Applied Photophysics Chirascan CD User manual

2.1 Updated January 2006
ADDING
NEW
DIMENSIONS TO KINETICS
Chirascan
Quick Start
This guide provides an introduction to the use of the
Chirascan CD Spectrometer.
It covers the running of simple experiments
and the basic use of the software.
ADDING
NEW
DIMENSIONS TO KINETICS
Applied Photophysics Limited
203/205 Kingston Road
Leatherhead KT22 7PB
United Kingdom
Tel: [phone]
(USA) 1- 800 543 4130
Fax: [phone]
Email: [email protected]
URL: www.photophysics.com

2.2 Updated January 2006
Contents
2.0 Introduction................................................................................................................3
2.1 Purging.......................................................................................................................3
2.2 Power-up....................................................................................................................4
2.3 Launching the software..............................................................................................6
2.3.1 Measuring a CD baseline and spectrum..................................................................7
2.3.1.1 The CD baseline...................................................................................................7
2.3.1.2 The CD spectrum...............................................................................................10
2.4 Viewing and manipulating spectra and traces..........................................................11
2.4.1 Viewing spectra ....................................................................................................12
2.4.2 Comparing spectra................................................................................................13
2.4.3 Manipulating spectra.............................................................................................14
2.4.4 Example of spectrum manipulation ......................................................................21
2.5 Saving data...............................................................................................................26
2.5.1 Converting data for use with third-party programs...............................................27
2.6 Printing.....................................................................................................................28
2.7 Operating notes and hints.........................................................................................28
2.7.1 Selecting Step Size (SS)........................................................................................28
2.7.2 Selecting Spectral Bandwidth (SBW)...................................................................28
2.7.3 Selecting pathlength and concentration.................................................................29
2.7.4 Selecting time per point ........................................................................................29
2.7.5 Nitrogen purge flow-rate.......................................................................................30
2.7.6 Measuring protein spectra.....................................................................................30
2.8 Example spectra.......................................................................................................31
2.8.1 Alcohol dehydrogenase.........................................................................................32
2.8.2 Bovine serum albumin..........................................................................................33
2.8.3 Cytochrome C.......................................................................................................34
2.8.4 Lysozyme..............................................................................................................35
2.8.5 Vitamin B12..........................................................................................................36
2.8.6 Tris(ethylenediamine) cobalt chloride ..................................................................37
2.8.7 Camphor sulphonic acid........................................................................................38
2.8.8 (R)-3-methylcyclopentanone ................................................................................39
2.9 Troubleshooting.......................................................................................................40
2.10 Notes......................................................................................................................41

2.3 Updated January 2006
2.0 Introduction
APL recommends that new users read this guide to familiarise themselves with the basic
operation of the instrument. The operations covered are start up of hardware and
software, use of the Chirascan instrument control and data acquisition software to
collect CD spectra and baselines, use of the graphics software to carry out common
operations on the data and to save data appropriately and conversion of data formats for
export to third-party programs.
2.1 Purging
Removal of air from the light path by purging with nitrogen is essential, not
optional. Failure to purge the instrument will result in the formation of ozone,
which is damaging to health and can cause deterioration of the optical components
in the instrument.
The instrument is purged using clean nitrogen that is distributed to the instrument via a
manifold that contains three gas flow controllers (see picture below). The gas pressure
regulator of the master supply should be set in the range 2bar < P < 6bar, in accordance
with the manufacturer’s recommendation. At first start-up, set the gas flow as high as
the supply pressure allows, with most flow through the monochromator (middle) flow
controller, and let the purge to continue for at least 15 minutes prior to attempting to
ignite the lamp (see next section). After this initial, high-flow purge, the controllers can
be set at 1 Lmin-1 -1 -1
, 3 Lmin and 1 Lmin for the lamp housing, monochromator and
sample housing respectively.
Three rotameters (gas-flow controllers) control nitrogen flow to the lamp housing,
monochromator and sample housing respectively, from left to right.

