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Applied Photophysics Chirascan LD Скачать руководство пользователя страница 15

Document 4207Q258 V3.00 January 2018

Page 15 of 30

Chirascan Couette Cell LD Accessory User Manual

Enter the settings as in Table

Signal

Linear dichroism

Mode

Spectrum

Wavelength range

700 to 200 nm

Wavelength step

5 nm

Bandwidth

1 nm

Time per point

1 second

Table 2.1: spectrometer settings for determining the LD calibration factor

With the reference plate holder in the neutral position (Figure

), name the spectrum appropriately, e.g. “

LD

neutral

” and click

Acquire

to run an LD spectrum. Move the plate holder to the vertically up position: the

handle should be pointing up, and the short peg seated in the groove. N

ame the spectrum e.g. “

LD vertical

up

”, run a spectrum and repeat for the other three positions.

Figure 2.6: The reference plate holder in the neutral, vertical up, and horizontal right positions (left to right)

In the Pro-Data Viewer software, plot the five spectra. The two vertical spectra should be similar to each other
and of opposite sign to the two horizontal spectra, which should also be similar to each other.

Average the two vertical spectra and the two horizontal spectra, set the neutral spectrum as the baseline and
subtract the baseline from the two averaged spectra. One of these spectra should be positive and the other
negative. Multiply the negative spectrum by minus 1 using the Pro-Data Viewer software to convert it to positive
and average the two spectra which are now positive. The accepted absolute LD value at 250 nm is 0.03006,
and by convention the orientation direction is taken as being the parallel direction.

The LD calibration factor used to calculate the linear dichroism is set in the configuration file

LDConfig.conf

in the folder C:\Program Files (x86)\Applied Photophysics\Chirascan\Config

The file can be adjusted using a text editor such as Microsoft Notepad (Figure

). The calibration factor is

listed as

CalFac

and is shown circled in the figure. You will need administrator rights to adjust the factor.

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Содержание Chirascan LD

Страница 1: ... Business Park Leatherhead Surrey KT22 7BA United Kingdom Tel 44 1372 386 537 Fax 44 1372 386 477 Email info photophysics com Applied Photophysics Inc 100 Cummings Center Suite 440 C Beverly MA 01915 United States Tel 1 978 473 7477 Email info usa photophysics com www photophysics com ...

Страница 2: ...ell LD Accessory User Manual This document contains important safety information Read this document and the Chirascan V100 Spectrometer User Manual before attempting to install or use the Couette Cell LD Accessory Failure to do so could result in death or serious injury ...

Страница 3: ...ory body 18 3 2 2 Temperature control connections 19 3 3 The Couette cell 20 3 3 1 Components of the Couette cell 20 3 3 2 Mounting the rotor assembly 21 3 3 3 Loading the sample and mounting the stator 22 3 3 4 Checking the sample 23 3 4 Making Linear Dichroism measurements using the accessory 23 3 4 1 The Chirascan Pro Data control software 23 3 4 2 The SmartMotor control software 23 3 4 3 The C...

Страница 4: ...rein 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 2018 APPLIED PHOTOPHYSICS LIMITED ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF SHALL NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF ...

Страница 5: ...derate injury OTHER INFORMATORY INDICATORS USED IN THIS DOCUMENT The sign to the left is used to indicate a situation which if not avoided could result in damage to the instrument HAZARD INDICATORS USED ON THE SPECTROMETER OR ITS ACCESSORIES Note that these hazard indicators may be either colored as below or as black and white The sign to the left is a general hazard indicator indicating the prese...

Страница 6: ...PD detectors used with the Chirascan spectrometers operate 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 Do not allow any item including any component of the alignment jig or the a to come into contact with the front end of the detector while the detector is connecte...

Страница 7: ...gen purge gas The Couette Cell LD Accessory has no purge gas requirements additional to those of the Chirascan spectrometer Circulating water For temperature control the Couette Cell LD Accessory requires a circulating water flow rate of about 200 300 ml per minute To achieve this a pressure of approximately 0 2 0 3 bar 3 to 4 5 p s i is needed For further information on suitable circulators pleas...

