Determination of light absorption pathlength in a vertical-beam photometer

• US 5,959,738 A
• Filed: 07/01/1997
• Issued: 09/28/1999
• Est. Priority Date: 07/25/1994
• Status: Expired due to Term

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Hafeman, Dean G., Chow, Calvin T.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): Vierra-Eisenberg, Jason D.
• Application Number:
  US08/886,711
• Time in Patent Office: 819 Days
• Field of Search: 356/381-83, 356/51, 356/319-320, 356/414, 356/418-419, 356/434, 356/436, 250/373, 250/339.05, 250/339.12
• US Class Current: 356/440
• CPC Class Codes: G01F 22/00 Methods or apparatus for measuring volume of fluids or fluent solid material, not otherwise provided for
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/3151 using two sources of radiation of different wavelengths G01N21/33 - G01N21/39 take precedence

Abstract

Disclosed are photometric methods and devices for determining optical pathlength of liquid samples containing analytes dissolved or suspended in a solvent. The methods and devices rely on determining a relationship between the light absorption properties of the solvent and the optical pathlength of liquid samples containing the solvent. This relationship is used to establish the optical pathlength for samples containing an unknown concentration of analyte but having similar solvent composition. Further disclosed are methods and devices for determining the concentration of analyte in such samples where both the optical pathlength and the concentration of analyte are unknown. The methods and devices rely on separately determining, at different wavelengths of light, light absorption by the solvent and light absorption by the analyte. Light absorption by the analyte, together with the optical pathlength so determined, is used to calculate the concentration of the analyte. Devices for carrying out the methods particularly advantageously include vertical-beam photometers containing samples disposed within the wells of multi-assay plates, wherein the photometer is able to monitor light absorption of each sample at multiple wavelengths, including in the visible or UV-visible region of the spectrum, as well as in the near-infrared region of the electromagnetic spectrum. Novel photometer devices are described which automatically determine the concentration of analytes in such multi-assay plates directly without employing a standard curve.

Claims

1. A photometric method for determining an optical pathlength of a sample having an analyte dissolved or suspended in an aqueous solvent wherein a relationship between light absorption of the analyte and the optical pathlength of the sample are unknown, the method comprising:

measuring a first light signal resulting from transmission of light, having a first predetermined wavelength, through the aqueous sample, and

measuring a second light signal resulting from transmission of light, having a second predetermined wavelength through the aqueous sample, and

determining the optical pathlength of the sample from a predetermined relationship between the first and second light signal, and the optical pathlength of the aqueous solvent, wherein the predetermined relationship between the first and second light signals are measured at at about 988-1008 nanometers and about 900-910 nanometers respectively.

2. The method of claim 1 wherein the first and second wavelength are in a near-infrared region of an electromagnetic spectrum of from 750 nanometers to 2500 nanmometers wavelength.

3. A photometric method of determining an optical pathlength of a sample having an analyte dissolved or suspended in an aqueous solvent wherein a relationship between light absorption of the analyte and the optical pathlength of the sample are unknown, the method comprising:

measuring a difference between a first and a second light signal resulting from transmission of light of a first and a second predetermined wavelength, respectively, through the sample, and

determining the optical pathlength of the aqueous sample from a predetermined relationship between the difference and a light absorption pathlength of the aqueous solvent, wherein the predetermined relationship between the first and second light signals are measured at at about 988-1008 nanometers and about 900-910 nanometers respectively.

4. A photometric method for determining either a concentration of an analyte, absorbance of an analyte per unit light absorption pathlength, or fraction of light transmitted by an analyte per unit light absorption pathlength, wherein the analyte is suspended or dissolved in a liquid aqueous solvent such that a relationship between light absorption of the analyte and the light absorption pathlength of the analyte are unknown, the method comprising:

measuring a first, a second and a third light signal resulting from, respectively, a first, second and third predetermined wavelength of light transmitted through substantially identical optical pathlengths within a sample and wherein a difference between the first and second light is related to the optical pathlengths and the third light signal is related to both the concentration of the analyte and the optical pathlengths;

measuring a fourth and fifth light signal resulting from, respectively, the first and second wavelengths of light transmitted through a predetermined optical pathlength of the aqueous solvent;

determining a difference in the first and second light signals and a difference in the fourth and fifth light signals; and

further determining either the concentration, the absorbance at the third wavelength per unit absorption pathlength, or the fraction of light transmitted at the third wavelength per unit light absorption pathlength, from the predetermined light pathlength, the third light signal, the difference in the first and second light signals and the difference in the fourth and fifth light signals wherein the first and second wavelengths are 985-1008 nanometers and about 900-910 nanometers respectively.

