Transcript Document

Calibration Tutorial: Discovery32
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Tutorial Objectives:
•
•
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Review basic features of DataViewer
Obtain calibration constants from a
external BSA protein standard
Get constants using RI/15deg/90deg
Get constants using UV/15deg/90 deg
Brief intro to Rh data
1.
2.
3.
4.
5.
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7.
8.
Name
Phone no.
Company
CDROM version
Software version
Computer Operating System
PDI Hardware being used.
Brief description of the problem.
Or PHONE
Ph. 508-520-8765
Fx. 508-520-8772
062801wrj-004
Experimental Conditions
Standard: BSA (2 mg/mL)
cat# Sigma P-0834
Column: YMC-Pack Diol-300, 300 x8 mm ID, 5 um silica
cat.# DL30S05-3008WT
Eluent: Phosphate buffered saline
120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer
Sigma Diagnostics #1000-3
Flow rate: 1 mL/min
Injection vol.: 100 uL
Order of detection: FIRST UV @ 280 nm SECOND LS (15 deg, 90 deg, Rh) THIRD RI
The light scattering detectors were built inside a Waters 410 RI ( PD 2020DLS inside)
The UV was a Waters 484 Tunable Absorbance Detector which was plumbed to the Waters 410 with the shortest
0.007” ID peek tubing . A PDI low dispersion inline filter was placed between the column and the first detector. The inline
Filter was fitted with a 13 mm diam., 0.2 um Supor membrane.
Data acquisition: The UV channel was assigned to the the spare input channel. Acquisition Rate was 1 Hz
(sample interval 1 second), with a collection fraction of 0.1.
Backgrounder
This tutorial is intended to be a quick “graphically oriented” approach to obtain calibration constants for your
HPLC/Light scattering (LS) detection system. The separation mode is typically based on size exclusion and is
typically called size exclusion chromatography (SEC) for aqueous based columns and solvents and gel
permeation chromatography (GPC) for non-aqueous based solvents and columns.
The following slide lists the key attributes of the calibrant and the chromatographic system that contribute to
reliable calibration constants. These detector constants are required, along with knowing the response factors for
the concentration of the standard for determining accurate molecular weights and their distributions when using
laser light scattering detection.
Although there are several approaches that have been implemented for calibration of laser light scattering
detectors, the PDI detection system uses the external standard approach as it is straight forward and can be
done with eluents that are compatible (if not identical to your actual running method). Even if you selected a
standardization mode that was chemically dissimilar to your actual running method. [ e.g. calibration was
performed using a polystyrene standard on a GPC column running tetrahydrofuran (THF) and the actual running
method was running a SEC column with a phosphate buffered saline (PBS) eluent ] the calibration constants
would virtually be the same (provided the conditions on the following page were observed).
This tutorial will first provide an overview of the new Discovery32 user interface which is dramatically different to
the former PDI data analysis program “PrecisionAnalyze”. Calibration for RI and LS is performed first with a
bovine serum album (BSA) chromatogram, followed by UV and LS.
The approach is to select your calibration mode, establish baselines for each of the traces, integrate the
standard peak and determine the calibration constants.
What constitutes a good standard?
The Standard
1. The material is of known Mw
2. Known concentration
3. Monodispersed (Mw/Mn 1.00 to 1.01)
4. An isotropic scatterer*.
5. Should preferably be free of aggregations
6. The dn/dc of the standard is required (if not known, it can be determined using the RI detector)
7. The extinction coefficient of the material at the monitoring wavelength is required when using UV as the
concentration source.
The Chromatography
1. Standard should chromatograph as a single peak.
2. Recovery of standard should be 100%
3. Enough material must be injected to obtain good signals from both LS and concentration source detectors.
4. If aggregates exist, the standard peak must be resolved from the aggregations.
5. The standard peak must also be resolved from terminal peak (small Mw impurities).
The Eluent
1.The refractive index of the eluent at the temperature of the detector cell is required when using 15 degree LS in
the Mw calculations.
