Abstract:
A method involving the automatic, online dilution of polymer and/or colloid solutions, such that, when the diluted polymer stream flows through suitable detectors, non-equilibrium processes, such as polymerization, degradation and aggregation, can be monitored. The dilution involves a reacting or stock solution of polymer and/or colloid, and at least one solvent. The online dilution technique can also be used to assess the effects of solvent quality and other solutes on polymer/colloid characteristics and reactions, and also permits equilibrium characterization of polymers/colloids by making a single stock solution of the polymer/colloid.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This is a continuation-in-part of my co-pending U.S. patent application Ser. No. 08/969,386, filed Nov. 13, 1997 now U.S. Pat. No. 6,052,184, which is incorporated herein by reference. 
     Priority of my U.S. Provisional Patent Application Serial No. 60/031,095, filed Nov. 13, 1996, incorporated herein by reference, is hereby claimed. 
    
    
     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 &lt;S 2 &gt; 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 &lt;S2&gt; z  and second virial coefficient A2. 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, use of the present invention in conjunction with HTDSLS allows simultaneous counting and characterization of 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 an 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 useful if the polysaccharide concentration changes in time). 
     The method whereby simultaneous, absolute characterization of polymers and number counting of large particles is carried out, is described in U.S. patent application Ser. No. 08/969,386. To optimize the technique, one should make the sample liquid flow relative to the irradiating laser beam (or other light source) in the scattering chamber, so as to produce countable scattering spikes as each large particle passes through the detected portion of the illuminated volume (the ‘scattering volume’), while ensuring, via correct design of the optical and electronic detection system, that there is on the average less than one large particle in the scattering volume at any given time. This allows the scattering level to recover to the baseline scattering of the pure polymer between the scattering spikes due to the large particles, so that the polymer can be absolutely characterized. The fraction of baseline time termed herein ‘clear window time’, and is detailed mathematically in ref. 5, wherein the method has recently been demonstrated. In this demonstration, it was first shown that useful characterization of a polymer solution could be made even in the presence of a large amount of particulate contamination. The contaminant was a known amount of 2 micron latex spheres introduced in increasing amounts to an aqueous polymer solution containing the polymer poly(vinyl pyrrolidone), or PVP. Secondly, the ability to simultaneously make absolute characterization of the polymer while the change in time of the large particle population was monitored was demonstrated by monitoring the growth of  E. Coli  bacteria amidst an aqueous solution of PVP polymer. 
     2. General Background of the Invention 
     SLS is currently used for three main purposes in academic, medical and industrial research and development, and industrial quality control; 1) to characterize useful averages of mass, mean square radius of gyration and second virial coefficient for unfractionated particles in equilibrium, using traditional procedures, 2) to characterize heterogeneous populations of particles which have been fractionated by techniques such as size exclusion chromatography and 3) to follow time-dependent processes. As examples of each purpose: 1) A new biological macromolecule or microstructure is isolated, or a new polymer is synthesized, and its average macromolecular characteristics are determined by SLS. A manufacturer of synthetic polymers, for water treatment, paints, coatings, adhesives, etc., would use SLS for quality control of their product. 2) A synthetic or biological polymer sample contains a wide variety of molar masses (polydispersiiy), and it is desirable to determine the mass and dimension distribution using a fractionation technique coupled to SLS. The purpose of this can be for fundamental research into a biological mechanism, to aid development of new products, to establish quality control specifications of new products, or to assess the effects of different chemical or physical treatments on the product, etc. 3) It is desired to determine how quickly a polymer degrades under attack by such agents as enzymes, heat, radiation, ultrasound, etc., and this can be determined by TDSLS. This will guide studies in developing new pharmacological inhibitors or promoters, or resistant plastics, or biodegradable materials. A central problem of great economic interest, in which TDSLS can be used is to have an on-line method for determination of the build up of molecular weight during industrial polymerization processes. A further use of the present invention will be in the simultaneous measurement of SLS and particle counting in heterogenous solutions. 
     Clearly, SLS and TDSLS have a wide range of applications, including, but by no means limited to products such as pharmaceuticals, foodstuffs, resins, plastics, coatings, inks, adhesives, liposomes, cosmetics, water treatment and paper making chemicals, paints, additives, plasticizers, microencapsulation structures, etc. 
     Current technology generally consists of a transparent, hollow sample cell, usually of glass or quartz, into which a scattering sample is introduced and through which a light beam (usually from a laser) is passed. The scattered light then passes through the walls of the transparent cell, where photodetectors or fiber optic pick-ups are placed. The signal from the detected scattered light is then processed and the properties of the scattering sample deduced. Such systems require that sample be introduced into the cell, remote from the main sample batch itself. A disadvantage of a transparent sample cell is that it creates interfaces between incident light and the sample which produce unwanted stray light or ‘glare’. This stray light or glare, constitutes one of the major pitfalls and nuisances in the actual practice of SLS. Such cells are also relatively expensive and require fairly precise alignment for proper performance. 
     The present invention includes a (preferably miniaturized) submersible probe which can be brought into the sample, rather than vice versa. No transparent cell need intervene between the sample solution, the incident beam and the optical detectors. This reduces the SLS instrument to a small probe, which can be thought of now as a simple lab probe, like that of a pH or conductivity meter, to be used simply and routinely. The probe portion is relatively inexpensive to fabricate, easy to align, and can even be made to be disposable. The photodetectors, signal processing, etc. are normally remote from the probe. It is anticipated that such an SLS/TDSLS probe is substantially more economical, versatile and easy to use than currently available systems. The present invention can be used in a variety of modes for both time-independent and time-dependent measurements; 1) Submersible mode, in which the probe is submerged in a vessel containing sample solution, such as a beaker, test tube, vat, reactor, etc. 2) Fill mode, in which small amounts of sample liquid (about 3 microliters to 30 milliliters, for example) can be simply pipetted, scooped, or otherwise transferred into the probe body. 3) Flow mode, in which by means of an integrally flanged pump, or hydraulic connection to a tube with flowing sample, the sample liquid flows through the invention. This can be used for unfractionated samples, including those undergoing time-dependent processes (polymerization, degradation, cross-linking, etc.), or samples solutions fractionated by SEC or other means. 4) Insert mode in which a standard glass vial or cell containing sample liquid is inserted into the probe body, instead of filling the probe body, immersing it, or flowing the sample through it. This would be used instead of any of the other three modes, when, for example, the sample may be deleterious for the chamber, because of causticity, gelling, precipitation, etc. It can also be convenient and valuable when many samples are independently prepared and are to be measured separately, and/or when the state of prepared samples in sealed cells is to be checked periodically, without disturbing the sample due to flow, pipetting, etc. The demands of a wide variety of users can be satisfied by simply changing low cost, optical probe assembly, since the detection, electronics, computer interfacing and basic software are the same. 
     Brief Comparison With Other Light Scattering Devices 
     The present invention is distinct from other light scattering devices. For example: U.S. Pat. No. 4,616,927 (Phillips, Reece and Wyatt) and U.S. Pat. No. 5,305,073 (Ford) describe the use of highly polished, optically transparent cell for absolute light scattering measurements. The current invention requires no optically transparent cell. Neither ofthese inventions are submersible, nor can either be considered as a ‘probe’ which can go into the sample liquid being measured. Neither, hence, can fulfill the probe function ofthe current invention, and both are also less versatile, and more costly in general. A submersible light scattering probe is presented by U.S. Pat. No. 5,350,922 (Bartz), but is designed for relative measurements of fairly turbid media (e.g. muds in suspension in water). They collect scattered light, indiscriminately, from 0 to 180° scattering angles, and hence cannot perform absolute light scattering on samples requiring exact specification of the scattering angle (their system could work for Rayleigh scatterers, i.e. for sizes much smaller than the incident radiation wavelength; since they are chiefly looking at particulates in suspension, which are generally very large, this condition would not be expected to be met). Furthermore, their device does interpose an optically transparent medium between the light source and the sample liquid and the detector and the sample liquid. In addition, that device cannot be used in either ‘fill mode’ nor flow mode, and hence is also considerably less versatile. None of these inventions mentioned have the versatility and interchangeability of the present invention. 
     The use of simultaneous multi-angle detection is shown in U.S. Pat. No. 3,850,525, “Simultaneous Multiple Measurements in Laser Photometers”. 
     The following patent documents are incorporated herein by reference: U.S. Pat. Nos. 3,850,525; 3,954,342; 4,265,535; 4,363,551; 4,548,500; 4,616,927; 4,995,514; 5,129,723; 5,155,549; 5,235,179; 5,305,073; 5,350,922; 5,434,667; 5,638,174; and Great Britain patent application Ser. No. 2166234. Also incorporated by reference are the following papers: Florenzano, Strelitzki and Reed,  Macromolecules , vol.31, pp.7226-7238,1998, “Absolute, On-line Monitoring of Molar Mass during Polymerization Reactions”; Strelitzki and Reed,  Journal of Applied Polymer Science , vol. 73, pp. 2359-2368, 1999, “Automated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometry”; Schimanowski, Strelitzki, Mullin, and Reed, “Heterogeneous Time Dependent Static Light Scattering”,  Macromolecules , (in press—copy attached). 
     Online methods of determining polymer and/or colloid properties are becoming increasingly important in both academic and industrial situations. One pressing need in the polymer industry is for automated, online systems that monitor polymerization reactions in bench scale, pilot plant, and full scale reactors. Numerous empirical means are typically used, including viscometric and hydrodynamic sensors, but none provide absolute, online measures of polymer Mw and polydispersity. Because most polymerization reactions are run at high concentration of reacting monomers, and because virtually all physical methods for determining Mw and other intrinsic properties of individual polymers require highly dilute solutions, it is not generally feasible to make such absolute methods directly on the reaction liquid. Rather, dilution must occur. Prior art in making dilutions of polyimer solutions for LS and viscometric characterization, is to achieve this manually, a time-consuming, tedious process, which can only yield data points widely separated in time. Although automatic dilution has been standard practice for many years in instruments such as automatic chemical titrators, which characterize equilibrium properties of solutions, the inventor is unaware of such a practice for polymer/colloid solutions, where totally different detectors need to be used, and a wide array of properties, phenomena and reactions can be monitored online. Ref. 6 details the first use of the online dilution technique in conjunction with LS, viscometric, refractometric and ultra-violet absorption detectors for monitoring polymerization reactions. 
     Other areas of application for the automated online dilution technique include monitoring degradation and fermentation reactions, stability of solutions, and robotic automation of equilibrium characterization for polymers/colloids. 
     The main idea behind the current invention is the automatic dilution of a stock of polymer and/or colloid solution so that equilibrium and/or non-equilibrium properties can be determined online. This involves withdrawing a fraction of material from the polymer stock solution vessel, at the same time that other liquid(s) is (are) drawn from another vessel(s). The automatic dilution can take place by a variety of methods. In its simplest form, one or more hydraulic ‘T’ fittings (or a single multiple-port fitting) with at least two capillaries or tubes can be used. The lengths and internal diameters of the capillaries or tubes can be chosen so as to establish the fraction of material that is automatically and continuously drawn from each vessel. The mixing takes place within the ‘T’ junction, or that of a multi-port fitting. Other methods include using programmable mixing pumps, set to withdraw specified fractions from two or more vessels, or binary, tertiary, quaternary or more complex pumps, which incorporate the mixing and high pressure outlet pumping capabilities in one unit. Such pumps are traditionally used to form gradients of solvents for use in chromatographic techniques such as high pressure liquid chromatography (HPLC), but the inventor is unaware of their use for the characterization of polymer solutions as set forth herein. 
