Note: Descriptions are shown in the official language in which they were submitted.
CA 02346358 2001-04-06
Method and Device for the Rapid Liquid Chromatographic Separation
of Substance Mixtures and for the Identification of Substances
Specification
The invention relates to a process and a device far the
rapid liquid-chromatographic separation of mixtures of sub-
stances and identification of substances according t:o the
preambles of claims 1 and 5.
For example, the pharrnaceutical research frequently encoun-
ters the problem of isolating pharmaceutically active sub-
stances from mixture~~ of substances. Thus, extracts of
natural products or mixtures of substances produced by com-
binatorial chemistry a:re being rested for potential activ-
ity. Using mixtures of: substances found to have activity,
attempts are then made to isolate the active substances by
means of complex sepax-ation procedures. Thereafter, the in-
dividual substances of the mixture isolated in this way are
subjected to an activity test once more. The active indi-
vidual substances being found are investigated for their
structure so as to exclude active substances which might be
already well-known. On.e drawback in this process is that
when testing mixtures of substances, the activity of indi-
vidual substances may be suppressed by superposition ef-
fects, leaving these substances undetected. Another draw-
back is possible simulation of activity by superposition
effects, followed by a cost-intensive and useless search
for these supposed active substances in the mixture of_ sub-
stances. Ultimately, it is disadvantageous that substances
already well-known are. excluded only after performing at
CA 02346358 2001-04-06
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least two tests for biological activity and after complex
isolation procedures, which is very costly. As a rule,
large quantities of material are required to perform these
tests, i.e., separations have to be carried out on a prepa-
rative scale. With respect to capital investment, however,
preparative units are more expensive compared to analytical
units. Also, preparative units use considerably higher
amounts of solvents and buffer substances during separa-
tion, rendering their operation costly and, in addition,
giving rise to major problems with disposal and to environ-
mental pollution.
The invention is based on the obj ect of providing a device
and a process for tree liquid-chromatographic separation,
isolation and identification of substances in an analytical
and semi-preparative :range, by means of which testing for
activity of mixtures of: substances is rendered unnecessary,
and which enable separating mixtures of substances and iso-
lating and identifying the individual substances more rap-
idly than is possible up to now.
Said object is accomplished through the characterizing
parts of claims 1 and 5.
Advantageous developments are specified in the subclaims.
The invention has various advantages. Double testing of
substances, namely, beforehand in the mixture of substances
and after isolation, is no longer necessary. According to
the invention, the complex and costly, and in part faulty
first activity test of the mixtures of substances can be
omitted. Instead, fol7_owing combined isolation and identi-
fication, only potentially new active substances are sub-
jected to further tests. One can do without costly treat-
ment of substances already well-known, as is common prac-
tice up to now. The input of time and cost for detecting a
CA 02346358 2001-04-06
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new active substance c:an be reduced substantially. In addi-
tion, this procedure is more reliable because the test re-
sults on unknown individual substances are unequivocal and
also, all of the active substances present in the mixture
will be detected.
The mixtures of substances to be investigated are processed
in a two-stage separation wherein, because the separating
columns and solid-pha:~e extraction columns (collecting col-
umns) are connected with the pump unit in a fashion accord-
ing to the invention, parallel separation of multiple frac-
tions from the first separation step is possible in the
second chromatographic: separation stage. Consequently, this
apparatus works substantially more rapid and thus, more
economical as compared. to well-known two-stage apparatus.
The individual substances are identified using per se known
direct computer-controlled comparison of chromatograms and
spectra obtained from detectors, and of the retention range
from the first separation step and the retention time from
the second separation step with information on well-known
substances in a data. base. Ultraviolet absorption, mass
spectrometry, light scattering, fluorescence, infrared
spectroscopy, and nuclear magnetic resonance spectroscopy
are possible as principles of detection and identification.
It is also possible to use additional identificatian pa-
rameters such as source and origin of the sample. Because a
smaller number of tests are required to identify the sub-
stances in the mixture and exclude substances already well-
known, the plant can be dimensioned for analytical and
semi-preparative scales. Analytical and semi-preparative
units are much more economical with respect to initial cost
and operation compared to preparative units commonly used
so far. Owing to the reduced consumption of solvent and
buffer substances, the process and device according to the
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invention are environmentally beneficial as a result of re-
duced amounts of waste.
