Abstract:
A method is provided for determining the concentration of a solution having a predetermined osmotic pressure and/or for generating a solution with a predetermined osmotic pressure. The method is performed by placing a sample of a solution into a sample cell, the sample cell including therein a membrane supported therein; sealing the sample in the sample cell; introducing gas at a predetermined elevated pressure substantially equal to the predetermined osmotic pressure; and generating a dialyzate by allowing the solution to dialyze through the membrane until equilibrium is achieved and no more dialyzate passes through while the sample cell is maintained at the predetermined elevated pressure, the sample remaining in the sample cell having an osmotic pressure substantially equal to the predetermined elevated pressure; collecting the dialyzate of said generating step; and weighing the dialyzate of said collecting step to determine the concentration of the solution remaining in the sample cell.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    The present application is related to U.S. application Ser. No. (Docket No 80131), filed Herewith, by Dr. David C. Boris, and entitled, “METHOD AND APPARATUS FOR MEASURING OSMOTIC PRESSURE,” and to U.S. application Ser. No. (Docket No. 80378), filed Herewith, by Dr. David C. Boris, and entitled, “METHOD FOR MEASURING CHANGES IN OSMOTIC PRESSURE.” 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates generally to osmometry and, more particularly, to measuring the osmotic pressure of complex solutions including, but not limited to, emulsions, dispersions, and charged and uncharged polymer solutions.  
         BACKGROUND OF THE INVENTION  
         [0003]    A variety of methods and devices are known for measuring the osmotic pressure exerted by solvent molecules diffusing through a semipermeable membrane. Commercially available devices for measuring the osmolality of solutions via membrane osmometry include the 4400 series colloid osmometers made by WesCor (Logan, Utah) and the type 1.00 Knaur membrane osmometer. Other devices for measuring the osmolality of solutions through vapor pressure osmometry are commercially available. An example of this type of device is VPO model 070 made by UIC Inc. (Joliet, Ill.). Still another known osmometer device operates by measuring freezing point depression. An example of this type of device is the Precision Systems Inc. (Natick, Mass.) Osmette XL product line. Other known osmometer variants include isopiestic vapor equilibrium osmometers and submerged dialysis bag osmometers. In addition, osmolality has been determined by measuring boiling point elevation. The vapor pressure osmometer, boiling point elevation, freezing point depression, and isopiestic methods measure the oncotic pressure (the osmotic pressure exerted by colloid in the solution) of the solution, that is, the osmolality of the solution including the contribution of low molecular weight components such as salts. The present invention is directed to methods for measuring the osmotic pressure of solutions, excluding the contribution of small molecules.  
           [0004]    Membrane osmometers and the dialysis bag techniques measure the solution equilibration across a semi-permeable membrane, thus excluding the direct contribution of small permeable molecules. One example of a membrane osmometer is taught in U.S. Pat. No. 4,150,564, titled “OSMOMETER FOR COLLOID OSMOMETRY,” by Wayne K. Barlow, et al., Apr. 24, 1979. The present invention is an improvement upon existing membrane osmometer designs, but relies on the same basic principle of establishing an equilibrium across a semi-permeable membrane. Typical membrane osmometers employ pressure transducer technology to directly measure the evolved osmotic pressure difference between a reference cell and the sample solution across a semi-permeable membrane. In the present invention, transducer technology is not required for measuring the osmotic pressure. Typical commercial osmometers are designed to minimize the sample volumes by introducing the sample into a meandering channel above the semi-permeable membrane that maximizes surface contact while minimizing sample volume. This meandering channel geometry is eliminated in the present invention because it limits the usefulness of commercial osmometers to low viscosity, non-fouling solutions and is particularly unsuited for complex solutions (dispersions and emulsions).  
           [0005]    Dialysis bag techniques, (Essafi, W.  Structure Des Polyelectrolytes Fortement Charges,  PhD thesis, Universite Pierre et Marie Curie, Paris, 1996) involve filling a semi-permeable dialysis bag with the sample solutions of unknown osmotic pressure and immersing it in a large volume of solution of known osmotic pressure. The sample changes concentration until an osmotic equilibrium is established. Then the sample is removed and its concentration at that known osmotic pressure is determined using other techniques (spectrophotometrically or gravimetrically). The present invention may be used in a mode of operation similar to this. In this mode of operation the sample solution is allowed to equilibrate to a known imposed air pressure. Then the concentrated sample is removed and the concentration determined separately. However, this method of operation is not the optimal nor is it the preferred embodiment of the method of the present invention.  
