High efficiency packed column supercritical fluid chromatography

A method and device are disclosed for performing supercritical fluid chromatography in packed columns of at least about 0.5 mm inside diameter. The column provides at least 50,000, preferably at least 100,000, theoretical plates with pressure drop across the column of at least 25 bar. The outlet pressure is controlled by the back pressure regulator. The column may comprise a plurality of individual columns in series. Separation is performed at a rate of at least 25 plates/min, preferably at least 100 plates/min.

BACKGROUND OF THE INVENTION 
This invention relates to packed-column supercritical fluid chromatography 
(SFC) and improvements therein. 
SFC has been used in chromatographic analyses for many years. See for 
example T.H. Gouw et al., J. Chrom., Vol. 68, pages 303-323 (1972) using 
SFC in packed columns. It has also long been known to carry out SFC 
analyses in open-tubular or capillary columns as described in Novotny et a 
1., U.S. Pat. No. 4,479,380. 
SFC is a generic name for a type of chromatography using mobile phases that 
are dense gases. In SFC, the mobile phase is a fluid subjected to 
temperatures and pressures generally near its critical point. Fluids at 
those conditions have densities much closer to liquids but often exhibit 
greater solute diffusion characteristics than liquids. In SFC, the solvent 
strength changes with density and pressure drops create density and 
retention gradients. 
Gas chromatography (GC) is in general known to exhibit very high resolving 
power which enables the analysis of complex materials of volatile 
compounds. However, only generally stable, volatile compounds can 
conveniently be analyzed by GC and it is not ordinarily easy to 
dramatically change selectivity. On occasions, some samples have been 
subjected to chemical derivatization to allow the use of GC. This, 
however, is not always a complete solution. Some compounds react more 
fully than others and this variable conversion rate may make the result 
less certain. Multiple reactions might be required to get all the solutes 
in a sample to be volatile and stable enough for GC. Those solutes 
separated, however, are not the original solutes in the sample thereby 
further decreasing certainty in the results. 
Liquid chromatography (LC) is known to enable the analyses of labile and 
relatively nonvolatile compounds, but it is generally regarded to be a low 
resolution analytical method. There are relatively few selective or 
sensitive detectors that work well with the liquid mobile phases typically 
used in LC. As a consequence, selectivity adjustment is often used in 
place of efficiency to resolve components in a mixture. Often, complex 
samples are not easily resolved by LC. It may require a complex sample to 
be split into multiple parts with each part undergoing different 
pretreatments and analytical methods. This can be expensive and 
time-consuming. 
SFC is often regarded as being intermediate between GC and LC. In general, 
packed-column SFC is superior to open-tubular SFC in achieving shorter 
analysis times; however, open-tubular SFC columns may provide a higher 
number of theoretical plates at the same pressure drop. 
Many have believed that the maximum permissible pressure drop may determine 
when packed columns may be used for rapid analysis and when capillary 
columns are needed for high efficiency separations. See, for example, 
Novotny et al., U.S. Pat. No. 4,479,380. It has also been believed that 
when pressure drop across the column becomes too high it may lead to 
increased capacity factors and therefore to broader peaks. See Mourier et 
al., Chromatographia Vol. 23 No. 1 Jan. 1987 pp. 21-25. In an article by 
Schoenemakers et al., Chromatographic Vol. 24, pp. 51-57, 1987, the 
effects of column pressure drop using packed SFC columns is discussed. A 
plot in FIG. 1 of theoretical plate efficiency [N] versus pressure drop 
for three tests suggests that efficiency is at a maximum of about 25,000 
plates at about 20 bar and decreases gradually at higher pressure drops 
above about 25 bar. 
Gere, Anal. Chem. 54, 736-740 (1982), reported the use of SFC in small 
particle diameter packed columns at high pressure drop, for example, 184 
bar in FIG. 3. However, at that pressure drop, the column efficiency was 
reported to be 18,750 theoretical plates. 
