JPH01182736A - Method of analyzing flow fractionation in sedimentation field - Google Patents

Abstract

PURPOSE:To enable accurate measurement even in a region, where relative holding is small, by using a particle, whose diameter and density difference with a developing liquid are known as a standard sample, performing sedimentation-field flow fractionation analysis, and obtaining a fractogram. CONSTITUTION:A particle, whose diameter dp and density difference DELTArho with a developing liquid are known, is used as a standard sample. Sedimentation-field flow fractionation analysis is performed, and a fractogram is obtained. Constants A and phi in the expression are determined based on a relative holding R, which is obtained from the fractogram. In the expression, W is the channel width of a column, D is the diffusion coefficient of the developing liquid, (k) is Boltzmann's constant, T is absolute temperature and G is centrifugal acceleration. The diameter and the density of the particle of an unknown sample are obtained from the fractogram based on the expression by using the determined constants A and phi.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an analytical method using the principle of sedimentation field flow fractionation.

[Prior Art] When particles in a fluid are placed in a centrifugal force field, they receive a force corresponding to the mass of the particles and tend to settle toward the outer wall.

When the particles are as small as submicrons, Brownian interlocking is possible, and the self-diffusion force increases in proportion to the concentration gradient created by sedimentation, forming a particle cloud where centrifugal force and self-diffusion force are balanced.

Furthermore, when a fluid flows through a narrow gap (channel), the flow velocity in the portion that is in contact with the wall surface is slower than in 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 -F F F uses these properties to separate and fractionate fine particles in a fluid.
(S edlsentation F 1eldF
low F lactlonatlon).

FIG. 2 is a diagram showing an example of a conventional rotating column for 5-FFF. This column has an inlet 23 and an outlet 24, as shown in FIG. 2(a), and is composed of an inner ring 21 and an outer ring 22 cut between the two. A separation groove 25 is dug from end to end on the outer peripheral surface of the inner ring 21, and a seal groove 27 is further provided so as to surround this separation groove 25. This seal groove 27
After embedding the sealing material 28 in the inner ring 21
is inserted into the outer ring 22 as shown in cross-section in FIG. 2(b). This allows inner ring 2
A channel 29 is formed in a portion of the separation groove 25 surrounded by the outer ring 22 and the outer ring 22 . The wedge 30 pushes the inner ring 21 apart and separates the inner ring 21 and the outer ring.
It is used to integrate the ring 22.

The channel 29 has a width W (approximately 250 μm) and a height B
(about 25mm) is the entire circumference (for example, the circumference is about 58es)
It is uniform throughout. Located in close proximity at each end of this channel 29 are an inlet 23 for injecting fluid into the channel and an outlet 2 for removing fluid from the channel.
There are 4. FIG. 2(c) shows the injection port 23. Outlet 249
The arrangement of the separation groove 25 and the seal groove 27 is shown.

FIG. 3 is a block diagram of the entire apparatus including this rotating column. In FIG. 3, reference numeral 1 denotes the rotating column shown in FIG. 2, which is rotated at high speed by a drive mechanism (not shown). A rotary joint 1 and a rotary joint 2 for connecting to a fixed flow path are installed on the rotating shaft portion. The developing liquid pressurized by the pump 3 is passed through the four-way valve 4. It is injected into the column 1 via the sampling valve 5 and the rotary joint 2. The developing solution flowing out of the column 1 is sent to a detector 6 such as a UV detector via a rotary joint 2 and a four-way valve 4.

As mentioned earlier, the developing solution flowing through the channel of the column has a parabolic flow velocity distribution that is maximum at the center and attenuates toward both walls, and the microparticles in the developing solution that are subjected to centrifugal force in this channel are It exhibits a unique concentration distribution determined by its diameter and density, and moves from the inlet to the outlet with a unique movement speed within the channel. Therefore, those with the lowest relative density (difference in density between the developing solution and the particles) Δρ and the cube of the particle diameter dp (relative mass) flow out of the column and are separated.
By detecting this with a UV detector, a fractogram as shown in FIG. 4, for example, can be obtained.

The relative retention R is defined as the non-retention capacity v with respect to the particle retention capacity Vr.
When defined as the ratio of o, Giddings et al. expressed R as the following formula (Anal, Cheap, 46.1
917 (1974)).

R-Vo/Vr -6λ (Coth(2λ)-1-2
λ)-6λ-12λ +12λ(eλ-1)-1...
(1) λ-6kT/πΔρ・dp3・GW...(2
) Here, k is the Boltzmann constant, T is the absolute temperature, Δρ is the density difference between the developing solution and the particles, G is the centrifugal acceleration, and W is the channel width.

Usually, in actual analysis, a W-250 μm column is used, and water containing about 0.1% of a surfactant is used as a developing solution. Then, an unknown sample is injected from the sampling valve 5 to obtain a fractogram, and Vo/Vr (= R) of the peak of interest is determined from this fractogram.
From this value of R (1).

Based on equation (2), the relative mass Δρ·dp3 of the particle corresponding to the peak of interest is determined. If the density of the unknown sample is known, the particle diameter can be determined from this relative mass, and if the particle diameter is known, the density can be determined. Furthermore, by performing the measurement twice with different types of developing solution, both the density and particle size of the unknown sample can be determined.

