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The invention relates to apparatus for measuring the optical
characteristics of transparent or semitransparent substances in liquid or
semiliquid form and in particular, to such apparatus which may make
measurements in the field of high pressure, liquid chromatography, in
which separations are accomplished with the utilization of columns.
Basic separations in liquid chromatography are produced by injecting the
samples to be analyzed into a liquid stream above a separating column
consisting of a tube-like container filled with adsorbents. The separation
of these samples under investigation occurs due to capillary action and
other phenomena well known in the modern practice of chromatography.
Liquids, solvents, or buffers carry the samples through the column, and as
media elutes from the exit of the column, the appearing solvents or
buffers contain fractional components of materials of the originally
injected substances. These fractions are optically detectable if their
flow is directed through a flow-through cuvette, having an optical
entrance and exit window which permits passage of a light beam. This light
beam will change intensity when it is intercepted by passing fractions.
Passing fractions, due to their characteristics, are indicated more or
less by different spectral frequencies as is well known in the art of
spectrophotometry.
In liquid chromatography fractions carried by solvent flow are, however, in
the main very minute and exhibit only extremely small absorption or
transmission changes, making their detection very difficult. The solvents
or buffers used, as well as many of the compounds separated, are often of
insufficient chemical stability over given time periods to be measurable
on a reliable basis. As a consequence, certian nonrepeatable errors are
present from one liquid chromatogram to another. It is therefore desirable
to measure the characteristics of buffers or solvents at the same time as
the characteristics of the separated materials are measured. A double beam
approach, permitting the utilization of a reference and sample cuvette,
has been found to be desirable. Since, as has been mentioned above,
absorption or transmission changes in sample quantities as small as 5
microliters require accurate and practical presentation and since these
absorption changes may amount to only a few ten thousandths of optical
density (O.D.), long-term stability of the total system becomes a major
objective in the solution of the measuring problem. It is therefore an
object of the invention to provide such apparatus wherein the light
utilized for measurement finds minimal impairment by optical or mechanical
components and provides optical characteristics of similar nature in the
reference and sample beams over long time periods. Since continuously
adjustable UV and visible radiation is required to find most favorable
absorption maximas for eluted media by the employment of monochromatic
light produced by a dispersing element, such as, for example, a refraction
grating, stability of the light source and uniformity of radiant flux
impinging onto the cuvettes or flow cells become criteria of utmost
importance.
To obtain and maintain the aforementioned stability, one of the objects of
this invention is to obtain this uniformity and stability which is
attainable with the conventional approaches used in the field of
spectrophotometry which employ light sources, entrance slit illumination
optics, monochromator with exit slits, exit radiation collimators, and
rotating or modulating optical choppers to provide radiation for reference
and sample measurements. In such conventional approaches, radiation
emerging from the exit slits of monochromators having an elongated
configuration, has to be condensed and optically treated to produce a
fairly well collimated beam of a size suitable to pass through cuvettes
with 1-mm aperture and 10-mm pathlength. This treatment of the beams
exiting from the standard monochromator slits results in light loss and
low energy passing through cuvettes with the effect that considerable
amplification of the detector signals is necessary. This results in
unstable and noisy signals and recording.
Since at least eight optical components are normally employed in
conventional spectrophotometers of the double beam type and since these
components consist of a condenser optic between the lamp and
monochromator, a 45.degree. reflector to reach the monochromator entrance
slit, a major collimator in the monochromator (one if Littrow arrangements
are used and two if Ebert arrangements are used), a dispersing element
consisting of a prism or grating, a condenser and collimator optic after
the exit slit of the monochromator, a beam splitter or chopper arrangement
consisting normally of two standing and two chopping or oscillating
mirrors (of the latter, one standing and one chopping mirror exclusively
serves for the reference and one pair of similar ones serves for the
sample channel to reach reference and sample cuvettes alternately), a
great number of unlike surface areas and areas of possible mechanical
instability, which make detection of minute differences between samples
and references extremely difficult, are introduced. As a consequence, a
certain error is present in all existing monochromatic readout systems
unless steps are taken to minimize the optical inconsistencies and
mechanical instabilities. A further object of the present invention is to
provide apparatus which will eliminate the systems errors and reduce the
number of optical components used for the production of two identical
spectra to illuminate reference and sample to a minumum of three, and to
replace choppers utilizing rotating or oscillating optical surfaces for
the purpose of dividing radiation into reference and sample beams by two
individual detectors or by a nonoptical reciprocating vane and signal
gating for maximum readout stability.