Страница 8: ... dichroism PMT Photomultiplier tube rad s 1 radians per second a measure of angular velocity rps revolutions per second a measure of rotational speed HYPERLINKS This document contains hyperlinks between references for example the Contents 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 tex...

Страница 9: ... may be induced for example by stretching a polymer film by the application of an electrical field or by alignment of deformation in a shear field The accessory uses a Couette cell to generate a shear field The sample is contained in the annular gap between two concentric quartz cylinders the outer of which the rotor is rotated about its cylindrical axis while the inner the stator is stationary Th...

Страница 10: ...n in blue the light path in violet The black arrows indicate the standard direction of rotation of the outer cylinder which is anticlockwise counterclockwise when viewed from above Figure 1 3 Schematic of the Couette cell top view is shown on the right with the light path in violet The sample is therefore sheared between the two cylinders in the cell at a rate proportional to the angular velocity ...

Страница 11: ...omogenous as possible so that all parts of the sample are experiencing almost the same shear rate and the error introduced by using a single value shear rate is minimized A coincidence The factor Ro Ro Ri is equal to 19 1 for a 9 55 mm diameter rotor and a 0 25 mm gap Since the conversion from revolutions per second rps to radians per second rad s 1 is to multiply by 2 the conversion factor from r...

Страница 12: ...ear as in Figure 2 1 Figure 2 1 The interior of the sample chamber after removal of accessories 2 2 LD calibration The accessory needs to be calibrated before being used for linear dichroism measurements If the accessory is supplied with a new spectrometer then the calibration will have been carried out by the Applied Photophysics Production Department as part of the build and testing process If i...

Страница 13: ...onent of the alignment jig or the accessory to come into contact with the front end of the detector while the detector is connected and powered up as this may cause an electrical short leading to serious injury to the user and damage to the detector Install the alignment jig as in Figure 2 4 Place the mounting plate of the jig over the support plate in the sample chamber The plate seats down with ...

Страница 14: ...uette Cell LD Accessory User Manual Figure 2 4 The alignment jig mounted in the spectrometer sample chamber Select Linear Dichroism from the drop down list in the Signal panel of the Chirascan control page Figure 2 5 Figure 2 5 The Signal panel for Linear Dichroism ...

Страница 15: ...tra The two vertical spectra should be similar to each other and of opposite sign to the two horizontal spectra which should also be similar to each other Average the two vertical spectra and the two horizontal spectra set the neutral spectrum as the baseline and subtract the baseline from the two averaged spectra One of these spectra should be positive and the other negative Multiply the negative...

Страница 16: ...libration factor is calculated as New calibration factor present calibration factor x 0 03006 measured LD value By convention the parallel direction for the Couette cell is taken as being the flow direction i e the horizontal direction of the alignment jig If the LD calibration factor gives the correct absolute value but the incorrect sign change the sign of the calibration factor Input the new fa...

Страница 17: ...h a viewing window so that the sample can be seen during and after loading to ensure that filling is correct and the sample is free from bubbles The sample is illuminated by an LED controlled by a rocker switch and the light is reflected upwards by an angled mirror so that the sample can be seen If required the temperature of the accessory can be controlled by circulating water that passes through...

Страница 18: ...onding pegs on the support plate When the plate is seated down secure it in place by tightening the two captive thumbscrews positioned at the right front and left rear of the mounting plate The detector can now be re installed The snout of the detector slides into the cowling which rests against the accessory body The cowling has a gas purge inlet which connects to the Y connector at the rear of t...

Страница 19: ... outside this range unless the standard fittings have been replaced with high temperature alternatives by Applied Photophysics There is no direct temperature control on the Couette Cell Accessory However the temperature can be controlled by an external a water circulator with a flow rate of about 200 300 ml per minute To achieve this a pressure of approximately 0 2 0 3 bar 3 to 4 5 p s i is needed...

