Diode Array Detectors

Photodiode Array

Advanced Detection Technologies for Compound Identification

1

Diode Array Detectors

Photodiode Array

PDA Optics Diagram

Lamp

Mirrors Lens

Flow Cell Slit

Grating

Diodes

Advanced Detection Technologies for Compound Identification

Waters 21,661

How the Diode Array Works The  of light striking the diode is determined by its position relative to the stationary grating

Diodes discharged

Diodes recharging

Liquid from column

Dr. Shulamit Levin,

1-4

Diode Array Detectors

Extraction of 3D Data

The Data is 3D

nm

Abs

nm

Abs

nm

200 0.00 201 0.01 202 0.02 203 0.03 -

200 201 202 203 -

0.00 0.01 0.02 0.03

200 0.00 201 0.01 202 0.02 203 0.03 -

200 0.00 201 0.01 202 0.02 203 0.03 -

-

-

-

-

Abs

nm

Absorbance

Absorbance

Chromatogram

Abs

2

Spectrum Time

Wavelength

996 PDA Spectrum Index Plot

Coelution of 2 Peaks

DNPH Derivatives 0.25 ng Each Peak Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm 440.00 420.00 400.00 380.00

440.00 420.00 400.00 380.00

360.00 340.00 320.00 300.00

360.00 nm 340.00 320.00 300.00

280.00 260.00

280.00 260.00

A

B

Coelution detection at a single wavelength Coelution is the sum of absorbance of 2 peaks A and B

AU

nm

1

0.0006 0.0006 0.0004 0.0004

AU

AU

0.0002

0.0002

Elution Time

0.0000

0.0000 4.00

Dr. Shulamit Levin,

6.00

8.00

10.00

12.00 Minutes

14.00

16.00

18.00

5-8

Diode Array Detectors

Spectrum Index Plot 300.00

Chromatographic Resolution & Coelution Detection

Spectra at apex and inflection points are displayed

tR(1)

t0 w1

nm

250.00

tR(2)

Rs = t R(2) - tR(1) 1/2 (w1 + w 2)

w2

Spectrumat maximum impurity is different

200.00 0.40

R=0

Maximum Impurity 0.20

R=0.3

R=0.7

R>1.0

AU

R=0 Purity Angle not effective; Match Angle useful R=0.3to R=0.7 Purity & Match Angle useful

0.00 2.20

2.40

2.60

2.80

3.00

R>0.7 Match Angle not useful

Minutes

Photodiode Array Technology Spectral Analyses Library Matching Compound identification Coelution detection Peak Purity Analysis Peak purity/peak homogeneity Coelution detection

Dr. Shulamit Levin,

Importance of Spectral Analyses Library Matching Compound identification Coelution detection Peak Purity Analysis Peak purity/peak homogeneity Coelution detection

9-12

Diode Array Detectors

Major Peak and Minor Peaks Analyte

PROBLEM: Locate all impurities

sin j =

Purity verification

AU

0.004 0.002

Bij2

Unknown

Absorbance

Impurities Resolved Coeluting

0.006

?

sj A)i 2

N

Library identification Standard

0.008

=

i =1

Absorbance

0.010

?i 1 (Bij  N

ood quality spectral formation is important for:

0.000 Time

-0.002 0.00

1.00

2.00

3.00

4.00

5.00

6.00

Peak Purity analyzes all spectra (minimum 15) within a peak Apex spectrum is the reference spectrum

7.00

Minutes

240.00

280.00

nm

Dr. Shulamit Levin,

320.00

53 degrees is a large spectral difference

Theophylline Dyphylline

Similar spectra for structurally related compounds

Absorbance

Absorbance

200.00

Time

Spectral Contrast 10 Degrees

Spectral Contrast 53 Degrees Ethylparaben EthylPaba

Time

Matching compares the unknown apex spectrum of the peak with a reference spectrum in a library

