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WO2002025235A1 - Procede et dispositif optique - Google Patents

Procede et dispositif optique Download PDF

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Publication number
WO2002025235A1
WO2002025235A1 PCT/GB2001/004013 GB0104013W WO0225235A1 WO 2002025235 A1 WO2002025235 A1 WO 2002025235A1 GB 0104013 W GB0104013 W GB 0104013W WO 0225235 A1 WO0225235 A1 WO 0225235A1
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WO
WIPO (PCT)
Prior art keywords
detector
frequency
radiation
component
container
Prior art date
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Ceased
Application number
PCT/GB2001/004013
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English (en)
Inventor
David Murray Goodall
Edmund Thomas Bergstrom
Malcolm Driffield
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BTG International Ltd
Original Assignee
BTG International Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BTG International Ltd filed Critical BTG International Ltd
Priority to AU2001284293A priority Critical patent/AU2001284293A1/en
Publication of WO2002025235A1 publication Critical patent/WO2002025235A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J4/00—Measuring polarisation of light
    • G01J4/04—Polarimeters using electric detection means
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G01N2030/746—Optical detectors detecting along the line of flow, e.g. axial

Definitions

  • the present invention relates to apparatus and methods of analysis of optically active materials. In particular, but not exclusively, it relates to a polarimetric detector for high performance liquid chromatography.
  • HPLC High performance liquid chromatography
  • polarimetry measures OR
  • CD circular dichroism
  • a polarimetric detector In a polarimetric detector ( Figure 1) unpolarised monochromatic light is passed through a polariser which produces PPL. The resultant beam is passed through the chiral sample and the plane of polarisation is rotated. A second polariser, which is at right angles to the first, is mechanically rotated until the light is detected by a photodiode detector. The OR is the angle by which the analyser is rotated to receive maximum light intensity at the photodiode detector.
  • This system has a very low signal to noise (S/N); because the analyser is mechanically controlled it is difficult to rotate by very small increments, and very small OR changes are difficult to measure.
  • S/N signal to noise
  • a lock in amplifier (LIA) is used to detect the signal at the frequency determined by the Faraday modulator.
  • the S/N is improved as all the noise is cut out except at the frequency at which measurement occurs.
  • One object of the present invention is to provide a method which will compensate for changes in laser output, transmission through the sample and degree of depolarization.
  • a detector for use in a polarimeter the polarimeter having a modulation means for modulating radiation at a first frequency, characterised in that the detector detects an output signal having a first component at the first frequency and a second component at a second frequency, and processes the two frequency components to determine the optical rotation of the radiation.
  • the second frequency has a value dependent on the first frequency, preferably the second frequency is twice the first frequency.
  • the optical rotation may be determined by taking the ratio of the frequency components.
  • the detector may comprise two phase sensitive detectors, one phase sensitive detector for detecting and measuring the first frequency component and the other phase sensitive detector for detecting and measuring the second frequency component.
  • an apparatus for use in the analysis of optically-active materials comprising a radiation source, container means for a sample of material under examination through which radiation from the source passes to a detector, polariser means disposed between the radiation source and the container means, analyser means disposed between the detector and the container, modulation means to modulate the rotation of the polarisation of radiation from the radiation source, characterised in that the detector detects an output signal having a first component at a first frequency and a second component at a second frequency, and processes the two frequency components to determine the optical rotation of the radiation.
  • the modulation means may be located between the polariser and the container or located between the container and the analyser.
  • a polarimeter comprising a source of radiation, modulation means to modulate radiation at a first frequency and a detector, characterised in that the detector detects an output signal having a first component at the first frequency and a second component at a second frequency, and processes the two frequency components to determine the optical rotation of the radiation.
