US2912895A - Spectrophotometry process - Google Patents
Spectrophotometry process Download PDFInfo
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- US2912895A US2912895A US417215A US41721554A US2912895A US 2912895 A US2912895 A US 2912895A US 417215 A US417215 A US 417215A US 41721554 A US41721554 A US 41721554A US 2912895 A US2912895 A US 2912895A
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- plate
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- absorption
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
- G01N21/03—Cuvette constructions
- G01N21/0303—Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment
Definitions
- Fig. 1 is a sectional elevational view showing apparatus capable of use in the practice of my invention, and illustrating an initial step of the operation
- Fig. 2 is a corresponding view illustrating a subsequent step
- Fig. 3 is a transverse sectional view on the line 3-3, Fig. 1.
- the length of light path through the absorbing solution is obviously A plus B.
- Figure 3 shows a transverse sectional view at the level of the light beam (3-3 in Figure 1), with the displace ment plate in position.
- the cell is not moved during removal of the displacement plate, and provided the latter is positioned in a plane perpendicular to the light beam and is larger than the light beam, the accuracy of results obtained should be dependent only on the precision of determining the change in light density produced by removal Hence even scratched test tubes can be used. Furthermore, because the change in density is determined only by the characteristics of the for exact uniformity of size.
- the plate should be in a plane perpendicular, or normal to the light beam. Rotation of the plate from the plane will produce (1) variation in the reflectance at entrance and exitsurfaces, and (2) variation in the length of solution displaced due to the longer slanting path of the light beam through the plate. Factors conerned in these two sources of error are (l) refractive index of the liquid, n (2) refractive index of the displacement plate, 12 and (3) angle of incidence, 1'.
- reflection does not change appreciably for small deviations in the position of the displacement plate.
- Displacement spectrophotometry offers the opportunity for making accurate relative optical density measurements in unselected test tubes, which can be calibrated for certain volumes, used in the water bath, employed fo dilution to volume without transfer, and then placed directly in the photometer.
- Displacement plates have been employed previously in optical cells but only for the purpose of decreasing thickness of the liquid layer,
- the maximum thickness of displacement plate that can profitably be used will be determined by several considerations.
- the plate should be enough wider than the light beam used that stray light will not escape around the edges. Furthermore, the width of the plate should be enough to avoid reflection from the edges. Within the limits of the circular cross section of the tube, the thicker the plate is, the narrower it will have to be.
- the plate displaces solution, care will have to be taken that liquid is not caused to run over when it is inserted. If dilution to a mark is made, part of the contents can be spilled into the sink after mixing, to reach a safe level. Thickness of the plate will determine the amount of displacement, and hence the amount of liquid that can safely be left in the tube.
- the errors in displacement spectrophotometry include in general all those of other types, with one or two exceptions.
- An exception is the contribution to error made by surface dirt or scratches on the absorption cell surface. These do not, within limits, affect the accuracy of displacement spectrophotometry.
- Errors peculiar to displacement spectrophotometry include dirt, scratches, or bubbles on the displacement plate. These imperfections may cause a loss of uniformity if several plates are used. When a single plate is used surface imperfections that are constant in effect, such as scratches, should produce little error, whereas changing imperfections such as bubbles will cause errors even when only a single plate is used.
- Density difierences obtained as described are not proestates portional to concentration of solute. Plotting of density difference values against concentration gives a straight line which in general does notgo through the origin.
- test tubes can be used for quantitative absorption spectrophotometry by making density readings before and after removal of a flat plate of glass or other transparent material set in the tube to displace a constant depth of solution. Studies of the nature and magnitude of errors that may be encountered in the procedure show that with moderate precautions good accuracy can be anticipated.
- the steps comprising: displacing fluid within said container by inserting a displacement plate with smooth, parallel, plane surfaces, made of a material absorbing little of the radiant energy employed, at right angles across the radiant energy beam to produce within the fluid an optically substantly void space, with smooth, parallel, plane surfaces, transverse to the beam of radiant energy employed and wholly containing the cross section of said beam; measuring the radiant energy transmitted in said beam through said container, displacement plate, and undisplaced fluid; withdrawing the displacement plate, whereby the unknown fluid replaces the space occupied by the plate, thus producing an increase in the length of path of the said beam of radiant energy through said fluid corresponding exactly to the distance the beam previously traversed the optically substantially void plate, and thus causing a greater absorption of the radiant energy used
Description
Nov. 17, 1959 R. H. HAMILTON SPECTROPHOTOMETRY PROCESS Filed March 18, 1954 United States Patent This invention relates to improvements in spectrophotometry, and a principal object of the invention is to provide an improved process of displacement or absorption spectrophotometry as hereinafter set forth.