     The applications of the automatic dilution technique include, but are not limited to: 
     1) The monitoring of polymerization reactions (see U.S. patent application Ser. No. 08/969,386), whereby a small quantity of reacting solution is continuously and automatically withdrawn from the reactor, and automatically mixed with one or more solvents, so as to bring the concentration of polymer into a dilute-enough regime so that LS and auxiliary techniques can be used to make an absolute, online characterization of the polymers. When combined with a concentration detector (ultraviolet and/or visible spectrophotometer, refractometer, evaporative light scattering detector, or other) the polymer weight average mass, M w , and the root mean square radius of gyration, Rg, can be monitored online. In some cases, the degree of monomer conversion will also be monitored online. By adding a flow type viscometer, of either the single or multiple capillary type, the reduced viscosity of the polymer can also be measured online. Sometimes a flow-type viscometer could be used prior to dilution to determine total solution viscosity. Since the reduced viscosity measured is a viscosity average of the entire, normally polydisperse population, it can be combined with the M w  yielded by LS and the concentration detector(s) to give a useful online index of the polydispersity of the polymer as it is produced in the reactor. 
     2) Automated determination of equilibrium properties of unfractionated polymer solutions can be made. The method allows a single stock solution of polymer to be made and automatically diluted in steps or in a continuous gradient. Coupling of an LS detector then allows determination of M w , Rg, and the second and third virial coefficients A 2  and A 3 , respectively. If a flow type viscometer is added, the reduced viscosity of the solution can also be determined. This method has been a recently demonstrated in ref. 7 for the characterization of PVP in terms of M w , Rg, reduced viscosity and A 2 and A 3 . 
     This method can be of considerable utility when 1) A 2  and higher virial coefficients are to be determined. Size Exclusion Chromatography (SEC), because it operates at very low polymer concentration, does not permit such determinations. 2) The polymers are too large to be separated by the SEC columns and merely elute in the void volume. 3) The polymers might damage expensive SEC columns, or it is not known which columns can be used to separate the polymers. 
     Furthermore, the automated technique lends itselfnaturally to robotic automation, which can be of considerable utility in situations where high sample analysis throughput is needed. 
     3) By combining the automatic dilution technique with HTDSLS online monitoring of bioreactors can be accomplished. In many bioreactors a microbial species, such as a bacterial or yeast population, co-exists and interacts with a polymer population. This includes cases where polysaccharides (e.g. xanthan) are produced by bacteria and traditional fermentation where biopolymers are broken down by yeast or other organisms. Another instance is in paper and pulp processing, where large cellulosic particles are gradually degraded by acids and other agents. Yet another situation arises in polymer reactors, where cross-linked or highly entangled aggregates, as well as spherulites and other polymeric particles may be produced in addition to individual polymer chains. In all these cases, the automatic dilution coupled to HTDSLS and a concentration detector will allow the simultaneous absolute characterization of the polymeric population and number density counting of large particles, and how they evolve in time. 
     4) The automatic dilution technique can be used to assess the effect of different solvents on a particular polymer, or interacting polymer/polymer or polymer/colloid system online. For example, the inventor and co-workers recently demonstrated how the automatic dilution technique could be used, in conjunction with an online capillary flow viscometer, to study the electroviscous effect in polyelectrolyte solutions (unpublished results, data curves attached in FIG.  3 ). When a polyelectrolyte solution at a very low initial ionic strength, whose nominal value is that of an added simple electrolyte, is diluted with a stock solution of the same ionic strength, the dilution is actually not isoionic because the counterions of the polyelectrolyte contribute to the initial ionic strength, but are diluted as dilution with the fixed ionic strength stock occurs. Hence the total ionic strength (due to added simple electrolyte and the polyelectrolyte counterions) decreases as this type of dilution proceeds. The consequence is the type of maxima in reduced viscosity vs. polyelectrolyte concentration seen in FIG.  3 . 
     These data represent the first demonstration, to the inventor&#39;s knowledge, of a continuous, online determination of the electroviscous effect. Until now, prior art required manual dilutions, which yielded only a small number of relatively widely separated concentration points. 
     Many other applications of the automatic dilution technique in the context of testing solvent effects and interactions properties can be envisioned. For example, by using a total of three reservoirs—one with the stock concentration of polyelectrolyte, one with a pure water or low ionic strength solution, and the other with a high ionic strength solution—the complete ionic strength behavior of a polyelectrolyte at constant concentration could be determined in a single, automated experiment. Coupling LS and viscosity detectors will yield both the change in static dimensions and polymer hydrodynamics in response to the changing solvent composition. Again, until now, state-of-the-art has required tedious, time-consuming manual mixing of solutions to make such studies. 
     A further field of application is in the area of polymer/polymer, polymer/colloid, and colloid/colloid interactions. Again, using multiple reservoirs, it will be possible to automatically and continuously monitor how polymers and colloids interact as such factors as their concentration, solvent qualities, and additives (e.g. small molecules such as urea) change. This can be of considerable use, for example, in pharmaceutical screening, tests of the flocculating power of new water purification agents, precipitation tests for proteins, and general stability tests for aggregating systems. 
     Clearly, the online dilution and auxiliary techniques have a wide range of applications, including, but by no means limited to products such as pharmaceuticals, foodstuffs, resins, plastics, coatings, inks, adhesives, liposomes, cosmetics, water treatment and paper making chemicals, paints, additives, plasticizers, microencapsulation structures, etc. 
     Brief Comparison With Other Techniques 
     The inventor is unaware of any techniques which use the automatic dilution of polymer and/or colloidal solutions in order to make online characterization of non-equilibrium properties, such as polymerization or degradation. On the other hand, the typical detector train scheme used in conjunction with the invention is quite similar to detector trains used in Size Exclusion Chromatography (SEC). 
     In SEC a small quantity of a fixed concentration of polymer solution is injected into a column fractionation system, wherein a certain uncontrolled amount of dilution occurs, after which measurements of equilibrium properties are made. Some chromatography techniques also automatically vary the composition of the solvent that a polymer is eluted in. Again, these are for equilibrium determinations, and can never be considered as online means for characterizing reactions and other non-equilibrium processes in polymer solutions. 
     Attempts at measuring polymerization reactions in real-time are generally performed on the reaction solution, either within the reactor or on sample withdrawn from the reactor. None of these use, to the inventor&#39;s knowledge, the online dilution technique, nor do any use the online dilution technique coupled with LS and concentration detectors, in order to have a continuous, online record of M w  and associated quantities. 
     Incorporated by reference are the following papers: Florenzano, Strelitzki and Reed,  Macromolecules , vol. 31, pp. 7226-7238, 1998, “Absolute, On-line Monitoring of Molar Mass during Polymerization Reactions”; Strelitzki and Reed,  Journal of Applied Polymer Science , vol. 73, pp. 2359-2368, 1999, “Automated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometry”; Schimanowski, Strelitzki, Mullin, and Reed, “Heterogeneous Time Dependent Static Light Scattering”,  Macromolecules , (in press—copy attached). 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention is the first fully submersible SLS probe for absolute macromolecular characterization (as opposed to particle counting, nephelometry, dynamic light scattering, or relative concentration measurements). The optical assembly of the present invention can be completely immersed in the scattering medium. Thus, the present invention includes a scattering probe which can ‘go into’ the medium to be measured (e.g. into test tubes, production vats, etc.), and samples of the scattering medium need not be introduced into a transparent sample cell remote from the medium itself, as is done in current systems. In the present invention the probe can be submerged in a variety of harsh environments, as concerns temperature, pressure and solvents, and communicates to the remote electronic and signal processing portion via a harness containing fiber optic cables. 
     The present invention can be used in several distinct modes (immersion, fill mode, insert mode and flow mode), giving it wide versatility. The probe of the present invention is not constrained to be immersed in order to function. A small quantity of sample can also be placed in the optical assembly compartment for measurement in a ‘fill mode’. A sample in a transparent vial or cell can also be placed in the chamber or ring member for measurement. Also, the probe can be hooked into a flowing stream of sample liquid for use in different applications such as polymer separation (e.g. size exclusion chromatography), and on-line, unfractionated flows of polymers in a vessel in equilibrium, or undergoing polymerization, aggregation, cross-linking or degradation processes. 
     The present invention can respond to the needs of a wide variety of users and applications by simply changing the inexpensive optical assembly, since the detection, electronics, computer interfacing and basic software are all the same. For example, a miniature probe with a 10 microliter channel could plug into the same ‘detection/analysis’ back-end as a 50 milliliter optical probe designed for immersion at high temperatures. There is wide room for substitution of different diameter fibers with different acceptance angles, number of photodetectors on the ‘detection/analysis’ back-end, etc. 
     The present invention does not require a transparent sample cell for the scattering solution. Unlike all current SLS systems for absolute macromolecular and colloidal characterization, no glass or other transparent cell need intervene between the sample, the detection fibers and the fiber or lens used for introducing the incident beam. Major advantages which this confers includes avoiding the expense, maintenance and cleaning of transparent cells, and minimizing glare and stray light, because the optical assembly is preferably made from a very dark or black material, and hence does not have highly reflective glass and/or other dielectric surfaces causing spurious glare and reflections. 
     The optical probe portion of the present invention is preferably miniature in scale. Whereas other devices also use only small sample volumes, those devices require that the sample be pumped or injected in through appropriate plumbing. In the present invention, when used in the fill mode, small quantities of sample can be simply pipetted or dropped into the optical assembly compartment, where they reside during the measurement. 
     The probe can achieve both absolute calibration and self-cleaning simultaneously when immersed in a proper solvent, such as toluene. Furthermore, because of the direct immersion there are no problems with index of refraction corrections associated with cells which do not maintain cylindrical symmetry about an axis perpendicular to the scattering plane. Hence, well-known, non-proprietary standard calibration procedures can be used for each detector. 
     The versatile scattering chamber is very inexpensive to fabricate and, in some instances, can be even treated as disposable. This contrasts to the generally high cost of the scattering cell/detector assembly in prior art units. 
     Unlike existing SLS units, the use of fiber optic detectors and narrow beam focusing make the system quite insensitive to alignment. This has the significant advantage of allowing the unit to operate with a simple coarse alignment, whereas a high degree of alignment is normally required in existing systems. This is achieved because the acceptance cone of the fibers is fairly large (typically 9°) and the beam is collimated to usually less than 100 microns. Hence, at a remove of 3 mm from the fiber, the beam can be moved up and down approximately 0.5 mm for a 9° acceptance angle fiber, without significantly changing the amount of scattered light entering the fiber. 
     Properly minimizing the scattering volume with a focused beam and using fiber optic detectors and fast detection electronics allow unfiltered samples to be measured, even when no flow or other relative motion between sample and detector exists. This is a major advance, considering that SLS in conventional instruments only became reliable after chemical filtration technologies improved considerably. 
