US3661460A - Method and apparatus for optical analysis of the contents of a sheathed stream - Google Patents

Method and apparatus for optical analysis of the contents of a sheathed stream Download PDF

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US3661460A
US3661460A US67819A US3661460DA US3661460A US 3661460 A US3661460 A US 3661460A US 67819 A US67819 A US 67819A US 3661460D A US3661460D A US 3661460DA US 3661460 A US3661460 A US 3661460A
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stream
flow chamber
sample
sheath
light
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US67819A
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Alan H Elking
Warren Groner
Alex M Saunders
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Bayer Corp
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Technicon Instruments Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells

Definitions

  • ABSTRACT There is provided a method and apparatus for optical analysis by photometry of a substance flowing in a liquid stream within a coaxial sheath stream of a transparent liquid.
  • the sheath stream flowing in the same direction, entrains the inner stream so as to confine it concentrically.
  • a photometer is used which includes a light source on one side of the sheathed stream in a position to direct light onto the inner stream which is cylindrical, the outer or sheath stream also being cylindrical.
  • the photometer also includes a light detector externally of the sheathed stream in an angular position to detect the photometric results of impingement of light on the contents of the inner stream. Refraction of light at the interfaces of the sheath stream is compensated by varying the radius of the inner stream through the control of the flow of one stream with reference to the other.
  • the concept also includes the narrowing of the sheathed stream to a very small diameter in which it is confined by a wall structure in the area of examination.
  • the present invention effectively tends to obviate the aforementioned difficulties, particularly in dark field light-scattering photometric techniques.
  • Another object includes the narrowing of the sheathed stream to a very small diameter in which it may be confined by a wall structure in the area of examination, and the avoidance in this area of any more volume of the sheathed stream than necessary to carry the sample. This tends effectively to reduce any ambient fluid which might obscure the sample.
  • a further object is to provide equal and opposite refraction of light in photometric analysis of a cylindrical sheathed stream by varying the radius of the inner stream within limited but practical ranges through the control of the relative flow of the inner and outer streams.
  • a flow chamber having an elongated passageway portion provided with an outlet end and at the other end a tube inlet of substantially smaller diameter extending concentrically a distance into the passageway portion and terminating forwardly in a free tube end.
  • The'flow chamber further comprises a second inlet in the passageway portion at a location a distance rearwardly from the free tube end.
  • the flow cell further comprises a restriction in the aforementioned passageway portion intermediate the aforementioned outlet end thereof and the free tube end, which defines a round opening, the internal surfaces of the passageway portion of the tube being circular.
  • a photometer is provided downstream from the upstream extremity of the restriction of the passageway portion and includes a light source, on one side of the sheathed stream in a position to direct light'onto the sample stream, and a light detector externally of the sheathed stream in an angular position to detect the photometric results of impingement of light on the contents of the sample stream.
  • a further provision is made for controlling the relative flow of the streams which controls the radius of the sample stream, so that the last named radius may be varied and thereby compensate for refraction of light at the interfaces of the sheath stream.
  • FIG. 1 is a diagramatic view of apparatus for optical analysis by photometry including a flow chamber, embodying the invention
  • FIG. 2A is a view of the flow chamber of FIG. 1 illustrating it in longitudinal horizontal section;
  • FIG. 2B is a longitudinal elevational sectional view of the flow chamber
  • FIG. 3 is an enlarged sectional view taken on line 3-3 of FIG. 2B, illustrating the sheathed stream within the flow chamber;
  • FIG. 4 is a further enlarged, fragmentary view in section taken on line 4--4 of FIG. 2B, illustrating the sheathed stream within the flow chamber and indicating how refraction of light at the interfaces of the sheath stream is compensated;
  • FIG. 5 is a view similar to FIG. 2A illustrating in vertical section a modified form of the flow chamber.
  • FIG. 1 there is shown the principal elements of apparatus for optical analysis by photometry of a substance flowing in a liquid stream within a coaxial sheath stream of a transparent liquid, the illustration being by way of example only.
