WO2001046679A1

WO2001046679A1 - Method and apparatus for analyzing samples in a clinical analyzer using coherent radiation

Info

Publication number: WO2001046679A1
Application number: PCT/US2000/035037
Authority: WO
Inventors: Noureddine Melikechi; Jenny Maria Juliane Magnes
Original assignee: Applied Optics Center Of Delaware, Inc.
Priority date: 1999-12-22
Filing date: 2000-12-21
Publication date: 2001-06-28

Abstract

A method for determining the quantity of a chemical species in a liquid sample contained in a sample container using a pair of spaced apart prisms to reflect a coherent radiation light beam a multiple number of times through the liquid sample and relating absorption of the beam to the amount of chemical species in the liquid sample.

Description

METHOD AND APPARATUS FOR ANALYZING SAMPLES IN A CLINICAL ANALYZER USING COHERENT RADIATION

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for analyzing liquid samples, particularly for analyzing biological sample liquids such as urine, blood serum, plasma, cerebrospinal fluid and the like. In particular, the present invention provides means for analyzing a liquid sample by measuring absorption of coherent radiation transmitted through the liquid using an adjustable pathlength.

BACKGROUND OF THE INVENTION

Fully automated clinical diagnostic analyzers are commercially available to perform chemical, and immunoassaying of biological samples such as urine, blood serum, plasma, cerebrospinal fluid and the like. Generally, chemical reactions between an analyte to be measured in the sample and reagents used during the assay result in a signal that can be measured by the instrument, and from this signal the concentration of analyte in the patient sample is calculated. A popular format for making such measurements in clinical analyzers employs spectrometric techniques in which absorption of radiation transmitted through the sample is measured at a wavelength which depend on the absorption characteristics of the analyte to be measured. For example, a commercially available halogen light source may be used in combination with a series of colored glass optical filters to provide absorption measurements centered around wavelengths ranging from 400 to 750 nanometers (nm).

Analyzers like those described above perform satisfactorily with relatively large patient samples, for instance in the 100-300 microliter (μl) range, partly because the sample volume is sufficiently large to generate reaction signals that can be distinguished with acceptable error levels. However, for many reasons including minimizing invasive impact on patients, it is desirable to obtain diagnostic measurements with smaller volume patient samples. Accordingly, an important design feature in improved clinical diagnostic analyzers is the ability to obtain accurate clinical measurements on sample volumes, for example in the 10-50 μl range. Tunable diode laser absorption spectroscopy is a known technique for detecting gas phase molecular and chemical species. Because the sensitivity of detection increases with increasing pathlength that a beam of interrogating radiation traverses a sample containing an analyte to be measured, sample test cells are designed with appropriate reflectors and dimensions to pass the radiation a multiple number of times through the sample to increase the effective interrogating pathlength. Since various test samples may have a wide range of analyte concentrations, it is advantageous to provide an absorption analyzer having a readily variable effective interrogating pathlength.

U.S. Pat. No. 3,524,066 describes an apparatus employing a semiconductor light source to provide a beam of coherent monochromatic radiation which is passed through a sample cell containing a selected medium having therein an known gas or liquid to be detected. The sample cell is designed to contain a selected quantity of the medium which is to be analyzed for the presence of a gas or liquid and comprises a pair of optical flats on each of the ends of the cell transverse to the direction of the laser beam. A portion of the optical flats is provided with reflective surfaces so that the laser beam is subjected to a series of internal multiple reflection, thereby increasing the effective length traversed by the beam. The gas or liquid to be detected is detected by selecting the light source so that the wavelength of emitted radiation corresponds closely to an known absorption band of the gas or liquid and measuring absorption of the radiation transmitted through the medium.

U.S. Pat. No. 5,173,749 discloses a non-planar multipass cell for spectroscopic measurements of a gas sample. A tunable diode laser is used to detect impurities in gases useful in semiconductor processing. U.S. Pat. No. 5,818,578 uses a similar technique in combination with a polygon shaped sample cell to achieve size reduction advantages.

