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Considerations in Peak Purity Measurements

Shulamit Levin Analytical Chemistry Department Medtechnica

Dr. Shulamit Levin

Photodiode array Detector

Liquid from column


Dr. Shulamit Levin

The Data is 3D behind every point in the chromatogram hides a spectrom!

nm 200 201 202 203 -

Abs 0.00 0.01 0.02 0.03

nm 200 201 202 203 -

Abs 0.00 0.01 0.02 0.03

nm 200 201 202 203 -

Abs 0.00 0.01 0.02 0.03

nm 200 201 202 203 -

Abs 0.00 0.01 0.02 0.03

Dr. Shulamit Levin

Extraction of 3D Data
XY plane = Chromatogram ; ZY plane = spectrum

Chromatogram
Absorbance

1 2
Time

Absorbance

Spectrum

Wavelength
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Coelution of 2 Peaks
A

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

B AU

Elution Time
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Chromatographic Resolution & Coelution Detection

t R(1) Rs = t0
w1

t R(2)

tR (2) - t R(1)
1/2

(w 1 + w 2 )

w2

R=0

R=0.3

R=0.7

R>1.0

R=0 Purity Angle not effective; Match Angle useful R=0.3 to R=0.7 Purity & Match Angle useful R>0.7 Match Angle not useful
Dr. Shulamit Levin

Peak Purity and Spectral Matching Principles:


Spectral contrast angle:

Purity verification
Apex

sin j =

( B ij s j Ai ) 2

Library identification
Standard Unknown

i =1

B ij 2

i =1

Absorbance

0 deg 90 deg

Time

Absorbance

0 Sin 1

Time

Time

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

Matching compares the unknown apex spectrum of the peak with a reference spectrum in a library
Dr. Shulamit Levin

Spectral Contrast Angle = 53 Degrees Very large difference


Ethylparaben EthylPaba

Absorbance

53 degrees is a large spectral difference

200.00

240.00

nm

280.00

320.00
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Spectral Contrast 10 Degrees


Theophylline Dyphylline

Absorbance

Similar spectra for structurally related compounds

230.00

250.00

270.00

290.00

310.00

nm
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Spectral Contrast 0.5 Degrees


Very similar spectra, CH2 difference Spectral Contrast can differentiate these spectra

Methylparaben Ethylparaben

Absorbance
200.00

240.00

nm

280.00

320.00

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Very Similar Spectra


Analyte and 2 impurities Spectra from 200 to 300 nm
Absorbance
210.00

230.00

250.00

nm

270.00

290.00

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Detection of Spectral Fine Structure Requires 1.2 nm Resolution

Absorbance

1.2 nm

Analyte and one impurity spectra from 245 to 275 nm 1.2 nm resolution

246.00

254.00

262.00

270.00

nm
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CAROTENOIDS - Extracted from leaves Spectrum Index presentation


Peak10 - 25.341 CART3 - 18.763 CART6 - 28.982 Peak12 - 30.828 CART4 - 22.994 CART5 - 24.687 CART1 - 2.577 CART7 - 31.220 Peak4 - 13.706 CART2 - 4.227 Peak5 - 16.726 Peak7 - 19.089 Peak14 - 31.858 35.00 Peak3 - 10.244

VIS Spectra of the peaks

500.00

400.00

nm
0.40 0.30

Zoomed Chromatogram

5/6 3
15.00

7 8 9

AU

0.20 0.10 0.00 5.00 10.00

10

4
Minutes
20.00

25.00

30.00

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An Example for Pure Peak


Spectra collected from Peak 9
28.757 28.802 28.851 28.900 29.000 28.950 29.043 29.093 29.142 29.208

350.00

400.00

450.00

nm

500.00

550.00

0.02

28.982

Purity Auto Threshold


AU

10.00 8.00

Peak is Pure

0.01

4.00 2.00

0.00 28.50 28.60 28.70 28.80 28.90 29.00 29.10 29.20 29.30 29.40 29.50

0.00

Minutes

Purity Angle 0.284

Purity Threshold Purity Flag 0.551 No

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Degrees

6.00

An Example for non Pure Peak


Purity Plot of Peak 4 - Not Pure
Purity Angle 1.885 Purity Threshold 0.404 Maximum Impurity 17.078 Purity Flag Yes

0.04

0.03 AU

16.726

Purity Auto Threshold

30.00

0.02 10.00 0.01 0.00 16.40 16.50 16.60 16.70 16.80 16.90 17.00 17.10 17.20 17.30 17.40

0.00

Minutes
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Degrees

20.00

An Example for non Pure Peak


Spectra Selected from Peak 4

Peak tail

16.950 17.000 17.050 17.100 17.150 17.200

}
400.00 450.00

16.500 16.550 16.600 16.650 16.700 16.750 16.800 16.900

350.00

nm

500.00

550.00

0.05 AU

Peak tail
0.00

16.00

16.50

Minutes

17.00

17.50

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Beware of Peak Integration- where the peak starts or ends!


Effect of Integration Events on Peak Purity Results
Purity Angle 0.297
0.05 AU

Purity Threshold USP Tailing 0.380 1.057

Symmetric and pure!

0.00

Th = 200 PW = 45
16.40 16.60 16.80 17.00 17.20 17.40 17.60

Minutes

Purity Angle 2.259 0.05 AU

Purity Threshold USP Tailing 0.410 1.438

Purity Angle 1.682 0.05 AU

Purity Threshold USP Tailing 0.401 1.415

Asymmetric and not pure!

Asymmetric and not pure!

0.00

Th = 10 PW = 45
16.40 16.60 16.80 17.00 17.20 17.40 17.60 Minutes

0.00

Th = 50 PW = 45
16.40 16.60 16.80 17.00 17.20 17.40 17.60 Minutes

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Peak is asymmetric but pure!


Name GBPN Purity Angle 0.217 Purity Threshold 0.383
7.507 7.534 7.577 7.636 7.708 7.768 7.470

Extracted chromatograms

200.00

220.00

240.00

nm

260.00

280.00

0.03 0.02 AU USP Tailing = 2.33

0.01 0.00 7.40 7.60 7.80 Minutes 8.00 8.20

Dr. Shulamit Levin

Nucleoside analogs Enantiomers - Identical UV Spectra 3D Plot


0.030 0.025 0.020 AU 0.015 0.010 0.005 0.000

250.00 300.00 350.00 9.00 10.00 11.00 12.00 Minutes 13.00 14.00
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Nucleoside analog Enantiomers - Identical UV Spectra


Spectra collected from the two peaks
11.085 11.185 11.285 11.302 11.402 11.502 11.685 11.785 11.885 11.918 12.002 12.102 12.202
250.00 0.010 300.00 nm 350.00

AU

0.005

0.000
11.00

Minutes

12.00

13.00

14.00

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Non-Linearity Effects
Spectra collected at low portions of the peak are different than those at around the apex
SI 1 SI 2 SI 3 SI 4 SI 5 SI 6 SI 7 SI 8 SI 9 SI 10SI 11SI 12SI 13 380.00

High Conc. Non Linear

nm 220.00
3.00 AU 2.00 1.00 0.00 7.00 7.50 8.00 8.50 9.00 Minutes 9.50 10.00 10.50

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Linear Range of Concentration


Spectra collected at low portions of the peak are identical to those around the apex
SI 1 SI 2 SI 3 SI 4 SI 5 SI 6 SI 7 SI 8 SI 9 SI 10 11 12 13 SI SI SI 380.00

Low Conc. Linear

nm 220.00
0.60 0.40 AU 0.20 0.00 8.00 8.50 9.00 Dr. Shulamit Levin

Minutes