Data Processing, Reviewing and Reporting
Alyssa Loo
Applications Consultant
Waters Singapore Solutions Centre
Agenda
Data Processing
Data Reviewing
Data Reporting
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Principle of Calculation
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Principle of Calculations
Processing standards:
− Obtain Elution volume from data
− Plot of log (M) versus f (Elution volume)
355000 43900 7
2890000 2800 5
Log(M)
4
0
Elution volume
0.00 10.00 20.00 30.00 40.00
Minutes
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Principle of Calculations
Processing unknowns:
− The signal is cut in slices (width dt)
− Each slice is characterized by
o Elution volume, Vi
o Area Hi.dt = niMi
− With the calibration curve Vi gives the MW in each slice, Mi
− ni for each slice is determined by the area and Mi
− Therefore Mn, Mw… can be calculated
Mn, Mw, Mz, I, ...
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Minutes
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Principles of Molecular Weight Distribution
Definition of MW averages :
− A polymer is made of species (chains) of varying lengths. Each chain is characterized by its
molecular weight, Mi, and its abundance ni. Then :
niMi niMi3 Mw
Mn Mz I
ni niMi2 Mn
niMi2 niMi4
Mw Mz+1
niMi niMi3
I=Mw/Mn Mn<Mw<Mz<Mz+1
Most of these values are necessary to characterize the polydispersity
These values can be calculated by different techniques, but GPC is the most convenient
and widely used method.
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Molecular Weight Averages/ Physical Property Correlations
Mn: Number average molecular weight
− can affect brittleness, flow and compression properties of the polymer.
Mw: Weight average molecular weight
− related to strength properties, and impact resistance
Mz: Z average molecular weight
− related to elongation and flexibility (Elastomers)
Mz+1
− related to die swell (Extrusion parameter)
PD: Mw/Mn, Polydispersity
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Molecular Structure of Polymers
24.00
MP
22.00
20.00
18.00 Mw Mn
16.00 PD = Mw / Mn
14.00
12.00
mV
10.00
Mz
8.00
6.00
4.00
Mz+1
2.00
0.00
-2.00 Largest Smallest
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Minutes
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Relative vs Universal Calibration
Which parameter controls the exclusion process ?
− Relative calibration: log(M) = f(Ve).
o The behavior of polymers in the columns depends on their molecular weight; Wrong ! (hydrodynamic
volume)
− The exclusion process is controlled by the volume in the solution. The elution volume
depends on the hydrodynamic volume
− The hydrodynamic volume depends on the molecular weight, the nature of the polymer and
the solvent
o PS and PVC with same MW will have different hydrodynamic volumes
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Relative Calibration
The calibration curve, log(M) = f(Ve) is not unique:
− Accurate molecular weight for PS are obtained through a PS calibration curve
− Accurate molecular weight for PVC are obtained through a PVC calibration curve
− Only a few polymers are available as narrow standards (PS, PMMA …)
− The relative calibration curve, log(M)=f(Ve) obtained with PS allows calculations relative to
PS
LogM vs e lution volume
6 PS
Log (M)
5 PMMA
3
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Elution volume
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Universal Calibration
Universal calibration: log(Vh)=f(Ve) or log([h] M)=f(Ve)
− The curve log(Vh)=f(Ve) is unique (Benoit law)
− Vh is the hydrodynamic volume, Vh is proportional to [h].M
− All standards are on the same curve
− One type of standard will generate a curve available for all unknowns
− Universal calibration for every type of polymer
Log[n]M vs elution volume
6 PS
Log[n]M
5 PMMA
3
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Elution volume
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GPC Calibration: Relative VS Universal
Relative Calibration – Generates a calibration curve that plots Log Mol Wt as a
function of Retention Time or Elution Volume
Universal Calibration – Generates a calibration curve that plots Log Hydrodynamic
Volume as a function of Retention Time or Elution Volume.