2. The viscosity in (poise) of the eluent at the temperature of the detector cell is required for Rh determinations.
*The Mw should be large enough to generate a sufficient light scattering signal but not so large that the 15 and 90
degree signals are not of equal intensity. For globular proteins non-isotropic scattering begins as the material
approaches 1 million daltons.
Typical protein standard for aqueous SEC: bovine serum albumin (66.5 kD)
Typical room temp GPC standard for THF solvents: polystyrene (96.4 kD polydispersity 1.01)
Procedures for Calibration (Get raw data for standard)
UV trace for peak standard
(first detection series)
Response
15 and 90 degree peak for
the standard (simultaneously
obtained and second in
series)
RI trace of standard peak (last
in series because of cell
backpressure limitations)
These peaks are offset by the amount of inter-detector
volume between the cells. The time differential is
dependent on the flow rate of the eluent, the volume
remain constant as long as the tubing connections
between detectors is not changed.
Time (Elution volume)
Outline of Procedures for Calibration (Establish baselines)
UV trace
15 degree static trace
Response
90 degree static trace
RI trace
Establish baselines for each of the peaks
Baseline for UV
Baseline for 90 degree static
Baseline for 15 degree static
Baseline for RI
Time (Elution volume)
Outline of Procedures for Calibration
(Get Interdetector volumes)
Offset zero (reference: LS detectors)
Neg. offset for UV
Pos. offset for RI
Response
After baselines the inter-detector volume (IDV) is
automatically determined by the “det. Cal constants”
routine. The algorithm requires baselines and integration
region encompassing the one concentration source
detector and one or more of the LS detectors. UV/LS and
RI/LS are determined separately as they have different
integration regions.
Integration Region for UV and LS
Integration Region for RI and LS
Time (Elution volume)
Outline of Procedures for Calibration
(Apply newly determined IDV’s)
Response
Offset zero (reference: LS detectors)
When the IDV have been properly determined the
concentration source detectors line up with the light
scattering detectors.
Time (Elution volume)
Response
Outline of Procedures for Calibration
(Regionally height normalize)
To better visualize the alignment , the traces
can be regionally height normalized .
Time (Elution volume)
Launch the program after installing onto computer
Double Click the Discovery Icon to launch program
Or
from the short cut found in
Start/Programs/PrecisionDetectors/Discovery32
DataViewer: Selecting a Directory
Discovery32 opens up into a DataViewer window. This
window allows you to ascertain running conditions for the
chromatogram and to get a preview of the files without
having to open each one up for examination. You may
select the directory to preview by clicking the “Change>>>”
button. A dialog box will appear where you may select the
directory of choice. The path of the directory will be shown
in the box to the right of the “Change>>>” button. You must
use the “Change>>” button to select a directory instead of
typing in the path box.
DataViewer: Previewing a File
This line was highlighted simply by clicking once on
the name. The chromatogram with all of the
collected channels of data are shown in the small
preview window (upper right hand corner).
DataViewer: Sorting Files
Date and time the file was collected.
Info description “sample”, “eluant”, “info”
etc are found starting here and scrolling
right..
Files may be sorted by
filename, eluant, sample
name, dn/dc or by date. The
run conditions for each file are
found in a scrollable row.
RLHSC represents the channels
collected
R = Rh (not RI)
L = (low) = 15 degree
H = (high) = 90 degree
S = (spare) = UV
C = (concentration) = RI
DataViewer: Tool Bar and Status Bar
TOOL BAR
STATUS BAR
The Tool bar and Status bar may be enabled or disabled
for viewing.
DataViewer: Changing Location and Shape of Toolbar
TOOL BAR can be
dragged from it’s
standard position and
can float as a separate
window which can be
reshaped to meet your
preferences.
The TOOL BAR can be
covered or hidden with
other Discovery32
windows, make sure
you place it in a
convenient corner for
best access.
