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Diode Array Detectors

Photodiode Array

Advanced Detection Technologies


for Compound Identification

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

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

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

AU

nm

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

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

?
sin

( B ij  s j Ai)

i= 1

0.00
2.40

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

Degrees

Peak Purity Plots can indicate


Peak homogeneity
Spectral homogeneity
Coeluting impurities
Spectral differences due to artifacts

Absorbance
Noise Angle
Purity Angle
Baseline

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

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

1.0

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

?? m 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

1.3 nm
5 nm
10 nm

1
0

Slit

Diodes

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

Linearity
greater
than 2 AU

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

NH

Simazine

C2H5

CH 3

N
CH NH C

CH 3
CH
CH 3

C NH
N

Propazine

Features and Advantages Optical Resolution

C
CH3
CH3

CH NH

N
C

NH

C2H5

Atrazine

FEATURES
Less than 2 nm optical and
dioderesolution

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 l
(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.

0.730

651471

PeakA

0.096

1.163

1.397

655846

PeakB

0.071

1.284

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

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

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.

0.740

660273

PeakA

0.042

1.203

1.590

666849

PeakB

0.026

1.347

9.023

560464

PeakC

0.079

1.839

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

1.777

4.95

1.960

0.27

2.743

028

3.143

0.06

9.927

0.93

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