Development of an Ultra-high Performance –Two-Dimensional –Liquid Chromatography (UHP-2D LC) Method for Synthetic Polymers2D-LC) Method for Synthetic Polymers

Lu Bai, Miroslav Janco, Edwin Mes, Lucia Asensi Bernardi, Kimy Yeung, James Alexander IVAlexander IV

Acknowledgements: Sven Claessens, Scott Wills, Dave Meunier, Kebede Beshah, Dean Lee

Analytical SciencesAnalytical SciencesDow Chemical Company

2D-LC Advantages and Challenges

• An increasingly used technique for analysis of synthetic polymers – more information on distributions (MWD, CCD, FTD t ) ti d i it f li tiFTD etc) : practiced in quite a few applications

• Total time required essentially proportional to the analysis time on the D2

• Short SEC columns often used but compromisedShort SEC columns often used, but compromised resolution

T l th ti l• Two examples on synthetic polymers

Mode combinations 1st Dimension x 2nd Dimension Columns Mobile phasesAnalysis time 

(min)Applications

RP‐temperature gradient interaction C18 × C18 Isocratic CH2Cl2/CH3CN 200 Branched polystyrenes (PS)

Survey of 2D‐LC Literature for Analysis Time

chromatogra phy (RP‐TGIC) × RP (LC‐CC)C18 × C18 Isocratic CH2Cl2/CH3CN 200 Branched polystyrenes (PS)

RP × RP C18 × carbon clad zirconia (CCZ) Methanol × acetonitrile 250 complex mixt. of oligostyrenes

RP × NP (HILIC) C18 microbore × aminopropyl silicagradient acetonitrile × isocratic ethanol‐dichloromethane‐water 120

Polymers: ethylene oxide‐propylene oxide (EO‐PO) 

(co)oligomers

RP x NP Supelco C18  column (150 Â 2.1 mm, 5 mm particles, 120 A ˚ pore size) x Hypersil bare silica column (150 4 6mm 3mm

ACN/THF x  n‐hexane in (non‐ 140 PS, PMMA, styrene‐MMA block 

RP x RP

RP x NP

RP x NP size) x   Hypersil   bare   silica   column (150 Â 4.6 mm,   3 mm   particles,   120 A ˚ pore‐size);   Nucleosil   bare   silica

stabilized) THF  140 copolymer

NP (LC‐CC) × SEC Alltech Platinum Silica × HSPgel‐RT MB‐L/Mchloroform/diethylether × 

chloroform ‐Polymer: degradation product of poly(bisphenol A)carbonate 

(PC)

NP × SEC Hypersil “bare” silica× PLgel Isocratic 48% ACN in DCM × THF 90Polymer characterization: poly(methyl methacrylate) 

(PMMA)

NP × SEC Hypersil “bare” silica ×Mixed‐C Isocratic THF–hexane × THF 240 Polymer: polystyrene (PS)

NP and SEC

NP × SEC Hypersil  bare  silica × Mixed C Isocratic THF hexane × THF 240 Polymer: polystyrene (PS)

LCCC and SEC LC @ near critical x SEC

Hypersil “bare” silica columns (150 mm × 1.0 mm i.d., 3 �m parti‐ cles; 100 ˚ A pore size; ThermoQuest, Breda, The Netherlands) x 

One or two 50 mm × 4.6 mm i.d. PLgel columns (Polymer Laboratories, 5 �m particles with 100 ˚ A pore size and/or 6 �m 

oligoPore particles with 100 ˚ A pore size, 25 ◦C)

ACN/DCM x THF 120 (1.5) hydroxyl‐functional PMMA polymers.

RP × SEC NovaPak silica × HSPgel Gradient × THF 300analysis of a series of styrene‐

methylacrylate (SMA) copolymers

RP x SEC Zorbax Eclipse XDB‐C8 column 80‐Å pores x PLGel Mixed C H2O/ACN‐THF x THF 240 poly(styrene)‐co‐poly(methyl methacrylate) latex used in 

coating formulations

NP ‐ TGIC x RP ‐ isocratica diol‐ bonded silica column (Nucleosil, 7 µm, 100 Å, 250 × 7.8 mm i.d.) x   C18‐bonded  silica  column (Kromasil, 5 µm, 100 Å, 

150 × 4.6 mm i.d.)iso‐octane/THF x ACN/DCM 1200 (12) polystyrene‐block‐

polyisoprene   (PS‐b‐PI) 

bare silica column (Nucleosil, 3 µm, 100Å, 50 × 2.1mm i.d.)/C18 PMMA and PBMA homo‐ and

