JPS60198458A - Column for chromatography - Google Patents

Abstract

PURPOSE:To increase the limiting flow rate for supplying a fluid sample, and to make quick analysis by using hydroxy-apatite [Ca10(PO4)6(OH)2] having a specific grain size range as a stationary material 3 of column chromatography. CONSTITUTION:A stationary material 3 is packed in a column 1 of chromatography and a fluid sample is supplied thereto through a sample port 5. The sample is separated by the column 1 and is discharged through an outflow port 6. Hydroxy-apatite crystal particles are used as the stationary material. The grain size thereof is made to contain small grain sizes below <=10mum stokes equiv. diameter or even if such small particles are contained therein, the mixing rate thereof is made <=1%. The preferable grain size is in the stokes grain size range of about 20-100mum. The limiting flow rate for supply is therefore increased if the hydroxy-apatite having such grain size is used and since the sepn. accuracy is good, a molecular sepn. of DAN.RAN, etc, and sepn. of the high molecular components in blood are efficiently effected.

Description

Detailed Description of the Invention Column chromatography of the present invention (Column Chr.
The present invention relates to a chromatography column using a column (separation tube) such as a straight tube, a spiral column, or a U-shaped column packed with a solid substance as a stationary phase.

More specifically, hydroxyapatite (Galo (PO,)6 (OH)2) is used as the solid stationary phase material.
This invention relates to an improvement in a column packed with a group of crystal particles.

Hereinafter, for convenience, the above hydroxyapatite crystal particle group will be referred to as HA crystal particles, and the column filled with the crystal particles will be referred to as HA crystal particles.
It is abbreviated as A column.

Chromatography using an HA column was developed by Nopel Prize winner Chiselius et al.
, S. Hjerten, and O. Levin, Ar.
ch, Biochem, old 0phys,, rib, 132 (
1115El)), which has a sensitive resolution, has been used as a means for separating and purifying proteins, nucleic acids, etc. ever since then.

By the way, as long as the HA column is used with the flow rate (flow rate per unit cross-sectional area in the column/unit time) of a fluid sample in the column as a liquid or gas kept at or below a certain critical flow rate level, Although the flow of the fluid sample in the column continues and is stable, the supply pressure of the fluid sample into the column is further increased to increase the flow rate of the fluid sample in the column.
Even if you try to forcefully increase the flow rate above the critical flow rate level specified in 2 above, the flow rate of the fluid sample in the column will drop significantly below the critical flow level, or the flow of the fluid sample in the column will almost stop. An event occurs that makes it impossible to perform chromatography.

Therefore, chromatography utilizing HA columns is inherently limited in the maximum flow rate of fluid sample that can flow into the column up to the critical flow level described above. However, the conventional H
The critical flow level of the A column was quite low. Therefore, in practice, chromatography using conventional HA columns has the disadvantage that it often takes a considerable amount of time to perform one chromatography.

The purpose of the present invention is to significantly increase the critical flow level of such an HA column compared to conventional ones, thereby enabling rapid chromatography. In a chromatography column packed with particle groups,
For use in chromatography, the crystal particle group is characterized in that it does not substantially contain small-diameter crystal particles with a Stokes equivalent spherical diameter of 10p, m or less, or even if it does, the contamination rate thereof is 1% by weight or less. The column is the gist.

That is, the present inventor tried various ideas and experiments to raise the critical flow level of the HA column as much as possible, but
Among these, as mentioned above, by pre-separating the HA crystal particles packed in the column, etc., it is possible to substantially exclude small-diameter crystal particles with a Stokes equivalent spherical diameter (hereinafter abbreviated as ST) of 10 gm or less. treated, or even if it does contain it, the contamination rate is kept below 1% by weight,
The present invention was completed based on the finding that when the HA column was used, the critical flow rate level of the HA column was significantly higher than that of the conventional column.

In the above, 10 JLm was added to the HA crystal particles packed in the column.
If 1% by weight or more of small-diameter crystal particles smaller than ST is included, is the effect of raising the critical flow rate level low?
Or not at all.

Furthermore, the average particle diameter of the HA crystal particle group used in the present invention, which does not substantially contain small-diameter crystal particles of 10 ILmST or less, or even if it does, has a mixing rate of 1% by weight or less, is 20 gm5.
T or more/100gm5T or less, preferably 30pLm
Preferably above ST and below 60#LmST.

If the average particle diameter is too large, the mixing rate of coarse particles will increase, and the gaps between the particles in the optical*HA crystal particle group in the column will increase, and at the same time, the variation in the size of the gaps will also increase. Chromatographic separation may be affected by non-uniformity in the flow of the fluid sample within the column, or by breaking the thermodynamic equilibrium between the fluid sample flowing within the column and the packed HA crystal particles during the quasi-static chromatographic process. will decrease.

