DOUBLE BEAM PHOTOMETER FOR MEASURING FLUID SAMPLES

Abstract: A double beam photometric flow cell (12) for high pressure liquid chromatography having illuminated sample and reference passageways (26 and 28) therein, the sample passageway (26) receiving flowing fluid sample fractions for analysis. Regulating apertures (30 and 36) control the relative amount of light passed through the passageways (26 and 28) such that the light energy through the reference passageway (28) exceeds that through the sample passageway (26). This reduces the contribution of the reference path (28) to 'shot noise' in the photometer output signal.

Description: DOUBLE BEAM PHOTOMETER FOR MEASURING FLUID SAMPLES Background of the Invention 1. Field of the Invention The present invention relates generally. to measurement of fluid samples and, more particularly, to optical measurement of such samples in a photometric flow cell. The invention is particularly suited for use in a liquid chromatographic system where a stream of successive fluid sample fractions " eluted from a chromatographic""column is flowed through -a photometric flow cell for analysis of individual sample fractions. 2. Description of the Prior Art Double beam photometers have long been used in fluid flow systems to optically measure fluid sam¬ ples. In one common form, such photometers comprise a flow cell body having a pair of light transmissive passageways — a sample passageway configured to re¬ ceive a fluid sample ahd a reference passageway con- figured to receive a reference fluid or no fluid at all. Light from a source is split into a sample beam and a reference beam and passed through the correspond¬ ing sample and reference passageways and hence through any sample or reference fluid therein. A light detec- tor intercepts light emerging from the respective passageways to derive a ratio measurement of sample and reference beam energy and hence a measure of a charac¬ teristic (usually absorbance) of the sample. A princi¬ pal advantage of a double- beam system is its inherent ability to reject common mode noise effects in the two paths, and hence to generate an output signal exhibit¬ ing a high common mode rejection ratio. In high pressure liquid chromatography (EPLC), double beam flow cell photometers have been adapted to receive and optically measure a stream of successive fluid-saasple fractions eluted from a chrσma- tographic column. The sample fractions (carried in a solvent matrix) are [typically minute in volume (e.g. 100 μl-1.0 ml), closely spaced, low in concentration, and are flowed at high ressur up to as high as about • 40 atmospheres in the flow cell. In designing 4α»ble beam flow cells for HPLC applications, coήfigαra iQns are desired which measure sample fractions with maxiπrnn resolution and sensiti- vity. Resolution ,i&. an indication of the system's ability to distinguish-* measurements of successive, closely spaced flowing samples, i.e. to distinguish the "peaks '11 generated in th photometer output optical sig¬ nal representative of such samples. Sensitivity is an indication of the minimum concentration or amount of sample which must be present to be distinguished--as- • - sample from the baseline,noise of the photometer output signal. In these respects a common- prior flow cell " and reference passage- ways identical in size and shape. Apparently, identi¬ cal sample and reference passageways were felt to.be desirable to provide a symmetry or balance in the optics geometry of the ' flow cell to establish adequate common mode rejection and resolution for the system. As prior ' flow -cell designs have been per¬ fected, so-called "shot* noise in the photometer output signal has become a more significant factor. Shot noise is a high and law frequency noise signal super¬ imposed on the output optical baseline signal of the photometer which -is ^believed -to represent statistical variations in light beam intensity. The effect of the shot noise signal is to* decrease the sensitivity of the " photometer. - By decreasing, system sensitivity, shot noise impairs the system 1 s- ~ ability to measure samples of low concentrations. .Inre fect, then, the " perfor¬ mance of the prior. . flow cells, though improved, has become limited by shot noise. One possible approach for contending with shot noise is to increase the light energy level in the sample passageway by increasing the passageway diameter. While such effects a shot noise reduction, it is at the expense of a substantial de¬ crease in system resolution. Accordingly, a need exists -for a double beam photometer for receiving and measuring fluid samples without the drawbacks of the prior art. The present invention meets these needs. Summary of the Invention * The present invention resides in an improved double beam photometer for measuring fluid samples in a manner which overcomes the drawbacks of the prior art. The improved photometer exhibits high sensitivity to¬ gether with high resolution and high common mode rejec¬ tion. The improved double beam arrangement is achieved in a commercially practical form which is simple and inexpensive in construction and reliable in operation. To the foregoing ends, the present invention is embodied in a photometer of the type comprising: (1) a light source, (2) means defining first and second light trans issive pat s configured to receive light from the source and to pass the light therealong and through a fluid