  • a supply of transparent liquid for the sheath stream the source being illustrated as a flask from which passes tube 11 emptying into an elevated vessel 12 for the sheath stream.
  • the vessel 12 has an overflow outlet including a tube 13 to carry off excess liquid and return it to the source 10 by gravity.
  • a pump 14 is provided to convey liquid from the source 10 to the vessel 12 which forms a constant-level reservoir for the sheath stream liquid.
  • the bottom of the vessel is provided with a liquid outlet connected to a gravity feed tube 15 connecting the vessel 12 with a lower flow chamber indicated generally at 16.
  • the flowcharnber is gen'erally'of elongated tubular shape and, in the illustrated form, the tube 15 is branched so that the flowchamber hasa pair of liquid inlets 17 for the sheath stream which are arranged opposite one another in the side-.
  • the tube 18 is ar- 3, 1 960 without causing undue pulsationsofthe sample within ranged concentrically 'with reference to the longitudinal passageway of theflow chamber, the tube 18 being of substantially smaller outer diameter than the which it extends.
  • v v v The other'end of the flow chamber 16 is a discharge end passageway portion into connected to a tube 19 discharging into a suitable receptacle which-is here shown as a stoppered flask 20. Vacuum tube 21 is connect'ed.
  • the flow chamber 16 is structured largely of a transparent material such as a suitable glass, and has associated with a transparent region thereof a photometer including a light source 25 at one sideof the flow chamber and at the opposite'sidea'light detector 26 so that light from the sourcepasses through the-contents'of the flow chamber to the light detector in the illustrated form.
  • the light detector might be arranged at right angles to the path of light impinged on the sheathed stream todetect fluoresence, for example, in the contents of the flow chamber 16.
  • the light emitted from the light source andimpinged on the stream to be analyzed may be reflected backin the direction of the light source for a distance before being diverted vto the light detector convenientlylocated to receive such diverted light rays, and such a technique is also possible according to the invention.
  • the illustrated arrange 'ment of the light source and light detector permits analysis of a sample by its light transmitting characteristics or its light absorbing characteristics
  • the sample contains particles in suspension to be counted by a forward scattering of lightby the particles, that is, toward the light detector in the position shown, as they pass substantially one
  • Thesample stream may be fed to the inlet tube 18 intermittently or continuously and be pulled or pushed for introduc-- tion into the flow chamber.
  • U.S. Pat. No. 2,879,14l issued and; also by. light-scattering desired the sheath; stream, like thesample stream, may be pushed or, pulled by a pump for introduction into the flow inlet forthe sheath streamf, V. v 7
  • theflow chamber may haveasingle inlet for the sheath stream, such as one concentric with .the tube 18.
  • lt is. necessary, here, that the sheath streamhave a sufficient distance of travel fromjts point of entry to the free forward end of 'the sample tube l 8-'to completely fill the passageway portion and establishsteady state laminar flow surrounding the tube in order to properly entrain the sample stream injectedthereinto from .the'tube 18 on its passage toward the discharge end of the flow-chamber connected to chamber inlets 17, which may be considered together as an the tube 19, as will more readily apparent fromthe discussion hereinafter of other views of the drawings. 1
  • the entrained sample stream is reduced in diameter after leaving the tube 18, and the radius of the sample stream may be variedby-controlling the relative flow of the sample and sheath streams as previ ously indicated.
  • a restrictive device such as a valve or the like in the sample stream inlet line due to the riskof clogging.
  • a needle valve 28 maybe interposedin the sheath stream tube 15 intermediate the vessell2 and the chamber inlet 17 tocontrol the inlet flowof the sheath stream'to the flow chamben-Aresistor may be used in place of the valve 28, ifv desired.
