Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
©2009 Waters Corporation | COMPANY CONFIDENTIAL©2011 Waters Corporation
A5 PDA Acquisition, Processing and Reporting
Peak Purity & Library Search
Rune Buhl Frederiksen, Nordic Customer Education Manager
©2011 Waters Corporation 2
Content
Presentation:— Detector Optics
— 2996/2998
— Maximizing signal-to-noise
— Important Instrument Method Parameters
— Understanding PDA Spectral Contrast Theory
Live Demo— Adding Peak Purity and Library Search Calculations to a PDA
processing Method
Exercise
©2011 Waters Corporation 3
Optics Assembly Light Path (2998)
©2011 Waters Corporation 4
2996/2998 Comparison
2996 2998
2D Data Collection Yes Yes
Simultaneous 2D & 3D Data Coll. No Yes
Absorbance Compensated data Coll.
No Yes
Noise Specifications 30uAU 10uAU
Drift Specifications 1mA/Hour 1mA/Hour
©2011 Waters Corporation 5
Maximizing Signal-to-Noise
The Lamp Optimization Software automatically maintains a minimal noise level throughout the useful life of the lamp. It automatically selects an appropriate exposure time for the photodiodes based on lamp energy, mobile phase and wavelength range for the analysis. This optimization is performed before each injection and requires no operator adjustments to the detector.
Spectral Exposure Optimization maximizes the signal across the entire wavelength range. The relative intensity of the Deuterium lamp spectrum varies widely across the wavelength range of the 2998 instrument. Analyses that require spectral ranges that span large portions of the spectrum may have reduced sensitivity in the lower energy regions. Spectral Exposure Optimization will improve signal to noise performance in lower energy regions of the spectrum while maintaining optimal performance across the range.
©2011 Waters Corporation 6
Spectral Exposure Optimization
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Dyes without Spectral Exposure Optimization
Dyes with Spectral Exposure Optimization
425nm530nm630nm
S/N Improvement1.2X 2.1X 2.6X
©2011 Waters Corporation 7
2998 PDA Console
The easy to use PDA Console allows for improved time management with an intuitive detector interface where major detector functions are located in single software location. A real-time status provides assurance that the detector is working correctly.
Further, a user can easily track hours of lamp operation, ignitions, and the lamp serial number.
©2011 Waters Corporation 8
Thermal Wander –How it works
A compensated reference wavelength collects wide-band absorbance data in a region of the spectra where no known analytes exist. It is used to reduce detector drift and wander, which can affect integration quality.
Empower software calculates the compensation value by averaging the absorbance values within a range of wavelengths that you select. It then subtracts that value from the absorbance value:
Abs-Comp(t) = Abs(t) - CRef(t) where
Abs-Comp = absorbance-compensatedAbs = absorbance
CRef = compensation referencet = elapsed time after start of injection
A starting and ending wavelength defines the compensation reference. CRef bandwidth must be ≥40 nm and ≤100 nm, and fall within a range of 190 to 800 nm.
©2011 Waters Corporation 9
General Tab
3D Data for Spectral Analysis
©2011 Waters Corporation 10
Optimal Data Rates
Reproducible integration requires 15 points across a peak.
Reproducible quantitation requires 25 – 50 points across a peak.
Why not collect as many data points as possible to always ensure good quantitation? ……….. Noise.
©2011 Waters Corporation 11
• Detectors have a base sampling rate.
• To get lower data rates, points are averaged resulting in some filtering.
• The noise at 20Hz and 5Hz will be different, even with the same filtering constant.
Data Rate Considerations
©2011 Waters Corporation 12
What is Digital Filtering and Why is it Important?
Digital Filtering is a mathematical algorithm applied to a data set that smoothes out higher frequency noise.
The desired result is reduced baseline noise with minimal impact on peak intensities so as to increase signal-to-noise.
The degree of filtering (time constant) is crucial, too much filtering can dramatically impact peak shapes and resolution.
©2011 Waters Corporation 13
2998 PDA Filter Time Constants
“Slow" refers to slowly eluting peaks which should not be distorted by additional averaging
"Fast" refers to fast eluting peaks which might be distorted, so the averaging window is reduced
Data Rate Slow (sec) Normal (Sec) Fast (sec)
1 4.000 2.000 1.000
2 2.000 1.000 0.500
5 0.800 0.400 0.200
10 0.400 0.200 0.100
20 0.200 0.100 0.050
40 0.100 0.050 0.025
80 0.050 0.025 0.0125
©2011 Waters Corporation 14
How to Select the Best Filtering Constant
Ensure that the data rate is set to collect 25 – 50 points.