     The present invention includes a submersible device, which measures relative light scattered at various angles from a large number of scattering particles, from which absolute macromolecular and colloidal characterization is made, via well known, non-proprietary calibration procedures and the well known procedures of Zimm and others. The device need not contain an optically transparent cell interposed between the scattering medium and the incident optic delivering the incident beam and the optical fibers used for detection. 
     The submersible absolute macromolecular characterization device described in the previous paragraph preferably consists of a completely solid or perforated, or striated or otherwise partially open solid piece, a ring member or a cylinder with a channel inside into which sample liquid enters upon immersion. In this device, polarized or unpolarized incident light (provided by a laser or any other source of visible or ultraviolet light) is led into the channel and spatially filtered with any suitable optical elements such as a tubular lens, miniature convex lens, flat window, fiber optic, irises, etc., or any suitable combination. The light so led in can undergo any necessary degree of collimation, including none, in order to make as narrow an incident beam waist in the detected scattering volume as desired. Scattered light detection is preferably achieved by fiber optic strands, or other fiber optic light conduits, which are exposed to scattered light in the channel, either by virtue of being recessed into the walls of the channel, being flush with the walls of the channel, or protruding into the channel. The degree of collimation of incident light and the diameter of the detecting fibers are combined to optimize the detected scattering volume for the particular sample to be measured. The transmitted incident light is preferably ‘dumped’ using any standard beam dump arrangement, such as a hole, Rayleigh horn, prism, etc. The channel is preferably black or blackened to reduce glare and stray light from the incident beam. The delivery and detection optical train elements are preferably gathered into a harness leading to the photodetectors, amplifiers and computer external to the light scattering probe. 
     Instead of the probe mentioned above which can be immersed in sampling liquid, a different probe can be provided, into whose channel, plugged at one end, rather, a small quantity of sample liquid can be transferred (e.g. by pipette, or by scooping) and therein reside while the scattering measurements are made. 
     Likewise, a third probe having suitable liquid flow connectors need not be immersed in sampling liquid; instead, through its channel the sample liquid can be made to flow for scattering measurements. 
     The submersible absolute macromolecular characterization device described above can consist of a ring member, not necessarily closed or circular (e.g. rectangular, elliptical, horseshoe, or any other shape capable of holding the light source fixed relative to the detection fibers (or photodetector when detection fibers are not used)) containing the incident beam delivery optics, beam dump and detection fibers, and which can be immersed directly in a sample liquid for scattering measurements. Alternatively, the submersible absolute macromolecular characterization device described above can consist of a ring member, not necessarily closed or circular (e.g. rectangular, elliptical, horseshoe, etc.) which can be placed inside of a chamber in a cell of appropriate dimension, so as to protect it from the liquid it is immersed in, ambient light or other factors, or to otherwise control how sample liquid reaches the ring member for scattering measurements. 
     The present invention includes a method whereby any of the devices described above, with appropriately small scattering volume, can be used to measure sample solutions which may contain significant numbers of large scattering contaminants by using fast enough photodetector response to identify, count and eliminate scattering intensity spikes produced by the contaminants, thereby enabling the recovery of the uniform scattering background due to the population of polymers or colloids in the sample. The sample may be either stationary or flowing to accomplish this. Very roughly, the number density of contaminant particles can be on the order of one per scattering volume, so that very tiny scattering volumes allow for relatively higher concentrations of impurity to be present. The identified spikes can be counted and used to assess the particle density of large particles in a solution, and how this number may change in time, as well as simultaneously determining the absolute uniform scattering from a population of polymer or colloids. 
     The present invention also includes a method whereby the flow mode of the present invention described herein can be used to measure, in real-time, the increase of the weight average molecular weight of polymers being produced in a solution of chemicals undergoing polymerization reactions. This method preferably includes the on-line dilution of the polymer containing solution to bring it into a concentration range where useful, absolute scattering can be measured. This range is where the quantity 2A 2 cM w  is preferably smaller than 1, but can actually be as much as 10. Such dilution can be achieved by the use of hydraulically pulling polymer solution and pure solvent through an hydraulic ‘T’ or other mixing chamber via a pump or other flow-causing device. A concentration sensitive detector is preferably installed in the line of fluid flow so as to determine in real-time the actual concentration of polymer in the diluted solution. Such a detector may be a refractive index monitor, ultraviolet or visible spectrophotometer, etc. 
     The present invention also includes a method whereby any of the devices herein described are used to monitor the changes in time of polymer solutions which are undergoing degradation, polymerization, aggregation, gelling, or phase separation. 
     The present invention also includes a method whereby any of the devices herein described are used to usefully characterize heterogeneous solutions, containing populations of both polymers or colloids and large particulate scatterers, whether either or both of these changes in time or not. 
     The present invention comprises a kit including light scattering devices of the type described herein, whereby a wide variety of optical probes (with widely varying dimensions, sample capacities, fiber optic types, numbers of angles) made of different materials to withstand different environments can be connected to the same ‘back-end’ of detection electronics, signal processing and data analysis. The kit can also include the detection electronics, signal processing and data analysis. 
     The present invention also includes a submersible light scattering probe for the absolute characterization of polymer and colloid solutions which includes a ring member made of a preferably dark, opaque material, having embedded therein a plurality of optical fibers which can be connected to optical detectors remote from the probe. The ends of the optical fibers are preferably in direct contact with the fluid being tested. Instead of submersing the probe in a fluid, fluid can be caused to flow through the probe, placed in the probe, or placed in a transparent vessel placed in the probe. Individual large scattering particles can also be detected, counted, and characterized at the same time absolute characterization of the polymer or colloid solution is performed. 
     This method preferably includes the on-line dilution of the polymer-containing solution to bring it into a concentration range where useful, absolute scattering can be measured. This range is where the quantity 2A2cMw is preferably smaller than 1, but can actually be as much as around 10 (or even higher). Such dilution can be achieved by the use of hydraulically pulling polymer solution and pure solvent through an hydraulic ‘T’ or other mixing chamber via a pump or other flow-causing device. A concentration sensitive detector is preferably installed in the line of fluid flow so as to determine in real-time the actual concentration of polymer in the diluted solution. 
     Such a detector may be a refractive index monitor, ultraviolet or visible spectrophotometer, etc. 
     FIG. 16 illustrates the scheme used by the inventor et al. (ref. 6) for the online monitoring of a poly(vinyl pyrrolidone), or PVP, reaction. 
     The present invention also includes a method whereby heterogeneous solutions, containing populations of both polymers or colloids and large particulate scatterers, can be characterized, whether either or both of these changes in time or not. 
     FIG. 17 shows a three vessel scheme, wherein one vessel contains the polymer or colloid to be characterized, and two other vessels are used, each of which contains different solvents. For example, the polymermight be electrically charged (i.e. a polyelectrolyte) and be dissolved in pure water in the first vessel, whereas solvent #1 might be pure water, and solvent #2 an aqueous solution containing salt. With such an arrangement it would be possible to maintain a fixed polymer concentration by pulling a fixed fraction from the first vessel, while the total salt concentration that the polyelectrolyte is subjected to is continuously changed from pure to very salty water (e.g. 4 molar NaCl). Since the concentration of polyelectrolyte is fixed, and known, a LS detector alone would furnish online information on how the polyelectrolyte conformations and interactions are changing as the solvent becomes more salty. Adding a viscometer would further indicate how the polyelectrolyte hydrodynamic properties are changing with salt concentration. 
     Similarly, other types of polymers and/or colloids could be in the first vessel, and solvent #1 could be of one type (e.g. pure water) and solvent #2 could be of another type (e.g. an alcohol or other solvent miscible in water). In this way the effects of changing solvent composition on the polymer and/or colloid could be continuously assessed online. Many other variations are possible, since the second solvent could also contain a polymer and/or colloid which interacts with the first polymer and/or colloid solution. The three vessel arrangement hence allows complete phase diagrams to be obtained online. Another area of use would be to determine under what solvent conditions globular polymers, such as proteins, become denatured into random coils. 
     Extension to more than three vessels is straightforward and is contemplated by the inventor. 
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
     For a further understanding of the nature, objects, and advantages of the present invention, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements and wherein: 
     FIG. 1 is a perspective view of the preferred embodiment of the apparatus of the present invention; 
     FIG. 2 is a side view of the preferred embodiment of the apparatus of the present invention immersed in a sample liquid; 
     FIG. 3 is a side view of the preferred embodiment of the apparatus of the present invention being used in a flow mode; 
     FIG. 4 is a perspective view of the preferred embodiment of the apparatus of the present invention being used in a fill mode; 
     FIG. 5 is a perspective view of the preferred embodiment of the apparatus of the present invention being used in an insert mode; and 
     FIG. 6 is a perspective view of an alternative embodiment of the apparatus of the present invention; 
     FIG. 7 is a schematic of how a diode laser might be incorporated into a base plate in a ring member version of the present invention; 
     FIG. 8 is a schematic representation of a ‘pinhole mode’ of detection; 
     FIG. 9 is a schematic representation of an ‘acceptance angle mode’ of detection; 
     FIG. 10 shows a fill mode Zimm plot for high molecular weight PVP irradiated with a 10 mW Argon ion laser and each angle calibrated to pure toluene; 
     FIG. 11 shows an immersion mode Zimm plot for unfiltered solutions of high molecular weight PVP (“1.3MD” PVP) irradiated with a 10 mW Argon ion laser, using 150 micron optic fiber in 3 inch diameter vessels of solution; 
     FIG. 12 shows a flow mode Debye plot for high molecular weight PVP at θ=90° irradiated with a 488 nm Argon ion laser, compared to the results of a Wyatt Dawn-F at 144° (633 nm He—Ne laser), with error bars; 
     FIG. 13 shows a flow mode measurement of a 0.5 mg/ml high molecular weight PVP (1.3MD PVP) solution with “contamination” by 10 micron latex spheres, using a 300 micron optic fiber at 90° and a 5 mW diode laser; 
     FIG. 14 shows a flow mode measurement of a polymerization reaction; 
     FIG. 15 shows the relative intensity converted to apparent mass (Kc/I) using equations (1)-(3), plotting approximate apparent mass versus real-time for PVP polymerization using a flow mode, using a 300 micron optic fiber at 900 and a 5 mW diode laser, and starting with 300 mg/ml VP diluted to about 6 mg/ml on-line; 
     FIG. 16 illustrates the scheme used by the inventor et al. (ref. 6) for the online monitoring of a poly(vinyl pyrrolidone), or PVP, reaction; 
     FIG. 17 shows a three vessel scheme, wherein one vessel contains the polymer or colloid to be characterized, and two other vessels are used, each of which contains different solvents; and 
     FIG. 18 shows typical online, electroviscous data for hyaluronic acid. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The preferred embodiment of the present invention is a submersible light probe  20  (see FIG. 1) including a ring member  21  made of a preferably dark, opaque material, having embedded therein a plurality of optical fibers  41  which can be connected to optical detectors  118  (see FIG. 7) remote from the probe  20 . The ends  44  of the optical fibers  41  are preferably in direct contact with the fluid  51  (see FIG. 2) being tested. In a first variation of the invention (see FIG.  3 ), fluid is caused to flow through the probe  20 . In a second variation (see FIG.  4 ), a base plate  81  is added so that the ring member  21  can contain a fluid to be tested. In a third variation of testing (see FIG.  5 ), a clear container containing fluid to be tested is placed through the ring member  21 . In yet another variation of the invention (preferably only when the probe is not submersible), photodetectors can replace the optical fibers. 