DataViewer: Loading a Chromatogram
The other way is to
go to File..Open or
Click the “Open Folder
Icon Button”
One way to load a
chromatogram is to double click
on the file in this box.
The file loaded will be
acknowledged by having the
line selected turn red AND
where the red ovals have been
placed.
DataViewer: Loading a Chromatogram (Continued)
Remember: If you single click another file, that
line will be highlighted with dark blue and an
UNLOADED chromatogram will be seen in the
preview window above (right). The actual loaded
file will be identified by the red highlighted text and
the places where the ovals are shown.
DataViewer: Viewing the Loaded Chromatogram
A processing window for the loaded
chromatogram can be accessed by
clicking this button.
This is the “chromatograms”
window. The sample name is the
only information the default
conditions will display unless the
report selection fields have been
properly checked. See next slide.
Report Selection: Placing File Name on Chromatogram
It is recommended that the following items be selected
in the Report Selection Menu.
It will show the file name on the chromatogram window.
If the “Add File Name to Footer” is not checked, you
can only determine what file you are looking at by the
file name in the DataViewer footer or in Summary
section of the Results window.
Report Selection: File name
The file name shown here will not
match the file name loaded if the
file that was loaded was “saved as”
with a different name. The original
name is shown here.
The “saved as” file name is shown
in the “status bar”
Helpful Hint 1: Optimizing Window Size
The “chromatograms” window is the only
resizable window. Making the window larger
makes it easier to perform functions such as
baseline and integrations.
Place the Tool bar over here and in full vertical
view. Then grab a corner of the chromatogram
window and drag it to a larger size. (Leave
enough room to see the tool bar).
(Note: Leave window open, if you close it and
decide to reopen it again, it will default back to
the original size)
Optional: Changing Display Settings
If you prefer to have a chromatogram window with a white
background follow the procedure below..
On the computer desktop go to
Start/Settings/Contol Panel/Display
The window (Display Properties) go to the Appearance tab select
“HIGH CONTRAST WHITE”.
Hint: A quick way to get to this screen is to right mouse
click the Windows desktop.
Optional: Changing Display Settings (high contrast white)
This is an example of what “high contrast white” looks like.
The Tool Bar
Open Overlay Display Window
Open Branching and Structure Window
Open Conventional GPC Window
These three
buttons will
be addressed
in a future
tutorial
Open Results Window
Open Calculation Set Window
These are the three buttons we
will need to use for calibration
Open Chromatogram Window
Start
Acquisition
Software
Print
Save
Open
If more than one copy or version of
Acquire32 is found, a dialog box
will pop up asking for your selection
Chromatogram Window
Each of the pop-up windows has click
on tabs where you can get to different
features. For the purposes of this
tutorial we will use only the “Operate
On Chromatograms” TAB
Set Up Calculations Window (Options)
SETTING UP TO CALIBRATE
Detector Calculations: RI/ Light Scattering
Mw Smoothing: None
Calculation Types: Mw Calculations (15 & 90 degree)
PD2020
Rh Calculations: Yes
Laser Wavelength: 809 nm (If you have Rh capability your
laser is 809 nm, if you do not have Rh capability you need
to refer to your Calcert document for the actual wavelength
of the laser used in your instrument). If you are uncertain or
believe your unit was upgraded, contact PDI and provide
them
Set Up Calculations Window (Detectors)
SELECTING THE DETECTORS TO DISPLAY
By clicking on the “Detectors” tab of the Setup calculation
window, you can select the detectors to display. To simplify
the viewing we will not visualize UV and Rh data when
performing baselines and integrations on the
chromatogram found in the “Chromatograms” window.
NOTE 1: The tabs are placed in order of how you would set
up your calculations. Options, Detectors, Constants and
finally Determine Cal Constants.
NOTE 2: If you select a channel and check both left and
right y-axis, the left axis is displayed only. If you select a left
y-axis for 90 and a right y-axis for RI, there will be two y
axes shown.