RP and SEC

NP/RP and TGIC

NP/RP‐TGIC x HT‐SECbare silica column (Nucleosil, 3 µm, 100 Å, 50 × 2.1 mm i.d.)/C18 bonded silica column (Nucleosil, 7 µm, 500 Å, 150 × 4.6 mm i.d.) x 

PolyPore (5 µm, 250 x 4.6 mm i.d.)

iso‐octane/THF or ACN/DCM x THF as low as 60

PMMA and PBMA homo  and copolymers, polyurethane 

samples

MTF and SEC MTF x SEC150 mm × 4.6 mm i.d. column packed with 0.1 to 1 �m 

polydisperse silica particles (Admatech, Aichi, Japan) x 10‐�m 106 Å PLgel particles

non‐stabilized HPLC‐grade tetrahydrofuran 360 branched polymers

UHPLC and UHPSEC UHPLC x UHPSECUPLC BEH C18, 130 pore size x UPLC BEH C18 w/ 130 pore size or 

BEH HILICACN/THF x THF 60 (1)

Poly(methyl meth‐ acrylate),   PMMA,   and   poly(n‐butyl   methacrylate),   PBMA, 

homopolymers,   as   well   as   (methyl   methacrylate)‐(n‐butyl methacrylate)   random   copolymers,   P(MMA‐co‐BMA), 

Example I: Polymer Dispersion Analysis with 2D-LC Step 1. Using UHP-SEC to Speed Up D2

Ultra Violet Response (mV)

571.82

514.19

456.56

398.94

341.31

283.68

Data File: 2013-13604-007_2014-06-02_23;24;00_01.vdt Method:

629.44

UVUV UVUV

V)

878.82

787.30

695.78

Data File: 2013-13604-007_2014-06-02_23;24;00_01.vdt Method:

970.34

Retention Volume (mL) 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

226.06

168.43

110.81

53.18

10.00 30.00

Component CRIRI ELSELS Component C

Component D

Refractive Inde

x Response (mV)

Retention Volume (mL) 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

604.27

512.75

421.23

329.71

238.20

146.68

55.16

10.00 30.00

Components A+B

12 L12 LEl ti V l

Component D

11 LL

Components A+BComponent D

12mL12mLElution Volume

Speed up D2: 2 min 1 min

1 1 mLmL

Original D2: SEC (conventional) Conditions:Column: high speed SDV LIM (50 × 20 ID mm, 5 µm) Mobile phase: THF; Flow rate: 6 mL/minSample concentration: ~ 16 mg/mLInjection volume: 100 μL

Updated D2: SEC (UHP-SEC) Conditions:Column: Acquity HILIC column (130 Å, 150 x 3 mm, 1.7 µm)Mobile phase: THF; Flow rate: 1 mL/min (back pressure ~6400 psi)Injection volume: 100 μL (back pressure ~6400 psi)Sample concentration: ~1.25 mg/mLInjection volume: 1, 3, 5, 10 μL.

Step 2. Revising D1 Gradient to Speed Up D1

Revising from 240 min 2.8 min 35 minOriginal D1 Conditions:Column: Supelco Ascentis Si (2.1 x 50 mm, 3 μm)Mobile phase: DCM-THF; Flow rate is 10 µL/min. In order to reduce the total run time, the flow rate was increased

Revised D1 Conditions:Column: Supelco Ascentis Si (2.1 x 50 mm, 3 μm)Mobile phase: DCM-THF; Flow rate: 0.02 (4 min-28 min) -0.04 mL/min; Runtime: 35 min.

before and after the peaks of interest to 0.2 mL/min and 0.4 mL/min, respectively.; Runtime: 240 min.

min) 0.04 mL/min; Runtime: 35 min.

Comparison: Conventional Vs. UHP-2D-LCConventional 2D-LC

Total runtime: 240 minUHP-2D-LC

Total runtime: 35 min

Component BComponents CComponents D

Component D

Components CComponents B

RI

(Identified by spiking)

RIComponent A Component A

Analysis time is much shortened by using sub 2 um particle size column and revising the

RTMWMW MWMW

. Analysis time is much shortened by using sub-2 um particle size column and revising the gradient

. Peak identification matched original data; product now in scale-up

Future Improvement includes increasing resolution of the 2D contour map by making more cuts. Future Improvement includes increasing resolution of the 2D contour map by making more cuts across each peak.

