Title of Invention

A SPECTROFLUOROMETER

Abstract

1. A spectrofluorometer comprising a light source, focusing means to collect light from the said light source onto the entrance slit of the excitation monochromator having a holographic gratings; means for rotating the said gratings, slit mechanism connected to the said monochromator to set the slit width for a selected band width of the monochromatic radiation coming out of the said exit slit of the said monochromator, a stepper motor driven cell holder mechanism accommodating a plurality of cells employed to place one of the said sample cells in the optical path where it is being iraradiated with the selected monochromatic radiation through a excitation system, emission optical system to collect the fluorescence radiation emitted by the said sample and focusing on to the said entrance slit of emission monochromator, a fluorescence detector producing a photocurrent proportional to the fluorescence radiations emitted by the said sample on operational amplifier to convert the photocurrent produced by the said fluorescence detector into a voltage signal, an absorption detector placed in the primary radiation path of the said sample to collect the transmitted light from the said sample, an interface circuit detector connected to the said absorption detector to convert the photocurrent produced into voltage signal, an analog multiplexer connected to another operational amplifier to select the outputs from either of the said two detectors, a converter to acquire the data from said detector interfacing circuits, a high voltage source supplying a variable high D. C. voltage required for photo-multiplier tube operation and a micro processor used for conversion control and display of various functions.

