there are 10,000 plates in 15 cm, since 1 &sgr; is 100 plates in the vicinity of the outlet, and the maximum concentration is 12 mg/g, the amount that can be processed is calculated as 0.36 mg. Since the calculated value is 0.5 mg when there are about 5000 plates, the calculation shows that the generally held view is substantially correct. For example, when there are 10,000 plates, based on calculation a column diameter of 242 mm is required to process 1 g. That is, a column with a length of 15 cm and a diameter of about 24 cm has to be used. In the same way, it is necessary to use a column with a diameter of 48 cm and a length of 15 cm for 4 g of sample, and a diameter of about 1 m (96 cm) and a length of 15 cm for 16 g of sample.
For example, in the case of a length of 15 cm and a diameter of about 24 cm, in order to make the column longer, when 20 &mgr;m particles are used rather than the normally used 5 &mgr;m particles without changing the a mount of a packing material , the length becomes 60 cm, and the diameter becomes 12 cm. Moreover, with 80 &mgr;m particles, the length increases four times to about 2.4 m, while the diameter becomes 6 cm. A diameter of 6 cm and a length of 2.4 m is quite a practical column size. In this case, when the reduced linear flow rate of the mobile phase is fixed, the linear flow rate at 80 &mgr;m is {fraction (1/16)} of the flow rate at 5 &mgr;m, and because the column length is increased by 16 times, it takes 256 times longer. However, even if the reduced linear flow rate is made 20 times, since the reduced plate height (h) is increased only about 4 times, the time required for analysis is after all in the order of 50 times. Therefore, if 10 samples can be arranged and developed on the column, separation can be carried out within 5 times of the processing time per sample.
Moreover, by increasing h 4 times the column length increases 4 times, and since the amount of a packing material also increases 4 times, 4 times the amount is processed in one cycle. However, since the length is increased 4 times, it takes 4 times longer. Therefore, if a large number of 4 g samples can be separated in succession then separation and purification can be carried out with the same resolution during a period of time 5 times that for the analysis of in the order of 0.5 mg of sample.
In a normal chromatography column the length in the direction of progress of the mobile phase is general
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