sample in at least one of the paths, and (3) means for measuring the relative intensity of the first and second beams upon emergence from the beam paths to derive a measure of the sample. Applicant has discovered that increasing the relative energy of light in the reference beam path to exceed that of the sample beam path reduces shot noise in the photometer output signal, thereby increasing system sensitivity, while maintaining the desired optical resolution and common mode rejection ratio of the photometer. In a preferred embodiment, the improved design is achieved by regulat¬ ing means comprising limiting apertures for light entering each optical path with the limiting aperture for the reference beam path being larger than that for the sample beam path- In the preferred embodiment, the sample and reference beam paths are through corresponding sample and reference passageways of a flow cell body. The regulating means comprises.limiting apertures for light entering each passageway, the limiting aperture for the reference passageway being larger than that for the sample passageway. The invention, further contemplates means cooperating with the light source to define a given area of the source from which light is passed to the sample and reference 'beam paths. Such an arrangement reduces parallax problems which can occur when the- areas of the source viewed by sample and reference are not coincident, and which in turn lead to poor common mode rejection if the source has spatial variations in intensity. Brief Description of the Drawing The figure is an optical diagram of a double beam photometer of the present invention as incorpor¬ ated in an EPLC s stem and illustrates in vertical cross section the flow cell of the photometer. Li it- ing rays of optical beam paths, of the photometer are illustrated in phantom outline. Description of the ^Preferred Embodiment As shown in the .drawing, the present inven¬ tion is embodied in a double beam photometer 10 com- prising a double beam flow cell 12 disposed in an opti¬ cal path between a light source 14 and an optical detector 16. The flow -cell is adapted to receive liquid sample fractions flowed under pressure from a liquid chromatograph column 18 through a supply conduit 20 and to discharge -the saaple after analysis through a discharge conduit 22. . discharge conduit is con- nected to a back pressure regulator (not shown) for establishing a requisite pressure in the flow path from column 18 through the flow cell 12. Typically, the discharge conduit is connected to a suitable waste receptacle or sump into which the fluid is flowed. The flow cell 12 comprises a generally cylin¬ drical body 24. A pair of generally parallel sample and reference passageways 26 and 28, respectively, extend between the ends of the body 24. Supply conduit 20 opens into one end of sample passageway 26 while discharge conduit 22 opens into the opposite end of the same passageway. With such an arrangement, sample fractions eluted from column- 18 are flowed under pres¬ sure through supply conduit 20 into sample passageway 26, through the sample passageway, and out discharge conduit 22 to waste. Reference passageway 28 may include similar supply and discharge conduits (not shown) at its opposite ends for flowing reference fluid into and out of the reference passageway. In- flow cell 12, however, such conduits are omitted since the flow cell is primarily employed for sample analysis without the presence of a reference fluid in the reference pas¬ sageway. The walls of sample passageway 26 are tapered conically outwardly in the direction of fluid flow from supply conduit 20 to discharge conduit 22. The walls thus define a opening or aperture 30 at the frustum of the conical passageway and a relatively larger opening or aperture 32 at the base of the cone. The reference passageway 28 is of generally cylindrical configuration. Body 24 forms an annulus 34 at the left end of the passageway which defines an opening or aperture 36 at such end. A" relatively larger opening or aperture 38 is defined by the cylin- drical wall at the opposite end of the reference pas¬ sageway. The left .end openings 30 and 36 of the re¬ spective sample and reference passageways are closed by a light transmissive window 40 sealed to the end of the flow cell body 24.. -Window 40 is also configured as a collimating lens for.light entering the sample and ref¬ erence passageways. The " opposite end of the sample passageway 26 is closed by a second light transmissive window 42. A clamp"(not: shown) retains windows 40 and 42 in place on the flow cell body 24 in a fluid-tight seal against the body. In the preferred embodiment of flow cell 12, the opposite or right hand end of the reference passageway 28 is open leaving the reference passageway exposed to the atmosphere. However, in the event the reference passageway were adapted to receive a reference fluid,, then*a.