Miroslav JANČO
Sample Preparation and GPC/SEC/APC
Method Development
1 The Dow Chemical Company, 400 Arcola Road, Collegeville, PA 19426
Page 2
Overview
A. Sample Preparation for GPC/SEC/APC Analysis
1. Sample Types
2. Solvents
3. Filters
B. GPC/SEC/APC Method Development
1. Eluent(s) / Mobile Phase(s)
2. Additives
3. Column(s)
4. Sample Loading
5. Flow Rate
6. Temperature
7. Standards and Calibration
C. Safety Considerations
D. Literature
Page 3
Sample Analysis Flow Diagram
Weighing
Dilution
Filtration
Eluents
Columns
Standards
Detectors
Data Collection /
ProcessingSample PreparationMethod Development /
Sample Analysis
Report Generation
Information
to CustomerSample Production
Collection / Sampling
Sample
Transport
Archiving
Modified from Ronald E. Majors, HPLC 2016 San Francisco
Page 4
Representative Sample Types
A. Solids:
B. Liquids:
Powders Pellets Pads Tapes Foams
Solutions, Oils Emulsions / Slurries Paints
C. Pastes, Waxes, and Creams:
Page 5
Myriad of Sample Prep Techniques
*Doug Raynie, Sample Prep Perspectives, LCGC N. America 34(3) 174–188 (2016)
Page 6
Representative List of Common GPC/SEC Solvents
A. Organic
1. Ambient T
Tetrahydrofuran (THF)
Chloroform (CHCl3)
2. Elevated T
Toluene
N,N-Dimethylacetamide (DMAc)
3. High Temperature (~140 - 210 oC)
Trichlorobenzene (TCB)
4. Specialty
Hexafloroisopropanol (HFIP)
B. Aqueous
Water
Aqueous buffers at different pH
Aq. buffers / Methanol mixed
solvents (up to 50% MeOH)
Polymer Handbook, 4th ed.; Editors: Brandrup, E. H. Immergut, and E. A. Grulke, Willey-Interscience, 1999
Page 7
Solvent Selection Guide: Polymers Soluble in Organic Solvents
Page 8
Solvent Selection Guide for Aqueous Soluble Polymers:
Filtration Step
The life of an HPLC column can be extended up to 46x by filtering samples prior to injection
www.pall.com/lab
A
B
C
Page 9
Page 10
Filtration Step
A. Organic solvents
1. PTFE
2. Nylon
B. Aqueous
1. PVDF
2. Cellulose
C. High Temperature
1. SS Frits
Diameter:
4, 13, 17, 25 and 30
mm ID
Pore Sizes:
0.1 µm
0.2 µm (APC)
0.45 µm (SEC)
1, 3.1 and 5 µm
Page 11
Membrane Chemical Compatibility Guide
Page 12
Automatization in Sample Preparation
Chemspeed Technologies
http://www.chemspeed.com
CTC Analytics AG
http://www.ctc.ch
Weighing station
Solvent selector (up to 5 solvents)
Temperature controlled shaker
Centrifuge
Filtration
Page 13
Steps in SEC/APC Method Development
Step 1:
Defining the Analysis Goal
Step 2:
Method Development Factors
Step 3:
Data Processing and Reporting
Page 14
Step #1: Defining the Analysis Goal
What is the driving analytical request?
A. High resolution
Optimize column porosity for target polymer
Maximize column length based upon flow rate, solvent
viscosity and system pressure
B. High throughput
Optimize column porosity for target polymer
Evaluate short columns taking full advantage of low
dispersion system
C. What is the polymer type?
Polymer composition
Single polymer, mixture, blend…
This will impact the method conditions and the system
configuration
Page 15
Step #2: Method Development Parameters
Column Selection
Sample Loading
Flow Rate
CalibrationEluent Selection
The eluent is selected with the following considerations:
Sample Solubility
Column Packing Compatibility
Viscosity
Additives
SEC System / Detection Compatibility
Cost
Page 16
Eluent / Mobile Phase Selection Criteria
Sample Solubility Need an eluent that can entirely dissolve the polymer sample and allow
complete sample elution (100% sample recovery) from the column set
Column Packing Compatibility No swelling/shrinking of column bed (stationary phase)
Viscosity What is the impact on the separation conditions?
