Photodiode Array
Photodiode Array
Lamp
Mirrors
Lens
Flow
Cell Slit
Grating
Diodes
Waters
21,661
Diodes discharged
Diodes recharging
Liquid from
column
1-4
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
Coelution of 2 Peaks
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
6.00
8.00
10.00
12.00
Minutes
14.00
16.00
18.00
5-8
Chromatographic Resolution
& Coelution Detection
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
0.00
2.20
2.40
2.60
2.80
3.00
Minutes
9-12
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
0.000
Time
-0.002
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Minutes
240.00
280.00
nm
320.00
53 degrees
is a large
spectral
difference
Theophylline
Dyphylline
Absorbance
Absorbance
200.00
Time
Time
230.00
250.00
270.00
290.00
310.00
nm
13-16
Methylparaben
Ethylparaben
Absorbance
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)
The shapes of
Spectrum Aand
Spectrum Bare
represented by
vectors
is the Spectral
Contrast Angle, the
difference between
Another option
spectral shapes
260
60.00
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
17-20
Purity Plot
Chemically Pure Compound
Degrees
Absorbance
Noise Angle
Purity Angle
Baseline
Minutes
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
21-24
Purity Plot
Chemically Pure Compound
2
Absorbance
Noise Angle
Purity Angle
2.0
Baseline
Peak Apex
Non-linear
Different
benzoic acid
concentrations
1.5
Absorbance
Degrees
1.0
Spectral shape
changes
0.5
0
0
Minutes
190.00
210.00
230.00
Compound Confirmation
Fail
Fail
Fail
290.00
Chromatographic and
Spectral Sensitivity:
large bandwidth
low resolution
Fail
Pass
270.00
Peak Homogeneity
Pass
250.00
nm
Pass
Library
Match
Fail
Linearity:
narrow bandwidth
Spectral
performances:
high resolution
25-28
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
1 nm
Diode
1 nm
light
beams
No
bunching
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
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"
1024 diodes
covering
190-950nm
0.7 nm/diode
Overall:
2 nm
Spectral
Performance
Diode 'bunching'
(software)
determinesthe
spectral
performance, 2 nm
29-32
Optical Performance
Quantitation
Major peaks
Minor peaks
Linearity
Optical Resolution
Sensitivity
Spectral Analyses
Library Matching
Peak Purity/Peak Homogeneity
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
40
30
Concentration
60
Wide
bandwidth is
non-linear
50
33-36
1.60
1.40
1.20
AU
214 nm is
on a
spectral
slope
227.4
nm
1.00
0.80
0.60
0.40
0.20
0.00
214
nm
Linearity
requires
good
optical
resolution
nm
Linearity
greater
than 2 AU
AU
2
1
1.2 nm
resolution
1
0 0
20
10
40
30
60
50
80
70
100
90
Concentration
37-40
Resolution
Resolution can be improved by:
1) using a small slit
Linearity
Optical Resolution
Sensitivity
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
41-44
Common Perceptions
Optical resolution
Diode or digital resolution
Absorbance
1.2 nm
resolution
Benzene
spectra
Absorbance
1.2 nm
Less resolution
at 3.6 nm vs.
1.2 nm
UV maxima
shifted
246.00
254.00
262.00
nm
270.00
230.00
250.00
270.00
nm
45-48
C
N
2H5 NH
CH 3
NH
Simazine
C2H5
CH 3
N
CH NH C
CH 3
CH
CH 3
C NH
N
Propazine
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
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
Linearity
Optical Resolution
Sensitivity
49-52
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
Chromatographic Sensitivity
Perceptions
Signal-to-Noise Ratio
0.2 AU
No
apparent
noise
2.8
3.0
3.2
Minutes
3.4
Noise
2.00
3.00
4.00
Minutes
53-56
0.0003
0.0002
AU
Waters 486
tunable UV
Peak = 0.0001 AU
0.0001
2.0
2.5
3.0
3.5
4.0
Minutes
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
3.20
3.60
Desethylatrazine
0.0004
0.0002
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
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
6:1
3:1
8:1
Increase S/N by
increasing peak height
Increase S/N by
decreasing noise
61-64
Lamp
Mirrors
Lens
Flow
Cell Slit
Grating
Diodes
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)
PROBLEM
4 Compounds
3 Peaks
7.247
0.20
0.730
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
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
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
4.00
6.00
8.00
10.00
12.00
14.00
69-72
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
*
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
73-76