     The purpose of the probe is to measure light scattering by particles in a fluid (static light scattering (SLS)). 
     It is believed that this is the first probe for SLS or TDSLS where light detectors (the optical fibers) are actually in the fluid, as opposed to being separated from the fluid by glass or some other media. 
     FIG. 1 is a perspective view of the minimal ring member version  20  of the present invention. FIG. 1 shows the essential layout of the ring member-version optical assembly  20 , with fiber optic detectors  41 , beam dump  32 , and a laser beam  31  entering a chamber  22  through a window  23 , either through local mounting and lensing, or via fiber optic transfer through one of the harness fibers. The ring member channel  22  may alternatively have a square or polygonal cross-section, instead of circular, which may be particularly useful for single or few angle detection. Such single or few angle detection may warrant simply mounting photodiodes on the sideof the chamber, rather than using fiber optics. The ring member o.d. and i.d can vary widely, depending on the application (specific dimensions for test versions are given in the “Experimental Verifications of the Invention”, below). The range of i.d. can be, for example, from about 2 mm to 50 cm, with the o.d. being determined by desired wall thickness, which can, 30 for example, range from about 1 mm to 10 cm. The length of the ring member can also, for example, vary from about 3 mm to 10 cm. Optionally a cowl  114  (see FIG. 7) made of rigid or flexible dark material can be placed over the ring member in any of its modes of operation to shield against ambient light. 
     FIG. 2 shows the immersion mode of the present invention. The ring member assembly  20  is attached to a handle  61 . The hollow handle  61  contains an optical harness  43 , which has been formried by drawing all the optical fibers  41  together. A sheath  45  on the outside of the ring assembly  20  protects the fibers  41  that are led into the harness  43 . A diode laser  62  can be mounted directly on or to the handle  61  for an integral optical assembly/light source version, or the beam can be led in through a fiber optic in the harness  43 . 
     FIG. 3 shows the flow mode of the present invention. Ring member assembly  20  is sandwiched between two end-pieces  71 , each of which has a hydrodynamically shaped flow channel  72 , and standard HPLC tubing and fittings  73  for liquid to be injected through the ring member assembly  20  via syringe, pump, etc. There are preferably O-rings  74  between the ring member  20  and the end-pieces  71 , and the three pieces are held together by through-bolts  75 , or a bracket. 
     FIG. 4 shows the fill mode of the present invention. Ring member assembly  20  can have a base plate  81  attached, so that sample solutions can be pipetted, scooped, or otherwise introduced into the channel, as with dropper  85 . A simple modification of ring member assembly  20  could involve not boring the channel  22  all the way through the ring member assembly  20  instead of using a removable base plate  81 . 
     FIG. 5 shows the insert mode of the present invention. A cylindrical vial or cell  92  containing sample solution  91  is simply inserted into ring member assembly  20 . This can be advantageous where the sample  91  may be damaging to the ring member assembly  20 , or where multiple samples are prepared and stored in vials and are to be measured individually on multiple occasions. 
     FIG. 6 shows the integral chamber version  100  of the probe of the present invention. By lengthening the ring member version, a one piece unit  100  can serve for both the flow chamber, to which HPLC connections are directly made, and for fill and immersion modes. Chamber o.d. and i.d. follow the ranges mentioned above, whereas the length for any given chamber can considerably exceed the ring member lengths; e.g., lengths can be from about 1 cm to 30 cm. Channel bore  102  can optionally be tapered. In FIG. 6, the laser input  31  can either be through lensing or via fiber. 
     FIG. 7 is a schematic of how a diode laser  62  might be incorporated on or into a base plate  161  in the ring member version  20  (applicable also to the chamber version  100 ). Also shown is an optional cowl or hood  114  to cover the ring member assembly  20  to reduce any effects of ambient light. Also shown is the overall schematic of the optical assembly attached via optical harness  43  to the photodiode/electronic assembly  111 , which then transmits scattering signals to a microcomputer  112 . If a remote laser is used, instead of on the base plate  161 , then the laser would normally be housed with the photodetectors  118 , and the beam led into the ring member assembly  20  or chamber  100  via a fiber in the optical harness  43 . In FIG. 7, a converging lens  63  is used to focus the laser beam. 
     FIGS. 8 and 9 are schematic representation of detection modes. The ‘pinhole mode’ (FIG. 8) occurs when the fiber  41  is not completely inserted into the through-hole  42  in the chamber wall, and the angle defined by the end  44  of the fiber  41  and the end of the hole is less than the acceptance angle of the fiber  41  in the particular solvent in which it is immersed. The “acceptance angle mode” (FIG. 9) is when said angle is larger than the acceptance angle of the fiber, which means the acceptance angle of the fiber itself will define the scattering volume  121 . 
     FIG. 10 shows a fill mode Zimm plot for high molecular weight PVP. 
     FIG. 11 shows an immersion mode Zimm plot for high molecular weight PVP. 
     FIG. 12 shows a flow mode Debye plot for PVP at θ=9°. 
     FIG. 13 shows a flow mode measurement of a 0.5 mg/ml high molecular weight PVP solution with ‘contamination’, by  10  micron latex spheres. The spheres were in a concentration of 40,000 particles/cc. It is possible both to count the number of spheres passing through the scattering volume, and obtain the absolute scattering due to the PVP, when using the program REEDFLO (see Appendix A of parent patent application Ser. No. 08/969,386) on DT2801a. Thus, the present invention can simultaneously conduct absolute macromolecular characterization of one substance and individual particle counting and characterizing techniques on another substance in the same fluid. 
     FIG. 14 shows a flow mode measurement of a polymerization reaction. Vinyl pyrrolidone monomer at 300 mg/ml at T=80° C. is polymerized using hydrogen peroxide initiator. The polymerizing mixture is withdrawn by a mixing pump, which dilutes the PVP to about 6 mg/ml. The diluted mixture is then pumped through the flow cell where the scattering is monitored continuously. Optionally, a concentration detector, such as an index of refraction detector, or ultraviolet or visible spectrophotometer, can be placed in the line of sample flow. 
     FIG. 15 shows the relative intensity converted to apparent mass (Kc/I) using equations (1)-(3). 
     The preferred embodiment of the present invention consists of an optical assembly  20 , from which a harness  43  of fiber optic cables  41  leads out detected scattered light to a remote photodetector and signal processing unit  111 ,  112 , and optionally brings in incident light. The signal processing unit  111 ,  112  is itself composed of standard components such as photodiodes  118 , photomultiplier tubes, amplifiers, discriminators, microcomputer  112 , etc. 
     The optical assembly  20  preferably consists of a solid material. The minimal version consists of a ring member  21  around which the fiber optic detectors  41 , incident beam input optics  31 , and beam dump  32  are arrayed (see FIG. 1 for this embodiment). The optical fibers  41  are either cemented into holes  42  in the ring member  21 , or are affixed with tiny optical fiber chucks (not shown), and are gathered into a ruggedized harness  43 , which is led to the photodetector assembly  111 ,  112 . The optical assembly  20  can be connected to a handle  61 , which may contain a laser  62 , and can be immersed directly in a sample solution  91  (see FIG.  2 ). The ring member  21  can also be mounted on a base plate  161 . The ring member  21  can also serve as a center portion for a segmented chamber, to the endpieces  71  of which are connected hydraulic fittings  73  for fluid to be pumped in and out through in the flow mode (FIG.  3 ). A small baseplate  81  can be attached to the ring member assembly  20  for fill mode use (FIG.  4 ), or the bore  22  in the ring member assembly  20  simply need not be perforated all the way through. For insert mode, a sample vial  92  can be inserted directly into the ring member assembly  20  (FIG.  5 ). In cases where ambient light might give detectable interference, the ring  20  can be covered with a simple cowl or hood  114  in both immersion, fill and insert modes. In the tests presented below, ambient light was not a problem, and no cowl or covering was used. 
     An integral chamber version  100  (see FIG. 6) can also be made, and consists of a hollow channel or wall  101 , normally cylindrical, but which may also have elliptical, square or polygonal cross section. The chief difference between the minimal ring version  20  and integral chamber version  100  is that the chamber  102  is simply longer than the ring chamber  22 , so that hydraulic fittings  73  can be directly connected. Furthermore, the extra length provides additional shielding from ambient light, and no cowl or other covering should generally be needed. 
     In either the ring version  20  or chamber version  100 , the internal diameter can be made over a wide range, depending on the application. Typically this diameter will run from about 1 mm to 20 cm. The total channel volume may range, for example, from about 3 to 50,000 microliters, with a preferred range of 10 to 1000 microliters. The wider the channel diameter the less problem there will be with stray light, but more sample solution will be required. In industrial settings, for example, where large volumes of sample are produced, and/or the samples are viscous, high volume cells may be a convenient solution, and pose the most robust and reliable means of achieving low stray light and highest ease of alignment. In situations where sample volume is scarce, e.g. in biotechnology research where only milligrams or less of substance is available, the channel will be made much narrower. Because the optical detection fibers  41  can plug into the same remote array  111  of photodetectors  118 , the only change in fabrication in meeting the demands of the high sample volume vs. the low volume user is in the low cost optical probe assembly  20 ,  100 . All photodetection, electronics, computer interfacing and basic software  111 ,  112  can remain the same. 
     In the walls of either the ring member or chamber versions, are seated an optical window  23 , lens, fiber, or other component for delivering the incident beam into the channel, as well as optical fibers  41  for detection of scattered light placed at any number of scattering angles, usually from about 10° in the forward direction to about 170° in the backscattering direction. A detection fiber can also be placed at the site of the beam dump (0°). The fibers  41  can be cemented into holes  42  in the chamber  22 ,  102 , or held in with tiny optical fiber chucks. Hence, the delivery element for the incident light and the optical fibers are in direct contact with the sample solution, or may be coated with a suitable transparent material, including glass, for protection against deleterious sample solutions. In the case for example where only a single or few angles are desired, small photodetectors (such as photodiodes) can be affixed directly to the outside wall of the chamber, thus eliminating the optical fibers  41 . 
     The body of the optical assembly in either ring member or chamber versions can be constructed of any material suitable to withstand the nature of the sample solution, such as stainless steel, black anodized aluminum, ceramic, Teflon, nylon, polycarbonate, or other plastics. The material is preferably opaque, preferably black or blackened, to minimize glare and stray light. 
     The power of the incident light is arbitrary, but will typically range from 0.1 to 100 mW. For good detectability and economy, the power range will preferably be from 0.25 to 50 mW. The wavelength can likewise fall anywhere in the visible or ultraviolet range. Since there are no requirements for coherence (unless a single mode optical fiber is installed optionally to collect light for dynamic light scattering, in which case a laser light source would be required), nor does the incident light have to be extremely monochromatic (a bandwidth of 50 nm would not be excessive), the light source does not have to be a laser. As such, conventional white light, broad band, or discrete line sources, such as arc lamps, light emitting diodes, vapor lamps and incandescent sources are all possible candidates for the incident light. By the same token, if a multiple wavelength source is used, it is possible to vary the scattering vector q q=4πn/λsin(θ/2) by introducing different discrete wavelengths and detecting at a single angle; e.g. by selecting wavelengths with a monochromator in front of a white light source and introducing these into the input optics. Using light from around 200 to 800 nm could yield a factor of four variation in q. This could avoid use of multiangle detection, and require only a single fiber optic for detection and single photodetector/amplifier. On the other hand, if both multi-angle detection and multiple wavelengths are used then, say, for wavelengths from 200 to 800 nm, and scattering angles from 15° to 170°, the factor of q can be varied by as much as a factor of 30. Appropriate collimation and/or focusing optics are usually needed to introduce the source beam into the channel. 