Additives How to identify and eliminate surface interactions to get purely SEC
mode separations?
System/Detection Considerations The solvent is compatible with your polymer, but what about the
chromatographic system?
APC Conditions:
Column set: 3BEH 45+125+450Å columns
(150x4.6 mm ID each) packed with 2.5 m
particles
Eluent: High Viscosity Solvent
Flow rate: 0.5 mL/min
Injection volume: 25L
Inj. C: 2.5mg/mL
Detection: UV @ 254nm
Page 17
Considering Eluent Viscosity for SEC/APC Separations
Solvent Viscosity (cP)
Acetone 0.32
Hexane 0.33
Acetonitrile 0.34
Heptane 0.39
Dichloromethane 0.39
Methyl ethyl Ketone 0.40
Ethyl Acetate 0.45
Isooctane 0.50
Tetrahydrofuran 0.55
Chloroform 0.57
Toluene 0.59
Methanol 0.60
Dimethylacetamide 0.77
Dimethylformamide 0.90
Water 1.00
Ethanol 1.00
Hexafloroisopropanol 1.02
Dimethyl sulfoxide 1.10
N-methylpyrrolidone 1.65
Isopropanol 2.30
High viscosity solvents will lead to:
High column backpressures
Lower mass transport and
May need higher column temperatures
14 600 psi
Page 18
Considering Eluent UV Cut Off Values
Solvent UV Cutoff (nm)
Acetonitrile UV 190
Pentane 190
Water 190
Hexane UV 195
Cyclopentane 198
Cyclohexane 200
Heptane 200
Isopropyl Alcohol 205
Methanol 205
Ethanol 210
2-Metoxyethanol 210
Methyl t-Butyl Ether 210
n-propyl Alcohol 210
Trifluoroacetic Acid 210
Tetrahydrofuran UV 212
n-Butyl Alcohol 215
1,4- Dioxane 215
Ethyl Ether 215
Solvent UV Cutoff (nm)
Ethylene Dichloride 228
1,1,2-Trichlorotrifluoroethane 231
Dichloromethane 233
Chloroform 245
n-Butyl Acetate 254
Ethyl Acetate 256
N,N-Dimethyl Acetamide 268
N,N-Dimethylformamide 268
Dimethyl Sulfoxide 268
Toluene 284
N-Methylpyrrolidone 285
Chlorobenzene 287
o-Xylene 288
o-Dichlorobenzene 295
1,2,4-Trichlorobenzene 308
Methyl Ethyl Ketone 329
Acetone 330
Methyl Isobutyl Ketone 334
Page 19
Considering Eluent Refractive Index Values
Solvent Refractive Index
Hexafloroisopropanol 1.275Methanol 1.329
Water 1.33Acetonitrile 1.344Acetone 1.359Ethanol 1.361Ethyl Acetate 1.37Hexane 1.373
Methyl ethyl Ketone 1.379Isopropanol 1.38Heptane 1.387Isooctane 1.404Tetrahydrofuran 1.408Dichloromethane 1.424Dimethylformamide 1.428Dimethylacetamide 1.438Chloroform 1.443N-methylpyrrolidone 1.468Dimethyl sulfoxide * 1.477Toluene 1.4961, 2,4 -Trichlorobenzene 1.571
Polystyrene– Blue
Polypropylene (Red)
Eluent:
TCB @ 140 C
THF
Toluene
Polydimethylsiloxane (PDMS)
Page 20
Additives: Why To Use Them?
