QUANTITATIVE DUAL-DYE PHOTOMETRIC METHOD FOR DETERMINING DILUTION IMPACT
First Claim
1. A method of measuring a liquid volume of a sample solution in a vessel, wherein the vessel includes a bottom and known dimensions, the method comprising the steps of:
- a. adding to the vessel a diluent including a known concentration of a diluent chromophore which absorbs light at a first wavelength;
b. measuring the absorbance of the diluent chromophore at the first wavelength;
c. adding a volume of the sample solution to the vessel, wherein the sample solution includes a known concentration of a sample solution chromophore which absorbs light at a second wavelength, and wherein the sample solution does not include the diluent chromophore;
d. mixing the diluent and the sample solution in the vessel to produce a mixture of the sample solution and the diluent;
e. measuring the absorbance of the mixture of the sample solution and the diluent at the first wavelength and at the second wavelength; and
f. calculating the volume of the sample solution added to the vessel based on the measured absorbances at the first wavelength and the second wavelength.
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Accused Products
Abstract
The invention provides ways to determine the impact of diluting a solution wherein the diluting may be carried out for any of a variety of purposes. In one embodiment, the method enables accurate volume dispensation calculations independent of meniscus shape. In another embodiment, the method enables accurate determination of plate washing efficiency. In yet another embodiment, the method enables the accurate determination of dilution ratio over a plurality of dilution steps. The methods described may be carried out using one or more systems arranged to perform the steps. A kit of the invention includes instructions for carrying out the steps of the methods and, optionally, one or more solutions suitable for conducting photometric measurements.
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Citations
27 Claims
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1. A method of measuring a liquid volume of a sample solution in a vessel, wherein the vessel includes a bottom and known dimensions, the method comprising the steps of:
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a. adding to the vessel a diluent including a known concentration of a diluent chromophore which absorbs light at a first wavelength;
b. measuring the absorbance of the diluent chromophore at the first wavelength;
c. adding a volume of the sample solution to the vessel, wherein the sample solution includes a known concentration of a sample solution chromophore which absorbs light at a second wavelength, and wherein the sample solution does not include the diluent chromophore;
d. mixing the diluent and the sample solution in the vessel to produce a mixture of the sample solution and the diluent;
e. measuring the absorbance of the mixture of the sample solution and the diluent at the first wavelength and at the second wavelength; and
f. calculating the volume of the sample solution added to the vessel based on the measured absorbances at the first wavelength and the second wavelength. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method of determining the dilution of a sample solution under dilution using a plurality of vessels, wherein the sample solution includes a first chromophore that absorbs light at a first wavelength and a second chromophore that absorbs light at a second wavelength, the method comprising the steps of:
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a. measuring in the sample solution the absorbance of the first chromophore at the first wavelength and the absorbance of the second chromophore at the second wavelength, wherein the sample solution is contained in a first vessel of the plurality of vessels;
b. transferring a target volume of the sample solution from the first vessel to a second vessel of the plurality of vessels;
c. mixing into the sample solution in the second vessel a target volume of a diluent, wherein the diluent includes the second chromophore at a concentration substantially equivalent to the concentration of the second chromophore in the sample solution;
d. measuring the absorbance of the first chromophore at the first wavelength and the absorbance of the second chromophore at the second wavelength in the second vessel; and
e. calculating a dilution ratio for the sample solution contained in the second vessel, wherein the dilution ratio represents the extent to which the sample solution has been diluted by the diluent mixed into the second vessel. - View Dependent Claims (9, 10, 11, 12)
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13. A method of determining the dilution of a sample solution diluted with a diluent, wherein the sample solution includes a known concentration of a first chromophore that absorbs light at a first wavelength, and a known concentration of a second chromophore that absorbs light at a second wavelength, the method comprising the steps of:
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a. transferring a target volume of the sample solution from a source into a vessel;
b. mixing into the sample solution in the vessel a target volume of the diluent, wherein the diluent includes the second chromophore at a concentration substantially equivalent to the known concentration of the second chromophore in the sample solution;