The invention will be :illustrated in more detail with ref-
erence to the drawings and embodiments:
Fig. 1 is a schematic: representation of the operating se-
quence of equilibrating in the first separation
step and washing of the feed column battery;
Fig. 2 is a schematic; representation of the separation of
a mixture of substances in the first separation
step and the .adsorption of fractions on the first
collecting column battery;
Fig. 3 is a schematic' representation of the separation of
a mixture of substances in the first separation
step and the adsorption of fractions on the second
collecting column battery;
Fig. 4 is a schematic: representation of the separation of
a mixture of substances in the first separation
step and the ,adsorption of fractions on the third
collecting column battery;
Fig. 5 is a schematic: representation of the equilibration
of the separation column batteries of the second
separation step;
Fig. 6 is a schematic representation of a parallel separa-
tion of adsorbed fractions in the second separation
step; and
Fig. 7 is a schematic: representation of the equilibration
of a collecting column battery.
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Fig. 1 through Fig. 7 representatively illustrate the con-
figuration and flow scheme of a device according to the in-
vention, including a ~~eparation column and three downstream
separation lines.
A pump unit. 2 comprised of three pumps 2.1 through 2.3 is
connected via 6-way 2-position valves 3.1 and 3.3 and 3-way
2-position 'valve 5.7 l..a a feed column battery 6, a separa-
tion column 10 for the first separation stage, and a second
separation stage comprised of three separation lines oper-
able in parallel, each of which having an upstream 6-way 2-
position valve :3.5, 3.6 and 3.7, respectively. In this way,
it is possible to convey the mobile phase in any desired
composition to any region of the device in a consecutive as
well as a parallel fashion.
Each separation line h.as a collecting column battery 7, 8
and 9 and a separation column battery 11, 12 and 13. Repre-
sentatively, the collecting column battery 7 includes the
collecting columns 7.1. through 7.6, and the separation col-
umn battery 11 includes the separation columns 11.1 and
11.2. The other two illustrated separation lines are iden-
tical in configuration. Other variants including more feed
columns 6.1 through 6.6 in the feed column battery 6, mul-
tiple separation columns 10, more than three collecting
column batteries 7, 8 and 9, each having more than six col-
lecting columns, and more than three separation column bat-
teries il, 12 and 13 having more than six separation col-
umns per battery are also possible.
The operating sequence of the process according to the in-
vention will be described in an exemplary fashion below.
Samples of mixtures of substances are dissolved in a sol-
vent and added with an adsorbent each time. Subsequently,
the solvent is removed using a rotary evaporator so that
the adsorbent covered with sample material achieves flow-
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ability properties. Th.e adsorbents covered with the mixture
of substances are fil7_ed into the feed columns 6.1 through
6.6 of feed column battery 6 and secured in feed column
battery 6. The subsequent program sequence steps are con-
trolled by a software.
The separation column 10 is equilibrated according to Fig.
1. In a parallel operation, the air is removed from the
feed column battery 6. Via pump 2.3, 3-way 2-position valve
5.7 and 6-way 2-position valves 3.1 and 3.3, the air is re-
moved with water from one of the dry-filled feed columns
6.1 through 6.6 to be injected next. At the same time, the
separation column 10 is equilibrated with a suitable mo-
bile solvent via pump 2.1 and 6-way 2-position valves 3.1
and 3.3.
Fig. 2 illustrates the separation of the mixture of sub-
stances in the first separation stage on separation column
and the subsequent adsorption of the fractions in a
separation line inc:Luding the collecting columns 7.1
through 7.6 of collecting column battery 7.
Once the air has been removed from one of the feed columns
6.1 through 6.6, the separation program is started. Ini-
tially, the 6-way ~;-position valves 3.3 and 3.5 are
switched in position. Via a low-pressure valve unit 1 in-
cluding the low-pressure valves 1.1 through 1.3, the compo-
nents of the mobile ~ohase can be fed into the system by
means of pump unit 2. The mobile phase is conveyed via the
low-pressure valve 1..1 of pump 2.1 and via pump 2.1, and
this system can be run both in an isocratic fashion and
with a gradient. Via 6-way 2-position valve 3.3 and 7-way
6-position valves 4.1; ~4.2, the mobile phase is conveyed by
pump 2.1 to that particular feed column 6.1 through 6.6
from which sample material is to be processed. The sample
to be separated is transferred from one of the feed columns
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6.1 through 6.6 to the separation column 10. Via a 6-way 2-
position valve 3.4 and. detector 14.1, the separated sample
components discharging from separation column 10 arrive at
T-piece 17, where water is mixed into the mobile phase via
pump 2.2 and 6-way 2-position valve 3.1. The amount of ad-
mixed water depends on the polarity of the substances to be
separated. Now, as the polarity of the mobile is increased
by the water, adsorption on collecting columns 7.1 through
7.6 of collecting column battery 7 is possible. Initially,
adsorption is effected on the collecting column battery 7
via 6-way 2-position valve 3.5, the collecting columns 7.1
through 7.6 being loadE=_d with fractions one by one.