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The lamp housing and monochromator are hermetically sealed, which means that once
the initial purge is complete, you should not need to repeat the high flow-rate purge,
unless the hermetic seals are broken (during service or lamp replacement) or if the
instrument has been left idle for many days.
NB It is essential that the nitrogen used to purge the instrument be clean. APL strongly
recommends the use of a combined molecular sieve (X13) and activated charcoal gas
trap, which will remove from the nitrogen stream all common impurities, which can
otherwise deposit on the internal optical surfaces. The cleaner the nitrogen, the longer
your Chirascan will maintain its far-UV performance.
2.2 Power-up
Ensure that the instrument is connected to the electricity supply and that it has been
purged according to Paragraph 2.1. Put the two black rocker switches (System and
Lamp) on the front panels to the on (I) position. The instrument will automatically
check its zero positions (you may hear the monochromator motors moving). Ignite the
Xe arc lamp by pressing the red Start button on the lamp control panel no sooner than
20 seconds after the lamp power supply has been switched on – this is to allow
sufficient time for the capacitors to charge up. If ignition is successful, the translucent
panel on the source housing adjacent to the control panel will glow yellow; if you do
not see a yellow glow, press the red Start button again; repeat until ignition is achieved.
The lamp will require at least 15 minutes to stabilise following ignition. Finally, ensure
that the computer, its monitor and printer are all powered up.
The System panel

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NB Once the lamp is struck, it is strongly recommended that it be left on for the
duration of the work planned for the day. Repeated powering up and powering down
will lead to instabilities (see previous paragraph) and will reduce the life of the lamp.
However, if it is necessary to restart the lamp after running, it must be allowed to cool
for about 15 minutes before re-ignition is attempted.
The light-emitting diodes (LED) on the System panel are labelled Status, Tx and Rx; Tx
and Rx will illuminate when commands are being transmitted or data is being received
respectively; the green Status LED should be constantly illuminated – if it is flashing, it
indicates that one or more of the electronics self-tests has failed. If this happens, try
restarting the electronics and if the problem persists, please contact APL technical
support.
The Lamp panel
The red LED on the Lamp panel indicates that power is present. The digital timer
records the number of hours that the lamp has been running; adjacent to the timer is a
recessed reset button: the timer must be reset when the lamp is changed.
On shut-down, it is recommended that only the lamp be extinguished by putting the
black rocker switch to the off (O) position. The electronics can be left switched on quite
safely. Once the lamp is extinguished, the nitrogen should be left on for a few minutes
while the lamp cools and because the lamp housing and monochromator are
hermetically sealed, there will be little or no loss of purging integrity overnight or even
over a weekend. Resuming normal nitrogen flow for 10 minutes after a weekend
shutdown is usually sufficient to re-establish a fully purged light path.

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2.3 Launching the software
On starting the computer, among other icons on the desktop, you will see two associated
with the Chirascan. They are called Chirascan and Chirascan Viewer. Double-click on
the Chirascan icon to launch the Pro-Data interface.
The Pro-Data interface
The Pro-Data interface enables you to configure the instrument and to manage
experiments. It is divided into a number of sub-panels - Signal, Baseline, Sample
Handling Unit, Monochromator, Sampling, Spectrum and Progress - in which are
grouped the parameters relevant to the control or management of that sub-panel’s