One of the problems encountered in laboratories using test tube type absorption cells for spectrophotometry is the securing and maintenance of sets of matched tubes of sufiiciently close characteristics. To circumvent this difficulty, displacement plates can be used toallow'displacement and replacement of layers of the colored fluid of uniform thickness. Critical study of the use of such plates shows a high degree of accuracy to be attainable. Results further indicate the possibility of eliminating need for transferring photometric solutions from the "Working test tubes in which previous analytical steps were carried out team optical set of test tubes in which photometric readings are made. Limitations of the method are discussed, and possibilities are considered for selection of suitable displacement plates.
The invention will be more readily understood by reference to the attached drawings, wherein: Fig. 1 is a sectional elevational view showing apparatus capable of use in the practice of my invention, and illustrating an initial step of the operation; Fig. 2 is a corresponding view illustrating a subsequent step; and Fig. 3 is a transverse sectional view on the line 3-3, Fig. 1.
In the usual absorption photometry of solutions, light of restricted wave length incident on the photocell or phototube is set to read unit intensity after passing through a cell containing solvent or solvent plus the amount of impurities in reagents (blank). Then, light intensity being maintained constant, an identical cell is substituted, containing solvent plus the light-absorbing molecules whose concentration is to be determined. The decrease in light intensity is noted and the concentration of the solute is calculated from the absorption produced by knownconcentrations.
The same results can be obtained by the addition to the light path of layers of solution of constant thickness. Such addition can be accomplished by removal of adisplacement plate immersed in the solution. By way of example, reference may be had to the drawings, Figure -1 of which shows an ordinary test tube 1, somewhat wider than the beam 3 of monochromatic light, or light of restricted wavelength presented by a spectrophotometer; The tube, containing a solution 4 the optical density of which it is desired to determine, is centered in a stable position in the light beam of the spectrophotometer. A displacement plate 2 shown in longitudinal section through the narrower dimension, and being a rectangular strip of plate glass or similar substance as discussed herein, narrow enough to fit into the tube, and wider than the beam of monochromatic light, is set into the solution in such a way as to be perpendicular to the light beam. The length of light path through the absorbing solution is obviously A plus B. The distance L, being the thickness of the displacement plate, and the plate beingmade of a material of the displacement plate.
. 2,912,895 Patented Nov. 17, 1959 transparent to light of the wave-length used, does not contribute appreciably to the absorption of the light used. Provision is made in the use of the displacement plate to compensate for this small absorption of light in the distance Ly Figure 2 shows everything shown in Figure 1, in the .same position and under the same circumstances, except only that the displacement plate has been withdrawn. The length of light path thro'ughthe absorbing solution is now A plus B plus L. The change in length of the light path through the absorbing solution is obviouslyL. This distance is independent'of A, of B, and of A plus B. In elfect, by removal of the displacementplate, one has interposed into the light path a rectangular layer of absorbing solution of the samedimensions as to width and thickness as the removed displacement plate. Within the limitations discussed elsewhere, the effect of this interposed layer of solution willbe independent of the size of, and of irregularities in, the test tube 1. It thus becomes unnecessary to use expensive rectangular optical cells, or carefully selected tubes, to contain the absorbing solution, since one can by using a displacement plateas described always interpose in the light beam a constant, reproducible layer of absorbing solution. 0
Figure 3 shows a transverse sectional view at the level of the light beam (3-3 in Figure 1), with the displace ment plate in position.
For a given solution and wave length the effect of the displacement plate itself onlight transmittance will be constant. Either of two procedures can be followed: (1) With the displacement plate immersed at right angles to the light beam, light intensity is set to read 100% (unity). The plate is then removed, and light intensity is read after removal. (2) Light intensity is allowed to remain such that transmittance is close to unity (between and and transmittance is read exactly, before and again after removal of the displacement plate. The difierence inthe optical densities correspondingto .the two transmittances, corrected by a similar density difierence obtained with a blank, gives an optical density corresponding tothat of the layer of solution displaced by the plate.
It is possible to use the latter procedure because of the following mathematical relationship:
and D =log T /T =log T +log T The optical density change produced by removing the plate is Hence, even though T is much larger than unity (100%) if T and T fall on the scale, it is still possible, from the difference in their logarithms, to obtain the optical density value of the displaced solution.
