Comparative colorimeter

A comparitive colorimeter for use in the field which simultaneously compares the color densities of two liquid samples and designates the degree of difference. The apparatus includes two major subsystems, optical and electronic. The optical subsystem is designed to provide identical light beams through both the sample and standard solutions and to minimize the effect which imperfect sample tubes have on the output. The electronic subsystem includes a log conversion and differential amplifier circuit for generating a difference signal representative of the difference between the optical densities of the sample and standard solutions. The difference signal is evaluated by a comparative circuit consisting of a resistive chain having a plurality of node voltages and hex inverters connected to the node voltages. The hex inverters drive a series of LED indicator lights to designate the relative degree of difference between the optical densities of the sample and standard. A correction circuit is provided for referencing the difference signal to the threshold voltage of the hex inverters and for compensating for initial differences in the optical paths of the sample and standard solutions.

BACKGROUND OF THE INVENTION 
This invention relates to a comparative colorimeter, and more specifically 
to an instrument which simultaneously compares the color (i.e., optical) 
densities of two liquid samples and designates the degree of difference. A 
colorimeter is useful for conducting: 
(a) medical diagnostic tests based on enzyme immuno assays (EIA), e.g., the 
test for Leutenizing Hormone as an indication of ovulation in humans; 
(b) medical diagnostic tests based on Enzyme Multiplied Immuno assay 
Technique (EMIT), e.g., tests for drugs of abuse such as barbituates; 
(c) veterinary diagnostic tests based on either (a) or (b), e.g., 
progesterone level in dairy cows; 
(d) environmental tests based on either (a) or (b), e.g., residual 
Chlorodane levels after extermination around residences; and 
(e) determination of the concentration of certain chemicals based on 
absorption of a given wavelength of light, e.g., concentration of the 
herbicide Dynaseb.TM.. 
In the above-identified tests, it is generally not the absolute value of 
optical density of a solution which is of interest, but rather a 
comparison of a sample solution with a standard solution. Further, while 
all of these tests can be satisfactorily performed in the laboratory using 
a standard off-the-shelf spectrophotometric instrument, such instruments 
are delicate and expensive and are not adapted for use in the field. For 
example, a colorimeter is useful in the field to determine whether the 
residual level of a pesticide applied to a crop has declined sufficiently 
to allow safe entry of personnel into the field for harvesting operations. 
A known colorimeter used in the field performs separate quantitative 
determinations of the optical densities of a sample solution and a 
standard solution. The human operator then computes the difference between 
the quantitative determinations. This device has several disadvantages. 
First, since the operator is generally interested in only the relative 
difference between the sample and standard, it is a waste of effort to 
make a quantitative determination for each of the sample and standard. 
Further, since the quantitative determinations of the sample and standard 
are made at different times, there exists a large potential for error if 
either of the solutions changes in optical density with respect to time or 
temperature. 
It is an object of the present invention to provide a relatively low-cost 
comparative colorimeter for use in the field for comparing the optical 
densities of two solutions and indicating the relative degree of 
difference. 
It is another object of this invention to provide a comparative colorimeter 
for simultaneously evaluating the optical densities of sample and standard 
solutions prepared at the same time in order to eliminate any error caused 
by changes in the optical densities of the sample and standard with 
respect to time. 
It is a further object of this invention to provide an optical subsystem 
for a colorimeter which reduces the error caused by defects in the sample 
tube such as scratches, irregularities or striations in the tube wall, and 
lack of concentricity. 
It is still another object of this invention to provide an electronic 
subsystem, including low-cost digital integrated circuits, for quantifying 
the difference between the optical densities of the sample and standard 
solutions. 
It is a still further object of this invention to provide a correction 
circuit to compensate for variations in the threshold voltage of the 
digital IC components with respect to batch, time, temperature, and supply 
voltage. 
It is still another object of this invention to provide a correction 
circuit to compensate for differences in the optical paths of the sample 
and standard solutions. 
SUMMARY OF THE INVENTION 
The apparatus of this invention provides a means for simultaneously 
comparing the optical densities of two solutions and generating an output 
indicative of the degree of difference. The apparatus includes two major 
subsystems, optical and electronic. 
The optical subsystem is designed to provide identical light paths through 
two liquid samples disposed in optically transparent sample tubes and to 
minimize the effect which imperfect sample tubes have on the measurement. 
The optical subsystem includes a light source means for transmitting a 
pair of substantially equal light beams, one to each of a pair of 
photodetectors. Each of the two liquid samples are simultaneously and 
identically positioned between the light source means and an associated 
photodetector. The photodetectors generate a pair of optical density 
output signals representative of the amounts of light passing through the 
samples and reaching the photodetectors. The optical system is designed to 
spread the light over a substantial portion of each sample tube to 
minimize the effect of defects in the tubes. 
In one embodiment of the optical system of this invention, a collimating 
lens is positioned between the sample tube and light source and spaced 
slightly closer to the light source than the focal point of the lens so 
that the lens transmits a slightly divergent beam of light to the tube. 
The tube is spaced with respect to the lens so that the outer diameter of 
the divergent beam at the center plane of the tube is substantially equal 
to the inner diameter of the tube. The sample tube acts as a cylinder lens 
to focus the beam at a vertical focal line, and the photodetector is 
spaced slightly ahead of the focal line so that the beam strikes a 
substantial portion of the photodetector. The photodetector output is 
substantially independent of defects in the sample tube. 
