U.S. patent number 5,449,902 [Application Number 08/168,884] was granted by the patent office on 1995-09-12 for apparatus for directly coupling analytical column with mass spectrometer.
This patent grant is currently assigned to Hitachi, Ltd., Takeda Chemical Industries, Ltd.. Invention is credited to Yoshiaki Kato, Kouzi Onishi, Norio Tada, Yoshinobu Yoshimura.
United States Patent |
5,449,902 |
Onishi , et al. |
September 12, 1995 |
Apparatus for directly coupling analytical column with mass
spectrometer
Abstract
An apparatus for directly connecting an analytical column and a
mass spectrometer comprising a fixed member having at least four
holes which respectively introduce washing solution, eluate
containing a component eluted from the analytical column, desalting
solution and eluent for eluting the component, and a movable member
rotated with respect to an axis having at least four tubes around
the axis and mounting the four trapping columns, whereby the
trapping columns are respectively washed, trapped, desalted and
eluted in parallel. Furthermore, a common trapping column may be
used instead of the four trapping columns by controlling the
apparatus with four analytical modes.
Inventors: |
Onishi; Kouzi (Nishinomiya,
JP), Tada; Norio (Ikeda, JP), Yoshimura;
Yoshinobu (Ibaraki, JP), Kato; Yoshiaki (Mito,
JP) |
Assignee: |
Hitachi, Ltd. (Tokyo,
JP)
Takeda Chemical Industries, Ltd. (Osaka, JP)
|
Family
ID: |
26334885 |
Appl.
No.: |
08/168,884 |
Filed: |
December 16, 1993 |
Foreign Application Priority Data
|
|
|
|
|
Dec 17, 1992 [JP] |
|
|
4-336496 |
Jan 8, 1993 [JP] |
|
|
5-001626 |
|
Current U.S.
Class: |
250/288; 250/281;
250/282 |
Current CPC
Class: |
G01N
30/728 (20130101); G01N 35/085 (20130101); H01J
49/0431 (20130101); G01N 2030/027 (20130101); G01N
2030/8411 (20130101) |
Current International
Class: |
G01N
30/72 (20060101); G01N 30/00 (20060101); G01N
35/08 (20060101); H01J 49/04 (20060101); H01J
49/02 (20060101); G01N 30/02 (20060101); G01N
30/84 (20060101); H01J 049/04 () |
Field of
Search: |
;250/288A,288,282,281 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Biological and Environmental Mass Spectrometry, vol. 16, pp.
393-397 (1988). .
Biological Mass Spectrometry, vol. 21, pp. 305-314 (1992)..
|
Primary Examiner: Berman; Jack I.
Attorney, Agent or Firm: Kenyon & Kenyon
Claims
We claim:
1. An apparatus for directly connecting an analytical column and a
mass spectrometer comprising,
a fixed member having at least four holes therein, said holes
respectively introduce washing solution, eluate eluted from the
analytical column which contains a component of interest, desalting
solution, and eluent for eluting the component, and
a movable member having at least four tubes around an axis, said
tubes being changeably connected to the four holes by rotating the
movable member with respect to the axis, and mounting four trapping
columns which are respectively connected to one ends of the four
trapping columns, whereby said four trapping columns are
respectively washed, trapped, desalted and eluted in parallel.
2. An apparatus as defined in claim 1, wherein
said fixed member has further one hole connected to the mass
spectrometer and three drain holes, which are changeably connected
to the other ends of the four trapping columns in parallel by
rotating the movable member with respect to the axis.
3. An apparatus as defined in claim 1, wherein more than five
trapping columns are mounted on said moveable member, and at least
two of the columns are changeably connected to the hole for
sequentially introducing to the same column the washing solution,
the eluate, the desalting solution and the eluent.
4. An apparatus as defined in claim 1, wherein said four holes are
selectively connected to at least two of the trapping columns in
parallel.
5. An apparatus as defined in claim 1, wherein
said movable member further has a bypass tube between the tubes
connected to the trapping columns for bypassing a drain from the
fixed member.
6. An apparatus as defined in claim 1, wherein
said movable member further has bypass tubes provided between the
four respective tubes for bypassing to a drain from the fixed
member.
7. An apparatus as defined in claim 1, wherein
the flow directions of the washing solution and the eluate in the
trapping columns are respectively opposite to a flowing direction
of the eluent in the trapping columns.
8. An apparatus as defined in claim 1, wherein
said fixed member consists of first fixed member having three holes
for respectively introducing the washing solution, the eluate and
the desalting solution, and second fixed member having the hole for
introducing the eluent, and
said movable member is disposed between the first and the second
fixed members.
9. An apparatus as defined in claim 8, wherein
said first fixed member has further one hole connected to the mass
spectrometer and said second fixed member has three drain holes,
said other one hole and said three drain holes are respectively and
changeably connected to the other ends of the four trapping columns
by rotating the movable member with respect to the axis.
10. An apparatus as defined in claim 8, wherein
said movable member is installed trapping columns more than five,
and at least two of the columns are changeably connected to the
hole for introducing the same one of the washing solution, the
eluate, the desalting solution and the eluent.
11. An apparatus as defined in claim 8, wherein said four holes are
selectively connected to at least two of the trapping columns in
parallel.
12. An apparatus as defined in claim 8, wherein
said movable member further has a bypass tube between the tubes
connected to the trapping columns for bypassing to a drain from the
fixed member.
13. An apparatus as defined in claim 8, wherein said movable member
further has bypass tubes provided between the four respective tubes
for bypassing a drain from the fixed member.
14. An apparatus as defined in claim 8, wherein
the flow directions of the washing solution and the eluate in the
trapping columns are respectively opposite to a flowing direction
of the eluent in the trapping columns.
15. A controlling method for directly connecting an analytical
column and a mass spectrometer by using a trapping column and a
plurality of change-over valves, comprising the steps of
controlling the change-over valves with at least four modes of
(1) first mode for washing the trapping column and introducing
eluate from the analytical column to the mass spectrometer,
(2) second mode for trapping a component contained in eluate eluted
from the analytical column with the trapping column and washing the
mass spectrometer,
(3) third mode for eluting the component trapped in the trapping
column and introducing the component to the mass spectrometer,
and
(4) fourth mode for washing the trapping column and the mass
spectrometer, and for draining the eluate from analytical
column.
16. A controlling method as defined in claim 15, wherein
the flow directions of the washing solution in the trappping column
and of the eluate from the analytical column in the trapping column
are respectively opposite to a flowing direction of the eluent in
the trapping columns.
17. A controlling method as defined in claim 15, wherein
said first, second, third and fourth modes are respectively and
independently selected by changing over the valves.
18. An apparatus for directly connecting an analytical column and a
mass spectrometer, comprising:
a trapping column,
a plurality of change-over valves connected to the analytical
column and the trapping column, and
a controller for controlling said change-over valves, wherein
said trapping column being controlled by following modes;
(1) first mode for washing the trapping column and introducing
eluate from the analytical column to the mass spectrometer,
(2) second mode for trapping a component contained in eluate eluted
from the analytical column by the trapping column and washing the
mass spectrometer,
(3) third mode for eluting the component trapped in the trapping
column and introducing the component to the mass spectrometer,
and
(4) fourth mode for washing the trapping columns and the mass
spectrometer, and for draining the eluate from analytical
column.
19. An apparatus as defined in claim 18, wherein
the flow directions of the washing solution in the trap columns and
of the eluate from the analytical column in the trapping column are
respectively opposite to a flowing direction of the eluent in the
trapping column.
20. An apparatus as defined in claim 18, wherein
said first, second, third and fourth modes are respectively and
independently selected by changing over the valves.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for directly coupling
an analytical column used in a liquid chromatograph (herein after
called LC) or a flow injection analysis device (herein after called
FIA) with a mass spectrometer (herein after called MS), and more
particularly to an apparatus for successively trapping a component
of interest in a trapping column, washing (desalting) the trapped
component of interest, eluting the trapped component of interest
and washing the trapping column so as to transmit an eluate from
the trapping column to MS.
In the conventional LC/MS apparatus, the trapping, the
washing(desalting), the eluting and the washing processes are
performed as shown in FIG. 24.
That is, liquid sample is injected in solution of a mobile phase A
through an injection port by using a micro-syringe and is separated
according to components of the sample by analytical column 4.
Then, an eluate A eluted from the analytical column 4 is diluted
with solution of mobile phase B and is transmitted to a trapping
column TC, whereby a component of interest for analysis in the
eluate is trapped by the column TC and others in the eluate are
wasted to a drain DR.
After that, as shown in a central position of FIG. 24, only the
solution of mobile phase B such as water, flows in the column TC so
as to wash, that is, desalt the column TC and eluate D from the
column TC are wasted to the drain DR.
Then, as shown in a right-hand side of FIG. 24, solution of the
mobile phase C such as organic solvent etc. flows in the column TC,
whereby the components of interest trapped in the column TC are
successively eluted as eluate C and are transmitted to the mass
spectrometer so as to analyze mass of the components.
Meanings of the words frequently used hereinafter will be explained
as shown in a following table.
______________________________________ Words Meanings
______________________________________ solution of the eluent for
being analyzed by LC mobile phase A eluate A eluate eluted from the
analytical column 4 solution of the diluent for diluting the eluate
mobile phase B A, and washing (desalting) liquid for the trapping
column TC confluenced eluate mixed solution of the eluate A and the
mobile phases B eluate B the confluenced eluate eluted from the TC
eluate D the solution of the mobile phase B eluted from the TC
after washing (desalting) solution of the eluent for eluting the
mobile phase C components of interest trapped in the TC eluate C
eluate containing the components of interest eluted from the TC
______________________________________
Sample solutions analyzed by the LC or FIA generally contain
nonvolatile ionic substances, and solutions containing nonvolatile
salt and buffer substances are widely used as the solution of the
mobile phase A.
When such nonvolatile substances are used solely in LC or FIA, few
problems arise.
In the case of an LC/MS, the LC/MS may be used to sample gas,
liquid, ions etc., which must pass through a small aperture or a
capillary tube into a high vacuum region. In such a situation, the
nonvolatile substances may be deposited around the small aperture
or inside of the capillary tube, so that deposits clog them.
Therefore, this problem has prevented the use of mobile phases
containing nonvolatile substances in LC/MS apparatuses.
The Japanese laid-open Patents Nos. 3-175355(1991), 62-138753(1987)
and 62-19758(1987) show an apparatus which traps component of
analyte in a trapping column, the components of interest are washed
(desalted) with solution of the mobile phase B, and the components
of interest are eluted with a solution of the mobile phase C in
order to solve the above problem.
FIG. 3 shows a block diagram of a conventional system for washing,
desalting, trapping and eluting the components of interest as shown
in the above Japanese laid-open Patent.
Numeral 1 shows a solvent of the mobile phase A containing the
nonvolatile buffer which is transmitted by a pump 2, and a sample
solution is injected through a sample injection port 3 by a
micro-syringe. The sample solution is separated according to the
components thereof in a analytical column by the solvent of the
mobile phase A so as to successively elute from the analytical
column and to be detected by a detector 5.
Then, eluate A from the analytical column 4 is desalted by a
desalting system 60. Desalting process in the desalting system 60
is performed by changing the flow path using a plurality of
valves.
At first, after trapping the components of interest in a trapping
column 61, the components of interest are washed, that is, are
desalted with the solution of the mobile phase B such as water.
Then, the component of analyte is eluted by the solution of mobile
phase C which does not contain nonvolatile substance and is
transmitted to a mass spectrometer 8 or a fraction collector.
In the system shown in FIG. 3, there is a problem as that the
component being desalted may be analyzed, but other components
contained in the sample are wasted to the drain DR and are not
analyzed.