Start with the inverse of the sampling rate
Filtering Time Constant = 1/Sampling Rate
Is increased sensitivity needed? Is baseline noise interfering with integration?
↑ Filtering Time Constant
Is increased resolution needed?
↓ Filtering Time Constant
©2011 Waters Corporation 15
2D Channels
Absorbance
Absorbance Compensation — Compensation Reference
Maxplot
Wavelength subtraction
Wavelength addition
Ratio
©2009 Waters Corporation | COMPANY CONFIDENTIAL©2011 Waters Corporation
Understanding PDA Spectra Contrast Theory
©2011 Waters Corporation 17
Agenda
Spectral Contrast
Threshold Calculations
Spectral Resolution
Peak Impurity and Peak Spectral Homogeneity
UV and Chromatographic Limitations
Multiple Pass Peak Purity
Software Examples
©2011 Waters Corporation 18
Spectral Contrast
The Spectral Contrast measures the shape difference between two spectra.
Spectra are baseline corrected by subtracting interpolated baseline spectra between peak baseline liftoff and baseline touchdown.
Spectra are converted into a vector in n dimensional space.
Vector lengths (concentration) are minimized using least-squares solution.
The vectors are moved into a two dimensional plane and the angle between them is measured.
An angle of 0 degrees means the spectral shape is identical and an angle of 90 degrees indicates no spectral overlap.
©2011 Waters Corporation 19
nm
Spectral Contrast
The shapes of Spectrum A and Spectrum B are represented by vectors
θ is the Spectral Contrast Angle which is the difference between spectral shapes
Spectrum A
Spectrum Bθθ
200.00 240.00 280.00 320.00
Spectrum BSpectrum A
Absorbance AU a
t 240
nm
AU at 280 nm
©2011 Waters Corporation 20
Spectral Contrast
Spectral Contrast 53 Degrees
200.00 240.00 280.00 320.00nm
EthylPabaEthylparaben
Abso
rban
ce
©2011 Waters Corporation 21
Spectral Contrast
Spectral Contrast 10 Degrees
230.00 250.00 270.00 290.00 310.00nm
TheophyllineDyphylline
Abs
orba
nce
Similar spectra for structurally related compounds
©2011 Waters Corporation 22
Spectral Contrast
Spectral Contrast 0.5 Degrees
200.00 240.00 280.00 320.00nm
MethylparabenEthylparaben
Abso
rban
ce
Very similar spectra, CH2 difference
Spectral Contrast can differentiate these spectra.
©2011 Waters Corporation 23
Threshold Calculations
The Threshold Angle is comprised of two parts: First, The Detector Noise Angle is calculated from the chromatographic baseline and is inversely proportional to the peak height.
Spectrum A
Spectrum B
Noise
θAbs
orba
nce
The Noise Region in gray forms a constant cylinder of uncertainty around the vector.
A vector drawn from the origin to the edge of the cylinder creates the noise angle.
The shorter the vector (lower concentration) the larger the noise angle.
©2011 Waters Corporation 24
Threshold Calculations
Second, The Solvent Effect corresponds to the constant portion of the Threshold Angle, it accounts for solvent effects and photometric errors.
The solvent effect can be accurately measured from a chemically pure standard, running six replicates and taking the highest obtained purity angle.
Auto-threshold will use a look-up table based on peak height for the solvent effect part of the threshold calculation.
©2011 Waters Corporation 25
25
Definition of Threshold
Threshold = Noise (Calculated by Empower)— Noise Angle is a measure of the spectral noise in a chosen area
of the chromatogram — Noise Angle is increased when the conc. of the compound is
decreased Threshold = Solvent Angle (Determined/estimated by the user)
— Solvent Angle is a numerical compensation for non-ideal conditions caused by the chromatography/Solvents
Threshold = Noise + Solvent Threshold = Auto Threshold (can only be used with peak purity
calculation, Calculated by Empower)— Determines the solvent angle for each peak in the
chromatogram— Calculates the noise angle based on the noise interval you have
selected and the peak spectrum— Computes the threshold angle for each peak in the
chromatogram as the sum of its noise and solvent angles
©2011 Waters Corporation 26
26
Determination of the Solvent Angle for peak purity calculations
Make 6 injections of a pure standard, Absorbance should not exceed 1 AU.
