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A simple automatic colorimeter with digital results adapted to computer processing
CLINICA CHIMICA ACTA
A
SIMPLE
ADAPTED
AUTOMATIC
239
COLORIMETER
TO COMPUTER
F. J. LANCEE,
B. E. A. GLASER,
WITH
2. KOLAR,
T. L. LIEM
Department ofChenzical Pathology, Rotterdam Medical Faculty, Rottevdam (The Netherlands) (Received
DIGITAL
RESULTS
PROCESSING
AND
B. LEIJNSE
University Hos$ital Dijkzigt,
April 24, 1969)
SUMMARY
We designed an automatic calorimeter based upon the principle of Authenrieth’s classical apparatus. With a double beam system, making use of electronical and mechanical devices, a very stable apparatus was developed. The chief advantage of this simple automatic calorimeter is that the result of the analyses appears in digital form, the numerical value corresponding with the concentration in the units desired. Thus, calculation can be omitted, and after transformation in the ASCII code, the results can be printed on a teletype (33 TB) or they can be fed directly into a computer.
INTRODUCTION
The increasing workload of analyses a clinical chemical laboratory has to perform daily makes intensive automation of laboratory techniques compulsory. Generally, the results of the analyses are not available in digital form, but have to be calculated from analog information, for example from recorder registrations. This is time consuming. Most clinical chemical investigations are based on a calorimetric method. Therefore, we have developed an automatic calorimeter which provides the results of the analyses in a digital form, adapted to computerised administration. This automatic calorimeter is based on the principle used in Authenrieth’s classical apparatus. A beam of light passes through a sample cuvette and a similar beam of light through a wedge-shaped reference cuvette. The latter can be moved in a plane perpendicular to the direction of the beams of light. The sample cuvette is filled with the solution of which the concentration has to be determined. The reference cuvette contains a solution of known concentration. In Authenrieth’s system the wedge-shaped cuvette is moved manually till the intensities of the two beams of light, after passage through the cuvettes, are equal, according to visual judgement. As has been stated, the light intensities of the beams entering the cuvettes are equal. When the light intensities of the emergent beams are equal too, then the extinctions in the two cuvettes are Clin. Chim. Acta, 25 (1969) 23g-qsz
LANCEE et d.
240
also identical. At that point, according to the law of Lambert-Beer, the product cjf concentration (c$) and optical path length (d,) through the reference cuvette equals that of the sample cuvette (c8 x d, = cr x d,). The wedge shape of the reference cuvette causes a direct proportionality between the optical path length and the distance (I) which this cuvette has to be shifted from the zero position to the point where extinctions are equal (ds = fx I). Hence, the concentration of the sample and the distance covered by the reference cuvette
d, (Cr=fxlxc,
=f’l
)
are directly proportional. The multiplication factor (f’) depends on (I) the concentration in the reference cuvette (2) the optical path length through the sample cuvette and (3) the proportionality factor (f) of the reference cuvette, which is dependent on the transmission between the reference cuvette and the motor. Thus, a properly selected set-up enables one to express the distance covered by the reference cuvette as a quantity with a numerical value equal to the sample concentration. In this way, calculations can be omitted. INSTRUMENT
The calorimeter
operates according to the double beam principle (Fig. I). Light
originating from a IOO W, IZ V tungsten lamp (L) fitted in a water-cooled holder, falls upon a lens (achromatic, 8 dioptrics) perpendicularly (LEI). The lens has been placed at a distance of about 13 cm from the centre of the lamp. The parallel beam of emer-
Fig. 1, Schematic representation
RCw of the calorimeter.
ging light falls upon an adjustable horizontal slit (S) of about I mm height. The beam of light passing through this slit is interrupted by a chopper (C) rotating at 2500 rev./ min, allowing the light beam to fall in turn upon the movable reference cuvette (RC) and upon the sample cuvette (SC). Sample cuvette and reference cuvette are both similarly wedge shaped, but the sample cuvette is truncated. The cuvettes are fitted perpendicularly to the direction of the light beam. The emergent beams of light from cuvettes fall upon another lens (LEz), similar to the first one. The converging beams of light pass through an interference filter (IF) and sequentially fall upon the surface of a phototube, Telefunken F.Z.-9012. The current output generated by the light on the photodiode contains a DC and an AC component. Clin.
Chim.
