Note: Descriptions are shown in the official language in which they were submitted.
~1~2Z6~
Narrow Bore Micro particle Column Packing Process
And Product
This invention relates to a process for packing
chromatographic columns and the resulting product and
more particularly relates to a process for packing a
narrow bore micro particle packed chromatographic
column for use in gas or liquid chromatography and the
resultant product.
The trend in chromatography has been to move
to higher pressures and smaller diameter columns for
efficient solvent utilization in high performance
liquid chromatography (HPLC) and for high column
efficiency in gas chromatography (GO). In addition,
the desire to obtain greater resolving power can be
realized by using long length columns. As a result
of these developments complex mixtures may be effect
lively separated while using smaller amounts ox
mobile solvent in LO and minimum analysis time in
GO. For a discussion of these developments see F.
J. Yang, "Fused-Silica Narrow-Bore Micro particle-
Packed-Column High-Performance Liquid Chromatography",
Journal of Chromatography, v. 236, p. 265 (1982).
The results of these developments is that new
chromatographic apparatus and more powerful cremate-
graphic techniques are being provided.
Whereas in gas chromatography the highest resow
lotion and speed of analysis has been obtained using
narrow bore open tubular columns (see, e.g., P. F.
Bent, Iota et at., "Silica Chromatographic Column",
US. Patent No. 4,293,415), in liquid chromatography
the highest resolution has been obtained primarily
with narrow bore micro particle packed columns. This
latter circumstance for liquid chromatography is due
to the fact that small particles (e.g., <10 m)
can be packed efficiently in long lengths of narrow
I
~2~6~
bore columns. For a discussion of developments in
HPLC as contrasted to developments in gas cremate-
graph see, for example, F. J. Yang, I'Narrow-~ore
Microparticle-Packed Column High-Performance Liquid
Chromatography", J. Chromatography, Proceedings, The
VI International Liquid Column Chromatography Con-
ferenCeCIq~
Since narrow bore micro particle packed columns
offer advantages in performance for HPLC and even for
GO, it is therefore required that satisfactory column
and packing materials and techniques be developed to
pack such columns. Older, gravitational methods of
packing columns have not proven satisfactory for narrow
bore columns. See G. H. Lathe, et at., "Separation of
Substance and Estimation of Their Relative Molecular
Sizes by the Use of Columns of Starch in Water", Boo-
chemical Journal, v. 62, p. 665 (1950) and P. Flodin,
"Methodological Aspects of Gel Filtration with Special
Reference to Desalting Operations, Journal of Chrome-
tography v. 5, No. 2, p. 103 (1961~. One technique
for packing narrow diameter columns is to pack a con-
ventional glass column and then heat and draw the
column to a narrower diameter. LO stationary phases
have then been bonded in situ. See M. Novotny, et
at., Packed Micro capillary Columns in High Performance
Liquid Chromatography", Anal. Chum., 50 271 ~1978).
The products formed by this technique are different
from conventional packed columns in that the columns
have low ratios of column diameter to particle size,
in the range of about 2-3. The disadvantage with this
approach is that due to the relatively large particle
sizes used, the performances of the packed microcapil
lazy columns is markedly inferior to columns of con-
ventional diameter of 4 to 5 mm which are packed with
m particles. Particle sizes smaller than 30 m
cannot be packed using this technique due to clogging
and the difficulty in obtaining uniform packing. As
may be appreciated, it generally becomes difficult to
pack progressively narrower columns with micro particles
since the inner diameter of the columns begins to
approach the particle size so that non-uniform packing
and clogging of the columns may occur or the product
may have high resistances to flow of solvent. And
as column lengths are increased, the problem is exacter-
bated.
Other column packing techniques used for packing to 5 mm ID LO columns are also known These include
the balanced slurry packing developed by R. E. Majors,
Anal. Chum. 44 1722l 1723 (1972) and J. J. Kirkland,
J. Chromatogr. Sat., 9 206, 207 (1971) for LO packed
columns; and conventional high pressure air compression
dry bed packing employed with GO columns. For small
diameter columns, e.g., columns having inner diameters
of 500 microns or less, it is known to incorporate the
particles in a slurry and flow the slurry through the
column. For example, see the brochure, "LO Slurry
Packing Kit", Scientific Systems, Inc., State College,
PA 16801. The slurry packing technique is typically
practiced by achieving a flow of the slurry through the
column, stopping the flow, draining off the liquid and
retaining the particles then present in the column.
