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Determination of light absorption pathlength in a vertical-beam photometerDetermination of light absorption pathlength in a vertical-beam photometer description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060238764, Determination of light absorption pathlength in a vertical-beam photometer. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] This invention relates to the field of photometry. In particular, the invention relates to spectrophotometric methods and apparatus capable of determining the light absorption pathlength for various samples to be analyzed with a spectrophotometer. [0003] 2. Description of the Related Art [0004] The problem of an undefined light absorption pathlength in vertical-beam photometers has existed since the advent of vertical-beam photometers, i.e., for over 20 years. Substantial errors in determination by vertical-beam photometry of either relative optical pathlength or the concentration of analytes in a solvent contained in a sample-retaining device of unknown optical pathlength by prior art methods occur because of 1) substantial variation in solvent temperature, 2) substantial variation in the solvent composition, 3) substantial presence of materials in the samples which absorb light in the wavelength region where the optical pathlength of the solvent is being monitored and 4) optical aberrations which occur upon passing analysis light though the variable curved meniscus of samples having a liquid-gas interface. [0005] Photometry is a common measurement technique employed to monitor optical characteristics of samples. Customarily, samples contain an analyte species dissolved in a solvent at an unknown concentration. The concentration of the analyte in a sample may be determined by using a photometric device to measure the fraction of light absorbed by the sample at a specific wavelength (.lamda.). The value of .lamda. is usually chosen to be near the wavelength of light where the analyte absorbs maximally. According to the Beer-Lambert law, equation 1, absorbance is determined as follows: Absorbance .times. .times. ( A .lamda. ) = log .times. I o I = .lamda. 1 C ( 1 ) [0006] where I.sub.o is the incident radiation intensity, I is the intensity of light emerging from the sample, .epsilon..sub..lamda. is the molar extinction coefficient of the analyte dissolved in the solvent, I is the light absorption pathlength, and C is the concentration of absorbing analyte in the solvent. The value of I customarily is measured with a photometric apparatus, such as a photometer or spectrophotometer, equipped with a fixed light path sample-retaining device called a cuvette, such as a 1 cm light absorption pathlength cuvette. The sample-retaining device contains a sample comprised of analyte dissolved in a solvent. The value of I.sub.o is ordinarily measured with the same system (photometric apparatus, sample-retaining device and solvent except that no analyte is present in the solvent. Alternatively, I.sub.o may be measured in the absence of both the sample and the sample-retaining device (this value of I.sub.o is called an "air blank"). When an "air blank" is employed, a separate A.sub..lamda. measurement of the solvent and sample-retaining device gives a "solvent blank" absorbance value. A "corrected absorbance" value related to absorbance of the analyte is then obtained by subtracting the "solvent blank" from each absorbance measurement made on the samples comprised of analyte dissolved in solvent and contained in the sample-retaining device. These two alternative procedures and combinations thereof give mathematically equivalent results. Absorbance measurements made by either procedure allows unknown concentrations of the analyte to be determined by calculation according to Eq. 1, provided that .epsilon..sub..lamda. and I are known. [0007] A spectrophotometer is a photometric apparatus which employs an adjustable means to pre-select a desired portion of the electromagnetic spectrum as incident radiation. Usually spectrophotometers employ a monochrometer having a dispersive means, such as a prism or diffraction-grating, to provide continuously selectable, narrow, bands of light centered about the desired wavelength .lamda.. Most conventional photometers and spectrophotometers employ a horizontal light beam that traverses the liquid sample so as to avoid passing through a liquid-gas interface that is typically above the sample. With such horizontal-beam photometers, the geometry and optical pathlength within the sample is fixed for any given cuvette. Cuvettes for visible and ultraviolet light absorption measurements customarily have a 1 cm pathlength. Cuvettes with pathlengths between 0.1 cm and 10 cm are also common, however. With any such fixed pathlength cuvette in a horizontal-beam photometers, unknown concentrations C of the analytes may be calculated from absorbance measurements provided that the values of .epsilon..sub..lamda. and l are known. [0008] When either .epsilon..sub..lamda. and I is not known, values of C may be determined readily by employing