Multistation analytical photometer and method of use

BACKGROUND OF THE INVENTION

The invention described herein relates generally to photometers and more particularly to an analytical photometer for simultaneously determining the presence of a common substance in a multiplicity of samples wherein precipitates are removed from the samples prior to their 40 transfer to respective cuvettes. It was made in the course of, or under, a contract with the U.S. Atomic Energy Commission.

An analytical photometer for simultaneously determining the presence of a common substance in a multi- 45 plicity of discrete samples is described in copending application of common assignee and applicant Ser. No. 784,739, now Pat. No. 3,555,284. In that case a multiplicity of sample chambers or cuvettes were arranged within a centrifuge rotor to provide a rotary cuvette sys- .-9 tern. A central transfer disc facilitated the mixing and transfer of sample liquids and reactants directly to the cuvettes.

In some reactions of interest in the biochemical field the sample fluids and reactants form precipitates upon 55 mixing. Such precipitates interfere with the photometric measurement when transferred to a cuvette system. In the system described in Pat. No. 3,555,284 no means are available to prevent such transfer of precipitates and precipitating reactions cannot be properly accommodated. go

Protein-free filtrates are required for a variety of purposes including the estimation of acid-soluble nucleotides, free amino acids, and for a number of clinical measurements including blood sugar. The precipitant employed and the ratio between sample and reagent may vary widely gg in such tests and measurements. A sensitive test for complete precipitation is the absorbance of the supernatant in the ultraviolet. Incompletely sedimented protein contributes both absorption and scatter rendering such test inaccurate. 70

It is, accordingly, a general object of the invention to provide an analytical photometer capable of removing precipitates from samples prior to their transfer to respective cuvettes.

Another object of the invention is to provide a centrifuge rotor wherein sample liquids and reagents which form precipitates may be mixed and centrifuged in a single system to provide a precipitate-free supernatant.

Other objects of the invention will be apparent from an examination of the following description of the invention and the appended drawings.

SUMMARY OF THE INVENTION

In accordance with the invention an analytical photometer is provided wherein precipitates may be removed from a multiplicity of discrete samples by centrifugation prior to transfer of the samples to cuvettes in a rotary cuvette system. A central transfer disc is provided with a first series of chambers which separately retain precipitating solutions while at rest, and release the solutions to respective sedimentation chambers upon rotation. A third series of chambers receives the supernatant from respective sedimentation chambers by gravity flow when the transfer disc and cuvette system are brought to rest. The supernatant may then be transferred centrifugally to respective cuvettes in the rotary cuvette system surrounding the transfer disc by rotating the disc and cuvette system. Such arrangement facilitates the preparation and photometric measurement of precipitate-free supernatants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates a transfer disc and photometric system designed in accordance with the present invention.

FIGS. 2 through 6 illustrate the stepwise operation of the transfer disc shown in FIG. 1 in mixing, sedimenting and transferring liquid solutions.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 schematically illustrates an analyzer incorporating a transfer disc made in accordance with the invention. A pancake-shaped rotor assembly 1 comprises a bolt-flanged steel rotor body 2, glass rings 3 and 4, a slotted polytetrafluorethylene cuvette ring 5, polytetrafluorethylene retaining rings 6 and 7, and a steel bolted flange ring 8. Rings 3, 4, 5, 6 and 7 are compressed between rotor body 2 and flange ring 8 to form a multiplicity of radially' oriented cuvettes 9 in slotted cuvette ring 5. Spaced holes 10, axially aligned with cuvettes 9, are provided in rotor body 2, retaining rings 6 and 7, and flange ring 8 so as to provide axially extending passageways permitting passage of a light beam through the cuvettes. A two-piece, centrally positioned removable transfer disc 11 is provided with sets of chambers 12 for receiving sample liquids and reactants while the rotor is at rest. Chambers 12 comprise a plurality (only two shown) of sloping cylindrical cavities which are interconnected at their upper ends and separated by partitions 13 at their lower ends. Partitions 13 prevent mixing of the sample and reactant liquids when the rotor is at rest while permitting such liquids to pass to sedimentation chambers 14 when the rotor is spinning. An inwardly and downwardly extending passageway 15 leads from the bottom of each sedimentation chamber 14 to the top of a holding chamber 16 disposed below each sedimentation chamber. An upwardly and outwardly extending passageway 17 leads from the base of each holding chamber 16 to the radially innermost part of a cuvette 9. A removable transparent Lucite cover plate 18 is provided to minimize evaporation and prevent windage from deflecting liquids during centrifugal transfer. A conventional centrifuge drive motor 19 supports the rotor assembly 1 while rotating it.