[Problems to be solved by the invention] In the analysis using the above equations (1) and (2), the experimental results and the theoretical equation agree very well in the region where R is relatively large, but
In a region where R is small, a large separation occurs between the two. Therefore, it was inevitable that there would be large errors in the density and diameter of the particles determined in that region.

Furthermore, the present inventors have previously discovered that in order to increase the resolution of fractograms and obtain them in a short time, the channel width W should be made small and pure water containing no surfactant should be used as a developing solution. has been found to be advantageous, but
If W was made smaller and pure water was used, the separation between the experimental results and the theoretical formula described above would further expand, making quantitative analysis impossible. Therefore, high-speed, high-resolution measurements were practically impossible.

The present invention has been developed in view of the above-mentioned points, and aims to provide a sedimentation field flow fraction analysis method that can eliminate the above-mentioned problems by introducing a new theoretical formula.

Means for Solving Problem C] In order to achieve the above object, the sedimentation field flow fraction analysis method of the present invention uses particles whose particle diameter dp and density difference Δρ from the developing solution are known as standard samples to be analyzed in a sedimentation field. Perform flow fraction analysis to obtain a fractogram, determine the constants A and φ in the following formula based on the relative retention R determined from the fractogram, and use the determined A and φ to calculate the unknown sample based on the following formula. + (A/v)D114(1+φλ) exp(-λ
2 v21~1) λ-6kT/πΔρ・dp3・G
-W (where, W is the channel width of the column, D is the diffusion coefficient of the developing solution, k is the Boltzmann constant, T is the absolute temperature, and G is the centrifugal acceleration.) The present invention will be explained in detail below using the drawings. .

[Example] The present inventor has determined what is the cause of the isolation from the above-mentioned theoretical formula, and what corrections should be made to formulas (1) and (2) to ensure that the formula can well describe the experimental results. In order to investigate what can be obtained by applying the column temperature and flow rate, we used reliable standard samples.

We conducted repeated experiments paying careful attention to measurement conditions such as rotation speed.

First, using a column with a channel width W of 125 μm and a channel wall made of Teflon, 1) pure water, 2) 0.1% Emulgen AP150 [Kao] (oxyethylene-
Oxypropylene copolymer) aqueous solution, ■ 0.01% sodium dodecylbenzenesulfonate aqueous solution, ■ 0.01%
Amphitol 20BS [Kao Co (Lauryl Bedine)] Using four types of developing solution (laurylbedine), the flow rate and column rotation speed were maintained at 1.5 + al/min and 200 Orpm, respectively, and A-H with the density and diameter shown in the table below was prepared. Five types of standard samples (polystyrene latex (S) manufactured by Japan Synthetic Rubber Co., Ltd.
TADEX former X 029)) was measured.

Table 1 From the obtained fractogram, relative retention R is expressed as retention capacity V
It is determined as the ratio of the non-retentive capacity Vo to r, and the horizontal axis is the logarithm, and the density of the developing solution is 0°9982 g/cm.
Then, using each particle size and density, the relative mass Δρ・dp3
Figure 5 shows the experimental results plotted with the logarithm of this relative mass as the vertical axis. In FIG. 5, the broken line is a curve based on the theoretical formulas (1) and (2) given above for G1dd1ngSd.

As is clear from FIG. 5, the experimental results almost agree with the theoretical formula when R is large, regardless of the type of developing solution, but when R is small, isolation from the theoretical formula occurs.
In particular, it was reconfirmed that the isolation was greatest in the case of pure water. It can also be seen that the separation behavior changes depending on the type and concentration of the surfactant.

The change in separation behavior depending on the type and concentration of surfactant was thought to be a phenomenon dependent on the interfacial tension between the particle accumulation wall of the channel and the developing solution.
, 5 dyne/c++ methanol as a developing solution,
Teflon with a critical surface tension of 18 dyne/ca+,
The experiment was conducted by selecting polyimide of about 40 dyne/cm as the material for the particle accumulation wall. As a result, whether the material of the particle accumulation wall is wetted by the developing solution (methanol) (polyimide) or not (Teflon), the separation behavior will still result in isolation based on the theoretical equations (1) and (2). As a result, a direct causal relationship between its isolation and interfacial tension was denied.

Therefore, we revisited the process of deriving equations (1) and (2) and added further consideration. That is, equations (1) and (2) are shown in FIG. 6(a).
It is derived based on the assumption that the flow velocity distribution maintains a parabolic distribution up to the wall surface, as shown in , and the flow velocity becomes zero on the wall surface. This assumption does not cause any disadvantage in many cases. However, retention in the sedimentation field flow fraction becomes noticeable when the particle cloud remains near the wall surface.

For example, at W-125 μm and R-0,1, the particle cloud remains at the mean pressure Ml. Flow rate 1500μl/l1l
When experimenting with In, the linear velocity of particles at a position 2 μm from the wall surface is about 97 μm/see. On the other hand, the moving speed due to diffusion of the developing solution is 86 μm in the case of pure water. Therefore, near the wall surface, the contribution of diffusion cannot be ignored, and if diffusion is taken into account, the actual flow velocity distribution will be as shown in Figure 6(b), and it must be assumed that the flow velocity at the wall surface is not zero. I found out that this is not the case.

