©2012 waters corporation 1 · ir(fppy)3 n f f n au 0.06 n f f ir f f n f ir(fppy)3 isomer...

Waters UPC² Workshop Series

Applications


Lipids


UPC²/MSUPC²/MSPolar Lipids Mixture Rapid Screening Polar Lipids Mixture Rapid Screening Separation (Brain, porcine standards)Separation (Brain, porcine standards)Separation (Brain, porcine standards)Separation (Brain, porcine standards)

1.6x108

PC

1.2x108

Inte

nsity

8.0x107

LPC

SM

4.0x107

CERPG PE LPE

00


0.0

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

UPC² and LipidomicsUPC² and LipidomicsEvaluation Mouse Heart ExtractEvaluation Mouse Heart Extract

Glycerolipids(NH + adducts) PC

2.0x108

(NH4+ adducts) PC

ty

1.5x108

Inte

nsi

5.0x107

1.0x108

PE

SM

Cardiolipins

TAG

DAG

MAG

LPC

0.0

0 00 1 20 2 40 3 60 4 80 6 00 7 20 8 40 9 60 10 80 12 00

LP

E

PGPS


Minutes0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80 12.00

UPC² Polar Lipids Mixture UPC² Polar Lipids Mixture MS ESI+ SIR OverlayMS ESI+ SIR Overlayyy

1.2x108

496.3

= 4

96.3

9.0x107

LP

Cm

/z =

4

PC

m/z

=

Inte

nsity

6.0x107

4.2

82.2

= 6

48.5

2.4 731.5

813.6

6.4 815.6

3.0x107P

Em

/z =

464

PE

m/z

= 4

8

CE

Rm

/z =

PE

m/z

= 7

02

SM

m

/z =

S

M m

/z =

PG

m/z

= 7

66

SM

m

/z =

0.0

L LPP


Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Vitamins


Vitamin E OTC FormulationsVitamin E OTC Formulations

2.74

1

1.00

1.20

1.40

1.60

3.19

3

3.37

7

3.57

0

AU

0.00

0.20

0.40

0.60

0.80

Peak 1 α-TocopherolPeak 2 β-TocopherolPeak 3 γ-Tocopherol

2.74

6

6AU

0.80

1.00

1.20

1.40Peak 4 δ-Tocopherol

3.19

7

3.37

6

3.57

1

0.00

0.20

0.40

0.60

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Instrument ACQUITY UPSFC, PDA (293 nm), EMPOWER 3Column HSS C18 SB (3.0 100 mm, 1.8 µm)

Run time 5 minutesMobile phase Gradient 0.5% MeOH to 5% over 5 minutes

Fl t 2 5 L/ i


Flow rate 2.5 mL/minPressure 130 bar (1885 psi)

Temp 50 CInjection 1.0 µl injectionSample Contents of Vit E capsule in Hexane

Vitamin Vitamin E E Degradation Analysis Degradation Analysis g yg y

2.74

1

2.83

0

0.009

0.010

0.011

1.554 Peak 1

287.2339.4 371.4

AU

0.000

0.002

0.004

1.648 Peak 2

2.94

4

AU

0 002

0.003

0.004

0.005

0.006

0.007

0.008

282.4 367.8 387.0

AU

0.000

0.002

2.598 Peak 3229.2

268.2331 2

AU0.010

0.020

1.55

4

1.64

7 2.59

8

-0.003

-0.002

-0.001

0.000

0.001

0.002

Minutes1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00

331.20.000

2.742 Peak 4212.7

293.1

AU

0.00

2.00

2.829 Peak 5

293.1352.2 366.6

AU

0.00

0.02

0.04

2.943 Peak 6

AU 0 02

0.04


293.1

A

0.00

0.02

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

Vitamin A formsVitamin A forms

tate

- 1.20

Ret

inyl

Ace

t

0.90

1.00

1.10

0 60

0.70

0.80

tate

- 3.

354

Retin

ol -

3.82

6

AU

0.40

0.50

0.60

207

04 8Re

tinol

Pal

mit R

0.10

0.20

0.30


1. 1.3

1.79

71.