Preferably, the optical system further includes a combination 
filter/diffuser positioned between the sample tube and photodetector. The 
filter/diffuser includes a filter for selecting a predetermined wavelength 
of light and a light diffusing layer for averaging the effect of 
individual defects in the sample tube so that the photodetector output is 
less affected by such defects. Preferably, the light diffusing layer is 
disposed over the surface of the filter adjacent the photodetector. In a 
further preferred embodiment, a plurality of such combination 
filter/diffusers are connected in serial arrangement and the photodetector 
is positioned adjoining a light diffusing layer at one end of the series. 
In a second embodiment of the optical system of this invention, a first 
lens set is positioned between the sample tube and light source. The first 
lens set includes a first collimating lens disposed adjacent the light 
source and having a focal point at the light source, and a first 
converging lens disposed adjacent the sample tube and having a focal point 
at the center of the sample tube. A second lens set is positioned between 
the sample tube and photodetector. The second lens set includes a second 
collimating lens disposed adjacent the sample tube and having a focal 
point at the center of the tube, and a second converging lens disposed 
adjacent the photodetector and having a focal point at the photodetector. 
Because in this second embodiment the light rays pass perpendicularly 
through the tube and the tube has no focusing effect, refraction is 
reduced and irregularities in the tube do not affect the focus point. 
Thus, the accuracy of this second embodiment is less dependent on the 
spacing between the lenses and tube than that of the first embodiment. A 
filter may be positioned between the two lenses of the second lens set, 
and because the light passes through the filter in parallel rays, there is 
minimal reflection at the filter surface and thus minimal light loss. 
The electronic subsystem of this invention provides a high level of 
accuracy and stability despite the stringent cost limitations imposed by 
the intended application. In particular, the circuit utilizes low-cost 
digital components to achieve a quantified output indicative of the degree 
of difference in optical densities of the two samples. A correction 
circuit is provided to compensate for variations in the threshold voltage 
of the digital IC components and to compensate for differences in the 
optical paths of the two samples. 
The electronic subsystem includes a differential circuit means for 
generating a difference signal representative of the difference between 
the optical density output signals of the photodetectors. The system 
further includes a comparative circuit including means for comparing the 
difference signal to a plurality of predetermined references and a means 
for indicating the relative value of said difference signal with respect 
to said predetermined references. 
In a preferred embodiment, the current outputs of a pair of photodiodes are 
logarithmically converted to voltage signals and the resultant voltage 
signals are differentially amplified to produce a difference signal. The 
difference signal is sent to a resistive chain including a plurality of 
nodes, each node having a different predetermined reference voltage. A 
plurality of digital gate means each having the same threshhold voltage 
are each connected to one of said nodes. The outputs of the gate means 
activate a plurality of indicator means to indicate the relative value of 
the difference signal with respect to the predetermined reference 
voltages. 
In addition, a correction circuit is provided for referencing the 
difference signal to the threshold voltage of the gate means. The 
correction circuit includes an additional gate means from the same batch 
used in the comparative circuit. The output of the correction circuit, 
which is referenced to the threshold voltage of the gate means, is applied 
to the comparative circuit so that the difference signal is referenced to 
the threshold voltage. Further, the correction circuit includes a zeroing 
mechanism to compensate for differences in the light paths of the two 
sample solutions.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 is a perspective view of the comparative colorimeter of the present 
invention. A housing 10 contains the optical and electronic subsystems of 
this invention. A pair of sample tube holders 11, 12 consist of tubular 
bores disposed vertically in the housing and having closed lower ends and 
open upper ends in the top wall 13 of the housing. The holders snugly 
engage two cylindrical sample tubes 5, 6 which contain the sample and 
standard solutions respectively. A row of five indicator lights 15, 16, 
17, 18, 19 is provided on the front wall 14 of the housing. An on-off 
button 20 is provided on top wall 13 of the housing. The housing is made 
of a nonbreakable plastic such as acrylonitrile-butadiene-styrene polymer. 
The device shown in FIG. 1 is compact and portable, being 5" (127 mm) 
wide, 2.5" (64 mm) high, 3" (76 mm) deep, and weighing about 200 grams. 
Typically, sample tubes 5, 6 are made of polystyrene and have outer 
dimensions of 12 mm in diameter and 75 mm in length, and an inner diameter 
of 9 mm. 
The comparative colorimeter of this invention consists of two major 
subsystems, optical and electronic. The important features of the optical 
subsystem include: 
(a) it must provide equal light paths through both of the sample tubes; to 
the extent that the light paths are not identical, then the ratio of light 
going through them must be constant despite: the buildup of dirt and 
contamination; thermal effects; warping or flexing of the printed circuit 
board or other structural members; mechanical shock and abuse; and aging 
of various components, e.g., light bulb and filters; 
(b) it must provide adequate light intensity at the photodiodes under all 
operating conditions; 
(c) it must minimize the effects of stray or ambient light; and 
(d) it must minimize the effect of imperfect sample tubes. 
In a laboratory spectrophotometric instrument, the samples are held in 
square cuvettes with ground and polished plane sides so that the sample 
container contributes little or no uncertainty to the measurement. In the 
field it is desirable to use inexpensive and disposable cylindrical 
plastic test tubes. However, these test tubes may contribute significantly 
to the uncertainty of the measurement. A typical plastic tube suffers 
three principal defects: 
(a) it may be scratched to a greater or lesser degree, depending on the 
care with which it is handled; these scratches tend to scatter and diffuse 
light, making the light path indeterminate; 
(b) it may contain striations or irregularities in the wall of the tube, 
which will refract light in an indeterminate way; and 
(c) the tube's exterior and interior may not be concentric, so that the 
actual path length and direction of a light ray passing through the sample 
depends on the orientation of the tube. 
