U.S. patent number 3,847,550 [Application Number 05/345,420] was granted by the patent office on 1974-11-12 for differential chromatographic method.
Invention is credited to W. Wilson Pitt, Jr., Charles D. Scott.
United States Patent |
3,847,550 |
Scott , et al. |
November 12, 1974 |
DIFFERENTIAL CHROMATOGRAPHIC METHOD
Abstract
A method for carrying out differential chromatography by
simultaneously introducing samples and eluent into at least two
parallel chromatographic columns, photometrically monitoring the
outflow from the columns, electronically subtracting the output of
one photometer from that of another and plotting the difference as
a function of time.
Inventors: |
Scott; Charles D. (Oak Ridge,
TN), Pitt, Jr.; W. Wilson (Oak Ridge, TN) |
Assignee: |
|
Family
ID: |
23354970 |
Appl.
No.: |
05/345,420 |
Filed: |
March 27, 1973 |
Current U.S.
Class: |
436/161;
73/61.53; 422/70; 436/64; 210/659; 422/91 |
Current CPC
Class: |
G01N
30/467 (20130101); G01N 30/861 (20130101); G01N
30/96 (20130101); G01N 2030/201 (20130101) |
Current International
Class: |
G01N
30/00 (20060101); G01N 30/46 (20060101); G01N
30/20 (20060101); G01n 033/16 (); G01n 033/18 ();
G01n 031/08 () |
Field of
Search: |
;23/23B,232C,253R,23R
;210/31C,198C ;73/61.1C |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Serwin; R. E.
Attorney, Agent or Firm: Horan; John A. Zachry; David S.
Hardaway; John B.
Claims
What is claimed is:
1. A method for carrying out differential chromatography comprising
the steps of:
flowing eluent through at least two substantially identically
packed chromatographic columns at substantially the same flow
rates;
simultaneously introducing substantially equal volumes of samples
into the eluent flow streams;
maintaining said columns at substantially the same temperature;
photometrically monitoring the outflow from each column by
photometer means;
differentially comparing the output of one photometer with that of
another photometer; and
plotting the difference in said outputs so as to produce a
differential chromatogram.
2. The method according to claim 1 wherein said columns are
identically packed by forcing a slurry of chemical separation
particles into said columns at a velocity which is greater than the
natural settling velocity of said particles.
3. The method according to claim 1 wherein said columns are packed
with chemical separation particles selected from the group
consisting of ion exchange resins and selective sorbents.
4. The method according to claim 3 wherein said particles are
within a size range of 10 to 40 microns.
5. The method according to claim 4 wherein said particles are
within a size range of 10 to 15 microns.
6. The method according to claim 1 including the further step of
plotting individual chromatograms for each of said columns.
7. The method according to claim 1 wherein said eluent increases in
concentration over time.
8. The method according to claim 1 wherein said samples are two
different urine specimens.
9. The method according to claim 1 wherein said columns and flowing
eluent are maintained at a pressure greater than atmospheric.
Description
BACKGROUND OF THE INVENTION
Ion exchange chromatography has proven to be an extremely useful
tool in many areas of technology. It is useful both as a means for
separating molecular constituents, as in the separation of the rare
earths, and as a means of chemical analysis. In the field of
chemical analysis, ion exchange chromatography has proven to be
invaluable in the analysis of physiologic fluid and in the analysis
of water pollutants.
In analyzing physiologic fluids, such as urine, abnormalities of a
particular sample can be detected by comparing the chromatogram of
the sample with that of a normal or standard sample. Also the
effects of drugs or other medication on the abnormal chemical
makeup may be visually observed by comparing before and after
chromatograms. Thus, in the field of physiologic fluid analysis,
ion exchange chromatography is valuable as a diagnostic aid as well
as an aid in determining treatment efficacy.
In water pollution control, it has become necessary to identify and
quantify refractory organic constituents that can result in
chlorinated residuals. The efficiency of a sewage treatment process
can be determined by comparing chromatogram samples taken before
and after treatment. Also the purity of the treated product may be
ascertained by comparing a chromatogram of the treated product with
that of an acceptable standard. The usefulness of ion exchange
chromatography as an analytical tool in water pollution control is
thus evident.
However, many of the fluids which can be used to produce
chromatograms are composed of a great number of constituents.
Urine, for example, has over 700 molecular constituents and over 50
molecular species have been identified in secondary sewage
effluent. On top of the number of different chromatogram peaks
which must be compared in analyzing samples, there is a problem in
that chromatograms from different ion exchange columns or even the
same column with a different eluent history will not produce peaks
at the same point in time. Therefore, conventionally produced
chromatograms are not superimposable for purposes of comparison.
Thus, the job of comparing chromatograms is tremendously tedious
and time consuming.
SUMMARY OF THE INVENTION
It is thus an object of this invention to provide a process whereby
chromatograms may be quickly and easily compared.