938

2.44

0

3.21

8

4.64

4

5.80

7

6.37

6

0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Separation Vitamin A forms (Zoom)Separation Vitamin A forms (Zoom)

0.060

p ( )p ( )

1.20

7

0.045

0.050

0.0551.

304

0 030

0.035

0.040

5 6

AU

0.020

0.025

0.030

97 0.41

1

6 43

1.15

5

1.51

686 1.

797

.938

3 2.44

0

86 6

3.21

826

8

55 4 1 493

.644

07 0 6.37

6

3

0.005

0.010

0.015


0.29

0.79

6

0.94 1.58 1

2.15

3

2.88

3.02

6

3.2

4.05

4.14

4

4.39

14.

4 44.

701

4.93

75.

064

5.45

8

5.67

95.

80

6.01

0

6.17

7

6.69

3

0.000

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

Fat Soluble Vitamin MixturesFat Soluble Vitamin Mixtures

0.060

0.040

0.050

AU 0.030

0.010

0.020

0.000

Mi t0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00


Minutes

Red - MS TIC (ESi+)Blue - UV at 317 nmBlack - UV at 262 nm

OLEDs


OLED Separation and Impurity OLED Separation and Impurity ProfilingProfiling

Organic Light Emitting Diode (OLED)

gg

(Cathode)

( d )(Anode)

(Glass board)


OLED Degradation Investigation OLED Degradation Investigation with LC/UV/MS with LC/UV/MS

FlrpicStructural Structural Isomers

Flrpic

α-NHP


TCTABAlq

Normal Phase LC Separation

UPC² Organic Light Emitting Diodes UPC² Organic Light Emitting Diodes OLEDsOLEDs

Ir(fppy)3Area = 0.4%Rt = 0.1%

α NHP

FF

1.00

9

1.42

10.075

0.100

TCTAArea = 1.1%Rt = 0.1% Balq (a)

Area = 1 1%

α-NHPArea = 0.4%Rt = 0.1%

N

N

FIr

N

F

1.92

0

2.02

8

2.47

1

AU

0.050

Area = 1.1%Rt = 0.1%

N

F F

0.025

0.000

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00


UPC² OLEDsUPC² OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

0.12

1.019 Peak 1

258.4

1.01

9

0.09

225.4

0.054

0.072

AU

0.06

AU

0.036

0.03

373.4

0.018

nm220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

0.00


Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

03

9.0x1071.004 Peak 1 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

763.92

2.8x107

1.00

7.2x107

761.95

ensi

ty

2.1x107

nten

sity

5.4x107

Inte

7.0x106

1.4x107

In

3.6x1070.0

m/z744.80 748.60 752.40 756.20 760.00 763.80 767.60 771.40 775.20 779.00

1.8x107


Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Standards and SpectraStandards and SpectraFlrpicFlrpic and MS ES+ m/z valuesand MS ES+ m/z valuespp