Provided'the cell is not moved during removal of the displacement plate, and provided the latter is positioned in a plane perpendicular to the light beam and is larger than the light beam, the accuracy of results obtained should be dependent only on the precision of determining the change in light density produced by removal Hence even scratched test tubes can be used. Furthermore, because the change in density is determined only by the characteristics of the for exact uniformity of size.
Ideally the plate should be in a plane perpendicular, or normal to the light beam. Rotation of the plate from the plane will produce (1) variation in the reflectance at entrance and exitsurfaces, and (2) variation in the length of solution displaced due to the longer slanting path of the light beam through the plate. Factors conerned in these two sources of error are (l) refractive index of the liquid, n (2) refractive index of the displacement plate, 12 and (3) angle of incidence, 1'.
Take n=n /n Also take E= /n sin i and reflected light incident light;
Fresnels formula can be modified to give R as a function of n and of the angle of incidence:
R: E-cos i) E I-sin 'i tan E+cosi (E-l-sin 2' tan 7!) Assuming for n the value 1.33, and for n the value 1.55, n becomes 1.165, and the above formula gives values for fraction of light reflected at the entrance surface, at varying values of i, as shown in Table I under R.
Table I.Vdriatin in light transmittance with angle of incidence, due to reflection R, and length of light path, L
Angle of Incidence, R L
As will be seen, reflection does not change appreciably for small deviations in the position of the displacement plate.
The situation is different, however, with respect to the change in length of the path of the refracted ray through the displacement plate with rotation.
Taking D as the optical density of a layer of the displaced liquid equal in thickness to the displacement plate set normal or perpendicular to the light ray, and taking D, as the apparent or false optical density of a thicker layer of liquid corresponding to the longer path of the refracted ray through the rotated plate, we can define the resulting positive error, L, as
of refraction of the light ray corresponding to the angle of incidence, i, that v Displacement spectrophotometry offers the opportunity for making accurate relative optical density measurements in unselected test tubes, which can be calibrated for certain volumes, used in the water bath, employed fo dilution to volume without transfer, and then placed directly in the photometer. Displacement plates have been employed previously in optical cells but only for the purpose of decreasing thickness of the liquid layer,
.for use with liquids of high optical density. Their use in ordinary test tubes as described here may make desirable readjustment of the concentrations of light-ab sorbing molecules to secure an optimum optical density range. This change can be produced by using a larger aliquot of the sample, by lesser dilution of the final colored solution, or in some cases by using a difierent wave length of light at which the absorption is greater.
The maximum thickness of displacement plate that can profitably be used will be determined by several considerations. The plate should be enough wider than the light beam used that stray light will not escape around the edges. Furthermore, the width of the plate should be enough to avoid reflection from the edges. Within the limits of the circular cross section of the tube, the thicker the plate is, the narrower it will have to be.
Because the plate displaces solution, care will have to be taken that liquid is not caused to run over when it is inserted. If dilution to a mark is made, part of the contents can be spilled into the sink after mixing, to reach a safe level. Thickness of the plate will determine the amount of displacement, and hence the amount of liquid that can safely be left in the tube.
The errors in displacement spectrophotometry include in general all those of other types, with one or two exceptions. An exception is the contribution to error made by surface dirt or scratches on the absorption cell surface. These do not, within limits, affect the accuracy of displacement spectrophotometry.
Errors peculiar to displacement spectrophotometry include dirt, scratches, or bubbles on the displacement plate. These imperfections may cause a loss of uniformity if several plates are used. When a single plate is used surface imperfections that are constant in effect, such as scratches, should produce little error, whereas changing imperfections such as bubbles will cause errors even when only a single plate is used.
It can be shown that it is not necessary to achieve precision in setting the plate at right angles to the light beam, but that good accuracy can be attained by approximating the proper position. Sufficient accuracy can be attained by inspection, without elaborate precautions. Out of twenty consecutive times in which the plate was set in place by inspection and in which the angle of deviation of the plate from normalcy was then measured, in no instance did the deviation exceed 2. At such values of i, the error from this source is negligible.