The degree to which these imperfections contribute uncertainty is highly 
dependent on the details of the optical design. 
In the prior art design shown in FIG. 4, a narrowly collimated or focused 
beam of light from a lamp 200 passes through a sample tube 201 and then 
through a lens 202 which focuses the beam on a photodetector 203 which is 
sensitive to a predetermined wavelength. A screen 205 having an aperture 
206 is disposed between the lamp and tube to prevent stray light from 
reaching the photodetector. A filter 204 is disposed between the lens and 
photodetector for selecting light of a predetermined wavelength. In this 
prior art design, it is desirable to pass a very narrow beam of light 
through the center of the tube only so that the light beam passes through 
a sample area having a roughly uniform thickness. This is required where a 
quantitative determination of absorbance is being made and it is necessary 
to know the dimensions of the sample through which the beam passes. 
However, individual defects in the area of the sample tube through which 
the beam passes have a significant effect on the photodetector output. 
All of the above-mentioned defects in the plastic sample tubes affect the 
measurement results of this prior art design very severely. Typically, a 
measurement in absorbance may contain an error of 0.05 to 0.1 as a result 
of tube defects alone. The amount of error is highly dependent on the 
individual tube under test and its orientation. 
Two designs have been found to be highly effective at minimizing the errors 
caused by sample tube defects. These designs are shown in FIGS. 2 and 3. 
A first embodiment of the optical subsystem is shown in FIG. 2. A pair of 
photodetectors 30, 31 are positioned at opposite ends of an axial line. A 
light source means 32, such an incandescent lamp, is centered at the 
midpoint of the axial line. A first sample tube 5 and a second sample tube 
6 are identically positioned on the axial line on opposite sides of the 
lamp, between the lamp and an associated photodetector. A collimating lens 
33A(33B) and a screen 34A(34B) having an aperture 35A(35B) are identically 
positioned on either side of the lamp between the associated sample tube 
5(6) and lamp 32. A filter/diffuser 36A(36B) is positioned between the 
associated sample tube 5(6) and associated photodetector 30(31). 
The object of this first design is to spread the illumination over 
substantially the entire width of the sample tube so that the net light 
transmitted to the photodetector is averaged over many scratches and 
defects and is minimally dependent on the lack of concentricity of the 
inner diameter and outer diameter of the tube. While the operation of the 
optical system with respect to sample tube 5 is described herein, the 
operation with respect to sample tube 6 is identical. 
The collimating lens 33A is positioned between the sample tube 5 and light 
source 32 and spaced slightly closer to the light source than the focal 
point of the lens so that the lens transmits a slightly divergent beam to 
the sample tube. The screen 34A prevents stray light beams from reaching 
the photodetector 30. Further, the height of the aperture 35A is 
restricted to prevent light from hitting the meniscus of the liquid sample 
in the tube or the bottom of the tube and reflecting into the 
photodetector. The sample tube 5 is spaced with respect to the lens 33A 
such that the outer diameter of the divergent beam at the center plane of 
the tube is equal to or slightly less than the inner diameter d of the 
tube. The sample tube acts as a cylinder lens to focus the beam at a 
vertical focal line and the photodetector is spaced slightly ahead of the 
focal line so that the beam strikes a large area of the photodetector. 
This insures a photodetector signal of large amplitude and averaged value. 
The combination filter/diffuser 36A disposed between the sample tube 5 and 
photodetector 30 consists of a filter glass 39 for selecting a 
predetermined wavelength of light and a diffusive sheet or film 40 
covering the surface of the filter glass adjacent the photodetector. For 
example, film 40 may be Mylar.TM. film frosted on both sides and about 
0.006" (0.15 mm) thick. Because scratches and defects in the sample tube 
cause light to bounce in a random manner, the diffusive sheet 40 is 
provided to produce an averaging effect so that the photodetector output 
is less dependent on defects in the sample tube. Preferably, the 
photodetector 30 is sensitive to the same predetermined wavelength as the 
filter glass 39. 
A preferred embodiment of the filter/diffuser is shown in FIG. 2A. Two 
pieces of filter glass 41, 43 and two sheets of diffusive film 42, 44 are 
sandwiched together and disposed adjoining a photodetector 45. Diffusive 
sheet 42 is sandwiched between filter glasses 41 and 43, and diffusive 
sheet 44 is sandwiched between filter glass 43 and photodetector 45. This 
filter/diffuser is preferred because the addition of a second diffusive 
film separated from the first aids in a more uniform diffusion of the 
light. The use of a second filter glass is necessary in most cases in 
order to limit the bandwidth of the light transmitted. Further, adjoining 
the photodetector 45 to diffusive layer 44 eliminates light loss and 
increases the amplitude of the photodetector output. 
A second embodiment of the optical subsystem of this invention is shown in 
FIG. 3. Again, the design spreads the light beam over a large area of 
possible defects in the tube. A first lens set 50A includes a pair of 
diametrically opposed lenses 51A, 52A positioned on an axial line between 
the sample tube 5 and light source 32. Collimating lens 51A is disposed 
adjacent the light source and has a focal point at the light source. 
Converging lens 52A is disposed adjacent the sample tube and has a focal 
point at the center of the sample tube. A second lens set 54A includes a 
second pair of diametrically opposed lenses 55A, 56A positioned on the 
axial line between the sample tube 5 and photodetector 30. Collimating 
lens 55A is disposed adjacent the sample tube and has a focal point at the 
center of the sample tube and converging lens 56A is disposed adjacent the 
photodetector and has a focal point at the photodetector. 