1.345 Peak 2 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune

743.93

745.90

Inte

nsity

1.8x106

2.7x106

3.6x106


743.97

745.91

Inte

nsity

2.4x106

3.6x106

4.8x106


743.90

745.90

746.97

Inte

nsity

2x106

3x106

4x106

m/z=745.9 m/z=745.9 m/z=763.9

ES+

20

9.0x107

0.0

9.0x105

m/z734.40 737.10 739.80 742.50 745.20 747.90 750.60 753.30 756.00 758.70

0.0

1.2x106

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00

0

1x106

m/z739.20 741.30 743.40 745.50 747.60 749.70 751.80 753.90 756.00

1.922 Peak 6 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune763.93

2.8x107


2.4x107


2.8x107

1.00

3 1.92

2.73

0

7.2x107

761.97

765.08

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

m/z757.80 759.60 761.40 763.20 765.00 766.80 768.60 770.40 772.20

743.96

747.05

Inte

nsity

0.0

6.0x106

1.2x107

1.8x107

m/z738.00 740.00 742.00 744.00 746.00 748.00 750.00 752.00 754.00 756.00

3.605 Peak 8 - SQ 1: MS Scan 1: 300.00-1000.00 ES+, Centroid, CV=Tune2 94


761.95

765.01

Inte

nsity

0.0

7.0x106

1.4x107

2.1x107

m/z759.60 760.80 762.00 763.20 764.40 765.60 766.80 768.00 769.20

m/z=763.9 m/z=763.9 m/z=746.0 m/z=727.9 m/z=806.0

.604

Inte

nsity

3.6x107

5.4x107725.97

727.94

729.01

Inte

nsity

0.0

1.6x106

3.2x106

4.8x106

6.4x106

m/z722.40 723.80 725.20 726.60 728.00 729.40 730.80 732.20 733.60 735.00

804.01

805.99

806.96

Inte

nsity

0.0

1.2x106

2.4x106

3.6x106

4.8x106

m/z802.80 803.70 804.60 805.50 806.40 807.30 808.20 809.10 810.00

1.34

5

1.44

8

1.60

01.

643

3.

4.10

2

1.8x107

3.6x10


Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy)3 Degraded Sample)3 Degraded Sample(( ppyppy) g p) g p

0.12

1.01

9

0.09

Ir(fppy)3

N

FF

N

AU

0.06

N

F

F

Ir

F

F

N

F

Ir(fppy)3Isomer

Ir(fppy)3 -2FIr(fppy)3 -F1.

935

6

0.03

N

F

F

Ir

N

FN

F

F

N

F

Ir

N

F

F

N

F

F

I (f )3 F

Ir

N

F

N

F

1.36

0

1.46

3

1.61

9

2.74

6

3.62

2

4.12

4

0.00

Ir(fppy)3–FIosmers

N

F F


Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

UPC²/MS OLEDsUPC²/MS OLEDsIrIr((fppyfppy))33 Degraded SampleDegraded Sample(( ppyppy))33 g pg p

10 X Reduction in runtime over traditional NPLC method

4 major constituents separated

Seven impurities identified

NP was 70-minute run with co-elutions NP was 70-minute run with co-elutions


USP Method Conversions


UPCUPC22 vs. Normal Phase vs. Normal Phase HPLCHPLC


Normal Phase Method Conversion Normal Phase Method Conversion EstradiolEstradiol to UPCto UPC2™2™

30.8

56

0 .0014

0.0016

0.0018

0.0020

Normal Phase HPLCCost per run ~ $5.89

Compound RT %Area S/N Unk Impurity 6 24 0 006 2 9

35.8

19

AU

0 .0004

0.0006

0.0008

0.0010

0.0012

Unk. Impurity 6.24 0.006 2.9Unk. Impurity Not Found --- --- Unk. Impurity 10.86 0.01 2.7Unk. Impurity Not Found --- --- Unk. Impurity 20.85 0.018 3Unk. Impurity 26.63 0.021 3.2Estradiol 30.86 99.87 ---

6.23

7

10.8

55

20.8

50

26.6

32

-0.0004

-0.0002

0.0000

0.0002

M inutes0.0 0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Main Impurity 36.81 0.077 9.2

Compound RT %Area S/N

ACQUITY UPC2

Cost per run ~ $0.05

pUnk. Impurity 2.26 0.012 3.4Unk. Impurity 2.59 0.004 1.9Unk. Impurity 3.34 0.01 3.1Unk. Impurity 5.66 0.006 1.7Unk. Impurity 6.15 0.016 5.5Unk. Impurity 8.13 0.013 3.1


p yEstradiol 8.81 99.89 ---Main Impurity 9.99 0.046 16

Chiral Separations


Chiral Comparison of Chiral Comparison of ACQUITYACQUITY UPCUPC22

vs. Normal Phase vs. Normal Phase HPLCHPLC


UPC² Enantiomeric Excess UPC² Enantiomeric Excess Determination of Benzyl Mandelate Determination of Benzyl Mandelate yy

R R 7 28

SFC chromatogram of R- and S- benzyl mandelate at 0.20 mg/mL of each enantiomer.