SUMMARY OF THE INVENTION 
It has been found that relatively nonvolatile and labile compounds can be 
rapidly separated using packed SFC columns in accordance with this 
invention with a combination of good sensitivity and efficiency. It is 
unexpected that these achievements are obtained by this invention which 
involves the use of a packed SFC column structure with an inside diameter 
of at least about 0.5 millimeter and a length sufficient to provide for at 
least about 50,000 theoretical plates with a pressure drop in excess of 
about 25 bar controlled by a back pressure regulator at the outlet. This 
invention is more particularly pointed out in the appended claims and 
described in its preferred embodiments in the following description with 
reference to the accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION 
This invention involves the use of packed column structures for SFC which 
have an inside diameter (i.d.) greater than about 0.5 millimeters. The 
upper limit on column i.d. can vary widely with column i.d. extending to, 
for example, 1 meter for commercial scale chromatography as used in 
preparations of fine chemicals or in biotechnical engineering. The i.d. 
range may usually be from about 0.5 millimeters to 20 centimeters, 
preferably from 1 millimeter to 10 centimeters. 
The column structure may be one continuous packed SFC column but preferably 
is made up of a plurality of individual columns connected in series as 
shown in FIG. 1. The number of such columns is not particularly critical 
but often will be between about 3 to about 20. Suitable columns are those 
packed columns used in LC, for example, HP.RTM. Hypersil Column 
799/6SI-574, which has an i.d. of 4.6 millimeters, a length of 200 
millimeters, and is packed with silica packing material having a 5 micron 
average particle diameter. 
The column structure is packed with a suitable SFC packing material, e.g., 
silica, alumina, or other known, suitable materials. The packing may be 
totally porous, pellicular or beads with a porous coating of a hard 
(nonporous) interior. Known bonded phases may be applied to the particles. 
The packing particles will usually have an average particle diameter 
between about 0.5 micron to 50 microns, preferably about 1.5 microns to 10 
microns. 
The length of the column structure is based on the desired number of 
theoretical plates which, in turn, is related to packing particle 
diameter. For rough calculation, the minimum single plate height (h min.) 
is equal to two times the average packing particle diameter (h min=2 
.times. particle diameter). For example, if a particle diameter is about 5 
microns, the single plate height is about 10 microns. If a column of about 
30,000 theoretical plates is desired, the length will be about 
30,000.times.10 microns = 300,000 microns = 30 centimeters. 
The desired number of theoretical plates should be at least 50,000 with no 
true upper limit but usually not much greater than about 1 million. A 
preferred range is about 100,000 to about 500,000 theoretical plates. 
As previously pointed out this invention enables rapid separation using 
packed SFC columns. Separation rates of at least about 50 plates/second, 
preferably about 100 plates/second can be obtained. It is expected that 
rates of 500 plates/second or higher can be realized. 
Referring now to FIG. 1, a fluid supply 10 which can be a supply of 
liquified compressed gas such as carbon dioxide, is connected to a mass 
flow control pump 12 which has a modifier reservoir 14 for the purpose of 
producing binary mixtures of modifier dissolved in the fluid. The pump 12 
feeds the supercritical fluid at the desired mass-flow rate through fixed 
loop injector 16 into the inlet of individual packed SFC column 18. The 
sample to be separated is introduced by syringe 15 through the fixed loop 
injector 16. A suitable pump is a HP.RTM. Modified Model 1050 isocratic 
pump. A suitable fixed loop injector is a Rheodyne Model 7410 with a 
microliter flow loop. Since the Model 1050 pump can ordinarily handle only 
one fluid at a time, two modified pumps are used, one for the fluid and 
the second for the modifier. 
The outlet from column 18 passes into the inlet of column 20. Note that 
column 20 is set off in FIG. 1 by broken brackets with a subscript (n) 
signifying that there can be a plurality of individual columns 20 
connected in series. Ordinarily, (n) may vary between about 1 to about 18 
or more. The outlet of column 20 passes into the inlet of final individual 
column 22. The group of individual columns 18 to 22 are located within 
oven 24 which maintains the temperature necessary for near supercritical 
operation. A suitable oven is HP.RTM. Model 5890 Chromatographic oven. 