Set Up Calculations Window (Constants) 1 of 2
The Run RI and Run UV constants shown on the left
side are the current constants being applied to the
chromatogram. These constants are stored with the
file that is opened. The right side constants are the
instrument values. These constants are not stored with
the file but are stored in Discovery32. Every time a
new file is opened with this program, the same
instrument constants will also appear. When you
calibrate a file or update constants through the
Determine Cal Constants routine, both left and right
side columns are updated.
If you wish the new constants to be associated with
data files to be collected. These constants can be
copied and pasted in the Calibration fields found in
PrecisionAcquire32.
Calibration constants can be manually entered into the fields if you
have the information available. The right arrow (RI)buttons when
clicked copy the contents of the Run RI side to the Instrument RI
side. The left arrow (RI) buttons transfer the constants from the
Instrument RI side to the Run RI side. The Run UV arrow buttons
perform similar functions. The “update” button activates every time a
manual change occurs. When you click “update” ,a dialog box will
pop up asking if you wish to save settings or cancel..
Set Up Calculations Window (Constants) 2 of 2
IMPORTANT: The Cal solvent index is the RI constant
of the solvent that was used during a calibration. It does
NOT update when the solvent index is changed in the
Results summary page. It will only update if a new
calibration has taken place and will obtain the new
value from the eluent RI field of the “Determine cal
constants” field.
If you are doing Mw calculations using two angles and
the solvent has changed for the method, you must enter
the NEW solvent index here also.
If you recently updated the cal constants for a file that
is currently opened (run side column) and you wish to
have these same values applied to another set of files
with older constants. Simply click this button and select
up to 20 files (at a time) by holding the ctrl button down
and clicking the file names you wish to modify. A dialog
box will confirm how many files were modified.
Alternatively, if you modified constants through the
“determine cal constants routine” the new constants
will also be found in the instrument side column. You
can then transfer the instrument constants to the run
constants side by clicking the left pointed arrows.
Setup Calculation Window (Determine Cal Constants)
Upon viewing this screen, all the
constants are blank here. This
occurs if you have not established
baselines and integration regions
for the chromatograms. Lets
establish baselines and integration
regions so that we can obtain new
constants.
Chromatogram Window: Establishing Baselines (1 of 3)
Right mouse click (once or twice) until a
pop-up selection box appears. Select
Set baseline. This dialog box then appears.
Deselect
Spare Detector then press OK.
Continue onto next slide
NOTE:
Low Angle Scatter = 15 degree Channel
High Angle Scatter = 90 degree Channel
Spare Detector = (Typically reserved for UV)
Chromatogram Window: Establishing Baselines (2 of 3)
start
end
The pointer converts to a cross-hair icon. Starting with a flat
portion on 90 degree and RI before the peaks, click and hold
the left mouse button down and drag it to a similarly flat region
to the right of the peaks. Release the mouse. If you, like what
you have selected, move your cursor to a region between the
two vertical black lines (start and stop of baseline
determination) and right mouse click. If you do not like the
selection, right mouse click outside that region.
Chromatogram Window: Establishing Baselines (3 of 3)
If you want to move a baseline marker.
Hold the CTRL button down then left mouse
click and hold the marker you wish to move.
Drag the marker to where you wish to move it
to and release.
Baseline markers shown
Chromatogram Window: Normalizing Region
Before selecting integration regions for calibration, it
is better to normalize the heights of the various traces
of the standard peak.
Right mouse click (once or twice) until a pop-up
selection box appears. Select
Normalize Region. Select the region you want to
normalize (exclude negative peaks).
Similar to what you did with baseline selection. Right
mouse click inside the vertical black lines to select.
Chromatogram Window: Zooming on Normalized Region
Take your cursor (pointer) . Hold the left
mouse button down and drag diagonally
across the region you wish to zoom into.
