JPS63200802A - Settling field fluidization two-phase partition chromatography - Google Patents

Abstract

PURPOSE:To obtain the title settling field fluidization two-phase chromatography capable of realizing two-phase partition chromatography, by using a rotary column utilizing centrifugal force to carry out countercurrent partition chromatography based on the principle of settling field fluidization fractionation. CONSTITUTION:The different kinds of fluids immiscible with each other are introduced into the rotary column utilizing centrifugal force to form two phases under the settling field, and a sample is separated and analyzed by utilizing the partition at the interface between the two phases. Concretely, selector valves 5 and 6 are controlled to introduce a developing soln. into the rotary column 11 by pumps 3 and 4 from liq. tanks 1 and 2, the sample is simultaneously introduced from a sampler 7, and the two phases are drawn off into a load resistance 9 and an UV detector 13. Samples having <=10<6>mol.wt. can be separated and analyzed by using the settling field fluidization fractionation device in this settling field fluidization two-phase partition chromatography, and the partition charomatography can be applied.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention is directed to field flow fractionation (Field Flow Fractionation).
This invention relates to a new sedimentation field flow two-phase partition chromatography that effectively performs countercurrent partition chromatography using the principle of actlonation.

[Conventional technology]

When particles in a fluid are placed in a centrifugal force field, they receive a force proportional to their mass. If this force is greater than the self-diffusion force of the particles, the particles will settle and separate from the liquid layer, forming a solid layer. However, if the particles are small, they will not settle completely and will form a layer where centrifugal force and self-diffusion force are balanced. Furthermore, when fluid flows through a narrow gap, the flow velocity at the portion in contact with the wall surface is slower than at the center, and takes on a parabolic flow velocity distribution toward the center of the gap. When a centrifugal force field is applied perpendicularly to this gap, particles in the fluid will move through the gap at a specific flow velocity at a position where centrifugal force and self-diffusion force are balanced. S-FFF (Sedimentation FFF) uses these properties to separate and fractionate fine particles in a fluid.
yield flow fractionation (sedimentation field flow fractionation using a centrifugal force field).

Regarding such sedimentation field flow fractionation, the present applicant has already made various proposals so far, and one example thereof (Jitsuka Sho 61-182443) will be explained. FIG. 3 is an assembly diagram showing an example of the configuration of a sedimentation field flow fractionation column, and FIGS. 4 and 5 are partial views of FIG. 3.

In FIG. 3, first, one end of the spacer 33 is fixed in the spacer mounting recess 40.40 of the variable diameter ring 32 with a screw, and the other end is semi-fixed.Next, the diameter of the variable diameter ring 32 is reduced. Compress the column and
The variable diameter ring 32 is inserted and arranged within the base 34. After that, when the tightening ring 31 is inserted into the variable diameter ring 32, the tapered surface of the tightening ring 31 aligns with the variable diameter ring 32.
As the tapered surface of the variable diameter ring 3 descends, the variable diameter ring 3
2 are forcibly expanded by the action of the shaft and firmly pressed against the inner wall of the column base 34 via the spacer 33. At this time, the fluid injection pipe 38 and the outflow pipe 39 of the variable diameter ring 32 are connected to the notch 35a of the tightening ring 31,
35a and is fixed at a position facing the insertion hole 35.35. Then, by inserting the bolt 45 into the screw hole 36.42, the tightening ring 31 and the column base 3 are connected.
4, and connect the swage type connector 46 to the injection pipe 38.
and connected to the outflow pipe 39.

Therefore, as shown in FIG. 4, a spacer 33 is sandwiched between the variable diameter ring 32 and the column base 34, and the thickness of this spacer 3 becomes the width W of the channel groove. The width W of this channel groove is selected in the range of 50 μm to 300 μm. Therefore, the opposing surfaces of the variable diameter ring 32 and column base 34 are mirror-finished. Selection of W can be easily achieved by replacing spacers 33 with different thicknesses and variable diameter rings adapted to the spacers 33. Further, since the spacer 33 is fixed to the variable diameter ring 32, the fluid introduction/outlet can be accurately set at the narrowest position of the gap of the arrow-shaped cutout 47. As a result, the cut portion 37 of the variable diameter ring 32 and the fluid injection tube 38
The column can be provided as close as possible to the outflow pipe 39, and the length of the column can be increased. Further, since the tightening ring 31 and the column base 34 are fixed by the bolts 45 on the bottom side of the column base 34, the thickness of the side wall of the heavy column base 34 can be reduced.