In the second embodiment, because the light rays pass perpendicularly 
through the sample tube and the tube has no focusing effect, 
irregularities of the tube do not affect the focal point. Thus, the space 
between the lenses and sample tube is less critical in this design. 
Furthermore, in the second embodiment a filter 57A can be placed between 
the two lenses 55A, 56A of the second lens so that the light rays pass 
through the filter as parallel rays. This minimizes refractions at the 
surface of the filter and thus reduces light loss. 
In FIG. 3, the first and second lens sets 50B and 54B between the lamp 32, 
sample tube 6 and photodetector 31, are identical to those of 50A and 54A 
previously described. 
To evaluate the effectiveness of the designs shown in FIGS. 2 and 3, in 
comparison with the prior art design of FIG. 4, a series of measurements 
was taken with one particular sample tube filled with a sample solution. 
Eight different orientations of the tube were tried for each design to 
find the range of readings resulting from the one sample and tube. The 
resulting inaccuracy can be expressed as an error in absorbance as 
follows: 
EQU .DELTA.A=log[(I.sub.max -I.sub.min)/2I.sub.av ] 
where 
I.sub.max =maximum light intensity resulting from the series of readings 
for a given sample, tube and optical design; 
I.sub.min =minimum light intensity resulting from the series of readings 
for a given sample, tube and optical design; and 
I.sub.av =average light intensity resulting from the series of readings for 
a given sample, tube and optical design. 
Design 1 was that shown in FIG. 2 with lens 33A having a diameter of 5/16" 
and a focal length of 3/8". Lens 33A was spaced 0.27" from lamp 32, screen 
34A, having an aperture 0.125".times.0.275", was spaced 0.40" from lens 
33A, tube 5 was spaced 0.35" from screen 34A, and photodiode 30 was spaced 
0.65" from tube 5 (all distances are from center point of element). The 
filter/diffuser used was that shown in FIG. 2A wherein 41 was a 2 mm thick 
filter glass, model GG-435 by Schott, imported by ESCO Products, Inc. of 
Oak Ridge, N.J., and 43 was a 4.0 mm thick filter glass, model 5-58 by 
Corning Glass Works of Corning, N.Y. Each filter glass had a diameter of 
5/16". The diffuser sheets 42 and 44 were Mylar.TM. film, each 0.006" 
thick and frosted on both sides. The photodiode was adjoining sheet 44. 
Design 2 was that shown in FIG. 3 with lenses 51A, 52A, 55A and 56A each 
having a diameter of 3/8" and a focal length of 1/2". The first lens set 
was spaced 0.5" from lamp 32, tube 5 was spaced 0.5" from the first lens 
set, lens 55A was spaced 0.5" from tube 5, lens 56A was spaced 0.25" from 
lens 55A, and photodiode 30 was spaced, 0.5" from lens 56A (all distances 
are from center point of element). Two filter glasses, the same as 41 and 
43 described in design 1 (but without diffusive sheets 42 and 44) were 
used. In design 2, the distance between the lenses and sample tube could 
be changed up to 1/16" (1.6 mm) without changing the photodetector reading 
significantly. In comparison, in design 1, the sample tube had to be 
spaced within 5/1000" (0.13 mm) with respect to the lens and with respect 
to the axial line extending between the lamp and photodetector to insure a 
consistent photodetector reading. 
The prior art design was that shown in FIG. 4 with lens 202 having a 
diameter of 5/16" and a focal length of 3/8". Screen 205 had an aperture 
206 of 1/10" in height. The same two filter glasses described for design 2 
were used. 
In all designs an incandescent lamp was used, model 158X sold by Gilway of 
Woburn, Mass. The sample tube was made of polystrene having a length of 75 
mm, an outer diameter of 75 mm, and an inner diameter of 9 mm. The 
photodiode was model S-1133 sold by Hamamatsu of San Jose, Calif. 
The following values of .DELTA.A were obtained for the prior art design and 
designs 1 and 2 of the present invention: 
______________________________________ 
PRIOR ART DESIGN .DELTA.A = .05 
DESIGN 1 .DELTA.A = .01 
DESIGN 2 .DELTA.A = .004 
______________________________________ 
Thus, it has been shown that both design 1 and design 2 of this invention 
give substantially better results than the prior art design. While it 
would appear that design 2 is preferable over design 1, other factors such 
as cost, size, complexity and the required accuracy of construction 
mitigate in favor of design 1. 
The electronic subsystem of the present invention, as shown in FIG. 6A, 
performs the following functions: 
(a) the current outputs ID.sub.1 and ID.sub.2 of the two photodiodes PD1 
and PD2 are logarithmically converted to voltage signals; 
(b) the two voltage signals are differentially amplified at A3 to produce a 
difference signal E.sub.0 ; 
(c) the difference signal E.sub.0 is compared to a plurality of 
predetermined reference voltages with a series of voltage comparators, 
consisting of digital gate means G.sub.1 -G.sub.5 and resistive chain 
R12-R17; 
(d) the digital gate means drive a plurality of indicator LEDs 1-5 
(corresponding to indicator lights 15-19 in FIG. 1) to indicate the 
relative value of the difference signal with respect to the reference 
voltages; and 
(e) a correction circuit is provided so that the difference signal E.sub.0 
is referenced to the threshold voltage E.sub.T of the digital gate means. 
