Elution at Critical Point of Adsorption: pToward Reliable Separation by Molecular Heterogeneities

Yefim Brun*, Chris Rasmussen, Brian McCauleyy

DuPont Central Research & Development*yefim.brun@dupont.com

GPC/SEC and Related Techniques, Philadelphia, 2015

2Molecular properties of polymers & other nanostructures

End-groups TopologyMolar mass,size and shape

telechelic combstarlinear

All polymerslinear

Chemical Composition and Microstructure

statistical alternating block gradientCopolymers

Biomolecules,nanoparticles

Size, shape, surface chemistry

nanoparticles, micelles, vesicles

Molecular heterogeneities (distributions): strong effect on end-use properties

Synthesis Molecular Structure Property

3Liquid chromatography for polymer characterization

Dissolution Separation Detection Data Reduction

Separation media

• porous particles (HPLC, UPLC) • non-porous particles (HDC)• monolithic columns• capillary

Interaction Polymer ChromatographySize Exclusion Chromatography Interaction Polymer Chromatography IPC

Size Exclusion ChromatographySEC

separation by everything but sizeseparation by molecular size

2D-ChromatographyIPC/SEC, IPC/IPC, SEC/IPC

4Interaction Polymer Chromatography (IPC)

Separation by everything but size

• Liquid Adsorption Chromatography (solvent gradient)

• Liquid Precipitation Chromatography (solvent gradient)• Molar mass

Ch i l iti

• Temperature Gradient Interaction Chromatography (isocratic)

•Chemical composition Blends, copolymers

• Chain blockiness • Liquid Chromatography at Critical Conditions (isocratic)

Stat. and block-copolymers

•Type and number of

• “Barrier” approaches (isocratic or gradient)

• Gradient Elution at Critical Point of Adsorption (gradient)

functional groupsTelechelic polymers, star-polymers, …

• Chain tacticity

• Temperature Gradient Interaction Chromatograph (isocratic)

• Chain tacticity

• Isotopic composition

5Different modes of isocratic separation

Elution of Narrow Polystyrenes on Symmetry® C18

Mobile phaseTHF THF- ACN (45/55)

474

5,570

Mobile phaseTHF 474

THF ACN (45/55)

474

710,000

9,100 18,100

5,570

tliqti

El ti ti t i (fl t 0 5 l/ i )

2.6 3.0 3.4 3.8 4.2 4.6 5.0 4 6 8 10 12

18,100i

Elution time tR, min (flow rate 0.5ml/min)

THF suppresses non-polar interaction

SEC mode in 100%THF : bigs elute first

Non-polar interaction prevails

Adsorption mode at 45%THF : bigs elute lastSEC mode in 100%THF : bigs elute first Adsorption mode at 45%THF : bigs elute last (or do not elute at all)

6Different modes of isocratic separation

Elution of Narrow Polystyrenes at Symmetry® C18

474THF ACN (48/52)

Critical Point of Adsorption (CPA)6.0

Elution at different compos. (THF,%)

S C474THF-ACN (48/52)

og M

5.5

6.0

50SEC LACLCCC

37,900 Lo

4.5

5.0 100 CR= 48% 45

3.5

4.0 80 40

186,000tliq 3.0

3.5

VliqV0

70ti

tR, min (flow rate 0.25ml/min)3 4 5 6 7 8 9 10 11 12 13

Transition mode: MW independent elutionElution volume VR (mL)

0 1 2 3 4 5 6

Transition mode: MW-independent elutionat = cr (48%THF)

Brun, Alden, J. Chrom. A 2002

7How to achieve CPA?

M h i f ti th h th i ht b l b t i t f i t tiMechanism of separation through the right balance between various types of interaction

Steric Interaction (SEC)

Critical point of adsorptionIPC(LCCC)

Adsorption Phase Transitiondso pt o(HPLC)

Phase Transition(Precipitation LC)

LCCC: LC at critical conditions, or LC at critical point of adsorption (CPA)

E lEnthalpy gain

Entropy lossMutual compensation effect : MW-independent elutionCPA should be found experimentally for each system

8Liquid Chromatography at Critical Conditions (LCCC)