U.S. Pat. No. 5,485,276 discloses a multi-pass optical cell for use with collimated radiation having a wavelength equal to the absorption wavelength of a species being monitored in a gas such as ambient air. A plurality of mirrors are placed on opposing sides of an air flow cell to cause the beam to be reflected transverse to the flow of air being monitored.

Accordingly, from a study of the different approaches taken in the prior art to the problems encountered with clinical analyses of small volume samples, taken with the challenges of maintaining a high level of detection and, at the same time, minimizing the physical size of an analyzer, there is a need for an improved approach to the design of clinical analyzer. In particular, there is a need for a method to provide an analyzer having increased sensitivity when analyzing samples of small volume. Further, it is desirable to provide such an analyzer with the ability to measure samples conveniently, without concern for issues like closed containers and sample tube types and sizes. Moreover, it is advantageous to provide an absorption analyzer having an easily adjusted pathlength of interrogating radiation to increase analyzer sensitivity in the instances of low analyte concentrations or very small sample volumes.

SUMMARY OF THE INVENTION

Many of these disadvantages to the prior art are overcome by using the apparatus and/or methods of this invention. This invention provides a method for determining the quantity of a chemical species in a liquid contained in a sample container by passing a coherent beam of interrogating radiation a number of times through the mixture and relating absorption of the beam to the amount of chemical species in the sample. More particularly, the present invention is directed at a method for measuring the presence of a chemical species or analyte in a liquid sample suspected of containing said species by propagating a collimated coherent interrogating radiation beam toward a first prism and a second prism spaced apart from one another to define an open sample receiving area therebetween. The first prism and second prisms are shaped and positioned so that the radiation beam is reflected within said prisms and caused to make a multiple number of passes through the sample receiving area. The first prism and second prisms are variably positioned relative to one another so that the multiple number of passes through the sample receiving area may be conveniently changed to accommodate samples with different sample volumes and analyte concentrations. The prisms may also be inclined relative to one another to interrogate different portions of a sample, thereby overcoming errors due to inhomogeneities in the sample. The liquid sample container is disposed within the sample receiving area so that the radiation beam passes through the liquid sample a number of times corresponding to the number of passes through the sample receiving area. The sample container may be moved within the sample receiving area away from the interrogating radiation beam to vary the number of radiation beam passes through the sample, hence varying the effective pathlength without repositioning either of the two prisms. The intensity of the radiation beam is detected after passing through the liquid sample the multiple number of times, and the wavelength of the radiation beam is selected so that the radiation beam is at least partially absorbed by the chemical species. By passing the radiation beam an optimum multiple number of times through the liquid sample, sensitivity and signal-to- noise advantages are obtained so that low levels of chemical species may be detected or so that analytical tests may be performed on samples having a small volume. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detailed description thereof taken in connection with the accompanying drawings which form a part of this application and in which:

FIG. 1 is a schematic plan view of a multi-pass coherent light chemical analyzer in which a first embodiment of the present invention may be used to advantage;

FIG. 2 is an amplified view of a portion of FIG. 1;

FIG. 3 is a schematic plan view of a multi-pass coherent light chemical analyzer in which a second embodiment of the present invention may be used to advantage;

FIG. 4 is a schematic plan view of a multi-pass coherent light chemical analyzer in which a first number of passes of coherent light may be employed in with the present invention;

FIG. 4a is a schematic plan view of the multi-pass coherent light chemical analyzer of FIG 4 in which a different number of passes of coherent light may be employed;

FIG. 5 is a plot of absorption data for chemical species useful within the chemical analyzer of FIG. 1;

FIG. 6 is a plot of absorption data obtained for multiple radiation passes using the chemical analyzer of FIG. 1;

FIG. 7 illustrates reflection corrected absorption data obtained for multiple radiation passes using the chemical analyzer of FIG. 1; and,

