Nordic User Meeting 2013

Empower3 FR2 PDA

Instrument, Processing and

Reporting using Purity and

Library search functions by

Ronald Haas

Service Data Specialist/Certified Trainer

Central Europe

Content

Create Method Set

— Instrument Method

Develop Processing Method

— Extract Chromatogram (Save in Method Set)

— Use Peak Purity and Library search

Create a PDA Report Method

Method Set with

Instrument Method

Login:System/Manager

Creating an Instrument Method in a Method Set

or Develop Method in Run Samples

Or

Create an Instrument Method

Creating an Instrument Method

PDA Detector Parameters

PDA detector output

PDA Chromatogram and Spectrum Display

Polychromatic light

PDA Detector Parameters

Resolution is a diode bunching

PDA Detector Parameters

The sampling rate and is set in relation to the width of the smallest peak.

A peak should have at least 20 to 50 data points for Quantitation and 12 to 15 for qualification.

Too many data points results in more noise.

Ch1: 15

80 p/s

40 p/s 20 p/s

10 p/s 5 p/s

1 p/s

Ch1: 16

80 p/s

Empower integration

Correct Integration

PDA Detector Parameters

The UV Blocking Filter is a physical filtering that occurs within the detector. When the UV Blocking filter is enabled, the UV light is filtered prior to entering the flow cell. The filter inhibits possible degradation to the sample. Some samples are very sensitive to UV light at low wavelengths.

Interpolate 340nm Region

Lamp (Deuterium) Spectrum

PDA Detector Parameters

The Waters 996 PDA Detector recharges each diode and reads the recharging current one diode at a time. The interval between two readings of an individual diode is the exposure time. The 996 requires a minimum of 11 ms to sequentially read all of the diodes. The exposure time has a range of 11 to 500 ms. The following is an example of how exposure time works if it were set to 50 ms: Recharges diode 1 and reads the recharge current. Recharges diode 2 and reads the recharge current. Sequentially recharges and reads the current for all remaining diodes. This sequence takes 11 ms. The detector waits 39 ms (50 ms - 11 ms) and then recharges diode 1 and reads the recharge current.

PDA Detector Parameters

Real Time Moving Averaging

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14

Time

Pe

ak

In

ten

sit

y

Filters the last data point in a filter window

Effect of Filtering on Peak Shape and Noise

Peak shape suffers

Best s/n

PDA Detector Parameters

Resolution is again diode bunching

Use 2D Channels only if you do not need 3D or want to use a different Resolution diode bunching (smoothing)

PDA Detector Parameters

The Median Baseline Filter (MBF)

— A processing tool for alleviating baseline curvature

— The MBF operates during acquisition in real time on the chromatogram

whose baseline is to be corrected. The MBF automatically extracts the

baseline, smoothes it, and then subtracts the smoothed baseline from the

original chromatogram. The result is a chromatogram with greatly reduced

baseline curvature.

MBF On

MBF Off

Median Baseline Filter

•The median becomes poorly defined with clusters of similarly sized peaks that have a cluster width approaching 15% of the run time. Consequently, the median baseline filter may cause baseline artifacts when used with clustered peaks. •Because the median baseline filter uses 15% of run time as its filter width, the real-time display of an median baseline filter channel is delayed by the filter width. The control panel shows a count-down clock indicating the data delay. At the end of the run time, the entire chromatogram is stored in the database. •Restrictions: The median baseline filter should not be used when:

•the peak width exceeds 5% of the run time. •the separation is isocratic. •the baseline is flat. •peak clusters approach the filter width of 15% of run time.

PDA Detector Parameters

To reduce detector drift, select the Absorbance - Compensated data mode, enter the wavelength of interest, and enter the resolution. Specify the compensation range in the channel at the bottom of the 2D Channels tab. Select a region of the spectra where no known analytes exist, and define a starting and ending wavelength for the compensation range. The average of the absorbance values within the specified wavelength range is then subtracted from the absorbance value. The bandwidth must be ≥40 nm and ≤100 nm, and fall within the range of 190 nm to 500 nm.

