JPS5970963A - Method of filling narrow diameter particle column and column - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of packing a chromatography column and its resultant. In particular, it relates to a method and the resultant product for packing narrow diameter columns used in liquid chromatography (LC) or gas chromatography (GC) with fine particles.

For highly efficient solvent utilization in high performance liquid chromatography (HPLC) and gas chromatography (
Due to high column efficiencies in GC), the chromatographic trend has shifted towards high pressure and small diameter columns. Furthermore, the desire to obtain greater separation power has also been realized through the use of longer columns. As a result of these developments, complex mixtures can be efficiently separated using moving solvent indicators in LC or with minimal analysis times in GC. A discussion of these developments can be found in F.
J. Yang, “High performance liquid lithography of fused silica narrow particle packed column 07
caNarrow-Bore Mlcropartlc
le-Packed-Column High-Per
formation Liquld Chromatog
raphy, Journal of Chromato
graphy, vol. 236, p. 265 (1982)). These developments have resulted in new chromatographic equipment and more powerful chromatographic techniques.

In GC, narrow diameter open tubular columns (U.S. Pat. No. 4,2
93,415), while in LC the best separations were obtained primarily using narrow diameter, particulate packed columns. This success in LC is due to the fact that long, narrow diameter columns can be efficiently packed with small particles (eg, 10 μm or less).

A discussion of the evolution of HPLC compared to the evolution of GC can be found in F. J. Yang, ``Narrow-Bore Microparticle Column
partlcle -PackedColumn Hi
gh-Performance Liquid Ch
romatography.

J, Chromatography, 6th National Liquid Column Chromatography Society M, 1982).

1 (PLC and GC) Narrow-diameter fine-particle-packed columns show excellent performance, so it is desirable to develop satisfactory columns, packing materials, and packing techniques to pack such columns. Packing columns The old gravity action method was unsatisfactory for narrow diameter columns. One technique for packing narrow diameter columns was to pack a regular glass column and then pull the column to reduce the diameter. There are techniques to narrow the LC stationary phase to be bonded in situ.M, PVio
Packed Microcapil such as votony
lary Columns in HLgh Perf
ormanceLiquid Chromtogra
phy (Anal, Chem, 50 271 (1
978)). The results formed with this technique have a low column diameter to particle size ratio (approximately 2 to
It differs from a normal packed column in that it has a range of 3).

A disadvantage with this method is that the performance of prepacked capillary columns with relatively large particle sizes is significantly inferior to conventional columns of 4-5 mm diameter packed with 5 μm particles.

With this method, particles with a size of 30 μnt or less cannot be filled due to clogging, and uniform packing is difficult.

It has become increasingly difficult to pack narrow diameter columns with fine particles as the column internal diameter approaches the particle size. This results in non-uniform packing and clogging of the column, resulting in high resistance to solvent flow.

The problem worsens as the column length increases.

Other column packing methods are known for packing LC columns within 4-5 mm. It includes an equilibrium density many-one packing method. R, E, Majors' Anal,
Chem.

44 1722.1723 (1972) and J, J, Klrkland J, C on LC columns.
hromatogr, Sc l, 920 (3,
207 (1971). The method then includes a conventional high pressure air compression dry separation packing method used with a GC column. For small diameter columns, such as those having an internal diameter of 500 microns or less, it is known to incorporate the particles into a slurry and run the slurry through the column. The slurry filling method is typically accomplished by the following steps. These steps include flowing the slurry through the column, stopping the flow, draining the liquid, and retaining particles present in the column.

A study of this technology evaluated the relationship between packing rate and column dimensions. For example, Pa of Y, Kato et al.
ccking of Toyopearl Column
For Get Filtration (J, Chr.
omatography, vol. 205, p. 185, 198
1. Vol. 206, p. 135, 1981). It has been shown that semi-constant power packing with variable flow rate and speed is suitable for packing columns for cell filtration. For example, Packi such as Y, Kato, etc.
ng ofToyopearl Colourns F
or Gel Filtration, 111 +
Sem1-Con5tant Pregsure Pa
cking (J, Chromato-graphy
, Vol. 208, p. 71, 198], ).

