HPLC light scattering detector for biopolymers

• US 5,250,186 A
• Filed: 10/09/1992
• Issued: 10/05/1993
• Est. Priority Date: 10/23/1990
• Status: Expired due to Term

• Current Assignee: Cetus Corporation (Novartis Ag)
• Original Assignee: Cetus Corporation (Novartis Ag)
• Inventors: Cunico, Robert L., Kunitani, Michael G., Dollinger, Gavin D.
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US07/959,112
• Time in Patent Office: 361 Days
• Field of Search: 210/656, 210/85, 210/198.2, 204/180.1, 204/299 R, 422/69, 422/70, 436/161, 436/171, 436/177, 356/366, 356/368, 356/369
• US Class Current: 210/656
• CPC Class Codes: G01N 15/0205 by optical means
• CPC Class Codes: G01N 15/04 Investigating sedimentation of particle suspensions
• CPC Class Codes: G01N 2021/4726 Detecting scatter at 90°
• CPC Class Codes: G01N 21/532 with measurement of scattering and transmission
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A high angle light scattering detector using classical Rayleigh scattering. A high intensity arc light source, filtered to leave only one wavelength illuminates a flow cell. Through the flow cell, very small particles such as biological proteins flow in solution after separation by HPLC or some other means. A UV detector generates data regarding the weight concentration of the eluting particles and a scattered light detector collecting scattered light at angles of approximately 90° generates a scattered light signal. The incident light intensity is also measured. The average molecular weight is then computed using the scattered and incident light data, the weight concentration data and a simplified mathematical relationship from which the size factor P and the viral coefficients have been eliminated.

Claims

1. An apparatus for determining the molecular weight of biological proteins eluting from a liquid chromatography column, comprising:

a mercury arc lamp light source having a characteristic curve relating output light intensity to wavelength;

a glass flow cell having a small volume flow channel having a volume approximately equal to 10 microliters which is coated with an index matching material to approximately match the refractive index of said glass to the index of refraction of the solvent eluting from said column and in fluid communication with the output of said liquid chromatography column, an input window, for providing an aperture through which light from said light source can shine on fluid flowing in said flow channel carrying said biological proteins, and having an output window for providing an aperture through which light scattered by said biological proteins at high angles can leave said flow cell, said input and output windows being optically coated with an index matching material to approximately match the index of refraction of said glass to the index of refraction of air;

a filter interposed between said light source and said input window for filtering out substantially all light other than a selected wavelength;

focussing means for focussing the image of said light source on said input window so as to minimize scattered light;

a photomultiplier light detector having a characteristic curve relating the magnitude of the output signal therefrom to the wavelength of light detected, and located such that light scattered by said biological proteins exiting from said output window causes said light detector to output a signal proportional to the intensity of said scattered light, and wherein said filter filters out all light except a wavelength at or near where the product of said characteristic cures for said arc lamp and said photomultiplier tube is a maximum;

concentration detector means in fluid communication with fluid exiting said liquid chromatography column embodying said biological proteins for detecting the weight concentration of said biological proteins and for generating a signal proportional to said weight concentration; and

means for receiving said signals proportional to said scattered light intensity and said weight concentration, for calculating said molecular weight of said biological proteins based upon the relationship

space="preserve" listing-type="equation">I.sub.s /I.sub.o =C B M.sub.w

where

M_w =the weight averaged molecular mass of the biological proteins which caused the scattered light,

I_s /I_o =the intensity of the scattered light, and

B=an empirically measured constant which depends upon the optical performance of the system and which is determined by putting at least two biological proteins of different known molecular weights through said liquid chromatography column and measuring the resulting scattered light intensity from each which results for each known molecular weight and plotting the linear function which results from the points (I_s /I_o, M_w) for the known weight averaged molecular mass of the biological proteins.

2. 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_s /I_o =the intensity of the scattered light at the selected angle;

C=the wight concentration of said particles in said solvent stream;

M_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.

3. 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° and

145°

, 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_w =the weight averaged molecular mass of the scatterer particles

a size parameter which corrects the relationship for the effects of multiple intraparticle scattering;

A₂ and A₃ =the second and third viral coefficients respectively, and

C=the weight concentrationof the scatterer particles,

and wherein ##EQU15## where P(θ

)^-1 =the inverse size factor,

n=the index of refraction,

R_g =the radius of gyration of the scatterer particles,

θ

=the scattering angle between the incident light and the scattered light, and

λ

=the wavelength of the incident light

and wherein the R₉ may be ignored as substantially zero for small particles having sizes less than λ

_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_g and M_w, calculating M_w by substituting for R_g in equation (2) herein the expression in M_w which is equivalent thereto and substituting equation (2) herein as thus rewritten into equation (1) herein for P(θ

) and automatically solving for M_w using a digital computer.

4. The apparatus of claim 3 wherein said means for illuminating includes a mercury arc lamp having a characteristic curve of output light intensity versus wavelength and a filter, and wherein said means for measuring said scattered light intensity includes a photomultiplier tube having a characteristic curve relating output signal magnitude versus wavelength, and wherein said filter filters out all wavelength except a wavelength at or near the wavelength at which the product of said two characteristic curves is a maximum.

5. 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

space="preserve" listing-type="equation">I.sub.s /I.sub.o =BcM.sub.w

where

I_s I_o =the scattered light intensity at a high angle between 35°

to 145°

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_w =the weight average molecular mass.

6. The apparatus of claim 5 wherein the particle sizes are limited so as to be equal to or smaller than λ

_o /4 where λ

_o is the wavelength light incident upon said flow cell.

7. The apparatus of claim 5 wherein said filter filters out all wavelengths except a wavelength which is located so as to generate maximum output signal from said scattered light detector.

8. The apparatus of claim 5 further comprising a polarizer located so as to prevent all light having other than vertical polarization from reaching said flow cell.

9. The apparatus of claim 5 wherein said flow cell has a scattering volume through which said particles flow of approximately 10 microliters.

10. The apparatus of claim 5 wherein said scattered light detector is a photomultiplier.

11. The apparatus of claim 10 wherein said photomultiplier is optimized for the blue-green wavelength and said filter filters out all wavelengths except a wavelength in the blue-green portion of the spectrum.

12. The apparatus of claim 10 wherein said photomultiplier is cooled.

13. The apparatus of claim 10 wherein said photomultiplier is a photon counting device.

14. The apparatus of claim 5 wherein said light source is a mercury arc lamp.

15. The apparatus of claim 5 wherein said light source is a Xenon arc lamp.

16. The apparatus of claim 5 wherein said light source is a laser.

17. The apparatus of claim 16 wherein said laser has blue-green output light.