Страница 20: ...ette cell 3 3 1 Components of the Couette cell The demountable Couette cell consists of a rotor assembly and a stator assembly The rotor assembly is made up of the quartz outer cylinder a removable PEEK plug and a PEEK centralizing bush as shown in Figure 3 5 Figure 3 5 The rotor assembly dismantled left and assembled right Centralizing bush Cylinder Plug Circulating water connections ...

Страница 21: ...and assembled right 3 3 2 Mounting the rotor assembly Assemble the components of the rotor assembly as in Figure 3 5 and mount the assembly into the well in the accessory body as shown in Figure 3 7 it should press down into place without the use of strong force When it is fully seated down the top of the assembly should not be above the upper surface of the accessory body Figure 3 7 The rotor ass...

Страница 22: ...hat the cylinder is secured in the assembly there is no need to use strong force all that is needed is for the cylinder not to slip in the collar Insert the inner cylinder gently into the outer cylinder but do not screw down the clamping plate at this point Do not screw down the clamping plate unless the locking collar is released This is to avoid the stator being forced against the rotor plug on ...

Страница 23: ...page select Linear Dichroism Figure 3 9 From then on operation of the instrument is similar to that when making CD measurements for details of which see the main Chirascan User Manual Remember that if you intend to measure the absorbance simultaneously with the LD spectrum then you need to take an air baseline with the Couette cell removed and then to measure the absorbance of the buffer at zero s...

Страница 24: ...indicates no command sent The toolbar icons are used to set the displayed features they also duplicate some of the functions on the Command Pane The Caption bar indicates the current Rotational Speed rps and Shear Rate 1 s The Status bar indicates the status of the motor Ready Running or Stopped 3 4 3 The Command Pane Entries are made on the Command Pane Figure 3 11 Figure 3 11 The Command pane En...

Страница 25: ...un clockwise by ticking the Reverse box To start the motor click the Start button When the Caption bar shows that the set velocity has been reached begin the data acquisition on the Pro Data Chirascan control page To move to another velocity enter the required velocity in the Max velocity box and click the Start button To stop the motor click the Stop button The motor will decelerate to zero at th...

Страница 26: ...nel Figure 3 9 An important point to note is that any LD response will distort the CD signal giving an incorrect CD measurement Always check that there is no LD response when making a CD measurement while shearing 3 5 2 Making a CD measurement using a rectangular cell CD measurements can be made using a short pathlength 1 mm rectangular cell without removing the whole accessory from the spectromet...

Страница 27: ...shear rates the flow regime may become turbulent The transition from laminar to turbulent flow is described by the Reynolds number Re which is the ratio of inertial to viscous forces i e Re γ R2 where is the density and is the viscosity of the liquid The value for the critical Reynolds number for the Couette system is given in some textbooks as greater than 50 000 4 but this is a value calculated ...

Страница 28: ...n Table 3 1 Figure 3 13 LD spectra for calf thymus DNA at various shear rates Sample Calf Thymus DNA Concentration 0 13 mg ml Buffer Water Wavelength range 180 to 360 nm Wavelength step 1 nm Bandwidth 1 nm Time per point 0 5 s except shear rates 120 and 240 s which were 1 s Pathlength 0 5 mm total Temperature Ambient 20 5 C Repeats 3 shear rates 0 to 30 s 1 2 shear rates 60 to 10 200 s 1 Shear rat...

Страница 29: ...ith any part of the accessory body Do not clean the body with organic solvents Use only a soft cloth and water or a mild detergent solution The Couette cell can be removed from the accessory body and dismantled for cleaning Clean the quartz components in the same way as a standard quartz CD cell 3 9 References 1 L Velluz M Le Grand and M Grosjean Optical Circular Dichroism Verlag Chemie 1965 page ...

Страница 30: ...or corrosive chemicals to come into contact with the Couette Cell LD Accessory as these may cause damage to the instrument clean up any spillages immediately To clean the Accessory use a soft cloth with a dilute solution of mild detergent in water The Couette Cell LD Accessory requires no special maintenance or storage conditions Keep the Accessory clean and dry remove the sample after use and sto...

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