To eliminate / suppress:
Polymer / surface (column packing)
Polymer chain interactions
Typical additives: LiBr, Triethylamine, Formic Acid, Acetic
Acid, Trifluoroacetic Acid, Ammonium Acetate…
Courtesy of PSSCourtesy of Agilent
Page 21
Step #2: Method Development Parameters
Column Selection
Sample Loading
Flow Rate
CalibrationEluent Selection
After information is gathered in Step 1, columns are
selected based on:
Surface chemistry
Particle size
Pore size
Column length
Page 22
GPC/SEC/APC Column Requirements
Modern GPC/SEC/APC columns must fulfill a variety of properties regarding:
• Physical requirements
•Mechanical stability: (pressure, temperature, pH...)
•Large pore volume
•Small interstitial volume
• Chemical requirements
• Well defind surface (homogeneity)
• Mo adsorbtion or repulsion
• Excellent solvent compatibility (no swelling/shrinking)
• Technical requirements
• Column hardware: solvent / corrosion resistant
• Clearly labeled, easy to handle
• Clear documentation
Page 23
Analytical Column Selection Criteria
Column Packing/Surface Chemistry:
Inorganic (bare and surface modified Si based)
Organic (PS-DVB, DVB, PMMA, PVOH, etc.)
Hybrid (BEH)
Particle size:
(1.7 – 2.5 – 3 – 5 – 10 – 20 µm)
Smaller particles for higher resolution
Larger particles to avoid shear degradation of very high MW components
Pore size:
Depends on molecular weight range of sample
Avoid exclusion of sample
Maximize pore volume in required separation region
Column Length/ID (Column Format):
Guard (30-50 x7.5-7.8 mm ID
Analytical (300 x 7.5-7.8 mm ID)
Preparative (300 x 25 mm ID)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
102
103
104
105
106
Vt= 1.9mL
Vp=1.05mL V
g=0.6mL
Vc= 2.5mL
log M
Retention time, [min]
Vo=0.85mL
VR = Vo + KDVp, KD=0-1
~3K – 80K
Page 24
SEC Column (150x4.6 mm ID) Characteristics
Page 25
Particle Size Effect
Smaller Particle Size :
Higher Resolution
Lower band dispersion
Higher number of theoretical plates
Higher column back pressure Risk of shear induced elongation and degradation
Lower mechanical stability (large pore size) of the beads
Particle Size Typ. Applications
20µm Ultra High Molar Mass Polymers (106 -107 Da)
10µm High viscosity eluents
5µm Low viscosity eluents
3µm Oligomers, Proteins
1.7-2.7µm APC Applications (dimer-2M Da)
Particle Size Impact on Ɖ of PS 11.6K standard
1K
30K
Useful separation range
Page 26
As particle size decreases, the
calculated dispersity becomes
closer to the reported Ɖ value,
and thus provides greater
accuracy of M data.
PS 11.6K
BHT
10 µm
5 µm
3.5 µm
1.7 µm
Particle
size
Average M data of PS 11.6K standard
Mw Mn Ɖ
10 11 100 9 700 1.14
5 11 400 10 900 1.05
3.5 11 500 11 200 1.03
1.7 11 400 11 100 1.03
Page 27
SEC Conditions:
Columns: (a) BEH 200 SEC
column (150x4.6 mm ID)
BEH packed with 1.7 m
particles and (b)
conventional SEC column
(250x4.6 mm ID) packed
with 5µm PS-DVB particles
Eluent: THF, Flow rate: 1.0
(a) and 0.5 mL/min (b)
Injection volume: 5L
Inj. C: 250ppm/component
Detection: UV @ 254nm
580
3K
11
.6+
7K
30
+2
2K
10
0+
66.3
K
a) APC
b) Conventional SEC
APC and Conventional SEC of 8 Component PS MixtureU
V d
ete
cto
r re
sp
on
se
, [A
U]
Retention time, min
0 2 64
Page 28
Individual Pore Size vs Mixed Bed Columns
Individual Pore Size Columns Mixed Bed / Linear columns