     In many applications use of a laser may be preferred. A laser source would preferably be around 200-1000 nm, and more preferably 450 to 780 nm, where the majority of economical, low power, commercial lasers operate. The laser beam is preferably focused at or near the center of the hollow channel, although an uncollimated, or reduced and re-collimated beam will also work. The beam waist can range from the diffraction limit of Gaussian beams (λf/D, where λ is the incident wavelength, D the unfocused laser beam waist diameter and f the lens focal length) typically on the order of 1 to 200 microns, up to a 2 mm unfocused beam. The preferred beam waist diameter will depend on the intended application, and would be given as an option to a potential user of the invention, according to their needs. For example, measurement of dilute solutions of small, clean solutions would tend to use a wider beam waist, whereas concentrated solutions containing significant stray scatterers would preferably use a very highly collimated beam. Use of a highly focused beam and detectors defining a small scattering volume allows less probability of finding large particles in the scattering volume at any instant. When a large particle enters, either with the sample stationary or under flow, a large spike is produced which can then be recognized and discriminated against, in order to recover the absolute scattering from the desired scatterers. Sufficiently fast detector response allows spikes to be identified, counted (for purposes of large particle counting), and eliminated, to recover the desired background scattering. 
     The method of delivering the beam can be directly through an optical window on the chamber, via a tubular transfer lens, such as the endo-index type, or via an optical fiber, either flexible or rigid, with such lenses, pinholes and other light handling components as is necessary to deliver the beam in focused or collimated fashion, with the desired beam waist, and with a minimum of glare and stray light. If the beam is delivered by optical fiber, the laser can be remote from the optical assembly. Alternatively, the laser can be mounted directly to the optical assembly (FIG.  7 ). 
     Directly across from the incident beam is a beam dump  32  for the incident beam  31  to minimize ‘glare’ and stray light. This beam dump  32  may be of any standard type, ranging from a hole, to a ‘Rayleigh horn’, to a complete sub-system involving coated or un-coated lenses, and/or prisms, mirrors, a photodetector, or other optical components. 
     The optical fibers  41  may be of the multimode variety, whose inside diameter may range from 10 to 1000 microns, the smaller sizes being preferred where highly scattering samples are being measured, or for subsequent use with dynamic light scattering. In fact, a single, relatively large fiber diameter may be selected, such as 500 microns, and a rotatable, annular mask can be affixed to the channel wall, which would have varying diameter pinholes for defining the field of view of each optical fiber. Alternatively, the cell interior may be permanently outfitted with sets of different diameter fibers, spaced closely about each selected scattering angle, all of which could be continuously monitored. The fibers themselves can be of virtually any commercial or research grade. They must be chosen, however, so as to be compatible with the solvent and sample conditions where the invention will be applied. Where toluene is used, for example, the fibers must withstand that solvent, so glass core fibers with glass cladding and buffer would be preferred, or some similar substitute, such as glass core with CPE (chloropolysulfatal ethylene) jacket from Belden corporation. 
     The way the optical fibers  41  are attached to the cell  21 ,  101  helps to define the scattering volume. If the fibers reach through the cell to the surface of the channel (chamber)  22 ,  102 , then the scattering angle will be defined by both the acceptance angle of the fiber in the particular solvent the cell contains, and the beam waist. Definition of the scattering volume in this way can be termed the ‘fiber acceptance angle mode’. If the fiber  41  is recessed back into a hole  42  in the chamber to the point where the angle subtended by the two ends of the cylindrical hole  42  is less than the acceptance angle of the fiber  41 , then detection can be said to be in the ‘pinhole mode’. The difference in detection modes is shown schematically in FIGS. 8 and 9. 
     The optical harness  43  leads all the detection fibers to a remote bank  111  of photodetectors  118 . The fibers  41  can be coupled to their respective detectors  118  by inserting them into permanently aligned quick connect optical fiber connectors, as are commercially available (e.g. Newark Corp. or Amphenol Corp.), positioned in front of the detector surfaces. 
     The optical assembly can be used in several modes. In one of its submersible modes, the assembly  20 , with no additional modifications, can be directly submerged into a sample solution  91  contained in a test tube  92 , industrial tank, etc. As a remote, fill mode unit, the channel may be capped at one end (or the channel simply does not have to be bored completely through), which allows a small quantity of sample to be pipetted, scooped, or otherwise introduced into it and reside in it, remotely from the main sample supply, if desired. Each end of the channel may also be outfitted with a coupling to accept a fluid flow, so that the assembly may also be used in flow mode, such as for monitoring, optionally with on-line dilution, unfractionated polymers degraded or produced in a vat, fractionated polymers from Size Exclusion Chromatography, capillary hydrodynamic fractionation, etc. In this mode of operation it may be desirable to hydrodynamically taper the interior to optimize the flow past the plane of the optical fibers and incident beam. The invention can also be used in insert mode, whereby samples in sealed cells or vials can simply be inserted into the ring member or chamber, in the traditional fashion. In this case, one returns to the common situation in which there is a transparent cell between the sample, incident beam and detection optics. 
     The invention can be simultaneously cleaned and absolutely calibrated by use of an appropriate solvent such as toluene, whose absolute Rayleigh scattering ratio is known. The probe is immersed in the solvent, or the solvent made to flow through it for cleaning purposes. At the same time, the solvent scattering is monitored, and when it reaches a steady value, this is used for determination of the absolute calibration factors for each detection fiber. 
     As regards the minimal ring member version, it can be used submersibly on its own or become a central portion of a three piece unit. This may be desirable for purposes where quick interchange of optical assemblies to different specifications, cleaner or newer units are made, etc. 
     In both the ring member version  20  and integral chamber version  100 , an outer protective sheathing  45 , such as a ring member of plastic or metal may slip over the fiber optics  41  protruding externally from the ring member  21  or chamber wall  101 . Likewise, in all cases, the entire optical assembly, whether a ring member or chamber, can be placed within a completely enclosed housing, into which sample can be introduced either by flow or immersion. Such a housing may be desirable when the optical assembly needs special protection from a harsh (e.g. high temperature) environment, or is immersed in turbulent or otherwise potentially damaging or signal distorting liquids. 
     The present invention includes the aforementioned ring member or integral chamber SLS probe. The incident beam  31  is introduced into the device via optical window  23 , or a fiber optic and/or tubular lens and other optical elements, and scattered light is taken out via fiber optics  41  whose tips  44  are arrayed at various angles in the horizontal plane of the ring member  21  or chamber wall  101 . All the optical fibers  41  and elements are drawn together into an ‘optical harness’  43 , which is led to the ‘outside world’ through a hollow handle  61  on the device  20 . he optical fibers  41  carrying scattered light and issuing from the harness  43  are coupled to conventional optical detectors  118  (e.g. PIN or avalanche photodiodes, photomultiplier tubes, etc.), whose voltage or current signals are led to a conventional signal processing device and/or into a computer  112 . The optical probe portion consists essentially of a piece of material, preferably dark, with optical fibers and a few other inexpensive optical elements (such as borosilicate windows  23 ) attached into a harness. As such, the probe itself should be quite inexpensive and could even be disposable. The photodetectors  118 , signal processing and computer analysis portions of the instrument are remote and permanent (although quite portable), and represent the major cost. In some cases, especially where few angles are involved, and submersible operation is not a priority, photodetectors (e.g. photodiodes) can be mounted directly to the chamber, thus avoiding use of the fiber optic detectors. 
     In the submersible mode, calibration (and cleaning) can be done by merely immersing the probe in a calibration solvent, kept handy in a closed vessel. This could be toluene, or any other solution whose absolute Rayleigh scattering ratio is known. 
     The software in Appendix A of parent patent application Ser. No. 08/969,386 can serve as a basis for data reduction, analysis and display. Data can be collected and reduced either on a standard microcomputer, or by building a customized microprocessor based unit. The software can include programmed criteria for averaging scattering signals, identifying, counting and rejecting scattering spikes from large, stray scatterers, and informing the operator when signal collection is done. Software can access on-board libraries to inform the operator of likely phenomena occurring in the sample (e.g. aggregation, gelation, degradation), and problems such as poor solution quality (e.g. too much ‘dust’), presence of aggregates, or other anomalies. 
     Experimental Verifications of the Invention 
     I) Fill mode tests 
     A) Transfer Lens Version/Single Angle 
     A first prototype of the invention in the integral chamber version was made in order to assess whether absolute macromolecular characterization, in terms of molecular mass, was feasible. This is meant to be only a demonstration of the feasibility of the invention, not a highly precise absolute molecular mass determination nor critical comparison of the invention&#39;s performance with a commercial instrument. 
     Dextran of nominal mass 200,000-300,000 g/mole was selected for the measurement. It was mixed at 0.003 g/cm 3  in an aqueous solvent containing 0.1 Molar NH 4 NO 3  and 0.1% sodium azide for protection against bacterial contamination. There is nothing special about this particular solvent, and even pure water would have been adequate (since dextran is a neutral polymer and is not subject to the unusual physical effects that charged polymers display in pure water). 
     An optical unit was fabricated from a 1⅞″ inch long piece of, e.g., black nylon round stock of ⅝″ o.d. An inner, cylindrical channel of diameter 7.7 mm was bored concentric with the axis. The inner ends of the channel were tapped to accommodate standard ⅜″ plugs, barbs and other hydraulic fittings. Perpendicular to the cylinder axis, a hole was drilled to accommodate a 1.98 mm o.d. Endogrins® lens, obtained from Edmund Scientific Co. Straight across from this hole on the opposite side of the channel a larger diameter hole was drilled for use as a beam dump. At 90° to the incident light hole a small hole was drilled to accommodate an optical fiber with inner core 100 microns and cladding 140 micron o.d. The fiber was inserted into the hole in the channel, and was found to work best when protruding but slightly from the hole into the channel. Both the fiber and lens were secured in their holes with optical putty. The opposite end of the fiber, which was about two feet long, was secured remotely from the optical assembly into a fiber optic chuck from New Focus Co., and butted up against the photosensitive surface of a Hammamatsu photodiode with integral FET op-amp, contained inside a light-tight box, containing both the diode/FET and an additional standard operational amplifier stage. 
     The amplified signal was fed into a Nicolet 4094B digitizing oscilloscope, although any data collection device with a rate of 1 KHz or faster would have sufficed. Sampling at 1 KHz or faster allows spikes from diffusing impurity particles and fluctuating scattering levels to be recognized and rejected, leaving the desired signal from the polymer or colloid scatterers. In fact, spike and fluctuation rejection was used in this and other tests. 
     Light of wavelength 488 nm and approximately 20 mW was from a Coherent Corp. Argon ion laser, which had an output beam waist of about 2 mm. The light could be delivered either highly focused or uncollimated. For high focusing, a 5 mm lens with a focal length, f=5 mm from Edmund Scientific was placed external to the optical assembly, and led to a beam waist of about 1.5 microns. This was transferred into the channel of the optical assembly via the 1.98 mm Endogrinsg lens, which was 6 cm long. Alignment of the delivered beam with respect to the detection fiber optic at 90°, and signal maximization for this arrangement was achieved by using a solution consisting of a {fraction (1/40)} dilution of 190 Angstrom latex spheres from Duke Scientific, although any moderately scattering solution, such as water with a tiny drop of milk or coffee creamer powder, would be adequate. 
     The system was then tested by measuring, sequentially, the photodiode dark count (i.e. with no laser beam entering the optical assembly), the photovoltage with pure water, with a 3 mg/ml solution of dextran, and toluene. The various liquids were introduced into and removed from the cell with a long, glass pipette with a rubber suction bulb at one end. The photovoltages are listed below: 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Table of Photovoltages (accuracies are to about +/− 1 mV) 
               