AU 0.20

R S RS=7.28

0.00

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

SFC h t f R b l d l t t 2 / L

0.004 RS

SFC chromatogram of R-benzyl mandelate at 2 mg/mL.

0.02% impurity

AU

0.000

0.002


Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

UPCUPC2 2 SubSub--2µm 2µm ChiralChiral SeparationsSeparationsWarfarinWarfarin

2.21

8

2.32

0

0.20

0.30 Prototype #1 1.7 µm 2.1 X 50 mm

2

AU

0.00

0.10

AU

0.20

0.30 Commercially Available 5.0 µm 2.1 X 150 mm

2.88

7

3.37

7

0.00

0.10

2

0.30 Prototype #2 1.7 µm 2.1 X 50 mm

0.96

21.

041

AU

0.10

0.20

Prototype #2 1.7 µm 2.1 X 50 mm


0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Methamphetamine


MethamphetamineMethamphetamine

Many of the illicit drugs occur as optical isomers with different psychotropic activities For example

pp

activities. For example, methamphetamine is a widely abused chiral drug.

The S form has 5 times the psycho-stimulant effect than that of the R form

(S)HN (R)HN

S(+)-methamphetamine R(-)-methamphetamine

stimulant effect than that of the R form. Methamphetamine is easy to make and

its precursors are readily available in the form of over-the-counter (OTC) drugs.

The impurity profile and enantiomeric The impurity profile and enantiomeric ratio of methamphetamine can provide valuable information to law enforcement agencies regarding the precursors as well as the synthetic routes. well as the synthetic routes.

Current analytical methods, including GC and CE, often involve long run times or time-consuming sample work-up steps.


p

Optimized Chiral Separation of R, SOptimized Chiral Separation of R, S--methamphetaminemethamphetaminepp

Chiral separation was achieved in less than 5 min.less than 5 min.

This represents a significant improvement in speed of analysis compared to GC or CE.U

0.004 1 2Rs=1.84

A

0.000

Minutes0.00 1.00 2.00 3.00 4.00

Optimized SFC chromatogram of methamphetamine. 1: S-MA; 2: R-MA.


Separation of methamphetamine with Separation of methamphetamine with its precursors: the fingerprintits precursors: the fingerprintp g pp g p

The enantiomeric ratio of R/S methamphetamine and their methamphetamine and their precursors are valuable information for law enforcement agencies to determine

AU

0.002 1 2

3

4

geographical location and manufacturing sophistication.

Simultaneous separation of R, S-methamphetamine and their two

0.000

3

methamphetamine and their two common precursors in 6 min.

SFC UV chromatogram of a mixture of: 1: S

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00

SFC UV chromatogram of a mixture of: 1: S-MA; 2: R-MA ; 3: (1S,2S)-pseudoephedrine; and 4: (1R, 2S)-ephedrine.


Drug TestingDrug Testingg gg g

1000000S-MA

Meth. is commonly tested during workplace drug

600000

800000

ak A

rea

R-MA

Linear (S-MA)

Linear (R-MA)

during workplace drug screenings.

Laboratories are required to detect methamphetamine at

R² (S) = 0.998R² (R) = 0.998

0

200000

400000

Pea

40% of its confirmatory level of 250 ng/mL.

The calibration curves of R- and S-methamphetamine ranging 0

0 200 400 600 800 1000

Concentration (ng/mL)

S-methamphetamine ranging from 50 ng/mL to 1,000 ng/mL, using SFC APCI MS.

Calibration curves of R- and S-methamphetamine obtained using SFC APCI MS.


UPCUPC2 2 Drugs of Abuse Drugs of Abuse MethamphetamineMethamphetamine

CE/MS

15-20 min

pp

15 20 min

GC/MSS/N=11.6 S/N=10.2

UPC2/MS/MS (ESI) 6 min

10-12 min

with derivatization step

S/ 6 /

LC/MS/MS

40-60 min 5 ng/mL for each enantiomer (25 pg on column)


Simultaneous separation of Simultaneous separation of methamphetamine and amphetaminemethamphetamine and amphetaminep pp p

Since amphetamine is the main metabolite of methamphetamine metabolite of methamphetamine, to test positive for meth., amphetamine also has to be detected in the sample.

