contemporary liquid chromatography of polymers event/201… · size-exclusion chromatography...
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CONTEMPORARY LIQUID CHROMATOGRAPHY
OF POLYMERS
PETER SCHOENMAKERS
IF POLYMERS WERE
ANIMALS
A typical polymer
A stressed polymer
Deborah Debbie
A confined polymer
Lamda
A congressional polymer
Polymers don’t move much (they just wobble a bit)
The relatively small polymer moves faster than the very big one
Polymers may take (seemingly) forever to move in and out of confined spaces
Polymers are claustrophobic
They are happy in a big hole
Polymers are claustrophobic
They are happy in a big hole
They don’t mind small holes
Polymers are claustrophobic
They are happy in a big hole
They don’t mind small holes
They don’t like to sit tight
Column band broadening in size-exclusion chromatography Average pore size 130 Å
0
5
10
15
20
25
30
35
40
45
50
0 0.5 1 1.5 2 2.5 3
H(µ
m)
u (mm/s)
toluene (ca. 4A)PS 5 kDa (33 A)PS 10 kDa (49 A)PS 20 kDa (73A)PS 30 kDa (93 A)PS 52 kDa (126 A)
Column band broadening in size-exclusion chromatography Average pore size 130 Å
Similar results were observed by F. Gritti and G. Guiochon, Anal. Chem . 79 (2007) 3188-3198
Higher Performance LC
The last decade has seen a paradigm shift in LC From high-pressure (or high-performance) HPLC To ultra-high-pressure or ultra(-high)-performance U(H)PLC
ul·tra [ˈʌltrə, uhl-truh] adjective going beyond what is usual or ordinary;
excessive; extreme.
ul·tra [ˈʌltrə, uhl-truh] adjective going beyond what is usual or ordinary
excessive; extreme. HYPERFORMANCE
LC
H - HETP (plate height) A - eddy-diffusion coefficient B - longitudinal diffusion coefficient u0 - linear velocity C - resistance-to-mass-transfer coefficient dp - particle size Dm - diffusion coefficient of analyte in mobile phase
uopt m
pmp D
udC
uBDAdH 0
2
0++=
The Van-Deemter Equation
Liquid chromatographers want to use smaller particles
Because these yield lower plate heights at higher velocities
HPLC U(H)PLC
Constant plate count (N) L ÷ dp
F ÷ dp-1
tR ÷ dp2
VR ÷ dp ∆P ÷ dp
-2
Shorter columns Much shorter analysis times Smaller eluent volumes Much higher pressures
Smaller particles are desirable
Higher pressures
needed
HPLC U(H)PLC
Constant plate count (N) L ÷ dp
F ÷ dp-1
tR ÷ dp2
VR ÷ dp ∆P ÷ dp
-2
Much more heat generated Radial temperature gradients jeopardize performance Narrower columns are required for effective heat dissipation
Smaller particles are desirable
Higher pressures
needed
HPLC U(H)PLC
Constant plate count (N) L ÷ dp
F ÷ dp-1dc
2
tR ÷ dp2
VR ÷ dpdc2
∆P ÷ dp-2
Drastic reduction in extra-column volumes needed
About a factor 10 when comparing with 3 µm
particles or a factor 15 in comparison with 5 µm
particles
Smaller particles are desirable
Higher pressures
needed
Narrow columns needed
HPLC U(H)PLC
Constant plate count (N) L ÷ dp
F ÷ dp-1dc
2
tR ÷ dp2
VR ÷ dpdc2
∆P ÷ dp-2
Drastic reduction in extra-column volumes needed
Design of columns, connectors and instruments is especially critical for (slowly diffusing!) polymers
Smaller particles are desirable
Higher pressures
needed
Narrow columns needed
Size-based separations
Both reducing the tubing length and increasing the column volume are needed to obtain acceptable extra-column dispersion
The observed peak should reflect the actual molecular-weight distribution of the polymer the contribution of the sample dispersity (PDI)
should exceed 90% for reliable characterization!