Then release mouse
start
end
Selecting Integration Region
Right mouse click (once or twice) until a pop-up selection box
appears. Select Set Integration Region. Hold and drag the
cross hair cursor (left mouse button) over the monomer peak
region of BSA and release. ( You may have to re-zoom in after
this operation)
Integration
marks shown
Determine Cal Constants: (1 of 5)
In the previous chromatogram, baselines and integration of the main
peak have been performed. We can now proceed with obtaining
calibration constants.
FIRST:Check that all of the fill-in fields information are correct. The
Mol. Wt. entered in as the full number (e.g 66500 not 66.5 kD).
Make corrections where necessary.
SECOND:The quality factor should be 0.98 to 1.00 before
accepting the inter-detector volume. (Make sure you have
measured the flow rate to confirm that what is programmed into the
pump is actually what the pump is delivering.) If the value is lower
than 0.98, try reintegrating a wider or narrower integration region. If
it still did not improve, rerun the standard and or check to make
sure it is monodispersed (pure).
.
THIRD: If the quality factor is adequate then click ALL then click
Apply.
If the standard consisted of a solitary peak the calibration is
essentially completed. Since our BSA standard does have
measurable dimer, trimer and higher order aggregates we have to
expand our integration region to include the total area of these
peaks so that an accurate concentration constant (RI or UV) is
obtained . After which the monomer region is reintegrated to
optimize 15 and 90 constants.
Determine Cal Constants/ RI Constant (3 of 5)
FIFTH: Expand the integration region to include all
of the RI area (terminal peak at 12 minutes
excluded). The 2 mg/mL is distributed over RI
aggregates and monomer.
Determine Cal Constants/ RI Constant (4 of 5)
SIXTH: Go back to Det. Cal Constants after reintegrating the RI
peaks. Click Accept then click Apply
NOTE: PrecisionAnalyze will report RI
constants ranging from 75000 to 85000 and
are not compatible with Discovery32 which
typically has a RI constant around 3.
.
Determine Cal Constants/ 15 & 90 degree constant (5 of 5)
SEVENTH: Reestablish integration for monomer, then
proceed to complete the 90 degree constant. Select the
start region somewhat away from the trough between
the dimer and monomer peak as the dimer is tailing a
little into the monomer. Then accept the 15 and 90
degree constant. Then click apply.
We have now calibrated the RI, 15 and 90 channels.
Checking your results (1 of 2): Run Params
Calibration of the monomer BSA peak is correct for
RI/15 and RI/90
The entered dn/dc is 0.1670 the software suggests that
this value should be 0.1451 if the area of the monomer
peak was precisely 2 mg/mL. This is not the case as
the 2 mg/mL is distributed over the several peaks.
The entered concentration is 2 mg/mL, the calculated
concentration for the monomer peak is 1.7381 mg/mL
which is correct as that is the portion of monomer
distributed between aggregates.
By checking this box, Batch calculations will then read
66.5 K
Checking your results (2 of 2):Summary
Avg. Mw for BSA for the integrated slice.
The heart-cut of the monomer peak is
very monodispersed at 1.001
The peak (apex) molecular weight is
shown here
Field Information Update
NOTE:
An extra line of information has been included
n the Run Information window.
The original file name (source file) and its new name
(If it has been modified and saved as) is now displayed
together..
Checking the BSA Dimer peak for Mw
Integrate the center region for dimer of BSA and
check Mw. The calculated molecular weight for this
peak is 2.02 x that of the calculated Mw for monomer.
Checking the BSA Trimer peak for Mw
Integrate the center region for trimer of BSA and
check Mw. The calculated molecular weight for this
peak is 2.98 x that of the calculated Mw for monomer.
Checking the BSA Tetramer peak for Mw
Integrate the center region for tetramer of BSA and
check Mw. The calculated molecular weight for this
peak is 4.17 x that of the calculated Mw for monomer.
Determining Large Aggregation BSA
Integrate the full RI area region.