Acta,
25 (1969) 23g-z+’
,241
AUTOMATIC COLORIMETER
When the reference cuvette is moved from zero position to the level where the intensities of the emergent light from sample and reference cuvette are equal, a change can be observed in the AC component. When the intensities reach equality, the AC amplitude approaches the value of zero. However, when the intensity of the light emerging from the reference cuvette is greater than that from the sample cuvette, a phase shift results. This phase shift can be detected electronically and used as end point. In our equipment the reference cuvette is driven by a 375 rev./min motor, giving the cuvette a linear velocity of 7.5 cm/min. The starting position of the reference cuvette is adjustable as described under OPERATION AND RESULTS. The distance covered by the reference cuvette from the starting position to the point where the light intensities are equal is measured by counting magnetically the number of revolutions of the driving motor. The decimal count is transformed into the ASCII code by electronic conversion. Thus, the results of the determination are available in a digital form suitable to be printed on a teletype (33 TB) or to be fed directly into a computer. The position of the sample cuvette can be varied with respect to the beam of light, providing an adjustable optical path length (d,). Moreover, as a second variable, the concentration of the reference solution (cS) is also adjustable. A third, less flexible adjustment is by changing the transmission (factor f), between motor and reference cuvette. Since cr = f’l and fl = f x cs/dr,it is possible by adjusting es, d, and f, as described above, to obtain a digital output with a numerical value exactly corresponding with the concentration, expressed in the desired units. OPERATION AND RESULTS
The reference cuvette is filled with a solution which has an extinction value slightly greater than the expected maximum extinction of a sample. The sample
0
100
20.0
CT%
mmok/l
Fig. 2. The determination of creatinine. The calorimeter is adjusted in a way that the numerical value of the pulsesis equal to the concentration of creatinine in mmole/l.
242
LANCEE
et d.
cuvette is filled with the blank solution. Then the zero level of the reference cuvette is mechanically adjusted to the position where the intensities of the emergent beams of light from sample and reference cuvette are equal. For each kind of determination, reference cuvette, sample cuvette and reference solution should be repeatedly adjusted as described above, to calibrate the calorimeter. The sample cuvette can be emptied and filled automatically. After each analysis the reference cuvette returns to zero position. When the sample cuvette has been refilled with a new sample the reference cuvette starts to move to the position where the light intensities are equal. In 60 minutes, 30 analyses can be performed. We performed the determination of creatinine with the usual Jai% technique, making use of the calorimeter described. The results obtained are presented in Fig. 2. In this case the reference cuvette was filled with a bichromate-sulphuric acid solution. The peak wave length of the passing light and the half width of the interference filter used are 525 and 8 nm, respectively. ACKNOWLEDGEMENTS
We are greatly indebted to the Switching Laboratory of the Department of Electronical Engineering, Technical University at Delft. Our thanks are due to Mr. B. van der Meulen and Mr. J. van Veen and their collaborators of the Central Research Workshop (Head Ing. H. A. Bak), Rotterdam Medical Faculty for technical assistance. C&n. Chim. Acta, 25 (1969) 239-242
A
SIMPLE
ADAPTED
AUTOMATIC
239
COLORIMETER
TO COMPUTER
F. J. LANCEE,
B. E. A. GLASER,
WITH
2. KOLAR,
T. L. LIEM
Department ofChenzical Pathology, Rotterdam Medical Faculty, Rottevdam (The Netherlands) (Received
DIGITAL
RESULTS
PROCESSING
AND
B. LEIJNSE
University Hos$ital Dijkzigt,
April 24, 1969)
SUMMARY
We designed an automatic calorimeter based upon the principle of Authenrieth’s classical apparatus. With a double beam system, making use of electronical and mechanical devices, a very stable apparatus was developed. The chief advantage of this simple automatic calorimeter is that the result of the analyses appears in digital form, the numerical value corresponding with the concentration in the units desired. Thus, calculation can be omitted, and after transformation in the ASCII code, the results can be printed on a teletype (33 TB) or they can be fed directly into a computer.
INTRODUCTION
The increasing workload of analyses a clinical chemical laboratory has to perform daily makes intensive automation of laboratory techniques compulsory. Generally, the results of the analyses are not available in digital form, but have to be calculated from analog information, for example from recorder registrations. This is time consuming. Most clinical chemical investigations are based on a calorimetric method. Therefore, we have developed an automatic calorimeter which provides the results of the analyses in a digital form, adapted to computerised administration. This automatic calorimeter is based on the principle used in Authenrieth’s classical apparatus. A beam of light passes through a sample cuvette and a similar beam of light through a wedge-shaped reference cuvette. The latter can be moved in a plane perpendicular to the direction of the beams of light. The sample cuvette is filled with the solution of which the concentration has to be determined. The reference cuvette contains a solution of known concentration. In Authenrieth’s system the wedge-shaped cuvette is moved manually till the intensities of the two beams of light, after passage through the cuvettes, are equal, according to visual judgement. As has been stated, the light intensities of the beams entering the cuvettes are equal. When the light intensities of the emergent beams are equal too, then the extinctions in the two cuvettes are Clin. Chim. Acta, 25 (1969) 23g-qsz
LANCEE et d.