Further, as shown in FIG. 5, the fluid injection pipe 38 and the outflow pipe 39 of the variable diameter ring 32
a, 35a and is fixed at a position facing the insertion hole 35.35, but since the notch 35a is cut thinner than the thickness of the tightening ring 31, the lower end surface of the tightening ring 31 Compared to the case where a notch is provided in the entire radial direction of the spacer 33, the spacer 33 in this portion is firmly fixed, and the sealing performance can be improved. Further, by disposing a sheet member made of polyimide or Teflon-coated polyimide between the spacer 33 and the column base 34, the interface with the fluid can be made of a material different from that of the column base 34.

On the other hand, a method called centrifugal countercurrent partition chromatography (for example, chemistry, 41!,
No. 8, p. 512 - (1986) "Centrifugal countercurrent partition extraction method"
), which allows another liquid that does not dissolve in the liquid to pass through the liquid held in the container by centrifugal force in the form of droplets, and is based on the difference in the distribution of the sample. This is a method of performing separation analysis.

[Problem that the invention seeks to solve]

However, the sedimentation field flow fractionation described above is applicable to samples with a molecular weight of 106 or more and a particle size of 0.
For samples with a molecular weight of 0.01 μm to several tens of μm, and a sample with a molecular weight of 10” or less, the strength of the action field would require a size that is practically unrealizable.

Furthermore, the method using centrifugal (countercurrent) partition chromatography has a relatively low separation efficiency per unit time, and requires a long time to achieve high separation performance.

The present invention has been made in view of the above facts, and
It is an object of the present invention to provide sedimentation field flow two-phase partition chromatography that effectively realizes two-phase partition chromatography using the sedimentation field flow fractionation method.

[Means for solving problems]

For this purpose, the sedimentation field flow two-phase partition chromatography of the present invention is a sedimentation field flow two-phase partition chromatography that performs countercurrent partition chromatography using the principle of sedimentation field flow fractionation, and is a rotational separation using centrifugal force. Identifying the column, the fluid introduction means for introducing different types of fluids into the separation column, and the sample introduction means for introducing the sample into the separation column, and introducing different types of fluids that do not dissolve into each other into the separation column using the liquid feeding means. This method is characterized in that two phases are created in a sedimentation field, and the distribution at the interface of the two phases is used for separation and analysis of the sample.

[Effect]

In the sedimentation field fluidized two-phase partition chromatography of the present invention,
Different types of fluids that do not dissolve in each other are introduced into a separation column to create two phases under a sedimentation field, and the separation and analysis of the sample is performed using the distribution at the two-phase interface. Separation analysis can be performed using partitioning and partitioning at the two-phase interface.

〔Example〕

Examples will be described below with reference to the drawings.

FIG. 1 is a diagram showing an embodiment of the configuration of a flow system for sedimentation field fluidized two-phase partition chromatography according to the present invention, and FIG. 2 is a sectional view of a rotating column. In the figure, 1.2.8 and 14 are liquid tanks,
3 and 4 are pumps, 5 and 6 are switching valves, 7 is a sampler, 9 is a load resistor, IO is a rotating diode, 11 is a rotating column, 12 is a rotation controller, and 13 is a UV detector.

The sedimentation field flow two-phase partition chromatography of the present invention can also realize separation analysis of sample systems with molecular weights below 10'' using a sedimentation field flow fractionation apparatus, and its application fields include:
It is effective in all fields where cocurrent partition chromatography is applicable. As shown in Figure 1, the equipment is basically the same as the equipment used for sedimentation field flow fractionation, except for the following points. First, two phases (mainly liquid-liquid) are required for distribution. Therefore, a means for introducing this, namely two pumps 3.4 and a liquid tank 1.2, is required, and a means for separately taking out the two phases at the outlet from the rotating column 11 (separation column) is required. It is.

With this configuration, the switching valve 5.
6, a developing solution is introduced from a liquid tank 1.2 using a pump 3.4, a sample is introduced from a sampler 7, and the two phases are taken out to a load resistor 9 and a UV detector 13.

Next, the operating principle of the sedimentation field fluidized two-phase partition chromatography according to the present invention will be explained.

To aid understanding, a case is shown in which H2C is used as the main developing solution and CHCII is used as the partitioned phase. CHCl5 is d”-1,4985 and Hz 0(
7) d”-0,99982:3 Heavier than 2.

Now, the rotation of the rotating column 11 is stopped, and each switching valve 5 is
．． 6 in the state shown in the figure, pump 3 pumps C from liquid tank 1.
HClx is sent at a flow rate of 10 I 1 /+win, and at the same time Hl is pumped from the liquid tank 2 by the pump 4.

is sent at a flow rate of 1000 μN/sin.