A photometer light source and projecting means are

3,586,484 provided to project a light beam of constant intensity intersecting rotor assembly 1 at a point corresponding to the radial positions of cuvettes 9 and spaced holes 10'. The light beam, indicated by a broken line in FIG. 1, is aligned in such a manner so as to be transmitted through each hole 10 and cuvette 9 as they pass through the beam. The photometric light source comprises an incandescent lamp 20 with a reflecting mirror 21 disposed below the rotor assembly and oriented to reflect the light beam upward, substantially normal to the plane of rotation. Electronic photodetecting means 22 is disposed above rotor assembly 1 and aligned to receive light transmitted through the cuvettes during rotation. Photodetecting means 22 is designed to respond electronically with an output which is proportional to the intensity of the light transmitted from light source 20 through the cuvettes. Photodetector 22 comprises a photomultiplier tube disposed directly above the cuvette circle to receive all light transmitted upwardly through the axially aligned openings.

The remaining electronic components illustrated schematically in FIG. 1 include a proportional tachometer 23 which supplies a voltage signal proportional to the rotor speed to a ramp signal generator 24 which, in turn, provides a signal to a pulse scanner 25. A revolution detector 26 synchronizes the ramp signal frequency with the rotor speed. Pulse scanning means 25, synchronizable by the ramp signal generator frequency, responds proportionally to pulses originating in photodetecting means 22 and sorts the pulses therefrom as to origin. Pulse peak readout means 27 continuously and simultaneously indicates phototransmittance of the liquid contents in each cuvette. The design and operation of electronic components 23 through 27 are described in greater detail in corresponding U.S. patent application Ser. No. 784,719, now U.S. Pat. No. 3,514,613 of common assignee.

Operation of the invention may best be understood by reference to FIGS. 2 through 6 where a sectional view of transfer dies 11 is shown repeatedly to illustrate successive steps in a typical operation. As shown in FIG. 2, a particulate suspension or liquid solutions which when mixed form a precipitate are placed in chambers 12 while the transfer disc 11 is at rest. The transfer disc 11 is slowly accelerated to cause the liquids in chambers 12 to flow outward into sedimentation chambers 14 as shown in FIG. 3 where they mix and the precipitating reaction takes place. Rotation is continued at high speed until the particulate matter 30 separates and settles in the radially outermost part of sedimentation chamber 14. The precipitate-free supernatant 31, being of lesser density than the precipitate, remains on top of the precipitate as shown in FIG. 4. The transfer disc is then allowed to come to rest and the supernatant liquid 31 flows downwardly and inwardly through passageways 15 into respective holding chambers 16 as shown in FIG. 5. Centrifugal force prevents earlier passage of the supernatant through passageways 15, but as the transfer disc is brought to rest gravitational force predominates to cause the supernatant to flow through the downwardly extending passageways. As shown in FIG. 6, subsequent transfer of the supernatant from holding chambers 16 is accomplished by further rotation which causes the supernatant to flow outward through passageways 17. Although the sedimentation chambers 14 shown in the schematic views of FIGS. 1 through 6 are specifically adapted to retain precipitates which form firm adherent pellets upon centrifugation, they may be modified to include a further outwardly extending depression of narrow diameter to contain precipates which do not form such firm adherent pellets.

Example

One chamber in each of ten sets of chambers, corresponding to chambers 12 in FIG. 2, were filled with 200 μ1. of partially hemolyzed human plasma containing sufficient hemoglobin to give a bright color allowing detection of incomplete precipitation. The remaining ten chambers in the ten sets of chambers were filled with 200 /4. volumes of 0.4 M HC1O₄. The transfer disc was accelerated and decelerated rapidly to facilitate mixing and then spun for 15 minutes at approximately 6850 r.p.m. The transfer disc was then stopped to permit drainage of the supernatant into the holding chambers as described in reference to FIG. 5. From each holding chamber a 200 gl. sample was withdrawn, diluted with 5 ml. of distilled water and read against distilled water at 260 nm. in a one-cm. light path cell. In a series of ten samples spun in the transfer disc assembly, the absorbance measured was 0.186 with a standard deviation of 0.004 absorbance units. This provided a severe test of the system since the concentration of plasma in precipitating mixtures is usually much lower.

An additional series of experiments was run using alternate samples of dye and distilled water to check whether cross contamination occurred between adjacent sets of chambers. The series of steps described in reference to FIGS. 2 through 6 was performed to transfer the dye solutions and distilled water from chambers 12 to chambers 16. The contents of chambers 16 were examined and no trace of cross contamination found.

The principle of operation of the transfer disc described herein is also useful in instances where no precipitate is involved but where two or more solutions are to be mixed together, incubated for a long interval, and subsequently transferred to a cuvette rotor to be read. The transfer disc 11 described herein could also be used in combination with the system for measuring and holding samples described in copending application of common applicant and assignee Ser. No. 806,920.

The above description of one embodiment of the invention is offered for illustrative purposes only and should not be interpreted in a limiting sense. For example, the transfer dies 11 may be used alone or in conjunction with a rotary cuvette system as shown. When used alone, the transfer disc would more properly be referred to. as a rotor. The designation “transfer disc” is used herein inasmuch as its principal use as envisioned by applicant is in conjunction with rotary cuvette systems where it is used to transfer liquids to individual cuvettes. It is intended rather that the invention be limited only by the scope of the appended claims.