240
also identical. At that point, according to the law of Lambert-Beer, the product cjf concentration (c$) and optical path length (d,) through the reference cuvette equals that of the sample cuvette (c8 x d, = cr x d,). The wedge shape of the reference cuvette causes a direct proportionality between the optical path length and the distance (I) which this cuvette has to be shifted from the zero position to the point where extinctions are equal (ds = fx I). Hence, the concentration of the sample and the distance covered by the reference cuvette
d, (Cr=fxlxc,
=f’l
)
are directly proportional. The multiplication factor (f’) depends on (I) the concentration in the reference cuvette (2) the optical path length through the sample cuvette and (3) the proportionality factor (f) of the reference cuvette, which is dependent on the transmission between the reference cuvette and the motor. Thus, a properly selected set-up enables one to express the distance covered by the reference cuvette as a quantity with a numerical value equal to the sample concentration. In this way, calculations can be omitted. INSTRUMENT
The calorimeter
operates according to the double beam principle (Fig. I). Light
originating from a IOO W, IZ V tungsten lamp (L) fitted in a water-cooled holder, falls upon a lens (achromatic, 8 dioptrics) perpendicularly (LEI). The lens has been placed at a distance of about 13 cm from the centre of the lamp. The parallel beam of emer-
Fig. 1, Schematic representation
RCw of the calorimeter.
ging light falls upon an adjustable horizontal slit (S) of about I mm height. The beam of light passing through this slit is interrupted by a chopper (C) rotating at 2500 rev./ min, allowing the light beam to fall in turn upon the movable reference cuvette (RC) and upon the sample cuvette (SC). Sample cuvette and reference cuvette are both similarly wedge shaped, but the sample cuvette is truncated. The cuvettes are fitted perpendicularly to the direction of the light beam. The emergent beams of light from cuvettes fall upon another lens (LEz), similar to the first one. The converging beams of light pass through an interference filter (IF) and sequentially fall upon the surface of a phototube, Telefunken F.Z.-9012. The current output generated by the light on the photodiode contains a DC and an AC component. Clin.
Chim.
Acta,
25 (1969) 23g-z+’
,241
AUTOMATIC COLORIMETER
When the reference cuvette is moved from zero position to the level where the intensities of the emergent light from sample and reference cuvette are equal, a change can be observed in the AC component. When the intensities reach equality, the AC amplitude approaches the value of zero. However, when the intensity of the light emerging from the reference cuvette is greater than that from the sample cuvette, a phase shift results. This phase shift can be detected electronically and used as end point. In our equipment the reference cuvette is driven by a 375 rev./min motor, giving the cuvette a linear velocity of 7.5 cm/min. The starting position of the reference cuvette is adjustable as described under OPERATION AND RESULTS. The distance covered by the reference cuvette from the starting position to the point where the light intensities are equal is measured by counting magnetically the number of revolutions of the driving motor. The decimal count is transformed into the ASCII code by electronic conversion. Thus, the results of the determination are available in a digital form suitable to be printed on a teletype (33 TB) or to be fed directly into a computer. The position of the sample cuvette can be varied with respect to the beam of light, providing an adjustable optical path length (d,). Moreover, as a second variable, the concentration of the reference solution (cS) is also adjustable. A third, less flexible adjustment is by changing the transmission (factor f), between motor and reference cuvette. Since cr = f’l and fl = f x cs/dr,it is possible by adjusting es, d, and f, as described above, to obtain a digital output with a numerical value exactly corresponding with the concentration, expressed in the desired units. OPERATION AND RESULTS
The reference cuvette is filled with a solution which has an extinction value slightly greater than the expected maximum extinction of a sample. The sample
0
100
20.0
CT%
mmok/l
Fig. 2. The determination of creatinine. The calorimeter is adjusted in a way that the numerical value of the pulsesis equal to the concentration of creatinine in mmole/l.
242
LANCEE
et d.
cuvette is filled with the blank solution. Then the zero level of the reference cuvette is mechanically adjusted to the position where the intensities of the emergent beams of light from sample and reference cuvette are equal. For each kind of determination, reference cuvette, sample cuvette and reference solution should be repeatedly adjusted as described above, to calibrate the calorimeter. The sample cuvette can be emptied and filled automatically. After each analysis the reference cuvette returns to zero position. When the sample cuvette has been refilled with a new sample the reference cuvette starts to move to the position where the light intensities are equal. In 60 minutes, 30 analyses can be performed. We performed the determination of creatinine with the usual Jai% technique, making use of the calorimeter described. The results obtained are presented in Fig. 2. In this case the reference cuvette was filled with a bichromate-sulphuric acid solution. The peak wave length of the passing light and the half width of the interference filter used are 525 and 8 nm, respectively. ACKNOWLEDGEMENTS
We are greatly indebted to the Switching Laboratory of the Department of Electronical Engineering, Technical University at Delft. Our thanks are due to Mr. B. van der Meulen and Mr. J. van Veen and their collaborators of the Central Research Workshop (Head Ing. H. A. Bak), Rotterdam Medical Faculty for technical assistance. C&n. Chim. Acta, 25 (1969) 239-242