  • Figure 1 shows the basic instrumentation for a polarimetric detector
  • Figure 2 is a schematic diagram of a polarimetric detector in accordance with one embodiment of the invention
  • Figures 3 to 12 show test results of various experiments carried out using the methods of the invention
  • Figure 13 is a block diagram of a polarimeter
  • Figure 14 is a schematic of a detector according to one embodiment of the invention
  • Figure 15 is a schematic of a modulation circuit for providing the reference frequency to drive the Faraday modulator of Figure 1;
  • Figures 16 to 18 are graphs showing the test results of experiments carried out using the methods of the invention.
  • the signal at the photodiode detector has two AC components; one at the frequency detennined by the Faraday modulator and one at twice this frequency.
  • the root mean square (RMS) amplitudes of these signals are if and 2f respectively.
  • ⁇ 0 modulation angle due to the Faraday modulator
  • ⁇ 0 intensity of the PPL at the analyser
  • ⁇ OR caused by the chiral sample
  • Equation 4 shows that lf/2f ' is proportional to ⁇ but is independent of ⁇ 0 , so there will be no apparent change in the 1F/2F signal due to changes in the transmitted light intensity.
  • Figure 2 there is shown two lock in amplifiers, one to detect the If component of the signal and one to detect the 2f component.
  • Tinolux sulfonated aluminium phthalocyanine
  • the expected OR at 820 nm was 68 m° and the actual OR before any polystyrene was added was 65 m°.
  • Figure 5 shows the HPLC-OR trace for a 20 ⁇ l injection of 1% w/v fructose and 1% w/v sucrose.
  • the HPLC conditions were: 250 x 4.6 mm ODS 2 column, flow rate 1 ml min ⁇ m distilled water mobile phase and OR detection at 635 nm.
  • Figure 6 shows the transmitted light intensity (2f) during the separation of a 20 ⁇ l injection of 1% (w/v) fructose and 1% (w/v) sucrose.
  • Fructose was the first peak to elute.
  • the / 2/and //OR traces were virtually superimposed upon each other, but the //signal was more noisy than the lf/2f signal.
  • the RMS noise between 1 and 2 min was 12 ⁇ ° for the //signal and 5 ⁇ ° for the lf/2f signal. Two substantial decreases in the 2/ signal were evident which correspond to the elution of the sugar peaks.
  • Figure 7 shows the HPLC-OR-abs for a 20 ⁇ l injection of 1.2 mM warfarin.
  • HPLC conditions were: 250 x 4.6 mm Chiralcel OD column, flow rate 1 ml min "1 , 80 : 19 : 1 hexane : propan-2-ol : acetic acid mobile phase, absorbance detection at 254 nm and OR detection at 635 nm.
  • Table 2 Noise values for 20 ⁇ l injection of 1.2 mM warfarin. On the semi-preparative scale, for a 5ml injection of 9 mM warfarin, a signal of approximately 1 m° per enantiomer was seen (see Figure 8).
  • Figure 8 shows the HPLC-OR-abs for a 5 ml injection of 9 mM warfarin (6 mg per enantiomer). HPLC conditions were: 250 x 4.6 mm Chiralcel OD column, flow rate 0.5 ml min "1 , 80 : 19 : 1 hexane : propan-2-01 : acetic acid mobile phase, absorbance detection at 330 nm and OR detection at 635 nm.
  • OR/absorbance is directly proportional to the enantiomeric excess and allows the determination of when a single enantiomer is eluting.
  • an absorbance detector alone could be used to distinguish between the enantiomers.
  • Figure 9 shows the advantages of using OR/absorbance when the enantiomers are co-eluting in the absorbance trace.
  • Figure 9 shows the OR absorbance and absorbance trace for a 5 ml injection of 9 mM warfarin. The flat parts of the OR/absorbance at 15-25 min and 35-45 min indicate the elution of single enantiomers.
  • Tr ⁇ ger's base ( Figures 10 and 11)
  • the chirality of Tr ⁇ ger's base, 2 is due to the helical shape of the structure
  • Figure 10 shows the HPLC-OR-absorbance for a 20 ⁇ l injection of 1.87 mM Tr ⁇ ger's base.
  • HPLC conditions were: 250 x 4.6 mm DNBPG column, flow rate 1 ml min "1 , 99 : 1 hexane : propan-2-ol mobile phase, absorbance detection at 254 nm and OR detection at 635 nm.
  • Tr ⁇ ger's base The flat signal at 14-16 min and the equal and opposite signal at 25-28 min indicated the elution of single enantiomers.
  • the portion of baseline at zero OR/absorbance was the racemic mixture eluting.
  • FIGS 14 and 15 illustrate details of a detection method which can be used for the present invention.
  • phase sensitive detectors PSDs
  • PSDs phase sensitive detectors
  • two PSDs are used to simultaneously measure the turbidity and the optical rotation of scattering samples.
  • One PSD is locked in to twice the reference frequency (2f) to measure the transmittance and the other measures the If signal.
  • the optical rotation is given by the ratio of the If to the 2f signal.
  • a PSD needs a reference sine wave, N r sin( ⁇ r t+ ⁇ r ) (used to modulate the experiment), which it multiplies with the signal.