Therefore, the volume ratio of CHCl3 and Hgo in the column 11 is 1:100. Next, when the column 11 is filled with the developing solution, the liquid flow is stopped, and the rotation controller 12 is activated to rotate the column 11 at about 11,000 rpm. Then, as shown in the second (!I), CHCl5 and HgO
is separated into two phases, with H and O on the inside of the column and carbon on the outside.
Two uniform layers of HCl3 are formed with each layer having a thickness of 100:1. Therefore, the channel gap WIOμm~100μm
If the range is selected, the liquid-liquid interface between CHCII, l and H2C will occur at W/101 from the outer wall surface. In addition, the second
A modification of the outlet in Figure ta+ is shown in Figure 2).

When liquid feeding is resumed from such a state, the liquid flow within the channel gap becomes a laminar flow, and the flow velocity distribution primarily becomes a parabolic distribution. That is, the average flow rate of HzO < W w
*. >, the average flow velocity (V c, lcr z ) of CHCl3 localized near the outer wall surface is significantly slower. Therefore, in order to maintain the once generated H, O/CHC1s interface at the same level, the cHc1s by pump 3 must be
It can be seen that the amount of liquid to be sent for ls may be extremely small. On the other hand, if the area occupied by CHCIIz in the cross section perpendicular to the flow direction of the channel is 5CNCL2, then the flow velocity after the start of rotation is CHCZ, which is UCNCL=<VCMCL3)
・sc, Ict3 becomes, and H2O becomes UN! 11-
<Vgzo ) 5xto Taking these ratios, we get UCNCL 5CNCL3 <VCN
CL3 >UHzo S, Igo
<VHzo )1 <VcHcti
> 1 00 <Vwz. 〉 It becomes about 1/33. That is, it is assumed that the flow velocity distribution function is . X is the distance from the wall, and θ is the angle formed with the wall.

Then, -3(□) is obtained, where l is the position of the interface between CHCl3 and )1.0 expressed as a distance from the wall surface. Therefore, since I W 101, <VCNCL3n31 <V. . > 101 33
So, <Ucwct2> 1 1
1<Unto) 100
33 3300. Therefore, H,
O 1000/Ij! /win, CHCl can be O-3111/gin.

Now, when switching the switching valve 5.6 into such a system and injecting multiple types of phenols (for example, phenol, o, m, p-cresol) from the sampler 7, the two-liquid calculation of H3O and CHCe Distribution occurs. Therefore, due to the difference in partition coefficients, more cresol (cresol that partitions into CHCNs is retained in column 11), so phenol is eluted first, and cresol isomers are also eluted and separated in sequence according to the difference in their partition coefficients. .

The in-column separation efficiency increases as the area of the interface where distribution occurs relative to the total amount of liquid sent increases. It corresponds exactly to the particle size and bore size that govern the surface area of the packing in banked column chromatography, and to the tube diameter and tube length in the case of oven tube capillaries. In this system, as W becomes smaller, the ratio of the area of the interface to the amount of combined liquid increases. Therefore, W is 50 to 30 used in sedimentation field flow fractionation.
A value smaller than 0 μm is generally used.

Note that the present invention is not limited to the above embodiments, and various modifications are possible. Fast mobile phase (here H
For example, if polyethylene glycol is used instead of CHCl2 as the slow mobile phase, the relative flow rate will be , the velocity is significantly slower than normally expected at the position of the two-liquid interface in a parabolic flow velocity distribution (in extreme cases, the low-speed mobile phase is stationary relative to the high-speed mobile phase, i.e., it cannot be regarded as the stationary phase). In such a case, the pump 3 can be stopped and used during the execution of the separation analysis.

Further, a gas may be used as the high-speed mobile phase.

Furthermore, special sedimentation field flow fractionation is possible in the system of the present invention. For example, if the density of a particle is greater than that of the fast mobile phase and less than that of the slow mobile phase, the particle can move the two-liquid interface into a new integral This becomes a sedimentation field flow fraction with a wall surface. In this case, solute retention is compounded by a new effect that reflects the affinity between the solute and the slow mobile phase.

Next, we will examine how efficient the sedimentation field fluidized two-phase partition chromatography of the present invention is compared to conventional ones.