Therefore, the diffusion coefficient is p-0°512 at 25°C.
X 10-0-5a/see and 2.32X10-5c
Using n-proper tool and methanol, which are significantly different from Kasumi 2/see, as the developing solution, W-250 μ
Experiments were conducted using two types of columns: m and 125 μm.

The results are plotted in Figure 7, which shows 0 (250 μm
) and · (125 μm) are methanol, Δ (250 μm)
The size (125 μm) indicates n-brono, respectively. From this figure, it was confirmed that the relative retention R was significantly dependent on W and D.

Based on the above experimental results, the present inventor has developed the conventional theoretical formula (
It has been found that the experimental results up to now can be explained without contradiction by the following equation, which is obtained by adding the correction term Rd to 1).

R-Ro +Rd-(3)R
d function (A/V) D114 (1+φλ) exp(-λ w
/2(U>...(4) Here, RO is the conventional term, and λ is also the conventional term, and is expressed by the following formula.

Ro-6λ-12λ +12λ(eλ-1)-1λ-6
kT/πΔρ·dp3 ·GW In the above equation (4), A and φ are constants determined by experimental conditions. In the apparatus used by the present inventor, the values were K-1 and φ-3 in the system of pure water, methanol, and n-propertool.

The solid and dashed lines in Figure 7 represent the diffusion coefficients of methanol and n-propyl alcohol at 20°C.
μm2/see, 420μm2/see, W=12
For 5μm and W-250μ'm columns (3), (
4) The results of calculation using formula are plotted, and the solid line is W
-125 μm 1 broken line indicates the case of W to 250 μm, respectively. The experimental results shown as dots (0°.: methanol, Δ, m; n property tool) and the case of W-125 μm are also 25
It can be seen that there is good agreement even in the case of 0 μm.

FIG. 1 shows an example of a flowchart for carrying out the sedimentation field flow fraction analysis method of the present invention using such a new formula. Hereinafter, one embodiment will be described according to this flowchart.

■First, prepare multiple types of standard samples with known particle sizes and densities.

■Prepare a developing solution (eg, pure water) suitable for dispersing the standard sample and unknown sample.

■Mix multiple types of standard samples and measure the fractogram at a predetermined rotation speed.

(2) Based on the obtained fractogram, determine the relative retention R for at least two sample peaks to obtain R1 and R2.

■These R1, R2, (3), (4), (1).

(2) Determine constants A and φ from equation (3).

(4) Confirm the formula.

■ Obtain a fractogram for an unknown sample.

(2) Determine the relative retention Rx for the peak of the unknown sample.

(2) Using Rx, calculate the relative mass Δp-dp3 of the unknown sample from equations (3), (4), (1), and (2).

■If Δρ is known, calculate dp from this relative mass,
If dp is known, Δρ is determined from the relative mass.

In order to improve the accuracy of the constants A and φ, it is preferable to increase the number of data by increasing the number of standard samples or by changing the number of rotations to several types.

[Effects] As described in detail above, the present invention provides a sedimentation field flow fraction analysis method that allows accurate measurements even in a small R region.

In addition, even if pure water is used without using a surfactant, and W is smaller than the conventional 250 μm, the experimental results and the theoretical formula match and accurate measurement results can be obtained, resulting in high speed and high resolution. measurement becomes possible.

[Brief explanation of the drawing]

Fig. 1 is a diagram showing an example of a flowchart for implementing the method according to the present invention, Fig. 2 is a diagram showing an example of a conventional rotating column for 5-FFF, and Fig. 3 is a diagram showing the entire apparatus including this rotating column. Fig. 4 is a diagram showing an example of a fractogram.
Figure 5 shows the separation of the curve based on the conventional theoretical formula and the curve based on the fractograms obtained for five types of standard samples using four types of developing solutions. Figure 7 is a diagram showing the flow velocity distribution of the developing solution.
It is a comparison diagram between the results of experiments conducted on samples A-H using two types of columns, W-250 μm and 125 μm, and a curve based on a theoretical formula newly introduced in the present invention.

Claims (1)

[Claims] (1) Perform sedimentation field flow fraction analysis using particles with known particle diameter dp and density difference Δρ from the developing solution as a standard sample to obtain a fractogram, and obtain relative retention R from the fractogram.
A sedimentation field flow fractionation characterized in that the constants A and φ in the formula below are determined based on the equation below, and the particle diameter or density of the unknown sample is determined from the fractogram of the unknown sample based on the formula below using the determined A and φ. Analysis method. R=6λ-12λ^2+12λ(e^λ-1)^-^1
+(A/W)D^1^/^4(1+φλ)exp(-λ
＾2W＾2/2√D)λ=6kT/πΔρ・dp＾3・
G・W (Here, W is the channel width of the column, D is the diffusion coefficient of the developing solution, k is Boltzmann's constant, T is the absolute temperature, and G is the centrifugal acceleration.)