£urther window or an extension of window 42 would be "' providedr sealed to the right face of body 24 for closing this end. of the reference pas¬ sageway. ' - Light source 14 is a gas * discharge lamp having a generally U-shaped zone 44 of light emission at a selected wavelength. A mask 46 having an aperture 48 is positioned adjacent the lamp to define a given area 50 of the source emitting zone 44 from which light is passed by aperture*48 toward the flow cell 12. The light passed by aperture " 48 is received and collimated by lens/window 40 and passed thereby as first and second light beams along paths through respective sample and reference passageways 26 and 28. Light emerging from the flow cell passageways 26 and 28 impinges on detectors 16a. nd 16b, respectively, which, in turn, generate output current signals indicative of the light energy striking each detector. Optical analysis of the -flowing sample frac¬ tions is performed in.,a conventional manner by easur- ing an optical characteristic of each sample fraction (e.g. absorbance) at "the ^selected wavelength as the sample flows through passageway 26. In this regard, the output current signals of detectors 16a and 16b, representative of the energy of the sample and refer¬ ence beams, respectively, exiting passageways 26 and 28, are processed to compare (as by ratioing) the sample and reference beam energies to derive a measure of the sample alone. It will be noted that the sample and refer¬ ence passageways 26 and 28 are configured so as to pass the respective sample and reference light beams there¬ through without the light striking the * interior walls of the passageways. Such minimizes noise caused by light deflecting from the passageway walls toward the detectors. The conically tapered wall of the sample passageway 26 contributes to such a result. Thus, even if a stream of sample fractions exhibiting changes in refractive index is flowed through passageway 26, such refractive index variations do ' not cause spurious deflection of radiation from the passage walls or corresponding output signal noise. In reference pas¬ sageway 28, the entrance aperture 36 and the compara¬ tively larger exit aperture 38 cooperate to accommodate passage of a generally^ conical beam through the pas¬ sageway without the beam striking the passageway walls. As previously mentioned, a primary aspect or feature of the present invention is the significant reduction in shot noise superimposed on the optical baseline output signal of the photometer. In that regard, it has been discovered " that such significant reduction is achieved by regulating the relative light energy admitted through the reference and sample pas¬ sageways such that the light energy in the reference beam exceeds that of the sample beam. Moreover, such noise reduction is achieved while minimizing noise due to refractive index variations and while maintaining high optical resolution and a high common mode rejec¬ tion ratio. To the fo e9θin^.^ea s and in accordance with the invention, the ÷relat±-we. diameters of entrance apertures 36 and 30, g6spect_£vely, to the reference and sample passageways 28 controlled such that aperture 36 is larger?in d±aiaeter than aperture 30. The. apertures thus . Λβrve as -.means for regulating the relative energy of ϋght. in.--the beam paths such that more light energy is .admifcted into the reference pas¬ sageway 28 and passed to corresponding reference detec- tor 16b compared to the light energy reaching detector 16a through the sample passageway 26. In the preferred embodiment the diameter of- reference passageway aper¬ ture 36 is approximately 1-4 times greater than the diameter of sample passageway aperture 30. The significance of the increase in reference beam light, energy with respect to the sample beam energy is illustrated as follows. Statistical fluctua¬ tions in the output:- m Lent -of ; a photodetector such as 16a or 16b is represented by A o x o •• . where I Q equals the average d.c. output current of the photodetector and k equals -a constant independent of I 0 . When the output signal of the overall detector 16 is the ratio of the^ou ut cnrrents of the sample and reference beams, the total fluctuation in output signal of detector 16 is represented-by: where I equals the output current of sample beam de- tector 16a, I equals--the"output current of the refer¬ ence beam detector Iffl?, and R equals.the ratio of the output currents. Since the magnitude of these statis- tical fluctuations in the detector output is inversely - proportional to the square root of the output current, it appears advantageous to maximize the light energy in both sample and reference beams. However, the light 5 energy of the sample beam is limited by the sample passageway volume, which is limited by the volume of the sample measured, which is in turn limited by the resolution requirement of the photometer. Signifi¬ cantly, however, applicant has recognized that no such 10 limitation of light energy is required for the refer¬ ence beam. With respect to the foregoing, assume, for • example, that l r equals ' l s . In such case it can be shown by the foregoing equations that the total output 15 shot noise is about ' 41% greater than that., for the sample beam alone. -However,- if I is twice that- of- I , then the total output shot noise is only 22% greater than that of the sample beam alone. Moreover, if. I is - four times greater ' than I , then the total shot noise 20 is only 12% greater than that for the sample beam alone, and so on. Therefore, the result of increasing light energy in the reference beam relative to that in the sample beam is to correspondingly ^ decrease-the-opti¬ cal baseline shot noise in the detector output signal -, 25 without sacrificing the superior common mode rejection ratio or high sensitivity of the double beam system. In effect, increasing the energy in the reference beam relative to that of the sample beam