           [0006]    The prior art also suggests use of polymer solutions of known osmotic pressure to have as reference solutions (rather than solvent) using commercial osmometers or other direct force measurement techniques to allow for measurement of higher osmotic pressure solutions, (Rau, Donald C.; Parsegian, V. Adrian.  Direct Measurement Of Temperature-Dependent Solvation Forces Between DNA Double Helixes.  Biophys. J. (1992), 61(1), 260-71; Sidorova, Nina Y.; Rau, Donald C.  Removing Water From An EcoRI-noncognate DNA Complex With Osmotic Stress.  J. Biomol. Struct. Dyn. (1999), 17(1), 19-31). This method works reasonably well for extending the pressure range available on commercial osmometers but does not properly treat the Donnan equilibrium established for charged species thereby potentially leading to erroneous results with charged polymers. This method of extending the pressure range is not needed in the operation of the present invention as the reference solution is simply dialyzate.  
           [0007]    The present invention capitalizes upon advances in stirred cell dialysis chamber technology to improve osmometer design. Stirred cell dialysis chambers are available from, for example, Amicon, (Beverly, Mass.). The principal use of the dialysis chamber in the prior art is to either concentrate a sample solution or to remove small molecule impurities by exhaustive flushing with pure solvent. The stirred cell is designed to be dismantled easily for cleaning. It has a magnetic stirring rod suspended above the membrane to keep the solution well stirred and to sweep clean the surface of the membrane. It can withstand more than 75 psi of external pressure, far exceeding the pressure measurable in conventional membrane osmometers (˜1-3 psi).  
         SUMMARY OF THE INVENTION  
         [0008]    It is therefore an object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure.  
           [0009]    It is a further object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which allows for easy dismantling and cleaning so that complex fluids (the emulsions, dispersions, etc.) which would foul traditional meandering channel membrane osmometers can be measured.  
           [0010]    A further object of the present invention is to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which includes stirring the surface of the membrane continually thereby reducing surface fouling and removing bubbles that hamper the accuracy and reliability of prior art osmometers when used to measure complex fluids.  
           [0011]    Yet another object of the present invention is to provide an osmometer which keeps the sample well mixed thereby avoiding particle settling and the development of surface concentration gradients.  
           [0012]    It is a further object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure.  
           [0013]    Still another object of the present invention is to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which can be used for measuring osmotic pressure of high viscosity solutions (above about 100 cp).  
           [0014]    The foregoing and numerous other features, objects and advantages of the present invention will become readily apparent upon reviewing the detailed description, claims and drawings set forth herein. These features, objects and advantages are accomplished by providing a sample cell with a removable pressurizing lid through which a sample solution may be introduced into the chamber of the sample cell. The sample is introduced above a membrane residing in the sample cell with a meandering dialyzate cell positioned below the membrane. Pressurized gas is introduced into the sample cell via a pressure regulator. Once the sample is forced through the membrane and dialyzate begins to emerge through a transparent dialyzate tube from the meandering dialyzate cell, the pressure through the pressure regulator is reduced until flow ceases. A magnetic stirrer is used to continually sweep the surface of the membrane. The primary purposes of the meandering dialyzate cell are to provide a structure for collecting dialyzate with maximum surface area contact with the membrane (for fast equilibration with the sample cell) which minimizes the volume of dialyzate relative to the volume of sample in the sample cell. However, those skilled in the art will recognize that the present invention can be practiced without a meandering dialyzate cell. Instead, the sample cell can easily be provided with a support structure other than a meandering dialyzate cell to support the membrane therein. Similarly, the sample cell can also be provided with a different dialyzate collector (e.g. a funnel). The support structure for the membrane and the dialyzate collector do not have to be integrated as is the case with a meandering dialyzate cell. However, these alternative arrangements would likely (although not necessarily) increase the volume of dialyzate needed before a meniscus is achieved in the transparent dialyzate exit tube.  
           [0015]    The use of a pressure regulator and an accurate pressure gauge allow for precise control and measurement of the applied air pressure on the sample. By systematically varying the applied air pressure until the visually observed liquid meniscus of the dialyzate in the dialyzate tube is stationary, the osmotic pressure of the solution across the dialysis membrane is exactly balanced by the imposed air pressure. Further, the method and apparatus of the present invention allow for the progressive adding of known amounts of diluent to the sample solution and mixing the new dilutions directly in the sample cell. This is achieved practically by simply placing the entire cell on an accurate balance and weighing the amount of added solution before each osmotic measurement. Calibration of the apparatus of the present invention does not require equilibration of a sample. Rather, calibration is a simple and fast procedure based on accurately measuring the air-line pressure. This is in marked contrast to the difficulty of repeatedly calibrating transducers and properly seating membranes in prior art membrane osmometers. This leads to a significant advantage in the amount of time required to operate the apparatus.  