Further, FIG. 4 shows a system having a plurality of trapping
columns TC1, TC2, TC3 which are used by successively collecting
eluate from analytical column 4. This is accomplished by exchanging
trapping columns by change-over valves 62, 63 in order to analyze
multiple successive components of interest.
That is, the trapping columns TC1, TC2, TC3 are changed over when
the components of interest eluted from a analytical column 4 are
detected and thereby successively trapping the components of
interest. After every column finishes trapping the components, the
valves 62, 63 are changed over again, and the trapped components of
interest are desalted, then eluted by the solution of the mobile
phase C and transmitted to a mass spectrometer 8 or a fraction
collector.
Further, a system using only one trapping column with a plurality
of sampling loops was proposed.
In the conventional system as shown in FIG. 3, the component of
analyte trapped in the trapping column is desalted and eluted when
measuring the sample solution with LC, and therefore, it is needed
for the trapping columns to be pretreated before every measurement
of the liquid sample.
In order to analyze one objective component, it takes a time T as
follows;
T={measuring time by LC+desalting and eluting time+analyzing time
by the MS+pretreating time of TC}
Therefore, it takes so much time for analysis that it is difficult
to perform it quickly and automatically.
Further, in order to analyze one component of interest in the
conventional system as shown in FIG. 4, it takes a time T as
follows;
T={measuring time by LC+desalting and eluting time+analyzing time
by the MS+(pretreating time of TC) (number of components of
interest trapped in TC)}
Therefore, more time for analysis is needed when the number of the
components of interest is more than number of the TC. The analysis
by the LC as above is performed by using the solution of the mobile
phase A for nonvolatile substance, but the system becomes more
complicated and more expensive in the following cases;
(1) The solution of mobile phase used for the analysis contains
volatile substances.
(2) An analysis means is used which does not need separation by
analytical column.
(3) The MS needs a means for preventing an introduction of the
components of non-interest.
SUMMARY OF THE INVENTION
The present invention overcomes the above-mentioned problems of the
conventional analytical methods.
An object of the present invention is attained by providing a
device described as follows:
(a) At least one fixed member and a movable member which is rotated
with respect to an axis and is slidable to and connected to a fixed
member.
(b) The fixed member has at least four output holes therein.
(c) The movable member has four tubes around the axis and mounts at
least four trapping columns respectively connected to the tubes,
thereby the tubes are slidable and change over the holes so as to
successively connect to the tubes.
(d) The four output holes are respectively connected to the
trapping columns so as to perform four processes in parallel as
follows;
(1) washing the trapping columns,
(2) trapping a component of interest contained in an eluate eluted
from the analytical column,
(3) washing(desalting) the trapping columns, and
(4) eluting the component of interest from the i trapping
columns.
Furthermore in the present invention, the movable member may mount
more than five trapping columns thereon and at least two of the
trapping columns are changed over so as to perform the same one of
the process as the washing, the trapping of the component of
interest, the washing(desalting) and the eluting of the component
of interest.
Furthermore in the present invention, the movable member has a
bypass tube between the tubes connected to the trapping columns for
bypassing a drain from the fixed member.
Furthermore in the present invention, the flow direction of the
solution of the mobile phase A and B in the trapping columns are
opposite to the flow direction of the mobile phase C in the
trapping columns.
As stated above, the present invention is characterized by having a
movable member mounting thereon at least four trapping columns
which are processed with four processing modes as (1) washing, (2)
trapping, (3) washing (desalting), (4) eluting.
Further in the present invention, the above four processing modes
are improved so as to use the same trapping columns in common by
adding further modes processing thereto as follows;
(1) first analytical mode for washing the trapping column and
introducing eluate from the analytical column to the mass
spectrometer,
(2) second analytical mode for trapping a component of interest
contained in eluate eluted from the analytical column by the
trapping columns and washing the mass spectrometer,
(3) third analytical mode for eluting the component of interest
trapped in the trapping columns and introducing the component of
interest to the mass spectrometer, and
(4) fourth analytical mode for washing the trapping columns and the
mass spectrometer, and for draining the eluate from analytical
column.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a sectional view of an embodiment of change-over
valves in the present invention.
FIG. 2 shows a partial sectional view of a movable member and the
fixed member of FIG. 1.
FIGS. 3 and 4 show examples of a block diagram of a conventional
LC/MS system.
FIGS. 5 is a block diagram for showing a function which is a
premise of the LC/MS system in the present invention.
FIG. 6 is a partial perspective sectional view of an embodiment of
change-over valves in the present invention.
FIG. 7 shows an exploded view of the change-over valve in the
present invention.
FIGS. 8(A), 8(B), 8(C), 9(A), 9(B), 9(C), 9(D) show a change-over
system of the trapping column by the change-over valves in the
present invention.
FIG. 10 shows a operating flow view of the LC/MS system in the
present invention.
FIG. 11 shows a block diagram for showing another embodiment of the
LC/MS system in the present invention.
FIG. 12 shows an analytical processing view of the system shown in
FIG. 11 in the present invention.
FIG. 13 shows another example of a change-over system of the
trapping columns in the present invention.
FIG. 14 shows a flow chart of the system shown in FIG. 13.
FIGS. 15(a), (b), 16 and 17 show further another examples of a
change-over system of the trapping columns in the present
invention.
FIG. 18 shows a graph of an analytical process carried out by the
system in FIG. 15 in the present invention.
FIG. 19 shows a block diagram for showing a further embodiment of
the LC/MS system in the present invention.
FIG. 20 shows a graph of an analytical process carried out by the
system in FIG. 19 in the present invention.
FIG. 21 shows a block diagram for showing an embodiment of sample
concentration by a pre-column using change-over valve in the
present invention.
FIG. 22 shows a block diagram for showing an another embodiment of
a concentrate analysis by a precolumn using a change-over valve in
the present invention.
FIG. 23 shows another example of a changed over system of the
trapping columns in the present invention.
FIG. 24 shows the conventional processes carried out by LC/MS
apparatus.
FIG. 25 is a block diagram for showing another embodiment in the
present invention.
FIG. 26 is an embodiment of a LC/MS analytical system in the
present invention.
FIG. 27 is an embodiment of a first analytical mode of a LC/MS
analytical system shown in FIG. 26 in the present invention.
FIG. 28 is an embodiment of a second analytical mode of a LC/MS
analytical system shown in FIG. 26 in the present invention.
FIG. 29 is an embodiment of a third analytical mode of a LC/MS
analytical system shown in FIG. 26 in the present invention.
FIG. 30 is an embodiment of a fourth analytical mode of a LC/MS
analytical system shown in FIG. 26 in the present invention.
FIG. 31 is a schematic diagram for showing a change-over of the
analytical mode in LC/MS by using nonvolatile solution of the
mobile phase in the present invention.
FIG. 32 is a schematic diagram for showing a change-over of the
analytical mode in a case of front removing by using volatile
solution of the mobile phase in the present invention.
FIG. 33 is a schematic diagram for showing a change-over of the
analytical mode in a case of removing components of non-interest in
the present invention.
FIG. 34 is a schematic diagram for showing a change-over of the
analytical mode in a case of a sample concentration by a repetition
thereof in the present invention.
FIG. 35 is another embodiment of a first analytical mode of a LC/MS
analytical system of the present invention.
FIG. 36 is another embodiment of a second analytical mode of a
LC/MS analytical system of the present invention.
FIG. 37 is another embodiment of a third analytical mode of a LC/MS
analytical system of the present invention.
FIG. 38 is another embodiment of a fourth analytical mode of a
LC/MS analytical system of the present invention.
FIG. 39 is a further embodiment of a first analytical mode of a
LC/MS analytical system of the present invention.
FIG. 40 is a further embodiment of a second analytical mode of a
LC/MS analytical system of the present invention.
FIG. 41 is a further embodiment of a third analytical mode of a
LC/MS analytical system of the present invention.
FIG. 42 is a further embodiment of a fourth analytical mode of a
LC/MS analytical system of the present invention.
FIG. 43 is a block diagram of LC/MS of another further another
embodiment of the present invention.
FIG. 44 is a explanatory view of analytical modes for automatically
removing high concentration components.
FIG. 45 shows a liquid chromatogram of first experimental
example.
FIG. 46 shows a mass chromatogram of first experimental
example.
FIG. 47 shows a liquid chromatogram of second experimental
example.
FIG. 48 shows a mass chromatogram of second experimental
example.
FIG. 49 shows a liquid chromatogram of third experimental
example.
FIG. 50 shows a mass chromatogram of third experimental
example.
FIG. 51 shows a liquid chromatogram of fourth experimental
example.
FIG. 52 shows a mass chromatogram of fourth experimental
example.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 5 is a block diagram for showing the function of the LC/MS
system including desalting process, from the introduction of eluate
A to the recovery of eluate C in the present invention. The
analytical processes of the washing, the trapping, the
washing(desalting) and the eluting with respect to all components
of the eluate A collected from an analytical column 4 are
repeatedly performed by using the multiple trapping columns.
In the system shown in FIG. 5, the solution of the mobile phase A
transmitted from the column 4 by a pump 2 is delivered by changing
over four trapping columns TC1 to TC4 sequentially with eight
rotational changing valves RV1 to RV8.
The four processing steps of washing, trapping, washing(desalting)
and eluting are performed as follows; (First step)
All of the rotational change-over valves RV1 to RV8 are set as
shown in FIG. 5 at first. The sample solution injected in the
solution of the mobile phase A from a sample injection port 3 is
separated according to the components in the sample by an
analytical column 4. The solution 13 of the mobile phase B such as
water is transmitted by a pump 14 through a path of a resistance
column 7 and dilutes the eluate A so as to provide confluenced
eluate.
The confluenced eluate flows into a trapping column TC1 through a
rotational valve RV1 so as to trap the component of interest which
is in the confluenced eluate. Eluate B then flows into a drain DR1
through a rotational valve RV5.
Simultaneously, the trapping column TC2 is washed with the solution
13 of the mobile phase B transmitted through the change-over valve
RV2 by a pump 14, the trapping column TC3 is washed by the solution
10 of the mobile phased C transmitted through the change-over valve
7 by a pump 9, and the trapping column TC4 is washed with the
branched solution 13 of the mobile phase B.
Under a condition as stated above, all of the rotational
change-over valves are changed over so as to come into second step
when the trapping column TC1 finishes to trap the components of
interest. (Second step)
The rotational change-over valves are changed over 1 to 2 in RV1, 2
to 3 in RV2, 3 to 4 in RV3, 4 to 1 in RV4, 1 to 2 in RV5, 2 to 3 in
RV6, 3 to 4 in RV7, and 4 to 1 in RV8.
Thereby, the trapping column TC1 is washed(desalted) by the
solution 13 of the mobile phase B, and the trapping column TC3 is
washed with the solution 13 of the mobile phase B.
The trapping column TC4 (changed over from the trapping column TC1)
traps the next component of analyte in the confluenced eluate, and
all of the rotational change-over valves are again changed over
into third step when the trapping column TC4 finishes to trap the
next components of interest. (Third step)
The rotational change-over valves are changed over 2 to 3 in RV1, 3
to 4 in RV2, 4 to 1 in RV3, 1 to 2 in RV4, 2 to 3 in RV5, 3 to 4 in
RV6, 4 to 1 in RV7, and 1 to 2 in RV8.
Thereby, the trapping column TC1 is washed by the solution 10 of
the mobile phase C, then the components of interest trapped on the
first step are eluted and transmitted to a mass spectrometer 8 so
as to provide a mass spectrum.