− The hydrodynamic volumes are calculated using the standard’s molecular weights and its K
and alpha values (GPC modified universal) or intrinsic viscosity(s) from the viscosity channel
data (GPCV Benoit’s protocol)
− All standards are on the same curve
− One type of standard will generate a curve available for all unknowns
− Universal calibration for every type of polymer
GPC Calibration: Select “Relative”
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GPC Calibration: Narrow Standards
To build a calibration curve :
Narrow disperse standards are injected 6.4
6.2
6.0
− Polydispersity < 1.1 5.8
5.6
5.4
− Elution volume at peak height 5.2
5.0
4.8
− Curve Log(MW) = f(Ve) 4.6
4.4
4.2
Log Mol Wt
4.0
3.8
3.6
3.4
3.2
3.0
2.8
2.6
Calibration: 18 19 20 21 22 23
Time (min)
24 25 26 27
1. >10 MW standards 9100
2. Standards injected alone or as
a mixture 190000
3. Mixtures should have baseline
resolved 1100000
4. Long process! (But accurate)
Elution volume
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Data Processing
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Workflow for New Processing Method (PM)
FOR Standards FOR Unknowns
Open PM
Develop PM using Wizard
Change Integration to Broad
INTEGRATION TAB: Adjust Peak
Process Narrow Standards
Width & Threshold to ensure peaks
(Integrate, Calibrate)
are integrated
Save All Results and Calibration SLICING TAB: Add in the
Curve Component Name & RT
Process Broad Unknowns
(Integrate, Quantitate)
Save Processing Method and All
Results
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Data Processing
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Data Processing
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Data Processing
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Select Injections for Review
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Processing method wizard
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Processing method wizard
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Processing method wizard
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Processing method wizard
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Processing method wizard
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Processing method wizard
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Processing method wizard
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Processing method wizard
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View Processing Method
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GPC Processing Method
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GPC Processing Method
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Integration for Narrow Injections
Narrow for standards; Broad for samples
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Set Up of Calibration Parameters
Relative / Universal Calibration
Fit Type: 3th order and up for GPC
(Generally 3th or 5th order)
Calibration Order: Depends on input in
Component Editor. Typically High to Low
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Process Standards
***Standards & Samples must be processed separately due to
difference in integration: Narrow VS Broad ***
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Calibrate
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Calibrate
Continue for All Standards
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View Calibration Curve
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Curve with Operating Range
Vo Vt
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Ignoring points
File > Save All
To save all Results &
Calibration Curve
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Integration Tab (For Unknowns)
“Traditional” or
“ApexTrack”
Change these
Parameters!
Change to Broad
for Unknowns
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Integration Tab (For Unknowns)
Peak Width and Threshold must be set to ensure that the narrowest and broadest
peaks have correct baselines
Adjusting Peak Width
− If the integrated peak is too narrow (small width), then increase the peak width
Adjusting Peak Threshold (Baseline Noise)
− Threshold value is a slope measurement that the software uses to determine peak start and
peak end points during peak detection
− If it is too low, baseline noise will be integrated, so increase threshold
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Slicing Tab (Unknowns)
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Slicing Tab (For Unknowns)
Put in the name of the Component,
Retention Time and RT Window
This is to allow the Peak to be
detected and MW calculated.
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Slicing Tab (For Unknowns)
We will leave these as default as these are GPC/V-LS options.
FYI:
Rg Fit: Fit type used to calculate the fitted radius of gyration.
MW Fit: Fit type used to calculate the fitted MW, slice MW.
Intrinsic Viscosity Fit: Fit type of curve fitted to observed viscosity data
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Integration
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Quantification
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GPC Batch Processing
GPC Batch Processing can be done if the unknown peaks are consistently eluting at
similar RT
For a New PM, it has to be done separately because:
− Integration Parameters are different from narrow standards and broad unknowns. Applying
Integration Parameters meant for narrow standards to broad unknowns may cause
integration to be inaccurate
− Need to input Component into the Slicing tab so that Empower will quantitate the MW for that
peak
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Data Review
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Review Results
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Review of Data with Compare
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Comparison of 4 Replicate Injections
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Calculated Results
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Results and Distribution
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Select Distribution Table
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Filter Option for ‘Size’ of Slice Shown
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Data Reporting
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Reporting Results
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Selection of Report Method
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Selection of Report Method
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Distribution Plot (Overlay Report)
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