A5 PDA Acquisition, Processing and Reporting · An angle of 0 degrees means the spectral shape is...

©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


Optics Assembly Light Path (2998)


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


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.


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


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.


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.


General Tab

3D Data for Spectral Analysis


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.


• 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


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.


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


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


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


Agenda

Spectral Contrast

Threshold Calculations

Spectral Resolution

Peak Impurity and Peak Spectral Homogeneity

UV and Chromatographic Limitations

Multiple Pass Peak Purity

Software Examples


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.


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


Spectral Contrast

Spectral Contrast 53 Degrees

200.00 240.00 280.00 320.00nm

EthylPabaEthylparaben

Abso

rban

ce


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


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.



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.



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.


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


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.


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.


Spectral Resolution

230.00 250.00 270.00nm

Benzene

Less resolution at 3.6 nm vs 1.2 nm

UV maxima shifted


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


30

How do we use UV-spectra

Peak purity/peak homogeneity analysis

Peak identification/Library search


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


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.


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.


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



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




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



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

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