known concentrations of the analyte dissolved in the same solvent (i.e., "standards") and performing similar light-absorbance measurements on unknown concentrations of analyte dissolved in the same solvent and on the standards. The most common procedure comprises plotting A.sub..lamda. versus concentration of analyte in the standards (i.e., a "standard curve") and then comparing the results obtained with the unknown concentrations of analyte to the standard curve. This procedure allows determination of the unknown concentrations of analyte from the "standard curve". [0009] Vertical-beam photometers also measure light absorption in order to determine the unknown concentrations of analyte in samples. In vertical-beam, photometers, however, the light beam usually passes only through one wall of the sample-retaining device, through the sample, and then through the interface between the sample a surrounding gas atmosphere (which is usually air). The latter liquid-gas interface, the meniscus, is usually curved, the specific shape depending upon the interactions between the liquid sample and the gas and the side-walls of the sample retaining device. Depending upon the design of a particular vertical-beam photometer, the light beam may traverse the meniscus either before or after passing through the sample. In either case, the optical pathlength through the sample is not a constant value. Instead, the optical pathlength is related to the sample volume and the meniscus shape. The nature of the sample, the sample-retaining device surfaces, and gas each contributes to the shape of the meniscus, quantitatively affecting the optical pathlength through the sample. Thus, in vertical beam photometers, the value of l in Eq. 1 usually is unknown and is difficult to control reproducibly. [0010] Vertical-beam photometry has become a popular technique despite the disadvantage of not having a fixed optical pathlength through the sample. This popularity stems from the fact that the optical characteristics of a large multiplicity of samples may be analyzed with a vertical-beam photometer in a small period of time. Typically, vertical-beam photometers monitor the optical characteristics of samples disposed in the wells of, for example, 96 well multi-assay plates. The optical characteristics, such as light absorption or light scattering, of the samples contained within each well of such multi-assay plates may be monitored, typically, in 10 seconds or less, and generally in one minute or less. Vertical-beam photometers also allow repetitive measurements of such a multiplicity of samples to be made typically with intervals of 10 seconds or less (and generally in one minute or less) between each of a series of measurements. In such a way the kinetic properties, such as the rate of change in absorbance, of a plurality of samples may be monitored in a very short time. [0011] In vertical-beam photometry of the prior art, an approximated constant value of l is used for standards and unknowns. Concentrations of unknown analytes are determined, often with acceptable precision, by plotting "standard curves" using the approximated value of l and comparing the absorbance results obtained with unknown concentrations of analyte to the standard curve, as mentioned previously. [0012] The fact that a value of C may not be calculated directly from Eq. 1, but instead must be determined from a standard curve constructed for each analytical measurement, severely hinders the ability of vertical-beam photometric techniques. The additional time and expense required for preparing such standard curves for each analysis is often an onerous disadvantage to vertical-beam photometry. Thus, convenient, accurate, and precise methods and apparatus for determining optical pathlength of samples in vertical-beam photometers would be of great utility. [0013] Japanese Kokai Patent Application number Sho58[1983]-1679Y2 discloses that the unknown optical pathlength of vessels may be determined by dispensing a colored solution, with a known relationship between optical pathlength and color absorbance, into the vessels and determining the color absorbance of this solution. A similar method is taught in U.S. Pat. No. 5,298,978, issued Mar. 29, 1994. [0014] Additionally Japanese Kokai Patent Application numbers Sho 60(1985]-183560 and Sho 61[1986]-82145 disclose methods of determining relative optical pathlength of aqueous samples within different reactor vessels (contained in a common reactor) by measuring the optical density of the samples at two different wavelengths in the near-infrared wavelength region from 900 to 2100 nanometers. With clear quartz reaction vessels, the reference teaches that (A.sub.975-A.sub.900), (A.sub.1195-A.sub.1070), or (A.sub.1260-A.sub.1070) may be used to determine the relative optical pathlength through aqueous samples. For reactors made of synthetic acryl resins, where the resin has interfering absorption bands, the prior art teaches that (A.sub.970-A.sub.1070) or (A.sub.1280-A.sub.1070) may be used to determine relative optical pathlength of the samples. Once relative