Mass spectrometer

Details Mass spectrometer Mass spectrometer

1999-03-02

2001-02-13

Anderson, Bruce C. (Department: 2881)

Radiant energy

Ionic separation or analysis

With sample supply means

C250S281000

Reexamination Certificate

active

06188065

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention concerns a mass spectrometer combined with a sample separation apparatus used for separation and analysis of mixed biological samples, for example, sugar, peptide and protein.
In the field of analysis, an importance has been attached to the development of mass spectrometry for biological compounds at present. Since the biological compounds are usually dissolved as a mixture in a solution, development has been progressed to a mass spectrometer combined with the sample separation apparatus for separating the mixture. As a typical example, there can be mentioned a combined apparatus of capillary electrophoresis apparatus-mass spectrometer utilizing capillary electrophoresis for the separation of the sample. The capillary electrophoresis is excellent in the separation of the mixture but can not identify substances. On the other hand, the mass spectrometer has a high analyzing sensitivity and is excellent for the ability of identifying substances but analysis of the mixture is difficult. In view of the above, a sample is separated by the capillary electrophoresis apparatus and the separated sample is analyzed by the mass spectrometer. Thus, the mass spectrometer combined with the capillary electrophoresis apparatus is much effective for the analysis of a mixture.
An existent mass spectrometer combined with the capillary electrophoresis apparatus described above is described in
Analytical Chemistry,
Vol. 60, No. 18, Sep. 15, 1988, pp. 1948-1952. The existent mass spectrometer will be explained with reference to FIG.
13
. In the mass spectrometer of the prior art, an electrospray ionization method is used for ionization of a sample. A capillary
1
is a fused-silica capillary having an outer diameter of about several hundreds micrometer and an inner diameter of about several tens micrometer. The inside of the capillary
1
is filled with a buffer solution. A sample solution is introduced from one end
2
a
to the inside of the capillary
1
. After introduction of the sample solution, the end
2
a
is kept in a buffer vessel
4
filled with a buffer solution
3
. The other end
2
b
of the capillary
1
is inserted to the inside of a metal tube
5
. Generally, a flow rate of a buffer flowing through the capillary is small and it is often difficult to nebulize the sample solution stably and continuously. Then, a sheath liquid
6
is introduced in a gap between the capillary
1
and the metal tube
5
for assisting nebulization. When a high voltage is applied from a high voltage power source
7
a
between one end
2
a
of the capillary
1
and the metal tube
5
, since the end
2
b
of the capillary
1
is electrically connected by way of the sheath liquid
6
with the metal tube
5
, a high voltage is applied between both ends
2
a
and
2
b
of the capillary
1
. Thus, the sample is sent to the end
2
b
while undergoing electrophoretic separation in the capillary
1
.
The sample reaching the end
2
b
is mixed with the sheath liquid
6
and then electrosprayed by a voltage applied between the metal tube
5
and an opposing electrode
8
a
by power source
9
for a nebulizer. Ions relevant to the sample molecules are contained in droplets formed by the electrospray. The ions relevant to the sample molecules are entered through a sampling aperture
10
a
into a differential pumping region
12
evacuated by an evacuation system
11
a
and, further, enter a vacuum region
13
evacuated to a high vacuum degree by a vacuum system
11
b
. The ions entering the vacuum region
13
are subjected to mass separation in a mass analysis region
14
and the mass-separated ions are detected by an ion detector
15
. A detection signal from the detector
15
is sent by way of a signal line
16
to a data processing apparatus
17
and put to data processing to obtain a result of mass spectrometry for the sample substance.
In the existent mass spectrometer combined with the capillary electrophoresis apparatus described above, electrospray ionization is used for ionization of the sample. The electrospray ionization is a method of taking out highly polar substances such as protein or peptide present as ions in a solution as gaseous ions. Therefore, neutral substances not possessing charges in the solution can not be detected at a high sensitivity in the mass spectrometer combined with the existent capillary electrophoretic apparatus. Since such neutral substances include, for example, amines in various kinds of medicines and neutrotransmitters, it is extremely important to analyze electrically neutral samples for the study in the field of biotechnology or medicine.
Further, as one of methods for separation of samples by capillary electrophoresis, micellar electrokinetic chromatography has been known. In the micellar electrokinetic chromatography, micelles are formed by adding a surfactant to a buffer solution, and a neutral substance not having charges is separated by utilizing the difference of distribution when each of the sample compounds is distributed in the micelles. Also in this case, for extending an application range of the mass spectrometer combined with the capillary electrophoresis apparatus, it has been desired for the development of an apparatus capable of analyzing, at a high sensitivity, neutral substances having no charges in the solution.
Further, the ion intensity obtained by the existent electrospray ionization method is approximately given by the following equation
Electrophoresis
, Vol. 14, 1993, pp. 448-457:
I(A
+
)∝V(A
+
)/V(C
+
)  (1)
where I(A
+
) represents a signal intensity of ion A
+
as an object of analysis, V(A
+
) represents a flow rate of ion A
+
to be analyzed, and V(C
+
) represents a flow rate of contaminant ions other than ion A
+
to be analyzed. Accordingly, for attaining mass spectrometry at a high sensitivity by using the electrospray ionization method, it is important to remove contaminant ion C
+
in the sample solution.
On the other hand, in the capillary electrophoresis method, a method of adding a salt at high concentration in a buffer solution for electrophoresis is generally used for preventing sample molecules from adsorbing on wall surfaces or the like. Accordingly, since contaminant ions (for example, Na
+
, K
+
) formed by dissociation of the salt are contained in a great amount in the ions obtained by electrospray, the denominator: V(C
+
) in the formula increases remarkably to reduce the signal intensity of the ion as an object of the analysis. Accordingly, in the existent mass spectrometer employing electrospray for the ionization of the sample, it was difficult to obtain a signal of the ion as an object of analysis at a sufficient intensity.
Further, in micellar electrokinetic chromatography, analysis is effected by forming micelles of a surface active agent such as SDS (sodium dodecyl sulfate) in a buffer. For forming the micelles, it is necessary to add a surfactant at a concentration exceeding a critical value (critical micelle concentration) in the buffer. Under micelle-forming conditions, cations and anions liberated from the surfactant are present in a great amount as contaminant ions in the buffer. Therefore, in the existent apparatus using the electrospray ionization method, measurement of the sample molecular ions is difficult by the effect of the contaminant ions.
With the reasons described above, it has been strongly demanded for providing a mass spectrometer combined with a sample separation apparatus such as a capillary electrophoresis apparatus improved so as to less undergo the effect of the salt in the buffer.
SUMMARY OF THE INVENTION
A first object of the present invention is to provide a mass spectrometer capable of separating an electrically neutral substance present in a solvent which was difficult to be ionized by an existent electrospray ionization method and analyzing the same at a high sensitivity.
A second object of the present invention is to provide a mass spectrometer capable of using, t

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