           [0016]    The preferred method of the present invention for determining the concentration of a solution having a predetermined osmotic pressure and/or for generating a solution with a predetermined osmotic pressure starts from a concentrated sample with a higher osmotic pressure than the desired predetermined osmotic pressure. A second method is given below for sample solutions which need to be concentrated to increase the osmotic pressure to the desired predetermined osmotic pressure. The basic idea is to measure the osmotic pressure of a series of gradual solvent dilutions of the concentrated sample, performed in the sample cell, until a final sample solution with the desired osmotic pressure is achieved. This is done by first placing in the sample cell a more concentrated sample with higher osmotic pressure than the desired predetermined osmotic pressure. The sample cell  12 , of course, includes a membrane  16  supported therein. The sample is then sealed in the sample cell  12  and gas is then introduced at a predetermined elevated pressure. The osmotic pressure of the sample can then be determined by varying the applied gas pressure until the dialyzate meniscus is stationary. Next the sample cell  12  is placed upon a balance and a known weight of diluent is added directly to the sample cell  12  so as to dilute the sample  64  to a concentration calculated to be slightly more than the desired osmotic pressure. A simple linear method of estimation of the amount of diluent to add based upon equating the ratios of concentrations to the ratio of measured and desired osmotic pressure will typically suffice. The diluted sample is resealed and the osmotic pressure is again measured. This process is repeated until the osmotic pressure desired is achieved. The known dilutions allows for the calculation of the final concentration from the initial concentration of the concentrated sample. This is the fastest procedure.  
           [0017]    If the added diluent ever lowers the osmotic pressure of the sample below the desired osmotic pressure then the sample  64  is placed in the sample cell  12  including therein a membrane  16  supported therein. Then the remaining steps of the method are: sealing the sample  64  in the sample cell  12 ; introducing gas at a predetermined elevated pressure substantially equal to the predetermined osmotic pressure; and generating a dialyzate by allowing the solution to dialyze through the membrane  16  until equilibrium is achieved and no more dialyzate passes through the membrane  16  while the sample cell  12  is maintained at the predetermined elevated pressure, the sample remaining in the sample cell  12  having an osmotic pressure substantially equal to the predetermined elevated pressure; collecting the dialyzate of said generating step; and weighing the dialyzate of said collecting step to determine the concentration of the solution remaining in the sample cell. Even when a slight over dilution occurs the process is reasonably fast because the over diluted sample has an osmotic pressure close to the desired osmotic pressure. This is important since using dialysis to concentrate a sample can be quite a time consuming process compared to the fast measurement process just described.  
           [0018]    If the sample available has a lower osmotic pressure than desired (a lower concentration) then the following more time consuming method must be used. This alternative method of the present invention for determining the concentration of a solution having a predetermined osmotic pressure and/or for generating a solution with a predetermined osmotic pressure is performed by placing a sample  64  of a solution which has a lower osmotic pressure than the desired predetermined osmotic pressure in the sample cell. The sample is placed into the sample cell  12 , the sample cell  12  including therein a membrane  16  supported therein; sealing the sample  64  in the sample cell  12 ; introducing gas at a predetermined elevated pressure substantially equal to the predetermined osmotic pressure; and generating a dialyzate by allowing the solution to dialyze through the membrane  16  until equilibrium is achieved and substantially no more dialyzate passes through the membrane  16  while the sample cell  12  is maintained at the predetermined elevated pressure, the sample  64  remaining in the sample cell  12  having an osmotic pressure substantially equal to the predetermined elevated pressure; collecting the dialyzate of the generating step; and weighing the dialyzate of the collecting step to determine the concentration of the solution remaining in the sample cell  12 . This is a more time consuming method because the sample must dialyze to a higher concentration. It is unavoidable, however, if the sample  64  needs to be concentrated.  
           [0019]    Generating a solution with a predetermined osmotic pressure was simply not possible with prior art osmometers. The methods of the present invention to generate such a solution and to determine its concentration are faster than even the mere measurement of the osmotic pressure of an unknown sample using prior art osmometers.  
