User Manual Chirascan CS/ORD
Contents
1. Measure A measurement will be made and the newly calculated calibration value will be displayed in the Calibration Value box 8 To accept the new calibration value click the Apply button This will overwrite the previous value To reject the new value and continue with the previous one click Cancel To input a different value type this directly into the box 3 OPERATION Once the CS ORD accessory has been set up and the calibrated as described in Section 2 operation of the spectrometer for ORD measurements is the same as that for CD measurements which is fully described in the main Chirascan User Manual It is recommended that all optical rotation measurements are background corrected to remove any artifactual features resulting for example from cell birefringence Document 4207Q112C03 01 Page 15 of 152. qseedeeseveuses EE A nteesesesaesno lt yous E E E 14 FOPERA TION E 15 FIGURES Figure 1 1 the CS ORD accessory analyser prism sssssssssssssssssseeeeeeene nnne nennen nnn nnn nnns nane 10 Figure 1 2 overlaid spectra of Absorbance blue CD red and ORD green sseeeeeseesssessss 11 Figure 2 1 sample chamber prepared for mounting the CS ORD accessory sseeseesseeeeeeree 12 Figure 2 2 Tue analyser prs HEU CIS eccreenri an quond Diva Ru V PhambNURER S tf oC NIME E E dUR CUNG Kran SUO IRR RM ROS 12 Figure 2 3 CS ORD accessory correctly assembled in the sample chamber cceeeeccceeeeeeeeseeeeeeeeeesaeeeeeeeeees 13 Figure 2 4 the Signal Dallel axxssmitarcerdeteinasesbpk teuitenisavtbnstudubisaaentinex2e kb aE1vE2 rx d EE ED ined UY bsvxFEEYAR suddhectstetnoradssaednntuebes 13 Figure 2 5 the ORD Calibration dialog DOX llsssesseesesseeeeeeeeennnnn nnne nnne nnn rn nenne nsns naar nnn nnns 15 Document 4207Q112C03 01 Page 9 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual OPPO e OPPO OPEC ESOC OP ee eee eee 1 INTRODUCTION 1 1 Optical rotatory dispersion The Chirascan CS ORD accessory is designed to be used with the Chirascan or Chirascan plus spectrometer to measure the optical rotatory dispersion of a sam
3. AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences User Manual Chirascan CS ORD Chirascan Series Optical Rotatory Dispersion Accessory February 2012 Document 4207Q112C03 01 0 H8 Photophysics Bee eJ Applied Photophysics Ltd 21 Mole Business Park Leatherhead Surrey KT22 7BA UK Tel UK 44 1372 386 537 Tel USA 1 800 543 4130 Fax 44 1372 386 477 Applied Photophysics was established in 1971 by The Royal Institution of Great Britain WWW pD hotop hysics Com Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual CORREO EERE EEE EEE HEHE EHH HH HHH This page is intentionally left blank Document 4207Q112C03 01 Page 2 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual This document contains important safety information Read this document and the Chirascan series User Manual before attempting to install or use the CS ORD accessory Failure to do so could result in death or serious injury Document 4207Q112C03 01 Page 3 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual USE OF THIS DOCUMENT This document is intended to inform the operator of Applied Photophysics CS ORD optical rotatory dispersion accessory on its design installation and operation with either the Chirascan or Chirascan plus spectrometer It is assumed that the
4. NT THESE INCLUDE THAT THE LICENSEE MAY NOT SELL RENT LOAN OR OTHERWISE ENCUMBER OR TRANSFER LICENSED SOFTWARE IN WHOLE OR IN PART TO ANY THIRD PARTY FOR A FULL COPY OF THE LICENCE PLEASE CONTACT APL OR SEE THE SOFTWARE INSTALLATION DISC Chirascan and Chirascan plus are trademarks of Applied Photophysics Ltd Microsoft and Windows are registered trademarks of Microsoft Corporation in the United States and other countries Sigma Aldrich is a registered trademark of Sigma Aldrich Co Missouri United States All other trademarks or registered trademarks are the sole property of their respective owners Document 4207Q112C03 01 Page 4 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences m n CS ORD Accessory User Manual HAZARD AND OTHER INDICATORS HAZARD INDICATORS USED IN THIS DOCUMENT AWARNING The sign to the left is used to indicate a hazardous situation which if not avoided could result in death or serious injury Document 4207Q112C03 01 Page 5 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual ESSENTIAL SAFETY INFORMATION MAKE SURE THAT YOU HAVE READ AND UNDERSTAND ALL THE SAFETY INFORMATION CONTAINED IN THIS DOCUMENT BEFORE ATTEMPTING TO INSTALL OR OPERATE THE OPTICAL ROTATORY DISPERSION ACCESSORY IF YOU HAVE ANY QUESTIONS REGARDING THE OPERATION OF THE ACCESSORY PLEASE CONTACT APL TECHNICAL SUPPORT SECTION AT THE ADDRESS SHOWN ON THE FIRS