2222columnextracolumnPDIobserved −++= σσσσ
Extra-column contribution (%) for different system configurations
PS molecular weight, Da
2.1 x 50 mm column, using
Column Manager (CM)
2.1 x 50 mm column tubing
length reduced
(avoiding CM)
2.1 x 150 mm column,
tubing length reduced
(avoiding CM)
4.6 x 150 mm column,
tubing length reduced
(avoiding CM)
two 4.6 x 150 mm columns, tubing length
reduced (avoiding CM)
92 (toluene) 87 73 42 6 31990 95 51 16 2 0.5
30320 90 37 14 3 1.552400 94 55 22 6 3523000 102 68 35 12 62061000 90 39 12 12 7
Awkward compromise
Heat dissipation High pressures imply much heat. Narrow columns are required to avoid radial gradients (and excessive band broadening) Slow diffusion Wide-bore columns are required to avoid excessive extra-column band broadening For polymers the best compromise may be 4.6 mm i.d. Polymer detectors (light scattering, viscometry) for UHPLC or not yet available
HPSEC Columns: 3×250 mm (10-µm particles) 7.5 mm i.d. PL-Gel Mixed B Mobile phase: THF 1 mL/min
UPSEC Column: 150 mm (1.7-µm particles ) 4.6 mm i.d. Acquity UPLC BEH C18 Mobile phase: THF 0.5 mL/min
1270 1370 1470 1570 1670t (sec)
1.8 2 2.2 2.4 2.6 2.8 3t (min)
Size-exclusion chromatography of polyurethanes
10 times faster 20 times less eluent much better resolution
YES, WE CAN! Perform HP-SEC separations Perform UHP-SEC separations
The pace of change
in 30 min
in 3 min
1.5
2.5
3.5
4.5
5.5
6.5
7.5
0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26
log
M
Velution, mL
Hydrodynamic chromatography 1.7-µm particles
SEC pore sizes 70 – 300 Å
UPSEC Calibration curve
PS standards; Mobile phase THF, 0.5 mL/min Column Acquity UPLC C18, 100 × 2.1 mm I.D.
1.5
2.5
3.5
4.5
5.5
6.5
7.5
0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26
log
M
Velution, mL
SEC pore sizes 70 – 300 Å
UPSEC Calibration curve
PS standards; Mobile phase THF, 0.5 mL/min Column Acquity UPLC C18, 100 × 2.1 mm I.D.
Other columns are now also available for UPSEC
Petra Aarnoutse et al. – SCM-7
Molecular-weight limit is determined by pore size of stationary phase
Fast size-based UPLC polymer separations
Separation in the SEC region column 150 x 4.6 mm, flow rate 1.85 mL/min,
Pressure 660 bar (system limit at this flow rate)
AU
0.000
0.010
0.020
0.030
0.040
0.050
0.060
Minutes 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
toluene PS 980
PS 2970 PS 7000 PS 19,880
PS 52,400
Molecular weight limit is determined by onset of molecule deformation
Separation in the HDC region column 150 x 4.6 mm, flow rate 1.85 mL/min,
Pressure 660 bar (system limit at this flow rate)
PS 52 kDa
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Minutes
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70
PS 96 kDa PS 197 kDa
PS 523 kDa PS 1,112 kDa
6 s 20 s
E. Uliyanchenko et al. J. Chromatogr. A 11 (2011) 1508
Polymers are subject to stress
y
x
Shear rate = γ = dvx/dy
Effect of particle size
vx,opt ÷ dp-1
y ÷ dp
γ ÷ dp-2
Stress
The extent of stress can be described by Deborah numbers (De). Polymers may undergo flow-induced stretching during HDC separations The transition from coil to stretched state of a polymer occurs around De = 0.5
Polymer deformation
D. Hoagland, R. Prud’homme. Macromolecules 1989, 22, 775-781 skip
Where: k – constant which depends on the packing structure; v – superficial solvent velocity (flow per unit area of empty bed) dp – particle diameter Φ – Flory-Fox parameter (2.5·1023 mol-1) η – solvent viscosity rG – radius of gyration of the polymer R – gas constant T – temperature
Deborah number
RTr
dv G