• General principles• Isocratic separation

• Some components elute at CPA and some – in adsorption or size exclusion modes

• Examples Blends of polymers: separation by chemical composition, tacticity, isotopic

iticomposition

Telechelic polymers: separation by type of functional groups

Block-copolymers: separation by length of individual blocksp y p y g

9LCCC: separation by end-groupsLi PEG (MW 40K) ith diff t d S t ® C

Critical point of adsorption (CPA): 50% ACN in H2O

Linear PEG (MW=40K) with different end groups on Symmetry® C4

mono-brominated PEGPEG standard with 2 OH ends

∆t = 0.13

Minutes1.80 1.85 1.90 1.95 2.00 2.05 2.10 2.15 2.20 2.25 2.30 2.35 2.40 2.45 2.50 2.55 2.60

tliq tli + ∆t(q)tliq tliq + ∆t(q)

10

P l i h diff MW d diff d (b i d h h i i d)

LCCC: separation by end-groupsPolystyrenes with different MWs and different end-groups (bromine- and phosphonium-terminated)

1200

Column: SymmetryShield™ C8; CPA: 49% THF in ACN

900

1000

1100

1200

40K

mV

700

800

900

28K

400

500

600TBP-PS Br-PS

28K

100

200

300

10K0

Minutes7.00 7.50 8.00 8.50 9.00 9.50 10.00

10M. Byrd, Y. Brun, Macromolecules, 2005

11LCCC: Practical limitations for isocratic separation

• Establishing CPA is tedious: set of narrow polymer homologous with different MWsto be run at different eluent compositions/temperature

• Often should be reestablished even for known polymer/system combination• Limited selection of conditions for non-CPA component:

in adsorption mode – irreversible adsorption, in SE mode – pore volume• Very sensitive to experimental cond., e.g. flow rate, temp., solvent/sample composition:

even tiny deviations from CPA may result in peak splitting limited mass recoveryeven tiny deviations from CPA may result in peak splitting, limited mass recovery,especially for high polymers, sample break-through

N P k C C l LCCC f 186K PSFlow rate (ml/min): Nova-Pak C18 Column(60A, 4 km, 3.9 x 300 mm)THF-ACN (49/51)

LCCC of 186K PSat different flow rates

0.5 0.250.125

Minutes0 5 10 15 20 25 30

• Often limited to oligomers/low-M polymer separation in wide-pore columns

12Barrier Techniques

• Liquid Chromatography under Limiting Conditions (D. Berek, et al) of solubility (LCS): sample > SOL, eluent < SOL

of adsorption (LGA): sample > CR, eluent < CRp ( ) sample CR, eluent CR

of desorption (LCD): sample < CR, eluent > CR

Separation of block copolymers fromparent homopolymers in LC LCD bytwo barriers(Roller, et al, Anal. Chem., 2014)( )

• SEC-Gradients method (W. Radke)• Completely eliminates break-through effect• Limitations:

• Separation is limited to pore volume• Separation is limited to pore volume• Local solvent gradient depends on viscosity, column hydrodynamics• Can be sensitive to chromatographic conditions ( flow rate,

injection volume, sample concentration

13

Narrow polystyrenes at Symmetry® C18

Gradient elution at critical point of adsorption

ACN – THF gradient (0 -100%) over 10 min

9,10050

oligomers LAC Gradient elution at CPA

n)50

40 Eluent gradient

TH

F,%

) CR

190 00043,900

CR = 48% THF

40

(at e

lutio

n

30

2,890posi

tion( 190,000355,000710,000

1,260,000

30

g

10

20

Elu

ent C

omp

10

20

086543 7

E

6.5Log M

02.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Elution time, min (flow rate 1 ml/min) Log M

Transition to CPA occurs faster (at lower M) for:

CPA (if it exists) can be established in 1 run!

Brun, J. Liq. Chrom., 1999Brun, Alden, J. Liq. Chrom., 2002Brun, Foster, JSS, 2010

( ) narrow pores steep gradient high column selectivity

14Gradient elution of narrow polystyrenes on Nova-Pak® C8

Gradient elution at redissolutionGradient elution at CPA

8055

HF)

CR

40

60

Eluent gradientEluent gradient

35

45

(%

TH

2025

35

14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5

0

14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5

15

MeOH – THF (0 -100%) over 10 minACN – THF (0 -100%) over 10 min

* < d t i t*sol < CR CR does not exist

Brun, Alden, J. Liq. Chrom 2002

15Different modes of gradient separation

Elution of polystyrenes at Nova-Pak® C8

X – THF (0 -100%) over 10 min )

53

63

g

(%TH

F)

Elution at redissolution

X: MeOH ( ) - elution at redissolution43

CR

Elution at CPA

ACN ( ) –elution at CPA

23

33 LAC

13

32.0 4.0 6.0 8.0

Log M

Only elution at CPA is MW-independent!