5. T PAGE OF THIS DOCUMENT OBSERVE ALL SAFETY LABELS AND NEVER ERASE OR REMOVE SAFETY LABELS PERFORMANCE OF INSTALLATION OPERATION OR MAINTENANCE PROCEDURES OTHER THAN THOSE DESCRIBED IN THIS USER MANUAL MAY RESULT IN A HAZARDOUS SITUATION AND WILL VOID THE MANUFACTURERS WARRANTY The CS ORD detector operates at high voltages and can produce a shock leading to serious injury or death Do not connect or disconnect the detector from the spectrometer unless the spectrometer is powered off The spectrometer is powered by the mains electricity supply which can produce a shock leading to serious injury or death Do not connect or disconnect the instrument from the mains supply unless the supply is powered off at source Ensure all communications and electrical connections are made before powering on the spectrometer Exercise care during operation and do not operate units with their covers removed Operate the spectrometer using only the cables provided Never operate a spectrometer with damaged cables AWARNING The spectrometer may be equipped with a light source 150 watt xenon or mercury xenon arc lamp that produces intense ultraviolet radiation that can irritate the eyes and may impair eyesight Never look directly at the light source Do not open the lamp housing while the lamp is operating or immediately after it is powered off Do not attempt to install the CS SF3 or any of its components unless the lamp is powered off or the lamp shutter is
6. closed Do not allow the skin to be exposed to UV radiation Document 4207Q112C03 01 Page 6 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual CS ORD INSTALLATION AND OPERATIONAL REQUIREMENTS Environmental and electrical requirements The CS ORD has no environmental requirements additional to those of the spectrometer Operation of the CS ORD is controlled by the spectrometer electronics and there are no additional electrical requirements Bench space The CS ORD has no bench space requirements additional to those of the spectrometer Nitrogen purge gas The CS ORD has no purge gas requirements additional to those of the spectrometer Servicing Servicing of the CS ORD should only be undertaken by qualified personnel If you are in any doubt at all please contact the Applied Photophysics Technical Support Department at the address given on the front of this User Manual Document 4207Q112C03 01 Page 7 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences GLOSSARY The following abbreviations may be found in this User Manual APD APL CD ORD PEM PMT SCP SHU HYPERLINKS Avalanche Photodiode Applied Photophysics Ltd Circular dichroism Optical rotatory dispersion Photoelastic modulator Photomultiplier tube Spectrometer control panel Sample handling unit CS ORD Accessory User Manual This document contains hyperlinks between references for example the Content
7. er Manual eee ee eee eee eee eee eee eee eee eee 2 INSTALLATION 2 1 Mounting the detector To install the detector room must be made within the Chirascan sample chamber for the analyser prism Remove any nitrogen purge equipment before or after the sample position and disconnect the water supply connectors Loosen the two retaining screws that hold the cell holder mounting plate in position and move the cell holder to the left i e towards the monochromator end of the sample chamber The inside of the ESHU should now appear as in Figure 2 1 Figure 2 1 sample chamber prepared for mounting the CS ORD accessory Loosen the detector locking ring withdraw the detector from the normal CD position slip the analyser prism assembly over the end of the detector inside the sample chamber and secure the analyser to the ORD detector using the grub screw on top with the fine adjuster and locking screw of the fine adjuster both accessible for adjustment Figure 2 2 I Qe e NN KIN 09 7 ow WAS S KUN or of Laan iy ND QU ET Coarse rotation __ locking screw A gt 4 Fine adjuster Grub screw Figure 2 2 the analyser prism fittings Document 4207Q112C03 01 Page 12 of 15 AppliedP n otophy SICS Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual DIT The cell holder can now be put back in place Replace the cell holder mounting plate and tighten the retaining screws Reconnect the
8. or circular dichroism measurements On current instruments April 2011 onwards the same detector may be used for both although it needs to be configured and calibrated for each mode This should be done by a qualified APL engineer The accessory consists of a linearly polarising analyser prism on a rotatable mount with standard PMT detector The photoelastic modulator PEM of the Chirascan is automatically configured by the instrument software as a dynamic half waveplate to output alternating vertical and horizontally polarised light The analyser prism is placed after the sample and before the detector at a 45 angle the null point to the alternating vertical and horizontal light When there is no optical rotation of incoming light the 45 linearly polarised component will have the same intensity for both the vertical and horizontal polarisations If optical rotation does occur then the intensity of the 45 component will alternate with the modulation of vertical and horizontal polarised light The difference in signal can be obtained by using a lock in amplifier at twice the frequency 100 kHz of the PEM and the detected AC DC signal ratio can be used to calculate the rotation The Chirascan software then converts the DC and AC signals into optical rotation reported in millidegrees m or 1 1000 of a degree Document 4207Q112C03 01 Page 11 of 15 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory Us
