Guide to using the AVIV 62DS CD spectrometer
M.Hanlon, B.A.Wallace
CONTENTS
SECTION
1......General
1.1....Access
1.2....Training
1.3....Booking usage of the CD
1.4....Logging usage of the CD
1.5....Reporting
2......Operation
2.1....Start-up procedure
2.2....Completion of the data form
2.3....Defining parameters
2.4....Saving data files to disk
2.5....Repeat scans
2.6....Shut-down procedure
2.7....Optimizing conditions
2.8....Sample handling
2.9....Simple temperature control
3......Data analysis
3.1....File conversion
3.2....Processing/analysis for
secondary structure
APPENDIX
I.....Example CD users form
II....Example log form
III...Full list of command keys
1. GENERAL
1.1 Access.
Prior approval is required for projects wishing to use the CD instrument.
There is a form available (see appendix I) for this that must be completed
together with a 1/2 page summary and submitted to BAW in order to be
given a
userID and permitted to join the users queue.
1.2 Training.
Currently, every investigator must be designated an 'approved user',
following
a training session. This may be arranged through B.A.
Wallace (BAW).
1.3 Booking usage of the CD.
A diary is available in the Spectroscopy Lab (EB20), situated on the
CD
instrument. Approved users may sign up for a maximum of two days, in
advance.
Fridays are normally reserved for maintenance but may occasionally
be
available.
Unless a start time is stated it will be assumed that a 10am start
is
required. If a user is more than 1 hour late or the CD has not been
booked,
then use is on a first come first served basis.
1.4 Logging usage of the CD.
The CD logbook is situated on top of the instrument in EB20. This must
be
completed and signed after every session. Please make a clear note
of any
problems you encounter in the use of the CD.
In addition the usage of cells must be logged in the appropriate section
of
the logbook. Again, please make a clear note if you cause or notice
any damage
to the cells.
1.5 Reporting.
In December of each year all users will be requested to provide a copy
of all
publications produced which used the Spectroscopy Lab facility, and
a 1/2 page
summary of all the projects done and results obtained must be submitted.
Please note, this is important for our on-going funding status, and
failure
to comply will mean loss of user privileges until submission.
2. OPERATION
2.1 Start-up procedure.
The CD must be powered up at least 30 minutes before beginning experiments.
2.1.1 Turn valve lever no.1 (on top of the nitrogen tank) full on to
start
the nitrogen supply. Adjust the pressure using the blue regulator knob,
until
the indicators at the left front panel of the CD reach the purple marks
(about
20psi on the valve pressure gauge.
2.1.2 Switch on the lamp power breaker at the front centre of the instrument;
turn on the coolant water supply by pushing both yellow valve levers
down.
Check that the pressure indicated by the gauges connected to the blue
filter
unit are reading between 20 and 60 psi. If not, then pressure may be
adjusted
via the green valve to the right of the filter unit.
2.1.3 Nitrogen tank pressure should be checked using the pressure gauge
to
the rear of the tank. A pressure of about 1.2 bar should be aimed for.
Pressure may be controlled by adjustment of valve levers 2 and 3.
THIS MUST BE CHECKED CONTINUOUSLY THROUGHOUT THE RUN
2.1.4 Leave the system to flush through with nitrogen for at least
15 minutes.
2.1.5 The 'lamp ready' indicator should be glowing. If not, check the
water and nitrogen pressures are sufficient
Press the red 'push to
start' button, on the left hand CD panel, to fire the lamp.
The series of red :EDs above it should go on.
2.1.6 Turn on the CPU power breaker at the front centre of the CD. Type
'y'
submit/queue=lj4_116_text user2:[user.xtal1]cdlog.frm
The example data form in Appendix II contains details regarding completion
2.3.1 shift-F8
2.8 Sample handling.
2.9 Simple temperature control.
3. DATA ANALYSIS
3.1 File conversion.
APPENDIX I. EXAMPLE CD DATA FORM
RUN # CD/ABS DISKFILE3__BM10JS1______VAXFILE4_as disk__REPS5__5__
DISKFILE________________VAXFILE__________REPS_______
......................ETC.
1. This field refers to the name of the diskette on which you
APPENDIX II. CD PROCESSING FORM
Name:_______________ Date:___________ Page:_________
Spectrum Name:____________________
Averaged filename (base):_________________________________ # base
PC disk label __________
APPENDIX III. FULL LIST OF COMMAND KEYS
Basic Instrument Functions - The Most Used
F6 ......... Create setup mode, copying from current screen.