Example (1) Three types of HA crystal particles A and BΦC, which are used to fill conventional HA columns, were obtained, and the size distribution of each crystal particle was examined. - HA of B among the above for strength wood invention
By separating the crystal grain group as the original particle group and removing small-diameter crystal particles of 10 gm5T or less, the product substantially does not contain the small-diameter crystal particles, or even if it does, the contamination rate is 1 weight. % or less of HA crystal particles were prepared.

This was used as a particle group and its particle size distribution was investigated. I''-4
The particle size distributions of seed particle groups A-D are shown in Figure 1A-
I) and shown in Table 1.

Table 1 (2) Among the above four types of HA crystal particle groups A-D, A and B-
As shown in Fig. 2, C is filled in a round pipe column container 2 made of glass with an inner diameter of 1 cm (inner cross-sectional area of about 0.79 cm2), and a round pipe column container 2 made of stainless steel with an inner diameter of ICl 11 is packed in the case of C. Effective long sentences in each =
A straight tube type HA column 1 of 30c+o was constructed and designated as columns A-B-C-D, respectively. 3 is a group of packed HA crystal particles, 4.
4 is a filter, and 5 and 6 are a fluid sample supply port and an outflow port, respectively.

A dilute phosphate buffer solution was supplied as a fluid to each of the columns A to D above, and the flow rate in the column of the fluid [m/(min・0
．． 79cm2)) was actually measured. The results are shown in column I of Table 2.

(3) Next, the peak flow rate, that is, the critical flow rate level for each column, was investigated by gradually increasing the pressure of fluid introduced into each column A to D and actually measuring the change in the flow rate in the column as a result. The results are shown in the TI column of Table 2. Note that a plunger pump was used to supply fluid to column D, since the critical flow level of this column is high.

Table 2 From (2) and (3) above, it can be seen that the HA column D according to the present invention is significantly superior in wave body flowability-critical flow rate level compared to the conventional columns A to C.

(4) Each of the four types of HA crystal particle groups A to D has an inner diameter of 0.
Packed into a stainless steel column for high performance liquid chromatography (HPLC) with a 6C1L effective length of 10 cm, lysozyme and cytochrome C, which are typical globular proteins, are mutually separated using the HA column. The sample solution supply pressure to the column was selected to be 20 to 25 kg/cm2.

However, in all cases of columns packed with particle groups A-B-C, the flow of the sample liquid in the column stopped immediately after the sample liquid supply started, making it impossible to perform chromatography.

On the other hand, particle group D, that is, does not substantially contain small-diameter crystals of 10 ILmST or less, or even if it does, the mixing rate is 1
In the case of the HA column according to the present invention packed with HA crystal particles of less than 8.9 mJL/wt.
(min/0.79 cm2) (flow rate converted to column inner diameter of 1 cm) was continuously and stably circulated, and a clear chromatogram (a) as shown in Figure 3 was obtained. In addition, we separately measured the critical flow level of the HA column and found that I
B, 7m viewing/(min・0.79cm2) (column inner diameter 1)
(Flow rate calculated in terms of fat) was excellent.

The chromatogram (A) in Figure 3 shows ultraviolet absorption at a wavelength of 280 nm, that is, 280 nm O, (Optica
(1den+1ty). This chromatography was also performed by applying a linear concentration gradient of phosphate buffer. Graph (b)
The phosphate ion molar concentration was simultaneously monitored by refractive index measurement.

As described above, the HA column of the present invention has a significantly higher critical flow rate level than conventional ones, and also maintains the original high resolution due to HA crystal particles, thus enabling high-speed and highly accurate chromatography. For example,
It is extremely effective and suitable as a column for chromatography in fields such as genetic engineering and medical research for separating molecules such as DAM and RAM, separating high molecular components of blood, and other fields.

[Brief explanation of the drawing]

FIG. 1 is a particle size distribution graph of various HA crystal particle groups, FIG. 2 is a longitudinal cross-sectional view of an example of a straight column column, and FIG. 3 is an example of a chromatogram. 1 is a column packed with HA crystal particles, 2 is a column container, 3
4 is a filter, 5 is a fluid sample supply port, and 6 is a fluid sample outlet.

Claims (2)

[Claims] (1) In a chromatography column packed with hydroxyapatite crystal particles as a solid stationary phase material, the hydroxyapatite crystal particles packed in the column are essentially small-diameter crystal particles with a Stokes equivalent spherical diameter of 10 p-m or less. A column for chromatography, characterized in that it does not contain it, or even if it does, its contamination rate is 1% by weight or less. (2) The average particle diameter of the hydroxyapatite crystal particles packed in the column is 20 pLm or more and 1100 IL or less in Stokes equivalent spherical diameter, preferably 3f) #Lm or more.
The chromatography power column according to claim 1, which has a power of 80 #Lm or less.