c. measuring the absorbance of the first chromophore at the first wavelength and the absorbance of the second chromophore at the second wavelength in the vessel; and
d. calculating a dilution ratio of the sample solution from the source, wherein the dilution ratio represents the extent to which the sample solution of the source has been diluted by the diluent mixed into the vessel. - View Dependent Claims (14, 15, 16)
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17. A method of determining the dilution achieved in carrying out a dilution protocol using a sample solution and a diluent, wherein the sample solution includes a first chromophore that absorbs light at a first wavelength and a known concentration of a second chromophore that absorbs light at a second wavelength, the method comprising the steps of:
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a. adding to a first vessel of a first set of a plurality of vessels a target volume of a first sample solution, wherein the first sample solution includes a first known concentration of the first chromophore and a known concentration of the second chromophore;
b. adding to a first vessel of a second set of the plurality of vessels a target volume of a second sample solution including a second known concentration of the first chromophore and the known concentration of the second chromophore, wherein the second known concentration of the first chromophore of the second sample solution is higher than the first known concentration of the first chromophore of the first sample solution, and wherein the target volume of the second sample solution added to the first vessel of the second set is substantially equivalent to the target volume of the first sample solution added to the first vessel of the first set;
c. carrying out a first dilution protocol step comprising;
i. mixing into the first vessel of the first set a first target volume of a diluent, wherein the diluent includes the second chromophore at a concentration substantially equivalent to the known concentration of the second chromophore in the first sample solution and the second sample solution;
ii. mixing into the first vessel of the second set the first target volume of the diluent; and
iii. measuring the absorbance of the first chromophore at the first wavelength and the absorbance of the second chromophore at the second wavelength in the first vessel of the first set;
d. carrying out a second dilution protocol step comprising;
i. transferring a target volume of the mixture of the first sample solution and the diluent of the first vessel of the first set to a second vessel of the first set;
ii. mixing a second target volume of the diluent into the second vessel of the first set;
iii. transferring a target volume of the mixture of the second sample solution and the diluent of the first vessel of the second set to a second vessel of the second set;
iv. mixing the second target volume of the diluent into the second vessel of the second set; and
v. measuring the absorbance of the first chromophore at the first wavelength and the absorbance of the second chromophore at the second wavelength in the second vessel of the second set; and
e. calculating a dilution ratio of the dilution protocol based on the absorbance measurements made in steps c.iii and d.v, wherein the dilution ratio represents the extent of dilution occurring between the first dilution protocol step and the second dilution protocol step. - View Dependent Claims (18, 19, 20, 21)
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22. A method of determining plate washing efficiency, comprising the steps of:
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a. placing a diluent in a first vessel to a level establishing a diluent height, wherein the diluent includes a known concentration of a diluent chromophore that absorbs light at a first wavelength, and wherein the first vessel has known dimensions;
b. measuring the absorbance of the diluent chromophore in the first vessel at the first wavelength;
c. calculating the pathlength of light through the diluent in the first vessel based on the measured absorbance of the diluent chromophore and a known absorbance per pathlength of the diluent chromophore, wherein the pathlength of light through the diluent is equal to the diluent height;
d. adding to a second vessel a target volume of a sample solution, wherein the sample solution includes a known concentration of a sample solution chromophore that absorbs light at a second wavelength, wherein dimensions of the second vessel are substantially equivalent to the known dimensions of the first vessel;
e. adding a wash solution to the second vessel and removing at least some of a mixture of the wash solution and the sample solution from the second vessel to establish a mixture height in the second vessel, wherein the mixture height is substantially equivalent to the diluent height;
f. measuring the absorbance of the sample solution chromophore in the second vessel at the second wavelength; and
g. calculating a dilution ratio for the sample solution contained in the second vessel, wherein the dilution ratio represents the extent to which the sample solution has been diluted by the wash solution added into the second vessel. - View Dependent Claims (23, 24, 25, 26, 27)
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Specification