Full Text

The present invention relates to a spectroflourometer, the main embodiment of which lies in it's capability to perform the function of two instrument's in one.The spectrofluorometer of the present invention can be used as a spectrophotometer through a simple mode selection. BACKGROUND OF THE INVENTION Spectroscopy has been defined the study of the absorption and I emission of light and other radiation, as related to the wavelength of the radiation. The spectroscopic methods are helpful in almost all technical fields, especially for identifying constituents and processes in any source that emits light. With spectroscopic methods one can achieve various aims which might vary from identifying an element in a mixture to determine the structure of an unknown molecule. Besides these, the spectroscopic techniques have also found place in unraveling the scientific principles behind the structure of atom and various properties exhibited by atoms of different elements. The Florescence forms an essential aspect of spectroscopy, where the main principle behind the fluorescence is that when the light energy is incident on a substance the molecules of the substance in ground state absorb light and transit into excited state. The molecules loose portion of the absorbed energy as vibrational energy and transit to a lower vibrational level with no radiatior emitted and return to the ground state emitting some optica] energy known as Fluorescence. Since portion of the light absorbed is lost in vibrational relaxation, the fluorescence emitted by the substance has longer wavelengths than the incident light 'energy. The intensity of the fluorescence emitted is proportional to the optical energy absorbed and thus to the concentration of the substance. The spectroflourometers have been used to measure the fluorescence spectra and help interpret the results. In a spectrofluorometer, the desired wavelength of exciting light is selected by a monochromator or filter (called primary monochromator or filter) between the light source and the sample. The wavelength of light to be measured is selected by a second optical component (called secondary monochromator or filter) between the sample and a photodetector. The output of the photodetector, a current which is proportional to the intensity of the fluorescent light, is amplified to give a reading on a meter. In operation, a standard sample is placed in the instrument and the sensitivity adjusted to a desired reading. Unknown samples and a blanks are then read. The net readings (blank reading subtracted) of the standard and the samples are in the same proportion as their concentration, permitting the calculation of sample concentration by simple ratios. SUMMARY OF THE INVENTION The present invention relates to a spectroflourometer where the novelty resides not only in the fact that the spectroflourometer is more efficient, but can also act as a spectrophotometer by a mere selection of panels. The special features of the present spectroflourometer are that the invented spectroflourometer's optical geometry is configured in such a way that it performs two instrument's function in one. It can be used as a spectrofluormeter and also as a spectrophotometer through a simple mode selection. Accordingly the present invention relates to a spectroflourometer comprises a light source, focusing means to collect light from the said light source onto the entrance slit of the excitation monochromator having a holographic gratings; means for rotating the said gratings, slit mechanism connected to the said monochromator to set the slit width for a selected band width of the monochromatic radiation coming out of the said exit slit of the said monochromator, a stepper motor driven cell holder mechanism accommodating a plurality of cells employed to place one of the said sample cells in the optical path where it is being iraradiated with the selected monochromatic radiation through a excitation system, emission optical system to collect the fluorescence radiation emitted by the said sample and focusing on to the said entrance slit of emission monochromator, a fluorescence detector producing a photocurrent proportional to the fluorescence radiations emitted by the said sample on operational amplifier to convert the photocurrent produced by the said fluorescence detector into a voltage signal, an absorption detector placed in the primary radiation path of the said sample to collect the transmitted light from the said sample, ar interface circuit detector connected to the said absorptior f^c.he^r signal, an analog multiplexer connected to another operational amplifier to select the outputs from either of the said two detectors, a converter to acquire the data from said detector interfacing circuits, a high voltage source supplying a variable high D. C. voltage required for photo-multiplier tube operation and a micro processor used for conversion control and display of various functions. DESCRIPTION OF THE ACCOMPANYING DRAWINGS The present invention can be explained explicitly with reference to the accompanying drawings, wherein:- Fig. 1. shows the instrument block diagram of spectroflourometer. Fig. 2. depicts the optical layout of spectroflourometer. Fig. 3. shows the EHT power supply block diagram for the spectroflourometer. Fig, 4. shows the CPU block diagram of the spectroflourometer; Fig. 5. depicts the stepper motor controller block diagram for the spectroflourometer. and Fig. 6. shows the power supply block diagram of the spectroflourometer. DETAILED DESCRIPTION OF THE INVENTION The block diagram of the invented Spectroflourometer of