The trapping column TC2 is washed and desalted with the solution 13
of mobile phase B and the trapping column TC3 traps the third
components of interest eluted from the analytical column 4. The
trapping column TC4 is washed(desalted) with the solution 13 of the
mobile phase B. All of the rotational change-over valves are
changed over into fourth step. (Fourth step)
The rotational change-over valves are changed over 3 to 4 in RV1, 4
to 1 in RV2, 1 to 2 in RV3, 2 to 3 in RV4, 3 to 4 in RV5, 4 to 1 in
RV6, 1 to 2 in RV7, and 2 to 3 in RV8.
Thereby, the trapping column TC4 is washed by the solution 10 of
the mobile phase C, then the components of interest trapped in the
second step are eluted and transmitted to a mass spectrometer 8 so
as to provide a mass spectrum.
The trapping column TC1 is washed with the solution 13 of mobile
phase B and the trapping column TC2 traps the fourth components of
interest eluted from the analytical column 4. The trapping column
TC3 is washed(desalted) with the solution 13 of the mobile phase
B.
All of the rotational change-over valves may be changed over
periodically by using a timer.
In the apparatus shown in FIG. 5, as the plurality of trapping
columns are processed in parallel in order to wash, trap,
wash(desalt) and elute the components of interest, following
problems in the conventional system should be improved upon;
(1) It is difficult to desalt and analyze many components of
interest.
(2) It takes a long time for analytical processing.
However the system described above needs so many valves so as to
damage its high quality separating function and also makes it
expensive.
FIG. 1 shows a sectional view of an embodiment of a change-over
valve 100 for improving the above problems in the present
invention.
The changing-over valve 100 shown in FIG. 1 eliminates many
rotational valves and can simultaneously perform at least four
process of trapping, washing(desalting), eluting and washing in
parallel. Thereby, complexed flow paths are simplified and many
rotational valves are eliminated so as to make the production cost
of the apparatus lower.
In FIG. 1, a movable member 36 fixed on a rotational axis 21 by a
screw 37 is rotated by a motor through a universal coupling 30.
Seal members 38, 34, formed from a material which has a high
chemical resistance, low friction coefficient and does not wear
easily, are fixed on an upper portion and a lower portion of the
movable member 36 with fixing pins 351, 352. The movable member 36
is disposed between fixed members 391, 392 through seal member 38,
34, and the fixed members 391, 392 are respectively fixed on
supporting plates 261 to 263 and supporting pole 271, 272 with
screws 25, 32.
The rotational axis 21 is supported on the supporting plates 261,
262 with bearings 241, 242 and is rotated by sliding on both
surfaces of the seal members 38, 34.
There are provided n number of input holes and n number of output
holes at equal distances on outer circumference of sections a-a',
d-d' of the fixed members 391, 392. In the same way, there are
provided m number of input holes and m number of output holes at
equal distances on outer circumference of sections b-b', c-c' of
the movable member 36. Where both of the numbers n and m are four,
angles between the input holes and angles between the output holes
are respectively 90 degrees.
The input holes and the output holes are formed with a same
structure and are connected to thin tubes 441 to 442 therein with
setscrews 401, 404.
FIG. 2 shows an enlarged partial view of the input and output holes
shown in FIG. 1.
Thin tube 492 is provided at inner end portion of the input hole in
the fixed member 39 and thin tube 491 is provided at inner end
portion of the output hole in the movable member 36. The thin tubes
491, 492 are provided so as to contact with respective sliding
surfaces 502, 501 of the seal member 38 and are connected with each
other through a thin tube 51 provided in the seal member 38.
Thereby, solution flows from the tube 441 to the tube 442.
Spring washer 23 and nut 22, as shown in FIG. 1, make the movable
member 36 and the fixed members 391, 392 firmly contacted through
the respective seal members 34, 38 and no leakage of the solution
arises at the sliding surfaces.
In the same way, the movable member and the fixed member are firmly
contacted at all of the input and the output holes. In FIG. 1, a
trapping column 43 is connected between the thin tubes 442 and 443
and the solution flows through a path of the thin tube 441 the
setscrew 401--the setscrew 402--thin tube 442 the trapping column
43--the thin tube 443--the set screw 403--the setscrew 404--thin
tube 444.
As the number of the trapping column are increased according to
subject and needs of the analysis, m number of the trapping columns
more than n may be mounted on the movable member 36.
EXAMPLE 1
A basic operation of the change-over valve in the present invention
will be explained using FIGS. 6 to 10.
FIG. 6 shows a partial sectional view of the change-over valve 100
having four input holes and four output holes on the movable member
36 and the fixed members 391, 392 respectively. As the input and
the output holes are respectively four, each of the trapping
columns is changed over when the movable member 36 rotates 360/4=90
degree. A hole a1 on the fixed member 391 is connected to a hole a2
on the movable member 36 and a hole a3 on the movable member 36 is
connected to a hole a4 on the fixed member 392.
FIG. 7 shows an exploded view of the above change-over valve 100 in
the present invention.
FIG. 8(A) shows the movable member 36 mounting the trapping column
in the present invention, FIG. 8(B) shows a flow path in the
change-over valve 100, and FIG. 8(C) shows a simplified figure of
FIG. 8(B).
As shown in FIG. 8(B), each of the four trapping columns are
respectively connected between paired holes, a2-a3, b2-b3, c2-c3
and d2-d3 and the solution flows in the hole a1 flows out of the
hole a4 through a path of a2-TC1-a3.
The flow path a1-a4 is arranged to trap the analytical component of
interest by the trapping column, flow path b1-b4 is arranged to
wash so as to desalt the component of analyte in the trapping
column, the flow path c1-c4 is arranged to elute the component of
analyte trapped in the trapping column by back-flushing it, and the
flow path d1-d4 is arranged to washing the trapping column by the
solution of the mobile phase B such as water before trapping the
next component of analyte.
Therefore, when the movable member 36 is rotated by every 90
degrees, the trapping columns are newly arranged allowing the four
processing steps to be simultaneously performed in parallel and
such change-over conditions are shown in FIGS. 9(A), 9(B), 9(C) and
9(D).
FIG. 10 shows a situation of the desalting in the condition shown
in FIGS. 9(A), 9(B), 9(C), 9(D).
A detector 5, provided after the analytical column 4, detects
components P1, P2 as shown in upper portion of FIG. 10 and a case
for desalting the components P1, P2 will be explained. The movable
member 36 is rotated at times of t0, t1, t2, t3, t4 so as to change
over the trapping columns and change processing mode from (A) to
(D).
For example in the case of the trapping column TC1, the processing
is performed in order of the trapping, the desalting, the eluting
and the washing, and in the case of the trapping column TC2, the
processing is performed in order of the desalting, the eluting, the
washing and then the trapping. In the same way, other processing
relating to other trapping columns is performed, and the processing
of the four trapping columns are performed by being either one step
ahead or behind the respective adjacent column.
The components of interest P1, P2 are trapped by the trapping
columns which are in the trapping processing when the components of
interest P1, P2 are detected by the detector 51. Therefore, the
component of analyte P1 is trapped by the trapping Column TC1 at
the processing mode (A), then desalted at the processing mode (B)
and is eluted by at the processing mode (C) so as to be detected as
P1 by a detector 51. In the same way, the component of analyte P2
is trapped by the trapping column TC4 at the processing mode (B),
is eluted at the processing mode (D) and is detected as p2 by a
detector 51.
The bottom of FIG. 10 shows a chromatogram detected by the detector
51 and peaks P1, P2 are more sharp than peaks P1, P2 because of an
effect of the back-flush.
Another embodiment of the present invention using the above
change-over valve 100 will be explained next.
EXAMPLE 2
FIG. 11 shows a block diagram of another embodiment of the LC/MS
system of the present invention, in which components of
non-interest in eluate A from the analytical column 4 are drained
so as to avoid contaminating the system.
The solution of the mobile phase A is injected sample solution
through a sample injection port 3 by a pump 2 and is separated by
the analytical column 4 according to components of interest and
detected by the detector 5. A flow path of the eluate A from the
analytical column is changed over by a six way change-over valve 6
and is indicated by a solid line or a dotted line as shown in FIG.
11. A port 1c is sealed.
The eluate A is transmitted to a T-shaped tube 70 through 1a, 1b of
the six way change-over valve 6 and is diluted with the solution of
the mobile phase B transmitted by a pump 12 in order to effectively
trap the analyte by increasing polarity thereof, and confluenced
eluate is provided from input hole a1 of the change-over valve
100.
The analyte in the confluenced eluate is trapped by the trapping
column TC1 and the eluate B from the trapping column TC1 is drained
from the output hole a4 into a drain DR2.
The solution of the mobile phase B is a branch-ratio thereof,
determined by a resistance column 7 so as to be branched to a flow
path b1, d1, and washes the trapping columns TC2, TC4, and wasted
into drains DR3, DR4 respectively through b4, d4. A needle valve
may be used instead of the resistance column 7.
The solution 10 of the mobile phase C is transmitted by a pump 9,
elutes the analyte trapped in the trapping column TC3, and eluate C
containing the analytes are transmitted to a mass spectrometer 8
through output hole cl so as to generate a mass spectrum.
Just before components of non-interest are eluted from the column
4, the change-over valve 6 is changed over to a state shown in the
dotted line and the eluate A from the column 4 is wasted into a
drain DR1. Thereby, the components of non-interest are not
introduced into the change-over valve 100 and mass spectrometer 8,
and the contamination in the system may be avoided.
FIG. 12 shows an analytical processing view of the system in FIG.
11 in the present invention.
At the top of FIG. 12, a chromatogram of LC from the detector 5 is
shown, a component Px is generated at time interval from t1 to t2
and eluted components Py and Pz are generated at time interval from
t3 to t4.
The change-over valve 100 is rotated so as to be changed over with
every 90 degrees in a predetermined cycle, for example one minute,
whereby trapping columns as shown by column number in FIG. 12 are
set in the flow paths of (a1-a4), (b1-b4), (c1-c4), (d1-d4).
Therefore, the trapping column TC2 is set in the flow path (a1-a4)
at the time interval from t1 to t2 when the component Px is eluted.
At the time interval from t2 to t3, the trapping column TC1 is set
in the flow path (a1-a4) and the trapping column TC2 moves to the
flow path (b1-b4) so as to be washed and desalted. At the next time
interval from t3 to t4, the components Py and Pz are trapped by the
trapping column TC4, simultaneously the trapping column TC1 moves
to the flow path (b1-b4) so as to be washed and desalted and
further the trapping column TC2 moves to the flow path (c1-c4) and
the component Px is eluted by back-flush and is fed into the MS 8
so as to detect peak value px as shown at the bottom of the FIG. 12
by the detector 8.
In the same way, the analytes represented by peak values Py, Pz are
trapped by the trapping column TC4 and finally eluted so as to
generate one peak pyz. As shown in FIG. 12, the peak shape from the
detector 8 is more sharp than those from the detector 5.
As stated above, the components eluted from analytical column are
continuously desalted, eluted, washed and trapped without failing
to analyze any component and a clear mass spectra of the components
of interest are obtained.
EXAMPLE 3
In the system shown in FIG. 11, in the case that the peak value Py,
Pz before fractionation are contained in one sampling cycle, the
number of the peaks of the components of interest from the detector
8 does sometimes not correspond to that of the components eluted
from analytical column.
In order to avoid such non-correspondence, the time between
change-overs should be shorter. But, there is needed time for
introducing into the inside of the trapping column other solvent
types in order to desalt and wash, and further it is needed a
constant time for completely removing the salt from the trapping
column. Therefore, there is a limitation of the time for
change-over the valve.
In the embodiment shown in FIG. 13, the flow paths of the
change-over valve 100 are increased and multiple trapping columns
are provided in the flow path which for the steps that need are
time for processing such as desalting, washing etc.