D, Is h l l %Developmen
to of Technique forMlniat
Urization of High-Perform
ance Liquid Chromatography
(J, Chromatography, 144
Vol. 157, p. 1977), the inner diameter was 0.5 mm;
A tube made of PTFE tube with an outer diameter of 1.0 mm was prepared by slurry filling technology. A column was selected that was many times longer than the column ultimately required.

The stationary phase is suspended in a suitable solvent as a slurry provided in a small bottle. A 250 microliter airtight syringe is connected to the tubing and filled with the solvent used to prepare the slurry. The lower end of the tube was dipped into the slurry, a syringe was attached to the microfeeder, and the slurry was aspirated at the upper end of the tube by manual or electrical actuation of the feeder. The lower end of the tube was then tightly plugged with a small amount of quartz wool to stop the filler from escaping. The microfeeder was activated manually or electrically to release the solvent.

The resulting column has the poor stability of a packed separation bed and is easily deformed at high speeds and high column inlet pressures. These are high pressure liquid chromatography (HPLC)
) is not suitable for

One object of the present invention is to provide a method for packing narrow diameter, high efficiency columns selected from the following materials: fused silica, glass, stainless steel, glass-lined stainless steel.

Another object of the invention is to uniformly pack a narrow diameter chromatography column along its length with microparticles having a diameter ranging from 3 μm to 10 μm for liquid chromatography and from 3 μm to 100 μm for gas chromatography. The goal is to provide a method to do so.

Another object of the invention is to provide a narrow diameter chromatography column packed with fine particles having uniformly low porosity and a stable packed separation bed. SUMMARY OF THE INVENTION It is an object of the present invention to provide a column having an end for connecting to an injector and an end for connecting to a detector interface that provides optimum column efficiency and packing separation method stability. An alternative method for packing diameter chromatography columns is provided. A fusible column with an internal diameter of 500 μm or less is selected. A slurry of mobile solvent and particles of a specific size is formed within the reservoir. In the case of liquid chromatography, the particle size range is 3 μm to 100 μm. An end restrictor is located at the end of the column to allow flow of mobile solvent and restrict passage of particles from the column. When the slurry is stored in the column, the slurry is flowed under pressure into the column. A two-step pressure sequence is used.

First, a separated bed of particles is filled and formed, and then the separated bed is uniformly compressed. In this way, the initial pressure is maintained for an initial period of time (preferably no more than 10 minutes).

During the second period, the initial pressure is increased to the maximum pressure. The resulting column is a stable, loosely packed column with a high number of plates per unit length.

[N”l of preferred embodiment: light]

The primary goal when packing a column is to have a reproducible uniform distribution of the packing material along both the length and transverse directions of the column. Such uniformly packed columns tend to have high minute capacity and are used for high speed analysis. As previously mentioned, gravity dry filling and slurry filling methods have been used. Since the column diameter is becoming smaller (for example, 50C+z1m
(below), it has become increasingly difficult to fill reproducibly. When conventional slurry filling methods are used,
Particularly difficult to fill are particles smaller than 5 μm, where wall effects lead to poor uniformity, resulting in low porosity and non-uniform packing density.

In the development of narrow internal diameter (narrow diameter) particulate columns for HPLC, the various classifications of columns are shown in Table 1.8 Each type has its own range of column internal diameter, particle size, and flow rate. have. Capillary columns, although difficult to fabricate, are economical to operate because they require only a small amount of packing material and a small amount of solvent. When packing narrow diameter columns, it is necessary to pack them uniformly over the entire length of the column. Additionally, packing too tightly at any point along the column must be avoided.

Otherwise, the close packing may unduly restrict the flow rate of mobile solvent through the column during operation.
For high efficiency separation, it is desirable to have a uniform packing density along the diameter of the column. The properties of the column packed according to the method of the invention are as follows.

(a) A long, narrow diameter column is efficiently packed with particles on the order of 3 μm.

(b) These columns have sufficiently low porosity to allow efficient separation.