Improvements in Baseline Wander with Reference Compensation

Some chromatographic methods or laboratory conditions can

result in baseline wander

— Gradient changes in the method

— Significant temperature fluctuations in the laboratory

Reference compensation may reduce the baseline wander

improving integration, especially of low level analytes

Chromatogram @ 230nm Chromatogram @ 230nm with Compensation Enabled

PDA Detector Parameters

Saving the Instrument Method

Method Set Wizard

PDA

Processing Method

Develop Processing Method

Tea

Extract Chromatogram and scroll the wavelength icon for an optimum chromatogram. Next Create a Processing Method with the Wizard.

When you clear peak width and threshold empower calculates a new optimized value for every chromatogram

Peak Purity

What does those numbers mean?

Peak Purity Theory

All Peak Spectra are one by one compared with the Spectrum of the Peak Apex. Here the spectra of a pure peak alike. In the explanation they are completely different. The explanation starts with a spectrum of only 3 wavelengths

λ3

λ1

λ2

α β

Mean

Every data point between Threshold start and end has a mobile phase spectrum. With the least square method the difference of the first wavelength is calculated. It is repeated for all the wavelengths and then the mean is calculated. This Mean value is converted to a distance and placed on the smallest vector. This creates an angle β. The difference between the 2 spectra has an angle α.

If α is larger then β, then the 2 spectra are not the same. If α is smaller then β, then the 2 spectra are the same.

Close the Processing Method Window,open Result Window and tile it vertically. When the Purity line comes above the Auto Threshold line, there the peak is not Pure.

Impurity

Peak Purity tab in Proc. Mth.

Go To > View: Processing Method Layout Purity

Library Search tab in Proc. Mth.

Library Search

Solvent Angle

ThBr Pur Thresh:2.295-2.115=0.180 Caf Pur Thresh:0.560-0.377=0.183 Libray Solvent Angle: 0.18 or 0.2

Retention Time Presearch: Empower only matches the Library Spectra within the entered window of the Ret. Time of the peak

Show Library Spectra in the table

Spectrum Index Plot

At the end Save Processing Method and the Preferences. Close Review and Process COOP Sample Set

Possible routine Window layout

Routine Analysis

PDA

Report Method

Add overlay library match. Combine the 2 tables , show only known comp. Add White Graphics to remove Legend of the Purity Plot Save the Report.

Other Report Groups

Is absolute retention time

Workshop Create a Processing Method Use: Project: NUT_PDA Method Set: Tea (System Name: Alliance_PDA, Instrument Name: Tea) Sample Set: COOP 1. Take Sample Set in Review 2. Select Standard 3 3. Open Method Set 4. Extract Wavelength 272 nm 5. Create Processing Method with the Wizard

1. Integration Start .9 min till end chromatogram 2. Do not clear PW and Thresh 3. Use Area, Amount and Linear Cal Curve 4. Peak Names: Theobromine and Caffeine 5. Ext Std. Method 6. No Library Search yet 7. For Peak Purity use interval time between 8 and 10 min. 8. Save Processing Method in the Method Set 9. Look at the Purity tab of the Processing Method. 10. Verify the peak purity for all injections, look at the Result Window graphics 11. Create a Library Method using Standard 2 12. Add the spectra to the Library 13. Add Spectra name column to the Spectra table and move and rename it together with the info column 14. Look at the Library tab in the Proc.Mth, select the Library and determine the solvent angle with the purity

noise and noise + solventangle. Select the retention time presearch. 15. Save the Processing Method. 16. Select the Spectrum Index Plot and overlay the library. 17. Save the Preferences. 18. Exit Review, Process the Sample Set and Review the Result Set. 19. Scroll through the injections looking at the chromatogra, Peak table, Spectrum Index Plot and Result

Window for the Purity. 20. If there is time, create a Report Method using the slides in the presentation.