In FIG. 13, two trapping columns are provided in the flow paths for
desalting b1-b4 and the flow path for washing d1-d4, and these two
processing steps in the two trapping columns are simultaneously
performed in parallel. Therefore, six trapping columns are provided
in the four flow paths. That is, the trapping processing is
performed in the flow path d1-d4 through the trapping column TC1,
the desalting processing is performed in the flow path b1-b4
through the two columns TC2, TC3 in parallel, the eluting
processing is performed in the flow path c1-c4 through the trapping
column TC4, and the washing processing is performed in the flow
path d1-d4 through the two columns TC5, TC6 in parallel. The
change-over valve 100 is rotated 360/6=60 degree next by next so as
to change over the flow paths.
The linear velocity of the solution flow in each of the parallel
two columns is equal the flow in the other single columns. The
processing speed of each columns in this embodiment can be half of
that in the embodiment having four trapping columns.
Therefore, the time chart for showing analytical process of the
trapping columns TC1, TC2, TC3, TC4, TC5, TC6 is as shown in FIG.
14 and those trapping columns take one cycle for the trapping, two
cycles for the desalting, one cycle for the eluting and two cycles
for the washing. Thereby, the peak values Py, Pz as shown at the
top of FIG. 12 are respectively trapped in the different trapping
columns each other and the peak pyz are separated.
EXAMPLE 4
As shown in FIG. 11, the six way change-over valve 6 is provided
after the analytical column 4 and the detector 5, whereby the
components of non-interest are selectively removed. But the six way
change-over valve 6 is very expensive and it's preferable to avoid
contamination. The embodiment shown in FIG. 15(A), 15(B) improves
such problem.
Multiple bypass tubes are provided on the change-over valve 100 and
the components of non-interest are drained to the outside through
the bypass tubes so as to bypass the trapping column. Such bypass
tubes may be provided in the change-over valve 100.
Thin tubes 21, 22, 23, 24 for bypassing are respectively provided
between trapping columns TC1, TC2, TC3, TC4 as shown in FIG. 15(A)
and the flow paths of the thin tubes 21, 22, 23, 24 are constructed
as shown in FIG. 15(B). The trapping columns are arranged in the
flow paths a1-a4, b1-b4, c1-c4, d1-d4 in the same way as in FIGS.
8, 11.
The flow paths u1-u4, v1-v4, w1-w4, x1-x4 may be used for other
flow systems and input and output holes on the fixed member are
sealed or may not be provided from the first.
Change-over systems of the flow paths in the FIGS. 15(A), 15(B) is
shown in FIGS. 16 and 17.
In FIG. 16, the change-over valve is rotated by 90 degree
sequentially, and the change-over valve is operated in the same way
as the cases in FIGS. 9, 11.
Further in FIG. 16, the change-over valve is rotated by 45 degree
from the state shown in FIG. 16, the state of the change-over valve
becomes as a state shown in FIG. 17, and all of the four flow paths
a1-a4, b1-b4, c1-c4, d1-d4 are changed into a bypass state.
Therefore, for example, when the eluate A flows into the flow pass
a1-a4 from the analytical column 4, the nonobjective component is
drained to the outside through the drain without being trapped by
the trapping column, and after the nonobjective component is
drained to the outside, the normal flow path for the processing
system of the trapping, the desalting, the eluting, and the washing
as shown in FIG. 16 is reestablished.
FIG. 18 shows the process operation stated above. A chromatogram of
LC detected by the detector 5 is shown at the top of FIG. 18, and V
and Px are components of non-interest which should be removed and
P1, P2, P3 are components of interest. The components of interest
trapped by the trapping column are eluted by the solution of the
mobile phase A containing much nonvolatile buffer.
The bottom of FIG. 18 shows a change-over mode of the change-over
valve 100 and code "C" means a removing mode of the component of
non-interest and code "S" means a sampling mode of the component of
interest. One vertical solid lines means a rotation of 45 degrees
of the change-over valve 100 and two vertical solid lines means a
rotation of 90 degrees of the change-over valve 100.
At the same time as injecting the sample solution, the change-over
valve is changed over into the mode C and then at a time t1 after
finishing the elution and voiding said volume components the
change-over valve 100 is rotated 45 degree so as to be the sampling
mode S and the change-over valve 100 is rotated 90 degree next by
next and the sampling process is performed.
Then, at a time t5 just before a peak value Px arises, the
change-over valve 100 is rotated over 45 degrees and is changed
over to be in the mode C, and at a time t6 when the component Px is
finished eluting, the valve 100 is again rotated 45 degree so as to
come back to the sampling mode. Then, after the sampling are
repeated by changing over the valve 100 sequentially and at a time
t10 when all of the analysis are finished, the valve 100 is rotated
45 degree so as to be changed to be in the removing mode C to
remove the components of non-interest, and all of the components of
non-interest are drained to outside through the drain. Further,
conditioning of the analytical column 4 may take place at this
time.
FIG. 19 shows a block diagram of LC/MS system based on the above
explained embodiments having function as follows;
(1) Continuous analyzing of multiple components of interest in the
solution of the mobile phase containing non-volatile components
(2) Directly introducing eluate A of LC by volatile solution of the
mobile phase into the MS,
(3) Removal of the components of non-interest, and
(4) Analysis by flow injection.
Process for performing the above function will be explain as
follows;
(1) Process for desalting and removing the component of
non-interest,
The solution of the mobile phase A which consist of carrier solvent
transmitted by the pump 2 and the sample solution is injected
thereto and is separated by the analytical column 4 according to
the components therein and is detected by the detector 5. The
eluate A from the detector transmitted through ports 1a, 1b of a
six way change-over valve 6 is transmitted to a tee 70, is diluted
with the solution 13 of the mobile phase B transmitted from the
pump 12 so as to form the confluenced eluate and is then
transmitted to the flow path a1-a4 of the change-over valve 100.
The dilution ratio of the confluenced eluate is determined by the
resistance column 7 and the resistance column 7 may be substituted
with a needle valve etc.
Further the solution 13 of the mobile phase B is supplied to the
flow paths b1-b4, d1-d4 etc.
The flow path c1-c4 is used for eluting the components of interest
trapped in the trapping column and the components of interest are
eluted by backflushing the solution 10 of the mobile phase C
transmitted by the pump 9 into the trapping column. The eluate C
from the port cl is transmitted to the MS 8 through port 1c, 1d of
the six way change-over valve 6 so as to be mass-analyzed. The
desalting process is performed by changing over the valve 100 in
the same way as shown in FIG. 9. The components of non-interest are
removed by using the bypass flow path of the changeover valve 100
in the same way as shown in FIGS. 16, 17, 18.
(2) Process for directly introducing eluate A of LC into the MS by
use of volatile solution of the mobile phase.
There is no need to perform the processes for trapping, desalting,
eluting etc. in the case of the solution of the mobile phase which
does not contain non-volatile components and the eluate A from the
analytical column 4 is directly transmitted to the MS 8. Therefore,
the six way change-over valve 6 is changed over shown as dotted
lines and the eluate A is transmitted to the MS 8 through the path
of 1a, 1g, 1f, 1d. At this time, the path of the change-over valve
100 may be either in the mode for removing the component of
non-interest or in the mode S for sampling and in the both mode all
of the flow paths are washed with solvent so as to prevent plugging
and contamination of the flow paths.
(3) Process for flow injection analysis,
In the case for analyzing by optimizing the analytical condition,
flow injection analysis without the analytical column is widely
used.
In this flow injection analytical mode, all of the flow paths of
the change-over valve 100 are set to be in the bypass mode C. The
solution 10 of the mobile phase C injected the sample solution from
the sample injection port 31 is transmitted by the pump 9 from port
c4 to port cl through the thin tube of the change-over valve 100,
and further transmitted to the MS 8 through path of 1c, 1d shown as
solid lines in the six way change-over valve.
At this time, the eluate A is wasted to the drain DR1 by the pump 2
through the path of ports 1a, 1b, tee 70, path a1-a4 in the bypass
mode. Further the pump 12 is operated so as to wash the system.
As stated above, conditioning of the analytical column 4 is
performed while the MS is operated.
EXAMPLE 5
FIG. 20 shows an analytical process of the apparatus for
concentrating trace components in post-column in the present
invention.
At first, the six way change-over valve 6 is changed over shown as
solid lines so as to be changed in the mode C, the sample solution
is injected in the injection port 3 at time I1 and component of
non-interest V is removed.
Second, the change-over valve 100 is rotated 45 degrees so as to be
changed over to be in the sampling mode S at time t1 just before
the component of interest Px is eluted from analytical column, and
the component of interest Px is trapped by the trapping column TC1,
then at time t2 when the trapped eluate is finished eluting, the
change-over valve 100 is rotated back 45 degree so as to be in the
mode C for removing the components of non-interest. Then the
process from the time-interval I1 to t2 is repeated and the
concentrated components of interest are trapped in the trapping
column TC1. Subsequently the processes of desalting, eluting for
analyzing are taken place. As stated above, as desired trace
component in the mixture are concentrated after being eluted from
the analytical column, the trace component is analyzed in high
sensitivity.
EXAMPLE 6
FIG. 21 shows a block diagram for showing an embodiment of sample
concentration by using a pre-column before injection into the
analytical column 4 in the present invention.
In order to concentrate multiple trace components, the
concentration of the components is performed before being
transmitted to the analytical column 4, and after that the
analytical column separates the trace components, thereby a
sensitive analysis becomes possible.
The carrier solvent 1 transmitted by the pump 2 is divided into two
paths by tee 73 and is transmitted from one of the paths to the tee
70, and other of the paths is connected to the input hole C4 of the
change-over valve.
The solution of the mobile phase A transmitted to the tee 70 is
diluted by the solution 13 of the mobile phase B, and the component
of interest therein is trapped by the trapping column TC1. The
eluate B is wasted to the drain DR1 through hole a4 and the
resistance column BP. The resistance column BP is provided in order
to make the pressure of the column TC1 balanced to the that of the
other flow path. The sample injection process as above is repeated
so as to make the components of interest in the trapping column TC1
concentrated.
After finishing the concentration of the components of interest,
the change-over valve 100 is changed over in the same way as shown
in FIGS. 9, 10 and the components of interest trapped in the
trapping column TC1 are desalted and eluted.
The trapping column TC1 is moved into the flow path of c1-c4 so as
to make the components of interest flow into the analytical column
4 and the components of interest are separated and detected by the
detector 5.
FIG. 22 shows another block diagram for concentrating components of
interest using the pre-column. The solution 13 of the mobile phase
B injected the sample solution injected through the injection port
3 is transmitted to the flow path a1-a4. The sampling injection as
above is repeated so as to fully concentrate the component of
analyte in the trapping column TC1.
Then, the change-over valve 100 is changed over to the flow path
b1-b4 so as to wash the trapping column TC1. Further, the
change-over valve 100 is changed over to the flow path c1-c4 so as
to elute the concentrated component in the trapping column TC1 by
using the solution 1 of the mobile phase A transmitted by the pump
2 and the eluate from the trapping column TC1 is transmitted to the
analytical column 4 so as to analyze the components.
Furthermore, as the change-over valve has a plurality of the same
trapping columns, the concentrated component by the trapping column
TC3 may be analyzed by using LC while the concentration operation
is performed in the trapping column TC1 so that the efficiency of
the analysis is improved more remarkably than the method using only
one trapping column.
In the above system, the sample solution is injected in the
solution 13 of the mobile phase B, and alternatively, the sample
solution may be injected directly by using a syringe.
Further, an auto-sampler may be used and the analyzer such as a
post column desalting system shown in FIG. 19 or the MS may be
used.
EXAMPLE 7
FIG. 23 shows a block diagram for showing another embodiment of the
change-over valve in the present invention. A number of channels of
the trapping columns are mounted on the movable member 36 in FIG.