FIG. 8 shows the results of signal-to-nose measurements obtained for multiple radiation passes using the chemical analyzer of FIG. 1. DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows schematically' the elements of a multi-pass coherent light chemical analyzer 10 comprising a collimated coherent light source 12 emitting a collimated coherent light beam 14 that is incident upon and reflected within a first prism 16 so as to make a first pass through a cuvette 18 containing a sample to be analyzed and positioned within a sample receiving area 19, shown in dashed lines and located between the first prism 16 and a second prism 20, spaced apart from prism 16. After passing through the cuvette 18, the beam is incident upon and reflected within the second prism 20 so as to make a second pass through cuvette 18. First prism 16 and second prism 20 are sized and positioned so that the coherent light beam 14 is caused to make a multiple number of passes through cuvette 18. Four such passes are illustrated in Fig. 1, however this is done for illustrative purposes only, the number of passes being adjusted as necessary to achieve a desired level of sensitivity as described hereinafter and illustrated in FIG. 4. After beam 14 makes a multiple number of passes through cuvette 18, as better seen in FIG. 2, the beam 14 is reflected a final time within either of the two prisms 16 or 20 and exits either prism 16 or 20 and is incident onto a mirror 22 which directs beam 14 to a primary optical detector 24, for example a photo diode. Mirror 22 is included for convenience in arranging the various elements of analyzer 10 as in an alternate arrangement, detector 24 could be placed to directly intercept beam 14 after the final reflection within either of the two prisms 16 or 20.

Fig. 2 further illustrates a particular embodiment wherein prism 16 was a 1-inch side, UV Fused Silica Prism # 10SR20, and prism 20 was a VHnch side UV Fused Silica Prism # 05SR20, both obtained from Newport Corporation (Irvine, CA). The multi-pass coherent light chemical analyzer 10 has coherent light beam 14 entering first prism 16 at point 11 and reflecting through total internal at points 13 and 15 of first prism 16 exits prism 16 at point 17, then propagates a first time through cuvette 18 before entering second prism 20 at point 19. After entering second prism 20, beam 14 is totally internally reflected at points 21 and 23 so as to exit prism 20 at point 25 and propagates a second time through cuvette 18. First prism 16 and second prism 20 are sized and positioned so that the process of coherent light beam 14 entering a prism, reflecting from surfaces thereof, exiting the prism and propagating through the test sample cuvette continues a multiple number of times. In an exemplary embodiment, like that of Fig. 2, after propagating the second time through cuvette 18, beam 14 re-enters first prism 16 at point 27 and is reflected at points 29 and 31 so as to exit prism 16 at point 33 and propagate a third time through cuvette 18. As shown, after propagating the third time through cuvette 18, beam 14 reenters second prism 20 at point 35 and is reflected at points 37 and 39 so as to exit prism 20 at point 41 and propagate a fourth time through cuvette 18. After propagating the fourth time through cuvette 18, beam 14 reenters first prism 16 at point 43 and is reflected at points 45 and 47 so as to exit prism 16 at point 49 and be directed towards mirror 22. In practice, a commercially available optical software design application, such as Zemax (Tuscon, AZ)may be used to efficiently determine sizes and geometrical shapes of prisms 16 and 20 so that a desired number of multiple passes of a coherent light beam through a test sample cuvette positioned between the prisms may be achieved.

Fig. 3 shows an alternate exemplary embodiment of the present invention in which a beam splitter 26 is placed between collimated coherent light source 12 and first prism 16 so that a portion of beam 14 is reflected towards a secondary optical detector 28 to provide means for monitoring or adjusting the amplitude of beam 14.

Cuvette 18 typically comprises a container containing a chemical mixture having a chemical species or analyte therein that is known to absorb light within a wavelength range and that is desired to be quantified in terms of density. An important feature of the present invention is the use of spaced apart prisms so that cuvettes of different shapes and sizes may be readily positioned within the air space therebetween, it only being required that the cuvette have opposing surface portions that are essentially transparent to the interrogating radiation; i. e.; that the cuvette have apertures that do not significantly absorb the interrogating radiation.