0.003

1

2

34

Simultaneous chiral separation of amphetamine and methamphetamine in less than 5 minutes

AU

minutes. 0.000

Minutes0.00 1.00 2.00 3.00 4.00 5.00

SFC UV chromatogram of a mixture of amphetamine and methamphetamine. 1: S-MA; 2: R-MA ; 3: S-AMP; and 4: R-AMP.

Minutes


Paracetamol


ParacetamolParacetamol and Process Impuritiesand Process Impuritiespp

A mixture of paracetamol is analysed by UPC² and UPLC.

Sample Composition:

S1 S2

Code Compounds Mw(g.mol‐1) me1 (mg) Vtot (ml) EtOH Vµl S1 Vtot (ml) mg/ml

A 4 hydroxyacetophenone 136 50 10 20 10 0.010

B p‐nitrophenol 139 50 10 20 10 0.010

C 2 hydroxyacetophenone 136 50 10 20 10 0.010

D 4 chloroacetanilide 169.6 50 10 20 10 0.010

E acetanilide 135 50 10 20 10 0.010

F phenacetin 179 50 10 20 10 0.010

G 3 aminophenol 151 50 10 20 10 0 010G 3 aminophenol 151 50 10 20 10 0.010

H 4 aminophenol (paracetamol) 151 100 10 60 10 0.060

I 2 aminophenol 151 50 10 20 10 0.01

S2 i H O f UPLC d i H f UPC²


S2 in H2O for UPLC and in Heptane for UPC²

Method ParametersMethod Parameters

SFC UPLC UPC²

System: H-CLassColumn: BEH RP18 2.1x50mm

1.7µm

System: UPC²Column: BEH 3.0x100mm

1 7 1.7µm

T°: 55°CF: 0.9 ml.min-1

Solvent A: Water 10mM acetate mmoni m pH 5

1.7µm T°: 40°C

F: 2ml.min-1

Solvent : Methanolammonium pH:5Solvent B: Acetonitrile

Vinj: 1µL

So e t et a oVinj: 2µl

time (min) % CO2 % MeOH Flow (ml.min‐1)

0 98 2

time (min) % A % B Flow (ml.min‐1)

RUN

0 100 0 0.9

3 73 27 0 9

RUN

0 98 2 2

0.4 98 2 2

2 93 7 2

2.4 93 7 2


RUN 3 73 27 0.9

3.5 100 27 0.9

2.6 80 20 2

2.7 98 2 2

UPLC Results @ 260nm UPLC Results @ 260nm @@

0.14 H

0.10

0.12

AU

0.08

0 0

A

0.04

0.06

0.00

0.02 G DCB

FEI


Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

UPC² Results @ 220nm UPC² Results @ 220nm

HH

A

C

B

C

DEFG

I


UPC² Results @ 260nm UPC² Results @ 260nm

H

A

C

B

C

DEFG

I


UPC² UPC² vsvs UPLCUPLC

H

H

A

CG

B D E FG

I

0.10

0.12

0.14H

A

AU

0.04

0.06

0.08

G DC

BF

A

EI


0.00

0.02

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

G CFEI

Analgeasics


AnalgesicsAnalgesicsgg

A mixture of analgesics are analysed by UPC² and UPLC.

Sample Composition

S1 S2

Code Compounds Mw(g.mol‐1) me1 (mg) Vtot (ml) EtOH Vµl S1 Vtot (ml) heptane mg/ml