23, and for example the eight trapping columns are mounted wherein
the same kind of the trapping columns are alternately arranged.
That is, when two kind of the trapping columns such as first group
of (TC11, TC12, TC13, TC14) and second group of (TC21, TC22, TC23,
TC24) are provided, one of the first group of (TC11, TC12, TC13,
TC14) and one of the second group of (TC21, TC22, TC23, TC24) is
alternately arranged on the movable member 36.
Each of the first group of (TC11, TC12, TC13, TC14) is used for a
reversed-phase column ODS and each of the second group of (TC21,
TC22, TC23, TC24) is used for ion-exchange column, and the first
group may be used as a reverse-phase chromatography and the second
group may be used as a ion-exchanging chromatography.
By rotating the change-over valve 100 every 90 degrees, either one
of the reverse-phase chromatography and the ion-exchanging
chromatography is used and by rotating the valve 100 45 degrees
once and thereafter by rotating the valve every 90 degree, another
one is selected, and therefore any one of the reverse-phase
chromatography and the ion-exchanging chromatography may be
selected and used whenever the operator wants.
In the above embodiments, LC is connected to the input side of the
valve 100 and the MS is connected to output side of the valve 100,
but FIA may be connected to the input side and other analyzing
device may be connected to the output side.
Instead of the detector 5, an ultraviolet spectrometer or
fluorescence spectrometer may be used.
The change-over valve 100 is used as a fraction c collector and the
eluate A is substituted into solvent which is easily processed
after being desalted so as to simplify the processing
thereafter.
As explained above in the present invention, at least four trapping
columns corresponding to four processing steps are used on the
movable member, but herein after in the present invention, improved
four processing modes make it possible to use a single common
trapping column instead of the above four trapping columns.
FIG. 25 shows a schematic block diagram of an embodiment of such
invention.
In FIG. 25, numeral 9' means an ion source of the MS; 21', 22',
23', 24' respectively three way change-over valves; 25' desalting
system for non-volatile salt; 27' controller for LC; 91', 92',
respectively flow paths. Other numerals which correspond to those
in the former figures represent the same elements as in the former
figures.
The system shown in FIG. 25 uses the following three analytical
systems (1), (2), (3) by changing over the respective three way
change-over valves 21', 22', 23', 24' which are controlled by the
LC controller 27'.
(1) Analytical system by the volatile solution of the mobile
phase,
In this case, the sample solution is injected from the injection
port 3 and the three way change-over valve 21' is changed over so
as to transmit the solution to the analytical column 4 and
components of the solution are separated. At this time, the three
way change-over valves 22', 23' are changed over so as to flow the
eluate A to the flow path 91'.
Then the eluate A containing analytes is fed to the ion source 9'
of the MS through the three way changeover valves 23', 24', whereby
the eluate A containing the components of interest is directly
transmitted to the ion source 9' of the MS by bypassing the
desalting system 25' and the analytes so as to provide a mass
spectra.
(2) Analytical system of the non-volatile solution of the mobile
phase by desalting,
The solution 1 of the mobile phase A containing the non-volatile
buffer and the non-volatile salt is transmitted by the pump 2. The
sample solution is injected through the sample injection port 3,
and the change-over valve 21' is changed so as to transmit the
sample solution to the analytical column 4, thereby the analytical
components are separated by the analytical column 4.
The valve 22' is changed over so that the separated components with
the eluate A are transmitted to the desalting system 25' and
further transmitted through the desalting system 25'
The components of interest are once trapped in the trapping column
12' (not shown in the figure) in the desalting system 25', and
washed so as to be desalted. The trapped components of interest are
eluted by the solution of the mobile phase C (not shown in the
figure) which does not contain the non-volatile salt. The eluate C
containing the components of interest is transmitted to the ion
source 9' of the MS by changing over the valves 23', 24' so as to
be ionized and provide a mass spectrum.
(3) Flow injection system,
In this case, the sample solution injected from the sample
injection port 3 is transmitted to the ion source 9' of the MS from
the three way change-over valves 21' with the carrier solvent, that
is the solution 1 of the mobile phase A, through the flow path 92'
and through the three way change-over valve 24' so as to be ionized
and provide a mass spectrum.
In this way as explained above, the three analytical systems are
easily performed by changing over multiple three way change-over
valves.
EXAMPLE 8
FIG. 26 shows a practical embodiment of LC/MS analytical system in
the present invention. FIGS. 27 to 30 show a characteristic four
modes, that is, first, second, third and fourth analytical mode, in
the LC/the MS analytical system.
FIG. 31 is a schematic diagram for showing a change-over of the
analytical mode in LC/MS by using non-volatile solution of the
mobile phase in the present invention.
FIG. 32 is a schematic diagram for showing a change-over of the
analytical mode in a case of removing void volume component by
using volatile solution of the mobile phase in the present
invention.
FIG. 33 is a schematic diagram for showing a change-over of the
analytical mode in a case of removing components of non-interest in
the present invention.
FIG. 34 is a schematic diagram for showing a change-over of the
analytical mode in a case of a sample concentration by a repetition
thereof in the present invention.
In FIGS. 26 to 30, numeral 6' means solution of the mobile phase A;
7', 10' respectively pumps; 8', 18' respectively tees; 9' an ion
source of the MS; 11' solution of the mobile phase C; 12' a
trapping column; 19' branched resistance column; DR1, DR2, DR3, DR4
respectively drains; V-1, V-2 multi-way change-over valves; 1a, 1b,
1c, 1d, 1e, 1f, 1g, 1h respectively ports of the valve V-1; 2a, 2b,
2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 2k, 2l port of the valve V-2; 30'
to 45' respectively thin tubes forming flow paths of the solutions
connected between various constructive members.
As shown in FIG. 26, the solution 1 of the mobile phase A for being
analyzed stored in an eluate storage chamber is transmitted by the
pump 2. The sample solution is injected from the sample injection
port 3 by syringe etc. and transmitted to the analytical column 4
in the solution 1 of the mobile phase A. The sample solution is
eluted according to the components contained therein from the
analytical column 4. The eluted components are detected by the
detector 5 and transmitted to the port 2i through thin tube 31 and
the valve V-2.
The thin tube may be any of a stainless tubes having a diameter
from 0.1 to 1.0 mm, and preferably is a stainless tube having a
diameter of 0.5 mm.
The valve V-1 is a eight way change-over valve and the valve V-2 is
a twelve way change-over valve, of course, multi way change-over
valve having more ports than eight or twelve ports may be used
instead of them, and the valves V-1, V-2 are changed over by a
motor controlled manually, by CPU or by LC controller (not shown in
the figure). Flow paths changed over by the valves are alternately
changed between a state I shown by solid lines and a state II shown
by dotted lines.
As the system shown in FIG. 26 has two valves and each valve has
two states I and II, the system are used in four states combined
with the two states of the valves.
Then, the analytical modes (1), (2), (3), (4) of the system will be
explained.
(1) First analytical mode,
In this mode, the valve V-1 is in the state I and the valve V-2 is
in the state I as shown in FIG. 27 in which connecting states
between the every ports are shown by solid lines.
For example, the ports 1a and 1b of the valve V-1 are connected,
but the ports 1a and 1h are not connected.
The eluate A eluted from the analytical column 4 is transmitted to
the ion source 9' of the MS through a path of the detector 5 the
thin tube 31' the ports 2i, 2j, the thin tube 41', the ports 1f,
1e, the thin tube 38', the ports 2a, 2b, the thin tube 37', and is
ionized so as to finally provide a mass spectrum.
The solution 11' of the mobile phase C for eluting, which does not
contain non-volatile salt, such as water and acetonitrile mixed
with 1:9 ratio, for example, is transmitted by the pump 10' so as
to be drained to the outside through a path of the thin tube 36',
the ports 2c, 2d, the thin tube 35', the ports 1h, 1g, the thin
tube 42', and the solution 11' of the mobile phase C is used for
washing the flow paths in the first analytical mode.
The solution 6' of the mobile phase B such as water, for example,
is transmitted to the tee 8' by the pump 7' so as to be branched,
and the solution 6' in one path branched from the tee 8' is drained
to the outside through the drain DR 3 through a path of the thin
tube 39', the ports 21', 2k', the thin tube 40' and the solution 6'
in other path branched from the tee 8' is further branched by the
tee 18' after its flow rate has been limited by the branched
resistance column 19.
The solution 6' in one path branched from tee 18' transmitted to
the ports 2h, 2g through the thin tube 32', but stops at the port
2g as the port 2g is sealed. The other solution 6' in other path
branched from the tee 18' flows into the trapping column 12' with a
direction as shown by an arrow in the figure through a path of the
thin tube 33', the ports 1c, 1d, the thin tube 45'.
The eluate D from the trapping column 12' is wasted to the outside
through the drain DR1 through a path of the thin tube 43', the
ports 1a, 1b. The flow rate between the flow paths 32' and 33' is
adjusted by the branch resistance column 19' and it may be
substituted by the needle valve.
As stated above, the eluate A is continuously transmitted to the
ion source 9' of the MS so as to provide a mass spectrum. The
trapping column 12' is washed by the solution 6' of the mobile
phase B which flows in a direction as shown by an arrow in the
figure and the solution 11' of the mobile phase C for eluting
washes the flow paths.
(2) Second analytical mode,
In this mode, the valve V-1 is in the state I, and the valve V-2 is
in the state II and the components of interest are trapped by the
trapping column 12'.
In FIG. 28, the eluate A eluted from the analytical column 4 is
transmitted to the thin tube 32' through a path of the detector 5,
the thin tube 31', and ports 2i, 2h.
The solution 6' of the mobile phase B is mixed with the eluate A at
the tee 18' through the branch resistance column 19' and dilute the
eluate A so as to provided the confluenced eluate. The confluenced
eluate is transmitted from the thin tube 33' to the valve V-1 and
further transmitted to the trapping column 12' with a direction as
shown by an arrow in the figure through the ports 1c, 1d, and the
thin tube 45'.
The components of interest dissolved in the confluenced eluate are
directly trapped with the trapping column 12'. The confluenced
eluate other than trapped components is wasted to the outside
through the drain DR 1 through the thin tube 43', and the ports la,
lb. The solution C of the mobile phase C for eluting which does not
contain the non-volatile salt is transmitted to the ion source 9'
of the MS by the pump 10', through a path of the thin tube 36, the
ports 2c, 2b, the thin tube 37' and wash the ion source 9' of the
MS and the flow path thereof.
The solution 6', of the mobile phase B from the path branched from
the tee 8' is drained through a path of the thin tube 39', the
ports 21, 2a, the thin tube 38', the ports 1e, 1f, the thin tube
41', the ports 2j, 2k, and the thin tube 40'.
As stated above, the components of interest eluted from the
analytical column 4 are trapped by the trapping column 12', and the
ion source 9' of the MS is washed by the solution 11' of the mobile
phase C, and then the third analytical mode is applied. (3) Third
analytical mode,
In this mode, the valve V-1 is in the state II and the valve V-2 is
in the state I and the components of interest trapped by the
trapping column 12' in the second mode is eluted and thereafter
introduced into ion source 9' of the MS.
In FIG. 29, the eluate A from the analytical column 4 is wasted to
the outside through the drain DR 2 through a path of the ports 2i,
2j, the thin tube 41', the ports 1f, 1g, and the thin tube 42'.
The solution 11' of the mobile phase C for eluting which does not
contain non-volatile salt is transmitted by the pump 10' and flows
into the trapping column 12' with a direction as shown by an arrow
in the figure through a path of the thin tube 36', the ports 2c,
2d, the thin tube 35', the ports 1h, 1a, and the thin tube 43'. The
flow direction of the solution 11' of the mobile phase C in the
third analytical mode is reverse to those of the solution 11' in
the first and second modes.