It is known in absorption spectroscopy that the intensity of radiation transmitted through a test sample is described by Beer's Law and is directly proportional both to the concentration of analyte within the sample being tested and to the pathlength of radiation within the sample. Consequently, given a certain size of a sample cuvette, prisms 16 and 20 may be readily designed so that effective interrogating optical path-lengths equal to a multiple number of times the single pass diameter of the cuvette may be achieved. The absorption path-length is selected by taking into consideration the test sample size and/or analyte concentrations being encountered and the sensitivity of the radiation detector 24 employed. In an exemplary embodiment, a test sample cuvette having a single pass diameter of 1 cm and a sample volumes between about 10 μl and 30 μl, in combination with a optical prism assembly designed to produce four optical passes through the cuvette, has been found to be effective in measuring analyte concentrations in the 10^"6 mg/mL range when a DET 110 photodiode (from Thorlabs, Newtown, NJ) with sensitivity 0.3 amp/watt at a wavelength of about 532 nm is employed. Obviously larger sample volumes could be analyzed using the present invention most likely producing results with correspondingly higher signal strengths. In accordance with the present invention, the multi-pass coherent light chemical analyzer 10 is intended to be used in absorption spectroscopy where the chemical species to be measured within the test sample displays a peak absorption within a particular range of wavelengths. Previously, it was popular to employ an incoherent light source emitting a broad spectrum of radiation wavelengths inclusive of the range of peak absorption of the analyte being measured. A filter was interposed between the light source and the test sample to reduce the interrogating bandwidth to be more like the peak absorption range; such filtering reduces the interrogating radiation intensity and thus the sensitivity of the analyzer to low levels of analyte. The present invention employs coherent laser radiation produced for example by diode lasers or by a helium neon lasers having wavelengths selected to be within the peak absorption ranges of several analytes, including instances where wavelengths are altered by non-linear optical means to also be within the appropriate peak absorption ranges.

Clinical tests are routinely performed using prepared quantities of chemical reagents in combination with automated analytical equipment to carry out prescribed analytical procedures that provide qualitative measurements of various analytes in body fluids like blood, serum and urine. Enzyme-linked immunoassays have achieved widespread use for the measurement of clinically important analytes. There are many different types of enzyme-linked immunoassays, such as sandwich immunoassays and competitive and noncompetitive heterogeneous immunoassays.

Enzyme-linked immunoassays often utilize an enzyme-labeled antibody specific for the analyte of interest. The enzyme-labeled antibody binds to an analyte of interest in a sample and the enzymatic activity of either the bound enzyme-labeled antibody or the free enzyme-labeled antibody is measured by reacting the enzyme with a substrate to produce a detectable chromophore. Affinity columns containing immobilized analyte are often used to separate the free labeled antibody from the bound labeled antibody so that the enzymatic activity of the bound labeled antibody can be measured. Virtually any enzyme that can be coupled to an antibody and can react with a substrate to produce a detectable chromophore can be used in enzyme-linked immunoassays. Importantly, different analytical procedures are often designed to employ a same enzyme so that various analytical procedures are based on monitoring absorption within a common range.

To illustrate the advantages in processing efficiency afforded by the present multi-pass, coherent light analyzer 10, consider a first example in which analyzer 10 is used to measure digoxin, a cardiovascular drug, in serum and plasma. Digoxin test results are used in the diagnosis and treatment of digoxin overdose and in monitoring levels of digoxin to ensure appropriate therapy. The test protocol uses an immunoassay technique in which free and digoxin-bound antibody-enzyme species are separated using magnetic particles. The assay chemistry is optimized for measurement of β-galactosidase activity and begins by mixing an antibody conjugate reagent with patient's serum or plasma. The antibody conjugate reagent utilizes the F(ab 2 fragment of the antibody to eliminate interference from rheumatoid factor. Digoxin in the sample is bound by the F(ab 2 - β-galactosidase in the antibody conjugate reagent. Magnetic particles coated with the digoxin analog ouabain are added to bind free (unbound) antibody-enzyme conjugate. The reaction mixture is then separated magnetically. Following separation, the supernatant containing the digoxin-antibody-enzyme complex is transferred and mixed with a substrate, of chlorophenol- β-D- galactopyranoside (CPRG). The β-galactosidase (β-gal) portion of the digoxin-F( ab')2- β- galactosidase complex catalyzes the hydrolysis of CPRG to chlorophenyl red (CPR) chromophore. The change in absorbance at 577 nm due to the formation of the CPR chromophore is directly proportional to β-galactosidase activity and the amount of digoxin in the test sample.

