Fluid sprinkling – spraying – and diffusing – With means to vibrate or jiggle discharge
Reexamination Certificate
2002-12-24
2004-12-07
Tapolcai, William E. (Department: 3744)
Fluid sprinkling, spraying, and diffusing
With means to vibrate or jiggle discharge
C239S102200, C239S690100
Reexamination Certificate
active
06827287
ABSTRACT:
BACKGROUND OF THE INVENTION
The present application is directed to droplet ejection, and more specifically, to the generation and transmission of droplets to analytical instruments such as mass spectrometers.
Mass spectrometers are an analytical tool concerned with the separation of molecular (and atomic) species according to their mass and charge. More particularly, it is an analytical tool used for measuring the molecular weight (MW) of a sample. For large samples such as biomolecules (e.g., analyte molecules), molecular weights can be measured within an accuracy of 0.01% of the total molecular weight of the sample, i.e., within 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. More commonly, the accuracy is about 0.05% or 20 Da with a good measurement and multiple charge states present. This is sufficient to allow minor mass changes to be detected, e.g., the substitution of one amino acid for another. Often, however, there are substitutions for amino acids that do not significantly alter the mass (i.e., isoleucine and leucine have the same mass), these substitutions may pose problems for any mass spectrometer. For small organic molecules, the molecular weight can be measured to within an accuracy usually sufficient to confirm the molecular formula of a compound. To achieve such measurements, a mass spectrometer of sufficient resolution will of course be required.
Mass spectrometers are particularly useful for analyzing the products of chemical reactions, since they can identify specific products by their mass signature. However, large complexes, such as protein-ligand interactions have been difficult to detect using conventional mass spectrometry methods. This is in part due to the fact the introduction and charging of the sample is a relatively violent process, which tends to break up larger molecules into fragments. Fortunately, new sample introduction techniques have been developed, such as electrospray ionization (ESI), which are much less violent, making it possible to now detect protein ligand complexes directly in mass spectrometers. Such mass spectrometers are ESI-quadrupole mass spectrometers, one in particular being ESI time-of-flight of mass spectrometers (ESI-TOF mass spectrometers).
There are numerous types of mass spectrometers, in addition to those known as time-of-flight. However, the basic components are similar. As shown in
FIG. 1
, mass spectrometer
10
can be divided into three fundamental parts. First, an ion source
12
ionizes the molecules of interest, then a mass analyzer
14
differentiates the ions according to their mass-to-charge ratio, and finally a detector
16
measures an ion beam current. Each of these elements can take many forms and are combined to produce a wide variety of mass spectrometers with specialized characteristics.
The analyzer and detector of the mass spectrometer, and often the ionization source, are maintained under high vacuum
17
to provide the ions a path to travel from one end of the instrument to the other without hindrance from other molecules. The entire operation of the mass spectrometer, and often the sample introduction process, is under a system controller
18
.
As may be apparent from the foregoing, an important aspect of mass spectrometry is sample introduction into the instrument
10
. The sample inlet
19
is the interface between the sample and the mass spectrometer. One approach is to place a sample on a probe which is then inserted, usually through a vacuum lock, into the ionization region of the mass spectrometer
10
. The sample can then be heated to facilitate thermal desorption or undergo any number of high-energy desorption processes used to achieve vaporization and ionization.
Capillary infusion is often used because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques, including, but not limited to, gas chromatography, liquid chromatography (LC), high pressure liquid chromatography (HPLC) or capillary electrophoresis (CE).
Up until twenty years ago, practical techniques for interfacing liquid chromatography (LC) with available ionization techniques were not available. A major problem prohibiting this interface was getting the sample stream from the liquid chromatographic process into the mass spectrometer without losing vacuum while also ionizing the sample. However, new ionization techniques, such as the previously mentioned electrospray ionization, allow liquid chromatography/mass spectrometry processes to be routinely performed. One configuration of a liquid chromatography/mass spectrometry design
20
is shown in FIG.
2
. More particularly, a pump
22
moves a sample liquid to chromatographic separation columns
24
wherein the liquid sample is separated into a series of components. The liquid sample is then sent to a capillary spray nozzle or needle
26
, and electrospray
28
ionization processes
28
are performed
28
. The ionized sample is then introduced to mass analyzer
30
, and detected by detector
32
for generation of output signals
34
.
As previously noted, the ionization source in this example is an electrospray ionization system. A more detailed view of such a system is shown as electrospray configuration
40
of FIG.
3
. In this design, a liquid sample
42
, which may be provided from the liquid chromatography process of
FIG. 2
moves through a metal capillary or needle
44
, which has an open end with a sharply pointed tip, such as the end of a syringe. This tip is attached to a voltage supply
45
of between approximately 1.5 kv to 4 kv, depending upon the implementation. The end of the tip faces a counterelectrode plate or cylinder electrode
46
held at a voltage lower than the tip voltage to generate a voltage gradient. As the voltage in the liquid is applied, the liquid becomes charged, generating a force sufficient to expel the liquid from the capillary tip. As the liquid sample
42
pushes from the tip, a shape described as a Taylor cone is developed. At the very end of the cone, the droplets push away from one another into a fine spray
48
, at times called a plume. It is to be appreciated that a capillary LC process uses pump pressure to expel the fluid.
Depending on the electric field used, the charges may be positive or negative. The droplets may contain both solvent molecules, as well as analyte (sample) molecules and may be less than 10 micrometers across. The droplets move across the electric field, and with the assistance of a flow of N
2
gas, provided by gas inlet
50
, neutral solvent molecules are evaporated from the droplets. As the droplets become smaller, and the total charge on the droplets stays the same, so the concentration of charge increases. Eventually, at what is known as the Rayleigh limit, Coulombic repulsion overcomes the droplet surface tension and the droplet explodes. The Coulombic explosion forms a series of smaller, lower-charged droplets. This process of shrinking followed by explosion is repeated, until eventually the analyte (sample) molecule is stripped of solvent molecules, and is left as a charged ion. These ions are then moved through capillary
52
, which is in a differently pumped region
54
. A skimmer
56
may be provided to further refine the sample.
A major instrumental challenge with electrospray ionization is the interface between the ion source (which may be at atmospheric pressure) and the mass spectrometer (at a high vacuum, one example value may be about 10
−6
torr; it is to be appreciated however that this value is instrument dependent). This problem is addressed by the use of a pinhole aperture
58
around 10-100 micrometers. In this design, the created ions are drifted (with the help of the electric field) towards the aperture. The emitted drops are then, as previously noted, analyzed in the analysis section and then provided to a detector for generation of output signals.
It is to be appreciated
Bank Steven J.
Elrod Scott A.
Ali Mohammad M.
Fay Sharpe Fagan Minnich & McKee LLP
Palo Alto Research Center Incorporated
Tapolcai William E.
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