           [0020]    For improved accuracy the height of both the dialyzate liquid column in the dialyzate tube and the sample reservoir are recorded and used to correct for the known hydrostatic pressure difference. The entire apparatus is placed on a hot plate for temperature control. This method is very fast, approximately 4-50 times faster than other methods. Because of the speed of measurement of the present invention, the osmotic pressure of chemical reactions can be monitored as a function of time or temperature in the sample cell. This feature potentially opens new areas of research for instance in medicine, or polymer chemistry where the appearance or disappearance of reactants is accompanied by an osmotic pressure change. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0021]    [0021]FIG. 1 is a schematic cross-sectional view of the osmometer device of the present invention;  
         [0022]    [0022]FIG. 2 is a top plan view of the meandering dialyzate cell;  
         [0023]    [0023]FIG. 3 is a perspective view of the frame in which the osmometer device of FIG. 1 resides during operation;  
         [0024]    [0024]FIG. 4 is a graph showing a comparison of osmotic pressure versus concentration data on Polystyrene Sulfonate obtained using the present invention and compared directly to data using a conventional prior art membrane osmometer; and  
         [0025]    [0025]FIG. 5 plots osmotic pressure data from three complex solutions not amenable to conventional prior art osmometry techniques including a silver halide containing gelatin photographic dispersion, a carbon particle slurry and a high viscosity charged polymer solution. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0026]    Turning first to FIG. 1, there is shown a schematic perspective view of the osmometer  10  of the present invention. The osmometer  10  includes a sample cell  12 . Sample cell  12  includes a chamber body  14  which is preferably made of polysulfone and is preferably transparent. An Amicon 8400 stirred cell dialysis chamber serves well as sample cell  12 . Residing in chamber body  14  is membrane  16 . Many semi-permeable membranes are available commercially which work well. For aqueous solutions a Diaflo ultrafiltration membrane YM10 (10K Mw cut-off) was effectively used for membrane  16 . Other membranes could be chosen with tighter (or looser) pores to selectively measure the osmotic contribution of lower (or higher) molecular weight fractions of the sample solution. Many of the commercially available membranes are suitable for organic solvents. The Diaflo YM10 membrane is suitable for most organic solvents as well, excluding Amines, phenols and solutions with pH less than 3 or greater than  13 . This is not a limitation of the device, other membranes are commercially available which circumvent all of these limitations. Membrane  16  is supported on meandering dialyzate cell  18 . Meandering dialyzate cell  18  is retained in chamber body  14  by means of base plate  20  which threadably engages chamber body  14 . An O-ring  22  provides a seal between chamber body  14  and meandering dialyzate cell  18 . There is a circumferential lip  24  in the interior surface of chamber body  14 . Circumferential lip  24  provides residence for support bracket  26  which preferably includes three radial spokes  28 . Extending down from support bracket  26  is stir rod axle  29 . Rotatably mounted on stir rod axle  29  is stir rod blade  30 .  
         [0027]    Press fit onto the top of chamber body  14  is lid  32 . A seal between lid  32  and chamber body  14  is provided by means of O-ring  35 . Attached to lid  32  is bushing  34  which aligns with bore  36  in lid  32 . Extending from bushing  34  is pressurized gas conduit  38  for which pressurized gas is supplied from a pressurized gas source  40 . Mounted in pressurized gas conduit  38  is a pressure regulator  42  and a pressure gauge  44 . Lid  32  is also provided with an L-shaped bore  46  in which a pressure relief valve  48  is mounted. Pressure relief valve  48  is manually operated by means of handle  50 .  
         [0028]    There is a bore  52  into meandering dialyzate cell  18  which communicates with one of radial channels  55  (see FIG. 2) in the top surface of meandering dialyzate cell  18 . The top surface of meandering dialyzate cell  18  also includes a series of concentric channels  57  therein. Bore  52  aligns with bore  54  through chamber body  14 . Coupling  56  mounts to chamber body  14  at bore  54  and transparent dialyzate exit tube  58  extends therefrom.  
         [0029]    When in operation, sample cell  12  resides in frame  60  (shown in a perspective view in FIG. 3) which is preferably open on at least two sides thereof to permit observation of sample cell  12 . Frame  60  is made of metal (preferably steel) and insures that lid  32  is retained on chamber body  14  when sample cell  12  is pressurized via pressurized gas conduit  38 . Frame  60  is supported on magnetic stirring/hot plate  62 . An exemplary magnetic stirring/hot plate  62  that may be used in the practice of the present invention is a Coming Hot Plate/Stirrer model PC-351. Because frame  60  is metallic, it acts as a good heat conductor for heating sample cell  12 .  