Alt B ...... Make this data the Baseline (tags data set with B).
Alt F8 ..... Toggle dynode voltage track mode (prompt A)
Other basic functions.
AVIV Model 62DS CD Spectrometer - List of Functions - V 4.1h - 07/29/93
Key Function Notes:
General Controls
Replot Current Screen - These cause view changes to take effect.
Overlay Another Data Set On the Current Screen
Change Current Data Set - Put some other data in memory on the screen.
Transfer Data To and From Disk
Disk and File Related Functions
Plot and Screen Appearance Controls
Mode Changes - These work in setup mode only - choose mode before starting.
Alt K ...... Enter Kinetics mode. (time on x axis)
Data Recording Start & Stop In All Modes - copies current screen
on start
Controls To Set Up For Data Collection - work on any screen
Controls To Set Up For Data Collection - must be in setup mode
Repeat Collection Controls - current screen is repeat template
Auxilliary Data Sets - data recorded in addition to main data
You may move freely between data sets during recording without loss.
Instrument Manual Controls (some work only in setup mode)
F1 ......... set bandwidth, if B mode on will move slit
Ctrl F5 .... set dynode servo operating level - changes full scale
Temperature Controls (need a programmable bath or thermoelectric holder)
Temp. derivative tolerance for "stable" (deg C/interval).
Maintenance and Troubleshooting Features
in response to the prompt to start the instrument. Also, type
prompted for your subdirectory and log file.
2.1.7 Leave the system for at least 15 minutes to allow the operating
temperature and lamp to stabilize.
2.2 Completion of the user's data form.
Copies of the data form (see Appendix II) may be obtained through the
Department VAX:
of the form.
2.3 Defining parameters.
Make sure you have placed a formatted 3.5in diskette in the drive of
the CD
PC.
The following is a procedure for setting basic parameters only.
It must be done when .setup is shown on the upper right part of the
screen.
This screen can be reached by using PgUp or PgDn or F6.
A fuller list of functions may be found in Appendix III.
Set path for saving files. You must send them direct to your diskette
by
typing: shift-F8
'a:'
check the disk directory with shift-F9 and then return to get back
2.3.2 F3
Set the scan step. Generally, a scan will be run in steps of 0.2nm.
However,
in certain cases such as optimising conditions a step of 1.0nm may
be
preferred as this will be quicker though less accurate.
2.3.3 Set the scale axes.
up-arrow
Set the upper limit of the y-axis.
down-arrow
Set the lower limit of the y-axis.
For most proteins these values will be 20 and -20 respectively.
2.3.4 Set the wavelength range.
Set the lower and upper values for the x-axis (i.e. wavelength)
respectively. NEVER SET THE LOWER VALUE TO LESS THAN 180NM AS THIS
COULD
DAMAGE THE INSTRUMENT.
When done hit
2.3.5 shift-F10
This will start the data recording. Note that this key may also be
used to
stop data recording mid-scan if necessary.
2.3.5.1 Manual scan: to check that the dynode and signal are in the
correct
range, go to wavelength manually with alt-F6. BE CAREFUL NOT TO DRIVE
THE CD TO WAVELENGTH BELOW 190NM OR THE INSTRUMENT MAY BE DAMAGED.
IF YOU MAKE A TYPING ERROR, REBOOT QUICKLY WITH ALT-CTRL-DEL.
2.3.6 During the recording you should note the dynode voltage on your
data
form.
2.4 Saving data files to disk.
After each scan you should save your data files to disk (n.b. you should
have
already specified a path for saving these, see 2.3.3.):
2.4.1 Use PgUp or PgDn to get to the last CD scan. This will be 'scan.ads'
(N.B. the filename of the screen currently displayed is shown in the
top right
of the screen). Use alt-R to rename the data file to that on your data
sheet.
2.4.2 Hit alt-S to save the data.
2.4.3 Hit alt-D to delete that data from the hard disk.
2.4.4 If you have collected an absorption scan at the same time (the
default),
you will find it called 'absorb.ads' when you PgUp/PgDn. Rename it
with
the extension '.abs' using alt-R and save as above.
2.5 Repeat scans.
2.5.1 shift-F5
Set the time between repeat scans if a delay is necessary. Default
is 0 - that
is, the next scan will begin immediately.
When prompted: 'Make non-zero to keep all repeat scans', respond with:
1 ret
When prompted to 'save scan name..' you should provide the name that
appears
on your data form (i.e. of the form bm10js1) but without any extension.
