HPLC light scattering detector for biopolymers

A method of determining the average molecular weight of particles in a solution, comprising:

• generating incident light having a first wavelength selected to cause a scattered light detector to output a maximum signal;
• directing said incident light on a solution containing said particles;
• generating incident radiation having a second wavelength suitable for measuring Uv absorption;
• directing said incident radiation at said second wavelength at a solution containing said particles;
• measuring the scattered light intensity for high angle scattered light at said first wavelength and the intensity of light incident on said solution at said first wavelength;
• measuring the intensity of transmitted light at said second wavelength which passed through said solution and the intensity of incident light at said second wavelength falling on said solution;
• calculating the weight concentration of said particles using the values of the intensity of incident light and said transmitted light at said second wavelength using a predetermined relationship;
• calculating the weight averaged molecular weight of said particles in said solution according to the relationship ##EQU13## where I.sub.s /I.sub.o =the intensity of the scattered light at the selected angle;
• C=the wight concentration of said particles in said solvent stream;
• M.sub.w =the average molecular weight of said particles;
• and
• B=a constant which can be empirically determined for each system by calibrating the system with toluene or by measuring the relative scattered light intensity for each of at least two particle samples having known average molecular weight.

An apparatus for generating signals from which the average molecular weight of particles in solution may be calculated, comprising:

• means for illuminating the particles in solution with a single wavelength of light;
• means for measuring the intensity of light scattered from said particles at a selected angle between 35.degree. and 145.degree., said scattered light intensity being measured relative to the intensity of light incident on said particles and for outputting a signal indicative of said relative scattered light intensity;
• means for measuring the weight concentration of said particles in solution and for outputting a signal indicative thereof;
• and
• calculation means for receiving said signals indicative of weight concentration and relative scattered light intensity and for calculating the weight averaged molecular weight of said particles using the relationships ##EQU14## where K=an optical constant relating wavelength, refractive index of the solution and change of refractive index of the solution over time among other things and which can be measured empirically for any selected system by using at least two types of particles of known weight averaged molecular weight to calibrate the system;
• R=the specific Raleigh constant
• M.sub.w =the weight averaged molecular mass of the scatterer particles
• a size parameter which corrects the relationship for the effects of multiple intraparticle scattering;
• A.sub.2 and A.sub.3 =the second and third viral coefficients respectively, and
• C=the weight concentrationof the scatterer particles,
• and wherein ##EQU15## where P(.theta.).sup.-1 =the inverse size factor,
• n=the index of refraction,
• R.sub.g =the radius of gyration of the scatterer particles,
• .theta.=the scattering angle between the incident light and the scattered light, and
• .lambda.=the wavelength of the incident light
• and wherein the R.sub.9 may be ignored as substantially zero for small particles having sizes less than .lambda..sub.o /4 such as substantially all bioproteins of interest, and wherein for larger particles about which sufficient information is known to look up in a table the relationship between R.sub.g and M.sub.w, calculating M.sub.w by substituting for R.sub.g in equation (2) herein the expression in M.sub.w which is equivalent thereto and substituting equation (2) herein as thus rewritten into equation (1) herein for P(.theta.) and automatically solving for M.sub.w using a digital computer.

An apparatus for generating signals from which molecular weight or mass of a particle in a solution stream eluting from a liquid chromatography column can be determined, comprising:

• a light source;
• a filter for limiting the light from said light source to a single known wavelength which is substantially longer than the size of the particles;
• a flow cell for receiving said stream and having an input window for receiving light from said filter to illuminate said stream and having an exit window through which light scattered from said particles at high angles of approximately ninety degrees exits said flow cell;
• a scattered light detector for receiving said scattered light at high angles exiting said exit window, and having a band of maximum sensitivity and having an output at which is output an electrical signal proportional to the intensity of said scattered light;
• means for receiving at least a portion of the light from said filter incident upon said flow cell and for determining the intensity of said light and outputting an electrical signal proportional thereto;
• receiving light from said light source;
• means for detecting the concentration of said particles in said solution and outputting a signal proportional thereto;
• and
• calculation means for receiving said electrical signals proportional to the scattered and incident light intensities and the weight concentration and for calculating the average molecular weight of said particles using the relationship
• I.sub.s /I.sub.o =BcM.sub.w
• where
• I.sub.s I.sub.o =the scattered light intensity at a high angle between 35.degree. to 145.degree. relative to the intensity of the incident light
• B=a predetermined optical constant which is experimentally determined for any particular optical system by measuring the scattered light intensities for at least two types of particles having known molecular weights, and
• C=the weight concentration of the particles in solution, and
• M.sub.w =the weight average molecular mass.