  • the chamber includes an elongated tubular body 30 of transparent glass material having diametrically opposite .side' arms in alignment with one another providing the inlets 17 for the sheath'stream, in which arms nipple fittings may be received, respectively, for connection to the flow chamber and are of approximately the: same internal after another in a stream intermediate the light source and the Mar. 24, 1959 discloses-(the disclosure'of which is incor-U porated herein by reference) a suitable automated sample supply apparatus, not shown, which has provision for holding a plurality of samples, which may be used.
  • the samples of a se- I ties in tubing may be separated from one another by a seg:
  • the longitudinal passageway portion 32 continues tothe rear end of the flow chamber and is closed by a plug 34 through the center of which, extends'the mid-portion of the sample stream inlet tube 18, so that the tube Isis concentrically supported in fluid-tight relation thereby in thepassageway portion 32.
  • the forward free end 36 of the tube 18 extends beyond the inlet arms 17 and terminates in the passagewayportion 32 whichis circular in cross section.
  • the cylindricaltube 18 which has an outer diameter substantially smaller than that Of the passageway portion 32 may have an inner diameter of approximately 0.0195 inch and be con- 7 stituted by hypodermic tubing or of a small-diameter tube structure of a non-corrosive metal. lts'fluid passageway is circular in cross section.
  • outer wall surfaces of the tubular body are thinned and flattened, as at 44, on opposite sides thereof in opposing relation to the light source and the light detector to minimize light refraction at the outer surface of the tubular body 30 and to permit the aforementioned optical elements 25, 26 to closely approach the fluid contents of the restricted passageway portion 42, as indicated in FIGS. 2A and 3.
  • This close working distance of the optical elements from the fluids contents may be approximately 0.010 inch, and permits use of a high numerical aperature condenser and objective.
  • the portion of the tubular body 30 of the flow chamber in the light path may be immersed in optical oil for a refractive index match.
  • the restriction 42 in the tubular body 30, which may be 0.010 inch, is illustrated as extending through the thickened discharge end 40 (FIG. 2A) of the body 30 received in one end of a sleeve 41 the other end of which receives a nipple fitting 43 for connection to the discharge tube 19 (FIG. 1) for discharge of the composite liquid stream from the flow chamber into the waste receptacle or trap 20 shown in FIG. 1.
  • the illustrated vacuum pump 22 connected through tube 21 to the receptacle 20 effectively discharges the flow chamber, and it has been found that the pump 22 may be run at varying vacuums without changing the radial dimension of the sample stream within the sheath stream in the examination area provided by the restricted passageway portion 42 of the flow chamber. 4
  • the diameter of the sample stream may be, in a particular instance, depending on the relative flow of the sample and sheath streams, approximately 0.003 inch. Also, by way of example, the sample stream may equal 9 per cent of the total flow. In such circumstances a total flow of the combined streams through the flow chamber of 5 ml per minute, or 3 millionths of a cubic foot per second, equal a Reynolds number of5 10.
  • the flow chamber in the modified form of the invention illustrated in FIG. 5, has an elongated block-like body 51 vertically arranged provided through its upper end with a pair of converging bores or sheath stream inlets 52 respectively receiving nipple fittings 54 for connection to the respective branches of a sheath stream supply tube similar to the tube previously described with reference to FIG. 1.
  • These inlets 52 converge, as shown, in a central, longitudinal bore or passageway portion 56 of the body 51, extending through the lower end thereof.
  • a central longitudinal bore intermediate the sheath stream inlets 52 which bore, communicating with the passageway portion 56, receives a sample stream inlet tube 58, similar to the sample stream inlet tube 18, previously described.
  • the tube 58 extends through a supporting plug 60, provided in an enlargement of the lastmentioned bore, and the tube 58 is concentrically arranged in the passageway portion 56, with its lower free end 62 terminating a distance upwardly from the lower end of the passageway portion 56, as illustrated in F IG. 5.
  • a restriction is formed in the lower discharge end of the passageway portion 56, the restriction in this instance being formed by a plug 64 inserted for support in the lower end of the passageway portion 56, the plug 64 having a central opening 66 defined by a funnel-like surface.