The indicator lights or LEDs 15-19 of FIG. 1, which correspond to LED's 1-5 
of FIG. 6A respectively, indicate the degree of difference in optical 
density between the samples in the tubes. If the sample and standard 
solutions have equal optical densities, then a middle green LED 17 turns 
on to indicate the same. If the solution of tube 5 has a lesser optical 
density than the solution of tube 6, when the difference exceeds a first 
predetermined level the comparator will trigger a right center red LED 18 
and simultaneously extinguish the green LED, and if the difference exceeds 
a larger second predetermined level, then a right red LED 19 will light. 
At no time is more than one indicator LED on. If the solution of tube 5 
has a greater optical density than the solution of tube 6, then the left 
center red LED 16 and left red LED 15 will light in the same manner as 
described for those to the right of center. A typical configuration is for 
the right center LED 18 (and left center LED 16) to come on at 
.DELTA.A=0.03 O.D. (difference in absorbance between the tubes is 0.03), 
and the right LED 19 (and left LED 15) to comes on at .DELTA.A=0.1 O.D. 
The operating voltage V.sub.REG for the lamp and the electronic subsystem 
is stabilized at 2.000 volts by the supply regulator circuit shown in FIG. 
6B. The circuit includes amplifier A4, capacitors C1, C2, C7, C8, 
resistors R1, R2, R3 (variable), R4, R7, R22, R23, transistors Q1, Q2, 
battery V+, and internal regulator V.sub.REF. A range of battery voltages 
V+ from 3.0 to 2.1 volts can be accommodated without degrading 
performance. Lamp life is extended substantially by operating the lamp 32 
at a constant 2.0 volts. 
The regulator circuit is built around one section of a amplifier A4 
specifically intended for voltage regulation. A4 in this preferred 
embodiment is amplifier U4 LM10CLN sold by National Semiconductor of Santa 
Clara, Calif. The LM10CLN drives transistors Q1 and Q2 as required to 
regulate V.sub.REG to exactly 2.000 volts. An internal regulator provides 
a constant V.sub.REF of 0.2 volts. V.sub.REG is divided down to 0.2 volts 
and then compared to this reference. V.sub.REF is also used to determine 
the operating points of the operational amplifiers A1 and A2 as shown in 
FIG. 6A. 
To compute the difference signal E.sub.0, the two diode currents ID.sub.1 
and ID.sub.2 are logarithmically converted to voltage signals and the two 
voltage signals are differentially amplified. The stability of the 
logarithmic conversion and differental amplification is such that an 
overall electronic error budget of 0.005 O.D. is not violated over the 
life of the instrument. 
FIG. 5 shows the log conversion and differential amplifier circuit for 
generating the difference signal E.sub.0. Light from light source 32 which 
passes through sample tube 5 and strikes photodiode PD1 generates a 
current ID.sub.1. Likewise, light from light source 32 which passes 
through sample tube 6 and strikes photodiode PD2 generates a current 
ID.sub.2. The photodiode currents can be expressed as: 
EQU I.sub.D1 =k.sub.1 g.sub.1 f.sub.1 I (1) 
EQU I.sub.D2 =k.sub.2 g.sub.2 f.sub.2 I (2) 
where I is the total intensity of light source 32, f.sub.1 and f.sub.2 are 
the fractions of light absorbed by the samples in tubes 5 and 6 
respectively, g.sub.1 and g.sub.2 are the fractions of light passing 
through tubes 5 and 6 which actually reach the photodiodes PD1 and PD2 
respectively, and k.sub.1 and k.sub.2 are the efficiencies with which the 
photodiodes PD1 and PD2 convert the light reaching their surfaces 
respectively. 
The diode currents I.sub.D1 and I.sub.D2 constitute optical density signals 
representative of the optical densities of the samples in tubes 5 and 6 
respectively. The diode currents I.sub.D1 and I.sub.D2 are logarithmically 
converted to voltage signals E.sub.1 and E.sub.2 by a pair of identical 
log conversion circuits shown in FIGS. 5 and 6A. Thus, amplifier A1, 
capacitor C11, and transistor Q4, arranged in parallel, convert I.sub.D1 
to E.sub.1, and similarly, amplifier A2, capacitor C12, and transistor Q5, 
arranged in parallel, convert I.sub.D2 to E.sub.2. A1 and A2 are from the 
same package and are identically powered by connection to the regulated 
supply voltage V.sub.REG and decoupling capacitor C3 (as shown for A2) and 
grounded (as shown for A1). Because the log conversion circuits are 
identical, the votages E.sub.1 and E.sub.2 may be expressed as: 
EQU E.sub.1 =Klog I.sub.D1 (3) 
EQU E.sub.2 =Klog I.sub.D2 (4) 
where K is a constant. 
The voltage signals E.sub.1 and E.sub.2 are differentially amplified at A3 
to produce a difference signal E.sub.0 as set forth below: 
EQU E.sub.0 =G(E.sub.2 -E.sub.1)+E.sub.c (5) 
where G is the amplifier gain and E.sub.C a correction voltage. The 
correction voltage E.sub.c is added to the positive input of A3 for 
reasons discussed hereinafter. The differential amplifier circuit includes 
differential amplifier A3, capacitor C4, and resistors R8, R9, R10, and 
R11, where R8=R10=R.sub.A and R9=R11=R.sub.B. The amplifier gain G=R.sub.B 
/R.sub.A is maintained constant through careful selection of the gain 
resistors. Selecting the value of the gain is also described hereinafter. 