2.5.2 shift-F6
Set the number of repeat scans. Generally 5 scans is chosen as this
allows
anomalous results to be discarded later when processing data (see Section
3).
2.5.3 shift-F7
This keystroke is required for starting data recording in repeat mode.
Data
recording may subsequently be stopped using shift-F7 then alt-F10.
2.5.4 You should save your data to disk after each set of repeats.
You will
not need to rename the scans, and the next scan will appear on the
display
as you delete the previous one. However, you will need to rename the
absorption scan, and rep_avg (the software automatically provides an
averaged
file of your data), if you wish to save them. Generally, it is not
recommended
that you save 'rep_avg' as a mathematically superior average is produced
by
the processing program (Super3). Remember to delete these files too.
2.6 Shut-down procedure.
2.6.1 Check first that you have saved your data files. You can do this
by
checking the contents of your diskette using
remaining files in memory, try to delete them with
saved a file that you attempt to delete then a message will appear
giving you
the opportunity to do so.
2.6.2 Hit ctrl-D to abort the CD program. (N.B Restart is done with
'go60' from the DOS prompt
2.6.3 Remove your data diskette from the drive and switch off the lamp
and CPU power breakers.
2.6.4 After about 10 minutes turn off the nitrogen supply at the tank:
push
all three levers to their closed positions. Turn off the water supply:
push
the two yellow levers all the way up.
2.7 Optimizing conditions.
The optimum CD signal-to-noise ratio will be obtained in the absorbance
range
between 0.5 and 1.5. This is a function of the concentration of your
sample
and the pathlength of the cell chosen. Given a specific concentration
therefore requires an appropriate choice of cell. Selection may be
made either
by recommendation or by carrying out test scans using various pathlengths.
In
practise this involves checking the dynode voltage recorded first for
your
baseline, and if that is o.k., next for your sample. This can be checked
in
manual operation mode as follows.
2.7.1 Check the dynode voltage (this appears on the middle left hand
side of
the screen) for your buffer and all additives at 190nm. This should
not exceed
450. If oit does then decrease the pathlength or choose a different
buffer.
2.7.2 If this is satisfactory, then check the dynode for your sample
at 190
and 195nm. Assuming an adequate signal is obtained then again the dynode
voltage should not exceed 500. If it does then you will need to select
a cell
of shorter pathlength.
N.B. If, when in manual mode you accidentally give a wavelength less
than 180nm or greater than 700, YOU MUST STOP THE PROGRAM IMMEDIATELY
USING
ctrl-D BEFORE THE MACHINE REACHES THAT VALUE OR ELSE DAMAGE WILL OCCUR.
From the above it is clear that for a given pathlength, concentration
may be
altered to achieve an optimum signal to noise.
2.7.3 It is also advisable to note the END dynode voltage (see example
CD data form) for your first scan and your final scan for a particular
sample.
If these values values differ by more than 10, this indicates a problem
such
as air bubbles or precipitation in your sample. This should be rectified
and
the experiment repeated.
2.8.1 If samples are suspected of containing any solid matter then
this should
be removed by filtration or centrifugation, otherwise artifacts due
to light
scattering may occur.
2.8.2 Cells should be cleaned meticulously before, during and after
use.
Rinsing in distilled water may be suitable during use. For non water-soluble
samples the cell may be rinsed with methanol, NEVER USE ACETONE. Cells
should
only be dried with suitable lens tissue. A can of dusting gas may be
used to
remove dust. After use the cell should be immersed in Helmanex cell
cleaning
solution for about 10 minutes, extensively rinsed in distilled water,
dried
and returned to its box, wrapped in lens tissue.
2.8.3 You should use the same cell for sample and baseline scans; the
cell
and cell holder must also be oriented in the same way, relative to
the beam.
The following just describes how to set a temperature for the sample
jacket
on the CD.
2.9.1 Switch on both the temperature control unit and the circulating
bath (located on the bottom right of the CD). Check that there is sufficient
water in the bath (the level should be within 25mm of the brim. There
is a
switch on the bath control panel which should be set for temperatures
less than+40oC
or greater than+40oC as appropriate. The digital readout indicates
the current
temperature of the bath (not the sample jacket). To adjust this, press
the
black button above the display and use the coarse and fine controls.
Note that
this temperature should always be set in the region of 12-15oC, no
matter what
the reqired sample temperature is.
2.9.2 Hit alt-T to enter temperature mode.
2.9.3 Hit shift-F1 to set the required jacket temperature. See the
display
on the left hand side of the screen. Wait until the set temperature
has
reached equilibrium (indicated by '=' to the right of the temperature).