First, we will discuss centrifugal countercurrent partition extraction (CPC), one of the conventional methods, in the literature [Chemistry, Vol. 41, No. 8, pp. 512-(19
Looking at the example described in 86)), the stationary phase! 15ml Mobile phase (Dil 360mj! ~ 12()0ml) Flow rate 4ml/sin Required time for separation 90 minutes to 300 minutes.Now, the particle size of the droplets in the stationary phase is 2W.
Assuming φ, Surface area 0.1257011 Volume 4. 2×10−”mj! Therefore, the total number of droplets and the total surface area of the two-liquid interface until the separation is completed are as follows.

When the mobile phase volume is 360ml, the total number of droplets is 360+ (4,2xlL")-85,7x
lO' The total interfacial area is! 0.77
00÷(4,2xlO-")-285,7x10' The total interface area is 35.9x10"cj.

On the other hand, in the sedimentation field flow two-phase partition chromatography of the present invention, the channel gap W=0.1 am.

B=20 mm, L-580-th floor, and the volume ratio of the mobile phase to the stationary phase is 20 (11). Here, it is assumed that the A stationary phase occupies 0.5% of the channel gap. Then, the volume inside the channel gap is v, = 0.01 x 2 x 58 - 1.16 cd 2 fi interface area (inside the channel) is S, palm 2 x 58 - 116 cd. Now, the total amount of mobile phase until the end of the official residence is 20 of vo.
Assuming that the total area of the effective interface is 5=2O3, -116x20 =2.32xlO',! Also, if the flow velocity is 0, 1 m//win,
The time required for separation is zoxv@+9.1-232m1n The amount of stationary phase is v, For chromatography, the amount of stationary phase was 129 minutes. The interfacial area of the two liquids is 1/4.6 to 15.5. The required time is 1/0.39 to 1.3, and the interfacial area per amount of mobile phase is 3.34 times.

Further, the theoretical plate number of the centrifugal countercurrent distribution extraction method is the number of multicapsule plates. This is because the inside of the microcapsule is agitated by the droplets, and it is thought that the concentration of distributed components in the stationary phase is uniform.

Therefore, in this case, the number of theoretical plates that can be expected is limited to 100 to several hundred plates. (Page 515 of the above-mentioned document) In contrast, in the sedimentation field fluid two-phase partition chromatography of the present invention, the solute zone only expands due to natural diffusion and non-average dispersion contribution, and the number of theoretical plates is 1000 or more, several tens of
00 stages can be easily obtained.

If the number of theoretical plates were to be the same, the time required would be considerably shortened, only a very small amount of stationary phase would be required, and the solute zone would be highly concentrated.

〔Effect of the invention〕

As is clear from the above description, according to the present invention, the distribution interface area for the feeding device can be increased, so that
Centrifugal partition chromatography with high separation efficiency can be realized. Also, the quantitative ratio of the slow mobile phase to the fast mobile phase,
Retention can be freely controlled by selecting the physical and chemical properties of the low-speed mobile phase. Therefore, separation and analysis of samples with a molecular weight of 10' or less, which has not been able to be handled up to now, can be realized using the sedimentation field flow fractionation device system. Furthermore,
Since you can freely choose the chemical properties of the integrated wall surface,
It is possible to realize a separation analysis method using a new separation mechanism that combines the effects of partition chromatography and sedimentation field flow fractionation.

[Brief explanation of the drawing]

1st ri! J is a diagram showing one embodiment of the flow system configuration of the sedimentation field fluid two-phase partition chromatography according to the present invention, FIG. 2 is a sectional view of a rotating column, and FIG. 3 is an example of the configuration of a sedimentation field fluid fractionation column. The assembly drawings shown in Figures 4 and 5 are 1 in Figure 3.
FIG. ■, 2.8 and 14...liquid tank, 3 and 4...pump, 5
and 6...switching valve, 7...sampler, 9...
Load resistance, 10...Rotation joint, 11...Rotation column, 12...Rotation control unit, 13...UV
Detector. Applicant JEOL Co., Ltd. Agent Patent Attorney Ryukichi Abe (2 others) Figure 1 21! Go to 5 Go to 9

Claims (1)

[Claims] (1) Sedimentation field flow two-phase partition chromatography that performs countercurrent partition chromatography using the principle of sedimentation field flow fractionation, in which a rotating separation column uses centrifugal force, and different types of fluids are introduced into the separation column. and a sample introduction means for introducing a sample into the separation column, and the liquid feeding means introduces different types of fluids that do not dissolve in each other into the separation column to create two phases under a sedimentation field, Sedimentation field flow two-phase partition chromatography, characterized in that separation and analysis of a sample is performed using the distribution at the two-phase interface.