  • the PSD output signal is just the product of the two sine waves which is two signals, one at the frequency ( ⁇ r + ⁇ s ) and the other at ( ⁇ r - ⁇ s ).
  • Analogue PSDs are being superseded by digital PSDs due to their superior performance with respect to harmonic rejection, output offsets, dynamic reserve, gain error and output filter stability.
  • a digital PSD digitises the signal, then carried out all the multiplications and low pass filtering digitally.
  • a PC fitted with a suitable data acquisition board could be configured as two PSDs that would extract the If and 2f signals from the same input signal. This would be a main advantage over the current system, where small output offsets from either the If or 2f signals can give large errors in the optical rotation measurement (1 f 2f) under conditions of high turbidity.
  • a test rig has been constructed to measure optical rotation using a laser and Faraday modulation, Figure 13.
  • the output from a 820 nm diode laser is adjustable and has been operated at ⁇ 10 nW for these experiments.
  • the polariser and analyser are made of a polarising glass; Polarcor is a mixed alkali borosilicate glass containing elongated, submicroscopic silver particles aligned along a common axis; when crossed they have a transmittance of- 0.0001.
  • Figure 14 shows how the signal from the photodiode (PD) is amplified and digitised.
  • a PC 150 MHz pentium
  • a data acquisition (DAQ) board ComputerBoards CIO-DAS802/16
  • the input sensitivities can be software selected ( ⁇ 1.25, ⁇ 2.5, ⁇ 5 or ⁇ IOV).
  • the maximum sampling rate of 100 kHz is always used to maximise dynamic range of the PSD.
  • An anti-aliasing filter has been designed and built to remove components above the Nyquist frequency of 50 kHz which would be undersampled (if undersampled, higher frequencies appear as lower frequency signals in the digital data stream).
  • the filter is a 6 pole Chebyshev low pass filter designed with 0.5 dB of ripple in the pass band and a cut off frequency of 5kHz; this gives 140 dB of attenuation at 50 kHz.
  • the filter was constructed using 1% tolerance polystyrene capacitors and metal film resistors and an AD 713 J quad precision high speed BiFET op-amp. The output from the filter is then digitised by the DAQ board in the PC.
  • the PC can carry out all the calculations at the required rate only if it is not having to do anything else, which is time consuming. This makes it necessary for the PC or the DAQ board to generate the reference signal so that the frequency and phase of the reference is automatically synchronised to the digitisation timing. There is then no need for the program running on the PC to have to monitor the reference.
  • Figure 15 shows how the reference is generated.
  • the DAQ board contains an 82C54 timer chip that contains three 16 bit countdown timers. Counters 1 & 2 are used by the board to pace the analogue to digital conversions; this means that the output of counter 1 is a 100 kHz signal which is locked in to the sampling rate.
  • This signal has been connected to the input of the spare counter and the counter's register loaded with the value 100; the result is a 1 kHz square wave output. There are always exactly 100 samples per period of this square wave.
  • a circuit has been built to convert this into a stable sine wave. The signal is first clamped by a zener diode to give a square wave with a very stable amplitude of 1.2 N which is low pass filtered to give a sine wave.
  • the filter is similar in design and construction to the anti-aliasing filter described above, except the cut off frequency component of the square wave; the result is a high quality sine wave.
  • a power amplifier (built using a TDA1514A IC) uses the reference sine wave to drive the Faraday modulator.
  • the polarimeter set-up has been calibrated with water and a 1% sucrose solution in a 1 cm cuvette. This gives a rotation change of 33.1 m° at 820 nm.
  • a variable attenuater made of two plastic polarising sheets placed in the optical path allows the performance to be tested under conditions of varying transmittance.
  • Figure 16 shows a baseline obtained with 100% transmittance and a detector rise time of 0.5 s (equivalent noise bandwith ⁇ 0.5 Hz). The rms short term noise calculated from this is 2 ⁇ ° ⁇ .Hz. On a longer timescale this test set-up suffers to some extent from drifting baselines and other glitches.
  • Figures 17 and 18 show the results of monitoring the optical rotation (the absolute value has been arbitrarily set by offsetting the analyser) while reducing the transmittance in stages from 100% to 0.1%, using the new system and test rig described here and the system currently in use for the mixed biopolymer studied (i.e. argon ion laser, crystal polarisers, two PSDs etc).

