Rapid Method Screening for High Resolution Aqueous SEC Measurements

Mike O’LearyInternational GPC Symposiumy p

Philadelphia 2015

©2015 Waters Corporation 1

Target Attributes: New Platform for SEC Polymer Characterizationy

Column– Low energy surface – minimal adsorptionLow energy surface minimal adsorption– High pore volume… and good mechanical strength– Range of pore size distributions

Good efficiency small particle size– Good efficiency - small particle size

Instrument– Capable of high pressure operation (1000 bar)– Low system dispersion– Accurate and precise mobile phase deliveryp p y– Range of solvent compatibility– Good vapor management

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The ACQUITY Advanced Polymer Chromatography (APC) Systemg p y ( ) y

Stable f ti

Flexible refractive

index detection

detection techniques

Low system dispersion

Rigid, solvent-resilient columns

Compatibility with

challenging Versatile column

Precise

challenging solvents management

Wide range of

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solvent management

gAPC

standards

ACQUITY Refractive Index DetectorFlow Cell

241410.3µL

ACQ-RI1.3µL1.3µL

1

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1 cm

BEH™ Technology ParticlesBridged EthylSiloxane/Silica Hybridg y y

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SEM Images: Pore-EnlargedBridged Ethyl Hybridg y y

45Å45Å 200Å200Å

450Å450Å 900Å900Å

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Low MW Standards PS MW: 266 - 15,000Columns: 2 x 45A APC XT 4.6x150 mm

0 28V1 = 15,700 + 4,920 + 1,250V2 9 130 + 3 470 + 474

4.20

0 21

0.28 V2 = 9,130 + 3,470 + 474V3 = 6,540 + 2,280 + 266

g M

ol W

t

3.60

AU

0 14

0.21

266 Dalton Marker

Lo

3.00

0.07

0.14

1 80

2.40

0.00

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1.80

Elution Volume1.76 1.98 2.20 2.42 2.64 2.86

Minutes2.25 2.50 2.75 3.00 3.25 3.50

Higher Resolution Data Faster- Life is good!g

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Column Chemistry- The story so fary

Maximize accessibility to pore structure, bondings are undesired

For organic separations, addition of small TMS “end-cap” (APC XT column) acceptable balance of minimizing surface activity and maintaining pore accessand maintaining pore access

For aq applications, strong salts and modifiers used minimize gsurface interactions

For this reason APC AQ columns have no surface bonding For this reason, APC AQ columns have no surface bonding

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Pullulan Standards- 100mM sodium nitrate/CH3CN : 80/20

ACQUITY APC AQ 200A 4.6x150mm

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PEG Standards: Beautiful Chromatography- Just the wrong kindg

194194

106590

414

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Impact of Temperature on Retentionp p

Analyte Mp 200Å XT 200Å AQ DVB Brand A

DVB Brand B

Percent retention time change: 30˚C vs 50˚C

y p Brand A Brand B

Polystyrene 44,200 -1.48 -1.76 -1.84 -1.87Poly(methyl methacrylate) 31,600 -2.49 -1.96 -1.90 -1.92

Poly (isoprene) 9930 -2.01 -1.82 -1.82 -1.89

Poly (ethylene glycol) 1340 -1.96 -34.99 -2.05 -1.87

Poly (4-vinyl phenol) 11 000 -3 38 Did not elute -2 01 -1 73Poly (4-vinyl phenol) 11,000 -3.38 Did not elute -2.01 -1.73

Poly (2-vinylpyridine) 1640 -2.38 Did not elute -1.62 -1.85Poly (dimethyl siloxane) 5940 -2.83 -1.65 -2.07 -1.94siloxane)

Polyurethane 100,000 -1.84 -2.09 -1.48 -3.52

Poly (vinyl acetate) 100,000 -1.89 -1.76 -1.42 -2.69

C ll l t t 30 000 1 42 2 10 1 29 0 44

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Cellulose acetate 30,000 -1.42 -2.10 -1.29 -0.44