16Elution at CPA vs. redissolution

Gradient elution of broad PS (Dow 1683) at Nova-Pak® C8

200

mV ACN THF gradient

120

160 ACN-THF gradient

<

80

0

MeOH-THF gradient

sol < cr

40

g

cr does not exist

015 16 17 18 19 20

Mi tMinutes

Brun, Alden, J. Chrom. A 2002

17Solvent gradient methods

Gradient elution at critical point of adsorption (CPA)Gradient elution at critical point of adsorption (CPA)separation by chemistry, topology

StericSteric

LACseparation by MW, chemistry, etc.

CPACPAredissolution

Adsorption Precipitation

Precipitation-redissolution chromatography: elution at dissolution point (GPEC)separation by MW and chemistry

Adsorption Precipitation

Steric

p y y

CPA does not exist:

redissolution

• irreversible adsorption• elution at redissolution

PrecipitationAdsorption

18Gradient elution at CPAA li tiApplications

• Separation of polymer blends by chemical composition

• Separation of telechelic polymers by functional groupsp p y y g p

• Separation of copolymers by chemical composition

• Separation of copolymers, including statistical, by microstructure, e.g. blockiness

• Copolymer purification by flash chromatography

• Separation of nanoparticles by surface chemistry

BenefitsBenefits• Easiest way to find CPA: just one gradient run!

• Broad range of eluents (including non-solvents)

• Practically no limitation on MW

• Usually full mass recovery

• Superb resolution and selectivity even at narrow pore or non pore columns• Superb resolution and selectivity, even at narrow pore or non-pore columns

• No peak splitting or some other chromatographic artefacts

Potential problemsp• Break-through effect

• Competition with dissolution mechanism in presence of non-solvent

19Effect of pore size on gradient elution

Narrow polystyrenes on different C18 columns in ACN – THF 10 min gradient

CPA depends on poreCPA depends on pore size through carbon loading

Stationary phase: S y ptype and distribution of functional groups, e.g. carbon loading per surface unit – main source of variability in LCCC

20

Chromatographic characterizations of polymer blend:

SEC vs. gradient elution ay CPA

polystyrene / styrene-acrylonitrile copolymer / styrene-butadiene copolymerChromatographic characterizations of polymer blend:

SEC: separation by size Gradient IPC: MW-independent elution

90

100Polystyrene

tion,

%

60

70

80

SBREluent Gradient

ent C

ompo

sit

40

50 St-AcElu

e

10

20

30

Minutes14 15 16 17 18 19 20 21 22 23 24 25 26 27

0

Minutes0 2 4 6 8 10 12 14 16 18 20

Isocratic separation by size in THF at SEC column set

MinutesGradient separation by composition in ACN-THF gradient at C18 column

Brun, Alden, J. Liq. Chrom., 2002

21Gradient elution at critical point of adsorptionGradient separation of blends: each component elutes at its CPAGradient separation of blends: each component elutes at its CPA

Separation by molecular parameters affecting CPA

Poly(alkyl methacrylate) blend (MW ~ 300K) at Symmetry® C18Poly(alkyl methacrylate) blend (MW 300K) at Symmetry C18

ACN – THF gradient (0 – 100%) over 30 minutes

20

24 PnHMA

PnBMAPEMA

16

PLMA

8

12

PMMA

4

8

0

255 10 15 20Elution time, min

Brun, Alden, J. Liq. Chrom., 2002

22Selectivity and resolution of gradient separation: narrow pores

Separation of ultra-high M ( > 106) polyacrylates at Nova-Pak® C18

PiBMA

PnBMA

iBMA/nBMA

ACN – THF (0 -100%) over 10 min

at Nova Pak C18

PiBMAPEAPMMA PEHA100

12 13 15

PMMAPnBA PnBMA

PEHA

80

100

(TH

F,%

)