The columns used for columns 18 to 22 can be the HP.RTM. Hypersil columns 
identified hereinabove. They can be identical in size and packing or they 
can vary in either or both respects as might be desired. For example, one 
could use 5 columns packed with silica and 5 columns packed with silica 
coated with C.sub.18 bonded phase, or one can use columns with different 
internal diameters. 
The outlet from final column 22 is passed to the ultraviolet (u.v.) 
detector 26 which produces the chromatogram. A suitable u.v. detector is 
HP.RTM. Model 1050 MWD with a modified flow cell. The outlet from column 
22 can also be passed to a GC detector through a fluid restrictor 30. A 
suitable GC detector is the flame ionization detector (FID). 
The outlet from the u.v. detector 26 passes through back-pressure regulator 
valve 32 and from there to suitable means for fluid recovery known in the 
art. The back-pressure regulator 32 effectively controls the pressure 
throughout columns 18 to 22 such that the fluid passing from the outlet of 
column 22 is still at or near a desired fluid density. This insures 
solvation throughout the entire column structure of the series of 
individual columns. In accordance with this invention the pressure drop 
from the inlet of column 18 to the outlet of column 22 will be greater 
than about 25 bar, preferably between about 50 to about 400 bar. 
The mobile phase can be either a fluid at or above its critical point or 
fluids that are subcritical but which dramatically change density when 
pressure is changed and change solvent strength depending on their 
density. This is discussed more thoroughly infra. 
The mobile phase should be a single phase throughout the columns. The 
mobile phase can be pure or modified fluids including tertiary fluids 
containing additives. As examples, pure fluids include: carbon dioxide, 
nitrous oxide, sulfur hexafluoride, fluoroform (CHF.sub.3), etc. and not 
limited to these. Modified fluids include: methanol, or other alcohols, 
acetonitrile, tetrahydrofuran, hexane and others mixed with one of the 
fluids mentioned under pure fluids above. Modified fluids can contain more 
than one modifier or more than one main fluid or both more than one 
modifier and more than one fluid. Tertiary fluids may include any of the 
mixtures under modified fluids above with the addition of polar additives 
such as trifluoroacetic acid, isopropylamine or a host of others mentioned 
in the literature. The useful concentrations of modifier are zero to 
approximately 50%, although higher concentrations may sometimes be useful. 
Additives tend to be used in much lower concentrations i.e. 10.sup.-5 M to 
a few percent. 
Comparisons between the operation of unpacked capillary columns and the 
packed columns described herein are instructive in understanding this 
invention. 
The optimum speed of a carrier gas through a chromatographic column is 
determined by the binary diffusion coefficient of the solute in the mobile 
phase and the diffusion path length in the column. Comparable diffusion 
path dimensions are: the inside diameter of a capillary and two times the 
outside diameter of packing particles. Thus, a 10 micron (i.d.) capillary 
column has roughly the equivalent efficiency to a packed column packed 
with 5 micron diameter particles. The optimum linear velocity would be the 
same and the number of plates per unit of length would be the same. This 
means that the speed and resolution would be the same. However, the amount 
of sample that could be injected might be very different. In a capillary, 
the maximum amount one can inject is proportional to the cube of the 
column inner diameter. Since this tends to be a very small number the 
amount one can inject is a very small number. A 10 micron i.d. capillary 
can tolerate injections no larger than a few hundred pico (10.sup.-12) 
liters. On the other hand, the maximum injection volume in a packed column 
is not related to any dimension of a single particle. Instead, the single 
most important dimension is the inner diameter of the column which can be 
many orders of magnitude larger than the particle diameter. On a 4.6 mm 
i.d. packed column, for instance, at least 5 .mu.l (and probably 50 .mu.l) 
can be injected. This is 10.sup.4 to 10.sup.5 times more volume than can 
be injected onto a capillary with the same efficiency per unit length. 
Thus, packed columns have at least the potential to detect orders of 
magnitude less concentrated solutes (similar mass detection but in much 
larger volumes). 