1 acetanilide 135 10 10 500 20 0.050

2 caffeine 194 10 10 1000 20 0.100

3 acethylsalicylic 180 10 10 1000 20 0.100

4 3 aminophenol 151 50 10 500 20 0.250

5 phenacetin 179 50 10 500 20 0.250

6 4 aminophenol (paracetamol) 151 100 10 250 20 0.250

7 2 aminophenol 151 50 10 500 20 0 2507 2 aminophenol 151 50 10 500 20 0.250

8 Acide salicylic 138 10 10 1000 20 0.100


Method ParametersMethod Parameters

SFC UPLC UPC²

System: H-CLassColumn: BEH RP18 2.1x50mm 1.7µm

T°: 60°C

System: UPC²Column: BEH 2 ep 3.0x100mm

1 7µm T : 60 CF: 0.9 ml.min-1

Solvent A: Water 10mM ammonium formate pH:3

Solvent B: MeOH

1.7µm T°: 40°C

F: 2ml.min-1

Solvent : MethanolVinj: 32µlSolvent B: MeOH

Vinj: 1µLVinj: 32µl

time (min) % CO2 % MeOH Flow (ml.min‐1)

0 99 1time (min) % A % B Flow (ml.min‐1)

RUN

0 100 0 0.6

3 64 36 0.6

RUN

0 99 1 2

0.5 99 1 2

3.5 60 40 2

4.0 60 40 2


3.5 100 0 0.64.1 99 1 2

UPLC Results 280nmUPLC Results 280nm

20

0.35

Caf

fein

e -1

.32

0.25

0.30

-0.7

57

heno

l -0.

958

phen

ol -

1.06

3

90 558 -1

.735

76

AU

0.15

0.20

ceta

min

ophe

n -

2-A

ceta

mid

oph

3-A

ceta

mid

op

ceta

nilid

e -1

.3

licyl

ic a

cid

-1.5

tyls

alic

ylic

aci

d

enac

etin

-2.

27

0.05

0.10

Ac Ac Sal

Ace

t

Ph

0.00

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00


UPC² UPC² ResultsResults @ 220nm Vinj: 2µL@ 220nm Vinj: 2µL@ j µ@ j µ

6

52

3

57

2

1

3

4

8


UPLC vs UPC²UPLC vs UPC²

0 25

0.30

0.352 UPLC

AU

0 10

0.15

0.20

0.25

0.00

0.05

0.10

Mi t0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

6 7 41 8 3 5

Minutes

5

6

7

0.70

20.60

0.50

UPC²

1

3

4

0.40

0.30

0.20


4

8

0.00 1.00 2.00 3.00 4.00

0.00

0.10

Polymer Additives


Polymer AdditivesPolymer Additivesyy

Lowinox 44B25

Irganox 1010

Tinuvin 328BHTIrganox 1330


Tinuvin P Tinuvin 312

UPC² ConditionsUPC² Conditions

Conditions:– Cosolvent: MeOH/MeCN 1/1Cosolvent: MeOH/MeCN 1/1

– Column: BEH 2EP 3.0x100mm 1.7µm

– Gradient: 1% for 1min, then ramp to 20% over 2.5min, 20% for 30s then re-equilibrationthen re equilibration

– Flow: 2.0mL/min

– Temp: 60°CP 120 b– Pressure: 120 bars

– Diluent: Heptane/Ethanol 9/1

– Concentration: 0.2mg/mL of each

– Detection: UV at 220nm


UPC² ResultsUPC² Results

5 min cycle time

836

ox 1

330

- 2.8

41

3.73

3

0.30

0.32

0.34

0.36

0.38

BH

T - 0

.407

Tinu

vin

P -

0.95

1

vin

328

- 1.2

25

Tinu

vin

312

- 1.8

gaar

d 44

5 - 2

.698

Irgan

o

351

Low

inox

44B

25 -

0.20

0.22

0.24

0.26

0.28

Tinu

v

Nau

Irgan

ox 1

010

- 3.3

AU

0.10

0.12

0.14

0.16

0.18

-0 02

0.00

0.02

0.04

0.06

0.08


-0.04

0.02

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00

FasterFaster GradientGradient

Faster gradient test – 3.5min cycle time

0.38

0.40

0.42

0.44

0.46

0.48

0.26

0.28

0.30

0.32

0.34

0.36

AU

0.14

0.16

0.18

0.20

0.22

0.24

0.02

0.04

0.06

0.08

0.10

0.12


-0.04

-0.02

0.00

Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50

UPLC Conditions UPLC Conditions

ACQUITY BEH C18; 2.1 mm x 50 mm (60°C)