230.00

250.00

270.00

290.00

310.00

nm

13-16

Diode Array Detectors

Spectral Contrast 0.5 Degrees

Very Similar Spectra Very similar spectra, CH2 difference

Methylparaben Ethylparaben

Analyte and 2 impurities

Absorbance

Spectral Contrast can differentiate these spectra

200.00

240.00

280.00

320.00

210.00

nm

230.00

250.00

270.00

290.00

nm

Spectral Comparison Spectrum A (apex)

Spectra from 200 to 300 nm

The shapes of Spectrum Aand

Peak Purity Plot

Choose one reference spectrum,

Spectrum Bare represented by vectors  is the Spectral Contrast Angle, the difference between

compare to it all other spectraon thepeak. Thecomparisonisdone by linear regretion to a straight line.

Another option

spectral shapes

260



60.00

Purity Angle at every data point

50.00 40.00

250

Spectra B(i)

240

Increases in Purity Angle are spectral differences

30.00

230 220

sin 

20.00

210 200

Impurity

10.00

N

? sin 

j

=

( B ij  s j Ai)

i= 1

0.00 2.40

N

?

2

B ij2

2.50

2.60

Minutes

2.70

Absorbance Purity Angle Noise Angle Baseline

i= 1

Dr. Shulamit Levin,

17-20

Diode Array Detectors

Purity Plot Chemically Pure Compound

Interpretation of Peak Purity Plots

5

Degrees

Peak Purity Plots can indicate Peak homogeneity Spectral homogeneity Coeluting impurities Spectral differences due to artifacts

Absorbance Noise Angle Purity Angle Baseline

0

Minutes

Purity Angle less than Noise Angle, ideal situation

Purity Plot: Mixture of 2 Compounds

Purity Plot: Mixture of 2 Compounds

Absorbance Noise Angle Purity Angle Baseline

10 Impurity

Impurity 10

Absorbance Noise Angle Purity Angle Baseline

Degrees

Degrees

20

0

0

Minutes

Purity Angle is greater than Noise Angle coelution on the front of the peak Dr. Shulamit Levin,

Minutes

Purity Angle is greater than Noise Angle - coelution near the peak apex

21-24

Diode Array Detectors

Purity Plot Chemically Pure Compound 2

Absorbance Noise Angle Purity Angle

Pure Benzoic Acid

2.0

Baseline

Peak Apex Non-linear

Different benzoic acid concentrations

1.5

Absorbance

Degrees

Effect of Concentration on Spectra

1

1.0

Spectral shape changes

0.5

0 0

Minutes

190.00

210.00

230.00

Purity Angle greater than Noise Angle Absorbance out of linear range at some wavelengths

Compound Confirmation

Fail

Fail

Fail

290.00

Chromatographic and Spectral Sensitivity: large bandwidth low resolution

Fail

Pass

270.00

Conflicts in Instrument design

Peak Homogeneity Pass

250.00

nm

Pass

Library Match Fail

Dr. Shulamit Levin,

Linearity: narrow bandwidth

Spectral performances: high resolution

25-28

Diode Array Detectors

Optical vs. Diode Resolution

Optical vs. Diode (Numeric) Resolution

IDEAL High optical resolution is 1 nm Light 1 nm

1 nm

Slit

Optical resolution affects linearity

Slit

1.2 nm Diode Resolution

Diodes

3 nm Optical Resolution

Diodes

996 Spectral Performance 1.2 nm Bandwidth

1 nm Diode

1 nm light beams

No bunching

Brand X Spectral Performance

Slit width, # diodes, and WL range (hardware) determine optical resolution: 1.2 nm

1nm slit, 2 nm 'bunch' nm ptical Resolution m light on >1 diode

0.7 nm diode resolution

3 diodes bunching

1 nm

1 nm ight beam

nm/diode (hardware) determine diode resolution, 1.2 nm

Slit 50 M allowing 1.2 nm bandwidith

Diodes (800-190) / 512 or1.2 nm per diode

Dr. Shulamit Levin,

Diode resolution (numeric resolution) is equal to wavelength coverage divided by diode number Hardware determines optical resolution,3 nm Optical resolution, determines the quality of spectra

