Liquid purification or separation – With means to add treating material – Chromatography
Reexamination Certificate
2000-05-26
2002-03-12
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
With means to add treating material
Chromatography
C210S656000, C210S175000
Reexamination Certificate
active
06355165
ABSTRACT:
FIELD OF THE INVENTION
The invention concerns a high pressure chromatographic separation system denoted herein as Matched Ion Polynucleotide Chromatography (MIPC) to distinguish it from traditional partitioning-based, reverse phase HPLC systems. In particular, this invention relates to MIPC chromatographic systems with improved, high precision column heater systems.
BACKGROUND OF THE INVENTION
Traditional chromatography is a separation process based on partitioning of mixture components between a “stationary phase” and a “mobile phase”. The stationary phase is provided by the surface of solid materials which can comprise many different materials in the form of particles or passageway surfaces of cellulose, silica gel, coated silica gel, polymer beads, polysaccharides, and the like. These materials can be supported on solid surfaces such as on glass plates or packed in a column. The mobile phase can be a liquid or a gas in gas chromatography. This invention relates to liquid mobile phases.
The separation principles are generally the same regardless of the materials used, the form of the materials, or the apparatus used. The different components of a mixture have different respective degrees of solubility in the stationary phase and in the mobile phase. Therefore, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, sees fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase sees fresh stationary phase and partitions into the stationary phase. Eventually, at the end of the column, there is no more stationary phase and the sample simply leaves the column in the mobile phase.
A separation of mixture components occurs because the mixture components have slightly different affinities for the stationary phase and/or solubilities in the mobile phase, and therefore have different partition equilibrium values. Therefore, the mixture components pass down the column at different rates.
Since chromatographic separations depend on interactions with the stationary phase, it is known that one way for improving separation is increasing the surface area of the stationary phase. The best separations are obtained when the interactions are low, i.e., the partitioning coefficient is low and the column is long, providing increased interactions.
In traditional liquid chromatography, a glass column is packed with stationary phase particles and mobile phase passes through the column, pulled only by gravity. However, when smaller stationary phase particles are used in the column, the pull of gravity alone is insufficient to cause the mobile phase to flow through the column. Instead, pressure must be applied. However, glass columns can only withstand about 200 psi. Passing a mobile phase through a column packed with 5 micron particles requires a pressure of about 2000 psi or more to be applied to the column. 5 to 10 micron particles are standard today. Particles smaller than 5 microns are used for especially difficult separations or certain special cases). This process is denoted by the term “high pressure liquid chromatography” or HPLC.
HPLC has enabled the use of a far greater variety of types of particles used to separate a greater variety of chemical structures than was possible with large particle gravity columns. The separation principle, however, is still the same.
An HPLC-based ion pairing chromatographic method was recently introduced to effectively separate mixtures of double stranded polynucleotides in general, and DNA in particular, wherein the separations are based on base pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al.,
Chromatographia
37:653 (1993); Huber, et al.,
Anal. Biochem.
212:351 (1993)). These references and the references contained therein are incorporated herein in their entireties. The term “Matched Ion Polynucleotide Chromatography” (MIPC) has been applied to this method by the Applicants as their understanding of the DNA separation mechanism has evolved. MIPC separates DNA fragments on the basis of base pair length and is not limited by the deficiencies associated with gel based separation methods.
Matched Ion Polynucleotide Chromatography, as used herein, is defined as a process for separating single and double stranded polynucleotides using non-polar separation media, wherein the process uses a counter-ion agent, and an organic solvent to release the polynucleotides from the separation media. Basic MIPC separations are complete in less than 10 minutes, and frequently in less than 5 minutes. For more difficult separations such as separations using isocratic solvent condition, the separation time will be longer. Effective isocratic and target zone elutions require precise control of the separation conditions.
As the use and understanding of MIPC developed, it was discovered that when MIPC analyses were carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes could be separated from heteroduplexes having the same base pair length (U.S. Pat. No. 5,795,976; Hayward-Lester, et al.,
Genome Research
5:494 (1995); Underhill, et al.,
Proc. Natl. Acad. Sci. USA
93:193 (1996); Doris, et al.,
DHPLC Workshop,
Stanford University, (1997)). These references and the references contained therein are incorporated herein in their entireties. Thus, the use of Denaturing HPLC (DHPLC) was applied to mutation detection (Underhill, et al.,
Genome Research
7:996 (1997); Liu, et al.,
Nucleic Acid Res.,
26;1396 (1998)).
DHPLC can separate heteroduplexes that differ by as little as one base pair. However, separations of homoduplexes and heteroduplexes can be poorly resolved. Artifacts and impurities can also interfere with the interpretation of DHPLC separation chromatograms in the sense that it may be difficult to distinguish between an artifact or impurity and a putative mutation (Underhill, et al.,
Genome Res.
7:996 (1997)). The presence of mutations may even be missed entirely (Liu, et al.,
Nucleic Acid Res.
26:1396 (1998)). The references cited above and the references contained therein are incorporated in their entireties herein.
The accuracy, reproducibility, convenience and speed of DNA fragment separations and mutation detection assays based on DHPLC have been compromised in the past because of DHPLC system related problems. Important aspects of DNA separation and mutation detection by HPLC and DHPLC which have not been heretofore addressed include the treatment of materials comprising chromatography system components; the treatment of materials comprising separation media; solvent pre-selection to minimize methods development time; optimum temperature pre-selection to effect partial denaturation of a heteroduplex during MIPC; and optimization of DHPLC for automated high throughput mutation detection screening assays. These factors are essential in order to achieve unambiguous, accurate, reproducible and high throughput DNA separations and mutation detection results.
A need exists, therefore, for an HPLC system which can separate DNA fragments based on size differences, and can also separate DNA having the same length but differing in base pair sequence (mutations from wild type), in an accurate, reproducible, reliable manner. Such a system should be automated and efficient, should be adaptable to routine high throughput sample screening applications, and should provide high throughput sample screening with a minimum of operator attention.
The basic MIPC separation process differs from the traditional HPLC separation processes in that the separation is not achieved by a series of equilibrium separations between the mobile phase and the stationary phase as the liquids pass through the column. Instea
Gjerde Douglas T.
Sutton John E.
Taylor Paul D.
Therkorn Ernest G.
Transgenomic Inc.
Walker William B.
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