(c) The uniform and stable particle distribution allows the column to be operated at high flow rates (01-20 μm 7 mm).

In a preferred embodiment of the method of the invention, the column is selected from materials such as fused silica, glass, stainless steel or slanted stainless steel.

This column is connected to a slurry storage facility of the type shown in FIG. Narrow diameter column 1 consisting of fused silica capillary 11
0 is inserted into the stainless steel tube 13. One end of the steel tube 13 fits into the coupler 12 and the other end fits into the coupler 16 and is held in place by the ferrule 15. Furthermore, a slurry reservoir 17 is inserted into the combiner 16 and is passed through the combiner 20 to the bong 7''P.
(not shown). The end of the column 10 is thus placed in adjacent relationship with the bottom of the slurry reservoir 17 through the ball tube 13. The lower portion of the wall 19 of the reservoir 17 is shaped to be in surface contact with the inner wall 14 of the coupler 16. In the preferred embodiment shown in FIG.
The inner wall of the wall 19 has a funnel shape, and the lower part of the wall 19 has a similar funnel shape.

At the end, a wall 19 abuts the upper end of the steel tube 13. The upper end of capillary tube 11 thus communicates only with slurry 18, which flows smoothly to the end of column 10 during filling. As shown in the figure, the diameter of the bottom of the funnel shape is column 10.
is preferably on the order of the inner diameter of the column 10, thereby avoiding flow impedance due to door effects and allowing the slurry to flow uniformly to the ends of the column 10 during packing.

During the packing process, a particle restrictor is connected to the downstream end of the column. As discussed below and as shown in the photographs of FIGS. 4a-48, uniformly low porosity packings can be obtained with particle restrictors. In contrast to the above-mentioned Ishii study, the flow of particles in the slurry under high pressure is stopped at the end of the column, and the particles are trapped and packed uniformly within the separation bed. During the packing process, the restrictor provides back pressure, allowing solvent to flow out the end of the column and retaining the packing material in the column. Typically the diameter of the filler material ranges from 3 to 10 μm for LC and from 3 to 100 μL for Gc.

Particle restrictors can take a variety of shapes.

As shown in Figure 2a, a wire 28 is inserted from the end of the column 25 into the second. Column 25 has an outer protective covering 26 along its length. The diameter of the wire insert 28 is also slightly smaller than the inner diameter of the column 25 so that solvent can exit the column via the annular opening 29. The annular opening 290 dimensions are similar to the column packing particles 27
small enough that it cannot pass through. Once the column is completely filled according to the method of the invention, the wire is removed and a porous stopper is inserted. The requirements of the stopper are that the mobile solvent will flow through it, yet the particles will be permanently confined within the column body.

A second type of flow restrictor is shown in Figure 2b. A thick-walled fused silica column 30 having an outer diameter approximately equal to the inner diameter of column 25 is provided with a small central cavity 32 . Column 30 is inserted into column 25 and secured to its end. The solvent flows through the central hollow part 32 of the column 3°, but the packed particles 27 flow through the central hollow part 32 of the column 3 because the central hollow part 32 is narrow.
The outflow of o is prohibited. The third type of restrictor is
Shown in Figure c. Column 25 is inserted into connecting pipe 33 .

The connecting pipe 33 has an inner diameter approximately equal to the outer diameter of the column 25, and is, for example, a Teflon pipe. A plug insert 31 is pressed against the end of the column 25.

A column or tube 34 is connected to the insert 31 as a detector intermediate. The opening through the plug insert 31 is sufficiently narrow to prevent particles 27 from passing through.

Instead of the plug insert 31 of FIG. 2c, a wire 39 may be inserted into the column 37 as shown in FIG. 2d.

Another type of restrictor is shown in Figure 2e. This is a variation that can be applied to any of the shapes described above. First, the column end or end restrictor 31. (or any other shape of restrictor), the large sized particles 35 are filled in the 7g interface. This large-sized particle 35 has a monkey-shaped opening (second a).
, Fig. 2a), the inner diameter of the column (Fig. 2'b) or the center of the stopper insert (j40tt ((Fig. 2c)).