  • the sheathed stream is injected downwardly into the ambient atmosphere for examination by a photometer, illustrated as comprising a light source 68, closely located at one side of the stream, and the light detector 70, located closely to the stream on the diametrically opposite side thereof, for a light path therebetween.
  • a photometer illustrated as comprising a light source 68, closely located at one side of the stream, and the light detector 70, located closely to the stream on the diametrically opposite side thereof, for a light path therebetween.
  • a funnel 72 to catch the streams shortly after they cross the last mentioned light path and collect them for drainage into a suitable receptacle therebelow, not shown.
  • the sheath liquid supplied to the inlets 52 of the flow chamber 50 picks up, in the passageway portion 56, the sample stream injected into the passageway portion 56 through the tube 58, in a manner to entrain it and accelerate the sample stream, narrowing it as the streams flow toward the restriction formed in the passageway portion 56 by the plug 64, wherein they are narrowed proportionately, thereby both being accelerated, all in a manner similar to the operation of the flow chamber 16 previously described.
  • the sheathed stream is unconfined after it exits from the flow chamber 50 through the plug 64 and passes across the light path between the light source 68 and the light detector 70. This makes possible extremely short working distances from the sheathed stream to the optical elements 68, 70, with attendant apparent advantages.
  • the radius of the inner or sample stream may be varied, as in the use of the previously described flow chamber 16, by adjusting the relative flow of the sheath and sample streams. This control of the relative flow of the streams may be accomplished in the previously described manner.
  • Another advantage of the form of the invention shown in FIG. 5 over that shown in FIG. 1, is that by injecting the sheathed stream into the ambient atmosphere for examination, a significant pressure drop may be avoided in the area of examination.
  • the flow chamber 16 may be oriented in any position so that the optical elements associated with the flow chamber may accordingly be positioned, that is, vertically or horizontally. Ifthe flow chamber 16 is arranged vertically with the flow directed upward, it is obvious that any air bubbles which might be caught in the chamber will be swept to the upper end thereof and out of the viewing area. Still another advantage of the form of FIG. 1, is that the sheathed stream is confined in the examination area so as to prevent splattering of the optical elements associated with the flow chamber as by an aberration in the sheathed stream.
  • the external surface of a cylinder such as the sheath stream will refract light if the substance with which it is in contact has a different index of refraction, such as air or glass.
  • This refraction may be compensated within workable limits including the indexes of refraction of the outer substance such as air or glass, the sheath stream and the sample stream.
  • Compensation is achieved if rays 74 (FIG. 4) parallel to the optical axis 75 reach the center line of the sample stream in parallel relation to the axis 75 of light from the aforementioned light source 25.
  • This compensation may be obtained by determining and establishing the correct radius of the sample stream with reference to the particular operating conditions prevailing at the time including the last-mentioned indexes of refraction.
  • the radial dimension of the sample stream must be such as to bring about equal and opposite refraction at the two interfaces of the sheath stream.
  • the index of refraction N, of the sample stream must be greater than the index of refraction N, of the sheath stream to obtain compensation of refraction at the interfaces of the sheath stream.
  • the sheath stream in the area of examination is unconfined
  • N 1.51; N, is l .36; N is approximately 1.38; and r, the radius of the sheath stream, is 0.005 inch: in solving for r, the radius of the sample stream, the following equation is used:
  • the sample stream is constituted by human. blood diluted with propylene glycol and in which the red cells are hemolyzed.
  • the red cell ghosts have the same index of refraction as the diluent so that the-red cells are rendered invisible, for the counting by photometric means, as aforesaid, of the white cells.
  • the photometer has a conventional ocular, not shown, to observe, with the human eye, the flow of the stream in the flow chamber so that it may be determined whether or not refractive errors have been compensated.
  • a conventional target, not shown, of the optical elements, projected through the flow chamber is brought into sharp focus at the particular sample stream radius which achieves compensation of light refraction as indicated above.