the present invention is shown in Figure 1. Each block of figure 1 is explained below with cross reference to the detailed figures indicated within the bracket of the block heading. BLOCK 1- Light Source A Xenon arc lamp is used as a source. This has high intensity-emission output throughout the spectral range of 200-900 nm. BLOCK-2 Source Optics A concave mirror is employed to collect the light energy from the Xenon arc lamp (1) and focus onto the entrance slit of excitation monochromater (3) . BLOCK 3. Excitation Monochromator This is the wavelength selector for excitation of the sample (5), This monochromator is having Czerny Turner configuration using two concave (3B and 3C) mirrors for collimation and decollimation. A holographic blazed grating (3D) is employed to disperse the polychromatic light falling on the entrance slit (3A) of this monochromator. The dispersed spectrum is focused onto the exit slit (3E) of this monochromator. The required wavelength is made available at the exit slit (3E) of this monochromator by rotating the grating (3D) in the appropriate direction by appropriate angle. The grating is rotated using a ' sine bar mechanism ' by which the linear movement of a micrometer is converted into the rotational movement of the grating table. A stepper motor driven gear mechanism is employed to drive the micrometer linearly. The bandwidth of the monochromatic radiation coming out of the monochromator is set by selecting the appropriate slit width. A stepper motor driven slit mechanism is employed to set the slit width for a selected bandwidth of the monochromatic radiation coming out of the exit slit {3E) of monochromator. Interference of higher order spectra on the selected first order spectrum is eliminated by introducing appropriate optical filter (3F) in the path of monochromatic radiation after the exit slit (3E) of the monochromator. A stepper motor driven filter wheel mechanism is employed to bring appropriate filters into the optical path. BLOCK 4 Excitation Optical System (Refer fig 2) An efficient optical system using both reflective and refractive optical components is employed to irradiate the sample. This optical system consists of the following optical components a. A plane mirror {4A) to direct the monochromatic radiation coming out of exit slit (3E) of excitation monochromator (3) towards the sample (5). b. A two element air spaced condenser (4B) , built with two planoconvex lenses, collects the above directed radiation and focuses onto the sample (5). BLOCK 5 Sample A stepper motor driven sample cell holder mechanism accommodating multiple cells is employed to place one of the sample cells in the optical path where it is irradiated with the selected monochromatic radiation, known as primary radiation, through the excitation optical system. The irradiate sample emits a secondary radiation known as fluorescence which is collected by the emission optical system placed at 900 to the primary radiation beam. BLOCK 6 Emission Optical System This optical system collects the fluorescence radiation emitted by the sample and focuses onto the entrance slit (7A) of emission monochromator (7). This optical system consists of a two element air spaced condenser built with planoconvex lenses. BLOCK 7 Emission Monochromator This is the wavelength selector which is tuned to the wavelength of the fluorescence emitted by sample. The configuration and construction of this monochromator is similar to that of excitation monochromator. BLOCK 8 Fluorescence Detector 'The low luminescence radiant power from the samples precludes the use of photodiodes. Therefore a PMT (photomultiplier tube) is used as a detector. PMT requires a high voltage power supply (14) ranging from 200 to -1000 Volts for it's operation. PMT produces a photocurrent proportional to the fluorescence radiation, emitted by the sample (5). BLOCK 9 Interface Circuit For Fluorescence Detector An operational amplifier, having ultra low bias current (fA), is configured as 'current to voltage converter' to convert the photocurrent produced by the fluorescence detector (8) into a voltage signal. The low photocurrent from the detector due to the low fluorescence light necessitates low bias current operational amplifiers. The voltage signal from the 'current to voltage converter' stage is buffered by another operational amplifier configured as non-inverting amplifier. Any offset voltage at the output of this non-inverting amplifier arising due to the offset voltage of individual operational amplifiers or dark current of the detector is nullified by feeding a voltage signal to the non-inverting amplifier. The output of this non inverting amplifier is fed to one channel of the Analog multiplexer (12) , This circuit operates on a dual power supply (23) BLOCK 10 Absorbance Detector A silicon photodiode is used for absorption measurements in the wavelength range of 200-lOOOm. This option makes the instrument more versatile to be used as a spectrophotometer. This detector is placed in the primary radiation path after the sample (5) . A collection lens (lOA) is placed in front of the detector to collect the transmitted light from the sample (5) and focus it onto the detector. A mechanical shutter (lOB) is provided in front of the detector which opens in the Absorbance mode and closes in the Fluorescence mode. This shutter (10 B) is operated by a stepper motor. BLOCK 11 Interface Circuit For Absorbance Detector An operational amplifier, having ultra low bias current (fA), is configured as 'current to voltage converter' to convert the photocurrent produced by the absorption detector (10) into a