9. ple based on the method of Shindo et al Applied Spectroscopy 43 pp 1471 1475 1989 The accessory uses the standard photomultiplier tube PMT or avalanche photodiode APD detector supplied with the spectrometer with the analyser prism shown in Figure 1 1 A j T wv i s Ozz oz oot at MT al blll AN Figure 1 1 the CS ORD accessory analyser prism Optical rotation is the rotation of linearly polarised light as a result of it passing through an optically active material and is a consequence of a difference in refractive index between the left and right circularly polarized components of the light linearly polarised light can be considered to be an equal combination of right and left circularly polarised light Measurement of optical rotation is used for the characterisation and quantification of chiral molecules in research and industry Optical Rotatory Dispersion is the variation in optical rotation with wavelength ORD and CD spectra both derive from the interaction of polarised circular or linear light with chiral molecules and are directly related each can be derived from the other using the Kramers Kronig transform Measurements of ORD and CD give essentially the same information but experimentally each technique has been favoured for particular applications CD is the higher resolution technique since it is only observed at wavelengths where the chiral molecule absorbs light This makes complicated spectra involving m
10. rement 1 Select the wavelength at which optical zeroing is to apply If you are working in spectrum mode you may wish to choose a wavelength somewhere in the middle of the required scanning range 2 Load the cuvette with a solvent blank and mount into the cell holder 3 Open the live display window by clicking on the HE icon in the Signal panel 4 To allow live adjustment of the analyser the lid open sensor which stops data acquisition when the lid is open must be defeated To do this open the lid and place a piece of card between the lid sensor in the right front corner of the sample chamber Make sure the PMT HT is less than 400 volts 5 Loosen the coarse rotation locking screw and rotate the analyser housing until the signal trace in the live display window approaches zero mdeg Then retighten the coarse rotation lock Use the fine adjuster to set the signal trace to zero on the live display Close the lid and check that the live display is still at zero 8 lt is advisable at this point to perform a background determination while the blank sample is still loaded see the main Chirascan User Manual for details 9 When the background determination has been performed replace the blank with the sample to be measured Optical rotation measurements can now be performed oe 9 2 4 Calibration For calibration of the CS ORD accessory Applied Photophysics Ltd recommends the use of a sucrose solution in water Sucrose C4 5H25O41 i
11. s available from many chemical companies including Sigma Aldrich product no 5016 and has a specific rotation at the sodium D wavelength 589 4 nm of 66 3 A 100 mg ml solution in a 10 mm pathlength cuvette will thus give an optical rotation of 663 m To perform a calibration ensure that the optical null position has been set as described in Section 2 3 and follow the procedure below 1 On the Sequencer panel of the Pro Data control software select Kinetics mode and on the Monochromator panel input the wavelength and bandwidth at which the calibration is to be performed 2 Onthe Trigger panel ensure that the trigger is set to Internal Collect a background point by clicking the Background button on the Background panel 4 Load the cuvette with a sample having a known optical rotation at the pathlength and wavelength selected 5 Click the Calibrate button in the Background panel The ORD Calibration dialog box will appear Figure 2 5 d Document 4207Q112C03 01 Page 14 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual ORD Calibration Desired ORD value mdeg 653 Calibration Value I Measure k Cancel Apply Figure 2 5 the ORD Calibration dialog box 6 Enter the known value of the optical rotation in m for your sample in the Desired ORD Value box The default value is 663 for a 100 mg ml sucrose sample in a 10 mm pathlength cuvette see below 7 Click