By substituting equations 1-4 into equation 5, the following equation for 
E.sub.0 is obtained: 
EQU E.sub.0 =KGlog[(k.sub.2 g.sub.2)/(k.sub.1 g.sub.1)]+KGlog(f.sub.2 
/f.sub.1)+E.sub.c (6) 
The ratio of photodiode efficiencies k.sub.2 /k.sub.1 is constant over time 
given equal wavelength of the incident light, equal temperature and equal 
reverse bias. Dark current will drift somewhat but is not a limitation in 
this application. 
The ratio g.sub.2 /g.sub.1 of optic path efficiencies depends on the 
details of the design and its implementation. If we assume perfect sample 
tubes and an unchanging rigid geometry, then the ratio g.sub.2 /g.sub.1 is 
fixed. 
Since the amplifier gain G is maintained constant through careful selection 
of gain resistors, and K is a constant, the entire first term in (6) is a 
constant C. Thus, the difference signal E.sub.0 can be represented as 
follows: 
EQU E.sub.0 =C+KGlog(f.sub.2 /f.sub.1)+E.sub.c (7) 
C can be compensated for by a simple additive offset applied at the input 
of the differential amplifier A3. This is accomplished by the correction 
circuit discussed hereinafter. The connection circuit further references 
the difference signal E.sub.0 to the threshold voltage E.sub.T of the hex 
inverters used in the comparative circuit so that E.sub.0 =E.sub.T when 
the optical densities of the two samples are equal. 
The difference signal E.sub.0 generated by A.sub.3 is sent to the 
comparative circuit shown in FIG. 6A, wherein E.sub.0 is compared to a 
series of predetermined reference voltages to determine to the relative 
value of the difference signal with respect to the reference voltages. The 
act of comparison is performed by a plurality of low cost digital 
integrated circuits. In the preferred embodiment, hex inverters such as 
the 74HC04 sold by Motorola of Austin, Tex. are used as the digital IC 
gates means. The hex inverters are used in the configuration shown in FIG. 
6A in order to economize on parts count and cost. 
The output from the differential amplifier E.sub.0 is divided by a 
resistive chain so that the voltage at each node of the chain is directly 
related to E.sub.0. The resistive chain consists of resistors R12, R13, 
R14, R15, R16 and R17 arranged in series between V.sub.REG and ground. 
Connected to each node is an input of one of hex inverter gates G1, G2, 
G3, and G4. Gates G1-G5 are all in the same package and each is connected 
to the regulated supply voltage V.sub.REG and to decoupling capacitor C5 
(as shown for G3) and to ground (as shown for G4). If the node happens to 
be above the switching threshold of the gate, which for the 74HC04 is 
about 1/2 of the regulated supply voltage V.sub.REG, then the gate output 
is low. Conversely, if the node is below the threshold, then the gate 
output is high. All of the gates are selected from a single package so 
they have very nearly the same threshold for a given temperature and 
supply voltage. 
The outputs of gates G1-G4 are connected to drive indicator LEDs 1-5. Thus, 
in addition to the resistive chain and hex inverter gages G1-G4, the 
comparative circuit further includes indicator LEDs 1-5, resistors R19, 
R20 and R21, diode D3, transistor Q3, and hex inverter gate G5 as shown in 
FIG. 6A. The indicator LEDs are arranged such that only one LED is on at a 
time. The operation of the comparative circuit is thus understood by 
determining its operation under three circumstances, when the optical 
densities of the two samples are equal, when the optical density of the 
first sample is greater than the second, and when the optical density of 
the second sample is greater than the first. 
Thus, when I.sub.D1 =I.sub.D2, i.e., the optical densities of the two 
samples are equal, then the voltage at TP4 (E.sub.0) will be E.sub.T. The 
gate outputs of G2 and G1 will be low so that the right center red LED 2 
and right red LED 1 will be off. The gate outputs of G3 and G4 will be 
high so that the left center red LED 4 and left red LED 5 will also be 
off. However, because the gate output of G5 and G3 will both be high, 
transistor Q3 will conduct and the green middle LED 3 will be on 
indicating that the two solutions have equal optical densities. 
By way of further example, if I.sub.D1 is greater than I.sub.D2 by up to a 
first predetermined value, e.g., 0.03 O.D., then the outputs of G2, G3 and 
G4 will be high and the output of G1 will be low. All LED's will be off 
except for the right center red LED 2 which will be on to indicate that 
the absorbence of the sample in tube 6 is greater than the absorbence of 
the sample in tube 5, but by no more than 0.03 O.D. If the absorbence of 
the sample in tube 6 is greater than that of the sample in tube 5 by at 
least a second predetermined value, e.g., 0.1 O.D., then the left red LED 
1 will be on, and all other LED's off. 
Similarly, if I.sub.D1 is less than I.sub.D2 by up to 0.03 O.D., then the 
outputs of G1, G2 and G3 will be low and G4 will be high. Thus the left 
center red LED 4 will be on to indicate that the absorbence of the sample 
in tube 5 is greater than the absorbence of the sample in tube 6 by no 
more than 0.03 O.D. Further, if the absorbence of the sample in tube 5 is 
greater than that of the sample in tube 6 by at least 0.1 O.D., then the 
left red LED 5 will be on and all other LED's off. 
The correction circuit provides both a zeroing mechanism to account for the 
fact that the two light paths and photodetectors are not truly identical 
and further provides a compensation factor to account for variations in 
the threshold voltage E.sub.T of the hex inverters used in the comparative 
circuit. The output of the correction circuit shown in FIG. 6A is a 
correction voltage E.sub.c which is applied to the positive input of A3. 