A still further object of the invention is to utilize a similar or the same
portion of the flux envelope emitted by a light source and to focus the
original source point of the light source in a dispersed manner in the
form of two defined identical spectra produced via one reflection-type
diffraction grating or refraction-type dispersing optic onto two similar
cuvettes or onto the unknown sample and reference areas. This is
accomplished by refocusing the light source either with a
multi-directional spherical or aspherical, corrected, refocusing,
reflection, mirror-type optic which is ground, polished, and coated with a
reflection surface or by refocusing by means of a multi-directional
transmission optic which reproduces two identical spectrally dispersed
images of the light source in the plane of the reference and sample
cuvette or on the reference or the sample area, after being reflected and
refracted by a reflection-type diffraction grating or refraction-type
dispersion optic.
By using the approach described above, possible light source energy
fluctuations, resulting in higher and lower radiant flux over short or
long durations, are equally expressed in the reference and sample beam. It
is therefore also an object of the invention to have light source
fluctuations transferred into separate sample and reference areas via only
one dispersion and one multi-directional refocusing optic for
instantaneous photoelectric comparison and formation of ratios between
sample and reference energy with a resulting ratio of 1 for the electrical
expression of a reliable zero for measuring purposes, since the energies
of the sample and reference will be almost identical.
This can also be expressed as follows. Light intercepted from the radiant
flux emitting area of the light source reaches two areas after dispersion
of two directional beams by one dispersing element. The two areas contain
the same energy. This energy can, however, differ in intensity due to
reduced flux over the lifetime of the source and due to deterioration of
the optical surfaces. Since identical areas of reflecting and/or
transmitting optics and the same area of a dispersion or refraction optic
are used, the reduction of energy will be equally expressed and the ratio
of energy between the sample and reference beam will be one to one if
these beams are unimpaired by energy-reducing or energy-adding substances,
such as absorbing or flourescing substances, regardless of the energy
level emitted by the light source or impaired by optical effects. As an
example, assume that the sample beam contains radiation to produce a
photoelectric signal of 5 V and the reference beam contains radiation to
produce 5 V at the beginning of a long-term test. The ratio of 5 V over 5
V equals 1. As the test proceeds, the light output of the source and the
dispersion and multi-directional refocusing optic deteriorates to yield
only 2 V in the sample beam and 2 V in the reference beam. The resulting
ratio of 2 V over 2 V equals 1. If the ratio of 1 is utilized to provide a
readout of transmission, the transmission of energy through an empty
sample cuvette will always be 100 percent of the energy available through
the empty reference cuvette or the optical density of the empty sample
cuvette will be zero before introduction of the sample to be measured,
regardless of actual light energy level emitted by the source or
transmitted by the system.
It is a still further object of the invention to provide apparatus wherein
an aperture within the light source or directly in front of the source,
limiting the size of such source, is refocused in the same manner into the
plane of the sample and reference beam by the same multi-directional
refocusing optic and only one dispersing element.
It is yet another object of the invention to provide a single reflecting
condensing and refocusing optic after the plane of reference and sample
cuvette mounting to intercept energy emerging from such sample and
reference cuvettes and to refocus such energies by means of
demagnification into an area of extreme proximity onto a single
photodetection element.
It is still a further object of the invention to employ two like sections
of the same reflecting element after the sample and reference cuvettes to
recombine and superimpose true images of the aperture, limiting the light
source size.
It is still another object of the invention to utilize the light energy
emerging from similar spectral areas of the spectra displayed in the
sample and reference plane to illuminate flourescent and refraction-type
cuvettes for photoelectric determination of flourescence or refractive
index changes in the measured media via the same apertures.
