Chemistry: analytical and immunological testing – Including sample preparation – Dilution
Reexamination Certificate
1999-09-23
2003-11-25
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Dilution
C436S034000, C436S164000, C436S166000, C436S052000, C436S053000, C436S085000
Reexamination Certificate
active
06653150
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable
REFERENCE TO A “MICROFICHE APPENDIX”
Not applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the absolute characterization of microscopic particles in solution. More particularly, the present invention relates to the absolute characterization of microscopic particles, such as polymers and colloids using static light scattering (SLS) and time-dependent static light scattering (TDSLS). In principle, the size range of detectability should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 2 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particles should run from about 500 g/mole to 10
14
g/mole, with useful measurability in the range of 500 g/mole to 10
9
g/mole, with a preferred range from about 1000 g/mole to 10
7
g/mole.
The preferred use of this invention is the determination of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. This is to be distinguished from turbidometric and nephelometric techniques, in which turbidity or relative scattering of solutions is measured and compared to relative reference solutions, in order to obtain concentrations of particles. The SLS technique employed refers to absolute macromolecular characterization, and not to determinations of concentrations of particulates with respect to specific relative calibrations, etc. This is also to be distinguished from devices which count and characterize single particles, although the present invention can count and characterize single particles, in addition to making SLS measurements. The least number of particles whose scattered light would be detected in the scattering volume (the volume of illuminated sample whose scattering is measured by a given photodetector) would be on the order of 20 and the maximum on the order of 4×10
17
, with the preferred range being from about 15,000 to 1.5×10
13
particles. In terms of concentration of solute (dissolved polymer or colloid) the range would be from about 10
−8
g/cm
3
(for very large particles) to 0.2 g/cm
3
(for very small particles) with the preferred range being from about 10
−6
to 10
−1
g/cm
3
. It should be pointed out that SLS in the absolute mode requires optically transparent solutions in which single, not multiple, scattering dominates. Many particle concentration detectors actually work in turbid solutions, which is a different range of conditions entirely.
SLS has proven to be a useful technique not only for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, but also for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass M
w
, z-average mean square radius of gyration <S
2
>
z
and second virial coefficient A
2
. Measuring the time-dependent changes in the scattered intensity allows calculation of kinetic rate constants, as well as deduction of kinetic mechanisms and particle structural features (e.g. refs. 2,3). TDSLS can be used to monitor polymerization and degradation reactions, aggregation, gelling and phase separation phenomena (e.g. ref. 4).
In addition to absolute SLS and TDSLS measurements, the present invention can also simultaneously count and characterize individual particles which are much larger than the principal polymer or colloid particles; e.g., the large particles may have a radius of 5 microns, whereas the polymer may have an effective radius of 0.1 micron. The large particles may represent a contaminant or impurity, or may be an integral part of the solution, e.g., bacteria (large particles) produce a desired polymer (e.g., a polysaccharide) in a biotechnology reactor. The number density of bacteria can be followed in time, and the absolute macromolecular characterization of the polysaccharide could also be made (an auxiliary concentration detector would also be necessary if the polysaccharide concentration changes in time).
The present invention involves automatic online mixing and/or dilution of solutions containing polymers and/or colloids in order to provide relative and/or absolute characterization of these microscopic particles in solution. In the following, the term ‘dilution’ will be used, because, whenever two or more solutions are mixed, as described herein, the solutes in each will become dilute. The automatic dilution is intended to replace the traditional prior art of manually diluting such polymer/colloid solutions in order to make characterizing measurements, and to extend measurement capabilities to novel situations, especially those involving non-equilibrium (that is, time-dependent) processes, such as polymerization, degradation, aggregation and phase separation. The method can be used in conjunction with a variety of detectors, such as static light scattering (SLS), time-dependent static light scattering (TDSLS), heterogeneous time dependent light scattering (HTDSLS), dynamic light scattering, refractometry, ultraviolet and visible spectrophotometry, turbidometry, nephelometry, viscometry and evaporative light scattering. The automatic, online dilution of polymer and/or colloid solutions will be shown to have broad applicability in many sectors. In referring to the ensemble of SLS, TDSLS and HTDSLS detectors and methods in the following, the term light scattering (LS) will be used for brevity.
In principle, the size range of detectability of the polymers and/or colloids should run from about 20 Angstroms to 100 microns, with useful measurability in the range from 20 Angstroms to 20 microns, and a preferred range from about 20 Angstroms to 5000 Angstroms. Stated in terms of molar mass, the detectable range of particle molar masses should run from about 500 g/mole to 1014 g/mole, with useful measurability in the range of 500 g/mole to 1011 g/mole, with a preferred range from about 1000 g/mole to 1010 g/mole.
This invention focuses on automated methods that are used to characterize equilibrium and non-equilibrium properties of solutions containing polymers and/or colloid particles. Characterization of polymers and colloids via LS detectors is in terms of average particle masses, static dimensions, interaction coefficients, and other properties, as well as their changes in time, when scattering is from a very large number of particles. When large colloidal particles are present, the use of the method in conjunction with HTDSLS also allows the determination of the number density of these particles, information on their dimensions, and, when the system is not in equilibrium, how these properties change in time.
SLS has proven to be a useful technique for characterizing equilibrium properties of microscopic particles, such as molar mass, dimensions and interactions, and TDSLS and HTDSLS for following time-dependent processes such as polymerization, degradation and aggregation. Measuring the time-independent angular distribution and absolute intensity of scattered light in the equilibrium cases allows the former properties to be determined, according to procedures set forth by Lord Rayleigh, Debye, Zimm and others (e.g. ref. 1). In particular, this invention can be used in conjunction with the well known procedure of Zimm to determine weight average molar mass Mw, z-average mean square radius of gyration <S2>
z
and second virial coefficient A2. Measuring the time-dependent changes in the scattered inte
Gakh Yelena
Garvey, Smith, Nehrbass & Doody, L.L.C.
Nehrbass Seth M.
The Administrators of the Tulane Educational Fund
Warden Jill
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