Fig. 3 illustrates the adsorption of additional fractions
on the collecting co7_umns 8.1 through 8.6 of collecting
column battery 8. Once all of the collecting columns of
collecting column battery 7 are loaded with fractions, the
6-way 2-position valves 3.5 and 3.6 switch the collecting
column battery 8 into the eluent stream. Now, the collect-
ing columns 8.1 through 8.6 are loaded with fractions one
by one.
Fig. 4 illustrates the adsorption of fractions on the col-
lecting columns 9.1 through 9.6 of collecting column bat-
tery 9. Once all of the collecting columns 8.1 through 8.6
of collecting column ibattery 8 are loaded with fractions,
the 6-way 2-position valves 3.6 and 3.7 switch the collect-
ing column battery 9 ~_nto the eluent stream. Now, the col-
lecting columns 9.1 through 9.6 are loaded with fractions
one by one. In the next. operating sequence step, the frac-
tions adsorbed on the three collecting column batteries 7,
8 and 9 are eluted in parallel and separated further on the
appropriately assigned separation column batteries 11, 12
and 13.
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The separation column batteries 11, 12 and 13 are equili-
brated prior to each separation. Fig. 5 illustrates the
equilibration of separation column batteries 11, 12 and 13.
For equilibration, the mobile phase is conveyed to separa-
tion columns 11.1 and 11.2 of separation column battery 11
via pump 2.1 and 6-waxy 2-position valves 3.1 and 3.5, re-
spectively. From there, the mobile phase is conveyed to the
waste via 6-way 2-posit: ion valve 3.4, detector 14.1, and a
fraction collector 15.1. In a parallel operation, the sepa-
ration columns 12.1 and 12.2 of the separation column bat-
tery are equilibrated via pump 2.2, the 6-way 2-position
valves 3.1 and 3.6, a detector 14.2, and fraction collector
15.2. Likewise, the separation columns 13.1 and 13.2 are
equilibrated via pump 2.3, the 6-way 2-position valve 3.7
and 3-way 2-position valve 5.7, a detector 14.3, and a
fraction collector 15.3 i.n a parallel operation.
Fig. 6 illustrates the parallel separation of fractions ad-
sorbed on collecting column batteries 7, 8 and 9, using the
separation column batteries 11, 12 and 13. To initiate the
separation step, the mobile phase is conveyed to the col-
lecting column battery 7 via pump 2.1 of pump unit 2 and
6-way 2-position valves 3.1 and 3.5. The first eluted frac-
tion from collecting column battery 7 (e. g. from collecting
column 7.1) is passed to separation column battery 11 via
6-way 2-position valve 3.5. There, one of the separation
columns 11.1 or 11.2 c:an be switched in, optionally in a
software-controlled f<~shion. Subsequently, the separated
components are passed to the detector 14.1 via 6-way 2-po-
sition valves 3.5 and 3.4. The software in the electronic
control unit assesses the signals by peak detection, di-
recting the separated components into the appropriate vials
of fraction collector 15.1. Simultaneously, time control of
the fraction collector 15.1 is also possible. Such time
control can be activated automatically in case no peak
passes the detector.
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In a parallel operation, the mobile phase is conveyed to
the collecting columr.~ battery 8 via pump 2.2 and 6-way
2-position valves 3.1 and 3.6. The first eluted fraction
from collecting column. battery 8 (e. g. from collecting col-
umn 8.1) is passed to separation column battery 12 via 6-
way 2-position valve 3.6. There, one of the separation col-
umns 12.1 or 12.2 can be switched in, optionally in a soft-
ware-controlled fashion. The separated components are
passed to the detector 14.2. In this case as well, the
software assesses the signals by peak detection, subse-
quently directing the separated components into the appro-
priate vials of fraction collector 15.2. Similarly, the
fraction collector 15.2 can be operated in a time-con-
trolled fashion. Such time control can be activated auto-
matically in case no peak passes the detector.
In parallel to the operations in two separation lines, the
third separation line is activated with respect to initiat-
ing the separation step. To this end, the mobile phase is
passed to collecting column battery 9 via pump 2.3 and
3-way 2-position valve 5.7 and 6-way 2-position valve 3.7.
The first eluted fraci=ion from collecting column battery 9
(e. g. from collecting column 9.1) is passed to separation
column battery 13 via valve 3.7. There, one of the separa-
tion columns 13.1 or 1.3.2 can be switched in, optionally in
a software-controlled fashion. The separated components are
passed to the detector 14.3. The downstream fraction col-
lector 15.3 is controlled as described above. After each
one of the first fractions has been processed in parallel,
the separation columzi batteries 11, 12 and 13 are re-
equilibrated (cf . , FicT,. 5) for preparation and in order to
separate the next fractions. Subsequently, the 7-way 6-po-
sition valves 4.3/4.4, 4.5/4.6 and 4.7/4.8 on the collect-
ing column batteries 7, 8 and 9 are switched over, so as to
enable processing of the second fractions as illustrated in
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Fig. 6. These operations are continued until all of the
fractions have been processed.