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  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

La présente invention concerne un dispositif destiné à être utilisé dans le cadre de l'analyse de substances actives du point de vue optique, comprenant une source de rayonnement, un système de réception destiné à un échantillon de substance en cours d'analyse, par lequel passe le rayonnement issu de la source, pour atteindre un détecteur, un système de polarisation disposé entre la source de rayonnement et le système de réception, un système d'analyse disposé entre le détecteur et le récipient, un système de modulation servant à moduler la rotation de la polarisation du rayonnement issu de la source de rayonnement. Selon l'invention, le détecteur détecte un signal de sortie ayant une première composante à une première fréquence et une seconde composante à une seconde fréquence et traite les deux composantes de fréquence pour déterminer la rotation optique du rayonnement.
PCT/GB2001/004013 2000-09-21 2001-09-06 Procede et dispositif optique Ceased WO2002025235A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001284293A AU2001284293A1 (en) 2000-09-21 2001-09-06 Optical method and apparatus

Applications Claiming Priority (2)

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GB0023227.2 2000-09-21
GB0023227A GB0023227D0 (en) 2000-09-21 2000-09-21 Optical method and apparatus

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WO2002025235A1 true WO2002025235A1 (fr) 2002-03-28

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006004731A3 (fr) * 2004-06-30 2006-04-13 Stheno Corp Systemes et procedes d'heterodynage chiro-optique
US7502111B2 (en) 2003-10-10 2009-03-10 Stheno Corporation Differential optical technique for chiral analysis
US7590196B2 (en) 2004-05-04 2009-09-15 Spectra Analysis, Inc. Chiral mixture detection system using double reference lock-in detector

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2393296A1 (fr) * 1977-05-31 1978-12-29 Mueller Arno Procede et appareil de determination quantitative de substances optiquement actives
US4498774A (en) * 1981-07-22 1985-02-12 Iowa State University Research Foundation, Inc. Micro-polarimeter for high performance liquid chromatography
US5012101A (en) * 1986-10-24 1991-04-30 National Research Development Corporation Optical apparatus and method
EP0805352A1 (fr) * 1995-11-16 1997-11-05 Matsushita Electric Industrial Co., Ltd. Procede et appareil d'analyse d'urine, procede de mesure de la rotation optique et polarimetre

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2393296A1 (fr) * 1977-05-31 1978-12-29 Mueller Arno Procede et appareil de determination quantitative de substances optiquement actives
US4498774A (en) * 1981-07-22 1985-02-12 Iowa State University Research Foundation, Inc. Micro-polarimeter for high performance liquid chromatography
US5012101A (en) * 1986-10-24 1991-04-30 National Research Development Corporation Optical apparatus and method
EP0805352A1 (fr) * 1995-11-16 1997-11-05 Matsushita Electric Industrial Co., Ltd. Procede et appareil d'analyse d'urine, procede de mesure de la rotation optique et polarimetre

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7502111B2 (en) 2003-10-10 2009-03-10 Stheno Corporation Differential optical technique for chiral analysis
US7590196B2 (en) 2004-05-04 2009-09-15 Spectra Analysis, Inc. Chiral mixture detection system using double reference lock-in detector
WO2006004731A3 (fr) * 2004-06-30 2006-04-13 Stheno Corp Systemes et procedes d'heterodynage chiro-optique
US7405826B2 (en) 2004-06-30 2008-07-29 Gibbs Phillip R Systems and methods for chiroptical heterodyning

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AU2001284293A1 (en) 2002-04-02

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