Overall: 1.2 nm Spectral Performance

Diode 'bunching' (software) determines the overall spectral performance, 1.2 nm

light beam

nm/diode(hardware) determine diode resolution, 0.7 nm

?? µm slit, allowing1nm bandwidth

Digital resolution, ncorrectly called "Spectral Resolution"

Slit width, # diodes, and WL range (hardware) determine optical resolution: 1 nm

1024 diodes covering 190-950nm 0.7 nm/diode

Overall: 2 nm Spectral Performance

Diode 'bunching' (software) determinesthe spectral performance, 2 nm

29-32

Diode Array Detectors

Photodiode Array Technology

Importance of Detector Linearity

Optical Performance

Quantitation Major peaks Minor peaks

Linearity Optical Resolution Sensitivity

Spectral Analyses Library Matching Peak Purity/Peak Homogeneity

Effects of Optical Resolution on Linearity

Optical vs. Diode Resolution

3 nm Optical Resolution

1 nm per diode is 1 nm diode resolution

3 3

1.3 nm resolution is more linear than 5 or 10 nm

2 2 AU

1 nm Diode

ight nm

Slit width determines optical resolution, 3 nm

1

1.3 nm 5 nm 10 nm

1 0

Slit

Diodes

0

20 10

Dr. Shulamit Levin,

40 30 Concentration

60

Wide bandwidth is non-linear

50

33-36

Diode Array Detectors

Benzoic Acid Spectrum

Technical approaches to gain in linearity

1.60 1.40 1.20

Increase optical resolution

AU

214 nm is on a spectral slope

227.4 nm

1.00 0.80 0.60

One forbidden technical approach: using a prism in place of a grating, since prisms are non linear

0.40 0.20 0.00

214 nm

220.00 240.00 260.00 280.00

Linearity requires good optical resolution

nm

Linearity 214 nm Benzoic acid

Other Causes of Non-Linearity

3

Linearity greater than 2 AU

2

AU

2 1

Second order effects Stray light Chemical interactions

1.2 nm resolution

1 0 0

20 10

40 30

60 50

80 70

100 90

Concentration

Dr. Shulamit Levin,

37-40

Diode Array Detectors

Photodiode Array Technology Optical Performance

Resolution Resolution can be improved by: 1) using a small slit

Linearity Optical Resolution Sensitivity

2) selecting a narrow bandwidth 3) Reducing the wavelength covering (nm/diode) 4) Enlarging the number of diodes Overall quality of optics design and manufacturing is a crucial factor

Resolution

Importance of Optical Resolution

Drawbacks: 1) Small slit: less energy means more noise 1) Reduce the wavelength range: lack of information in the visible 2) More diodes: smaller diodes means noisier signal (less energy on each diode) Quality of optics design and manufacturing: means important R&D plus QC efforts from the supplier

Dr. Shulamit Levin,

Differentiation of Spectral Differences Similar spectra Spectral fine structure Spectral Analyses Library matching Peak purity / peak homogeneity

41-44

Diode Array Detectors

Common Perceptions

Factors Affecting Spectral Resolution

Most UV spectra have very broad spectral peaks

Optical resolution Diode or digital resolution

Good optical resolution is only required when there is spectral fine structure

Spectral Resolution - 1.2 nm vs. 3.6 nm Analyte and one impurity spectra from 245 to 275 nm

Absorbance

1.2 nm resolution

Benzene spectra Absorbance

Spectral Fine Structure

1.2 nm

Slit width and bandwidth

Less resolution at 3.6 nm vs. 1.2 nm UV maxima shifted

246.00

254.00

262.00

nm

Dr. Shulamit Levin,

270.00

230.00

250.00

270.00

nm

45-48

Diode Array Detectors

UV spectra of Triazines with the Waters 996 detector C

C N 2H5 NH

CH 3

N

C

C

NH

Simazine

C2H5

N

CH 3

N

N CH NH C

CH 3 CH CH 3

C NH N

Propazine

Features and Advantages Optical Resolution

C CH3 CH3

CH NH

N

N C

C

NH

C2H5

Atrazine

FEATURES Less than 2 nm optical and dioderesolution

N

221.6 nm

220.5 nm 221.6 nm 221.6 nm

221.6 nm 220.5 nm

260.6 nm 261.8 nm

ADVANTAGES Differentiation of similar spectra Visualizing spectral fine structure Linearity 190 to 800 nm to 2 AU