             
          
           
               
                   
                   
                   
                   
                 Rayleigh 
                   
                   
                 app. M 
               
               
                   
                 volt. 
                 scattering 
                   
                 ratio, 
                   
                 app. M 
                 Wyatt Dawn F 
               
               
                 measured 
                 (mV) 
                 difference 
                 K 
                 I (cm −1 ) 
                 Kc/I 
                 (θ = 90°) 
                 (θ = 144°)** 
               
               
                   
               
               
                 Photodiode 
                 −65 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 dark voltage 
               
               
                 pure water 
                 −57 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 3 mg/ml 
                 −30 
                 I dex  − I water  = 27 
                 1.46 × 10 −7   
                 7.63 × 10 −5   
                 4.23 × 10 −6   
                 174,000 
                 191,000 
               
               
                 dextran 
                 −51 
                 I tol  − I dark  = 14 
                 NA 
                 3.96 × 10 −5 * 
                 NA 
                 NA 
                 NA 
               
               
                 toluene 
               
               
                   
               
               
                 dn/dc = 0.142 for dextran  
               
               
                 *This is the known Rayleigh ratio for toluene at T = 25° C. for λ = 488 nm.  
               
               
                 **This is the proper angle for comparison, since the Dawn-F was used with a 632 nm He—Ne laser, and the test chamber with a 488 nm Argon ion laser.  
               
             
          
         
       