substantially reduces the contribution of the reference beam to the 30 output shot noise level. In dimensioning the photometer 10 in accor¬ dance with the invention, aperture 48 is placed as close to light source 14 as feasible to define the given .source area 50 for viewing by both sanple and : 35 reference passageways. This reduces parallax problems which can occur when the areas of the source viewed by the sample and reference .passageways are not coincident and which, in turn, lead "to poor common mode rejection if the source has spatial variations in intensity. The beam dispersion angle 8--is- selected so as to minimize the interior volume...of flow cell sample passageway 26 to ensure adequate sam le-,resolution while assuring an adequate light throughput for minimum optical baseline noise. In this regard θ .is defined as 2 tan d, where f is the focal length of lens 40 at the wave¬ length of use (here 254 nm) and d is the diameter of • aperture 48. • ■ Once a* is--fixed, the size f aperture 48 is adjusted to view as.large an emitting zone 50 of the source 14 as feasibly which, completely fills the aper- ture with light. When this maximum dimension of aper¬ ture 48 is attained * then f will be a maximum fixed by the maximum aperture dimension and by θ. Maximizing f tends to minimize the parallax problem. While the nature and type of optical compon- ents in the foregoing system will vary depending upon the application and-type, of samples to be measured, one successfully operated system incorporated in a liquid chromatograph was constructed in accordance with the following specifications- Source 14 is a mercury lamp which emits at 254,n . -Aperture 48 of mask 46 is 0.125 inch in diameter. Lens -40 r has a focal length of 2.0 inches, an operating focal length at 254 nm of f = 1.787 inches and is--spaced from mask 46 by this dis- -tance f. Entrance opening 30 of sample passageway 26 is 0.05 inches in diameter while, in accordance with the present invention, entrance opening 36 of the reference passageway is 0.07 inches in diameter. Both passageways have a path .length through body 24 of 10 mm. θ or the beam dispersion in flow cell 12 is 3.96°. Detector 16a and 16b have =5 mm diameter light sensitive areas spaced 7 mm cen£er-fco-center. The distance from the rear or right end of flow cell 12 to the detectors is 10 mm. While photometer 10 has been illustrated with sample passageway 26 conically tapered and reference passageway 28 cylindrical, it will be understood that both could be conical or both could be cylindrical. Preferably conical beam paths through the passageways 26 and 28 are defined by the entrance and exit openings thereof. In another form, the passageways 26 and 28, instead of being generally parallel, are disposed at an angle such that their axes when " extended rearwardly toward source 14- meet or converge -so as" to view a limited or focal area of the source. In this form, the collimating function of lens/window 40 is unnecessary. While a preferred embodiment of the invention has been illustrated and described, it will be apparent that modifications may be made therein without de¬ parting from the invention defined in the appended claims.

Claims: What is claimed is: 1. A photometer for measuring a fluid sam¬ ple comprising: a light source; a body having first and second light: trans¬ missive passageways .configured to receive light from the source and to pass the light along first and second light beam paths through the corresponding first and second passageways,- at .least the first of the pas- sageways configured to receive fluid samples in the corresponding first.light-beam path; means for .measuring th relative intensity of the first and second light beams upon emergence from the first and second passageways " to measure the effect of a sample on the first light beam; and means for regulating the relative energy—of * light in the beam paths such that the light energy of the second light beam exceeds that, of the first light beam. 2, The photometer of claim 1 including: means cooperating with the light source to define a given area, of the source from which light is passed to the first and, second passageways. 3. The photometer of claim 1 or 2 wherein: _ the regulating means comprises limiting aper¬ tures for light entering each passageway, the limiting aperture for the second passageway being larger than that for the first passageway. 4. The photometer of claim 1 "further in- eluding conduit means.for connecting the first.passage- way to a chromatographic column for receiving fluid sample fractions flowedJfrα the column for analysis. 5. A photometer of claim 4 wherein the sec ¬ ond passageway is void of fluid. 6. The photometer for measuring a fluid sample comprising: a light source; means defining first and second light beam paths configured to receive light from the source and to pass the light therealong and through a fluid sample in a first of the paths; means for measuring the relative intensity of the first and second beams upon^emergence from the beam paths to measure the effect of the sample on the first light beam; and means for regulating the relative energy of light in the beam paths such that " the " light " energy " of " ~ the second light beam exceeds that of the first light beam.

Inventors: BERICK ALAN C
Applicants: BECKMAN INSTRUMENTS INC
Application year: 1982
Main classification: G01N21
IPC codes: G01N21, G01N30