Capillaries as narrow as 10 microns are seldom used because they are too 
difficult to make and use reproducibly. Instead, the most common SFC 
capillary is about 50 micron i.d. This column type has an optimum linear 
velocity 1/5th that of 5 micron packings (the diffusion path is 5 times 
longer so things must travel 1/5th as fast). Further, it takes a column 5 
times as long to get to the same total efficiency. Thus, the typical 50 
micron i.d. capillary column is 25 times slower, and up to 10.sup.5 times 
less sensitive than a packed column with the same efficiency containing 5 
micron particles. 
The present invention permits utilization of pre-existing components in an 
unexpected way. Standard columns manufactured for LC (described above) 
were simply connected in series using short lengths of capillary tubing. 
The entire stack was placed in the oven of a gas chromatograph and 
thermostated. Mixtures of methanol in carbon dioxide and pure carbon 
dioxide were used as the chromatographic mobile phases. Outlet pressure 
and flow rate were controlled. At 2 ml/min at the pumps, pressure drop 
across 11 columns (2.2 meters total length) packed with 5 micron particles 
was 100 to 165 bar depending on fluid temperature, pressure and 
composition. Temperatures from 28.degree. to 100.degree. C. were used 
although both higher and lower temperatures would be useful. Outlet 
pressures from 65 to 180 bar were tried but again a wider range would be 
useful. Flow rates were also varied from 0.5 to 4.5 ml/min (linear 
velocity from approximately 0.25 to 2 times optimum) and a wider range 
would still be useful. The fluid need not be precisely supercritical as 
pointed out hereinafter. 
The columns produced very high efficiencies. Each column used individually 
produced at least about 20,000 plates using the equation for plates: 
EQU N=6.28 (t.sub.R /W.sub.E).sup.2 
where N is theoretical plates, T.sub.R is the retention time of a solute 
W.sub.E is the area/height of that same peak. This area/height measure is 
similar to the peak width at half height but gives a more accurate 
estimate of peak distortions such as tailing. Most modern recording 
integrators use area/height instead of peak width at half height. This 
difference requires a small change in the constant (6.28 instead of 5.54 
used with width at half height). 
FIG. 2 is a chromatogram obtained using 10 HP.RTM. Hypersil LC columns 
(described hereinabove) in series. The sample was lemon oil. The mobile 
phase was 5% methanol in carbon dioxide at 60.degree. C. at 315 bar inlet 
pressure and 150 bar outlet pressure fed at 2 ml per minute. The fluid 
density in was about 0.9 and the density out was 0.8.Very sharp peaks were 
obtained as shown (NV-0006.D: MWDC, Sig=270, 4 Ref=450,80). Representative 
peaks A through G are shown in FIG. 2 and the theoretical plates were 
calculated using the formula N=6.28 (t.sub.R /W.sub.E).sup.2 as follows: 
A=79,000 plates, B=143,000 plates, C=141,000 plates, D=164,000 plates, 
E=223,700 plates, F=178,000 plates, and G=257,000. 
With pure carbon dioxide and modified carbon dioxide as the mobile phase, 
viscosity is much lower than with normal liquids. Optimum linear velocity 
is achieved with a flow at the pumps of 2 to 2.5 ml/min. Pressure drops 
averaged substantially less than 20 bar/column at these flow rates 
(compared to LC 1/4 the pressure drop at 3 times higher flow). At the same 
time efficiency was very high, averaging 100% of the theoretical. In a few 
isolated instances reduced plate heights as low as 1.43 have been 
observed. LC theories do not appear to fit packed column SFC very well. 
Surprisingly, efficiency did not degrade when multiple columns were 
connected in series. In LC, pressure drop is very high with only a few 
columns in series. As more columns are used, pressure drop tends to 
increase until it exceeds the pressure capability of the pumping 
apparatus. Since the pressure drop on one column is fairly high in LC, 
most workers tend to avoid connecting columns in series. Those who have 
tried have observed serious losses in efficiency. The sum of the 
efficiencies of each of the LC columns used separately is much higher that 
the total observed efficiency of the columns connected in series. 
Therefore, efficiency increases more slowly than expected when the column 
length is increased. However, analysis time is directly proportional to 
the column length, so it takes longer to get relatively poorer 
performance. 