Deliver:
Larger pore volume
High(er) resolution
Narrower/Limited
separation range
Deliver:
Wide separation range
Low(er) resolutionIdeal for use as scouting columns when MW of the
sample is unknown or for analysis of samples with
broad dispersity
4 5 6 7 8 9 10 11 1210
2
103
104
105
106
107
108
102
103
104
105
106
107
108
Linear S
Linear M
LInear XL
SDV
Mola
r M
ass [
Da]
Elution Volume [ml]
4 5 6 7 8 9 10 11 12
102
103
104
105
106
107
108
102
103
104
105
106
107
108
10 000 Å
100 000 Å
1 000 000 Å
10 000 000Å
50 Å
100 Å
500 Å
1 000 Å
SDV
Mola
r M
ass [
Da]
Elution Volume [ml]
Page 29
Combination of the SEC/APC Columns
Page 29
Use Column Combination to Enhance: RESOLUTION
Add column of identical pore size (Increase pore volume)
Doubling column length increases resolution by factor of 1.4
PSS SUPREMA 5µm, 100Å
(8x300 mm)
Sample: Protein (Monomer / Dimer)
PSS SUPREMA 5µm
2x 100Å (8x300 mm each)
Courtesy of PSS
Page 30
Combination of the SEC/APC Columns
Page 30
Use Column Combination to Enhance: SEPARATION RANGE
Add column of different pore size
Avoid column missmatch
Courtesy of Waters
µRIU
0.00
15.00
30.00
Minutes
0.00 1.50 3.00 4.50 6.00
3 Columns in series
45A + 125A + 450A
2 Columns in series
45A + 125A
Altering the Resolution of SEC separation
SEC Conditions:
Column set: 3PLgel (300x7.5 mm ID) columns packed
with PS-DVB gel, Pore Size: 102+103+104 A, Particle
Size: 5 m, Tc= 40 oC
Eluent: THF (HPLC grade fro J.T. Baker), Flow rate: 1
mL/min
Injection volume: 100 L, Inj. C: ~1 mg/mL/component
Detector: RI, TD = 40 oC
SEC Conditions:
Column set: 2PL Oligopore (300x7.5 mm ID)
columns packed with PS-DVB gel, Pore Size:
Oligopore, Particle Size: 6 m
Eluent: THF (HPLC grade), Flow rate: 1 mL/min
Injection volume: 100 L, Inj. C: ~2
mg/mL/component
Detector: ELSD, TNEB = 40 oC, TECH=110 oC
Polymer
Mw~700K, Mn~100K
Oligomer
Mw~1200, Mn~600
Page 31
Page 32
SEC/GPC Column Selection Guide (Waters)
http://www.psscolumnselector.com/
http://navigator.chem.agilent.com/
https://www.phenomenex.com/size-exclusion-
chromatography-column
Page 33
SEC/GPC Column Selection Guide (PSS):
The Magic Triangle from PSS...
(or the importance of the polarity of the packing material)
http://www.psscolumnselector.com/
Page 34
Column Choices Guide (Agilent)
Page 35
Care and Use of GPC columns
Operate column(s) in column packing compatible solvent only and do not exceedrecommended backpressure and temperature limits
Change solvents by flushing the column at 0.1ml/min (overnight) in the new solvent
Never use methanol or acetonitrile with PS-DVB gels
Polar solvents shrink the gel packing, causing voids
Note: Flush the complete system with solvent to be used before connecting
the columns to the system
Always keep the flow through the columns at low flow, 0.1mL/minute; do not letsystem stand idle
Increase flow at 0.1 mL/minute to the specified flow rates of the columns
For columns operated at high temperatures (140 - 150 oC) - purge the columns at0.1 mL/min overnight at ~80 oC and then ramp up to operating temperature overseveral hours
Store the column bank together in the solvent used
Keep end fittings on the column tight to keep the column packing from drying out
Be careful not to drop the columns (they are fragile)
Filter sample solutions!