2.9.4 Hit
Using a PC connected to the network insert you diskette and start up
FTP file
transfer protocol:
'ftp' ret
Log in when prompted.
Create a subdirectory in your account on the Department VAX.
create/dir [dirname] ret
Transfer the files:
send drive:dirname\filename.001 [dirname]filename.001 ret
Repeat this for each data file. You can use the up-arrow key to bring
up the
previous command typed and just edit the file extension.
If you have several files to transfer: in FTP, change the local directory
first (that is, to the directory on your diskette where your data files
are
stored:
lcd drive:\directory name ret
then change the remote directory (that is, to the subdirectory you
created
on the VAX):
cd [dirname] ret
Then
mput *.*
You will be prompted to confirm the transfer of each file.
Exit ftp (type bye) and log into your VAX account from a tektronics
terminal
(or PC with tektronics emulation). Set default to the subdirectory
containing
your data:
set def [.directory name]
To convert the files to analysis program format:
dir/col=1/output=z.list
This will create a file with the correct format called z.list containing
all
your data files in alphabetical order. If you only require specific
files to
be converted then follow the above command with the names of the appropriate
files.
Next type:
run user2:[user.xtal1]pctovax4
When prompted for file name enter z.list. Just hit ret when prompted
for
subdirectory.
Note that converted filenames will be terminated with a 'v': e.g.
bm10js1v.001.
When the conversion is complete you should delete then original data
files
and z.list from your subdirectory.
3.2 Processing and analysis for secondary structure.
To run the processing program:
run user2:[user.xtal1]super3
When prompted for a log file name give the parent name of your data
files (of
the form bm10js1). The .log extension will be added.
Type your initials when prompted for your name and select TEK for terminal
type.
3.2.1 All program options are abbreviated to three letter names.
Select ave to average your data files. Enter the number of files to
be
averaged, enter 1 for the baseline shift and YY for filetype. Note
that when
you enter the filenames they now end with a 'v'. Make a note of the
average
option but exclude this file. Note that you can only do this if you
have more
than three repeat scans. On completion respond 1 to put the average
in current
memory. To be on the safe side write this average to your subdirectory.
Use
the wri option and respond 20 for number of residues (this is just
an
arbitrary value) and 4 for number of parameters. When prompted for
a filename
give the parent name with .avg extension. Note, as an alternative to
this
files may be stored in temporary memory using the mem option, however
these
will be lost when the program is exited (or crashes).
3.2.2 Now the averaged spectrum must be smoothed. Use the smo option.
You may opt to smooth the spectrum several times if required, though
in
general one smoothing is appropriate. When prompted for number of points
try
35 as this is typical for a file collected at 0.2nm. Respond 1 to see
the plot
and check that the peaks have not been truncated. Depending on what
type of
terminal emulator you are using you may have to hit
into plot mode. If the peaks have been truncated then repeat, choosing
a
smaller odd number. When you have finished inspecting the plot choose
option
23 to return. Choose 1 to put this result in current memory and write
the file
out using the parameters above (3.2.1) but give a different file extension,
say .smo.
3.2.3 Repeat 3.2.1 - 3.2.2 for the baseline files.
3.2.4 Now the baseline file should be subtracted from the sample file,
using option sub. The sample file comes first. The number of files
to subtract
is 2, the integer for the first is 1 and for the second -1. If you
chose to
write the files out the you will need to use the rea option to read
them back
in. Or if they are in temporary memory then enter the unit number.
3.2.5 Next the files must be zeroed where there is no absorption. To
do
this, find the y average over the wavelength range 263-267nm using
the fin
option. Input the values just stated for the lower and upper values
respectively. The value returned is delta and you should make a note
of this
on your processing form.
3.2.6 This value should be subtracted form baseline corrected file
by
using the add option and inputting the negative of delta.
3.2.7 You can use the plo option to check that you data has been
correctly zeroed. You can also the inv option to look at the 263-275nm
region.
Type 1 to view the data and start the scan at 275nm. Keep typing 1
to scan the
data.
3.2.8 If you are happy with the data so far write the result out using
a .c extension after the parent filename.
3.2.9 Either now or later, if you prefer, the scale of the plot may
be
changed to mean residue elipticity, using the mul option. The constant
for
multiplication is (0.1xMRW)/(conc. x pl).
MRW is the mean residue weight: that is Mwt/no. of residues; conc should
be
given in mg/ml and pl is the pathlength in cm.