The small particles that make up the working area of the column are then flushed into the column according to the method of the invention, filling the entire length of the column. Small particles are effectively stopped by a layer of large particles. Restrictors t,i used to pack long columns:
, short columns (e.g. 4 tyn X2 mm,
], a column packed with Ottm particles). After filling is complete, the column restrictor is removed and the permanent restrictor is fitted in place.

The slurry storage tank V is preferably filled with a high concentration (greater than 20 particles/volume) of filler in a solvent such as methanol or acetone. The upper end of the reservoir is connected by a conventional coupler 20 to a high pressure pump, supplying the solvent under pressure as the mobile phase during filling.

The arrangement of storage tank J7 and column 10 is shown in FIG.

In this case, it is a downward filling. Preferably, the force shim 10 is positioned above the reservoir 17 in the top filling mode. This eliminates the possibility of fine particles settling into the storage tank and filling it non-uniformly. Stirring is preferred to ensure uniform filling of the slurry within the reservoir. Stirring is preferably accomplished by non-contact means such as ultrasonic means (not shown). The slurry in the reservoir is pressurized to an initial fill pressure. The initial reservoir pressure is (LL) column length,
(b) Depends on Sculpin internal diameter and particle size. 50Lyn
Pressures for columns with an initial length of 100 mL or more are selected according to Table 1.

50 0.5 5050
0.3 15050
0.2 30050 0.1
400200 05
200200 0.3 3
00200 0.2 4002
0 (10,1500) Once the initial pressure is achieved, the slurry V begins to flow into the cavity. The reservoir pressure and flow rate are maintained at the initial pressure for less than 10 min. The initial pressure is then gradually increased. or linearly, the pressure is increased from the range of 50 to 500 atm to 2,00 to 800 atm.The initial pressure is maintained for a predetermined period of time.The pressure is then increased for a predetermined period of time until the maximum pressure is activated. The mobile phase solvent flows through the column, and the 1st column is flushed and packed into the column.At the beginning of the period when the initial pressure is maintained, the flow rate is close to about Ice/min.The column is filled with packing material. When 11, the flow rate gradually decreases as t.Two-step pressure sequence (
(initial pressure, then maximum pressure) allows the separation bed to be formed uniformly, with the compression pressure asymptotically approaching the compression level associated with the maximum pressure. 2-step pressure Jli? Uniformity is achieved through order. Full compaction is achieved because the separation bed is formed at low pressure without turbulence and then the particles settle in place. If maximum pressure is used initially, non-uniformity will result along the length of the column. After 10-30 minutes of operation at maximum pressure,
I? The pump is stopped and the pressure in the column is gradually reduced stepwise or linearly. Since the pressure decrease is gradual,
``A significant backward force that drives out the 11th agent V'',
do not have. Next, the column 1o is removed from the tube 13 and separated from the slurry storage tank 17. The packed separation bed Q'1 is purified with chromatographic solvent to prepare the column for use in chromatographic analysis.

A somewhat denser, shorter column length and higher initial pressure may be used to pack the column. The non-uniformity introduced by any initial turbulence creates a high initial pressure limit. The process of the invention is carried out in the same way, except for the initial starting pressure selected according to Table 1.

25 0.5 30 (1250,3
400 250,2500 250,1600 500,5400 500,3500 500,2700 500,1800 It is possible to fill with such a high initial pressure.
Only short columns. This is because columns packed at such high initial pressures become dense and the flow of mobile solvent is impeded in the final length increments of the column.

Columns packed by the method of the invention according to Table 1 have low flow resistance. Flow resistance factor is 50-40
It is obtained in the range of 0 and determined by the following φ.

d;Δp φ=□−−− Uη　L Here, d, - particle size Δp - pressure gradient across the column U - Glandular transfer solvent flow rate η - Viscosity of mobile solvent L is two column length.