260.6 nm

220.00

240.00

260.00

280.00

300.00

216.00

220.00

224.00

228.00

nm

nm

Benefits of Good Optical Resolution

Photodiode Array Technology Optical Performance

Peak confirmation Confidence in compound identification Confidence in peak homogeneity with good peak purity analysis Good detector linearity Quantitation at high and low concentrations Spectral analyses Identification of major and minor compounds

Dr. Shulamit Levin,

Linearity Optical Resolution Sensitivity

49-52

Diode Array Detectors

Signal-to-Noise Ratio

Importance of Sensitivity Detection of Low Concentrations of Analytes Detection of impurities, metabolites, by-products and degradation products Quantitation

Signal-to-noise (S/N) is peak height to noise

6:1

3:1

8:1

Detection of Spectra at Low Concentrations Peak identification Peak purity / peak homogeneity

Chromatographic Sensitivity

Perceptions

Signal-to-Noise Ratio

Photodiode array detectors (PDA) are much less sensitive than variable wavelength detectors 0.001 AU

0.2 AU

PDA detectors are noisy

No apparent noise 2.8

3.0

3.2

Minutes

Dr. Shulamit Levin,

3.4

Noise

2.00

3.00

PDA detectors can not be used for quantitation of minor peaks

4.00

Minutes

53-56

Diode Array Detectors

Technological Advances in PDA Detectors

Waters 996 Chromatographic Sensitivity

New designs to improve signal-to- noise performance

0.0003

Waters 996 PDA Peak = 0.0001 AU

0.0002

Increased chromatographic sensitivity

AU

Waters 486 tunable UV Peak = 0.0001 AU

0.0001

Increased spectral sensitivity 0.0000

Enhanced software for improved performance 1.5

2.0

2.5

3.0

3.5

4.0

Minutes

High Sensitivity Chromatogram

Chromatographic Sensitivity Triazine herbicides at detection limit 0.0010

0.00006

0.0008

Simazine

0.00004

0.0006

0.00002

Peak height = 0.00007 AU 257 nm 1 sec filter

0.00000

AU

-0.00002 -0.00004 -0.00006 -0.00008 1.60

2.00

2.40

2.80

Minutes

Dr. Shulamit Levin,

3.20

3.60

Desethylatrazine

0.0004 0.0002

10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 Minutes

Conditions: Gradient: Phosphate-Acetonitrile Column Novapak2x 300mm Sample: 2 ppb each pesticide Injection: 150 µl (0.3 ng on column)

PDA Resolution: 1.2 nm, Acquisition: 200 to 350 nm, 2 spectra per second. Chromatogram extracted at 220 nm No smoothing or bunching

57-60

Diode Array Detectors

High Sensitivity Spectrum

UV spectrum for Simazine at 2 ppb 0.00024

220.5 nm

0.00020

Absorbance

AU 0.00010

268.9 nm

0.00007 AU 0.530 AU

339.0 nm

306.8 nm

0.00000 -0.00002

210.00 220.00

240.00

260.00

280.00 nm

300.00

320.00

340.00

230.00

250.00

270.00

290.00

nm

Millennium PDA Spectrum Review Plot - SampleName Mel2ppb, Vial 44, Inj. 1

Increase Signal-to-Noise Ratio Signal-to-noise (S/N) is peak height to noise

6:1

3:1

8:1

Increase S/N by increasing peak height

Factors Increasing Signal Increase sample concentration Increase injection volume Wavelength Low volume flow cell