2.7 Updated January 2006
function. In addition, there is an unlabelled panel for controlling shutter function and
detector high voltage.
2.3.1 Measuring a CD baseline and spectrum
NB When you put a sample into the sample chamber, it is inevitable that a small
quantity of air will be introduced to the low-volume light path within the purge tubes in
the sample chamber. It takes only a few seconds to re-establish an air-free environment
and you should not need to wait longer than that before starting your measurement.
2.3.1.1 The CD baseline
Every CD spectrometer has a CD baseline, caused by the interaction of circularly
polarized light (CPL) with the slight birefringence found in the PMT detector window
and to a lesser extent in the cuvette. To see the true CD spectrum of your sample, this
baseline must be measured and subtracted from the CD spectrum of the sample.
To measure the CD baseline, you must measure the CD spectrum of a blank. The blank
must be identical in every way to the sample that you want to measure, except that the
sample itself is absent. For example, if you intend to measure an aqueous solution of
lysozyme in a 0.2mm path-length rectangular cuvette, then the CD baseline is
determined using the same cuvette containing only water.
Signal panel in the Pro-Data interface
In the Signal panel, there is a drop-down menu that contains the main data collection
modes: choose Circular Dichroism. In Options, choose either delta A (delta absorbance)
or millidegrees, depending on the units in which you wish to express the CD
measurement. Select Absorbance if you wish to simultaneously measure the absorbance

spectrum of the sample. Note that Circular Dichroism, millidegrees and Absorbance are
selected by default.
Monochromator panel in the Pro-Data interface
Set the lower and upper wavelength limits of the scan range for your experiment in the
Monochromator panel of the Pro-Data interface – these will normally be the same for
both CD baseline and CD spectrum. For example, set Low to 180nm and High to 260nm
if you are measuring a typical protein. Set the wavelength step size in Step, typically
1nm or less, and click on Set. To the left of the monochromator wavelength bar, set the
required bandwidth. Typically, a bandwidth of 1nm is suitable for protein CD
spectroscopy.
Sampling panel in the Pro-Data interface
In the Sampling panel, set the time per point to the desired value. An approximate scan-
time is shown, which is approximately equal to the product of the time-per-point and the
scan range divided by the step size. Note that the scan time will have a direct effect on
the quality of the spectrum. If you are happy with this, with the blank in place in the
sample holder, go to the Signal panel and click on Baseline. A CD baseline will now be
measured; the display and data manipulation program Pro-Data viewer will
automatically start and the data will be displayed; the data are stored as a file called
Baselinexxxx.dsa (unless a different seed name has been specified – see later) in your
default working directory. The Auto Subtraction tick box becomes active at the
conclusion of a baseline scan: if you wish to subtract the baseline automatically from
subsequently measured CD spectra, tick Auto Subtraction.
NB. If Pro-Data viewer has been launched from the desktop independently of Pro-Data
itself by clicking on its desktop icon, then Pro-Data will not know of the existence of
the viewer – it is a separate program – and Pro-Data viewer will not respond to the start
of an experiment in the normal way.
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In general, when working at the instrument, it is recommended that you launch Pro-Data
Viewer from Pro-Data using the icon in the toolbar. This will ensure that local
communication is established, that Pro-Data viewer will work correctly as a real-time
display and that the data will be saved to a DataStore in the current working directory.
Baseline CD spectrum
You do not need to use the default name for the baseline – you can specify a seed name
by clicking on the Seed Names icon in the toolbar of the Pro-Data interface and
specifying seed names for the baselines and the spectra.
.
Type in seed names of choice
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2.10 Updated January 2006
2.3.1.2 The CD spectrum
Once you have measured the CD baseline, replace the blank with your sample. As the
conditions for the measurement of the sample will be identical to those for the blank, go
straight to the Spectrum panel of the SPC and click on Start. The CD spectrum will be
recorded under the conditions applied to the blank and stored as a file called
Spectrumxxxx.dsa, unless you have specified a different seed name (see previous
paragraph).
The program Pro-Data viewer will automatically display the data as it is collected; an
example is given below. Double-clicking anywhere in the graphics panel will autoscale
the display.
CD spectrum of lysozyme (raw data)
When measuring any CD spectrum on Chirascan, other properties are simultaneously
recorded. These include the voltage applied to the detector, the temperature and the
absorbance spectrum of the sample. They can be viewed by selection from the Window
menu.