pkDe
312.6 ηΦ⋅⋅=
Shear Rate
Size of the molecule
Samples: PS in THF, Column ID 2.1 mm, dp 1.7 µm, Temperature 250C
Deborah numbers (deformation above 0.5)
Polymer MW, DaFlow rate, mL/min
0.1 0.2 0.5 0.7 11,000,000 0.05 0.11 0.27 0.38 0.542,000,000 0.18 0.37 0.92 1.3 1.83,000,000 0.38 0.75 1.9 2.6 3.89,000,000 2.6 5.2 13 18 26
Very-high-molecular-weight polymers may be deformed under UHPLC conditions
Slalom chromatography (SC) – a tunnel effect
Smallest polymers elute first Large polymers elute (much) later Large polymers must adapt their shapes
Hydrodynamic chromatography Coiled molecules
Size-exclusion chromatography
Slalom Chromatography Stretched molecules
Mcrit Coil-stretch transition
PS standards. Column Acquity UPLC C18, 1.7-µm particles, pore sizes 70 – 300 Å, 100 × 2.1 mm I.D., Mobile phase THF, 0.5 mL/min
UP size-based separations – Calibration curve
1.5
2.5
3.5
4.5
5.5
6.5
7.5
0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26
log
M
Velution, mL
Two peaks for one “narrow” PS standard
1.5
2.5
3.5
4.5
5.5
6.5
7.5
0.12 0.14 0.16 0.18 0.2 0.22 0.24 0.26
log
M
Velution, mL
The first peak emphasizes the low-molecular-weight fraction Application: Polymer degradation studies
Stress resistance of polymers?
Deformation (reversible)
Degradation (irreversible)
PS standard (Mr = 3 MDa) injected on UPLC column 100 mm × 2.1 mm i.d.; 1.7-µm particles at different flow rates 0.1 mL/min (De=0.4) 0.2 mL/min (De=0.8) 1 mL/min (De=3.9)
Peaks were collected and re-injected at 0.1 mL/min
The obtained peaks were compared to the PS standard of similar concentration, which was not previously injected on the column
Degradation study of polystyrene 3 MDa
Degradation study of polystyrene 3 MDa 1. Eluted from UPLC 100 mm × 2.1 mm i.d. column (1.7 µm particles)
at different flow rates - 0.1 mL/min (De = 0.4)
- 0.2 mL/min (De = 0.8) - 1.0 mL/min (De = 3.9) 2. The peaks were collected and re-injected at a flow rate of
0.1 mL/min 3. The obtained peaks were compared to the original PS standard
PS
Degradation products of THF?
5 mL/min on 4.6-mm i.d. column 15 mL/min on 8-mm i.d. column
- 0.1 mL/min (De = 1.9) - 0.3 mL/min (De = 5.6) - 0.5 mL/min (De = 9.3) - 0.7 mL/min (De = 13.1) - 0.9 mL/min (De = 16.8)
Polymers re-injected at 0.1 mL/min
0.1 ml/min
0.3 ml/min 0.5 ml/min
0.7 ml/min
0.9 ml/min
Slalom Chromatography Y. Liu, W. Radke, H. Pasch. Macromolecules 2005, 38, 7476-7484
Degradation study of polystyrene 7 MDa
1.7-µm particles 2.1-mm i.d. column
- 0.1 mL/min (De = 5.1)
- 0.5 mL/min (De = 25.6)
PS standards re-injected at 0.1 mL/min. Normalized chromatogram
0.5 ml/min
Not injected before
0.1 ml/min
Degradation study of polystyrene 13 MDa
Evidence of chain degradation of very large PS standards (> 3 MDa) (using very small particles and very high velocities, De > about 5)
1.7-µm particles 2.1-mm i.d. column
Pressure-driven separation
Channel of molecular dimensions
Increased retention for branched polymers requires low flow rates and very narrow channels (Size of molecules, Rh > 0.4 Rchannel)
Rh
Molecular-topology fractionation (MTF) Branching-selective separation in very narrow channels*
Polymeric monoliths
* Meunier D.M., Smith P.B., Baker S.A., Macromolecules 38 (2005) 5313
skip
MTF×SEC of linear polystyrene standards
at 10 µL/min
1.3 2.6
3.7 MDa
Critical MTF×SEC of linear polystyrene standards at 30 µL/min
1.3 2.6 3.7 MDa
Critical MTF×SEC of “star-shaped” polystyrene
Linear PS
(2.6 MDa)
One-arm star (3.9 MDa)
Two-arm “star”
(5.2 MDa)
One single branching point in almost 40,000 monomeric units!