Flow rate: 1 ml/min

Gradient separation, water to acetonitrile

From 48% to 100% B in 2 minutes; 1 minute at 100% B and From 48% to 100% B in 2 minutes; 1 minute at 100% B and back to initial

0.5µl injection (0.25 mg/ml of each additive, @220 nm)


UPLC Results UPLC Results Polymer Additives (Industrial Grade)Mixture With 8 Compounds

uvin

P

328

30

0.90

1.00Ti

nuvi

n31

2

4B25

445

Tinu

vin

3

Irgan

ox13

3

U

0.60

0.70

0.80

in31

2

Tinu

v

in31

2

BHT Lu

win

ox44

Nau

gard

4

gano

x10

10

AU

0.30

0.40

0.50

Imp

1 Ti

nuv

Imp

2 Ti

nuvi

Impu

rity

c

Irg0 00

0.10

0.20


0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

UPLC Results UPLC Results

Polymer Additives (Industrial Grade)Mixture With 11 Compounds

vin

P

1081

x13

30

0.80

0.90Ti

nuv

inuv

in 3

12

x 44

B25

Irgan

ox

gard

445

Tinu

vin

328

10Irg

anox

168

U

0.50

0.60

0.70

p 1

Tinu

vin

312 Ti

purit

y a

2 Ti

nuvi

n 31

2

BHT

Luw

inox

Nau

g

purit

y c

Irgan

ox 1

01

Imp

1 Irg

afos

rg

anox

107

6Irg

afos

168

AU

0.20

0.30

0.40

Imp

Imp

Imp

Imp Ir

0.00

0.10

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00


UPC² vs UPLC UPC² vs UPLC 0.

407

P -

0.95

1

225

vin

312

- 1.8

36

2.69

8

Irgan

ox 1

330

- 2.8

41

nox

44B

25 -

3.73

3

0 2 4

0 . 2 6

0 . 2 8

0 . 3 0

0 . 3 2

0 . 3 4

0 . 3 6

0 . 3 8

2 4

6

81B

HT

- 0

Tinu

vin

Tinu

vin

328

- 1.2

Tinu

v

Nau

gaar

d 44

5 -

Irgan

ox 1

010

- 3.3

51

Low

in

AU

0 . 0 6

0 . 0 8

0 . 1 0

0 . 1 2

0 . 1 4

0 . 1 6

0 . 1 8

0 . 2 0

0 . 2 2

0 . 2 4 23

45

7

1

- 0 . 0 4

- 0 . 0 2

0 . 0 0

0 . 0 2

0 . 0 4

M in u t e s0 . 0 0 0 . 2 0 0 . 4 0 0 . 6 0 0 . 8 0 1 . 0 0 1 . 2 0 1 . 4 0 1 . 6 0 1 . 8 0 2 . 0 0 2 . 2 0 2 . 4 0 2 . 6 0 2 . 8 0 3 . 0 0 3 . 2 0 3 . 4 0 3 . 6 0 3 . 8 0 4 . 0 0 4 . 2 0 4 . 4 0 4 . 6 0 4 . 8 0 5 . 0 0

1 00 2

U 0 60

0.70

0.80

0.90

1.00 2

3

4

6

1 Ti

nuvi

n31

2

2 Ti

nuvi

n31

2

1

urity

c

AU

0.20

0.30

0.40

0.50

0.60

5 78


Imp

Imp

Impu

0.00

0.10

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

HAP


ResultsResults

Column : HSS C18 3x50mm 1.7µm100% CO2Pressure gradient : from 100 to 180

2

Pressure gradient : from 100 to 180

1

35

4 6

1 naphtalene2 acenaphtene3 fluorene


4 2methylnaphtalene5 phenantrene6 fluoranthene

Customer XCustomer XSample Sample –– UPC²UPC²

0.66

8

5.36

7

0.230

0.240

0.250

pp

M th 60 k

0 160

0.170

0.180

0.190

0.200

0.210

0.220 More than 60 peaks integrated (220 nm)— 30 with % area > 0.1%

— 11 with % area > 0 5%

21

AU

0 100

0.110

0.120

0.130

0.140

0.150

0.160 11 with % area > 0.5%

0.60

5

1.96

1 3.74

1

5.12

0 040

0.050

0.060

0.070

0.080

0.090

0.100

0.43

00.