Studies of this technique have evaluated the relation-
ship between packing velocity and column size, see Y.
Kayo, et at., "Packing of Toyopearl column For Gel
Filtration", J. Chromatography, v. 205, p. 185 [1981~,
v. 206, p. 135 [1981], and have shown that semi-constant
pressure packing with variable flow velocity may be
preferred for packing columns for gel filtration (see
Y. Kayo, et at., "Packing of Toyopearl Columns For Gel
~2~24~
Filtration, 111, Semi-Constant Pressure Packing", J.
Chromatography, v. 208, p. 71 (1981).
In D. Itch, et at., "Development of Technique
for Miniaturization of High-Performance Liquid
Chromatography", J. Chromatography, v. 144, p. 157,
1977, a column made of PTFE tubing of 0.5 mm I.D. and
1 0 mm OLD. was prepared by a slurry packing technique.
A tube several times longer than required for the
finished column was selected. The stationary phase
was suspended in a suitable solvent as a slurry, which
was placed in a small bottle. A 250 micro liter air-
tight syringe was connected with the tube and they
were filled with the solvent that was used to prepare
the slurry. The lower end of the tube was then dipped
into the slurry, the syringe was attached to a micro-
feeder and the slurry was sucked up to the upper end
of the tube by either manual or electrical operation
of the feeder. The lower end of the tube was then
plugged tightly with a small amount of quartz wool to
stop the packing material from leaking out. The micro-
feeder was operated manually or electrically to disk
charge the solvent. The resultant columns have a poor
packed bed stability and are easily deformed at high
flow rates and high column inlet pressures. They are
not suitable for high pressure liquid chromatography
(HPLC).
It is therefore an object of the present invent
lion to provide a process for packing narrow bore high
efficiency columns selected from the materials of
fused-silica, glass, stainless steel, or glass-lined
stainless steel.
It is another object of the present invention to
provide a process for uniformly packing a narrow-bore
chromatographic column over its length with micro-
particles having a diameter in the range of 3 m
~z~z~
. -5-
to 10 m-for liquid chromatography and 3 m to
100 m for gas chrome.~_yrphy.
It is a further object of the present invention
to provide a narrow-bore micro particle packed cremate-
graphic column of uniformly low porosity and stable packed bed.
It is another object of the present invention to
provide a column having ends for connection to the
injector and detector interface which achieves optimum
column efficiency and packed bed stability.
Brief Description of the Drawings
For a more complete understanding of the present
invention reference ma be had to the accompanying
drawings of embodiments of the present invention:
FIG. 1 is a cross-sectional view of a fixture
for packing a narrow-bore chromatographic column in
accordance with the process of the embodiment;
FIGS. aye are side cross-sectional views of
alternative end fittings for use in the end of a
narrow-bore chromatographic column when it is being
jacked in accordance with the process of the
embodiment;
FIG. 3 is a process flowchart describing the
column packing process;
FIG. pa is a reproduction of a photograph of an
entire column cross section;
FIG. 4b is a reproduction of a photograph of a
partial column cross section at the inlet end of the
column near the center of the column;
FIG. 4c it a reproduction of a photograph of a
partial column cross section at the inlet end of the
column near the wall;
Jo
LO
FIGS. Dow are reproductions of photographs o.
partial column cross sections at the outlet end of
the column near the wall;
FIG. 5 is a Van Diameter performance plot for a
3 m C18 bonded-phase particle 330 m I.D. narrow
bore micro particle packed column where the test
compound was porn.
FIG. 6 is a chromatogram showing the separation
of a mixture of polynuclear aromatic hydrocarbons.
FIG. 7 is a chromatogram showing separation
of an EPA priority pollutant PEA sample using a
320 m x 1 m, 3 m Cog reverse phase column.
Summary
A process for packing narrow bore chromatographic
columns is provided. A flexible column of inner die-
meter less than 500 m is selected. A slurry is
formed in a reservoir from a mobile solvent and par-
tides of specified diameter. For liquid cremate-
graph the particle size ranges from 3 m to 10 m;
for gas chromatography the particle size ranges from
3 m to 100 m. An end restriction is placed on the
end of the column to permit the flow of mobile solvent
and to restrict the passage of particles out the end
of the column. The reservoir is attached to the column
and the slurry is flowed under pressure into the column.