And it is still another object of the invention to provide light access to
the readout photodetector in an alternating mode for reference and sample
ratio determination via a reciprocating nonoptical shutter vane driven by
a unique stepper drive.
It is also one of the objects of the invention to utilize the stepper drive
and its characteristics for alternate signal interception and to overcome
conventional instabilities and nonreproducibilities caused by mechanical
imperfections, temperature dependencies of motor-magnets and coils via
exactly timed electronic gating of the signals, eliminating the leading
and trailing slopes, only utilizing the flat portion of the sample and
reference signal.
These and other objects, advantages, features, and uses will be apparent
during the course of the following description when taken in conjunction
with the accompanying drawing wherein:
FIG. 1 is an exploded view, in perspective, of an embodiment of the
invention;
FIG. 2 is an exploded view, in perspective, of a further embodiment of the
invention depicting a duplication of the reference and sample arrangement
resulting in two reference and two sample beams;
FIG. 3 is an exploded view, in perspective, of a still further embodiment
of the invention depicting a single reflecting condensing and refocusing
optic and sections thereof for energy interception and depicting a
nonoptical shutter vane on a reciprocating electromechanical drive;
FIG. 4 is a functional block diagram showing the principal electronic
elements for driving the stepping motor and the precision gating
electronics as well as the signal versus time graphs at selected points in
the circuit; and
FIG. 5 (on the same sheet as FIG. 3) is an elevational view, partly in
section, of a flow-through cuvette used in the apparatus of the invention
for flourescence detection.
In the drawing, wherein, for the purpose of illustration, are shown
preferred embodiments of the invention, and wherein like numerals
designate like parts throughout the same, the numeral 1 designates a
source of light (FIG. 1). This source of light is depicted as an arc
source which radiates through a full circle of 360.degree.. Since the full
circle of radiation cannot be intercepted by optics, a slit mask 7 is
mounted as closely as possible to the arc source to provide only
illumination to the multi-directional refocusing optic which consists of
components 2 and 3.
This multi-directional refocusing optic as depicted features a hinge
connection 2' to provide accurate grinding and polishing and to produce
identical radii for components 2 and 3. After overcoating of the polished
surfaces with reflecting material, reflection surfaces are produced which
permit one to illuminate an identical area on a grating 4 by either
portion of the multi-directional refocusing optics 2 and 3. The light
reaching the grating 4 is dispersed by this grating into spectra 8 and 9.
Light reaching the area of the projected spectra is restricted by aperture
plate 14 which contains two identical apertures 8' and 9' to permit only
selected portions of the spectra to reach the flow-through or
spectrophotometric cuvettes 5 and 6. Light passing through cuvettes 5 and
6 then reaches photodetectors 10 and 11 which are connected to ratio
circuitry 12 to compare the signals by producing ratios thereof for
display or readout recorders or readout 13. The grating 4 can be tilted
between pivot points 20 by arm 22 through an angle indicated by the arrows
25 using a follower pin 24 and a standard sine drive as is well known in
the construction of monochromators and whose details are not depicted
herein. The change of angle of grating 4 accomplishes the sweeping of
spectra 9 and 8 across the apertures 8' and 9'. If grating 4 is moved in
an angular fashion as indicated by directional arrow 25, two identical
spectra, produced by the same area of the grating and originating from the
same apex of the central rays originating from the source and directed
toward the grating by multi-directional refocusing optic 2 and 3, will
sweep across the two apertures 8' and 9' in aperture plate 14 for
selection of desired spectral areas to illuminate cuvettes 5 and 6.
FIG. depicts a sysyem very similar to that described and shown in FIG. 1
with two added components in the multi-directional refocusing optic
designated 35 and 36. The addition of these two components having similar
surfaces to those of components 2 and 3 permits one to illuminate a single
grating with two additional light beams reaching the same apex point on
grating 4 and being dispersed by grating 4 into two additional spectra 43
and 44 apertured by apertures 43' and 44' on aperture plate 14. The two
additional spectra reach photodetectors 41 and 42. The addition of these
two photodetectors depicts clearly one of the advantages of the invention
to produce several identical spectra in at least pairs of two as
designated by numerals 8 and 9 or 43 and 44 to provide a
spectrophotometric apparatus which contains only one grating but provides
several analyzing channels without employment of beam-dividing choppers
and originating from one radiating source. As clearly shown, cuvettes 5,
6, 39, and 40 can be utilized for spectrophotometric measurements and
comparison by the usual ratio circuitry and readout.