The components of interest trapped in the trapping column 12' in
the second analytical mode are eluted in a reverse direction to
that in trapping, desalting and washing.
The eluate C, containing the eluted components of interest, is
introduced to ion source 9' of the MS through a path of the thin
tube 45', the ports 1d, 1e, the thin tube 38' the ports 2a, 2b, and
the thin tube 37'. The analyte is ionized by the ion source 9' of
the MS and a mass spectrum is provided.
The solution 6' of the mobile phase B is transmitted by the pump 7'
and is branched by the tee 8'. The solution 6' in one path branched
from the T tube 8' is wasted through the drain DR 3 through a path
of the thin tube 39', the ports 2f, 2k and the solution 6' in other
path branched from the T tube 8' is wasted through the drain DR 1
through a path of the branch resistance column 19', the tee 18',
the thin tube 33', the ports 1c, 1b, and the thin tube 44'.
Therefore as stated above, the eluate A from the analytical column
4 is wasted to the outside, and the components of interest trapped
in the trapping column 12' are eluted by the back-flush of the
solution 11' of the mobile phase C. The analyte is introduced to
the ion source 9' of the MS so as to provide a mass spectrum and
the flow paths are washed by the solution 6' of the mobile phase B.
(4) Fourth analytical mode,
In this mode, the valve V-1 is in the state II and the valve V-2 is
in the state II and the trapping column 12' and the ion source 9'
of the MS are washed and the trapping column, if having trapped the
component, is washed and desalted while the component is adsorbed
onto the trapping column.
In FIG. 30, the eluate from the analytical column 4 is transmitted
to the tee 18' through a path of the thin tube 31', the ports 2i,
2h, the thin tube 32'.
The solution 6', of the mobile phase B is transmitted by the pump
7', and is branched at the tee 8'. The branched solution 6' is
transmitted to the T shape tube 18' and is confluenced with the
eluate A so as to dilute the eluate A. The confluenced solution is
wasted to the drain DR 1 through a path of the thin tube 33', the
ports 1c, 1b, and the thin tube 44'.
Further, the solution 6' of the mobile phase B in other path
branched from the tee 8' flows into the trapping column with a
direction shown by an arrow in the figure through the thin tube
39', the ports 21, 2a, and the thin tube 38', the ports 1e, 1d, and
the thin tube 45'.
The eluate D from the trapping column 12' is wasted to the drain DR
4 through a path of the thin tube 43', the ports 1a, 1h, the thin
tube 35', the ports 2d, 2e, the thin tube 34'. The solution 11' of
the mobile phase C is transmitted by the pump 10' and wash the ion
source 9' of the MS through a path of the thin tube 36', the ports
2c, 2b, the thin tube 37'.
Therefore as stated above, the eluate A from the analytical column
4 is wasted to the outside and the trapping column 12' is washed by
the solution 6' of the mobile phase B flows in a forward direction
and the ion source 9' of the MS is washed with the solution 11' of
the mobile phase C which does not contain the nonvolatile salt.
When the components of interest are trapped in the trapping column
12', the components of interest are washed without being eluted by
selecting the appropriate polarity of the solution of the mobile
phase B.
Next, various practical analysis will be explained by using the
combinations of the above stated first, second, third and fourth
modes referring to FIGS. 31 to 4.
(1) Process for introducing into the MS after desalting,
In FIG. 31, the chromatogram shown at the top thereof is a liquid
chromatogram detected by the detector 5, and code X means a
component of analyte and codes Y, Z, V, W mean analytical
components of non-interest.
The analytical modes shown at the bottom of the figure changed by
operating the valve shows the process from desalting to introducing
to the MS.
The process from desalting the component of analyte X in order to
measure using the LC/MS to introduce the component X to the MS will
be explained hereinafter.
At first, before analyzing the solution, the trapping column 12' is
processed in the fourth analytical mode by changing the valve for
the pretreatment.
Then, the sample solution is injected and is kept in the fourth
analytical mode till a time t1 just before the component X is
eluted. While at that time, the components of non-interest V, Y
eluted from the analytical column 4 are wasted to the outside with
the eluate A and simultaneously trapping column 12' is washed with
the solution 6' of the mobile phase B.
Then, on the time t1, the valve is changed so as to be in the
second analytical mode. The component of interest X is trapped by
the trapping column 12'. At a time t2 when the component X is
finished eluting, the valve is changed so as to be in the fourth
analytical mode.
It takes few minutes to perform the fourth analytical mode,
practically from 3 to 5 minutes, and the trapping column 12' is
washed and desalted by the solution 6' of the mobile phase B. While
the desalting process is performed, the component is kept to be
trapped in the column 12'.
Then, on the time t3, the valve is changed so as to be in the third
analytical mode and the components X trapped in the column 12' is
eluted by the back-flush of the solution 11' of the mobile phase C.
The eluate C containing the component X is introduced to the ion
source 9' of the MS so as to generate a mass spectrum.
On the time t4 when the component X is finished being introduced to
the MS, the valve is changed so as to be in the fourth analytical
mode again, and the trapping column 12' is washed with the solution
6' of the mobile phase B. In this fourth analytical mode, only the
component X is trapped in the trapping column 12' and the mass
spectrum is provided after desalting and eluting it. And all of the
components of non-interest V, Y, Z, W are wasted to the
outside.
(2) Process in the LC/MS by the solution of the mobile phase which
does not contain the non-volatile salt,
In FIG. 32, the chromatogram shown at the top thereof is a liquid
chromatogram detected by the detector 5, and the four components
denoted V, X, Y, Z are successively eluted from the analytical
column 4. The components X, Y, Z are the analytes and the component
V is the component of non-interest eluted by the void volume.
The changing over of the analytical mode by the valve changing
operation shown at the bottom of FIG. 32 shows the removal of
components eluted by the void volume.
In the case of reversed-phase chromatography, ionic compounds,
salts and compounds with high polarity are generally not held in
the analytical column and are eluted by the voided volume. Such
chemical compounds may plug the ion sampling aperture and the ion
source 9' of the MS may be contaminated when these components are
introduced into the ion source 9'.
If a mass-spectrum of the components is obtained, it is the
mass-spectrum relating to the mixture thereof and does not offer
any useful information. Therefore, by removing the components
eluted by the void volume and keeping the ion source 9' free from
contamination, the ion source 9' is kept clean for a long time and
capable of providing useful information.
In FIG. 33, L represents a component of analyte, O, P, Q represent
trace components, V represents a component eluted by the void
volume and the M, V are commonly the components of
non-interest.
In the case of analyzing impurities, after the main component M is
introduced into the ion source 9', the trace components O, P, Q may
not be detected because of the carry-over of the component M. When
the contaminating components such as M above are frequently
introduced in the ion source 9', the contamination of the ion
source 9' is occurs easily so as to make frequent cleaning
necessary. In order to improve such problem, the contaminating
components such as M should be wasted to the outside without being
introduced into the ion source 9'.
In FIGS. 32, 33, the component V eluted by the voided volume and
the contaminating components of no interest are wasted to the
outside in the fourth analytical mode. At this time, the ion source
of the MS is flushed with the solvent, that is, the solution 11' of
mobile phase C so as to be washed and the ionization of the ion
source 9' is prevented from being unstable.
After eluting the components of non-interest M, V, the first
analytical mode is provided and the eluate A from the analytical
column 4 is introduced into the ion source 9' and the mass spectrum
is obtained. In the case the trapping time of the component of
non-interest is known, the first analytical mode may be provided
based on the trapping time.
Referring to FIG. 32, the removing of the component V eluted by the
void volume will be explained.
At first, the fourth analytical mode is provided, and the sample
solution is injected through the sample injection port 3. The
fourth analytical mode is provided and the eluted components of
non-interest are wasted to the outside until time t1.
Then, the first analytical mode begins at time t1 and the eluate A
is directly introduced into the ion source 9' of the MS. The
components of interest X, Y, Z are successively ionized thereby
providing the mass spectra corresponding thereto. Further, the
fourth analytical mode begins at time t2, the LC/MS finishes
measuring and preparation of the washing of the columns and the
following analysis are performed.
In FIG. 33, examples such as the components eluted by the void
volume and the contaminating components of non-interest are
shown.
The fourth analytical mode begins and the sample solution is
injected. The fourth analytical mode is held till time t1 and the
component of non-interest V is wasted.
At time t1, being changed to the first analytical mode, the
component of analyte L is measured.
At time t2 just before the main component M is eluted, the fourth
analytical mode is started and is performed until time t3 when the
elution of the main components finishes, and the main component M
is wasted to the outside.
At the time t3, the first analytical mode is again started and the
components of interest O, P, Q are measured. At the time t4, the
fourth analytical mode is started and the preparation for the next
measurement is performed.
As explained above, the first and fourth analytical modes are
selectively and repeatedly performed, and the introduction and
removal of the components to the ion source 9' separated by the LC
are easily performed.
(3) Process for FIA,
In the case of analyzing pure compounds, it is enough to get these
mass spectra. In this case, it is not necessary to separate by
using the analytical column. Sometimes, the FIA is convenient and
is used in selecting a kind of the ionization of the sample
solution such as APCI and ESI, optimizing of the ionizing condition
and selecting a positive or negative ionizing mode.
The FIA system is obtained by providing an injection port 3' at a
portion of the flow paths of the solution 11' of the mobile phase C
as shown in FIG. 30.
As shown in FIG. 30, when the fourth analytical mode is set, the
analytical column 4 is always washed with the solution 1 of the
mobile phase A and the trapping column 12' is washed with the
solution 6' of the mobile phase B and further the ion source 9' of
the MS is washed with the solution 11' of the mobile phase C. The
sample solution is repeatedly transmitted to the ion source 9' of
the MS from the injection port 3' through the thin tube 36' so as
to obtain the mass spectrum easily and in a short time.
In the second analytical mode shown in FIG. 28, the FIA may be
performed in the same way as above. But, in the second analytical
mode, eluate A from the analytical column 4 is diluted by the
solution 6' of the mobile phase B and flows into the trapping
column 12'.
In the fourth analytical mode, the analytical column and the
trapping column are operated in the different paths and there is no
relation therebetween. Therefore, the fourth analytical mode is
suitable for FIA.
(4) Process for analyzing by concentrating the components of
interest,
When measuring by the LC/MS, the low concentration of the
components of interest makes it difficult to measure them
accurately.
Referring to FIG. 34, such process for concentrating the components
will be explained.
At first in the LC/MS, the sample solution is injected in the
fourth analytical mode. At time t1 just before the component of
analyte is eluted, the second analytical mode is started, the
trapping column 12' traps the component of analyte X. At time t2
when the elution of the component of interest X is finished, the
fourth analytical mode is changed to be processed. After the
elution of all the components except the components of interest is
finished, the sample solution is again injected.
As explained above, the fourth and second analytical modes are
alternately repeated so as to repeat the injection of the sample
solution, the components of interest X is repeatedly trapped in the
trapping column 12', and is concentrated. At the time t0 when the
trapping and the concentration of the component of interest X are
finished, the component of analyte X is eluted by back-flush and
introduced to the ion source 9' of the MS.
By repeating the injection of the sample solution, the band-width
of the component of analyte X trapped in the trapping column
becomes many times broader than observed when trapping once, but,
the broadening of the bandwidth may be fully suppressed by
considering the kind and length of the trapping column 12',
polarity of the solution of the mobile phase B, and dilution ratio.
The elution by the back-flush has an effect to cancel the
broadening of the band-width.