Acrylate D~ 6 nm << gR ~50 nm

60

posi

tion

( Acrylatecopolymers(core-shell)blend

g

20

40

uent

com

p

Elution time, min14 15 16 17 18 19 20 21 22 23 24

0

Elu

Brun, Alden, J. Liq. Chrom 2002

23IPC of copolymersSt ti ti l l• Statistical copolymer

Single CPA (as homopolymer)

CPA depends on chemical composition and microstructure (blockiness)

• Block-copolymer E h i di id l bl k h i CPA Each individual block has its own CPA

Retention in gradient elution depends on MW and composition

Could be separated by block-length in isocratic and gradient modes

• Effect of blockiness on retention in gradient elution Always increases retention Always increases retention

Elution time at same chemical composition: alternating < statistical<block-copolymer

• Practical implication• Practical implication Gradient elution allows for separation by chemical composition and microstructure of

statistical and block-copolymers

Y. Brun, J. Liq. Chrom., 1999Y. Brun, P. Foster, JSS, 2010

24Isocratic elution of statistical 2-EHA/n-BA (50/50) copolymers18,

CH3CH2CH2CH2

CHCH2

OOC

CHCH2

CHCH2

H3CCH2

CHCH2

CH3

OO

(THF,%):4 3

100 70 60 58 56 541 8

Isocratic elution on XTerra® C8 in THF/ACN mixtures

CPA

3

4.1

4.2

4.3

CR, stat = 60%THF1 - 8CPA

Log

M

3.9

4

t 3.81.0 2.0 3.0 4.0

Elution volume Ve (mL)Vliq

Elution time, min (flow rate 0.25ml/min)4 5 6 7 8 9 10 11 12 13 14 15

tliq

0

25Gradient elution of statistical 2-EHA/n-BA (50/50) copolymers

XTerra® C8, ACN – THF (0 -100%) over 10 min

60

70

LAC Gradient elution at CPA80

90

100 8

THF,

%)

50

t Elu

tion

50

60

70

23

5,6,7CR,stat

ositi

on

(T

CR, PBA

CR,PEHA

30

40at

10

20

30

40

PBA (33K)

PEHA (42K)1

2

4

luen

t Com

po

Elution time min (flow rate 1 ml/min) Log M

203.8 3.9 4.0 4.1 4.2 4.3

0

10

17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5

El

Elution time, min (flow rate 1 ml/min) Log M

CR, PBA = 42%CR,stat = 60% = 68%

Effect of MW on retention

CR,PEHA = 68%

26Gradient elution: theory vs. experiment

Stat. 2-EHA/n-BA copolymers at different C18 columns

ACN – THF (0 -100%) at different gradientsACN THF (0 100%) at different gradients

60

on

67Effect of pore size/carbon loadingEffect of gradient rate

on

50

55

HF)

at E

lutio

61

63

65

HF)

at E

lutio

45

50

(%

TH

57

59

61

(%

TH

40

3.8 3.9 4.0 4.1 4.2 4.3L M

55

57

3.8 4.0 4.2 4.4Log MLog M

Symmetry ® 300A

XTerra ® 127AColumns:5 min

10 minGradient time:Column: XTerra ® 127A

Nova-Pak ® 60A10 min15 min20 min

Gradient time: 10 min

27Gradient elution at CPA of TFE-based photoresists

Hexane-THF (0 – 100%) over 10 min, Nova-Pak® Silica

8 5

9.0

9.5

10.0Hexane THF (0 100%) over 10 min, Nova Pak® Silica

7.0

7.5

8.0

8.5 polarity

CF3

mV

5 0

5.5

6.0

6.5 OCH 2COH

CF3

NB-F-OH

3.5

4.0

4.5

5.0 NB F OH

TFE / NB-F-OH di-polymer

3.012.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0

TFE / NB-F-OH / t-butyl acrylate - high conversion, batch polymerization

TFE / NB-F-OH / t-butyl acrylate - low conversion, batch polymerization

CPA of each fraction depends on its chemical composition: separation byCPA of each fraction depends on its chemical composition: separation bychemical composition

28Separation by chemical composition: gradient elution at CPA

Chemical composition heterogeneit of eth lene meth l acr late copol mersChemical composition heterogeneity of ethylene-methyl acrylate copolymers from different processes: Autoclave vs. Tube

