Methods and Apparatus For Measuring the Concentration of a Substance in a Solution

• US 20080304048A1
• Filed: 11/28/2006
• Published: 12/11/2008
• Est. Priority Date: 11/29/2005
• Status: Active Grant

• Current Assignee: Cytiva Sweden AB (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences AB (GE Healthcare Technologies, Inc.)
• Inventors: Tormod, Stig
• Granted Patent: US 8,068,227 B2
• Time in Patent Office: Days
• US Class Current: 356/51
• CPC Class Codes: G01N 2021/0193 the sample being taken from a stream or flow to the measurement cell
• CPC Class Codes: G01N 2030/746 detecting along the line of flow, e.g. axial
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/33 using ultraviolet light G01N21/39 takes precedence
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/0624 Compensating variation in output of LED source
• CPC Class Codes: G01N 2201/0639 Sphere lens
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J

Abstract

The present invention relates to methods and apparatus for detecting and measuring the concentration of a substance in a solution, the substance having an absorption at 300 nm or less. The methods and apparatus have particular utility in detecting and measuring the concentration of proteins and nucleic acids.

Claims

1-25. -25. (canceled)

26. :

A method for measuring the concentration of a substance in a solution, said substance having an absorption at 300 nm or less, said method comprising;

i) separating said substance from one or more compounds present in said solution by chromatography;

ii) transmitting ultraviolet light of wavelength 300 nm or less from a light source through a band pass filter;

iii) passing said ultraviolet light emanating from said band pass filter through a beam-splitter;

iv) diverting a first portion of the light by the beam-splitter and quantifying the electromagnetic radiation of said first portion with a reference detector to obtain a reference value;

v) irradiating a known path length of the solution with a second portion of the light passing through the beam splitter;

vi) quantifying the electromagnetic radiation transmitted through the solution with a sample detector to obtain a sample value; and

vii) calculating the absorbance (A) from said sample value and said reference value;

wherein said light source is a light emitting diode which emits light of wavelength of 300 nm or less.

27. :

The method of claim 26, wherein the solution is selected from the group consisting of cell extract, cell lysate and cell culture or mixtures thereof.

28. :

The method of claim 26, wherein the solution is irradiated in a cell of known internal volume, said cell being transparent to light of wavelength of 300 nm or less.

29. :

The method of claim 26, wherein said reference detector and said sample detector is a UV sensitive photomultiplier or a UV sensitive photodiode.

30. :

The method of claim 26, wherein said substance is a protein or a peptide.

31. :

The method of claim 26, wherein said substance is a nucleic acid.

32. :

The method of claim 26, wherein said chromatography is selected from the group consisting of adsorption chromatography, affinity chromatography, high-performance liquid chromatography, ion-exchange chromatography, exclusion chromatography, gel-filtration chromatography, gel-permeation chromatography, hydrophobic chromatography, ionic-interaction chromatography, reverse phase chromatography, ligand-mediated chromatography and molecular sieve chromatography.

33. :

An apparatus for measuring the concentration of a substance in a solution, said substance having an absorption at 300 nm or less, comprising;

i) a cell of known path length for containing said solution, said cell being transparent to light of wavelength of 300 nm or less;

ii) a light source, wherein said light source is a light emitting diode which emits light of wavelength of 300 nm or less;

iii) a band pass filter;

iv) a beam splitter for dividing light into a first portion and a second portion;

v) a reference detector for detecting electromagnetic radiation diverted by said beam splitter;

vi) a sample detector for detecting the electro magnetic radiation transmitted through said solution; and

vii) a chromatographic separation means for separating the substance from one or more compounds present in the solution.

34. :

The apparatus of claim 33, wherein said cell is of known internal volume.

35. :

The apparatus of claim 33, wherein said reference detector and said sample detector is a UV sensitive photodiode or a UV sensitive photomultiplier.

36. :

The apparatus of claim 33, further comprising a collimating lens.

37. :

The apparatus of claim 33, further comprising a focusing lens.

38. :

The apparatus of claim 33, wherein said chromatographic separation means is a chromatography column or membrane.

39. :

The apparatus of claim 38, wherein said chromatography column or membrane separates the substance from said one or more compounds on the basis of chromatography selected from the group consisting of adsorption chromatography, affinity chromatography, high-performance liquid chromatography, ion-exchange chromatography, exclusion chromatography, gel-filtration chromatography, gel-permeation chromatography, hydrophobic chromatography, ionic-interaction chromatography, reverse phase chromatography, ligand-mediated chromatography and molecular sieve chromatography.