A two-step pressure sequence is used to first fill up
and form the bed of particles and then to uniformly
compress the bed. Thus, an initial pressure is main-
twined for an initial period of time, preferably less
than 10 minutes. Next, the pressure is raised from
the initial pressure to a maximuin pressure for a second
period. The product is a stable, yet loose packed
column having a high plate number per unit length.
I. '
~6;~49
Description of the Preferred Embodiments
The ultimate aim in packing columns is to obtain
reproducibly uniform distributions of the packing
materials both across and along the length of the
columns. Such uniformly packed columns will tend
to have high resolving power and be susceptible to
being used for high speed analysis. As discussed
previously, gravitational, dry packing and slurry
packing techniques have been employed. As column
diameters have become narrower, e.g., <500 m,
they become increasingly more difficult to pack in a
reproducible manner. Poor uniformity has resulted
due to wall effects when conventional slurry packing
techniques have been used. Micro particles smaller
than 5 m have been particularly difficult to pack
and non-uniform packing density as well as low
porosity has resulted.
As seen in Table I, in the development of narrow
bore micro particle packed columns for HPLC, certain0 column categories have emerged.
Table I
Column Particle Flow Rate
Designation ID (I m) Size to m) ( loin
Unpacked <50 -- 0.01
Micro capillary
Packed 50- 200 10-100 <0.1
Micro capillary
Packed 500-1000 5- 20 20- 00
Small-Bore
Packed 50- 500 3- 10 LO 0.1-20
Narrow-Bore 3- 100 GO
I
--8--
Each category has its own range of column I.D.,
particle size and flow rate. While potentially
difficult to fabricate, the micro capillary columns
require only small amounts of packing material and
are economical to operate since they only use small
amounts of solvent. In packing narrow-bore columns,
as emphasized elsewhere, it is necessary to pack
uniformly along the full length of the column; it
is also necessary to avoid packing the column too
tightly at any particular position along the column
since such tight packing could unduly restrict the
flow of mobile solvent through the column during
operation. In addition, it is also desired to obtain
uniform density of packing across the diameter of
the column so as to produce high efficiency swooper-
lion. The characteristics of the particular columns
packed in accordance with the process of the present
invention are (a) that long, narrow bore columns are
efficiently packed with particles as small as 3 m
and (b) that they have a low enough porosity to
permit efficient separation but yet (c) due to the
uniform and stable distribution of particles, the
columns may be operated with a high flow rate
(0.1-20 l/min.).
In the preferred embodiment of the process of
the present invention a column of I.D. in the range
of less than 500 m is selected from fused silica,
glass, stainless steel or glass-lined stainless steel
materials. The column is connected to a slurry
reservoir fixture of the type shown in FIG. 1.
Narrow-bore column 10 composed of fused silica cavil-
lazy tubing 11 is inserted into stainless steel tube
13. One end of tube 13 nests in union 12 and the
other end nests in union I and is held in place by
ferrule 15. Slurry reservoir 17 is also inserted
~2~;~49
into union 16; slurry reservoir 17 communicates with
a pump (not shown) through union 20. The end of
column 10 is thus inserted through tube 13 into an
abutting relationship with the bottom of slurry
reservoir 17. The lower portion of wall 19 of
reservoir 17 is shaped to fit flush with internal
wall 14 of union 16. In the preferred embodiment,
shown in FIG. 1, the interior wall of union 16 is
funnel-shaped at the center so the lower portion of
wall 19 is similarly funnel shaped. At its terminus,
wall 19 meets the upper end of tube 13. The upper
end of capillary tubing 11 therefore only communicates
with slurry 18 so that during packing the slurry
flows smoothly into the end of column 10. As shown,
the bottom of the funnel preferably has a diameter
comparable to the I.D. of column 10 so that impedance
to flow due to the door effect is avoided; during
packing the slurry flows uniformly into the end of
column 10.
During the packing process a particle restructure
is connected to the downstream end of the column.