A more serious source of error is the presence of inequalities in the displacement plates used. Ordinary polished plate glass can vary more than 2% in thickness over very short distances. Glass plates and even polished narrow-band-pass filters may deviate 1% or more in the parallelism of the two surfaces. Such lack of parallelism will affect the integrated thickness of the plate within the light beam. The limits of this variability in commercial plate glass differ with the method of manufacture, and should be leastwith drawn glass finished with larger polishing blocks. There is a possibility of error arising from local variations in the index of refraction of glass not prepared for optical purposes. These errors can be minimized by using a single displacement plate, which willhave to be wiped dry between tubes containing solutions with appreciable difierences in concentration. Cleaning would be facilitated by treatment of the surface with a silicone, or by making the plate from a transparent nonwctting plastic of suflicient resistance to chemicals. Care would have to be taken that air bubbles did no cling to such a surface on immersion.
It should not, however, be difiicult to prepare plates of glass, plastic, quartz, or other transparent material, of sufficiently uniform thickness and optical characteristics to be interchangeable. v
'Light absorption of the unused or undisplaced solution will act as an additional light filter, and may accentuate stray light effects, especially with wide band width. Thus, marked changes in the geometry of the containing vessel may affect the apparent density values obtained.
Density difierences obtained as described are not proestates portional to concentration of solute. Plotting of density difference values against concentration gives a straight line which in general does notgo through the origin. The same statement applies to the usual type of spectrophotometry, unless a blank is used, because of the constant contribution to each value by the blank absorption. It is customary to correct for this blank value, either automatically in the setting of the initial light intensity (setting the instrument for T=l00% with a blank), or by subtracting the density value of the blank from each of the other values. In displacement spectrophotometry this constant value is the resultant of the reagent blank plus the contribution of the plate itself (absorption by the glass, reflectance, and other factors). It can easily be determined by measuring the density difference produced by immersion of the plate in a reagent blank such as is usually prepared.
Ordinary, unselected test tubes can be used for quantitative absorption spectrophotometry by making density readings before and after removal of a flat plate of glass or other transparent material set in the tube to displace a constant depth of solution. Studies of the nature and magnitude of errors that may be encountered in the procedure show that with moderate precautions good accuracy can be anticipated.
I claim:
In the process of spectrophotometric determination of the concentration of molecules that absorb radiant energy in a fluid of concentration unknown as regards such molecules by the measurement of absorption of radiant energy from a narrow beam of radiant energy of suitably restricted wavelength passed transversely through a substantially transparent container of the fluid, the steps comprising: displacing fluid within said container by inserting a displacement plate with smooth, parallel, plane surfaces, made of a material absorbing little of the radiant energy employed, at right angles across the radiant energy beam to produce within the fluid an optically substantly void space, with smooth, parallel, plane surfaces, transverse to the beam of radiant energy employed and wholly containing the cross section of said beam; measuring the radiant energy transmitted in said beam through said container, displacement plate, and undisplaced fluid; withdrawing the displacement plate, whereby the unknown fluid replaces the space occupied by the plate, thus producing an increase in the length of path of the said beam of radiant energy through said fluid corresponding exactly to the distance the beam previously traversed the optically substantially void plate, and thus causing a greater absorption of the radiant energy used; measuring the radiant energy transmitted in said beam through said container and fluid with said displacement plate removed; the change in transmission of radiant energy being determined by the dimension of'the displacement plate along the axis of the beam of radiant energy used, and being reproducible because of the constant dimensions of the displacement plate, and being substantially independent of the shape and optical condition of the surface of the container of the fluid of unknown concentration, said change in transmission of radiant energy making possible calculation of the unknown concentration of energy-absorbing molecules being measured.