  • the photometric viewing area should be larger than the diameter of the sample team.
  • Apparatus for optical analysis of a sample stream including a liquid in a coaxial sheath stream liquid comprising: means defining a flow chamber. having an elongated passageway portion having a forward outlet end and at a rear end of said portion a tube inlet of substantially smaller diame ter than said passageway portion extending concentrically a distance forwardly into said passageway portion and terminating forwardly in a free tube end, the last-named means defining a second inlet in said-passageway portion at a location a distance rearwardly from said free tube end, and the lastnamed means also defining a restriction with a narrow sloping approach in said passageway portion intermediate said outlet end thereof and said free tube end, the internal andexternal surfaces of said free tube end and the internal surface of said passageway portion being circular in cross section, means for flowing a sample stream through said'tube inlet into said passageway portion, means for flowing a sheath stream into said second passageway inlet at a greater flow rate than the sample stream to en
  • said means controlling the relative flow rates of said streams comprises a device operative to restrict the flow of the sheath stream into the flow chamber.
  • said means for flowing the sheath stream into said flow chamber comprises a constant-level reservoir establishing a head of sheath stream liquid.
  • said means for flowing the sample stream into said tube inlet of the flow chamber comprises a pump upstream of the tube inlet.
  • said means controlling the relative flow rates of the streams comprises a valve operative to vary the flow of the sheath stream into the flow chamber.
  • said means for flowing the sample stream into said sample tube inlet of the flow chamber comprises a pump upstream of the tube inlet, said outlet end of the flow chamberbeing connected to a liquid trap throughan outlet conduit, and further comprising a vacuum pump 'operatively connected to the last-named outlet conduit.
  • V 8 Apparatus as defined in claim 1, wherein: the flow chamber is vertically arranged.
  • said means defining a flow chamber comprises a tubular body having said restriction of the passageway portion therein.
  • Apparatus as defined in claim .1, whereimsaid means defining a flow chamber comprises a tubular body having said restriction of the passageway portion therein, said restriction enclosing the sheathed stream where light from said source. is impinged on the sample stream.
  • a method for optical analysis of a sample stream including a liquid in a coaxial sheath stream liquid comprising: providing means defining an elongated passageway portion of circular cross section in a flow chamber which portion has at one end thereof a circular restriction; flowing a first laminar liquid stream into said passageway portion in a direction toward said restriction; concurrently flowing in the same direction a slower moving liquid stream of cylindrical cross section and of a substantially smaller diameter than said first stream, containing a sample, into the first stream concentrically so that the latter sheaths and entrains the second stream, accelerating and narrowing it as the streams approach said restriction in a concentric relation to one another; directing an impinging light beam of a photometer, in a direction transversely of the aforementioned direction of flow, onto the samplecontaining stream for photometric analysis of the sample by a light detector of the photometer, downstream from the upstream extremity of said restriction; and controlling the relative flow rates of the streams into said passageway portion, which controls the radius of the second or sample stream, so that
  • step of controlling the relative flow of the streams into said flow chamber comprises controlling said sheath stream.
  • the step of flowing the sheath stream into the flow chamber comprises flowing the sheath stream from a constant-pressure source.

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  • Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
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  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
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US67819A 1970-08-28 1970-08-28 Method and apparatus for optical analysis of the contents of a sheathed stream Expired - Lifetime US3661460A (en)

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AU3143671A (en) 1973-01-25
DE2142237A1 (de) 1972-03-02
NL7111164A (da) 1972-03-01
GB1321917A (en) 1973-07-04
FR2106060A5 (da) 1972-04-28
AU445066B2 (en) 1974-02-14
BE770541A (fr) 1972-01-27
DE2142237B2 (de) 1974-01-10
DE2142237C3 (de) 1974-08-15
CA936017A (en) 1973-10-30
CH552807A (de) 1974-08-15

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