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Dr. Shulamit L

Considerations in Peak Purity

Measurements

Shulamit Levin

Analytical Chemistry Department

Medtechnica

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Liquid from

column

Photodiode array Detector

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The Data is 3D behind every point in

the chromatogram hides a spectrom!

nm Abs

200 0.00

201 0.01

202 0.02

203 0.03

--

nm Abs

200 0.00

201 0.01

202 0.02

203 0.03

--

nm Abs

200 0.00

201 0.01

202 0.02

203 0.03

--

nm Abs

200 0.00

201 0.01

202 0.02

203 0.03

--

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Extraction of 3D DataXY plane = Chromatogram ; ZY plane = spectrum

WavelengthAb

sorbance Spectrum

Time

Absorba

nce

Chromatogram

1

2

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Coelution of 2 Peaks

AU

Elution Time

A

B

Coelution detection at a

single wavelength

Coelution is the sum ofabsorbance of 2 peaksA and B

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Chromatographic Resolution

& Coelution Detection

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

t0

t R(1)t

w 1 w

Rs =

tR (2) - t R(1)

1/2 (w1 + w2)

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Dr. Shulamit L

Peak Purity and Spectral Matching Principles:

Spectral contrast angle:

Absorbance

Time

Standard

Time

Unknown

Matching compares theunknown apex spectrumthe peak with a reference

spectrum in a library

Libraryidentification

Absorbance

Time

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

spectrum

Purity verification

Apexsin j ====

( Bij sj Ai )

2

i ==== 1

N

Bij

2

i ==== 1

N

0000 Sin 1111

0000 deg 90909090 deg

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Spectral Contrast Angle = 53 Degrees

Very large difference

200.00 240.00 280.00 320.00

nm

EthylPaba

Ethylparaben

Abso

rbance

53 degreeis a largespectral

difference

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Spectral Contrast 10 Degrees

230.00 250.00 270.00 290.00 310.00

nm

Theophylline

Dyphylline

Absorban

ce

Similar spectra fostructurally relatedcompounds

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Spectral Contrast 0.5 Degrees

200.00 240.00 280.00 320.00nm

MethylparabenEthylparaben

Absorb

ance

Very similarspectra, CH2difference

Spectral Contracan differentiatethese spectra

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Very Similar Spectra

210.00 230.00 250.00 270.00 290.00nm

Absorbance

Analyte and 2 impurities

Spectra from 200 to 300 nm

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Detection of Spectral Fine Structure Requires 1.2 nm Resolution

246.00 254.00 262.00 270.00

nm

Absorb

ance

1.2 nm

Analyte and one

impurity spectrafrom 245 to 275nm

1.2 nmresolution

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AU

0.00

0.10

0.20

0.30

0.40

Minutes5.00 10.00 15.00 20.00 25.00 30.00

CAROTENOIDS - Extracted from leaves

Spectrum Index presentation

CART1-2.577

CART2-4.227

Peak3-10.244

Peak4-13.706

Peak5-16.726

CART3-18.763

Peak7-19.089

CART4-22.994

CART5-24.687

Peak10-25.34

1

CART6-28.982

Peak12-30.82

8

CART7-31.220

nm

400.00

500.00

1 23

4

5/67

8 9

10

Zoomed Chromatogram

VIS Spectra of the peaks

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An Example for Pure Peak

Spectra 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 Flag

0.284 0.551 No

A

U

Degrees

0.00

0.01

0.02

0.00

2.00

4.00

6.00

8.00

10.00

Minutes

28.50 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

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An Example for non Pure Peak

Purity Angle Purity Threshold Maximum Impurity Purity Flag

1.885 0.404 17.078 Yes

Purity Plot of Peak 4 - Not Pure

AU

D e g r e e s

0.00

0.01

0.02

0.03

0.04

0.00

10.00

20.00

30.00

Minutes

16.40 16.50 16.60 16.70 16.80 16.90 17.00 17.10 17.20 17.30 17.40

16

.726

Purity

Auto Threshold

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AU

0.00

0.05

Minutes16.00 16.50 17.00 17.50

An Example for non Pure Peak

Spectra Selected from Peak 4

Peak tail

{nm

350.00 400.00 450.00 500.00 550.00

17.200

17.15017.10017.05017.00016.950

16.90016.80016.750

16.70016.65016.60016.55016.500

}Peak tail

f k i h h k d

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Beware of Peak Integration- where the peak starts or ends!Effect of Integration Events on Peak Purity Results

AU

0.00

0.05

Minutes

16.40 16.60 16.80 17.00 17.20 17.40 17.60

Purity Angle Purity Threshold USP Tailing

2.259 0.410 1.438

Th = 10 PW = 45

Purity Angle Purity Threshold USP Tailing

1.682 0.401 1.415

AU

0.00

0.05

Minutes

16.40 16.60 16.80 17.00 17.20 17.40 17.60

Th = 50 PW = 45

AU

0.00

0.05

Minutes

16.40 16.60 16.80 17.00 17.20 17.40 17.60

Purity Angle Purity Threshold USP Tailing

0.297 0.380 1.057

Th = 200 PW = 45

Symmetric and pure!

Asymmetric and not pure! Asymmetric and not pure!

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Dr. Shulamit L

Peak is asymmetric but pure!

AU

0.00

0.01

0.02

0.03

Minutes

7.40 7.60 7.80 8.00 8.20

nm200.00 220.00 240.00 260.00 280.00

7.4707.7687.7087.636

7.5777.5347.507

Extracted chromatograms

Name Purity Angle Purity ThresholdGBPN 0.217 0.383

USP Tailing = 2.33

Nucleoside analogs Enantiomers Identical UV Spectra

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Nucleoside analog s Enantiomers - Identical UV Spectra

3D Plot

0.000

0.005

0.010

0.015

0.020

0.025

0.030

A U

9.00 10.00 11.00 12.00 13.00 14.00

Minutes

250.00

300.00

350.00

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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

12.20212.10212.00211.91811.88511.78511.685

11.50211.40211.30211.28511.18511.085

Spectra collected from the two peaks

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Non-Linearity EffectsSpectra collected at low portions of the peak are different than those at

around the apex

AU

0.00

1.00

2.00

3.00

Minutes

7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50

nm

220.00

380.00

SI 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 Linea

Linear Range of Concentration

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Linear Range of ConcentrationSpectra collected at low portions of the peak are identical to those around

the apex

nm

220.00

380.00

SI 1 SI 2 SI 3 SI 4 SI 5 SI 6 SI 7 SI 8 SI 9 SI 10SI 11SI 12SI 13

AU

0.00

0.20

0.40

0.60

Minutes

8.00 8.50 9.00

Low Con

Linear