voltage signal. TJhe voltage signal from the 'current to voltage converter' stage is buffered by another operational amplifier configured as non inverting amplifier. Any offset voltage at the output of this non inverting amplifier arising due to the offset voltage of the individual operational amplifiers or dark current of the detector is nullified by feeding a voltage signal to the non inverting amplifier. The output of this non inverting amplifier is fed to another channel of the Analog multiplexer (12). This circuit operates on a dual power supply (23). BLOCK 12 Analog Multiplexer A multi channel analog multiplexer is used to select the outputs from either of the two detector interfacing circuits (Florescence or Absorbance) to Analog to digital converter (13). CPU (16) provides control signal to the analog multiplexer to select Fluorescence or Absorbance mode based on user instruction. BLOCK 13 Analog To Digital Converter (ADC) A 12 bit Analog to digital converter is used to acquire the data from the .detector interfacing circuits (9 and 11) through analog multiplexer (12). The analog signal acquired is converted into binary form. The output of the Analog to Digital converter is connected to the data bus of CPU (16). Since the instrument is of scanning type, the ADC should be of fast converting type. ADC's based on successive approximation technique provide an economical solution with a conversion time of few micro seconds ((s). The ADC used for data acquisition is a 12 bit Analog to Digital Converter using successive approximation technique with conversion time of 20-3 0 (s. Input range used for this ADC is (0-lOV) . BLOCK 14 EHT Circuit (Refer fig 3) This circuit supplies a variable High DC voltage from -200 to -lOOOV required for photo multiplier tube operation. A DC high voltage is generated by a quadrupler circuit configuration {14A) using diodes and high voltage capacitors with AC input of 250V derived from secondary winding of transformer (22) . This high voltage is applied through a resistor to the shunt element (14B) built with transistors. An operational amplifier is used as the control amplifier (14D) with the control input (14C) voltage of 0 to 8V (which gives the EHT a working of -200 to -1000 Volts) to control the shunt element (14B). A portion of the EHT voltage is fed back to the control amplifier (14D), through a resistor and capacitor network (14E), which is compared with the control input(14c). The difference between the fed back voltage and control input voltage is amplified by the control amplifier (14D) to drive the shunt element (14B). This provides an active voltage sharing circuit to shunt regulate the EHT at about 125 times the control input. The control input voltage is set by CPU (16) by sending an appropriate binary data through PPI {16C). This binary data is converted into corresponding analog voltage by a digital to analog converter {14C) and applied to the control amplifier (14D) of EHT circuit. BLOCK 15 Xenon Lamp Power Supply The power supply of the Xenon lamp produces a 25kV ignition pulse to establish the arc. A continuous current of 7.5 amp at 20 volts sustains the arc. BLOCK 16 CPU Circuit (Refer Fig 4) A microcompresser is used to perform various functions like data acquisition, data processing, control of stepper motors, user interface, data presentation on display and printer, CPU is interfaced to various input/output devices through Programmable Peripheral Interface ICs (16C). The instrument operating programme is permanently stored in an EPROM (16A). The data generated during instrument operation is stored in a IC (16B). This circuit is powered by digital power supply (24). BLOCK 17 Stepper Motor Controller (Refer fig 5) This instrument has number of stepper motor controllers to drive the various mechanism like 1. Grating drive 2. Sample holder 3. Monochromator slit 4. Filters 5. Shutters The CPU (16) initializes these motors to the homing positions when the instrument is switched on with the help of a 'homing sense' signal generated by the optical switches mounted on respective motors. The CPU (16) sends the control signals through PPI (16C) to respective transistor driver IC (17A) which drives respective motor. The 'homing sense' signal generated by the optical switch (17C) is also read through PPI (16C) by the CPU (16) to stop the control signals and thereby the motor. The 'stepper motor controller' circuit and the stepper motor are powered by stepper motor power supply (25). BLOCK 18 Key Board The user commands to the instrument are given through 18 key keyboard. The keys are arranged in 3x6 matrix. This matrix is interfaced to CPU (16) through PPI(16C).' BLOCK 19 Display The CPU communicates to the user through LCD module. This module can display 48 characters with 24 characters per one line. It is interfaced to CPU (16) through PPI (16C). BLOCK 20 Printer/RS 232C The results of the measurement can be printed on parallel interface dot matrix printer in the form of table or graph. The printer is interfaced to CPU (16) through PPI (16C) .The instrument can be controlled through PC. The CPU communicates to PC through serial communication port of the PC. BLOCK 21 Mains Input (Refer fig 6) The instrument is powered from a 23 0 ( 10% Vac, 50 HZ, 1 phase. BLOCK 22 Transformer (Refer fig 6) The transformer primary is powdered from mains supply (21). Multiple secondary windings with appropriate voltage and current ratings are provided to supply to various power supplies (14, 23,24,25). BLOCK 23 Analog Power Supply (+V, -V) (Refer fig 6) This supply is used to power the analog circuits & ADC. The offset voltage