As discussed subsequently and as Shannon the photo-
graphs of FIGS. aye, the restructure permits a
uniformly low porosity packing to be obtained. In
contrast with the Itch approach, described above,
the flow of particles in the slurry under high pros-
sure is stopped at the end of the column and the
particles are collected and uniformly packed in the
bed. During the packing process, the restructure
provides a back pressure and permits solvent to flow
out the end of the column but retains the packing
materials in the column. Typically, the packing
materials have a diameter in the range of 3 to
m for LO and 3 to logy m for GO.
I
--10--
The restructures may take several shapes. As
shown in FIG. pa a wire 28 is inserted up the end
of column 25 which has along its length an external
protective coating 26. Wire insert 28 is of a die-
meter which is slightly less than the inner diameter of column 25 thereby allowing solvent to flow out of
the column via the annular opening 29. The dimension
of the annular opening 29 is small enough so that the
column packing particles 27 will not pass through.
Once the column is fully packed in accordance with
the process of the present invention, the wire may
be withdrawn and a porous plug inserted. The require-
mint for the plug is that mobile solvent must flow
through it yet it must permanently restrict the
particles to the body of the column. A second type
of flow restructure is shown in FIG. 2b. A thick
walled fused silica column 30 having a small central
core 32 and whose outer diameter is approximately
equal to the inner diameter of column 25 is inserted
in and adhered to the end of column 25. Solvent
flows through the central core 32 of column 30 yet
the packing particles 27 are constrained from
passing out of column 31 due to the narrowness of
central opening 32. A third type of restructure is
shown in FIG, 2c. Column 25 is inserted into connect
ion tubing 33, e.g., Teflon tubing, whose inner
diameter is approximately the size of the outer
diameter of column 25. A plug insert 31 is forced
against the end of column 25. Column or tubing 34
is connected to the insert 31 for detector interface
in. The opening through plug insert 31 is suffix
ciently narrow to prevent the particles 27 from
passing through. In addition to the plug insert 31
of FIG. 2c, the insert may be a wire 39 in a column
37 as shown in FIG. Ed. Another type of restructure
~lZ2~ 9
is shown in FIG. ye. This is a variation which can be
applied to any of the configurations above 9 Here,
larger size particles US are first packed inside the
end of the column or at the interface with the end
restructure 31 (or any one of the above restructure
arrangements). The larger particles are selected to
be large enough so as to not pass through the opening
whether it is an annular opening (FIGS. pa, Ed), the
I of a column (FIG. 2b) or the central opening of
a plug insert (FIG. 2c). The smaller particles that
constitute the working portion of the column then are
flowed through the column in accordance with the pro-
cuss of the invention and fill up the length of the
column. The smaller particles are effectively stopped
by the layer of larger particles. The restructure used
in the packing process for long length columns can
also be a short packed column (e.g., 4 cm x 2 mm,
10~ m particle packed column). After packing is come
pleated the restructure packed column may be removed and
a permanent restructure end fitting put in place.
The slurry reservoir is preferably filled with a
high concentration (on the order of ~20% particles/
volume) of packing material in a solvent such as moth-
anon or acetone. The upper end of the reservoir is
connected by a conventional union 20 to a high pros-
sure pump for supplying the solvent under pressure as
a mobile phase during packing. The orientation of
reservoir 17 and column 10 may be as shown in FIG. l;
this results in downward packing. Preferably column 10
is located above reservoir 17 in an upward packing
mode so that the micro particles do not settle in the
reservoir and pack nonuniformly. To ensure uniform
packing the slurry in the reservoir is preferably
agitated, preferably by non contact means such as ultra-
sonic means (not shown). The slurry is then pressurized
I
-12-
in the reservoir to an initial packing pressure.
The initial reservoir pressure is proportional to
(a) the column length, (b) the column inner diameter,
and (c) particle size. The pressure is selected in
accordance with Table II for columns having an initial
length of 50 cm or longer.