     
     The Zimm equation for SLS, when q 2 &lt;S 2 &gt;&lt;&lt;1 is                Kc   I     =       1     M   app       =         1     M   w            (     1   +         q   2     &lt;     S   2          &gt;   z       3       )       +     2        A   2        c                 (   1   )                                
     where I is the excess Rayleigh scattering ratio from the polymer solution (the total scattering minus the pure solvent background). M app  is the apparent mass, defined as per the equation (i.e. it neglects the effects of finite 2A 2 c and &lt;S 2 &gt; z  effects). M w  is the weight averaged polymer mass, &lt;S 2 &gt; z  is the z-averaged radius of gyration, A 2  is the second virial coefficient, c is the polymer concentration in g/cm 3 , and K is given, for vertically polarized light,              K   =       4        π   2              n   2          (          n     /        c       )       2           N   A          λ   4                 (   2   )                                
     where n is the index of refraction of the sample solvent (n=1.33 for water), and λ=4.88×10 −5  cm, is the vacuum wavelength of the incident light. 
     The absolute scattering I was calculated according to                I        (   q   )       =           V        (   q   )       -       V   s          (   q   )               V   c          (   q   )       -       V   d          (   q   )                I   e        f             (   3   )                                
     where V(q) is the photodetector voltage from the sample scattering at wave vector q, V s (q) is the scattering voltage at q of the pure solvent in which the polymer or colloid is dissolved, V c (q) is the scattering voltage of the calibration solvent scattering at q, and V d (q) is the dark voltage of the photodetector at q. I c  is the known, absolute Rayleigh scattering ratio for the calibration solvent. For toluene at 25° C., I c =1.406×10 −5  cm −1  at 633 nm, and 4.96×10 −5  cm −1  at 488 nm. In equation 3, f is an optical correction factor, given approximately as (n sample solven /n calibration solvent ) 3 . This accounts approximately for the difference in field of view and detector solid angle for optical fibers in the chamber. 
     For water n=1.333 and for toluene n=1.494 so that f is approximately 0.71. 
     The results for the dextran are shown in the above table. The apparent mass of 174,000 (at θ=90°) is obtained from the invention and 191,000 from the Wyatt Dawn F (at θ=144°). At these angles, q 2  is approximately the same for each instrument. At any rate, R g  =225 Angstroms for this Dextran (as measured on the Dawn F), so that there is very little q 2  dependence over the visible light range. 
     The fact that the apparent mass from the invention is within 10% of the value of that obtained from an established instrument clearly demonstrates the feasibility of making absolute molecular mass determinations. Refinement of the instrumentation should make results even more accurate. At any rate, it is generally recognized in the SLS field that molecular weights of polydisperse samples are seldom accurate to more than a few percent. 
     B) Multiple Angles 
     A similar chamber (with no hydraulic fittings) was made except that it was outfitted with detection fibers at 70°, 90° and 135°, and two opposed  3  mm sapphire windows, glued into holes in the chamber, were used for beam ingress and egress. Toluene was used for absolute calibration at each angle. Zimm plot results from a solution of high molecular weight PVP are shown in FIG.  10 . Ten mW of argon ion laser power were used, and a 50 mm focal length lens as used to focus the laser beam through the window in the chamber. 
     II. Immersion Mode Test 
     An immersion cell was constructed from nylon roundstock of 16 mm outer diameter and 12 mm i.d. and 8 mm long. 150 micron optical fibers were glued in with epoxy at 45°, 90° and 150°, with their front surfaces at the level of the inner cell diameter face. Two 3 mm holes were cut in opposite ends of the cylinder, and were left empty for the tests (i.e. neither entrance window nor beam dump were used). The optical fibers leading to the remote detector were secured so that no additional bending or deformation of them occurred, since additional bending or deforming leads to large losses in transmitted light. A tubular stainless steel handle was attached to the cylinder to allow for manipulation. The cylinder was immersed in 3″ diameter beakers containing the test liquids, and the handle, protruding from the solution, was secured with a ringstand. 20 mW of Argon ion laser power were delivered in a beam from above the beakers, and a 50 mm focal length lens was used to focus the light in the center of the cylindrical chamber. 
     Scattering tests at the three angles were carried out using 0.2, 1.0, 1.5 and 2.0 mg/ml solutions of a high molecular weight polymer, PVP. A digitizing oscilloscope was again used to monitor the detected light at each angle, one at a time. These solutions were unfiltered. Identification and rejection of spikes from large impurity particles diffusing through the scattering volume and fluctuating signals from other causes allowed this unusual series of measurements on unfiltered solutions to be made. The scattering voltage of toluene at each angle was used to find the absolute calibration factor at each angle. FIG. 11 shows typical results. These compare quite favorably with the results for the fill mode example above (I-B). 
     III. Flow Mode Tests 
     A 3-piece flow cell was constructed out of nylon roundstock of 16 mm o.d The central portion was 8 mm long, with a 7 mm bore, and contained a single 300 micron fiber epoxied in at a scattering angle of 90°. Two 3 mm sapphire windows were mounted on opposite sides of the central bore, one for laser beam ingress, the other for egress. Endcaps of the same material and o.d. pressed on each side of the central portion and O-rings created a seal. Round aluminum plates outfitted with long bolts served to clamp the endcaps to the central piece. The endcaps each had a small hole drilled in them for fluid to reach the bore of the central portion, and each was outfitted with a standard GPC fitting, allowing attachment of standard PEEK (polyethyleneethyleneketone) HPLC (high performance liquid chromatography) tubing to allow liquid samples to be pumped in and out. 
     The basic construction of the center portion can be identical to that of the immersion cell, making the two ultimately interchangeable, or at least slight modular variations of each other. Also, these cells can easily become fill mode cells by simply adding a base plate (as in the drawings). 
     A) Debye Plot at a Single Angle 
     Solutions of high molecular weight PVP of concentrations 0.25, 0.5, 1.5 and 2.0 mg/ml were pushed through the cell manually with a syringe, at roughly 1 ml/min. The experiment was repeated several times and error bars obtained. Kc/I at θ=90° is shown in FIG. 12, along with the associated error bars, and a comparison with results from a Wyatt Dawn-F. Ten mW of argon ion laser power were used, and a 50 mm focal length lens was used to focus the laser beam through the window in the chamber. 
     B) Discrimination Against Large Particles 
     The present inventor wrote program REEDFLO (see Appendix A of parent patent application Ser. No. 08/969,386) to capture data through a DT2801-a analog-to-digital converter board and perform averaging and data storage functions. Maximum speed is about 40 microseconds per point with this board, and up to eight separate detectors can be monitored per board in the differential input mode. The idea was first tested as to whether the flow cell with small scattering volume could usefully measure both absolute polymer scattering levels and identify and count spikes from large particles. Ten mW of argon ion laser power were used, and a 50 mm focal length lens was used to focus the laser beam through the window in the chamber. The scattering volume was roughly 5×10 −7  cc. 
     To this end a mixture of 0.5 mg/ml PVP of molar mass around 10 6  grams/mole was mixed with Duke Scientific 10 micron latex spheres such that the sphere concentration was 4×10 4  particles per cc. This gave roughly an average of 0.02 particles per scattering volume. The solution was pushed through the cell manually using a syringe, roughly at a flow rate of 1 ml/minute. The 5 mW diode laser (wavelength=635 nm) was used as the light source. 
     FIG. 13 shows that the cell was capable of measuring both the homogeneous background scattering from the polymers, and both identify and count the number of large particles in the flowing sample. Given the pure solvent level shown on the drawing, it is hence possible to recover the absolute intensity scattered by the homogeneous polymer background scattering. A significant degree of contamination by large particles can hence be tolerated in this system. 
     C) Kinetics of Polymerization 
     The kinetics of polymerization were carried out in real-time using the flow cell. A 5 mW diode laser was used, and a 50 mm focal length lens was used to focus the laser beam through the window in the cell. A 30% solution of vinyl pyrrolidone (VP) monomer was mixed in water with 0.1% ammonia, and the solution heated to 80° C. The polymerization was initiated with 0.7% hydrogen peroxide. At high concentrations, such as 30% VP, there is very little change in light scattering intensity as polymerization proceeds (i.e. in eq. (1) 2A 2 c is much larger than 1/M w (1+q 2 &lt;S 2 &gt; z /3)). Hence the reaction solution must be diluted for TDSLS to be a useful monitor of M w  in real-time. To do this, concentrated reactant is withdrawn with a pump and mixed with solvent from a separate reservoir of pure solvent. This can be achieved by using a hydraulic ‘T’ one arm of which goes to the concentrated reaction solution, and the other to the pure solvent, with the mixed output being then pumped out by a pump and forced through the scattering flow chamber. It turned out that use of a programmable mixer was more convenient for mixing reactant and pure solvents. A standard ISCO (corporation) 2350 HPLC pump was used to pull mixed material from this pump and push it through the flow cell and refractive index (RI) detector, which was placed in series with the flow to measure the concentration, and any possible variations, of the diluted sample. For this experiment the reaction mixture, initially at 30%VP, was diluted so that the sample passing through the flow chamber was at 6 mg/ml. 
     FIG. 14 shows the results of a polymerization reaction in terms of scattered intensity in arbitrary units vs. time, whereas FIG. 15 shows the approximate apparent mass, obtained by eqs. (1)-(3). The apparent mass is simply I/Kc. For PVP of mass about 30 kD, there is no significant angular dependence, so q 2 &lt;S 2 &gt;˜0. Furthermore, A 2 ˜5×10 −4 so that at a PVP concentration of 0.006 g/cm 3 , 2A 2 cM w ˜0.18. Such a correction to the apparent mass, about 18%, is easily taken into account. 
     Preferably, optical fibers  41  are attached to ring member 21 with fiber optic light chucks, such as those commercially available from Upchurch Company. 
     FIG. 16 shows apparatus for an online measurement of M w , monomer conversion, total solute concentration and reduced viscosity during a polymerization reaction. The method and results are described in detail in Florenzano, Strelitzki and Reed,  Macromolecules , vol. 31 pp. 7226-7238, 1998, “Absolute, On-line Monitoring of Molar Mass during Polymerization Reactions”. In summary, vinyl pyrrolidone monomer at 200-300 mg/ml at T=60-80° C. was polymerized using hydrogen peroxide initiator. The polymerizing mixture is withdrawn by a mixing pump, which dilutes the PVP to about 6 mg/ml. The diluted mixture is then pumped through the light scattering, ultra-violet absorption, viscosity and refractive index detectors, whence the mentioned polymer properties are obtained online. 
     The reason the technique will not work for undiluted reactor liquid is detailed in the cited reference. In brief, at high concentrations of monomer and polymer, the total scattering from the solution will usually be dominated by inter-polymer effects, and will not accurately reflect the average molecular mass of the individual polymer chains, which is the desired quantity. Sufficient dilution, in this case, online, insures that the scattering is dominated by the Mw of the polymers, and not inter-polymer effects. 
     Automatic Characterization of Batch Solutions of Polymer 
     The two vessel scheme has been used by Strelitzki and Reed (ref. 7) to automate batch characterization of polymer solutions, in conjunction with refractive index, multi-angle LS and viscometric detectors. The advantages over the manual dilution methods have been detailed above. 
     Determination of the Electroviscous Effect 
     The two vessel scheme has also been used by Strelitzki and Reed (unpublished results) to investigate the electroviscous effect in polyelectrolyte solutions. To accomplish this, polyelectrolytes (hyaluronic acid, xanthan and poly(styrene sulfonate) were used) were dissolved at about 1 mg/ml in a low strength NaCl solution (these generally ran the range from 0M to 0.001M NaCl) and placed in the first vessel. A stock solution of salt at the same concentration as in the first vessel was placed in the second vessel, and the gradient programmer was set to perform a continuous dilution of the polyelectrolyte from its full concentration in the first vessel to zero, or vice versa. Because the original polyelectrolyte solution also contains the counterions of the polyelectrolyte, the actual ionic strength of the solution is higher than the nominal ionic strength due to the added NaCl. As dilution of the polyelectrolyte takes place with pure solvent of the same nominal ionic strength, the total ionic strength of the diluted polyelectrolyte solution actually decreases, since the counterion concentration decreases with dilution, which leads to the electroviscous effect. Typical online, electroviscous data for hyaluronic acid is shown in FIG.  18 . 
     Table of Abbreviations 
     A 2 =second virial coefficient (cm 3 ×Mole/g 2 ) 
     C=concentration (in g/cm 3 ) 
     FET=field effect transistor 
     g/cm 3 =grams per cubic centimeter 
     g/mole=gram per mole 
     He—Ne laser=Helium Neon laser 
     TDSLS=Heterogeneous time dependent static light scattering 
     HPLC=High Pressure Liquid Chromatography 
     kD=kiloDalton (1,000 grams per mole) 
     λ=wavelength 
     LS=light scattering 
     M=molarity 
     M w =weight average molecular mass (grams per mole) 
     mg/ml=milligram per milliliter 
     ml=milliliter 
     ml/min=milliliter per minute 
     mV=millivolt 
     mW=milliwatt 
     nm=nanometer 
     PVP=poly(vinyl pyrrolidone) 
     &lt;S2&gt;=mean square radius of gyration (in Angstrom 2 , nm 2 , or cm 2 ) 
     SEC=Size Exclusion Chromatography 
     SLS=Static light scattering 
     TDSLS=Time dependent static light scattering 
     VP=vinyl pyrrolidone 
     PARTS LIST 
     The following is a list of parts and materials suitable for use in the present invention: 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                 10 
                 optical assembly of the preferred embodiment of the present 
               