As explained above, the valve operation as above makes it possible
to desalt and analyze the sample solution, analyze the solution of
the mobile phase which does not contain the non-volatile salt and
removing the components of non-interest, analyzing by FIA, and
concentrating the trace components.
EXAMPLE 9
Further, another embodiment of the LC/MS using three sets of six
way change-over valves in the present invention will be explained.
FIGS. 35 to 38 show a analysis system of the LC/MS in the present
invention.
In FIGS. 35 to 38, V-1, V-2, V-3 mean six way valves; 3a, 3b, 3c,
3d, 3e, 3f mean ports of the six way change-over valve V-3; 80',
81', 82', 83', 84', 85', 86', 87', 88', 89' mean thin tubes.
The system shown in FIG. 35 corresponds to the first analytical
mode in the above example 8.
The solution 1 of the mobile phase A is transmitted by the pump 2.
The sample solution injected from the sample injecting port 3 is
separated by the analytical column 4 according to the components
thereof and is detected by detector 5.
Eluate A containing the analytes passing through the detector 5 is
introduced into the ion source 9' of the MS through the thin tube
80' through a path of the ports 1a, 1f of the valve V-1, the thin
tube 84', ports 1e, 1d, the thin tube 83' the ports 2d, 2c of the
valve V-2, the thin tube 87'. At this time, the trapping column 12'
is washed by the solution 6 of the mobile phase B transmitted by
the pump 7 through a path of the tee 8', the thin tube 85', the
ports 2a, 2b, the thin tube 86' with a direction shown by an arrow
in the figure. The port 3c in the valve V-3 is sealed.
In the first analytical mode, the components of interest eluted
from the analytical column 4 are directly introduced into the ion
source 9' of the MS so as to analyze them.
The system shown in FIG. 36 corresponds to the second analytical
mode in the above example 8.
The eluate A containing the components of interest eluted from the
analytical column is transmitted to the thin tube 85' through the
ports 1a, 1b of the valve V-1, the thin tube 81', ports 3e, 3d of
the valve V-3. Further, eluate A is confluenced with the solution
6' of the mobile phase B at the tee 8' on the way to the thin tube
85' and diluted by the solution 6' of the mobile phase B. The
confluenced solution flows into the trapping column 12' through a
path of ports 2a, 2b of the valve V-2, the thin tube 86' with a
direction shown by an arrow in the figure.
The eluate B containing the components which is not trapped in the
trapping column TC is wasted to the outside through the drain DR 1
through a path of the thin tube 88', ports 2e, 2f, the thin tube
89'. All the while, the ion source 9' of the MS is washed with the
solution 11' of the mobile phase C transmitted by the pump 10'
through a path of the thin tube 82', ports 1c, 1d, the thin tube
83', ports 2d, 2c, and the thin tube 87'.
In the second analytical mode, the components of interest eluted
from the analytical column 4 are trapped by the trapping column
12'.
The system shown in FIG. 37 corresponds to the third analytical
mode in the above example 8.
The solution 11' of the mobile phase C is transmitted by the pump
10' to the valve V-1. Then, the solution 11' is transmitted to the
valve V-2 through the ports 1c, 1d, the thin tube 83' and further
to the trapping column 12' through a path of the ports 2d, 2e, and
the thin tube 88 with a direction as shown by an arrow in the
figure.
The analytical components trapped by the solution 11' of the mobile
phase C is eluted by the back-flush. The eluate C containing the
analytical components is transmitted to the valve V-2 through the
thin tube 86' and then transmitted to the ion source 9' of the MS
through a path of the ports 2b, 2c, the thin tube 87'. All the
while, the eluate A from the analytical column 4 is wasted to the
outside through a path of the detector 5, the thin tube 80', ports
1a, 1b, the thin tube 81', ports 3e, 3f.
In the third analytical mode, the analytical component trapped in
the trapping column 12' is eluted by the back-flush and is
introduced to the ion source of the MS so as to provide the mass
spectrum.
The system shown in FIG. 38 corresponds to the fourth analytical
mode in the above example 8.
The solution 6' of the mobile phase B is transmitted to the valve
V-2 by the pump 7' from the tee through the thin tube 85. The port
3c of the valve V-3 is sealed. Further, the solution 6' of the
mobile phase B washes the trapping column 12' by flowing through a
path of ports 2a, 2b, and the thin tube 86' with a direction shown
by an arrow in the figure. The eluate D from the trapping column
12' is drained through a path of the thin tube 88', ports 2e, 2f,
the thin tube 89'.
The solution 11 of the mobile phase C is transmitted to the ion
source 9' of the MS by the pump 10' through a path of the thin tube
82', ports 1c, 1d, the thin tube 83', ports 2d, 2d, and the thin
tube 87'. Thereby the ion source 9' of the MS is washed.
In this analytical mode, the trapping column 12' may be washed and
the desalting of the analytical component trapped in the column may
be accomplished too.
In the example 9, the second, third and the fourth modes are
changed-over in the same way as the example 8, thereby it becomes
possible to trap and desalt the components of interest from the
analyzing system containing the non-volatile salts, and introduce
only the components of interest into the MS directly.
Further, the FIA is performed in the same way by providing a sample
injection port 3' on the thin tube 82 of the flow path for the
solution 11' of the mobile phase C and by injecting the sample
solution in the fourth analytical mode.
EXAMPLE 10
Further, another embodiment of the LC/MS using two sets of sixteen
way change-over valves in the present invention will be
explained.
FIGS. 39 to 42 show explanatory views of a system of the LC/MS in
the present invention.
In FIGS. 39 to 42, codes V-1, V-2 mean sixteen way change-over
valves; 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o,
1q ports of the sixteen way change-over valve V-1; 2a, 2b, 2c, 2d,
2e, 2f, 2g, 2h, 2i, 2j, 2k, 2l, 2m, 2n, 2o, 2q ports of the sixteen
way change-over valve V-2; 50' to 73' the thin tubes.
The four modes of the first, second, third and fourth modes may be
applied in the same way as in the examples 8 and 9.
In this example 10, the sample injection port 3 becomes independent
from the analytical column 4 at the fourth analytical mode, and the
FIA may be possible by using the sample injection port 3. That is,
the difference between this example and the examples 8 and 9 is
that the sample injection port 3 for separating the components in
the LC and the sample injection for FIA are the same one.
Such construction is useful in the case of a large system such as
an auto-sampler which is a large sample injection system because
there is no need to change distributing tubes for the solution.
FIG. 39 corresponds to first analytical mode.
The solution 1 of the mobile phase A is transmitted to the sample
injection port 3 by the pump 2 through a path of the thin tube 63',
ports 1q, 1p of the valve V1, and the thin tube 51'. The sample
solution is injected from the sample injection port 3 and flows
into the analytical column 4 through a path of the thin tube 64',
ports 1b, 1a, and the thin tube 50'.
The sample solution is eluted from the analytical column 4
according to the components thereof and after the components in the
eluate A from the column 4 are detected by the detector 5, the
components again flow into the valve V-1 through a path of the
ports 2n, 2m of the valve V-2, and the thin tube 56'.
Further, the components again flow into the thin tube 57' of the
valve V-2 through a path of ports 1j, 1i, and are introduced into
the ion source 9' of the MS through a path of ports 2a, 2b, and the
thin tube 73'.
The solution 6' of the mobile phase B is transmitted by the pump 7'
and is divided at the tee 8' to a branched solution flow through a
path of the thin tube 65', ports 2q, 2p, the thin tube 66' and a
branched solution flows through the branched resistance column 19',
and the two branched solution are confluenced again at the tee 18'.
The confluenced solution 6' of the mobile phase B washes the
trapping column 12' flowing through a path of the thin tube 59',
ports 1g, 1h, and the thin tube 58' with a direction shown by an
arrow in the figure.
After washing, the eluate D is wasted to the outside through the
drain DR 1 through a path of the thin tube 61', ports 1e, 1f, and
the thin tube 60'. The solution 11' of the mobile phase C is
transmitted by the pump 10' and is branched at the tee 20'. One of
the t paths branched the T shape tube 20' is transmitted to the
valve V-1 through a path of the thin tube 70', ports 2f, 2e, and
the thin tube 54', and wasted to the drain DR 2 through ports 1e,
1k, and the thin tube 55'. Other of the branched paths is wasted
through the drain DR 3 i ports 1n, 1m, the thin tube 53', ports 1d,
1c, the thin tube 62', and ports 2c, 2d.
In the first analytical mode, the eluate A from the analytical
column 4 is directly fed to the ion source 9' of the MS and all the
while the trapping column 12' and the thin tube are washed by the
solvent.
FIG. 40 corresponds to the second analytical mode.
The eluate eluted from the analytical column 4 flows into the tee
18' through a path of the ports 2n, 2p of the valve V-2, and the
thin tube 66'. The solution 6' of the mobile phase B is transmitted
by the pump 7' through the tee 8', and the branched resistance
column 19' and confluenced with the eluate A at the tee 18'. The
confluenced solution flows from the thin tube 58 to the trapping
column 12' through the thin tube 59', and the ports 1g, 1h with a
direction shown by an arrow in the figure.
After the component of analyte is trapped by the trapping column
12', the eluate B which contains non-trapped components is wasted
through the drain DR 1 to the outside through a path of the thin
tube 61', the ports 1e, 1f, and the thin tube 60'.
The solution 11' of the mobile phase C is transmitted by the pump
10' and divided into two paths at the tee 20'. The solution 11 of
the mobile phase C in one of the two paths is wasted through the
drain DR 4 to the outside through a path of the thin tube 70',
ports 2f, 2g, and the thin tube 69'. The solution 11' in other of
the paths is transmitted to the valve V-1 through the thin tube 52'
and further transmitted to ion source 9' of the MS through a paths
of the 1n, 1m, thin tube 53', ports 1d, 1c, the thin tube 62',
ports 2c, 2b, and the thin tube 73'.
The analytical component from the analytical column 4 is trapped by
the trapping column 12' in the second analytical mode and all the
while, the ion source 9' of the MS is washed with the solution of
the mobile phase C.
The system shown in FIG. 41 corresponds to the third analytical
mode.
The eluate A eluted from the analytical column 4 is drained through
the ports 2n, 2m, the thin tube 56', the ports 1j, 1k, the thin
tube 55'.
The solution 6' of the mobile phase B is transmitted by the pump 7'
and is divided into two paths at the tee 8'. The solution 6' of the
mobile phase B in one of the two paths is transmitted through a
path of the ports 2q, 2p, and the thin tube 66'. The solution 6' in
other of the paths is transmitted through the branched resistance
column 19' and the two branched solutions 6' are confluenced at the
tee 18'. The confluenced solution 6' is wasted through the drain DR
1 to the outside through a path of the thin tube 59', and the ports
1g, 1f.
The solution 11' of the mobile phase C is transmitted by the pump
10 and branched at the tee 20'. One of the branched solution 11 of
the mobile phase C flows into the trapping column 12' through a
path of the thin tube 70', ports 2f, 2e, the thin tube 54', the
ports 1l, 1m, the thin tube 53', the ports 1d, 1e, and the thin
tube 61'.
Whereby, the analytical components trapped in the analytical column
12' is eluted by the back-flush. The eluate containing the
analytical components introduced into the ion source 9' of the MS
through a path of the thin tube 58', ports 1h 1i, the thin tube
57', the ports 2a, 2b and the thin tube 73', and provide the mass
spectrum.
Other of the branched solution 11' of the mobile phase C washes the
sample injection port 3 through a path of the thin tube 52', the
ports 1n, 1p, the thin tube 51' and wasted through the drain DR 3
to the outside through a path of the thin tube 64, the ports 1b,
1c, the thin tube 62' and the ports 2c, 2d.
