Radiant energy – Ionic separation or analysis
Reexamination Certificate
2002-09-16
2004-05-18
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
C250S288000, C250S292000, C204S166000, C436S173000
Reexamination Certificate
active
06737640
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new electrospray ionization mass analysis apparatus and method thereof, wherein a sample solution is led to an electrospray ion (ESI) source and is ionized therein, and the ion generated therein is fed to an ion storage type mass spectrometer arranged in a highly vacuum space, where the ion is subjected to mass analysis.
BACKGROUND OF THE INVENTION
In recent years there has been a remarkable growth in biological researches over diversified fields. Especially, protein, peptide and DNA play an extremely important role in the living body, and have been the objects of study by a great number of research workers.
Generally, these organic compounds derived from living organism occur in a very small amount in a complicated matrix. There has been a growing demand for extract a very small amount of these biological organic compounds from the living body and analyzing them using a mass spectrometer directly coupled with liquid chromatograph LC/MS apparatus) with a high degree of sensitivity. The LC/MS apparatus is an apparatus for separate a mixture with a liquid chromatograph (LC) and providing qualitative and quantitative analysis using a—mass spectrometer (MS) with a high degree of sensitivity. Electrospray ionization (ESI) is typical ionization means used in the LC/MS. The ESI is an ionization technique used under atmospheric pressure and is known as providing soft and highly sensitive ionization. For this reason, this method has come to be used very often for biological analysis.
To ensure stable and highly sensitive measurement of a very small amount of components using the aforementioned ESI, some parameters must be optimized. One of these parameters is the flow rate for supply of the solution to the ESI ion source. To achieve highly sensitive measurement, the flow rate of the solution flowing through the ESI capillary tube must be kept within a certain range. In ESI, the optimum flow rate is said to lie in the range from 10 nL/min (10
−8
L/min) to several 1 &mgr;L/min (10
−6
L/min). If a solution is fed into the ESI capillary tube at a flow rate higher or lower than this level, the ESI will become unstable and anticipated highly sensitive measurement will not be achieved.
The flow rate of a conventional LC often used for analysis of a mixture is in the range from several mL/min (several 10
−3
L/min) to several 100 &mgr;L/min (several 10
−4
L/min). The flow rate of the semimicro LC is in the range from 100 &mgr;L/min (several 10
−4
L/min) to several 10 &mgr;L/min (several 10
−5
L/min). Since there is a big difference between the flow rate of the conventional LC and semimicro LC and the optimum ESI flow rate, both type of chromatography have been unable to be directly coupled with the ESI without solution being split. Needless to say, the splitting of solution will cause deterioration of sensitivity in measurement.
U.S. Pat. No. 4,861,988 discloses the art for improving the ESI to ensure that the aforementioned ESI can be applied to the conventional LC and semimicro LC of high flow rate. According to this art, the spray probe is improved to allow a stable spraying of a large volume of solution. As shown in
FIG. 12
, the ESI probe consists of two capillary tubes
30
and
32
having different outer diameters. The first capillary tube
30
having a smaller diameter is inserted into the second capillary tube
32
having a larger inner diameter. This configuration provides a coaxial ESI probe. A high voltage of 3 to 4 kV is supplied from the DC high voltage power supply
5
and is applied to the first capillary tube
30
. Sample solution is led into the first capillary tube. Nitrogen gas is fed to the space between the first and second. Sample solution is released into the atmosphere as minute charged droplet
6
by the mechanical force and action of electric field. The charged droplet is further pulverized by mechanical crushing due to the flow of gas and evaporation of solution. Ion is released into the atmosphere in the final stage. The generated ion is introduced into a high-vacuum mass spectrometer.
This method has brought about a drastic increase in the flow rate to be introduced into the ESI probe. The increase is from 1 mL/min (10
−3
L/min) to 100 &mgr;L/min (10
−4
L/min). This is just applicable to the flow rate of the conventional LC (several mL/min to several 100 &mgr;L/min) and semimicro LC (several 100 &mgr;L/min to several 10 &mgr;L/min). This method has come to be called “Ion Spray” or “Pneumatic Assisted Electrospray”.
U.S. Pat. No. 5,504,329 discloses another art of improving the ESI permitting measurement of still further minute components with high sensitivity. The art disclosed therein was later called Nanospray technique. After the tip of an extra-fine capillary tube made of glass having an outer diameter of about 0.2 mm and inner diameter of about 0.03 mm has been elongated by a burner or sharpened by etching, the nozzle tip is gold plated. The D.C. voltage of about 1 kV supplied from the high voltage source is applied to the tip of the nozzle. The flow rate of a sample solution from a nanospray device ranges from is several nL/min (several 10
−9
L/min) to 10 nL/min (several 10
−8
L/min). Measurement for more than one hour was enabled by only the sample sucked into the nanospray spray capillary tube. Accordingly, this nanospray technique has come to be used in combination with extra-low flow rate chromatography in CE (Capillary Electrophoresis); further, it has come to be used for extremely highly sensitive measurement of isolated components. The nanospray technique has enabled ESI measurement in the range of flow rate below 10 nL/min.
Table 1 shows the ESI art, improved ionization arts, optimum flow rate and compatible chromatography:
TABLE 1
No
Ionization
Optimum flow rate
Compatible chromatography
1
Ion Spray
1 mL/min to 100 &mgr;L/min
Conventional LC,
semimicro LC
2
?
Several 10 &mgr;L/min to
Micro LC
1 &mgr;L/min
3
ESI
1 &mgr;L/min to 10 nL/min
Capillary LC
4
Nanospray
1 nL/min to 10 nL/min
Capillary electrophoresis
The advent of Ion Spray, ESI and Nanospray has enabled selection of an ion source suited to each one of various types of chromatography. However, as shown in Table 1, there is no ESI ion source that is best suited to the micro LC from 10 &mgr;L/min to 1 &mgr;L/min. For this reason, the researchers have to use the ESI or Ion Spray for the micro LC, based on the understanding that the sensitivity and stability are not satisfactory.
For the micro LC, the flow rate of mobile phase is from 10 &mgr;L/min to 1 &mgr;L/min and the required amount of sample solution can also be reduced to the level as small as nL(10
−9
L/min). So its range of application is rapidly expanding to cover the field of analyzing the biological component. Needless to say, there has been a growing demand for ionization technique optimum to this field.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide an electrospray ionization mass analysis apparatus and the method thereof provided with an ESI ion source that can be directly coupled to the micro LC.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is led into a capillary tube under atmospheric pressure,
voltage or electric field is applied to the tip of this capillary tube, thereby allowing an ion to be generated by an electrospray ion source provided therein, and
generated ion is led to and stored in an ion storage type spectrometer disposed in a vacuum chamber so that a mass spectrum is obtained by subsequent mass sweeping. This electrospray ionization mass analysis apparatus is characterized in that
the voltage formed by AC voltage superimposed on the DC voltage or the electric field formed by AC electric field superimposed on the static electric field is applied to the tip of the aforementioned capillary tube, whereby electrospray ionization is carried out.
The present invention provides an electrosp
Hashmi Zia R.
Hitachi High-Technologies Corporation
Lee John R.
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