Alternately, an enzyme-linked immunoassay for cydosporin A may be performed using analyzer 10 by contacting a lysed whole blood sample containing cydosporin A with excess beta-D- galactosidase-labeled anti-cyclosporin antibody to form a reaction mixture containing a complex of cydosporin A with labeled antibody and free labeled antibody, separating free antibody from the reaction mixture by contacting the reaction mixture with a solid phase comprising an immobilized cydosporin on a solid support, separating the solid phase from the liquid phase, and measuring the amount of cydosporin A by measuring the amount of the bound beta-D-galactosidase label in the liquid phase by adding to the liquid phase CPRG as a beta-D-galactosidase substrate and measuring the amount of the CPR chromophore produced using the coherent light analyzer 10 of the present invention operating with a laser having output radiation of about 577 nm.

In another example, the coherent light analyzer 10 may be used to quantitatively measure prolactin in human serum and plasma. Measurement of prolactin concentrations is important for investigation of infertility and disorders of the hypothalamic-pituitary axis. Decreased prolactin concentrations are usually associated with general pituitary hormone deficiency (Sheehan's syndrome), presenting as inability to lactate following normal pregnancy. Elevated prolactin concentrations, hyperprolactinemia, is the most common human hypothalamic-hypophyseal disorder. It can be caused by prolactin-secreting pituitary adenomas; hypothalamic-pituitary dysfunction; and in response to a variety of drugs, exercise, and stress. Hyperprolactinemia may result in absent or abnormal menses, galactorrhea, infertility, decreased libido, and/or visual field impairment. Measuring prolactin with the coherent light analyzer 10 of the present invention involves an enzyme immunoassay based on the "sandwich" principle. The assay mixes test sample with chromium dioxide particles, :oated with monoclonal antibodies specific for prolactin, and conjugate reagent (b-galactosida:;e labeled monoclonal antibodies specific for a second binding site on the prolactin molecule). A particle/pralactin /conjugate sandwich forms during the incubation period. Next, the sandwich is washed to remove unbound conjugate and transfers the sandwich into a sample test pack. The test pack is then placed in the coherent light analyzer for measurement of prolactin in the sample. The bound b-galactosidase catalyzes hydrolysis of chlorophenol red-b-d- galactopyranoside (CPRG) to chlorophenyl red (CPR). The color change measured around 577 nm due to formation of CPR is directly proportional to the concentration of prolactin present in the patient sample.

Another feature of the present invention is the ability to automatically conduct a system performance evaluation by simply testing a control or calibrator solution containing a known amount of a dye which is known to absorb certain radiation intensities at certain wavelengths. During its initial operating set-up, analyzer 10 is calibrated by determining the output signal from detector 24 for a series of calibration test samples having a known values of different dye concentrations ranging from 0 mg/ml to a value which exceeds the maximum value expected to be found in patient test samples. Afterwards, a control test is used to check for proper functioning of the radiation source and diode measuring system. In the present embodiment of a multi-pass coherent radiation analyzer, as a matter of convenience described below so that a single laser radiation source 12 could be employed, an absorbance test was selected using a stable certified solution of cobalt sulfate (CoS0₄) and measures the difference in absorbance at two different wavelengths for CoS0₄ diluted as a sample and as a reagent; the difference must be within a nominal range of a properly operating analyzer 10.