12. s tables or references to Sections or Figures in the text and sources To follow a link place the cursor over the reference and use CTRL click Hyperlinks in the text are indicated by underlined blue font Document 4207Q112C03 01 Page 8 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual CONTENTS WzztedNIc BeGB Is 4 HAZARD AND OTHER INDICATORS ssssssssseeseeeeeeeeennnnnnnnnnn nnns nnnnai nnn n niis s ssnsa aris sisi sssesa iiis sri ss sn saa arn n nnns 5 ESSENTIAL SAFETY INFORMA HON zucchini cease te nue iate E aaa esp pin dese use gated nue ades et sd dcum hd duae 6 CS ORD INSTALLATION AND OPERATIONAL REQUIREMENTS sesessseseeeeeeeeeneen nennen nennen nnn 7 150 10 8 eepza 9 sie D 9 IM NN esdes 10 1 t Optical rotatory dispersi n MT m m T 10 T 2 Operating PNNGIDISG WOMEN 4eueoeeeaesee 11 2 INSTALLA HON Daana 12 2 1 MOUNUNG the d teClo RERO Rom 12 2 2 eei siio ailes eco S sorsra a ae ia EO ER 13 23 L00 Seung ODUCal AUN TE P 13 2A MO AO IA sect cers dees A EE E A
13. ultiple absorption bands easier to interpret Alternatively ORD measurements can be obtained at wavelengths where the chiral molecule being measured does not necessarily absorb light This means that ORD measurements can be made on materials with high concentrations or where absorption bands are obscured by solvent or buffer salt absorption Document 4207Q112C03 01 Page 10 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual Ny 98etrags8vqy 0 3 220 240 260 280 300 320 340 360 380 Wavelength Figure 1 2 overlaid spectra of Absorbance blue CD red and ORD green of the same solution of CSA The relationship between absorbance CD and ORD is illustrated in Figure 1 2 which shows overlaid spectra of Absorbance blue CD red and ORD green of the same solution of 1S 10 camphorsulphonic acid CSA at a concentration of 0 92 mg ml in water using a 1 cm pathlength cuvette The measurements were made using a Chirascan in CD mode and with the CS ORD accessory 1 2 Operating principles The CS ORD accessory enables the measurement of optical rotation circular birefringence on the Chirascan spectrometer The technique makes use of the existing optics in the Chirascan i e the main monochromator and photoelastic modulator to modulate the polarisation state of the light hitting the sample On older Chirascan spectrometers ORD measurements required a different detector from that used f
14. user of this document is familiar with the operation of the Chirascan or Chirascan plus and with Applied Photophysics Pro Data software In particular it is assumed that the user is familiar with the hazards associated with the operation of the spectrometer and has read the safety information contained in its User Manual Note that the CS ORD is normally used with the CS PCS single cell temperature control accessory and the some of the images of the interior of the spectrometer Sample Handling Unit show this unit in place The information in this document is subject to change without notice and should not be construed as a commitment by Applied Photophysics who accept no responsibility for errors that may appear herein This document is believed to be complete and accurate at the time of publication and in no event shall Applied Photophysics be held responsible for incidental or consequential damages with or arising from the use of this document COPYRIGHT 2012 APPLIED PHOTOPHYSICS LTD ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF SHALL NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER THE SOFTWARE PROVIDED WITH THE CHIRASCAN AND ITS ACCESSORIES PRO DATA CHIRASCAN PRO DATA VIEWER ETC IS THE PROPERTY OF APPLIED PHOTOPHYSICS LTD APL AND IS SUPPLIED UNDER LICENCE APL IS WILLING TO LICENSE THE SOFTWARE ONLY UPON THE CONDITION THAT THE LICENSEE ACCEPTS ALL THE TERMS CONTAINED IN THE LICENCE AGREEME
15. water and purge gas supplies Ensure that the soft tip of the analyser is in contact with the sample holder and tighten the detector locking ring The mounted accessory should appear as in Figure 2 3 Figure 2 3 CS ORD accessory correctly assembled in the sample chamber 2 2 Software configuration On the Signal panel of the Pro Data Chirascan control software select Optical Rotation from the drop down list Figure 2 4 Signal res Options AutaPM Absorbance Phili I Fluorescence Baseline Auto Subtraction Calibration Figure 2 4 the Signal panel 2 3 Zero setting optical null Before beginning optical rotation measurements it is important that the analyser polariser is correctly aligned so that a zero rotation signal is measured when there is blank sample present The CS ORD analyser housing provides both a coarse and fine adjustment Figure 2 2 and features in the software have been provided to facilitate the zero setting process Document 4207Q112C03 01 Page 13 of 15 Applied Ultrasensitive Spectroscopy for the Life Sciences CS ORD Accessory User Manual Note that the zero setting is exact only at a single wavelength since the cell birefringence and optical imperfections that result in baseline artifacts in ORD measurements are wavelength dependent Background correction should therefore also be used when making ORD measurements the same cuvette being used for zeroing baseline determination and sample measu
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