The resultant has a porosity of 0.5 or greater and is classified as a loosely packed column. For the LC column,
Small particles, for example 3-10 μm, are suitable fillers. For GC columns, particles in the range 3-100 μm are suitable. As shown in Figures 4a-4d, the resultant is:
The column has a substantially uniform porosity r along its entire length and diameter. 41] and 4 Cl:/, l &: u1, l'
1Z x 0.3mm inner diameter and filled with particles of 311n
The particle distribution at the inlet end of the column was observed using a scanning electron microscope (SEM). According to these figures,
It can be seen that there is no significant difference in the particle distribution between the center of the surface of Tsunoram~1 and the area near the wall.

A uniform particle distribution across the column diameter is evident. Figures 4d and 48 are scanning electron microscopy (SEM) views of the particle distribution at the outlet end of the column.

The SEM diagram shows that the particle distribution and particle size along the column diameter at the column end are the same. Therefore, the density and uniformity of the particle distribution shown in Figures 4a-b and 4c-d are the same. This indicates uniformity of packing along the length of the column. The full cross-sectional view of FIG. 4a confirms the uniformity of the ridges. This uniformity is in contrast to that of conventionally packed columns. In conventional methods, large particles tend to cluster along the walls because the walls of the column exert a greater tensile force on larger particles. This uniformity due to the present J is very advantageous. This is because no extra band broadening effects occur during sample analysis and the column becomes highly efficient. For column lengths of 50 cm or more, such loosely packed columns are preferred. They range from 0.1 to 2 o Mic CI of the mobile solvent during the performance of chromatographic analysis.
This is because it allows convenient flow rates on the order of IJ liter/min. Even if the porosity is high, the filler is not consolidated during use. This is because even though the column is operated at high pressure, due to the small cross section the total force applied to the separation bed is small. Such columns enable high-speed analysis. This is because high pressure and high flow rate can be obtained. Very high separation capacities are obtained because long columns can be packed with sub-3 μm particles for LC.

Some notable comparisons between prior art columns and columns according to the present invention are shown in Table 2.

Table IV Features Prior art Number of columns of the present invention
20,000-30,000 200,000 (4
, 6mm inner diameter, 25crn) (300μm inner diameter, 2
m) Peak volume f4 50 1
50-200 using solvent l(・07 minutes
2μt/min sample size 1m9107
1 g fused silica tubes with internal diameters ranging from 57 to 376 μm and lengths of 2 m were prepared using the filling method of the present invention.
0 μm CI8 stationary phase chemically bonded-ph
ase) filled with particles. Reversed phase octadecylsiloxane is chemically bonded to the particulate silica before packing into the narrow diameter column. The mobile phase was Einkratik (l5oc)
(ratic) 70-3o under conditions)
l) It was roux water. The separation capacity of the column is determined by Va in Figure 5.
n Deemter plot. Figure 5 shows spherical 3
Column packed with μm C11l-bonded silica particles (1 m
X3: 3 a μm inner diameter). It can be seen that the effect of flow velocity is not significant. 0.3~1.6 rta
For flow rates in the range of n 7 seconds, the column efficiency is
iif: kept high for bed uniformity.

In this flow rate range, the total number of column stages is 110,0.
It exceeded 00. This should be compared to 20.0 (10 column stages) of a conventional column.

The separation capacity of the column resulting from the method of the invention is illustrated in the chromatograms of FIGS. 6 and 7. FIG. 6 shows the separation of polycyclic aromatic hydrocarbon contaminants by a 320 μm x 2 m fused silica column packed with 311 mC, 8-bonded reverse phase silica particles according to the method of the present invention. The mobile solvent was 70% acetonate, water, and the flow rate was 1.8 μt/min. In the case of a 2 m column, the total number of column plates measured for pyrene (when k'=10) was 144.000 plates. 3 μm C, 144 for 8 columns
A single column with a total efficiency of ,000 plates has not been reported. FIG. 6 demonstrates the efficiency of the tank packing technique for packing long columns with small particles. Figure 7 shows the separation of PNA, a US Environmental Protection Agency priority pollutant, by a 320 μm x 1 meter fused column packed with 3 μm reverse-phase bonded silica particles according to the method of the present invention. It is something. The transfer φ11 solvent was 70% acetonitrile:water, (At, speed was 09
It was μl/min. The basis of the PNA mixture was obtained) (secondly, it should be noted that in a reversed-phase system with benzo(a) anthracene and 1 socratlc), the amount of chrysene hidden in the Q chrysen
Please note the separation from e). The advantages and solvent savings of long narrow-diameter tuberams filled with microparticles in 179 combined samples are evident.