This object as well as other objects is accomplished by
simultaneously introducing eluent into identically packed ion
exchange columns, flowing the eluent through the columns at
substantially the same flow rate, introducing equal volumes of
comparative samples into the eluent flow streams at substantially
the same flow rate, photometrically analyzing the eluent outflow
from the columns and electronically producing a differential
chromatogram from the output of the photometers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of a twelve-port valve used in
the sample loading process of this invention.
FIG. 2 is similar to FIG. 1 but the valve is in the operational
condition.
FIG. 3 schematically illustrates the apparatus used in the practice
of this invention.
FIGS. 4-7 are chromatograms produced according to the process of
this invention.
DETAILED DESCRIPTION
According to this invention, it has been found that the combination
of several critical steps can be used to produce a differential
chromatogram. As used within this disclosure the term "differential
chromatography" or "differential chromatogram" means the practice
of, or the charts produced by the practice of electronically
comparing the differences between chromatograms of similar samples,
which differ in the quantities of molecular constituents which are
present, and charting this difference as a function of time of
outflow from ion exchange columns.
The process of this invention may be carried out using simple ion
exchange chromatography or it may be preferably carried out using
the more sophisticated techniques of high pressure and eluent
gradient ion exchange chromatography, which techniques are well
known in the art.
As a critical part of this invention two columns must be
identically packed with ion exchange resin particles having a size
of about 10 to 40 microns but preferably within the range of about
10 to 15 microns. The particles may be either spherical or
irregular as long as they are within the operable size range. The
condition of identical packing cannot be achieved by gravity
packing of the columns but must be achieved by dynamically packing
the columns. As is explained in J. Chromatog. 42 (1969) 263-265,
dynamic packing is achieved by forcing a slurry of about 25 to 50
volume percent resin particles suspended in the intended eluent
column at a velocity which is greater than the natural settling
velocity of the resin. The packing efficiency is further improved
by filling the column with a liquid, such as water, to be displaced
during the process of dynamic packing. This prevents any air
pockets from forming during the packing process.
After the columns are packed, the individual flow rates of each are
determined by flowing eluent through each column with means which
are to be described below. If the flow rates of the two columns are
not within 2 percent of each other based on the faster flowing
column, a resistance is added to the eluent delivery line of the
faster flowing column. The resistance is in the form of a porous
metal disc held in a compression fitting through which the eluent
must flow.
Another critical part of the process of this invention comprises
maintaining both of the columns at the same temperature during the
chromatography process. This is best accomplished by using a common
constant temperature bath for both columns. However, other means
for maintaining both columns at the same temperature may also be
used.
As was previously mentioned, simultaneity of process steps on the
two columns is extremely critical for carrying out the process of
this invention. If the samples and eluents are not simultaneously
introduced into the columns, the chromatogram peaks will be "out of
phase," thus producing a completely unintelligible differential
chromatogram. For this purpose it has been found that a
double-ganged six-port valve as is shown in Clin. Chem. 18 (1972)
767-770 is useful for producing simultaneous introduction of
samples. This results in a 12-port valve, which is schematically
shown in FIGS. 1 and 2. FIG. 1 shows the valve in the sample
introduction position. Samples are introduced into ports 7 and 8,
thus filling loops 13 and 14 with equal volumes of samples. Eluent
is introduced into ports 3 and 4 and flows out of ports 1 and 2
through two ion exchange columns connected to ports 1 and 2.
Central shaft 15 can be rotated 60.degree., as shown in FIG. 2,
thus placing filled loops 13 and 14 into communication with eluent
flowing through ports 3 and 4 thus simultaneously introducing both
samples into their respective columns through ports 1 and 2. It
should be noted that this can be done without interrupting the
eluent flow through ports 3 and 4. The sample introduced through
port 8 ends up being ejected through port 2 into the ion exchange
column, and in a like manner, the sample introduced through port 7
is ejected through port 1 into the other ion exchange column.
FIG. 3 shows an apparatus arrangement for carrying out the process
of this invention. Reference numeral 17 represents eluent
introduction means. Such means may include means for introduction
of an eluent whose concentration varies with time, such as is used
in eluent gradient chromatography. For reference purposes the
eluent is fed through photometer reference cell 18 prior to being
split and introduced into the 12-port valve 16 described above.
This section of the line, prior to the split, may be provided with
high pressure means and valve relief means if desired. Eluent flows
through ports 1 and 2 into parallel ion exchange columns 19. The
two identically packed ion exchange columns 19 are maintained at
the same temperature by common bath 20.
The outflow from columns 19 contains separated constituents which
flow out in their respective order. The different constituents are
detected by photometers 21 and 22. Photometers 21 and 22 are
conventional dual-beam, dual-wave-length (254 and 280 nm)
photometers used in liquid column chromatography. A more complete
discussion of the photometers is found in J. of Chromatog. 51
(1970) 175-181. The output from photometers 21 and 22 is recorded
by recording potentiometers 23 and 24. The output from photometers
21 and 22 is also fed to recording potentiometer 25 which provides
a differential chromatogram. For the sake of simplicity, three
individual recording potentiometers 23, 24 and 25 are illustrated.