Gradient chromatogram (ELSD) Ch i l C iti Di t ib ti

Autoclave, 25% MA300

340

Gradient chromatogram (ELSD)

80

90

Chemical Composition Distribution

mV

180

220

260

Wt/d

MA

50

60

70

Tube 25%MA100

140

180 dW

30

40

Tube, 25%MA

20

60

12 13 14 15 16 17 18 19 20 210

10

0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40Retention Time (min)

MA content, wt. fractionBroad distribution of tubular product indicates significant compositional heterogeneity:

increased melting point melt strength

Y. Brun, M. Pottiger, Macrom. Symp., v. 282, 2009

g p g

29

On-line IPC-FTIR of EMA copolymers

Elvaloy 1125 at 32 minutesElvaloy 1125 at 27 minutes0 026

Composition through ratio CO@1730cm-1/ CH@2920cm-1

High MAEster C=O

Medium MA

Elvaloy 1125 at 27 minutesElvaloy 1125 at 30 minutes

0.0180.0200.0220.0240.026

banc

e

Low MA

Ethylene C-HMedium MA

0.0100.0120.0140.016

Abs

orb

0.0020.0040.0060.008

1800200022002400260028003000 1800 2000 2200 2400 2600 2800 3000 Wavenumbers (cm-1)

30Isocratic and gradient elution 2-EHA/n-BA block copolymers

A (2 EHA)A (2-EHA)CH3CH2CH2CH2

H CCH2

CHCH2

OOC

CHCH2

B (n-BA)OO

CHCH2

H3C

CH3

100F,%

)

12345, gradient: ACN – THF (0 -100%) over 10 minIsocratic: THF-ACN (68/32) (CPA of PEHA)

80

100

ion

(TH

F

PBA PEHA12345678CR, PEHA

PEHA1 2 3 4

56

7

PBA

40

60nt

Com

posi

tiEluent gradient

CR, PBA

478

0

20Elue

n

Block-copolymers do not have CPA: separation by length of individual blocks

Elution time, min17.6 18.2 18.8 19.4 20.0 20.6 21.017.0

0

Elution time, min (flow rate 0.25 ml/min)

4 5 6 7 8 9 10 11 12tvoidt0

31Effect of copolymer microstructure on gradient elution

Statistical and block- 2-EHA/n-BA (50:50) copolymers

Nova-Pak® Silica, XTerra® C18Hexane – THF (0 -100%) over 10 min

PEHA PBA

100

F,%

)

ACN-THF (0 -100%) over 10 min

100PEHAPBAPEHA PBA

Stat Block

80

ositi

on (T

HF

80

PBA

Stat. Block

Stat.(Mn 17107) (Mn 12127)

40

60

uent

Com

po

40

60

20

40

Elu

20

40

12.0 17.012.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.50

150

16 17 18 19 20 21 22

Blockiness always increases retention: separation by microstructure

32Effect of copolymer microstructure on gradient elution

Statistical and block styrene/MMA (50/50) copolymers

Nova-Pak® C18Nova-Pak® Silica, 18ACN-THF (0 -100%) over 10 min

PMMA SStat (91K)

Block (93K) Block (407K)100

Hexane – THF (0 -100%) over 10 min

PS (107K)

Block (93K) Block (407K)100

on (T

HF,

%)

PMMA PS( )

80

PS (107K)PMMA (103K)

Stat (91K)80

t Com

posi

tio

40

60

40

60

Elu

en

20

0

20

12 13 14 15 16 17 18 19 20

013 14 15 16 17 18 19 20 21 22 23

0

12

Retention of block-copolymers increases with MWBrun, Foster, JSS, 2010

33Control of transesterification in melt-derived copolymers

1.30 07

Blocky copolymer50 mol% F16

0 91.01.11.2

PTTA

U0.040.050.060.07

Statistical copolymer50 mol% F16

50 mol% F16

AU

0.60.70.80.9 A

0 000.010.020.03

0.30.40.5

0.00

Minutes16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

0.00.10.2

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Drysdale, Brun, McCord, Nederberg, Macrom. 2012