3.2.10 To calculate the percentages of each type of secondary structure
use the lin option. Enter the name of your file (the one with the .c
extension). For data taken at 0.2nm intervals the reference file required
is
user2:[user.xtal1]yangl. For data at 1.0nm intervals use
user2[user.xtal1]newyangs4. Note the average helix length for soluble
proteins
is 8 and for membrane proteins is 20. Use these values unless you know
the
correct value for your protein. If prompted then choose the constrained
analysis. When the analysis is complete you should make a note of the
percentages of secondary structures given for the normalized analysis
(and the
constrained analysis if necessary.). Also make a note of the NRMSD
value. If
this is <0.05 the data has an excellent correlation with the reference
data
set; 0.05 - 0.1 is a good correlation; greater than this and the correlation
is poor. Look at the fit when prompted.
3.2.11 When you have finished make sure you quit the program using
the
exi option.
DO NOT exit the program with ctrl-C as you will create many dummy files
in
your directory.
3.2.12 A log file will be automatically created containing a list of
all
the operations you carried out.
DATE_Todays date DISK1_BPCD1__ PAGE2_1/2
USER_Your initials
1 cd/abs SAMPLE6___myoglobin (Sigma, sperm whale, lot 34h7)__
__in 1uM NaCl pH 6.5__________________________
CONC:(est.)7_1mg/ml (determined)8______ METHOD9_____
^ RANGE10_190-300_ INTERVAL_0.2nm____ TC_____
PATHLENGTH (cm)__0.05______ TEMP11____
DYNODE:12400____ 450____ 500____550____600____END___
SAMPLE______________________________________________
______________________________________________
CONC:(est.)________(determined)________METHOD_______
^ RANGE__________ INTERVAL__________ TC_____
PATHLENGTH (cm)____________ TEMP______
DYNODE: 400____ 450____ 500____550____600____END____
have stored this CD data.
2. Pages should be numbered 1/1, 1/2 as appropriate so all log
sheets for a particular experiment can be tracked.
3. File naming convention:
first character : your initial (register what this will be
with MH);
second character: initial of sample, e.g. myoglobin;
third/fourth numeral(s): day of date;
fifth character: to represent date: a for Jan, b for Feb
etc.
sixth character: This will be B for baseline run or S for
sample.
seventh numeral: indicates which sample or baseline.
4. Generally the vaxfile name will be the same as the disk
file.
5. Number of repeat scans carried out.
6. The sample should be described SPECIFICALLY, including details
such as Lot No.
7,8,9: An estimated concentration should be included, though a
determination may not be required. If it is then the method should
be stated also.
10: The wavelength range selected should be included here,
together with the interval in the following field.
11: A field is provided for recording the temperature, if
temperature control is used, otherwise ambient temperature
is assumed.
12: In the dynode fields the wavelength corresponding to
these dynode voltages should be recorded (see Section 2.7 on
optimisation). In 'END' field note final dynode voltage, before
wavelength returns to its initial value
Sample____________________________________________________
Buffer____________________________________________________
Concentration______________ Ligands_______________________
MRW________________________ Pathlength____________________ Multiplication
Factor_____________________________________
Averaged filename (sample):_______________________________ # sample
files collected__________ used_______ ave shifts file
1:_____________ 5:____________ av. res.=
2:_____________ 6:____________
3:_____________ 7:____________
4:_____________ 8:____________ Smoothed filename
(sample):_____________ # points_______
files collected____________ used_______ ave shifts file
1:_____________ 5:____________ av. res.=
2:_____________ 6:____________
3:_____________ 7:____________
4:_____________ 8:____________ Smoothed filename
(base):_________________ # points_______ Subtracted
filename:______________________________________ Corrected
filename:_______________________ delta__________ Multiplied
filename:_________ Multiplication factor_______ Normalized
against:__________ Normalization factor________
date spectra taken_____________ processed_________________
AVERCD filename________________ scattering file [Y/N]________ CDMIN
filename_________________ lambda shift _____ hl_______ plot [Y/N]___
plot
character_________________________________
lineq results:
range (hl,shift) c? a b t r sum NRMSD
_____________________________________________________________
Alt K ...... Enter Kinetics mode.
Alt T ...... Enter Temperature mode
Alt W ...... Enter Wavelength mode.<>BR> F1 ......... Set bandwidth
for data collection. (Normally the
default is appropriate.)
F3 ......... Set x axis step size for collection. (Normally
0.2nm).