Fig. 7 illustrates the equilibration of the collecting col-
umns 7.1 through 7.6 of collecting column battery 7. In
this program sequence step, the collecting columns 7.1
through 7.6 are washed with water, thereby being prepared
for the next cycle. 'This is done in a sequential fashion
via pump 2.2, 6-way 2-position valves 3.1, 3.5, 3.6, 3.7,
and 7-way 6-position valves 4.3/4.4 of collecting column
battery 7. Equilibration of the collecting column batteries
8 and 9 is performed in an analogous manner. The 6-way
2-position valves 3.5 and 3.6 are switched in position, and
the collecting columns 8.1 through 8.6 are equilibrated via
pump 2.2, 6-way 2-position valves 3.1, 3.5, 3.6, 3.7, and
7-way 6-position valves 4.5/4.6 of collecting column bat-
tery 8. Subsequently, the 6-way 2-position valves 3.6 and
3.7 are switched in position, and the collecting columns
9.1 through 9.6 are equilibrated via pump 2.2, 6-way 2-po-
sition valves 3.1, 3.5, 3.6, 3.7, and 7-way 6-position
valves 4.7/4.8 of co~.lecting column battery 9. Following
this program sequence, the 7-way 6-position valves 4.1/4.2
of feed battery 6 are switched over to the next feed col-
umns (e. g. 6.2), and t=lze entire program cycle is restarted
(Sequence step 1: Equilibration of separation column 10 and
venting of feed column 6.2, illustrated in Fig. 1, etc.).
After processing the second sample, the next feed column
6.3 can be switched 7_I1 the eluent stream. Because sample
feed columns having undergone processing can be replaced by
new ones anytime, continuous operation with an unlimited
number of samples is possible.
During the first and second separation steps, chroma-
tograms, retention data and spectra are collected via de-
tectors 14.1, 14.2 and 14.3, processed directly in a. com-
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puter, and compared with data of known substances. Thus,
known substances can be identified and sorted out already
in on-line mode. In doubtful cases, additional data ob-
tained off-line following separation and isolation can be
used for identification.
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Reference list
1 Low-pressure
valve
unit
1.1 Low-pressure
valve
1.2 Low-pressure
valve
1.3 Low-pressure
valve
2 Pump
unit
2.1 Pump
2.2 Pump
2.3 Pump
3 6-way 2-position valve
3.1 6-way 2-positi.anvalve
3.3 6-way 2-positian valve
3.4 6-way 2-position valve
3.5 6-way 2-position valve
3.6 6-way 2-position valve
3.7 6-way 2-position valve
4 7-way 6-position valve
4.1 7-way 6-position valve
4.2 7-way 6-position valve
4.3 7-way 6-position valve
4.4 7-way 6-position valve
4.5 7-way 6-position valve
4.6 7-way 6-position valve
4.7 7-way 6-position valve
4.8 7-way 6-position valve
3-way 2-position valve
5.1 3-way 2-position valve
5.2 3-way 2-position valve
5.3 3-way 2-position valve
5.4 3-way 2-position valve
5.5 3-way 2-position valve
5.6 3-way 2-position valve
5.7 3-way 2-position valve
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6 Feed column
battery
5.1 Feed column
5.2 Feed column
5.3 Feed column
5.4 Feed column
5.5 Feed column
5.6 Feed column
7 Collecting column battery
7.1 Collecting column
7.2 Collecting column
7.3 Collecting column
7.4 Collecting column
7.5 Collecting column
7.6 Collecting column
8 Collecting column battery
8.1 Collecting column
8.2 Collecting column
8.3 Collecting column
8.4 Collecting column
8.5 Collecting column
8.6 Collecting column
9 Collecting column battery
9.1 Collecting column
9.1 Collecting column
9.2 Collecting column
9.3 Collecting column
9.4 Collecting column
9.5 Collecting column
9.6 Collecting column
Separation column
il Separation columm battery
11.1 Separation column
11.2 Separation column
12 Separation column battery
12.1 Separation column
12.2 Separation column
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13 Separation column battery
13.1 Separation column
13.2 Separation column
14 Detectors
14.1 Detector
14.2 Detector
14.3 Detector
15 Fraction colle,r.tor
15.1 Fraction collector
15.2 Fraction collector
15.3 Fraction collector
16 Waste
16.1 Waste
16.2 Waste
17 T-piece