At 30 µL/min this implies three hours
Classification of separation methods λ = (radius of polymer)/(radius of flow-through channel)
Polymers are religious
They hide from the devil ...
... and fly with the angels
weak eluent / poor solvent
strong eluent / good solvent
S-co-MMA (60)
S-co-MMA (40)
S-co-MMA (80)
S-co-MMA (20)
PS 200,000
PS 2,450
PS 7,000
PS 30,000
PS 900,000
PMMA 127,000
PMMA 840,000
PMMA 28,300
PMMA 6,950
PMMA 2,990
Chemical composition
Mol
ecul
ar s
ize
LCxLC (LCxSEC) of religious polymers
UHPLC×UHPLC setup
From Column 1 To Column 2
Waste
Loop 1
Loop 2 Pump 1 Pump 2
Column 1 Column 2
Detector Detector
2D pump
PMMA 65 k
PMMA 50 k
PMMA 24 k PMMA 14 k
PMMA 100 k
PMMA/PBMA 80/20 80 k
PMMA/PBMA 65/35 20 k
PMMA/PBMA 40/60 15 k
PMMA/PBMA 40/60 50 k
PMMA/PBMA 20/80 110 k
PBMA 18 k PBMA 57 k
PBMA 100 k
Chemical composition
Mol
ecul
ar s
ize
Sample - mixture: -poly(methyl-methacrylate) -poly(n-butyl-methacrylate) -poly(methylmethacrylate)-block-poly(n-butyl-methacrylate)
Time, min 22 18 14 10 6
UPLC×UPSEC of polymers
Awkward compromise Heat dissipation High pressures imply much heat. Narrow columns are required to avoid radial gradients (and excessive band broadening)
Slow diffusion of polymers Wide-bore columns are required to avoid excessive extra-column band broadening
Pre-column band broadening Post-column band broadening
UPLC “General Solution” Gradient elution is routinely used for separating complex samples
Weak-eluting sample solvents (weaker than the starting eluent) allow focussing of the analytes at the top of the column
Injection band broadening can be negated and only detection band broadening pertains
Breakthrough effect in gradient-elution LC of polymers (PMMA 34,500)
0
10
20
30
40
50
60
70
4 5 6 7 8 9 10Time (min.)
ELSD
resp
onse
30µl
20µl
15µl
10µl
Column: C8-silica (250 x 4.6 mm i.d.; 5 µm particles; 100 Å pore size); Gradient: from 38% to 100% THF in water(25)/MeOH (75) in 20 min.
Skip explanation
Adsorption
Critical %
Below critical (adsorption conditions)
Above critical (exclusion conditions)
Exclusion Injection Plug
Adsorption peak
Explanation of breakthrough effect
time
“Breakthrough” peak
Adsorption
Direction of flow
Isocratic LC 73/30 n-hexane/THF Silica column
Breakthrough effect in isocratic LC of polystyrenes
breakthrough
adsorption
Isocratic LC 73/30 n-hexane/THF Silica column
Breakthrough effect in isocratic LC of polystyrenes
Avoiding breakthrough effects in gradient LC of polymers
Eva Reingruber et al., J.Chromatogr.A 1217 (2010) 6595-6598
Avoiding breakthrough effects in gradient LC of polymers
Eva Reingruber et al., J.Chromatogr.A 1217 (2010) 6595-6598
Avoiding breakthrough effects in gradient LC of polymers
Eva Reingruber et al., J.Chromatogr.A 1217 (2010) 6595-6598
Copolymers from ethyl and benzyl diazoacetate
O
O
OR1
OR1
OOR1
OOR1
nN2
O
R1ORhI-precatalyst
- N2t =
30 min
N2
O
R2O
- N2
O O
O O
OR1 OR2
OR1 OR1
OOR2
OOR2
n
homo block gradient block
Eva Reingruber et al., J.Chromatogr.A 1255 (2012) 259-266
c
a b
d
Copolymers from ethyl and benzyl diazoacetate
The more peaks the merrier?
