Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2001-08-23
2004-03-16
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S42300F, C250S282000, C250S281000, C250S42300F, C073S023360
Reexamination Certificate
active
06707035
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for the introduction of selected chemical elements present in solid or liquid samples into an accelerator mass spectrometry (AMS) system or other analytical instrument.
AMS is a powerful tool for the ultra-sensitive detection of
14
C and
3
H in biological samples, with proven applicability to current problems in environmental toxicology and human carcinogenesis. For
14
C detection, AMS has 1000-fold higher sensitivity than liquid scintillation decay counting, thereby allowing the quantization of attomole (10
−18
mole), or smaller, samples. Almost all existing radiocarbon AMS systems require that the sample to be analyzed be introduced into the ion source as solid graphite. Graphitization is a lengthy process (typically taking 6-10 hours) and considerable skill is required to produce layers of uniform composition and thickness and to prevent sample contamination. For example see the paper by J. S. Vogel, K. W. Turteltaub, J. S. Felton, B. L. Gledhill, D. E. Nelson, J. R. Southon, I. D. Proctor and J. C. Davis Nucl. Instr. and Meth. B52 (1990) 524, incorporated herein by reference. Therefore, the analysis of biological samples by AMS requires highly specialized sample preparation procedures that are not compatible with standard chromatographs. This requirement has been a major impediment to the use of AMS in the biomedical sciences.
Liquid chromatography is the technique of choice for high performance separation of large, non-volatile or polar molecules such as proteins, carbohydrates, peptides, and oligonucleotides. The coupling of a liquid chromatograph (LC) to an AMS is particularly challenging because the interface must provide for the efficient conversion of biological molecules in a variety of solvents into CO
2
or H
2
, and must do so with high sample transfer efficiency, good peak shape retention, and minimal contamination with naturally occurring
14
C or
3
H from other sources such as solvents and previous samples.
To prepare a liquid-phase sample for AMS, the interface must efficiently convert the desired isotope into one or more gaseous compounds suitable for introduction into the ion source. Negative ion sources that allow the sample to be introduced as gaseous CO
2
(or H
2
) have been developed, and have been shown to have sufficiently low sample-to-sample memory for detection of
14
C at or near modern abundance. For example, see the paper by C. R. Bronk and R. E. M. Hedges, Nucl. Instr. Meth. Phys. Res. B29(1987) 45; also R. Middleton, J. Klein and D. Fink, Nucl. Instr. Meth. Phys. Res. B43 (1989) 231, incorporated herein by reference. In the ion source, CO
2
(or H
2
) is converted to C
−
(or H
−
) for injection into the accelerator mass spectrometer. The AMS ion source may produce positive ions as an intermediate step, as described in U.S. Pat. No. 5,438,194, entitled “Ultra-Sensitive Molecular Identifier”, by Koudijs et al. A sample chromatogram is illustrated in FIG.
1
. However, the applicability of GC-AMS is limited to volatile substances.
For some isotopes, such as
14
C, it is important to strip a large fraction of the solvent accompanying the analyte. The extremely low naturally occurring background of tritium lowers the concentration at which samples can be introduced before separation of analyte from sample matrix (e.g., solvent) becomes necessary. The natural abundance of
3
H (
3
H:
1
H) is ≦10
−15
, at least 3 orders-of-magnitude lower than the natural abundance of
14
C. The impact of the lower natural abundance of
3
H on AMS measurement capabilities can be seen from the following considerations. If it is assumed that the current produced by the AMS ion source is 25 &mgr;A, then the particle current of H
−
or C
−
is 10
16
ions/min. For
3
H detection, a transport efficiency of 50% and a natural abundance of 10
−15
yields a corresponding
3
H background of 5 cpm at the AMS detector. Detection of
3
H with SNR=10 in 1 minute would therefore require 105 cpm
3
H from analyte. In this example, the concentration of
3
H-labeled analyte is 2.2 pM (105 cpm
3
H÷0.5×10
16
cpm H, multiplied by 100 moles H/L) and the volumetric flow rate of sample introduced into the ion source is about 1 nL/min. It is clear from these numbers that, even at these very low sample concentrations and flow rates, accurate AMS detection of
3
H without solvent removal is possible with negligible contribution from naturally occurring background.
The limits on direct sample introduction for
14
C detection are more difficult to define, but are clearly more stringent. For
14
C, the natural abundance of 1.4×10
−12
gives a background count rate of 6,600 cpm under the same assumptions used above. Detection of the same number of
14
C atoms from analyte (105 cpm) yields a SNR=1.3. In order to obtain the same statistical accuracy of SNR=10, it would be necessary remove solvent to a level of about one part in 10
3
prior to AMS analysis. Alternately, higher flow rates of sample into the AMS could be used. In this example, about 900 cpm
14
C from analyte (in a background of 6,600 cpm from solvent) would yield a SNR=10 due to counting statistics alone, but it would be necessary to eliminate all other noise contributions at the level of better than 1.5%. For these reasons, accurate
14
C detection by AMS without solvent removal is extremely difficult.
U.S. Pat. No. 5,438,194, entitled “Ultra-Sensitive Molecular Identifier”, by Koudijs et al. discloses a system where a liquid or gas chromatograph is coupled directly to the ion source system of an AMS analyzer. However, there are no provisions for desolvation, and the molecular dissociation and ion formation occur in the same process in the ion source itself. In addition, the inventors disclose several designs for the ion source system. However, none of these disclosed designs include a provision for desolvation. At a relatively low solvent flow rate of 1 &mgr;L/min, the detection limit for
14
C without desolvation will be approximately 0.1 femtomole or higher. This is significantly greater than the target sensitivity for a LC-AMS system of detection of LC peaks containing one attomole (10
−3
femtomole), or less, of
14
C or
3
H.
An additional disadvantage of the direct coupling of a liquid chromatograph to the ion source systems described in U.S. Pat. No. 5,438,194 is that molecular dissociation and ion formation (positive or negative) occur in the same process. This coupling of the dissociation and ionization functions will most likely result in a significant dependence of conversion efficiency on input chemical form. The prior art mentions the possibility of dissociating the molecules by high temperature pyrolysis, but there is no detailed description of what compounds are to be formed and whether the dissociation and ionization functions will be separated in this case.
Systems coupling a liquid chromatograph through a conversion reactor to a standard mass spectrometer have been developed for IRMS. These systems include the “moving wire” system as described by R. J. Caini and J. T. Brenna, Anal, Chem. 65 (1993) 3497, and the chemical reaction interface mass spectrometry (CRIMS) interface, as disclosed by M. McLean, M. L. Vestal, Y. Teffera, and F. P. Abramson,
J. Chrom. A,
732 (1996), 189. The “moving wire” system has the disadvantage that only a small fraction of the LC eluent can be deposited on the wire, resulting in low analyte transfer efficiency to the IRMS. The CRIMS interface incorporates a Vestec “Universal Interface” (UI) to separate analyte from solvent. The UI is based on the formation of a highly focused particle beam using thermospray vaporization followed by a multiple-stage desolvation process (momentum separator). The UI operates at normal-bore HPLC flow-rates and uses a high He gas flow to carry the particle beam through the apparatus. The disadvantage of the particle beam desolvation approach for AMS is that exist
Hughey Barbara J.
Mehl John T.
Shefer Ruth E.
Skipper Paul L.
Tannenbaum Steven R.
Hashmi Zia R.
Lee John R.
Newton Scientific Inc.
Samuels , Gauthier & Stevens, LLP
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