               
                   
                 invention 
               
               
                 20 
                 ring member assembly of a first embodiment of the present 
               
               
                   
                 invention 
               
               
                 21 
                 ring member of the ring member assembly 20 of the first 
               
               
                   
                 embodiment of the present invention (such as nylon, 
               
               
                   
                 polycarbonate, anodized aluminum, kevlar or ceramic) 
               
               
                 22 
                 chamber of ring member 21 
               
               
                 23 
                 incident beam window of ring member 21 (e.g. Edmund scientific 
               
               
                   
                 borosilicate or sapphire circular windows) 
               
               
                   
                 (e.g., 5 mm diameter, 2 mm thick) 
               
               
                 24 
                 beam dump window of ring member 21 (same as 23, or similar) 
               
               
                 31 
                 incident beam (provided by, for example, a vertically polarized 
               
               
                   
                 5 mW diode laser commercially available from Lasermax Inc., 
               
               
                   
                 Rochester, NY) 
               
               
                 32 
                 beam dump (such as a window or prism followed by a Rayleigh 
               
               
                   
                 horn or a detection fiber) 
               
               
                 41 
                 optical fibers (such as optical fibers of 100, 150 and 300 micron 
               
               
                   
                 core diameter, commercially available from Polymicro 
               
               
                   
                 Technologies as parts FVP100110125, FVP150165180 and 
               
               
                   
                 FVP300330370, respectively.) 
               
               
                 42 
                 holes for optical fibers 41 
               
               
                 43 
                 optical harness (e.g. the fibers can be ‘braided’ together with 
               
               
                   
                 semiflexible plastic tubes and covered with a rugged sheath, such 
               
               
                   
                 as is commonly done for telecommunication fiber bundles) 
               
               
                 44 
                 ends of the optical fibers 41 
               
               
                 45 
                 outer protective sheathing 
               
               
                 51 
                 sample solution (for example 1 mg/ml Polyvinylpyrrolidone in 
               
               
                   
                 water) 
               
               
                 52 
                 container for sample solution 51 (glass beaker, for example) 
               
               
                 61 
                 handle for ring member assembly 20 (stainless steel, for example) 
               
               
                 62 
                 light source (such as a diode laser) 
               
               
                 63 
                 converging lens 
               
               
                 70 
                 flow mode assembly of the present invention 
               
               
                 71 
                 end piece of flow mode assembly 70 (made of nylon, ceramic, 
               
               
                   
                 anodized aluminum, or kevlar, for example) 
               
               
                 72 
                 hydrodynamic tapered flow channels in end pieces 71 
               
               
                 73 
                 HPLC tubing and fittings (e.g. Rainin Corp., or ISCO) 
               
               
                 74 
                 O-rings 
               
               
                 75 
                 retaining bolts 
               
               
                 80 
                 fill mode assembly of the present invention 
               
               
                 81 
                 base plate (made of plastic or anodized aluminum, for example) 
               
               
                 91 
                 sample solution (1 mg/ml polyvinylpyrrolidone in water, for 
               
               
                   
                 example) 
               
               
                 92 
                 container for sample solution 91 (glass, for example) 
               
               
                 100 
                 integral chamber assembly of the present invention 
               
               
                 101 
                 integral chamber wall (such as stainless steel, black anodized 
               
               
                   
                 aluminum, ceramic, Teflon, nylon, polycarbonate, or other plastics) 
               
               
                 102 
                 integral chamber 
               
               
                 111 
                 photodiode assembly (containing Hammamatsu Corp photodiodes, 
               
               
                   
                 for example) 
               
               
                 112 
                 computer for data collection and analysis (such as an IBM personal 
               
               
                   
                 computer clone such as a Starion 919 from Digital Equipment 
               
               
                   
                 Corp.) 
               
               
                 113 
                 strain relief loop 
               
               
                 114 
                 cowl 
               
               
                 115 
                 acceptance angle of fiber optic 41 in FIG. 8 
               
               
                 116 
                 acceptance angle of fiber optic 41 in FIG. 9 in water 
               
               
                 117 
                 acceptance angle of fiber optic 41 in FIG. 9 in toluene 
               
               
                 118 
                 optical detectors 
               
               
                 161 
                 baseplate 
               
               
                   
               
             
          
         
       
     
     References (Incorporated Herein by Reference) 
     1. Zimm, B. H. J. Chem. Phys., 16, 1093-1116 (1948) 
     2. W. F. Reed “Time-dependent light scattering from singly and multiply stranded linear polymers undergoing random and endwise scission”, J. Chem. Phys., 103, 7576-7584, (1995) 
     3. S. Ghosh and W. F. Reed “New Light Scattering Signatures from Polymers undergoing Depolymerization w. App. to Proteoglycan Degradation” Biopolymers, 35, 435-450 (1995) 
     4. W. F. Reed “Time-Dependent Processes in Polyelectrolyte Solutions”, invited chapter for Berichte der Bunsen-Gesellschaft special volume on Polyelectrolytes, 100, 6, 1-11, 1996 
     5. Ruth Schimanowski, Roland Strelitzki, David A. Mullin and Wayne F. Reed “Heterogeneous Time Dependent Static Light Scattering” ,  Macromolecules , in press (accepted Aug. 6, 1999 
     6. Fabio H. Florenzano, Roland Strelitzki and W. F. Reed, “Absolute, Online Monitoring of Polymerization Reactions”,  Macromolecules , vol. 31, no. 21, 7226-7238, 1998 
     7. Roland Strelitzki and Wayne F. Reed, “Automated Batch Characterization of Polymer Solutions by Static Light Scattering and Viscometry”, J. App. Polym. Sci., 73, 2359-2368 1999 
     All measurements disclosed herein are at standard temperature and pressure, at sea level on Earth, unless indicated otherwise. All materials used or intended to be used in a human being are biocompatible, unless indicated otherwise. 
     Attached as Appendix A to parent patent application Ser. No. 08/969,386 is data collection and storage software which can be used as a basis for more complex software to perform absolute macromolecular characterization and electronically filter out, count, and characterize large scattering particles. 
     As used herein, “large scattering particle” (LSP) means an individual particle which would produce scattered light greater than the noise level of the detector (in FIG. 13, for example, the noise level is around 0.04 V and the large scattering particles are indicated at about 12 seconds, 26 seconds, 38 seconds, and 46 seconds, in addition to other locations). A LSP could be unwanted impurities, aggregates of the polymer or colloid being studied, or an integral part of the solution. 
     The detectors and interface operate at a rate fast enough to resolve the residence time of a large scattering particle in the scattering volume. The interface between the photodetector and the computer can be a voltage-converting or a current-converting interface. 
     Preferably, the scattering volume is chosen such that the number of large scattering particles is small enough to not prevent absolute macromolecular characterization of the substance being studied, and preferably small enough to not significantly interfere with absolute macromolecular characterization of the substance being studied. For example, the average number of LSPs in the scattering volume can be less than 1000, preferably less than 500, more preferably less than 200, even more preferably less than 100, still more preferably less than 50, even more preferably less than 20, even more preferably less than 10, most preferably less than 5. The average number of LSPs in the scattering volume can be even 0 to 1. 
     The present invention is a relatively inexpensive, simple, versatile apparatus for use in SLS and TDSLS. 
     The size range of detectability can be, for example, 20 Angstroms to 100 microns. 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 transmission means for transmitting light from the light detection means to the photodetectors is preferably of a sufficient length and flexibility to allow the submersible probe to be submersed in the fluid to be sampled without submersing the photodetectors, and to allow the other probes to be remote from the photodetectors, which is helpful when the probe is to be used in harsh environments which might damage the photodetectors and associated electronics.] 
     As used in the claims, “light source” can refer to a window, lens, or optical fiber, for letting light in from a light generator, such as a laser. 
     All measurements disclosed herein are at standard temperature and pressure, at sea level on Earth, unless indicated otherwise. All materials used or intended to be used in a human being are biocompatible, unless indicated otherwise. 
     The foregoing embodiments are presented by way of example only; the scope of the present invention is to be limited only by the following claims.