Alt F6 ..... Set recording wavelength in kinetics or temp
mode.
Alt F10 .... Data recording start (and stop) in all modes.
Ctrl D ..... Exit this program.
Print Scrn . Print current screen on printer (a dot for dot
screen copy).
Alt H ...... Help - show this list.
Shift F8 ... See/set current data path.
Shift F9 ... Show disk directory.
Left ....... Set left x limit. (Usually the lower wavelngth).
Right ...... Set right x limit. (Thi sis usually the upper
wavelength). (can extend kinetics run time
during run)
Up ......... Set upper y limit, (usually magnitude of
spectrum). (can rescale any time - data is not lost
Down ....... Set lower y limit. just because it is off
scale.)
Home ....... Replot data using current limits (set by up,
down, etc. above).
Ctrl Home .. Reset to maximum data limits and replot (see all data
points).
Pg Up ...... Show next data set in memory (data sets are in
circular ring).
Pg Dn ...... Show previous data set in the memory ring.
Alt G ...... Go directly to the data set in the ring which you name.
Alt C ...... Perform baseline Correction on this data set (if b is
showing). (For display purposes only, proper correction should
be done during data processing).
Alt D ...... Delete this data set from memory.
Alt L ...... Load a data set into memory from disk.
Alt N ...... Add a Note to this data set (to blank a note
enter a space).
Alt R ...... Rename this data set (after a scan, before saving it).
Alt S ...... Save this data set on disk.
Shift F1 ... Set setpoint temperature (a temp controller must be
present)
Shift F3 ... Toggle temperature setpoint mode (prompt S, data
tagged "TE")
Shift F4 ... Toggle temperature recording mode (prompt T)
Alt F1 ..... Eecute a file of commands. (Ctrl C can interrupt
execution) Alt F3 ..... Execute again the last command
file entered with Alt F1
F7 ......... Set kinetics time constant or wave mode averaging time.
F8 ......... Fast access to files in ring, save, overlay, etc. see
below
End ........ Set y limits to y extrema within current x limits and
replot.
Ctrl End ... Toggle screen x limits common or separate for
each data set.
Alt 2 ...... Show statistics for the data points in view
(n,mean,slope,etc)
Alt 9 ...... Toggle show/average points with same x value (see all
noise).
Abbreviations Ctrl, Shift, Alt mean to HOLD DOWN the named shift key
and tap
the named key following. (I.e. there are 4 cases for some keys.)
"Toggle" controls alternate between two states - like on/off - and
take
effect immediately. Some require an acknowledgement by user.
"Set" controls give a prompt showing the current value and wait for
a new
value to be entered. Press enter without a new value if you do not
want to
make a change. You may use back arrow to correct errors.
Ctrl D ..... Exit program (asks first if all data is not
saved).
Ctrl Left .. Prints current data set on printer (as it would be
on disk).
Print Scrn . Prints current screen on printer (dot for dot
screen copy).
Alt H ...... Help - shows this list.
Shift F8 ... Set default data path (use floppy for all data).
Shift F9 ... Show disk directory.
Right ...... Set right x limit.
Up ......... Set upper y limit.
Down ....... Set lower y limit.
Home ....... Replot data using current limits (set by up,
down, etc. above).
End ........ Set y limits to y extrema within current x limits and
replot.
Ctrl Home .. Reset to maximum data limits and replot (see all data
points).
(View changes also take effect on a data set change.)
(Ctrl End allows selection of a mode where all data sets share the
same x limits, as last set by the operator, and y limits set as by
End
key)
Alt X ...... Overlay with limits of other data view (sets
different).
Alt Z ...... Overlay other with limits of this screen (sets
similar).
(this is a temporary overlay e.g. Home will erase it, also see F8
below)
Pg Up ...... Show next data set in memory (data sets are in
circular ring).
Pg Dn ...... Show previous data set in the memory ring.
Alt G ...... Go directly to the named data set in the memory ring.
Alt L ...... Load a data set into memory (name can include
drive and path).
Alt S ...... Save this data set (asks first if data of same name
on disk).
Alt D ...... Delete data set from memory if saved on disk
(else asks first).
Alt N ...... Add a Note to this data set (to blank a note
enter a space).
Alt R ...... Rename this data set (name can include drive and
path).
Shift F8 ... Set the default drive and data path.
Shift F9 ... Access all disk directories - uses full path name &
wildcards.
Alt 9 ...... Toggle show/average points with same x value (see
all noise).
Alt 0 ...... Toggle zero line on/off (draws/plots line if 0 is
in y range).