References Cited in the file of this patent UNITED STATES PATENTS 1,635,470 Exton July 12, 1927 1,877,501 EXton Sept. 13, 1932 1,954,925 Exton Apr. 17, 1934 2,073,223 Rose Mar. 9, 1937 2,645,971 Herbst July 21, 1953 2,844,066 Friel July 22, 1958
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US417215A US2912895A (en) | 1954-03-18 | 1954-03-18 | Spectrophotometry process |
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US417215A US2912895A (en) | 1954-03-18 | 1954-03-18 | Spectrophotometry process |
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3027799A (en) * | 1959-01-26 | 1962-04-03 | Brunswick Corp | Sample space adapters for spectrophotometers and the like |
US3279307A (en) * | 1963-01-21 | 1966-10-18 | Barnes Eng Co | Spectrum examination of substances |
US3526462A (en) * | 1967-08-17 | 1970-09-01 | Univ Delaware | Radiant energy absorption cell with a transversely movable wedge-shaped spacer block therein |
US3578865A (en) * | 1968-10-03 | 1971-05-18 | Mobil Oil Corp | Method and apparatus for photo-electrically monitoring the degradation of an oil stream |
US4762798A (en) * | 1985-12-31 | 1988-08-09 | Marshall Diagnostics, Inc. | Device and method for determining a characteristic of a fluid sample |
US5039224A (en) * | 1990-02-12 | 1991-08-13 | Rourke Patrick E O | Self-referencing remote optical probe |
FR2714183A1 (en) * | 1993-12-20 | 1995-06-23 | Lvmh Rech | Device for raising the level of a liquid in a cell, well or the like, multiwell plate equipped with this device, and use of this device or this plate for measuring the optical density. |
US20090059332A1 (en) * | 2007-09-05 | 2009-03-05 | Baker Hughes Incorporated | System and method for dual path length optical analysis of fluids downhole |
USD977142S1 (en) * | 2016-08-18 | 2023-01-31 | Sio2 Medical Products, Inc. | Vial |
US11697114B2 (en) * | 2015-12-11 | 2023-07-11 | Babson Diagnostics, Inc. | Centrifugation method separating serum or plasma from whole blood using a specimen container having a cap to retain blood cells |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US1635470A (en) * | 1922-06-23 | 1927-07-12 | William G Exton | Method of and apparatus for measuring turbidities and color of fluids |
US1877501A (en) * | 1932-09-13 | Trations | ||
US1954925A (en) * | 1931-08-12 | 1934-04-17 | William G Exton | Photo-electric measuring apparatus |
US2073223A (en) * | 1934-08-28 | 1937-03-09 | Hilton W Rose | Means for determining color density of liquids |
US2645971A (en) * | 1949-06-28 | 1953-07-21 | Rca Corp | Surface contour measurement |
US2844066A (en) * | 1951-08-17 | 1958-07-22 | Du Pont | Method of photometric analysis |
-
1954
- 1954-03-18 US US417215A patent/US2912895A/en not_active Expired - Lifetime
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US1877501A (en) * | 1932-09-13 | Trations | ||
US1635470A (en) * | 1922-06-23 | 1927-07-12 | William G Exton | Method of and apparatus for measuring turbidities and color of fluids |
US1954925A (en) * | 1931-08-12 | 1934-04-17 | William G Exton | Photo-electric measuring apparatus |
US2073223A (en) * | 1934-08-28 | 1937-03-09 | Hilton W Rose | Means for determining color density of liquids |
US2645971A (en) * | 1949-06-28 | 1953-07-21 | Rca Corp | Surface contour measurement |
US2844066A (en) * | 1951-08-17 | 1958-07-22 | Du Pont | Method of photometric analysis |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3027799A (en) * | 1959-01-26 | 1962-04-03 | Brunswick Corp | Sample space adapters for spectrophotometers and the like |
US3279307A (en) * | 1963-01-21 | 1966-10-18 | Barnes Eng Co | Spectrum examination of substances |
US3526462A (en) * | 1967-08-17 | 1970-09-01 | Univ Delaware | Radiant energy absorption cell with a transversely movable wedge-shaped spacer block therein |
US3578865A (en) * | 1968-10-03 | 1971-05-18 | Mobil Oil Corp | Method and apparatus for photo-electrically monitoring the degradation of an oil stream |
US4762798A (en) * | 1985-12-31 | 1988-08-09 | Marshall Diagnostics, Inc. | Device and method for determining a characteristic of a fluid sample |
US5039224A (en) * | 1990-02-12 | 1991-08-13 | Rourke Patrick E O | Self-referencing remote optical probe |
FR2714183A1 (en) * | 1993-12-20 | 1995-06-23 | Lvmh Rech | Device for raising the level of a liquid in a cell, well or the like, multiwell plate equipped with this device, and use of this device or this plate for measuring the optical density. |
WO1995017661A1 (en) * | 1993-12-20 | 1995-06-29 | Lvmh Recherche | Device for raising the liquid level in a well for measuring the density |
US20090059332A1 (en) * | 2007-09-05 | 2009-03-05 | Baker Hughes Incorporated | System and method for dual path length optical analysis of fluids downhole |
US7826050B2 (en) * | 2007-09-05 | 2010-11-02 | Baker Hughes Incorporated | System and method for dual path length optical analysis of fluids downhole |
US11697114B2 (en) * | 2015-12-11 | 2023-07-11 | Babson Diagnostics, Inc. | Centrifugation method separating serum or plasma from whole blood using a specimen container having a cap to retain blood cells |
USD977142S1 (en) * | 2016-08-18 | 2023-01-31 | Sio2 Medical Products, Inc. | Vial |
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