In the third analytical mode, the components of interest trapped in
the column are eluted by the back-flush and provide the mass
spectrum. Other flow paths are washed and the eluate A from the
analytical column 4 is wasted to the outside.
FIG. 42 shows a system corresponding to the fourth analytical mode.
The solution 1 of the mobile phase A is transmitted to the
analytical column 4 by the pump 2 through a path of the thin tube
63', the ports 1q, 1a, and the thin tube 50'. Then, the solution 1
of the mobile phase A is wasted through the drain DR 1 through a
flow path of the detector 5, the thin tube 67', the ports 2n, 2p,
the thin tube 66', the T shape tube 18', the thin tube 59', and the
ports 1g, 1f.
The solution 11' of the mobile phase C is transmitted to the tee
20' so as to be divided into two branched flows. The solution 11'
of one of the branched flows is wasted through the drain DR 4
through a path of the thin tube 70', the ports 2f, 2g, and the thin
tube 69'. The solution 11' of other of the branched flows is
transmitted to the ion source 9' of the MS through a path of the
change-over valve V-1, the ports 1n, 1p, the thin tube 51', the
sample injection port 3, the thin tube 64', the ports 1b, 1c, the
thin tube 62', the ports 2c, 2b, and the thin tube 73'.
The solution 6' of the mobile phase B is transmitted to the
trapping column 12' by the pump 7' through a path of the ports 2q,
2a the thin tube 57' the ports 1i, 1h, and the thin tube 58'. The
solution of the mobile phase B washes the trapping column 12' by
flowing as shown with an arrow in the figure.
After washing the column 12', the eluate D is wasted through the
drain DR 3 through a path of the thin tube 61', the ports 1e, 1d,
the thin tube 53', the ports 1m, 1l, the thin tube 54', the ports
2e, 2d, and the thin tube 72'.
In the fourth analytical mode, the trapping column 12' is washed
with the solution of the mobile phase B and the ion source of the
MS is washed with the solution 11 of the mobile phase C. The sample
injection port 3 is independently provided in the flow path of the
washing solution 11' of the mobile phase C which is separated from
the analytical column 4, thereby it becomes possible to provide the
FIA in the ion source 9' of the MS in the fourth analytical mode.
At this time, the analytical column 4 is washing with the solution
1 of the mobile phase A.
In the examples stated above, it becomes possible to obtain the
desalting of the sample solution, the direct analyzing by the
LC/MS, the removal of the components of non-interest, and the FIA
by changing over the four modes of the first, second , third and
fourth analytical modes.
EXAMPLE 11
FIG. 43 is a block diagram of LC/MS in another further embodiment
of the present invention and FIG. 44 is a explanatory view of
analytical modes for automatically removing high concentration
components in the LC/MS shown in FIG. 43.
In FIG. 43, numeral 26' means a comparator; 27' a LC controller;
28' a LC/MS. In FIG. 44, code O, P, Q means components of interest;
M main component; V component eluted by the void volume; and the
codes M, V the components of non-interest.
As shown in FIG. 43, the detector 5 is disposed in a rear position
of the analytical column 4 and monitors the components eluted from
the analytical column 4. The comparator 26' always compares an
output voltage Vs of the detector 5 with a comparing voltage Vr of
the comparator.
When the components having high concentration are detected, the
output voltage Vs from the detector 5 becomes higher than the
comparing voltage Vr, and the LC controller 27' outputs an order
signal to the LC/MS 28' so as to change into the fourth analytical
mode.
After finishing the elution of the components having high
concentration, the voltage Vs from the detector 5 becomes lower
than the voltage Vr, and then the LC controller 27' give the order
signal to the LC/MS 28' so as to return to the former mode.
Using FIG. 44, how to change the analytical modes will be
explained.
After setting the fourth analytical mode, the sample solution is
injected. The component V eluted by the void volume is wasted to
the outside. At time t1, the first analytical mode is performed so
as to measure the components of interest O, P.
When the component M having high concentration is eluted and the
output voltage Vs from the detector 5 becomes higher than the
voltage Vr of the comparator, the first analytical mode is
automatically changed into the fourth analytical mode at time t2.
In the case the output voltage Vs becomes lower than the comparing
voltage Vr at the time t3, the analytical mode returns to the first
analytical mode.
In this first analytical mode, the eluted component of analyte Q
may be measured after the main component M is eluted. At time t4,
the measuring by the LC/MS is finished and the analytical mode is
changed into the fourth analytical mode in order to perform a next
analytical process. In the example as above, the trapping time of
the components having the high concentration is not known, it
becomes possible to remove the component having the high
concentration and to prevent their carry-over.
EXPERIMENTAL EXAMPLES
The experimental examples by using above examples will be explained
hereinafter.
The sample used as the example is an anti-fungal having a molecular
weight of 331. The system used for the sample is the example 9 as
shown in FIG. 35 and the analytical conditions are shown in a
following table.
______________________________________ Item Conditions
______________________________________ analytical column 6 .times.
150 mm ODS column ion source 9' atmospheric pressure chemical
ionization trapping column 12 4 .times. 30 mm ODS.column detector 5
UV monitor detecting wave length 260
______________________________________
The sample is dissolved with the solution of the mobile phase for
analysis and the concentration thereof is 100 pl/ml. The sample
solution of 100 pl/ml (injection volume thereof: 10 .mu.g) is
injected from the sample injection port 3.
FIG. 45 shows a liquid chromatogram of first experimental example.
FIG. 46 shows a mass chromatogram of first experimental example.
FIG. 47 shows a liquid chromatogram of second experimental example.
FIG. 48 shows a mass chromatogram of second experimental example.
FIG. 49 shows a liquid chromatogram of third experimental example.
FIG. 50 shows a mass chromatogram of third experimental example.
FIG. 51 shows a liquid chromatogram of fourth experimental example.
FIG. 52 shows a mass chromatogram of fourth experimental
example.
EXPERIMENTAL EXAMPLE 1
FIGS. 45, 46 show the example in the case where all of the eluate
in the analytical system relating to the volatile solution of the
mobile phase is introduced to the MS.
The solution 1 of the mobile phase A is a solution mixed with
acetonitorile and aqueous solution 0.01M of ammonium acetate which
are mixed with a ratio 5:1.
The liquid chromatogram shown in FIG. 45 is outputted from the
detector 5 and the analyte are eluted at the time 8.78 minutes.
FIG. 46 shows a mass chromatogram of pseudo-molecular ion (M+H) of
m/z 332 detected by the LC/MS. This analysis of the component of
interest is performed with the first analytical mode. That is, all
of the eluate by the LC is introduced to ion source 9' of the
MS.
In the FIG. 46, numerals 4611 on upper right handside of the mass
chromatogram means a height of the peak, which corresponds to a
maximum current (mA) when the pseudo-molecular ion of m/z 332 is
detected.
EXPERIMENTAL EXAMPLE 2
FIGS. 47, 48 show the same chromatogram as in the FIGS. 45, 46 and
show experimental examples of the component of interest introduced
into the MS by removing the components eluted by the void
volume.
FIG. 47 shows a liquid chromatogram monitored by the detector 5,
and the component of analyte is eluted at the time 8.72 minutes and
the components of noninterest are eluted by the void volume at the
time 3.46 minutes.
The LC analysis is performed by injecting the sample after the
fourth analytical mode is set. The fourth analytical mode is held
until the time 7 minutes in order to remove the components of the
void volume which appeared at three minutes after injection. After
7 minutes passed, the analytical mode is changed to the first
analytical mode and the eluate A from the LC is introduced to the
ion source 9' of the MS.
As explained above, the salt etc. which is eluted by the void
volume are wasted to the outside and the components of interest are
introduced to the ion source 9' of the MS.
The FIG. 48 shows a mass chromatogram by the pseudo-molecular ion
of m/z 332 and the retention time of the components of interest
does not change if the valve is changed over.
EXPERIMENTAL EXAMPLE 3
FIGS. 49, 50 show the same chromatogram as in the experimental
examples 1, 2 and show experimental examples of the components of
interest trapped by the trapping column and introduced into the MS
after removing the components eluted by the void volume.
In FIG. 49, the solution 1 of the mobile phase A is an aqueous
solution of ammonium acetate and there is not any special necessity
to desalt the component in the LC/MS.
The experimental data shown in FIG. 49 is provided in order to
prove the functions of the system in the present invention.
The fourth analytical mode continues till 7 minutes and the eluate
A is wasted to the outside, and simultaneously the pretreatment of
the trapping column 12' is performed. The second analytical mode is
performed from 7 minutes to 9.8 minutes and the components of
interest from the analytical column 4 is trapped by the trapping
column 12'. The fourth analytical mode is again performed from 9.8
minutes to 15 minutes, and the components of interest are desalted
and the eluate A is wasted to the outside. After 15 minutes, the
third analytical mode is performed, and the component of interest
trapped in the trapping column 12' is eluted by the back-flush.
Here, the matter to which attention should be paid is that the
height of the peaks detected by the LC/MS in the experimental
examples 1, 2 as shown in FIGS. 46, 48 are respectively 4611 and
3925, and the peak in this experimental example is 7970. This data
means that the peak of the components of interest eluted by the
back-flush becomes more sharp than that of the components eluted
from the analytical column 4 and the height of the peak becomes
higher. It is very useful for measuring the components of interest
having very low concentration as the sensitivity of the apparatus
becomes higher. Such a system as above is applicable to the
volatile solution of the mobile phase.
EXPERIMENT EXAMPLE 4
FIGS. 51, 52 show chromatogram when using the non-volatile solution
of the mobile phase. The solution 1 of the mobile phase A is an
aqueous solution of methanol and potassium dihydrogenphosphate
0.05M mixed with a ratio 35:65 and the flow rate thereof is 1.2
ml/min.
The solution 11 of the mobile phase C for eluting the components of
interest trapped in the trapping column 12' is an aqueous solution
of acetonitrile of 90%. The solution 6' of the mobile phase B for
diluting is pure water.
The sample is dissolved by the solution 1 of the mobile phase A to
a concentration of 100 .mu.g/ml and the sample of 100 .mu.l is
injected. The analytical column is ODS 6.times.150 mm and the
trapping column is ODS 4.times.30 mm.
A chromatogram by the LC as above is shown in FIG. 51 and the peak
arises at 17.38 minutes under a condition as above.
The chromatogram by the desalting system in this experimental
example is shown in FIG. 52.
The fourth analytical mode is performed just after injecting the
sample solution until 16 minutes, and the eluate A from the
analytical column A is wasted to the outside and the trapping
column 12' is washed with the solution 6' of the mobile phase B,
that is, the water. After 16 minutes, the second analytical mode is
performed and the components of interest are trapped by the
trapping column 12'.
At 18.4 minutes when the elution of the component of interest is
finished, the fourth analytical mode is performed the component
trapped in the column 12' is desalted by flowing the solution 6' of
the mobile phase B, that is, the water. At 23 minutes, the third
analytical mode is preformed and the components trapped in the
trapping column 12' is eluted by the back-flush of the solution 11'
of the mobile phase C and introduced to the MS.
In this case, the height of the peak in the mass chromatogram is
9894 and becomes very sharp in the same way as in the analysis of
ammonium acetate solution. The peak height becomes more than twice
of the peak height 4611 in the case of directly introducing the
ammonium acetate solution to the MS in the experimental example
1.
The system explained in the experimental example 4 is applied to
the LC/MS which use both of the nonvolatile buffer and the
non-volatile salt and the sensitivity thereof is improved.
Further, every example and every experimental example are explained
relating to the system which directly connects the LC and the MS by
selecting the four modes of the first, second , third and fourth
analytical modes which are performed by easily changing over the
valves V-1, V-2, V-3 and connecting the flow paths 91', 92'
depending on the signal from the LC controller 27'.
* * * * *