34Separations by end-groups: isocratic vs. gradientOH

Cl 4-arm PEGs with OH- and Cl-ends

Isocratic elution (LCCC) Gradient elutionOH

Cl water-ACN at XTerra® C18

7000

water/ACN (60.1/39.9, v/v) water – ACN gradient: 20%ACN -100%ACN 350 0

500

600

number of chlorine ends 250

300

2 4number of chlorine ends

mV

300

400

4

number of chlorine endsm

V

150

200

100

200 123

4100

150

13

Minutes3 4 5 6 7 8 9

50

Minutes21 22 23 24 25 26 27 28

35IPC-MS w/LTQ_Orbitrap: 4-arm PEG

Theoretical predictionof gradient elution of PEGs with various end groupsPEGs with various end-groups

Peak3Peak1Peak4

Peak2

Peak543Peak3ea

123

N L :1 . 5 5 E 8T I C M S 0 5 2 7 1 1 _ P EG _ 1 0 2 _ 2 _ i n_ w a t e r _ 0 7

TIC0

1

0 Cl1 Cl

2 Cl3 Cl

3 0 3 2 3 4 3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0 5 2T i m e ( m i n )

0 Cl 4 Cl

Further details in C. Rasmussen talk

36Purification based on step-wise flash IPCP ifi ti f St/MMA l f id l h l

1st elution 2nd elution

Purification of St/MMA copolymer from residual homopolymers

PSremoval

Copolymerremoval

PMMAremoval

Final solution (PMMA stayed on the column)

window window window

Intermediate solution (PS removed)

Initial solution (PS, PMMA, copolymer)

Minutes15 16 17 18 19 20 21

ELSD Chromatograms in Hexane-THF Gradient on Silica columnMinutes

37Separating Nanoparticles by Surface Chemistry

Nanoparticle

Free polymer chains grafted within thegrafted within the monolith pores provide higher degree of interaction between

ti l d

Stationary Phase

nanoparticle and column

Stationary Phase

M lith l d b tt f ti l• Monolith column produce better recovery for nanoparticles

• Rigid surface provides minimal tangential nanoparticle/column interaction

• Currently commercially available monoliths from styrene-divinyl crosslinked gelcontain relatively short chains. Monoliths with grafted flexible chains are needed

S th i d t th M l l F d t B kl N ti l l b (D F S )• Synthesis done at the Molecular Foundry at Berkley National lab (Dr. F. Svec)

38Separation of decorated colloidal silica by degree of coating

Separation of colloidal silica coated with mix of phenyl- and aminophenylsilane

1:3

PhTMS / APhTMS ratio:

Mobile phase: toluene-DMAc gradient

APhTMS

PhTMS

3:1 1:1

Mobile phase: toluene DMAc gradientColumn: BMA/EDMA monolith grafted with PMMA chains (F. Svec, Berkley )

Si-OH9:1

13 14 15 16 17 18 19 20 21 22 23 24 25 26

Elution time, min

• Long and flexible polymer functional groups (bonded phase) produce “reversed” critical point of adsorptioncritical point of adsorption.

• Nanoparticle is “swallowed” by these chains through multiple attachment mechanismFirst reported at HPLC 2012

39Summary : isocratic vs. gradient separations at CPA

LCCC

C ld b d ith “i ti ”

Gradient elution at CPA

• Could be used with “isocratic” detectors (e.g. refractometer)

• All limitations of isocratic separation for non CPA component

Some limitations on choice of detectors

All benefits of gradient separation : broad range of eluents including nonfor non-CPA component

• Set of narrow polymer homologous

broad range of eluents, including non-solvent, practically no limitation on MW

CPA is determined with a single run of hi h MW b d l di it lwith different MWs needed to

establish CPA. Multiple isocratic runs at different mobile phase compositions /temperature

high MW broad polydispersity sample. Some solubility study may required to avoid separation by solubility

compositions /temperature

• CPA is often unreproducible even for the similar column and the same chromatographic conditions

Does not require prior knowledge of CPA: could be easily reestablished

chromatographic conditions

• Very sensitive to experimental cond., e.g. flow rate, temp., solvent/sample composition : fluctuations result in

Superb resolution, could be controlled by gradient rate and pore size, no peak splitting or many other chromatographiccomposition : fluctuations result in

peak splitting, limited mass recovery, especially for high polymers, sample break-through

splitting or many other chromatographic artefacts. Sample break-through could be suppressed by non-solvent

40