FIGs. 4 and 4a show how a variable number of radiation passes through a sample may be achieved using the multi-pass coherent light analyzer 10 by simply moving either of prisms 16 or 20 relative to the other prism. This is another key feature of the present invention in that it provides the ability to vary the absorption pathlength so that samples of widely different concentrations may be analyzed. FIG. 4 is illustrative of an embodiment in which second prism 20 is smaller than first prism 16 and is shaped and positioned relative to first prism 16 so that a total of four passes of coherent light beam 14 from coherent light source 12 travel through cuvette 18 positioned within sample receiving region 19. FIG. 4a is illustrative of a related embodiment in which second prism 20 is re-positioned to more closely mirror-image the position of first prism so that a total of ten passes of coherent light beam 14 from coherent light source 12 travel through cuvette 18 positioned within the same sample receiving region 19 as in FIG. 4. These two FIGs. 4 and 4a illustrate a key feature of the multi-pass coherent light analyzer 10 of the present invention by providing a method to easily vary the number of passes of light passed through a sample within cuvette 18 positioned within the sample receiving region 19 between the spaced apart prisms 16 and 20. In the instance of detecting an analyte known to have low concentrations within a sample contained in cuvette 18, an arrangement like that of FIG. 4a could be employed to increase the number of passes of light through the sample so that an increased pathlength is achieved, thereby providing an increased sensitivity assay methodology. Alternately, a lower power output lasers may be employed for the coherent light source 12 in multi-pass light analyzer 10 which has its prisms 16 and 20 arrayed so that a higher number of interrogating passes is achieved, like in FIG. 4a. In another embodiment, prisms 16 and 20 may be inclined relative to one another so that different portions of the sample may be interrogated by coherent light beam 14, allowing an operator to inspect for inhomogenities within the sample being tested. In a further embodiment, cuvette 18 may be positioned at different places within the sample receiving area 19 so that a different number of interrogating passes is achieved in a sample contained in the cuvette 18 without moving first prism 16 and a second prism 20 relative to one another, as illustrated by the dashed in FIG. 4c.

FIG. 5 is a plot of absorption data for the two chemical species identified above, CPR and CoS0₄, the former being illustrative of an enzyme-based chromophore detection protocol performed by the analyzer 10 and the latter being illustrative of a dye-based control solution useful in routinely ensuring the quality level of performance of analyzer 10. It is apparent from the overlap of the absorption curves that a single wavelength coherent radiation source, for example the second harmonic output of a Nd:YAG laser having a wavelength of about 532 nm may be employed to advantageously obtain data for the quantity of analyte in a test sample and to confirm proper operation and calibration using a control solution when CPR and C0SO₄ are selected to demonstrate the operation of analyzer 10.

It is important to note that these are the only functions required by an automatic clinical analyzer in order to have commercial viability; i. e., the ability to measure an amount of a chromophore indicative of the amount of an analyte of interest and the ability to measure a chemical control indicative of the operational integrity of the analyzer. It is well known that analyte detection schemes use chromophores other than CPR; however, in the inventive concept disclosed here, CPR is illustrative only and is not intended to be limiting. The basis of the present invention is the demonstrated ability of a multi-pass coherent radiation detection scheme to detect the amount of a chemical species which is related to the amount of an analyte present in a sample. The present invention is therefore applicable in any instance that a chemical species which is related to the amount of an analyte present in a sample is known to absorb radiation; the selection of an appropriate radiation wavelength being well within the skill of an artesian so that a multi-pass coherent radiation analyzer like analyzer 10 may be straightforwardly designed to detect a variety of analytes.