However, in actuality it has been found convenient to use a single
multipoint recording potentiometer. A six point potentiometer
(Minneapolis Honeywell Brown Electronik, Model Y) with a span of 10
millivolts has been found to be useful in the process of this
invention.
The process of this invention is, of course, not limited to use
with columns containing ion exchange resin; but may be used with
any chemical separation particles such as those disclosed in
application Ser. No. 306,062(70) commonly assigned herewith. Such
particles include not only ion exchange resins but also selective
sorbents such as silica, zirconia, hydroxylapatite and alumina. The
common ion exchange resins which constitute the preferred
embodiments of this invention include those comprised of styrene
cross-linked with divinyl benzene, as well as those containing
epoxy-polyamine, phenolic and acrylic lattices. Various functional
groups are attached to the above resins for use in the ion exchange
process. Such resins can be in either the cationic or anionic
state.
Having generally described the invention, the following example is
given as a further illustration of the process of this
invention.
EXAMPLE
Two chromatographic columns, 0.33 cm I.D. .times. 150 cm, were
fabricated from nominal 1/4 inch O.D. type 316 seamless, stainless
steel tubing and the stationary phase consisted of 12- to
15-.mu.-diameter, anion exchange resin (Aminex A-27, Bio Rad
Laboratories). This resin is a polystyrene -- divinyl benzene
copolymer with quaternary ammonium active sites. The resin was
first dynamically prepacked into a cartridge and then extruded as a
packed bed into the chromatographic columns using an eluent pump.
The columns were then operated through one complete chromatographic
sequence, after which the individual eluate flow rates were
determined and adjusted so that they were within 2 percent of each
other. This was done by adding flow resistance, in the form of a
porous metal disc in the eluent delivery line, to the column system
with the higher flow rate.
An automated dual-chamber gradient generation system was used to
generate the eluent buffer concentration gradient; and a single,
positive-displacement pump (MilRoyal D pump manufactured by the
Milton Roy Co.) was used to force the eluent through the columns in
parallel. The eluent was an ammonium acetate -- acetic acid buffer
(pH 4.4). The acetate concentration was 0.015 molar for the first
two hours. The acetate concentration was then linearly increased
from 0.015 molar to 6 molar over the next 28 hours. The final 2
hours of the 32-hour run was at 6 molar. Operating pressures of up
to 3,000 psi were used.
The two columns were enclosed in stainless steel jackets containing
a heating fluid that was temperature-controlled and circulated.
The twelve-port sample injection valve of FIG. 1 was used to allow
direct introduction of two samples simultaneously into the separate
columns. The external sample loops 13 and 14 were filled at ambient
pressure; then after the internal connecting ports had been
reoriented by turning the valve handle, the two samples were
simultaneously injected into the column eluent streams without
terminating the eluent flow.
The two column monitors were dual-beam, dual-wave-length, flow
photometers previously described. One flow cell in each monitor was
used as a reference cell in which the eluent passed prior to
entering the high pressure pump and the other cells were used for
the column eluate stream.
In each test, the column eluate flow rates were adjusted to 10.5
ml/hr., and the column temperature was varied from ambient
(25.degree.C .+-. 1.degree.C) during the initial 6 hours to
60.degree.C thereafter during the remaining part of the 32 hours of
a typical run.
The variation in column eluent buffer concentration during the run
(i.e., the concentration gradient) consisted of approximately the
first 21 ml of eluent that was 0.015 M in acetate, followed by a
linear increase to 6 M for the next 294 ml and then a final 21 ml
of 6 M in acetate.
The reproducibility of the two columns was determined by separating
two identical urine samples on the two columns. In this case (FIG.
4) the graphical representations of the absorbances of the column
eluates at 254 nm are almost superimposable, with the differential
signal giving only minor indications of difference. Only the first
9 hours of a 32-hour run are shown in FIGS. 4-7. In the other three
chromatograms (FIGS. 5-7) different samples are compared.
Differences between the urine of a normal subject (column No. 1)
versus that of a subject with a cancer (column No. 2) are notable
(FIG. 5), while the effects of drug therapy can be studied by
comparing the urine of a subject before (column No. 1) and after
(column No. 2) the ingestion of the drug (FIG. 6). Similarly, the
effects of a processing step can be evaluated by comparing a
process stream before and after the processing step. As an example,
chlorination greatly affects the molecular contaminants in the
effluent from the primary stage of a sanitary sewage plant (FIG. 7)
as indicated by both positive and negative changes in the
differential signal. Column No. 1 contained primary sewage effluent
before chlorination and column No. 2 after chlorination.
It is thus seen that the process of this invention provides a
method by which chromatograms may be readily compared and
differences quickly ascertained. By merely visually scanning the
differential signal, one is able to readily ascertain any
difference which exists between samples. These differences may then
be used to diagnose diseases or to determine the efficacy of a
treatment. While the invention has been described in terms of two
columns in parallel, it is apparent that more than two columns may
be operated in parallel so that more than one sample may be
compared with a standard.
* * * * *