         [0030]    Initially the pressurizing lid  32  is removed and the sample solution  64  is introduced into the chamber  14  above the membrane  16 . The gas delivered via pressurized gas conduit  38  can be air, nitrogen or a non-interacting (inert) gas. Preferably, pressurized gas source  40  can deliver gas at a relatively high pressure (80 psi). The air pressure applied to the sample solution  64  is controlled by the pressure regulator  42  which has the capability of smoothly varying the pressure over the entire desired range of measurement (0-10 psi for low pressure applications, 0.2-75 psi for high pressure applications). Pressure regulators with more or less sensitivity can be chosen based upon the osmotic pressure of the sample solution being measured and the desired accuracy. Two examples of pressure gauges can be used in the operation of the present invention are the Nullmatic 40-30 pressure regulator and the Ashcroft 40 psi pressure regulator. The applied pressure is measured on pressure gauge  44 . The accuracy and range of the osmometer  10  depends on the accuracy and pressure range of the pressure gauge  44  selected. A Capsuhelic 0-300 inch of water gauge has been used successfully for lower pressure solutions. Additional accuracy can easily be obtained by using any of a number of more expensive and accurate commercially available pressure gauges.  
         [0031]    The sample cell  12  plus solution  64  is weighed and then the lid  32  is sealed with the pressure release valve  48  open. The sealed sample cell  12  is then placed inside the metal pressure frame  60  and the pressure release valve  48  is closed. This frame  60  holds the lid  32  firmly in place under pressurization. The base of the frame  60  establishes good thermal contact between the magnetic stirring/hot plate  62  and the sample cell  12 . The heating element of the magnetic stirring/hot plate  62  is turned on and set to the chosen temperature setting. The entire apparatus can be submerged into a water bath (not shown) with a temperature sensor (not shown) for precise temperature control. Because of the large volume of sample solution  64  and the fast measurement time this step is often not needed in the practice of the method of present invention. The magnetic stir portion of the magnetic stirring/hot plate  62  is turned on thereby magnetically driving the angled magnetic stirring rod blade  30  which is preferably set to a moderate rate of stirring of about 1-2 revolutions per second. For clear solutions the surface of the membrane  16  is then visually inspected through the transparent sides of the chamber  14  for bubbles or debris.  
         [0032]    The pressure is raised initially to between 5 and 15 psi to wet the membrane  16  with the sample solution  64 . Once the sample solution  64  is forced through the membrane  16  and the dialyzate begins to emerge through the transparent dialyzate exit tube  58  such that there is a visible meniscus  66  therein, the pressure is reduced using the pressure regulator  42  until flow ceases. Pressure is reduced further until flow reverses direction and the dialyzate is drawn back into the meandering dialyzate cell  18  and ultimately back through the membrane  16  into the transparent sample chamber  14 . The meandering channel dialyzate cell  18  maximizes the surface area for equilibration while minimizing the volume of dialyzate and thus the amount of dilution of the sample  64 . This is a dramatic improvement over the commercial membrane osmometers that have meandering channel sample chambers which minimize the sample volume rather than the dialyzate volume. For fast and easy use a minimum dialyzate volume to sample volume ratio is preferable. For expensive samples a smaller cell can be selected which maintains the large sample volume to dialyzate volume ratio but reduces the dimensions of the entire cell. Finally, the pressure is varied carefully until the meniscus  66  in the dialyzate exit tube  58  holds substantially stationary, that is, stationary over a few minute time period. The osmotic pressure of the sample solution  64  is equal to the applied gas pressure read upon the pressure gauge  44  when the flow is substantially stationary, that is when equilibrium across the membrane is reached. Preferably, the osmotic pressure measured is then corrected for the slight hydrostatic pressure difference calculated from the difference in height of the liquid column in the dialyzate exit tube  58 , and the height of the sample surface  64  in the sample cell  12  (typically this correction is between 1 and 10 centimeters of water. This technique of measuring the hydrostatic pressure differential can be adapted into a method for achieving increased accuracy, particularly in low pressure applications (0-1 psi). Increased accuracy in low pressure applications can be accomplished by suspending the dialyzate tube vertically and measuring the difference in heights of the stationary meniscus  66  in the dialyzate tube  58 , and the height of the sample surface  64  in the sample cell  12 . The osmotic pressure is then calculated by correcting the gauge pressure for the hydrostatic pressure difference. Preferably, the step of measuring the difference in heights of the stationary meniscus  66  in the dialyzate tube  58 , and the height of the sample surface  64  in the sample cell  12  is performed at two or three applied pressures typically differing by a few centimeters of water (1-5 cm). The osmotic pressure is then calculated by correcting the gauge pressure for the hydrostatic pressure difference for each chosen pressure. The two or three osmotic pressure values obtained are then averaged. Typically the pressures chosen are only different by a few centimeters of water to avoid large columns of dialyzate in the vertical dialyzate exit tube which would require large dialyzate volumes.  