Example II: End-cap Distribution for Polymer ProductsChallenges with Separating by End-Cap Distributions

• MWD and End-cap distributions are correlated with application properties• Polyurethane backbone with A and B end-caps: three different end-capping possibilities in this product: AA, AB and BB

A B

*Ideal structure of the polymer

AA l

Co-elution of AB + BB polymers; molecular weight

polymer effects on retention

From residual A Impurity from sample

UPLC Conditions: water/THF, 0.2 mL/min on Acquity BEH C18, 1.7 µm, 2.1 mm X 50 mm

Offline 2D-LC Analysis of End-capping: SEC x UHPLCGPC Fractionation

[a.u

.]

MALDI-MS analysis of a low MW fraction

1012

6.8

1500

2000

Inte

ns. [

BB series

1164

2.6

500

1000 AB series

Dow Confidential Information

8000 9000 10000 11000 12000 13000 14000m/z

8

Leveraging APC in UHP-2D-LC Method DevelopmentLoops emptied to D2 : UHP‐SEC

Loop s filled from D1:UPLCValco 8‐port 2‐position valve; Loop Size10 μL

Modulation Period 0.5 min

D1 Conditions:Column: Acquity BEH C18, 1.7 μm, 1x100 mmFlow rate: 0.02 mL/minD t t UV@254 R ti 55 i

D2 Conditions:Column: Waters APC XT200, 2.5 μm,4.6 x 150 mm

/Detector: UV@254 nm; Runtime: 55 min. Flow rate: 1.8 mL/minDetector: ELSD; Runtime: 0.5 min.

9

Advanced Polymer Chromatography (APC)UHP SEC Analysis of 16 PS StandardsUHP SEC Analysis of 16 PS Standards

se, [

mV

] 1 13

0K56

0K 310K

200K

20K

98K

66K

30K

2K

~200 – 1M

tor r

espo

ns 12

44K

22

11.6

K

7000

250

00 220

(BH

T)

RI d

etec

t

32 170

580

2

UHP SEC Conditions:

Column set: 3 UHP SEC columns (150x4.6 mm ID) packed with BEH TMS particles,

Run time: 6 min

( )

Pore Size: 45+125+450Å, Particle Size: 2.5 µm, Tc= 40 oC

Eluent: THF (Certified grade from Fisher), Flow rate: 1 mL/min

Injection volume: 10 µL, Inj. C: ~0.07 mg/mL/component

Janco, M., Alexander, J., Bouvier, E., & Morrison, D. (2013). Ultra-high performance size-exclusion chromatography of synthetic polymers. J. Sep. Sci. , 36, 2718-2727.

Detector: RI, TD = 40 oC

Online 2D-LC Analysis of the Polymer Products Product 2Product 1

Pea

k 2

ntio

n Ti

me

Stro

nger

BB 72k 30k 16k134k

Pea

k 1

UPL

C R

eten

Inte

ract

ion:

S

AB

AA

AB

AAU

SEC Elution Volume Hydrodynamic volume: smaller

I

View with Inclination (Side view) View with Inclination (Side view)

AA AA

( ) ( )

11Residual A can also be detected but not shown in the zoom-in figures.

Correlating Volume% with Performance

AA

AB

A

BB

2 samples (#1 and #3) “different from control”, 2 of “close to control” samples (#2 and #4) d 1 “ diff t f t l” (#5) h

1 2 3 4 ctrl 5

#4), and 1 “very different from control” (#5) are shown

Volume% can be calculated from 2D charts

It shows the distribution of different end-capped polymers in the sample

The distribution pattern a very sensitive parameter to sample composition and correlate with performance of the samples.

Conclusions and Future Directions•UHP-2D-LC has shown great potential to improve throughput for 2D-LC analysis. Analysis time can be significantly reduced with these technologies.

•Analysis of polymer dispersion adopted some of the above mentioned tools and•Analysis of polymer dispersion adopted some of the above mentioned tools and was used to demonstrate the possibility of making 2D-LC a practical tool for industrial use. Analysis of another polymer product showed the high resolution separation of three end-capped types, providing a direct, visual means for evaluating the variants and correlate end-cap distributions with performance.

•A potential drawback with using these technologies is the narrower retention window on D1 possibly resulting in small number of slices per D1 peakwindow on D1 possibly resulting in small number of slices per D1 peak.

•Use of RI is still challenging if cycle time needs to be minimized

Th i l d l i b 2 l h D1 f h ff i• The next steps include evaluating sub-2 µm column on the D1 for the effects it has on resolution of the 2D contour map in the polymer dispersion analysis

Thanks for Your Attention!