Column materials for liquid and gas chromatography vessels with narrow diameter particles can be selected from the following materials:

Bath fused silica, glass, stainless steel and U if
This is Ruslindsdenless Steel. Requirements for the monthly fee (r, be inert, and be able to be molded to the required narrow diameter. Preferably, the molded to narrow diameter, 1. Occupy the small body iJI. It is flexible so that it can be spiral even if it is long.Furthermore, the material i,
It preferably cures a smooth inner surface and exhibits no wall effect. Fused silica has been found to be a suitable material. This is due to the extremely high degree of smoothness of the fused silica inner wall and the heat dissipation through that wall. Due to heat dissipation, there is no significant thermal gradient across the column. It is also ideal for direct interfacing to detectors such as fused silica, flame-based detectors, mass spectrographs, and Fourier transform infrared detectors. This is because the flow rate matches the flow requirements of these detectors. Columns packed according to the method of the invention have an inner diameter of less than about 500 μm. In the case of liquid chromatography, 10
It has a particle size of less than μm and a length of more than 10 crn. In the case of gas chromatography, it has a particle size of less than 100 μm. The particles can also be physically coated or bound to gas, liquid, liquid, or any type of phase useful for ion exchange chromatography.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of a reservoir for packing a narrow diameter chromatography column according to the method of the present invention. 2a-2 Q 1 (ffl k+, end of narrow diameter chromatography column packed according to the method of the invention:
FIG. 12 is a side tier view of some variations of end fittings used in tl. Fig. 3)-4(1':t1) is a flowchart for explaining the method of filling a silica column according to the present invention. Figs. :”+mm
) is a reproduction of a photograph of a cross section. Figure 4a shows the column total area -, <2.6 Figure 4C shows a partial cross-section near the wall at the column inlet end using SEM at a magnification of 2.4. ) near the wall and 11
1. The official cross section was taken by SEM. 'i' x 10'' times. Figure 4θ is the same as Figure 4d, but with 2.4 x 103
It is expressed in times. Figure 5 shows a narrow particle packed column (7) with an inner diameter of 330 μm packed with 3 μmO□8 stationary phase chemically bonded particles.
FIG. 3 is a Dθemter performance plot diagram. Test Compounds Figure 6 is a chromatogram showing the separation of a mixture of polycyclic aromatic hydrocarbons. FIG. 7 is a chromatogram showing the separation of bentose nucleic acid, a US Environmental Protection Agency priority contaminant, using a 320 μm x 1 m 3 ttm C,8 reverse phase column. [Explanation of main symbols] 10... Narrow column 11... Melting glue capillary tube 12. 16. 20. Coupler 13... Stainless steel tube 14... Inner wall 15... Ferrule 17... Slurry storage ( Coal 18, slurry 19, wall 20...coupler 25...column 26...protective coating 27...filling particles 28...wire 29...annular opening 30...column 31... - Plug insert 32...Hollow part 33...Connecting tube 35...Particle 37...Column 39...Wire Patent applicant Parian AssoA Inc. FIG, 3 FIG, 4a FIG, 4c FIG , 4b Fig, 4d FIG, 4e FIG, 6 → tR, hr FIG, 7 t,, MIN

Claims (1)