         [0033]    For measuring a dilution series the pressure relief valve  48  is open, the lid  32  is removed, the diluent is added and the sample is reweighed so that the new concentration is known. The lid  32  is reaffixed and put in the metallic frame  60 . Finally the pressure relief valve  48  is closed and the measurement method is repeated as described above. This entire process can be performed in just a few minutes.  
         [0034]    An alternate method of measuring the osmotic pressure of a sample solution is to set the pressurized gas conduit  38  to a fixed pressure using the pressure regulator  42  and pressure gauge  44  and then wait for the solution to dialyze down to the concentration corresponding to this pressure. The solution can then be removed and its concentration can be determined using other techniques (spectrophotometrically or gravimetrically for instance). This mode of operation allows for the preparation of solutions of known osmotic pressure. This is not the preferred mode of use because the dialysis process is quite time consuming, sometimes taking several hours to reach the chosen osmotic pressure.  
         [0035]    Looking next at FIG. 4, the accuracy of the present invention is demonstrated using some standard solutions from the prior art literature. Clearly the data generated using the method and apparatus of the present invention is in good agreement with the polystyrene sulfonate data measured using conventional membrane osmometry. Further, the highest concentration point obtained using the invention was beyond the viscosity and pressure range accessible using conventional membrane osmometry methods.  
         [0036]    Turning to FIG. 5, it can be seen that the osmotic pressure data was obtained for three complex solutions using the present invention. The osmotic pressure of these three complex solutions cannot be measured using prior art membrane osmometry techniques. The first solution is a 143 centipoise charged polymer solution made from mixing gelatin and a synthetic polyelectrolyte. This high viscosity solution cannot be measured using the meandering sample channel arrangement of commercial membrane osmometers because of the high backpressure needed to introduce the solution. In addition, this high osmotic pressure is slightly beyond the nominal operating limits of commercial membrane osmometers (although most transducers are still fairly linear in this range). The second solution is a carbon black slurry dispersed in gelatin. While the high measured osmotic pressure of this solution is slightly beyond the operating limits of conventional membrane osmometers, the more significant problem with this solution is that the introduction of such a carbon black slurry into commercial osmometers will foul the osmometer severely. The procedure for cleaning and recalibration of the osmometer after this solution requires several hours and makes these sorts of measurements time prohibitive as a practical matter. In contrast, in the operation of the present invention another solution can be measured within minutes. The third solution measured is a yellow photographic (silver halide) dispersion. This solution fouls conventional membrane osmometers and gives data which is not reproducible because of settling and surface plugging. These problems are avoided using the present invention because of the constant stirring of the solution and sweeping of the surface.  
         [0037]    As discussed above, those skilled in the art will recognize that the present invention has many advantages over prior art osmometers. For example, it should be appreciated that the present invention is easily dismantled and cleaned so that complex fluids (emulsions and dispersions) can be measured which would foul traditional meandering channel membrane osmometers. In addition, the magnetic stirring rod blade  30  that continually sweeps the surface of the membrane  16 , reduces surface fouling and removes bubbles that hamper the accuracy and reliability of commercial prior art membrane osmometers when used to measure complex fluids. Further, because chamber body  14  of the device is transparent, the upper surface of the membrane  16  may be observed to check for detrimental bubbles, deposits, etc. In addition, the magnetic stirrer keeps the sample well mixed, avoiding particle settling and the development of surface concentration gradients which can lead to inaccurate measurements in commercial membrane osmometers of the prior art when used with complex fluids (particularly dispersions).  
         [0038]    Sample measurements in the present invention are very fast, approximately 4-50 times faster than other prior art methods. Because of the speed of use of the present invention it can be used to measure the osmotic pressure for solutions that change as a function of time or temperature. In fact, chemical reactions can be initiated in the osmometer sample cell and then monitored as a function of time. Reactants can be added to the sample cell at the desired temperature, and a timer can be started. The sample cell  12  is sealed and placed on the magnetic stirring/hot plate  62  where the temperature is maintained and mixing is initiated. The sample cell  12  is pressurized and the pressure is varied until the dialyzate meniscus  66  in the exit tube  58  is substantially stationary, that is, there is substantial equilibrium across the membrane. The meniscus  66  may not become entirely stationary if the osmotic pressure of the reacting sample is changing too rapidly. The pressure, as read from pressure gauge  44 , required to keep the meniscus  66  stationary, is then recorded as a function of time. These measurements can be recorded repeatedly at a time step as small as 1-2 minutes to monitor the evolution of a chemical (or biological) reaction that changes osmotic pressure with time. Chemical and biological reactions that change osmotic pressure with time include an enormous class of reactions that use up or produce reactants.  