Mobile phase: THF; Flow rate: 0.6 mL/min; Detection: UV or ELSD

Let’s Try Something Else- Protein SEC column

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10% ACN in water 0.75 ml/min with one 150 mm 125 A BEH Diol column – PEG Stds

430

106

202633430

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Optional Solvent Select Valve

Fully compatible with the

p

ACQUITY APC supported solvents– 6 solvent lines

Enables automated solvent switching on the ACQUITY APC System– Solvent resilient ACQUITY APC

hybrid particles not susceptible to swelling with solvent switchingswelling with solvent switching

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GPC Method Development Screening Protocol- Completed in 4 hoursp

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Two 150 mm 125 A BEH Diol columnsPEG 106,202,430,633, , ,

µRIU 9.00

13.50

18.00

100% Water0.75ml/min

0.00

4.50

Minutes3.74 3.85 3.96 4.07 4.18 4.29 4.40 4.51 4.62 4.73

28.00

42.00

90% Water/ 10% Methanol0.75 ml/min

12300 psi at 40C

µRIU

-14.00

0.00

14.00

Minutes3.63 3.74 3.85 3.96 4.07 4.18 4.29 4.40 4.51 4.62

0.75 ml/min

24 00 90% W t / 10% A t it il

µRIU

6.00

12.00

18.00

24.00 90% Water/ 10% Acetonitrile0.75 ml/min

RIU

50 00

55.00

60.00

65.00

70.00

75.00

80.00

85.00

90.00

95.00

100.00

105.00

90% Water/ 10% Acetonitrilel/

0.00

Minutes3.74 3.96 4.18 4.40 4.62

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µR

-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

Minutes2.76 2.78 2.80 2.82 2.84 2.86 2.88 2.90 2.92 2.94 2.96 2.98 3.00 3.02 3.04 3.06 3.08 3.10 3.12 3.14 3.16 3.18 3.20 3.22 3.24 3.26 3.28 3.30 3.32 3.34 3.36 3.38 3.40 3.42 3.44 3.46 3.48 3.50 3.52

0.75 ml/minPrototype Diol (high coverage)

Utility of BEH Dioly

BEH particle structure enabling– Mechanical strength and solvent switching capability allows for fast

development/screening of conditions

Diol bonding very effective surface coverage

Small dp of Diol particles (1.7um) and high viscosity of water/organic mixtures result in pressures approaching system max, therefore limiting column selection flexibility

Options for reducing backpressure– Increase Temperature– Smaller length columns– Increase dp

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Is the resolution optimized?

How Does This Compare to the XT 45A Experiment? p

0 21

0.28

Operating range 200-5k Da

ACQUITY APC XT 45A 2x 4.6x150mm

AU

0.14

0.21

4741250

0.00

0.07

Minutes2.25 2.50 2.75 3.00 3.25 3.50

1250

60.00

80.00

Operating range 1k-30k Da?ACQUITY BEH Diol 125A 2x 4.6x150mm

26

282

238

194

150

µRIU

20.00

40.00 202430633

4k7k20k

©2015 Waters Corporation 19

326

0.00

Minutes2.10 2.40 2.70 3.00 3.30 3.60 3.90 4.20 4.50 4.80

Two 150 mm 125 A BEH Diol columns

4.30

4.40

4.50

4.60

3.70

3.80

3.90

4.00

4.10

4.20

3

Log

Mol

Wt

3 00

3.10

3.20

3.30

3.40

3.50

3.60

2.40

2.50

2.60

2.70

2.80

2.90

3.00

1.80

1.90

2.00

2.10

2.20

2.30

©2015 Waters Corporation 20

1.70

Retention Time2.20 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 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80

Summary and Next Stepsy p

This particular diol coverage has reduced the 125A porsity to effectively ~ 80-90Ay

45A particle bonding not achievable, but there may be “sweet spot” with 90A starting material

Effect of ligand density/surface coverage levels needs to be Effect of ligand density/surface coverage levels needs to be better understood

Alternative OH (and other) ligands to be investigated

Combination of the low dispersion instrument and robust BEH particle enables fast development experimentsparticle enables fast development experiments

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©2015 Waters Corporation 22