considerations in peak purity...
TRANSCRIPT
Dr. Shulamit Levin
Considerations in Peak Purity Measurements
Shulamit LevinAnalytical Chemistry DepartmentMedtechnica
Dr. Shulamit Levin
Liquid fromcolumn
Photodiode array Detector
Dr. Shulamit Levin
The Data is 3D – behind every point in the chromatogram hides a spectrom!
nm Abs
200 0.00201 0.01202 0.02203 0.03--
nm Abs
200 0.00201 0.01202 0.02203 0.03--
nm Abs
200 0.00201 0.01202 0.02203 0.03--
nm Abs
200 0.00201 0.01202 0.02203 0.03--
Dr. Shulamit Levin
Extraction of 3D Data XY plane = Chromatogram ; ZY plane = spectrum
WavelengthAbso
rban
ce
Spectrum
Time
Abso
rban
ce
Chromatogram
λλλλ1
λλλλ2
Dr. Shulamit Levin
Coelution of 2 PeaksA
U
Elution Time
A
B
Coelution detection at a single wavelength
Coelution is the sum of absorbance of 2 peaks A and B
Dr. Shulamit Levin
Chromatographic Resolution& Coelution Detection
R=0 R=0.3 R=0.7 R>1.0
R=0 Purity Angle not effective; Match Angle usefulR=0.3 to R=0.7 Purity & Match Angle usefulR>0.7 Match Angle not useful
t0
t R(1) tR(2)
w 1 w 2
Rs = tR (2) - t R(1)
1/2 (w1 + w2)
Dr. Shulamit Levin
Peak Purity and Spectral Matching Principles:Spectral contrast angle:
Abso
rban
ce
Time
Standard
Time
Unknown
Matching compares the unknown apex spectrum of the peak with a referencespectrum in a library
Library identification
Abso
rban
ce
Time
Peak Purity analyzes all spectra (minimum 15) within a peakApex spectrum is the reference spectrum
Purity verificationApex
sin θ θ θ θ j = = = =
( B ij −−−− s j A i ) 2
i = = = = 1
N
Σ Σ Σ Σ
B ij2
i = = = = 1
N
Σ Σ Σ Σ
0 0 0 0 ≤≤≤≤ Sin θ θ θ θ ≤≤≤≤ 1111
0 0 0 0 deg ≤≤≤≤ θ θ θ θ ≤≤≤≤ 90 90 90 90 deg
Dr. Shulamit Levin
Spectral Contrast Angle = 53 DegreesVery large difference
200.00 240.00 280.00 320.00nm
EthylPabaEthylparaben
Abs
orba
nce
53 degrees is a large spectral difference
Dr. Shulamit Levin
Spectral Contrast 10 Degrees
230.00 250.00 270.00 290.00 310.00nm
TheophyllineDyphylline
Abs
orba
nce
Similar spectra for structurally related compounds
Dr. Shulamit Levin
Spectral Contrast 0.5 Degrees
200.00 240.00 280.00 320.00nm
MethylparabenEthylparaben
Abs
orba
nce
Very similar spectra, CH2 difference
Spectral Contrast can differentiate these spectra
Dr. Shulamit Levin
Very Similar Spectra
210.00 230.00 250.00 270.00 290.00nm
Abs
orba
nce
Analyte and 2 impurities
Spectra from 200 to 300 nm
Dr. Shulamit Levin
Detection of Spectral Fine Structure Requires 1.2 nm Resolution
246.00 254.00 262.00 270.00nm
Abs
orba
nce 1.2 nm
Analyte and one impurity spectra from 245 to 275 nm
1.2 nm resolution
Dr. Shulamit Levin
AU
0.00
0.10
0.20
0.30
0.40
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00
CAROTENOIDS - Extracted from leavesSpectrum Index presentation
CAR
T1 -
2.57
7
CAR
T2 -
4.22
7
Peak
3 -1
0.24
4
Peak
4 -1
3.70
6
Peak
5 -1
6.72
6
CAR
T3 -
18.7
63
Peak
7 -1
9.08
9
CAR
T4 -
22.9
94
CAR
T5 -
24.6
87
Peak
10 -
25.3
41
CAR
T6 -
28.9
82
Peak
12 -
30.8
28
CAR
T7 -
31.2
20
nm
400.00
500.00
Peak
14 -
31.8
58
1 23 4