[Claims] 1. A method for filling a narrow diameter column with slurry, comprising the following steps a) to d): a) selecting a flexible column having an internal diameter of 5001 tln or less; b) plugging the outlet end of the column with an end restrictor to allow flow of mobile solvent and restrict passage of packing material; C) preparing a slurry from mobile solvent and packing material;
and d) flowing the slurry through the column under pressure according to steps +U) to a3); dl) applying an initial pressure and maintaining said initial pressure for an initial period; dl) said increasing the pressure from an initial pressure to a maximum pressure; and d3) maintaining said maximum pressure for a second period. 2. Scope of Patent No. 817 Requested Scope A slurry packing force method for a narrow diameter column for gas chromatography described in item 1, wherein the slurry preparation step includes a transfer solvent and a 3 μm to 100 μm
and a filler comprising particles having a diameter in the range of m. 3. Ir! A method for filling a slurry in a narrow-diameter column for liquid chromatography as claimed in claim 1, wherein the slurry preparation step comprises mixing a mobile solvent with a
and a filler comprising particles having a diameter in the range of m. 4. A method for filling a narrow diameter column with slurry according to claim 2 or 3, wherein the step of applying and maintaining the initial pressure includes applying an initial pressure selected from the table below. Column length (crn) Column inner diameter (corner) Initial pressure (atm) 50 0.5
505 (10,3150 500,2300 500,1400 2000,5200 2000,3300 2000,2400 2000,1500 5, A narrow diameter column slurry filling method as described in claim 4, wherein: A method in which the step of maintaining pressure is a step of maintaining a maximum pressure for a period of 10 minutes or more. 6. A method for filling a narrow diameter column with slurry as set forth in claim 4. 7. After the slurry conditioning step and before the step of flowing the slurry through the column under pressure, the step of agitating the slurry is added. 7. A method for filling a slurry in a narrow diameter column, wherein: the slurry stirring step comprises the step of stirring the slurry with ultrasonic waves. 8. A narrow diameter column according to claim 1. A slurry filling method, wherein: plugging the column outlet end includes plugging the column outlet end with a narrow diameter tube that restricts the flow of mobile solvent and restricts the passage of packing material. 9.0 i?''The method for filling a slurry in a narrow diameter column as set forth in claim 1, wherein the step of plugging the outlet end of the column includes plugging the outlet end of the column, thereby forming an annular passageway between the wire and the column to permit flow of mobile solvent and restrict passage of packing material. 10. A method for filling a narrow diameter column with slurry as described in item 1 of Patent No. 61, wherein the step of plugging the column outlet end includes applying a sleeve over the end of the column. and inserting a narrow-diameter plug into the sleeve so as to be adjacent to the end of the column. 11. A narrow-diameter column slurry according to claim 1. A method of packing, wherein plugging the column outlet end comprises: applying a sleeve over the end of the column; inserting an extension column into the sleeve to 12. A method according to any one of claims 8 to 11, comprising the steps of: placing a needle in an adjacent relationship with an end of the column; and inserting a needle into the end of the extension column. A method for filling a narrow diameter column with slurry, as described in , comprising: before flowing the slurry through the column and after plugging the outlet end of the column: setting a collection of particles having a larger diameter than the constituent particles of the filler in a portion adjacent to the end restrictor, thereby forming a stop layer of the filler reservoir; 13. A method for filling a narrow diameter column with slurry according to claim 1, wherein the step of selecting the linear column comprises fused silica, stainless steel or glass-lined stainless steel.
The method consists of selecting a flexible column from a steel column. 14 Claim [2[1] The method for filling a slurry in a narrow diameter column according to paragraph 13, wherein: the column selection step selects a flexible column of fused silica having a length of 50 Crn or more. A method consisting of steps. 15. A method for filling a slurry in a narrow diameter column with τ as set forth in claim 4, wherein: after maintaining the maximum pressure during the second period, the slurry in the column is A method that includes steps of gradually reducing the pressure of 16.% A narrow-diameter packed column manufactured according to any of the methods described in claims 1 to 15. 17. A narrow-diameter packed column manufactured according to steps a) to d) of: a) selecting a flexible column with an internal diameter of 500 μm or less; b) allowing the flow of mobile solvent and of the packing material. plugging the output end of the force ram with an end restrictor to restrict passage; C) preparing a slurry from mobile solvent and filler;
and d) flowing said slurry through said column under pressure according to steps d1) to d3) of: dl) applying an initial pressure and maintaining said initial pressure for an initial period; d2) said increasing the pressure from an initial pressure to a maximum pressure; and d3) maintaining said maximum pressure for a second period.