The avg. Mw is 74.5kD with a
polydispersity of 1.057
Looking at the Mw vs Elution Profile
The Mw vs Elution Profile helps to visualize the Mw
variations across a peak. Here both monomer and
dimer show very good consistancy as indicated also by
the polydispersity factor.
Calibrating the UV Detector and LS Channels (1 of 6)
Now that we have successfully calibrated the BSA
chromatogram using RI/Lightscattering, lets proceed to
complete the calibration using the UV/Lightscattering
option. Select “UV/Lightscattering Calculations”
Disable viewing the RI channel and enable the UV
channel. Check both left and right y-axis buttons for the
UV
Calibrating the UV Detector and LS Channels (2 of 6)
We only now need to establish baseline on the UV
trace since the other channels have been already
operated on.
Select only the “spare detector” which is the UV
channel. Then click OK. Select and drag the
cross hair cursor on a flat region before the first
lift off of UV baseline and well after the landing
on the UV trace. (3 minutes to 14 minutes.)
Calibrating the UV Detector and LS Channels (3 of 6)
Zoom in on the monomer/dimer region and reintegrate
the monomer region. Note that the UV trace is before
the LS channel which is what is expected if no time
correction is applied to the trace.
Make sure the UV Extinct (extinction coefficient
is correct and the quality factor for the IDV is
greater than 0.98. Click “ALL” then click Apply.
Calibrating the UV Detector and LS Channels (5 of 6)
Expand the integration region to include all of
the UV area, then go back to Setup
calculations/ Determine Cal Constants and
accept the UV/spare constant. Then click Apply.
(We already confirmed the fields in the
parameter boxes were correct)
Calibrating the UV Detector and LS Channels (6 of 6)
Finally reintegrate monomer region (exclude the
front liftoff and tailing landing).
Accept the 15 and 90 degree constants. Then
click Apply.
Checking Calibration for UV LS Channels (1 of 2)
Calibration of the monomer BSA peak is correct for
UV/15 and UV/90
The entered UV ext (extinction coefficient) is 0.6750,
the software suggests that this value should be 0.5716
if the area of the monomer peak was precisely 2
mg/mL. This is not the case as the 2 mg/mL is
distributed over the several peaks.
The entered concentration is 2 mg/mL, the calculated
concentration for the monomer peak is 1.6937 mg/mL
which is correct as that is the portion of monomer
distributed between aggregates.
Checking Calibration for UV LS Channels (2 of 2)
Avg. Mw for BSA for the integrated slice. Note it is not
exactly 66.5. While the calibration is based upon total
area of LS signal and UV (essentially batch type
calculation). The calculations performed on the peak is
slice by slice. If the curves of the LS and UV are not
perfectly coincident, the Mw will be calculated
differently.
The monomer peak is fairly monodispersed at 1.003
The peak (apex) molecular weight is shown here
Checking the BSA Dimer peak for Mw
Integrate the center region for dimer of BSA and
check Mw. The calculated molecular weight for this
peak is 1.95 x that of the calculated Mw for monomer.
Checking the BSA Trimer peak for Mw
Integrate the center region for trimer of BSA and
check Mw. The calculated molecular weight for this
peak is 3.05 x that of the calculated Mw for monomer.
Checking the BSA Tetramer peak for Mw
Integrate the center region for tetramer of BSA and
check Mw. The calculated molecular weight for this
peak is 4.28 x that of the calculated Mw for monomer.
Comparing UV and RI responses -plus Mw (UV) for full area
UV tracks very closely with RI,
but shows just a little less
response at the tetramer region
which accounts for the
somewhat higher calculation
for Mw as compared to RI.
Full integration of monomer and
Aggregations.
Looking at the Mw vs Elution Profile (UV based calculations)
Hydrodynamic Radius Data
Check these boxes to display Rh data.
Rh data is displayed as “plus signs”, the
radius in nm can be read off the y-axis.
The monomer size is in the 3.4 to 3.6 nm
Region. The expected size for BSA is 3.6
nm.