The correction circuit includes a hex inverter G6 from the same package as 
the hex inverters G1-G5 used in the comparative circuit. A trim 
potentiometer R18, having an output voltage E.sub.p, is provided along 
with resistors R25 and R26 and capacitors C9 and C10. 
The switching threshold of the hex inverter G6 is about one-half of the 
regulated supply voltage V.sub.REG. This logic gate is quite abrupt in its 
action and all of the gates in the package have very nearly the same 
threshold voltage E.sub.T for a given temperature and supply voltage. The 
transfer characteristic of the hex inverter is shown in FIG. 7. The slope 
of the transfer characteristic in the region of E.sub.T is the gain of the 
hex inverter if it is considered as an amplifier. With the gate output 
tied to the input, the input and output will stabilize at a value which is 
called the threshold or switching point E.sub.T. The absolute value of 
E.sub.T varies from one batch of devices to another, and varies according 
to time, temperature, and supply voltage. All of the inverters in a 
package have the same threshold within a few milivolts. By utilizing G6 in 
the correction circuit as an amplifier as shown in FIG. 6A, the difference 
signal E.sub.0 is referenced to the threshold voltage of G6. 
Another function of the correction circuit is to compensate for differences 
between the optical paths during the initial set-up procedure. For this 
purpose, the trim-potentiometer R18 is introduced to the circuit. The 
circuit will stabilize with the input voltage at E.sub.T if the trim-pot 
is adjusted to a value E.sub.p, where 
EQU (E.sub.p -E.sub.c)R.sub.d =(E.sub.p -E.sub.T)(R.sub.c +R.sub.d) 
EQU (8) 
where R26=R.sub.c and R25=R.sub.d. 
During initial calibration of the device, f.sub.2 is made equal to f.sub.1 
(equal samples) so that KGlog(f.sub.2 /f .sub.1)= 0. The trim-pot is 
adjusted until the voltage E.sub.0 =E.sub.T Now, if E.sub.T changes 
following calibration, E.sub.0 will follow the change in E.sub.T as set 
forth below: 
EQU E.sub.0 =E.sub.T +KGlog(f.sub.2 /f.sub.1) (9) 
Thus, if the value of E.sub.T drifts upward, the output E.sub.0 will also. 
At the same time, the voltage inputs for each of the comparator circuits 
will be going up by the same amount. The output of the differential 
amplifier is thus fully and exactly compensated for drift in E.sub.T. At 
the same time the initial differences in the optical paths are corrected 
with one trim-pot adjustment. 
While the incandescent lamp 32 shown in FIG. 6B is the preferred light 
source, alternatively, a pair of diametrically opposed LED's can be used 
as light sources, one to illuminate each of sample tubes 5 and 6. FIG. 6C 
shows a circuit for driving red LED 6 and red LED 7. 
While the invention is not so limited, the following table of values for 
the circuit elements shown in FIGS. 5, 6A, 6B and 6C are an example of an 
operative circuit according to this invention. 
TABLE 1 
______________________________________ 
A1 U1 25L2 CP 
A2 U1 25L2 CP 
A3 U4 LM10C LN 
A4 U4 LM10C LN 
C1 .01 uF 
C2 10 uF 
C3 .01 uF 
C4 .22 uF 
C5 1 uF 
C6 .01 uF 
C7 .1 uF 
C8 1 uF 
C9 .01 uF 
C10 .01 uF 
C11 100 pF 
C12 100 pF 
D3 CR1 1N6263 
G1 U3 74HC04 
G2 U3 74HC04 
G3 U3 74HC04 
G4 U3 74HC04 
G5 U3 74HC04 
G6 U3 74HC04 
Q1 PN2222 
Q2 TIP32C 
Q3 PN2222 
Q4 U2 LM394N 
Q5 U2 LM394N 
R1 470.OMEGA. 
R2 10K.OMEGA. 
R3 2K.OMEGA., 20 turn 
R4 9.1K.OMEGA.** 
R5 100.OMEGA. 
R6 100.OMEGA. 
R7 1K.OMEGA.** 
R8 7.68K.OMEGA.* 
R9 499K.OMEGA.* 
R10 7.68K.OMEGA.* 
R11 499K.OMEGA.* 
R12 9.1K.OMEGA.** 
R13 1.0K.OMEGA.** 
R14 1.0K.OMEGA.** 
R15 1.0K.OMEGA.** 
R16 1.0K.OMEGA.** 
R17 9.1K.OMEGA.** 
R18 10K.OMEGA., 20 turn 
R19 100.OMEGA. 
R20 10K.OMEGA. 
R21 100.OMEGA. 
R22 3.9K.OMEGA. 
R23 10K.OMEGA. 
R24 -- 
R25 100K.OMEGA.** 
R26 56K.OMEGA.** 
______________________________________ 
*ultra-stable 1% resistors, matched to .1%, 25 PPM 
**1% metal film resistors 
The operational amplifier U1 25L2 CP is sold by Texas Instruments of 
Dallas, Tex. The differential amplifier U4 LM10CLN is sold by National 
Semiconductor of Santa Clara, Calif. The diode CR1 1N6263 is sold by 
Hewlett-Packard of Palo Alto, Calif. The hex inverter U3 74HC04 is sold by 
Motorola of Austin, Tex. The transistors PN2222, TIP32C, and U2 LM394N, 
are all sold by National Semiconductor of Santa Clara, Calif. 