Table II
Column column Initial
Length (cm) ID (mm)Pressure (elm)
10~ 50 0.5 50
0.3 150
0.2 300
0.1 400
200 0.5 200
200 0.3 300
200 0.2 ~00
200 0.1 500
As the initial pressure is attained the slurry begins
to flow through the column. The reservoir pressure
and flow are maintained at the initial pressure for
not more than 10 minutes. Then the pressure is
raised in stops or linear fashion from the initial
pressure, in the range of 50 to 500 atmospheres, up
to a pressure of 200 to 800 atmospheres. During the
period the initial pressure is maintained, during the
period of pressure ramping, and during the period of
operation at maximum pressure mobile phase solvent
is flowing and particles are being swept into and
packed in the column. At the beginning of the period
the initial pressure is maintained, the flow rate
approaches 1 cc/minute. As the column fills up with
packing material the flow rate gradually diminishes.
The two-step pressure sequence (initial, then maxim
mum) allows the bed to form uniformly and then to be
I
compressed more tightly until the level of compress
soon associated with the maximum pressure is asymp-
tidally approached. The two-step sequence permits
uniformity to be obtained since the bed is formed
at non-turbulent lower pressures and then full come
press ion is achieved once the particles are in place.
If the maximum pressure were used initially, then
non-uniformities along the length of the column
could result. After operation at the maximum pros-
sure for ten to thirty minutes the pump is then turned off and the pressure is reduced gradually
through the column either stops or linearly.
Since the reduction in pressure is gradual there is
no significant backwards force to dislodge the
packing material. Column 10 is then removed from
tube 13 and thereby is disengaged from slurry riser-
void 17. The packed bed is then purged with a chrome-
to graphic solvent in order to ready the column to be
useful for chromatoyraphic analysis.
For packing columns somewhat more densely shorter
column lengths and higher initial pressures may be
used. The limit on higher initial pressures is
created by the non-uniformity that would be intro-
duped if there were initial turbulent flow. The pro-
cuss of the present invention is practiced in the
same manner except that the initial starting pressure
is selected in accordance with Table III.
~%~ I
-14-
Table III
Column Column Initial
Length (cm) ID (mm) Pressure (elm)
0.5 300
0.3 400
0.2 500
0.1 600
0.5 400
0.3 500
I 0.2 700
0.1 800
Only short columns can be packed with such higher
initial pressures since columns packed at such high
initial pressures would be more dense and the flow
of mobile solvent would be impeded at the last inane-
mental lengths of the column.
Columns packed by the process of the present
invention in accordance with Table II have low flow
resistance. Flow resistance factors are obtained in
the range of 50 to 400 as defined by 0:
= U
Err do = particle size,
p = pressure gradient across the column,
= linear mobile solvent flow rate,
= viscosity of the mobile solvent, and
L = column length.
The product has a porosity >0.5 and is classified as
a louse packed column. For LO columns small particles,
ego, 3 to 10 m, are the preferred packing materials;
for GO columns particles in the range of 3 to 100 m
are preferred. The products have a substantially unit
form porosity along the length and across the diameter
of the column, as shown in JIGS. Audi. Figures 4b
~Z2~
and 4c show the scanning electron-microscope (SUM)
views of particle distributions at the inlet end of
a 1 m x 0.3 mm ID 3 m particle packed column. The
Figures show that the particle distributions at the
center and near the wall of the column cross section
have no significant differences. The uniform duster-
button of particles across the column diameter is
evident. Figures Ed and ye show the scanning elect
iron microscope views of particle distribution at
the outlet end of the column. The SUM views show
that particle size and density of distribution across
the column diameter at -the column end are the same.
And the density and uniformity of the particle disk
tribution as shown in Figures Ahab and 4c-d are
the same. This indicates the uniformity of packing
along the length of the column. This uniformity is
corroborated by the full cross sectional view of
FIG. pa. This uniformity is contrasted with columns
packed by conventional techniques in which larger
particles tend to collect along the walls as the
walls exert higher drag forces on the larger par-
tides. This uniformity is advantageous because no
extra band spreading effects occur when samples are
analyzed and thus the columns are highly efficient.
For column lengths greater than or equal to 50 cm
such loose packed columns are preferred since they
permit mobile solvent to flow at reasonable flow
rates on the order of 0.1 to 20 microliters/min
during the performance of chromatographic analysis.
Even though the porosity is high, the packing ma-
trials are found not to settle with use. This is
due to the fact that even though the columns are
operated at high pressure the total force being apt
plied to the packed bed is small, since the cross-
sectional area is small. Such columns permit a fast
-16-
analysis to be accomplished because at high pressures
high flow rates can be attained. Because long length
columns can be packed with 3 em or smaller particles
for LO, very high resolution power can be obtained.