Eva Reingruber et al., J.Chromatogr.A 1218 (2011) 1147-1152
Breakthrough in second-dimension SEC?
One-dimensional SEC (solvent CHCL3)
Projection from LC×SEC
t0
Effect of sample solvent in SEC
Sample solvent CHCL3 / MeOH 60/40
Sample solvent CHCL3
40 µL 50 µL
62.5 µL 87.5 µL
Effect of transfer volume in LC×SEC
40 µL 50 µL
100 µL 150 µL
Effect of transfer volume in LC×SEC
SEC×LC vs. LC×SEC
HR-SEC possible in 1D
Possible 2D focusing
Possible sample clean-up
2tR not limited
2D breakthrough
2D gradients
1D overloading, adsorption
Limited choice of detectors
HR-gradient LC possible
Choice of detectors
2tR limited
Independent optimization
High sample capacity
Low-resolution 2D SEC
Possible 1D breakthrough
Possible 2D adsorption
Align separation selectivity with sample dimensions Excellent separations Structured, easily interpretable chromatograms Need to avoid 2D injection band broadening / breakthrough 1D effluent should not be too good a 2D eluent Need to avoid 2D adsorption 1D effluent should not be too bad a 2D eluent When using gradients the 1D effluent varies in time
LC×LC of polymers
SEC×LC vs. LC×SEC
HR-SEC possible in 1D
Possible 2D focusing
Possible sample clean-up
2tR not limited
2D breakthrough
2D gradients
1D overloading, adsorption
Limited choice of detectors
HR-gradient LC possible
Choice of detectors
2tR limited
Independent optimization
High sample capacity
Low-resolution 2D SEC
Possible 1D breakthrough
Possible 2D adsorption
Align separation selectivity with sample dimensions Excellent separations Structured, easily interpretable chromatograms Need to avoid 2D injection band broadening / breakthrough 1D effluent should not be too good a 2D eluent Need to avoid 2D adsorption 1D effluent should not be too bad a 2D eluent When using gradients the 1D effluent varies in time
LC×LC of polymers – major dilemma
We need to switch solvents
Fist-dimension peak profile
“Passive modulation” (as in LC×LC)
“Active modulation” (as in GC×GC)
Modulation
Loop
1
Detector
Waste
Injector
Pum
p 1
Pump 2 Lo
op 2
Col
umn
1
Col
umn
2
Column-based LC×LC
Loop
1
Detector
Waste
Injector
Pum
p 1
Pump 2 Lo
op 2
Col
umn
1
Col
umn
2
SPE
1 Detector
Waste
Injector
Pum
p 1
Pump 2
SPE
2
Col
umn
1
Col
umn
2
Column-based LC×LC with SPAM Stationary-phase assisted modulation
SPE
1 Detector
Waste
Injector
Pum
p 1
Pump 2
SPE
2
Col
umn
1
Col
umn
2
HYPERformance LC
HYPERFORMANCE
LCAndrea Gargano Michelle Camenzuli Anna Baglai Henrik van de Ven (Rudy Vonk) (Margaryta Ianovska) SCM-7
Conclusions
Despite some complications, UHPLC can have significant benefits for the separation of polymers SEC (Mr < 50 kDa PS) and HDC (Mr < 1 MDa PS) separations may be performed Very large molecules (Mr > 1 MDa PS) may be deformed, resulting in slalom chromatography Ultra-high-molecular-weight polymers (Mr > 3 Mda PS) may degrade in the column at high flow rates LC×LC is indispensable for polymer characterization Perhaps LC×LC can be improved using active modulation
Martin Lopatka (NWO) John Mommers (DSM)
THE END
Analytical-Chemistry / Forensic-Science Group I S
CM
SCM-7 Seventh International
Symposium on the Separation and Characterization of
Natural and Synthetic Macromolecules Amsterdam, The Netherlands
January 28-30, 2015
www.scm-7.nl