         [0039]    An alternative embodiment of the method of the present invention is to monitor the changes in osmotic pressure of a sample or ongoing chemical reaction as a function of time as the temperature is varied. This involves using a hot plate or water bath capable of varying the temperature of the sample. To measure the temperature one can affix a thermometer  70  or a thermocouple probe or other temperature sensing devices to the cell. In this case the sample (or reactants) are added at some prescribed temperature to the sample cell  12 , and a timer is started. The sample cell  12  is sealed and placed on the magnetic stirring/hot plate  62  (or in the water bath) where mixing is initiated in the sample cell  12  and the temperature is varied in some predetermined way as a function of time. Examples include ramping the temperature either upward or downward, or causing step changes in the temperature of the sample  64 . The sample cell  12  is pressurized and the pressure is varied until the dialyzate meniscus  66  in the exit tube  58  is nearly stationary. The meniscus  66  may not become entirely stationary if the osmotic pressure of the sample  64  is changing too rapidly. The pressure on the gauge  44  needed to keep the meniscus  66  stationary is then recorded as a function of time and temperature. This is the osmotic pressure. These measurements can be recorded repeatedly at a time step as small as 1-2 minutes to monitor the evolution of a chemical (or biological) reaction that changes osmotic pressure with temperature and time. These techniques would be particularly suitable for monitoring the progress of chemical or physical gelation reactions for example. The osmometer  10  of the present invention can clearly be used to measure the osmotic pressure of samples  64  which are unstable if held more than 5-10 minutes (for example due to chemical or colloidal instabilities) or change substantially within 5-10 minutes. Previously these types of measurements simply were not possible with commercial prior art membrane osmometers. This alternative embodiment can be used to monitor the changes in osmotic pressure of a sample as a function of time as the temperature is varied, not only for chemical reactions, but also for those changes in osmotic pressure resulting from, for example, phase changes, morphological changes, colloidal instabilities including Oswald ripening, aggregation, counter ion condensation, and micelle formation.  
         [0040]    Sample measurements in the device of the present invention are very fast, approximately 4-50 times faster than other prior art methods. Because of the speed of use of the present invention it can be used to measure the osmotic pressure for solutions that are unstable if held more than 5-10 minutes (for example due to chemical or colloidal instabilities). Previously these measurements simply were not possible with commercial prior art membrane osmometers. The present invention is faster because the sample volume is typically several hundred times larger than the dialyzate volume so that the change in concentration upon equilibration can be safely ignored allowing accurate measurements to be made immediately (1-5 minutes per sample). In contrast, typical prior art membrane osmometers require multiple flushes of each sample solution before concentrations stop changing and reproducibility is established (˜20-50 minutes per sample). A second reason that the device of the present invention allows for faster measurements is that new solutions can be mixed directly in the sample cell  12 . Accurate dilution series are made by starting from a sample solution  64  and progressively adding known amounts of diluent. This is achieved practically by simply placing the entire cell on an accurate balance and weighing the amount of added solution before each measurement. This sort of mixing and weighing is not possible in conventional art osmometers and thus requires the individual preparation of separate samples for dilution series. In addition, because the sample cell  12  is not mixed in conventional prior art osmometers, between each measured dilution several extra flushes are typically required to achieve stable measurements, further slowing down the measurement process. In the operation of the present invention, calibration does not require equilibration of a sample. It is simple and fast based on accurately measuring the air-line pressure. This is in marked contrast to the difficulty of repeatedly calibrating transducers and properly seating membranes in commercial prior art membrane osmometers. This leads to significant time savings in operation.  
         [0041]    Further, the accuracy of the device of the present invention is significantly greater than with prior art osmometers. The accuracy of the device of the present invention is determined by the air pressure gauge which can be controlled very precisely (to within millimeters of water if desired). Commercial prior art osmometers can only achieve 1-2 cm of water accuracy. The applicable pressure range which can be used in the operation of the present invention is determined by the sample cell maximum (which can withstand up to 75 psi), far higher than the conventional transducer technology used in prior art membrane osmometers which typically measure in a range of 1.5-3 psi).  