FIG. 3 depicts a configuration of the apparatus in which two channels, used
to determine ratios between sample and reference, can be served by one
single photodetector 19 by using an elliptical or other aspherical beam
combiner optic 17 or sections thereof as shown and designated with
numerals 18 and 18'. In this configuration beams 15 and 16, constituting
the central beams of spectra 8 and 9 reaching the cuvettes 5 and 6, are
alternately permitted by chopper blade 28 to reach photomultiplier 19.
Chopper blade 28 is driven by motor 27 in a reciprocating motion as shown
by arrows 28'.
It is also within the scope of the invention to use two such choppers or
one with two reciprocating blades and two photodetectors with the four
channel construction of FIG. 2.
FIG. 4 shows a sine wave generator 56 and driver amplifier 57 for
controlling the stepping or chopper motor 27 and chopper blade 28 to move
one step in a clockwise direction during the positive half wave to permit
beam 16 to pass and one step in counterclockwise direction to permit beam
15 to pass during the negative half wave. The detector 19 therefore sees
the signal obtained with the light beam 16 passing through cuvette 6 which
is marked as sample signal S in the graphs 58 and 59' which depict the
signals appearing at points 50' and 52', respectively. Since the inertia
of stepping motor 27 causes a time delay or a phase shift, the phase
shifter 55 delays or shifts the phase of the sine wave generated by 56 by
the same amount as the stepping motor delays its mechanical action. The
shifted sine wave at the output of phase shift 55 produces an accurately
timed pulse by means of the pulse generator 53 during the positive half
wave. The pulse generator 54 generates an accurately timed pulse during
the negative half wave. These pulses drive solid state switches 51 and 52
in a manner such that the signal wave from photodetector 19, which has
been amplified by amplifier 50, and as shown in graph 58, is conducted
only during the plateau portion of the signal R and S. The signal of graph
58 is at the same time separated into two signals which are shown in
graphs 59 and 59'. Graph 59 appears at point 51'. One signal channel
contains only R signals, and other signal channel contains only S signals.
The ON time of the drive pulses from generators 53 and 54 is identical,
therefore, the integrated area of the pulse R in graph 59 represents the
reference signal R, and the integrated area of the pulse S in graph 59'
represents the sample signal S, eliminating the influence of the
uncertainty of the leading and trailing slopes of the signals R and S as
shown in graph 58. The switching points of the switches 51 and 52 are
marked with the same numbers in graphs 58 and 59 and 59' to show which
portion of the wave of graph 58 has been transferred by the switches 51
and 52 as shown in graphs 59 and 59'. Ratio circuitry 12 contains ratio
electronics with outputs to feed readout devices well known in the art.
FIG. 5 is a view of a special version of a cuvette 5' for flourescence
investigation using the apparatus of the invention, in which the body of
cuvette 5' is formed of quartz or other suitable material through which a
liquid flow for detection and analysis is directed. This cuvette 5' is
embedded in a clear plastic or glass block 70 having a spherical,
aspherical, or elliptical configuration or surrounded by an elliptical,
spherical, or aspherical reflector to direct flourescence emerging from
the walls of cuvette 5' to photodetector 19 through a filter 32. The
propagation of rays, indicated as dotted lines, is designated by the
numeral 30. The cuvette 5' operates by exiciting the spectral energy,
originating out of beam 15 or 16, as illustrated in FIG. 1, through window
34. Flourescence samples, which have entered through port 31 and are
present in the main body portion 34' of cuvette body 5', are excited by
this entering radiation which cannot exit and reach photodetector 19
directly due to mask 71, will radiate and this radiant energy will be
reflected as described before.
While particular embodiments of the invention have been shown and
described, it is apparent to those skilled in the art that modifications
are possible without departing from the spirit of the invention or the
scope of the subjoined claims.
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