Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2003-02-11
2004-10-26
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S281000, C250S310000, C250S307000
Reexamination Certificate
active
06809316
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an electrospray ionization mass analysis apparatus and system thereof, wherein a sample solution eluting out of a low flow rate chromatograph such as a micro liquid chromatograph is led to an electrospray ion (ESI) source and is ionized therein, and the ions generated in this ion source are fed to a mass spectrometer arranged in a highly vacuum space, where the ions are 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 complicated matrices. There has been a growing demand for extracting 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 separating 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 ionsization 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 that determines the amount of solution to be supplied 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 &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 ionization will become unstable and anticipated highly sensitive measurement will not be achieved. U.S. Pat. No. 5,504,329 discloses an art for ESI improvement for providing highly sensitive measurement of a very small amount of components. 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.
In the micro LC field, the flow rate is extremely small, below several &mgr;L/min. and a big problem is raised by the dead volume of the LC parts and the pipe connection among the parts thereof. When the dead volume between the micro-column and detector is greater for the flow rate, the sample components separated by the micro-column will be dispersed and mixed among them, with the result that separation and sensitivity will be lost a substantially. Further, the dead volume between the LC pump and micro-column will cause a problem of the delay in gradient elution. This requires the dead volume to be minimized.
Gradient elution is a method for quick elution of the sample component by changing the composition of the eluent with the lapse of time. This gradient elution technique is improves the separation of the sample components. This improves the S/N ratio and reduces the measurement time at the same time. Accordingly, LC is used extensively.
In micro LC, even if the start of gradient is specified and multiple pumps have fed out solvent at a predetermined flow rate, a long time is required before the composition of the eluent is changed in the micro-column. This delay raises a problem. This is called a delay in gradient elution.
Assume that a pump
1
is now feeding out solvent A at 20 &mgr;L/min. Also assume that a pump
2
starts to feed out solution B at the rate of 0.2 &mgr;L/min. at a predetermined time. A mixer and a pipe regionrranged between pumps
1
and
2
and micro-column. If their volume is 5 &mgr;L, the delay of gradient will be 5/0.2=25 min. Namely, gradient is effectively started in the micro-column 25 minutes after the pump
2
started to feed solution B. This makes it difficult to ensure correct separation and analysis by micro LC. In order to improve this delay of gradient elution, it is important to reduce the size of the mixer and dead volume. The dead volume can be decreased by reducing the pipe diameter or pipe length. However, reduction of pipe diameter raises a new problem of easy clogging of the pipe. Especially when a biological sample is to be analyzed, a biological macromolecule such as sugar and protein present in the sample as well as NaCl and salts will cause clogging of the pipe. Further, separation of protein requires salt having a high concentration of 100 mM or more to be added to the mobile phase in many cases. This salt of high concentration is deposited in the dead volume of the pipe, with the result that the pipe is clogged in the final stage. Accordingly, the frequently used system in the micro LC is a micro LC system where A semimicro or conventional LC pump is used up to gradient solution feeding, and the eluent is split immediately before the inlet. A great volume (1 mL/min. to 0.1 mL/min.) of solvent is used up to the pump, mixer and pipe, so the dead volume among them can be ignored. In other words, the problem of delay in gradient elution has been solved. The split eluent at a very small flow rate (10 to several &mgr;L/min.) is led to the micro column through the injector. This method has a disadvantage that the greater part of solvent must be discarded by the splitter, but it solves the aforementioned problem of the delay in gradient resulting from dead volume, and ensures economical configuration of the system. For these merits, this method has come to be used over a wide range.
The Japanese Application Patent Laid-Open No. 06-13015 discloses ions implantation apparatus for evaluating a trouble such as equipment failure, displacement by comparing with the reference value the status value of a particular peak in a mass spectrum. The Japanese Application Patent Laid-Open No. 10-10109 discloses an apparatus for avoiding damage of the optical detector cell resulting from a clogged flow path in a mass analysis apparatus directly coupled with a liquid chromatograph, the aforementioned mass analysis apparatus being designed to ionize and detect the component leaching therefrom.
The micro LC wherein the solvent is split before the micro column can be said as an extension of the general-purpose LC and semimicro LC technology. So since the micro LC is capable of analyzing a trace quantity of sample, it is expected to find a widespread use in the field of biological technologies. According to this method, however, the major portion of solvent is split and discarded as waste, and the amount of solvent flowing into the micro column is no more than one hundredth to one tenth of the solvent supplied to the splitter. So even if the ESI capillary are clogg
Dickstein , Shapiro, Morin & Oshinsky, LLP
Hashmi Zia R.
Hitachi High-Technologies Corporation
Lee John R.
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