of the operational amplifiers used in the analog circuit is sensitive to the 'net difference' in the positive and negative supply rails applied to the circuit. If two independent regulators for positive and negative supplies are used, the difference in their difference in their drift characteristics causes a drift of the net difference in power supply rails which changes the offset voltage of the operational amplifiers. To avoid this problem a dual tracking regulator configuration is used where the negative supply tracks the positive supply regulating in zero change in the net difference in power supply rails. An AC voltage of 15 TO 18 V derived from secondary winding of transformer (22) is rectified, using a center tapped diode bridge rectifier (23A), and filtered with capacitive filter. An adjustable floating regulator {23C) is used to provide the positive power supply. A potential divider network (23D) using a matched pair of resistors is connected across the positive and negative rails of the power supply. The voltage from this potential divider network (23D) is compared with the power supply return line potential by an operational amplifier configured as an error amplifier {23E) to generate the error signal. This error signal is amplified by the error amplifier (23E) to drive a series pass transistor (23F) to provide regulation of the negative supply. A short circuit protection is provided by means of a transistor (23G). BLOCK 24 Digital Power Supply (VCC) (Refer fig 6) This supply is used for CPU circuit & ADC. An AC voltage of 8-10 V derived form secondary winding of transformer (.22) is rectified, using a diode bridge rectifier (24A), and filtered with capacitive filter (24B). A three terminal fixed voltage regulator (24C) is used to provide the regulated power supply. BLOCK 25 Stepper Motor Power Supply (Vm) (Refer Fig 6) This supply is used to energise a number of stepper motor used in the instrument to control various mechanisms. Since total load requirement is high (2-3 amperes) , the load is distributed to three voltage regulator circuits. An AC voltage of 15-18 V derived from secondary winding of transformer (22) is rectified, using a diode bridge rectifier (25A, 25D, 25G) and filtered with capacitive filter (25B, 25E, 25 H). Three terminal fixed voltage regulators (25C, 25F, 251) are used to provide the regulated power supply. WE CLAIM: 1. A spectrofluorometer comprising a light source, focusing means to collect light from the said light source onto the entrance slit of the excitation monochromator having a holographic gratings; means for rotating the said gratings, slit mechanism connected to the said monochromator to set the slit width for a selected band width of the monochromatic radiation coming out of the said exit slit of the said monochromator, a stepper motor driven cell holder mechanism accommodating a plurality of cells employed to place one of the said sample cells in the optical path where it is being iraradiated with the selected monochromatic radiation through a excitation system, emission optical system to collect the fluorescence radiation emitted by the said sample and focusing on to the said entrance slit of emission monochromator, a fluorescence detector producing a photocurrent proportional to the fluorescence radiations emitted by the said sample on operational amplifier to convert the photocurrent produced by the said fluorescence detector into a voltage signal, an absorption detector placed in the primary radiation path of the said sample to collect the transmitted light from the said sample, an interface circuit detector connected to the said absorption detector to convert the photocurrent produced into voltage signal, an analog multiplexer connected to another operational amplifier to select the outputs from either of the said two detectors, a converter to acquire the data from said detector interfacing circuits, a high voltage source supplying a variable high D. C. voltage required for photo-multiplier tube operation and a micro processor used for conversion control and display of various functions. 2. A spectrofluorometer as claimed in claim 1, wherein the said light source is Xenon arc lamp. 3. A spectrofluorometer as claimed in claim 1, wherein the said focusing means is a concave mirror. 4. A spectrofluorometer as claimed in claim 1, wherein the said monochromator having a Czerny Turner configuration is provided with two concave mirrors for collimation and decoUimation. 5. A spectrofluorometer as claimed in claim 1, wherein the said excitator optical system comprises a plane mirror to direct the monochromatic radiation coming out of slit of the said excitation monochromator towards the sample, and a two element air spaced condenser, built with two planoconvex lenses. 6. A spectrofluorometer as claimed in claim 1, wherein the said sample cell holder is driven by a stepper motor to enable the sample cell to come in optical path. 7. A spectrofluorometer as claimed in claim 1, wherein the said fluorescence detector is photomultiplier tube wherein the said interface circuit is an operational amplifier having ultra low bias current. 8. A spectrofluorometer as claimed in claim 1, wherein the said absorbance detector is a silicon photodiode. 9. A spectrofluorometer, substantially as hereinbefore described with reference to the accompanying drawings.

Documents:

1846-mas-1996 claims.pdf

1846-mas-1996 correspondence -others.pdf

1846-mas-1996 correspondence -po.pdf

1846-mas-1996 description (complete).pdf

1846-mas-1996 drawings.pdf

1846-mas-1996 form-1.pdf

1846-mas-1996 form-26.pdf

1846-mas-1996 form-4.pdf