Alt = ...... Toggle full screen mode (large, simple plots
without legend).
Alt - ...... Enter a global note for full screen mode (a
Alt N ...... Add a Note to this data set (to blank a note enter
a space).
Ctrl End ... Toggle screen x limits common or separate for each
data set.
F8 special mode subcommands to select display mode:
Alt 1 ...... Select normal screen mode.
Alt 2 ...... Select full screen mode. (same as Alt = toggle
above)
Alt 3 ...... Select overlay with note lines mode.
(F6 ......... create setup mode from current screen)
Alt T ...... Enter Temperature mode. (temperature on x axis)
Alt W ...... Enter Wavelength mode. (wavelength on x axis)
Alt F10 .... toggle scan (single data collection - also see
repeat)
Left ....... set wave or temperature scan left limit
Right ...... set scan right limit (can increase after kinetics
start)
Ctrl F3 .... see/set relative offset in effect. (Enter 0 to
cancel offset.)
Alt N ...... add a Note to this data set (copied to all
descendants)
F6 ......... create setup mode from current screen (to allow
changes)
F1 ......... set bandwidth for data collection
F2 ......... toggle full scale range (5x change) normally 500 md
or 100 md
F3 ......... set x axis step size for collection (negative ok for
temp.)
F7 ......... set kinetics time constant or temp. & wave
averaging time
Ctrl F5 .... set dynode servo operating level - changes full scale
rotation
Alt F6 ..... set recording wavelength in kinetics and
temperature modes
Shift F5 ... set minimum time between start of repeats (minutes) choose
to keep all repeats or just the average
enter name (without extension!) for scans to be kept under
Shift F6 ... set number of repeats to do (can change after
starting)
Shift F7 ... toggle repetitive scan (prompt R) - see Alt F10
(Alt F10 is disabled if R is on, turn off R first to
stop repeat mode)
Alt F7 ..... toggle oplevel (average current) track mode.
(prompt O)
Alt F8 ..... toggle dynode voltage track mode (prompt A)
Shift F4 ... toggle temperature recording mode (prompt T)
Shift F10 .. toggle external data recording (+5v to -5v)
(prompt X)
Ctrl F4 .... toggle slit width recording mode (prompt w)
But, these sets are not buffered like main data set is in kinetics
mode.
Multi-Wavelength Temperature and Changer controls - Ctrl F8
See and select current changer position. (if changer present)
Enable and disable cell positions for kinetics and temperature collection.
(Changer kinetics period set by F3, dwell time at each cell by F7.)
Enable/disable slow rotation of changer while temperature is settling.
Add or delete wavelengths (0-5 allowed) for multi-wave temperature
mode.
Data Manipulation - Operates on data set shown on screen.
Always makes a new set. You delete old data when and if you are ready
to.
The result data set shows all data points (as if Ctrl Home were done).
Alt A ...... Take antilog of this data set - new =
antilog(old+constant).
Alt B ...... Make this data the Baseline (tags data set with B).
Alt C ...... Perform baseline Correction on this data set (if b is
showing).
Alt D ...... Delete data set from memory if saved on disk (else
asks first).
Alt E ...... wEight this data set point by point with another data
set.
Alt F ...... "Flip", convert between Absorbance and %
Transmission data.
Alt I ...... Polynomial smoothing and derivative operations (see
Alt 7).
Alt 7 ..... Polynomial parameters: poly order, window size,
derivative.
Alt J ...... Peak picking using first derivative (see Alt 8 for
controls).
Alt 8 ..... Peak pick parameters: poly order, window,
thresholds, etc.
Alt L ...... Load a data set from disk (name can contain drive and
path).
Alt M ...... Multiply this data set by a constant - new =
constant*old.
Alt N ...... Add a Note to this data set (to blank a note enter
a space).
Alt O ...... Add a constant Offset to this data set - new =
constant+old.
Alt P ...... Add another data set in memory to this one - new =
other+old.
Alt Q ...... Subtract another data set in memory from this - new
=old-other.
Alt R ...... Rename this data set (name can have drive, path and
extension).
Alt S ...... Save this data set on disk under current name
(including path).
Alt U ...... Take logarithm of this data set - new = constant +
log(old).
Alt V ...... Take inverse of this data set - new =
constant/old.
Alt Y ...... Change Y axis label (show new units after
transformation).
Alt 1 ...... Extract data subset in view into a new data set.
Alt 2 ...... Simple descriptive statistics on data in view.
