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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
First Claim
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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 whereMw =the weight averaged molecular mass of the biological proteins which caused the scattered light,Is /Io =the intensity of the scattered light, andB=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 (Is /Io, Mw) for the known weight averaged molecular mass of the biological proteins.

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