In designing the electronic subsystem of this invention, it is necessary to 
set the gain of differential amplifier A3 by selecting appropriate values 
for the gain resistors R8, R9, R10, and R11. The selection is made so that 
the indicator LEDs 1-5 come on for predetermined differences in absorbance 
which are of importance for the test at hand. One method for setting the 
gain of A3 is to use known sample solutions which have been accurately 
measured in a laboratory spectrophotometric instrument. Alternatively, the 
required gain can be calculated. To do so, one must understand the 
difference between the concepts of absorbance and absorptivity. An example 
of the calculations required to set the gain of A3 is described 
hereinafter. 
The definition of absorbance is: 
EQU A=log I/I.sub.o 
where I.sub.o is the intensity of light transmitted when the sample tube is 
filled with completely clear fluid and I is the light transmitted when the 
actual sample is in place. See Skoog and West, "Principles of Instrumental 
Analysis," Saunders College, Philadelphia, Pa., 1980. 
If one compares two samples which transmit amounts of light I.sub.1 and 
I.sub.2, then the differential absorbance .DELTA.A is of interest where: 
EQU .DELTA.A=log I.sub.2 /I.sub.o -log I.sub.1 /I.sub. o =log I.sub.2 /I.sub.1( 
10) 
Beer's law relates light absorbance to the properties of the sample under 
test: 
EQU A=abc 
where a=absorptivity of the solution, b=path length, and c=concentration of 
the solution. 
Specific absorptivity is defined as: 
EQU .alpha.=ac (11) 
This is the measure of absorbance per unit path length, and is dependent on 
the sample's nature and concentration. Thus: 
EQU A=.alpha.b 
If one compares two different solutions in identical sample containers, 
then the differential absorbance is: 
EQU .DELTA.A=A.sub.2 -A.sub.1=b(.alpha..sub.2 
-.alpha..sub.1)=b.DELTA..alpha.(12) 
When a sensitivity of 0.03 units of absorbance is desired, what is really 
required is a specific differential absorptivity .DELTA..alpha.=0.03 since 
this is the quantity which is independent of the means of measurement. 
Combining equations (10) and (12) produces: 
EQU .DELTA..alpha.=(1/b).DELTA.A=(1/b)log(I.sub.2 /I.sub.1) 
It is convenient to use natural logarithms (ln), so that 
EQU .DELTA..alpha.=(1/2.3026b)ln(I.sub.2 /I.sub.1) (13) 
Further one must consider the path length b for a particular instrument. 
Thus, for the optical configuration of FIG. 2, one may consider as an 
approximation that the light rays pass directly through the sample tube 
without refraction. Then, it can be shown geometrically that the average 
path length through the solution is 
EQU b.sub.av =(.pi./4)d 
where d is the inner diameter of the sample tube. This is the value of path 
length which must be used in a calculation for specific differential 
absorptivity: 
EQU .DELTA..alpha.=[4/(2.3026.pi.d]ln(I.sub.2 /I.sub.1) (14) 
Equation (14) relates specific differential absorptivity to light 
intensities. In addition one needs to determine the relationship between 
light intensities and E.sub.0, the output of the differential amplifier. 
The characteristic of the LM394 log conversion circuit is that of an ideal 
silicon diode, for which 
EQU V.sub.f =(T/11,600)ln(I.sub.f /I.sub.s) 
where V.sub.f =forward voltage, T=absolute temperature .degree.K, I.sub.f 
=forward current and I.sub.s =saturation current for that diode. 
The two LM394s which are used in the two log amplifiers are housed in the 
same package and come very well matched from the factory, so that I.sub.s 
is the same for both. The forward current I.sub.f is just equal to the 
photodiode current caused by the incident light: 
EQU I.sub.f1 =I.sub.D1 
EQU I.sub.f2 =I.sub.D2 
The differential amplifier extracts the difference in voltage between the 
two log conversion circuits and amplifies it by a factor G (gain): 
EQU E.sub.0 =G(V.sub.f2 -V.sub.f1) 
EQU which reduces to: 
EQU E.sub.0 =(GT/11,600)ln(I.sub.D2 /I.sub.D1) 
Diode current I.sub.D is proportional to light intensity I at the diode: 
EQU I.sub.D2 .alpha.I.sub.2 
EQU I.sub.D1 .alpha.I.sub.1 
EQU so 
EQU E.sub.0 =(GT/11,600)ln(I.sub.2 /I.sub.1) 
Combining this result with equation (5): 
EQU .DELTA..alpha.=[4/(2.3026.pi.d)][(11,600 E.sub.0)/GT] 
EQU or 
EQU E.sub.0 =(2.3026.pi.dGT.DELTA.)/[(4)(11,600)] (15) 
For this example, we want to have .DELTA..alpha.=0.03 when the first red 
light (i.e., LED2 or LED4) comes on. The first red light comes on when 
E.sub.0 =0.100 volts, as is determined by the resistor chain to which the 
hex inverter inputs are attached. 
Given 
EQU .DELTA..alpha.=0.03 
EQU T=298.degree. K (room temperature) 
EQU d=9 mm=0.9 cm 
EQU E.sub.0 =0.100 volts 
Then equation (15) can be solved to give G=79.7 as the required gain. Thus 
we need to choose 
EQU R11/R10=R9/R8=79.7 
This calculation provides results as close as those which have been 
determined by experimental means. 
Although a preferred embodiment of the invention has hereinbefore been 
described, it will be appreciated that variations of this invention will 
be perceived by those skilled in the art, which variations are 
nevertheless within the scope of this invention as defined by the claims 
appended hereto.