The specification ox columns packed in accordance
with the process of the present invention including
their performance is reported in detail in F. J.
Yang, "Fused-Silica Narrow-Bore Micro particle
Packed-Column High-Per~ormance Liquid Chromatography",
J. Chromatography, v. 235, p. 265 (1982) at p. 266.
Some of the notable comparisons between prior art
columns and these columns are given in Table IV:
Table IV
Present
15 Feature Prior Art Invention
Column Plate 20,000 - 30,000 200,000
Number (4.6mm, ID, 25cm) (300 Jo m ID,
2 meters)
Peak Capacity 50 150 - 200
20 Solvent lcc/min. 2 l/min.
Utilization
Sample 1 moo 10 micro-
Size grams
Fused-silica tubing with I.D. ranging from 57 to
376 Jam and with lengths up to 2 m was packed with
3, 5 and 10 m Cog bonded-phase particles using
the packing technique of the present invention.
Reversed-phase octadecylsiloxane was chemically
bonded onto the micro particulate silica before packing
into the micro bore columns. The mobile phase was
70:30 acetonitrile-water under Socratic conditions.
~22t~
-17-
The resolving power of the column is indicated by the
Van Diameter plot of FIG. 5 for 1 m x 330 m I.D.
columns packed with spherical 3 m Cog bonded silica
particles. It shows no significant flow rate effect.
For the flow rates range between 0.3 and 1.6 mm/sec,
column efficiency maintains at its high value due to
the uniformity of the packed bed of the column. The
total column plate number exceeded 110,009 for the
flow-rate range studied. This compares with column
plate numbers of 20,000 for conventional columns.
The resolving power of the product of the pro-
cuss of the present invention is further shown by the
chromatograms of FIGS. 6 and 7. FIG. 6 shows the
separation of a mixture of polynuclear aromatic hydra-
carbons by a 320 m x 2 meter fused silica column packed by the process of the present invention with
3 m Cog bonded reverse phase silica particles.
The mobile solvent was 70% acetonitrile:H2O and the
flow rate was 1.8 l/minute. For the two meter
column, the total column plate number measured for
porn with k' = 10 was 144,000 plates. The total
single column efficiency of 144,000 plates has not
previously been reported for 3 m Cog column. It
demonstrates the effectiveness of the packing tech-
unique for packing long column with small particles In FIG. 7 an EPA priority pollutant PEA sample was
separated using a 320 m x 1 meter fused silica
column packed by the process of the present invention
with 3 m reverse phase bonded silica particles. The
mobile solvent was 70% acetonitrile:H2O and the flow
rate was 0.9 l/minute. Note that baseline resole-
lion of the PEA mixture was obtained. In particular,
note the separation of benzo(a)anthracene and cry-
sine which is normally concealed in Socratic reverse
phase system. The advantages of using long narrow-
I
-18-
bore micro particle packed column in complex sample
analysis and in solvent saving are evident.
The selection of column materials for narrow-
bore micro particle packed liquid and gas cremate-
5 graph may be made from the following materials fused silica, glass, stainless steel and glass-lined
stainless steel. The requirement is that the material
be inert and capable of being formed in the requisite
narrow diameters. Preferably, the materials, when
formed with narrow diameters, are flexible so that
long lengths can be coiled to occupy small volumes.
In addition, the materials have preferably smooth
inner surfaces and do not exhibit wall effects. It
has been found that fused silica is a preferred
material due to the extreme smoothness of its inner
walls and to its ability to dissipate heat through
the walls; with such heat dissipation there is no
significant temperature gradient across the column.
Fused silica columns also are ideal for interfacing
directly to detectors such as flame base detectors,
mass spectrographs and Fourier Transform Infrared
Detectors, since the flow rates are matched to the
flow requirements of these detectors. Columns
packed in accordance with the process of the present
invention typically will have an inner diameter less
than about 500 m. For liquid chromatography they
have a particle size of less than 10 m and a length
of more than 10 cm. For gas chromatography they
have a particle size of less than 100 Jo m. The
particles may be physically coated or have bonded to
them any types of phases useful for gas, liquid, gel
or ion-exchange chromatography.