Calculate peak purity based on threshold = Auto threshold.
If purity angle < threshold for all injections set solvent angle = 0, and use threshold = Auto thresholdIf purity angle > threshold for some of the injections, solvent angle should be used and threshold = Noise + solvent.
Calculating the solvent angle. Process all injections with threshold = noise. Solvent angle = largest difference between purity angle and purity threshold, and use threshold = noise + solvent.
©2011 Waters Corporation 27
27
Determination of the Solvent Angle for library search
Make 6 injections of a pure standard, Absorbance should not exceed 1 AU.
Create a library based on one of the injections .
Process all the other (5) injections with threshold = noise.
If match angle < threshold for all injections set solvent angle = 0, and use threshold = noise.If match angle > threshold for some of the injections, set solvent angle = largest difference between match angle and match threshold, and use threshold = noise + solvent.
©2011 Waters Corporation 28
Spectral Resolution
230.00 250.00 270.00nm
Benzene
Less resolution at 3.6 nm vs 1.2 nm
UV maxima shifted
©2011 Waters Corporation 29
Spectral ResolutionA
U
0.000
0.001
0.002
nm250.00 300.00
266.2
AU0.000
0.001
0.002
0.003
nm250.00 300.00
271.5
312.1
14.4 nm 1.2 nm
©2011 Waters Corporation 30
30
How do we use UV-spectra
Peak purity/peak homogeneity analysis
Peak identification/Library search
©2011 Waters Corporation 31
31
Abso
rban
ce
Spectra per peak
Peak Purity Peak Purity analysis analysis all all spectraspectra within a within a peakpeak
Apex spectrumApex spectrum is is the reference the reference spectrumspectrum
Peak Purity (homogeneity)
200.00 240.00 280.00 320.00nm
Abs
orba
nce
©2011 Waters Corporation 32
Peak Impurity and Peak Spectral Homogeneity
The Peak Purity Algorithm uses Spectral Contrast to compare all spectra within a peak to the Apex spectrum. The resulting Purity Angle is a weighted average of all of the calculated angles.
If the Purity Angle is less than the calculated Threshold Angle, within the noise of the system the peak is spectrally homogeneous.
If the Purity Angle is greater than the calculated Threshold Angle, there is something within the peak that can not be explained by noise. The peak is impure.
©2011 Waters Corporation 33
Spectral Resolution
Spectral Resolution or the ability to differentiate one UV spectrum from another.
The Waters PDA runs with a fixed 50micron slit, producing an optical resolution of 1.2nm.
For 1.2nm optical resolution a 200nm to 400nm range n = 166. i.e. Spectral Contrast uses 166 dimensions to describe the curve shape.
For 4.0nm optical resolution a 200nm to 400nm range n = 50. i.e. Spectral Contrast uses 50 dimensions to describe the curve shape.
©2011 Waters Corporation 34
34
Ekstraction of UV spectra in every data point
Temps
210.0 250.0 290.0nm
Apex
Abs
orba
nce
210.0 250.0 290.0nm
Inflection
Abs
orba
nce
BaseA
bsor
banc
e
210.0 250.0 290.0nm
AU
0.000
0.100
0.050
Peak Purity (homogeneity)
©2011 Waters Corporation 35
35
Ekstraction of UV spectra in every data point
Temps
210.0 250.0 290.0nm
Apex
Abs
orba
nce
210.0 250.0 290.0nm
Inflection
Abs
orba
nce
BaseA
bsor
banc
e
210.0 250.0 290.0nm
AU
0.000
0.100
0.050
Peak Purity (homogeneity)
©2011 Waters Corporation 36
UV and Chromatographic Limitations
R=0 Purity Angle not effective; Library Match Angle useful
R=0 R=0.3 R=0 R=0.3 R=0.7 R>1.0 R=0.7 R>1.0
R=0.3 to R=0.7 Purity & Library Match Angle useful
R>0.7 Library Match Angle not useful Chromatographic Resolution
©2011 Waters Corporation 37
UV and Chromatographic Limitations
The UV spectrum of different compounds can be identical.
The concentration of the impurity may be too low to detect.
Each of these three limitations become a trade off to the other two.
Ref: Detecting Coeluted Impurities by Spectral Comparison, Marc V.Gorenstein et al LC-GC Volume 12 Number 10 October 1994 pages 768-772