5/67
8 910
Zoomed Chromatogram
VIS Spectra of the peaks
Dr. Shulamit Levin
An Example for Pure PeakSpectra collected from Peak 9
nm350.00 400.00 450.00 500.00 550.00
29.20829.14229.09329.04328.95029.00028.90028.85128.80228.757
Purity Angle Purity Threshold Purity Flag0.284 0.551 No
AU
Deg
rees
0.00
0.01
0.02
0.00
2.00
4.00
6.00
8.00
10.00
Minutes28.50 28.60 28.70 28.80 28.90 29.00 29.10 29.20 29.30 29.40 29.50
28.9
82
PurityAuto Threshold
Peak is Pure
Dr. Shulamit Levin
An Example for non Pure Peak
Purity Angle Purity Threshold Maximum Impurity Purity Flag1.885 0.404 17.078 Yes
Purity Plot of Peak 4 - Not PureAU
Deg
rees
0.00
0.01
0.02
0.03
0.04
0.00
10.00
20.00
30.00
Minutes16.40 16.50 16.60 16.70 16.80 16.90 17.00 17.10 17.20 17.30 17.40
16.7
26
PurityAuto Threshold
Dr. Shulamit Levin
AU
0.00
0.05
Minutes16.00 16.50 17.00 17.50
An Example for non Pure PeakSpectra Selected from Peak 4
Peak tail
{nm
350.00 400.00 450.00 500.00 550.00
17.20017.15017.10017.05017.00016.950
16.90016.80016.75016.70016.65016.60016.55016.500
}Peak tail
Dr. Shulamit Levin
Beware of Peak Integration- where the peak starts or ends! Effect of Integration Events on Peak Purity Results
AU
0.00
0.05
Minutes16.40 16.60 16.80 17.00 17.20 17.40 17.60
Purity Angle Purity Threshold USP Tailing2.259 0.410 1.438
Th = 10 PW = 45
Purity Angle Purity Threshold USP Tailing1.682 0.401 1.415
AU
0.00
0.05
Minutes16.40 16.60 16.80 17.00 17.20 17.40 17.60
Th = 50 PW = 45
AU0.00
0.05
Minutes
16.40 16.60 16.80 17.00 17.20 17.40 17.60
Purity Angle Purity Threshold USP Tailing0.297 0.380 1.057
Th = 200 PW = 45
Symmetric and pure!
Asymmetric and not pure! Asymmetric and not pure!
Dr. Shulamit Levin
Peak is asymmetric but pure!
AU
0.00
0.01
0.02
0.03
Minutes7.40 7.60 7.80 8.00 8.20
nm200.00 220.00 240.00 260.00 280.00
7.4707.7687.7087.6367.5777.5347.507
Extracted chromatograms
Name Purity Angle Purity ThresholdGBPN 0.217 0.383
USP Tailing = 2.33
Dr. Shulamit Levin
Nucleoside analog’s Enantiomers - Identical UV Spectra3D Plot
0.0000.005
0.010
0.015
0.020
0.025
0.030
AU
9.00 10.00 11.00 12.00 13.00 14.00Minutes
250.00
300.00
350.00
Dr. Shulamit Levin
Nucleoside analog’ Enantiomers - Identical UV Spectra
AU
0.000
0.005
0.010
Minutes11.00 12.00 13.00 14.00
nm250.00 300.00 350.00
12.20212.10212.00211.91811.88511.78511.68511.50211.40211.30211.28511.18511.085
Spectra collected from the two peaks
Dr. Shulamit Levin
Non-Linearity Effects Spectra collected at low portions of the peak are different than those at
around the apex
AU
0.00
1.00
2.00
3.00
Minutes7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50
nm
220.00
380.00SI 1 SI 2 SI 3 SI 4 SI 5 SI 6 SI 7 SI 8 SI 9 SI 10SI 11SI 12SI 13
High Conc.Non Linear
Dr. Shulamit Levin
Linear Range of Concentration Spectra collected at low portions of the peak are identical to those around
the apexnm
220.00
380.00SI 1 SI 2 SI 3 SI 4 SI 5 SI 6 SI 7 SI 8 SI 9SI 10SI 11SI 12SI 13
AU
0.00
0.20
0.40
0.60
Minutes8.00 8.50 9.00
Low Conc.Linear