optical pathlength is known for each of the vessels of the reactor, then optical density values of analyte (measured at a third wavelength) may be normalized for variation in optical pathlength to obtain the relative concentration of analyte in each reactor vessel. Employing vessels with known concentrations of analyte allows one to determine the absolute concentrations of analyte within other vessels. [0015] There also exists need for methods and apparatus that may be utilized with samples that are dissolved in a variety of different solvents or in mixtures of different solvents. Because analytes are extremely diverse and may have diverse light-absorption properties, there exists no apparatus capable of determining concentration and optical pathlength of any analyte dissolved in various solvents or mixtures of solvents. Further complicating this situation is the extreme variability of concentrations of analytes from one sample to the next. SUMMARY OF THE INVENTION [0016] The instant invention provides a solution to the problem of undefined light absorbance pathlength in vertical-beam photometers. The invention provides methods and devices that are convenient to employ and that require minimal additional measurement apparatus. Thus, the cost associated with making such measurement is kept to a minimum. [0017] The invention further provides methods and apparatus for determining optical pathlength and sample concentration that furnish accurate and reproducible results. The results, determined by using the invention in vertical-beam photometers, are essentially interchangeable and indistinguishable from those obtained in horizontal-beam photometry. [0018] The invention also provides methods and apparatus for determining optical pathlengths between 1 millimeter and 1 centimeter in aqueous samples within vertical-beam photometers. The inventive methods and apparatus may be utilized with samples that are dissolved in a variety of different solvents or in mixtures of different solvents. [0019] Thus, in one embodiment of the invention, optical pathlength is determined in vertical-beam photometers by analyzing an optical property of the sample solvent which is dependent upon optical pathlength but independent of all relevant concentrations of all analytes which may possibly be contained within a sample solvent. [0020] In one aspect, the invention provides multi-channel photometric analysis devices for determining optical characteristics of analytes in sets of liquid-containing samples having unknown optical pathlengths. These devices comprise [0021] a. a first sample holder for holding the sets of liquid-containing samples in one or more substantially vertical optical channels; [0022] b. a means for positioning the sets of samples in the optical channels; [0023] c. a light source means, a wavelength selection means, and a light distribution means which cooperate to transmit light substantially vertically through the samples, wherein the light comprises a first calibration wavelength, a second calibration wavelength and an analyte-measuring wavelength, wherein the first and second calibration wavelengths are different and within the near infrared wavelength region of from 750 to 2500 nanometers and provide characteristic light signal values for each liquid sample, wherein there exists a predetermined relationship between the light signal values and the optical pathlength through the samples and wherein the analyte-measuring wavelength provides a characteristic analyte light signal value related to the concentration of analyte present in each sample; [0024] d. a detector for determining measured light signal values from light transmitted through each sample at the first and second calibration wavelengths and the analyte-measuring wavelength; [0025] e. a means for determining a measured relationship between the light signal values; [0026] f. a means for determining from the measured relationship and the predetermined relationship a correction factor related to the optical pathlength through each sample; [0027] g. a means for determining from the correction factor and the analyte light signal value, a ratio relating the analyte signal value to the optical pathlength in each of the samples. [0028] The invention also encompasses vertical-beam photometric devices for measuring the rate of change in optical characteristics of samples contained in sample sites disposed on an assay plate, the device comprising: [0029] a wavelength selection means for selecting a first wavelength band of light from a first wavelength range ad for selecting a second and a third wavelength bands of light from within a second wavelength range; Continue reading about Determination of light absorption pathlength in a vertical-beam photometer... Full patent description for Determination of light absorption pathlength in a vertical-beam photometer Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Determination of light absorption pathlength in a vertical-beam photometer patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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