FIG. 6 is a plot of absorption data obtained for multiple radiation passes using analyzer 10 to detect a chromophore associated, for example, with any of the above assays. As expected, the absorbance increases in direct proportion to the number of passes through the cuvette 18 containing a test sample, in this instance a solution of CPR at a concentration of 1.33 x 10^"5 in 0.1 M Na₂HP0₄, pH = 8. Obviously, when analyzer 10 is designed to achieve a larger number of radiation passes, a greater amount of absorbance occurs within the test sample, thereby producing a larger signal variance that may be advantageously used to design assays of greater sensitivity. Alternately, such multiple pass coherent radiation detection schemes could be employed instead of conventional assays which require signal amplification methods like a Rabin cascade when detecting very low levels of analytes like Free T4 and thyroid-stimulating-hormone. Additionally, such multiple pass coherent radiation detection schemes could be employed when small sample volumes are desired to be tested.

FIG. 7 illustrates reflection corrected absorption data obtained in the instance that multipass analyzer 10 is designed for seven radiation passes through cuvette 18 and wherein reflections from the cuvette 18 are normalized so as to not affect accuracy of measurements. In order to achieve such seven pass measurements, the relative positions and/or dimensions of both the first prism 16 and the second prism 20 are modified, using for example, optical design software like that mentioned hereinbefore and as illustrated in FIG. 4, or ese the sample may be moved within the sample receiving area away from the interrogating radiation beam to vary the number of radiation beam passes through the sample, hence varying the effective pathlength without repositioning either of the two prisms as illustrtaed in Fig. 4c. The top curve (a) illustrates absorbance when reflections from the cuvette containing the test sample are neglected. In this instance, the incident radiation density was first measured without a cuvette 18 in the analyzer 10. Next, cuvette 18 containing a test sample was replaced between first prism 16 and second prism 20 and the transmitted radiation density was measured, thereby enabling absorbance to be determined. Measurements were made at wavelengths between about 570 nm and 600 nm by selecting various sources of laser radiation. The middle curve (b) illustrates the effects of reflections that take place at surfaces of cuvette 18 as radiation is passed through the cuvette 18. In this instance, the incident radiation density was first measured without a cuvette 18 in the analyzer 10. Next, an empty cuvette 18 not containing a test sample was placed between first prism 16 and second prism 20 and the transmitted and reflected radiation density was measured, thereby enabling absorbance to be determined. The bottom curve (c) illustrates the effects of reflective losses or the absorption measurements within the measured radiation density as a consequence of reflections from the cuvette containing the test sample. Curve (c) is obtained by subtracting curve (b) from curve (a). When making actual multi-pass radiation absorbance measurements, the effects of a "reflection- normalization" curve like curve (c) would be considered to improve the accuracy of such analysis. Obviously, reflection losses may also be minimized by employing well known optical coating techniques on the various optical elements used to demonstrate the present invention.

FIG. 8 is a plot of absorbance of CPR at 1.33 x 10^"6 M as a function of laser radiation over a smaller radiation range than in previous Figures and illustrates a further advantage of using multiple radiation passes as compared with a more conventional analytical scheme where a single absorption radiation pass is employed, even in the instance that the radiation is of a coherent nature. In the instance that the absorption of CPR is measured for a sample having concentration 1.33 x 10^"6 M, again using a 1 cm single pathlength, the signal-to-noise ratio obtained when 7 passes are made through the sample has been measured as 24.3. In contrast, the signal-to-noise ratio obtained when a single pass is made through the sample has been measured as 6.0. In order to conduct such single pass measurements, it is only necessary to replace the second prism 20 with the detector 24. However, as seen in FIG. 8, in the instance of multi-pass systems, the actual absorbance achieved with 7 passes is roughly 7 times greater than the absorbance achieved with a single pass, and the signal-to-noise ratio obtained when 7 passes are made through the sample is about 4 times greater than the signal-to-noise ratio obtained when a single pass is made through the sample. This improvement in signal-to-noise value is not as large as the improvement in absorbance because in practicing present invention, increasing the interragating radiaiton pathlength by a factor of, for example, 7 times, also increases the signal by a corresponding factor of about 7 times (ignoring reflection losses), however, the noise level is not correspondingly increased as noise originated from stray light, detector dark currents, etc.