The number of peaks that can be separated is proportional to the square 
root of the number of plates. This means that the number of plates must be 
increased by 4 times in order to separate 2 times more peaks. In LC such a 
doubling of the number of resolved peaks is very difficult because it 
requires that the column be made 4 times longer and the pressure drops 
tend to be excessive. The general solution has been to accept that LC is a 
low efficiency technique and concentrate on selectivity adjustment to 
resolve specific solute pairs. Complex mixtures tend to be broken down 
into multiple parts and each part analyzed separately. 
In LC, normal phase columns are usually not very efficient, they become 
less efficient when they are connected in series, and the ability to 
increase total efficiency is very limited. In the use of SFC in this 
invention, none of these limitations appear to apply. Each column is 
extremely efficient. There is no loss in efficiency when the columns are 
connected in series. Pressure drops across each column are minimal. Large 
total pressure drops do not appear to affect peak shapes adversely. The 
total number of plates can be increased by at least an order of magnitude 
allowing more than 3 times more peaks to be separated. From the 
experiments conducted, it appeared likely that columns with at least 
400,000 plates could be made. A typical normal phase LC column exhibits 
10,000 plates which can baseline separate about 17 peaks per decade of 
partition ratio (k')(i.e., 16.7 peaks between k'=1 and k'=10, and 16.7 
peaks between k'=10 and k'=100, etc.). With 400,000 plates one can 
separate 105 peaks per decade of k' (105 peaks between k'=1 and k'=10, 
etc.) 
There is not unanimity in the art with respect to definition of the term 
SFC. Some workers have insisted that all subcritical operation is LC and 
not part of SFC. This is not always correct. There are large areas of 
temperature and pressure that are subcritical but the fluid behaves 
exactly the same as supercritical fluids. SFC is the generic name for a 
type of chromatography using mobile phases that are dense gases. Under 
International Union of Pure and Applied Chemistry (IU) definitions, the 
fluids used are sometimes liquids and sometimes gases. However, these 
definitions are not very descriptive in the region of interest. The fluids 
can be subcritical or supercritical provided that the fluid (like carbon 
dioxide) acts much like a gas at high densities, using the definition of a 
gas as a fluid that expands to fill the volume available. The expansion is 
accompanied by a drop in pressure and density. A liquid is a fluid that 
does not expand to fill the available volume but has a characteristic 
volume at a specific temperature and pressure. Fluids that are near the 
supercritical region can be technically defined as liquids under IU 
definitions but behave more like a gas using the above definition. Using 
fluids in this region results in chromatography different from LC. The 
most noticeable difference involves the compressibility of the fluid and 
the non-uniformity of the solvating power of the fluid across the column. 
In LC, the entire column is operated under essentially uniform conditions. 
The solvent strength is everywhere the same. Even in gradient elution LC 
where the composition of the fluid is changed vs time the rate of change 
is generally small enough so that the solvent strength of the mobile phase 
is nearly constant across the column. In SFC, the solvent strength changes 
with density and pressure drops create density and retention gradients. 
Both subcritical and supercritical fluids can create such gradients. It is 
this kind of behavior without losses in efficiency that applies to the 
process and apparatus of this invention. 
Several advantages are realized in using this invention. The high 
efficiency of capillary GC can be achieved with the selectivity adjustment 
and sample capacity of LC. By using packed columns instead of capillaries, 
this invention permits large volume, full-loop injections to be made into 
a large i.d. packed SFC column, thereby eliminating the problems of poor 
precision and area reproducibility common with capillary columns. 
Large-volume injections allow lower concentrations to be detected by some 
detectors (e.g., mass detectors) leading to analyses with higher 
sensitivity. Finally, outlet pressure can be controlled using a back 
pressure regulator instead of a fixed restrictor as used on most capillary 
columns. This enables the use of pumps in the flow control mode leading to 
greater reproducibility of retention time over a longer period of time and 
operation at optimal linear velocity. 
While the invention has been described and illustrated with reference to 
specific embodiments, those skilled in the art will recognize that 
modifications and variations may be made without departing from the 
principles of the invention as described hereinabove and as set forth in 
the following claims and their full range of equivalence.