450

0.46

10.

488

0.85

30.

869

1.83

7

2.04

92.

220

2.27

7

2.49

82.

645

2.71

32.

818 2.

867

2.94

12.

973

3.00

53.

070

3.11

83.

180

3.30

73.

319

3.34

23.

371

3.43

43.

472

3.55

73.

589

3.80

43.

831

3.88

63.

929 3.96

34.

118

4.14

4 4.24

84.

296

4.34

94.

381 4.

476

4.54

14.

586

4.68

74.

704

4.74

74.

766

4.81

14.

836

4.95

64.

997

5.03

95.

164

5.30

15.

479

5.57

1

5.89

85.

969

6.21

16.

270

6.51

9

6.93

1

7.50

5

-0.010

0.000

0.010

0.020

0.030

0.040


Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Zoom on Zoom on baselinebaseline (220nm)(220nm)

0.009

( )( )

Noise is at very low level

0.005

0.006

0.007

0.008

U 0.000

0.001

0.002

0.003

0.004

AU

-0.004

-0.003

-0.002

-0.001

0.000

-0.008

-0.007

-0.006

-0.005


-0.009

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

UPLC IUPLC I--Class SeparationClass SeparationBEH Shield RP18; 2.1mm X 100 mmBEH Shield RP18; 2.1mm X 100 mm;;

0.80

1.00 Analytical conditions:— Eluent A: Formic acid 0.1%

AU

0.20

0.40

0.60 — Eluent B: Acetonitrile

— 25% to 100% B in 15 min at 0.5 ml/mn

— Column temperature: 35C0.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

0.10

Column temperature: 35C

More than 60 peaks integrated (285 nm)— 31 with % area > 0.1%

AU

0.04

0.06

0.08 — 12 with % area > 0.5%

0.00

0.02

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00


Minutes

MethodMethod Station vs. UPC²Station vs. UPC²

6.0e+1

Method Station Viridis 2EP 5µm

AU

2.0e+1

4.0e+1

0.66

8

7

0 . 2 4 0

0 . 2 5 0

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

0.0

0

5.36

7

0 . 1 4 0

0 . 1 5 0

0 . 1 6 0

0 . 1 7 0

0 . 1 8 0

0 . 1 9 0

0 . 2 0 0

0 . 2 1 0

0 . 2 2 0

0 . 2 3 0 UPC²Acquity BEH 2EP 1.7µm

0.60

5

1.96

1 3.74

1

5.12

1

AU

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0


0.43

00.

450

0.46

10.

488

0.85

30.

869

1.83

7

2.04

9

2.22

02.

277

2.49

8

2.64

52.

713

2.81

8 2.86

72.

941

2.97

33.

005

3.07

03.

118

3.18

0

3.30

73.

319

3.34

23.

371

3.43

43.

472

3.55

73.

589

3.80

43.

831

3.88

63.

929 3.96

3

4.11

84.

144 4.24

84.

296

4.34

94.

381 4.

476

4.54

14.

586

4.68

74.

704

4.74

74.

766

4.81

14.

836

4.95

64.

997

5.03

9

5.16

4

5.30

1

5.47

95.

571

5.89

85.

969

6.21

16.

270

6.51

9

6.93

1

7.50

5

- 0 . 0 1 0

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

M in u t e s0 . 0 0 0 . 5 0 1 . 0 0 1 . 5 0 2 . 0 0 2 . 5 0 3 . 0 0 3 . 5 0 4 . 0 0 4 . 5 0 5 . 0 0 5 . 5 0 6 . 0 0 6 . 5 0 7 . 0 0 7 . 5 0 8 . 0 0 8 . 5 0 9 . 0 0 9 . 5 0 1 0 .0 0

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