In the prior art, it is known to use signal-to-noise enhancing techniques employing optical choppers and phase-sensitive amplification to increase the sensitivity of the analyzer 10 and to minimize the deleterious effects of external noise signals that may originate from room light, dark currents, etc. The use of standard signal-enhancing or noise reduction techniques like these are intended to be encompassed within the present invention without further explanation so as to improve the signal-to-noise ratio of analyzer 10.

It is to be understood th.it the embodiments of the invention disclosed herein are illustrative of the principles of the invention and that other modifications may be employed which are still within the scope of the invention. Accordingly, the present invention is not limited to those embodiments precisely shown and described in the specification but only by the following claims.

Claims

What is claimed is:

Claim 1. A method for measuring the presence of a chemical species in a liquid sample suspected of containing said species, the method comprising: propagating a collimated coherent radiation light beam having a known intensity and a known wavelength through the liquid sample a multiple number of passes; and detecting the intensity of the radiation beam after said beam makes said multiple passes through the liquid sample said number of times, wherein the wavelength of the radiation beam is selected so that the radiation light beam is at least partially absorbed by said chemical species.

Claim 2. The method of claim 1 for measuring the presence of a chemical species in a liquid sample wherein propagating the collimated coherent radiation light beam through the liquid sample comprises spacing a first prism and a second prism mutually apart to define an open sample receiving area therebetween, said first prism and second prisms being shaped and positioned so that the radiation light beam is reflected within and between said prisms to provide said multiple number of passes.

Claim 3. The method of claim 2 for measuring the presence of a chemical species in a liquid sample wherein the multiple number of passes is changed by moving the first or second prisms relative to one another.

Claim 4. The method of claim 2 for measuring the presence of a chemical species in a liquid sample wherein the multiple number of passes through the liquid sample is changed by moving the sample within the sample receiving area between the first and second prisms.

Claim 5. The method of claim 1 wherein the chemical species is a chromophore.

Claim 6. The method of claim 1 wherein the chemical species is a dye.

Claim 7. The method of claim 1 wherein a laser diode is used as a source for propagating the collimated coherent radiation light beam.

Claim 8. The method of claim 4 wherein the wavelength of the collimated coherent radiation beam is about 532 nanometers.

Claim 9. The method of Claim 2 wherein the chromophore is chlorophenyl red.

Claim 10. The method of claim 3 wherein the dye is cobalt sulfate.

Claim 11. The method of claim 1 wherein the radiation beam passes through the liquid sample between four and seven times.

Claim 12. The method of claim 1 wherein said liquid sample is from about 10 microliters to 30 microliters in volume.

Claim 13. A multi-pass coherent light chemical analyzer comprising: a source for producing a collimated coherent radiation beam propagating in a known direction; a first prism and a second prism spaced apart to define an open sample receiving area therebetweeen, said first prism and second prisms being shaped and positioned so that the propagating radiation beam is reflected within said prisms and thereby caused to make a multiple number of passes through the sample receiving area; a radiation detector positioned to intercept the radiation beam after said beam propagates a multiple number of passes through the sample receiving area; and, a liquid sample container disposed within said sample receiving area and containing a chemical species of interest, wherein the wavelength of the radiation beam is selected so that the radiation beam is at least partially partially absorbed by said chemical species.

Claim 14. The apparatus of claim 13 wherein the chemical species is a chromophore.

Claim 15. The apparatus of claim 13 wherein the chemical species is a dye.

Claim 16. The apparatus of claim 13 wherein a laser diode is used as a source for propagating the collimated coherent radiation beam.

Claim 17. The apparatus of claim 16 wherein the wavelength of the collimated coherent radiation beam is about 532 nanometers.

Claim 18. The apparatus of claim 13 wherein a photodetector is used in detecting the intensity of the radiation beam after the beam passes through the liquid sample.

Claim 19. The apparatus of claim 13 wherein the radiation beam passes through the liquid sample between four and seven times.

Claim 20. The apparatus of claim 13 wherein said liquid sample is from about 10 microliters to 30 microliters in volume.