5. A photometric method for determining either a concentration, an absorbance per unit light absorption pathlength, or a fraction of light transmitted per unit absorption pathlength for an analyte suspended or dissolved as a sample in an aqueous solvent wherein neither an optical pathlength or the sample nor its relationship to light absorption of the analyte are known, the method comprising:

measuring a first, a second and a third light signal resulting from, respectively, a first, second and third predetermined wavelength of light transmitted through the sample wherein the first and second wavelengths are 985-1008 nanometers and about 900-910 nanometers respectively,

measuring a fourth and fifth light signals resulting from, respectively, the first and second wavelengths of light transmitted through a predetermined optical pathlength of a first reference liquid containing the solvent alone, measuring a sixth, seventh, and eighth light signal resulting from respectively, the first, second and third predetermined wavelength of light transmitted through a predetermined light pathlength of a second reference liquid having the analyte suspended or dissolved in the solvent and determining either a concentration, an absorbance per unit light absorption pathlength, or a fraction of light transmitted per unit light absorption pathlength, of the analyte from the predetermined light pathlength and the first, second, third, fourth, fifth, sixth, seventh, and eighth light signals.

6. A photometric method for determining optical pathlength of an aqueous liquid sample, containing analyte in a solvent, and disposed on a multi-assay plate, the method comprising:

measuring a difference in an amount or fraction of light transmitted, at two different wavelengths, through a reference liquid consisting of the solvent, and contained in a liquid-containing means of known optical pathlength;

measuring a difference in the amount or fraction of light transmitted through the aqueous liquid sample at the first and second wavelengths, wherein for each wavelength the light passes substantially vertically through a meniscus of the sample and wherein the analyte is at concentration unknown with respect to the second reference aqueous liquid wherein the first and second wavelengths are 985-1008 nanometers and about 900-910 nanometers respectively,

determining the optical pathlength of the aqueous liquid sample in a multi-assay plate by relating a difference so measured in the liquid sample to the difference so measured in the reference aqueous liquid and to the known optical pathlength wherein the aqueous liquid and the multi-assay plate are maintained at substantially the same temperature.

7. A method for determining a concentration of an analyte in an aqueous liquid sample containing a solvent and disposed in a multi-sample assay plate comprising:

measuring a difference in the amount, or fraction of, light transmitted through a first and a second reference aqueous liquid for each of two different wavelengths wherein the first reference liquid consists of the solvent substantially without the analyte and the second reference liquid consists of the analyte dissolved or suspended in the aqueous solvent and wherein the first and second reference liquids are retained in a liquid containing means of known pathlength;

measuring the amount, or fraction of, light transmitted through the second reference aqueous liquid at a third wavelength;

measuring the amount, or fraction of, light transmitted at the first, second and third wavelengths, substantially vertically through the sample wherein the analyte is present in the aqueous solvent at a concentration unknown with respect to the first or second aqueous liquids, and

determining the concentration of the analyte in the aqueous liquid sample by relating the difference to an amount, or fraction of, light transmitted through the sample at the first, second and third wavelengths wherein the first and second wavelengths are 985-1008 nanometers and about 900-910 nanometers respectively.

8. A photometric device comprising

a light source for providing at least a portion of a first wavelength range,

a wavelength selection means for providing light of a first, a second, and a third wavelength within the first wavelength range,

a sample retaining means for retaining one, or more cuvettes of predetermined light absorption pathlength,

a sample retaining means for retaining a multi-assay plate,

a light-transmitting means for transmitting at least the first and second wavelength through the cuvette(s),

a light-transmitting means for transmitting at least the first, second and third wavelength through sample sites on the multi-assay plate,

photodetector means for detecting the light transmitted through the cuvette(s) and the light transmitted through the sample sites,

a means for relating the light transmitted through the cuvette and the light transmitted through the sample sites additionally comprising a means for determining light absorption pathlength of the samples contained in the multi-assay plate from the relative transmission of the first and second wavelengths of light through the samples and through the cuvette, additionally comprising a means for maintaining the multi-assay plate and the cuvette at substantially the same temperature, wherein the light is transmitted substantially horizontally through the cuvette, so that the light need not pass through a gas-liquid interface when the sample is a liquid, and wherein the light is transmitted substantially vertically through samples contained in the multi-assay plate, so that the light generally will pass through a gas-liquid interface when the samples retained in the multi-assay plate are liquid.