Alt 3 ...... Shift Alt A, M, O, U, V, Y, & 5 to x axis
operation next time.
Alt 4 ...... Up to 4th order polynomial transform - new =
a4y^4+a3y^3...+a0
Alt 5 ...... Divide this data set by a constant - reverse of Alt
M
Alt 6 ...... Divide another data set into this one - new =
old/other
Ctrl F3 .... See/set data offset. (prompt Z shows if offset
non-zero)
F7 ......... set time constant, control idle data smoothing
Ctrl PgDn .. miscellaneous controls, see list below
F5 ......... toggle constant bandwidth mode (prompt B)
Alt F5 ..... go to slit width entered (only if B mode off) - see
F5
Alt F6 ..... go to wavelength entered
rotation
(full scale range shown just above data "FS=xxxx")
Shift F1 ... set setpoint temperature (need temperature
controller)
Shift F2 ... set temperature equilibration time delay (need
controller)
Shift F3 ... toggle temperature setpoint mode (prompt S) (need
controller)
Shift F4 ... toggle temperature recording mode (prompt T)
(data recorded when temperature was controlled are
tagged "TE") Supplemental Temperature Controls
Ctrl F7 .... Temperature control sampling interval (decimal
minutes).
Extra derivative intervals beyond minimum 4 for "settled".
Temp. error iir smoothing filter multiplier (per interval).
Fraction of smoothed error to add to next target. (tracking)
(If setpoint on (S-F3), temperature control offsets bath temperature
a little
to make thermometer read the specified temperature. Else no offset.)
Thermoelectric controls (appear on Ctrl F7 if thermoelectric installed)
Set jacket overshoot temperature (deg C) (to hasten cell settling)
Set jacket overshoot time (sec) (after which correct setpoint restored)
Choose thermometer temperature to show (j)acket, (p)robe, or (s)ink
Enable/disable simultaneous recording of probe temperature
Controls in all modes. (bath or thermoelectric)
Begin scan (tight) temp. tolerance (deg C).
In scan (loose) temp. tolerance (deg C).
Set the maximum rate that setpoint will change at. (can generate ramps)
Alt F2 ..... repaint the screen (recover after error message)
Alt F4 ..... home all motors then return to current wavelength
Alt F9 ..... Energy test mode toggle (prompt E) - move slit to
get 500 V
Ctrl F1 .... set home wavelength (maintenance feature for
calibration)
Ctrl F2 .... set dynode mode (maintenance feature - leave at 0)
set fixed dynode mode setpoint (maintenance
feature)
Ctrl F5 .... set dynode servo operating level - changes full scale
rotation
Ctrl F6 .... set dynode servo integral constant - .2
set dynode servo derivative constant - .15
set dynode servo gain constant - 70 (if
oscillates try 40)
Ctrl PgDn .. access to width error flag criteria
set dynode voltage high and low limits
set number of d/a conversions per servo update, data
limits
set oplevel stringency (tolerance for #, range 0-8)
set error flag stringency (tolerance for *, 0-4. Error
delay)
set whether to return to starting x value after data
collection
set basic full scale range in milidegreees (100)
set ratio of second full scale range to basic
range (5)
set oplevel compensation scale (1) and offset (0) (v/v)
set modulator retardation scale (.0025) and ,
offset (0) (v/nm)
temperature servo integral time
temperature servo derivative time
temperature servo gain
temperature servo enable/disable
Ctrl PgUp .. toggle display of servo error flags (explain * by
data)
flag labels explanation (2 rows under flags, upper is group, lower
is
error)
group - p: pmt dc, s: signal, v: dynode voltage
error - x: constant dynode voltage changed to keep pmt dc within
range
(error flags listed below are stored separately for each group)
u: underrange, signal below measurable range
o: overrange, signal above measurable range
w: data range too wide (may be excess noise)
h: some conversions high (may be excess noise)
l: some conversions low (may be excess noise)
The dynode voltage l and h flags mean that it is outside normal
range
A # by the data indicates that the pmt dc level is out of range.
A servo attempts to maintain the pmt dc level at the given setpoint
by
changing the dynode voltage. The pmt dc level is normally about 1
volt,
but it can be changed with the Ctrl F5 key while in setup mode.
It is normal for the # to flash while the dynode voltage is adjusted
to compensate for changes in absorbance or energy. A persistently high
pmt dc level may indicate a light leak.
Either a # or a * will inhibit data collection in wave or temp modes.
Recording WILL continue in kinetics mode, but the DATA MAY NOT BE VALID.