         [0042]    Finally, the present invention enables the determination of the osmotic pressure of high viscosity solutions. Meandering channel osmometers of the prior art have small sample volumes and small sample channels and due to high backpressure are unusable with high viscosity solutions (above about 100 cp).  
         [0043]    In the preferred embodiment, transparent dialyzate exit tube  58  has been described herein as extending from a coupling  56  attached to the chamber body  14  at bore  54 . Those skilled in the art will recognize that a variety of different structural arrangements are available for providing and attaching the transparent dialyzate exit tube  58 . Transparent dialyzate exit tube  58  can even originate inside chamber body  14  so long as the meniscus can still be viewed. As such, dialyzate exit tube  58  will be attached, either directly or indirectly, to some portion of sample cell  12 . The dialyzate exit tube  58  must, however, be transparent and should be oriented at any angle from horizontal to a configuration where a large portion thereof is oriented vertically upward. By vertically upward, it is meant that the dialyzate exit tube  58  can be oriented vertically with the end thereof which is open to atmosphere residing at a higher elevation than the remainder of exit tube  58 .  
         [0044]    In the description of the method and alternative methods of the present invention, the sample or solution is described as being introduced to the sample cell  12  above the membrane  16 . Those skilled in the art will recognize that a variety of configurations are available for sample cell  12  which may not be in agreement with the traditional meaning of the terms “above” and “below”. For example, the sample cell may take the shape of a U-shaped cylinder with the membrane positioned perpendicular to the cylindrical axis of the U-shaped cylinder almost anywhere along the length thereof. With that in mind, it is appropriate to think of the sample cell  12  being divided by the membrane  16  such that there is a sample side of the membrane  16 , (that side of the membrane  16  where the sample or solution is added to the sample cell  12 ), and a dializate side of the membrane  16 . For the purposes of this application “above” is intended to mean on the sample side of the membrane  16 .  
         [0045]    The present invention has far reaching potential uses. It allows for easy measurement of osmotic pressure of complex fluids and could be used as a method for screening drugs for activity in osmotically implicated biological phenomena such as blood pressure changes, arthritic swelling of joints and excessive ocular pressure. It makes possible the study of the evolution polymerization reactions, and colloidal instability phenomenon. The osmotic pressure gives a direct measure of the appearance or disappearance of macromolecular charged species. This sort of research has been hampered by the problems of commercial prior art osmometers overcome by this invention.  
         [0046]    From the foregoing, it will be seen that this invention is one well adapted to obtain all of the ends and objects hereinabove set forth together with other advantages which are apparent and which are inherent to the invention.  
         [0047]    It will be understood that certain features and subcombinations are of utility and may be employed with reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.  
         [0048]    As many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth and shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.  
       Parts List  
       [0049]    [0049] 10  osmometer  
         [0050]    [0050] 12  sample cell  
         [0051]    [0051] 14  chamber body  
         [0052]    [0052] 16  membrane  
         [0053]    [0053] 18  meandering dialyzate  
         [0054]    [0054] 20  base plate  
         [0055]    [0055] 22  o-ring  
         [0056]    [0056] 24  circumferential lip  
         [0057]    [0057] 26  support bracket  
         [0058]    [0058] 28  radial spokes  
         [0059]    [0059] 29  stir rod axle  
         [0060]    [0060] 30  stir rod blade  
         [0061]    [0061] 32  lid  
         [0062]    [0062] 34  bushing  
         [0063]    [0063] 35  o-ring  
         [0064]    [0064] 36  bore  
         [0065]    [0065] 38  pressurized gas conduit  
         [0066]    [0066] 40  pressurized gas source  
         [0067]    [0067] 42  pressure regulator  
         [0068]    [0068] 44  pressure gauge  
         [0069]    [0069] 46  L-shaped bore  
         [0070]    [0070] 48  pressure relief valve  
         [0071]    [0071] 50  handle  
         [0072]    [0072] 52  bore  
         [0073]    [0073] 54  bore  
         [0074]    [0074] 55  radial channels  
         [0075]    [0075] 56  coupling  
       Parts List—continued  
       [0076]    [0076] 57  concentric channels  
         [0077]    [0077] 58  transparent dialyzate exit tube  
         [0078]    [0078] 60  frame  
         [0079]    [0079] 62  magnetic stirring/hot plate  
         [0080]    [0080] 64  sample solution  
         [0081]    [0081] 66  meniscus  
         [0082]    [0082] 70  thermometer