Increase S/N by decreasing noise

Dr. Shulamit Levin,

61-64

Diode Array Detectors

PDA Optics Diagram

Lamp

Mirrors Lens

Flow Cell Slit

Grating

Factors Affecting Noise Optics bench design Lamp energy Wavelengths Mobile phase Resolution Filter

Diodes

Each component in the optics path will affect noise

Technical approaches to gain in chromatographic sensitivity

AU 0.10

0.00 5.00

Sophisticated approaches: optimize the optics design: minimum dispersion, good focus of light on the diodes lamp optimization software (eliminate the need for different slits)

Dr. Shulamit Levin,

PROBLEM 4 Compounds 3 Peaks

7.247

0.20

0.730

enlarge slit width (decrease resolution) change optical resolution (affects spectrum) diode bunching (affects spectrum) noise smoothing (affects peak shape and height) reference wavelength substraction (loss of information in the subtracted band)

1.397

Traditional approaches:

Method Development #1

#

RetTi me (min)

Area (uV*sec)

Minutes

10.00

Match SpectrumNam e

Matc h Angl e

Matc h Thre sh.

1

0.730

651471

PeakA

0.096

1.163

2

1.397

655846

PeakB

0.071

1.284

3

7.247

1019807

PeakC

0.883

1.640

65-68

Diode Array Detectors

Peak tracking

Standards run only once for library

0.00

10.00

5.00

D

AU 0.10

0.00

Mobile phase changed to shorten run time

0.743

0.20

5.627 5.910

9.023

9.773

AU 0.10

Mobile phase changed 4 peaks identified

D

1.590

0.20

Method Development #3 1.277

0.740

Method Development #2

Minutes

2.00

4.00

6.00

8.00

Minutes #

RetTi me (min)

Area (uV*sec)

Match SpectrumName

Matc h Angl e

Matc h Thre sh.

1

0.740

660273

PeakA

0.042

1.203

2

1.590

666849

PeakB

0.026

1.347

3

9.023

560464

PeakC

0.079

1.839

4

9.773

434562

PeakD

0.516

2.747

# 1 2 3 4

Ret Time (min)

Area (uV*sec)

0.743 1.277 5.627 5.910

Match Spectrum Name

652303 654077 366935

PeakA PeakB PeakD PeakC

682223

Match Angle

Match Thresh.

0.139 0.125 1.455

1.161 1.274 2.366

0.369

1.649

Stability Test at 12 Weeks

Stability Test at 8 Weeks

Impurity 1

Impurity 2 Analyte

4.416 0.085

*

0.05

0.03

0.01 0.00 2.0

4.0

6.0

8.0

10.0

0.03 ANALYTE

Unknown

0.02

Impurity2

AU

0.04

12.0

Unknown

210

230

250

270

290

nm Unknown

AU 0.02

14.0

0.01

Minutes

Minutes

ANALYTE

1.021 6.424

Impurity2

Impurity 1 Unknown

Absorbance

Purity Angle Impurity 1

0.04

Impurity 1

0.05

Peak Flag

0.00 2.00

Dr. Shulamit Levin,

4.00

6.00

8.00

10.00

12.00

14.00

69-72

Diode Array Detectors

Stability Data at 12 Weeks Ret #

Match

Time

Area

Spectrum

Match

(Min)

%

Name

Angle

Flag

0.792

*

1

1.777

4.95

2

1.960

0.27

3

2.743

028

4

3.143

0.06

5

9.927

0.93

6

Peak Purity Using Photodiode Array Detection

11.193 93.50

Impurity 1

Purity Angle

Flag

33.261

Spectral homogeneity or peak homogeneity

* NEVER Chemical Purity Impurity has absorbance Impurity is present in high enough concentration Impurity is spectrally different from the analyte

6.461 Impurity 2

6.542

12.879 25.868

Analyte

0.154

*

0.092

Positive Compound Identification and Monitoring Integrated PDA with Mass Detector enables: PDA peak purity to investigate peak homogeneity UV and mass spectral information to be used from the same run for positive compound identification UV monitoring of separation for diagnostic purposes and quantitation

Dr. Shulamit Levin,

73-76