Page 36
Step #2: Method Development Parameters
Solvent Selection
Sample Loading
Flow Rate
CalibrationColumn Selection
The amount of polymer injected in the column can
impact the fidelity of the separation. The two factors
that contribute to this are:
Concentration
Injection volume
Page 37
Sample Loading Criteria
Polymer Concentration
Mass overload on column will lead to a loss of resolution
Need to balance this with sensitivity, particularly if looking
for lower level contributors
Sample Molar
Mass
g/mol
Typical
Concentration,
weight to volume
[mg/mL], (%)
< 10 000 2 (0.2%)
10 000 – 1 000 000 2-1 (0.2-0.1%)
> 1 000 000 0.2-0.5 (0.02-0.05%)
Courtesy of PSS
Page 38
Sample Loading Criteria
Injection Volume
Too large of an injection volume can also lead to mass
overload
Large sample volumes increase the peak volumes which
contributes to a loss in resolution
20µL
200µL
Number of Analytical
Columns
Guideline Injection
Volume
[µL]
4, 5 200
3 100
2 50
1 20
Page 39
Step #2: Method Development Parameters
Solvent Selection
Sample Loading
Flow Rate
CalibrationColumn Selection
Flow rate is often not evaluated in GPC method development.
However, it is an important method variable that impacts:
Speed of analysis
Separation efficiency
High Speed SEC Conditions:
Column: PSS High Speed SEC column (50x25 mm ID) packed with 5 m PS-DVB particles, pore size: linear M
Eluent: THF
Flow rate: 6.25 mL/min
Injection volume: 100 L
Inj. C: 1mg/mL
Detection: RI
Disadvantages:
High solvent consumption, compromised resolution of separations
Fast SEC
Page 40
Courtesy of PSS
Page 41
Rapid SEC
Rapid SEC Conditions:
Column: PL Rapid SEC column (100x10 mm ID) packed with 5 m PS-DVB particles, pore size: linear M or L
Eluent: THF
Flow rate: 1, 2 or 3mL/min
Injection volume: 100 L
Inj. C: 1mg/mL
Detection: UV@254nm
Disadvantages:
Compromised resolution of separation Courtesy of Agilent
Step #2: Flow Rate
Courtesy of Waters
AU
0.000
0.010
0.020
0.030
0.040
AU
0.000
0.010
0.020
0.030
0.040
AU
0.000
0.010
0.020
0.030
0.040
Minutes
0.00 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 10.50 11.00 11.50 12.00 12.50
8.4K
8.4K
8.4K
67K
67K
67K
579K
579K
579K
7.52M
7.52M
7.52M
200 µL/min
400 µL/min
600 µL/min
Poly(styrene) standard
7.52 M distorts with
increased flow rate –
Slalom Chromatography
Page 42
Page 43
Step #2: Method Development Parameters
Solvent Selection
Sample Loading
Flow Rate
CalibrationColumn Selection
Ensuring that you are adequately calibrating your bank of columns
is an important variable in method development. You need to
consider:
• Which standards?
• How many standards?
• Calibration type
• Frequency of calibration
Page 44
Readily Available GPC/SEC Standards
A. Organic
1. Ambient T
Poly(styrene) (PS)#
Poly(methyl methacrylate)#
(PMMA)
Poly(vinyl chloride)
Poly(dimethyl siloxane)
Poly(tetrahydrofuran) (PTHF)
2. High Temperature (~140 - 210 oC)
Polyethylene (PE)
A. Aqueous
Polyacrylic acid (PAA)
Polymethacrylic acid
(PMAA)
Polyethylene glycol/oxide
(PEG/PEO)%,#
Polystyrene sulphonate
Dextran
Pullulan
Poly(vinyl Pyridine)
% - can be used for both organic and aqueous systems
# - as individual standards and mixes
SEC Charts of PS Standards and Calibration Curve
SEC Conditions:
Column set: 3PLgel (300x7.5 mm ID) columns packed with PS-DVB gel, Pore Size: 102+103+104 A,
Particle Size: 5 m, Tc= 40 oC
Eluent: THF (HPLC grade from J.T. Baker), Flow rate: 1 mL/min
Standards: PS1 MIX A, MIX B and PS 162
Injection volume: 100 L
Inj. C: ~1 mg/mL/component
Detector: RI, TD = 40 oC
Page 45
APC of 16 PS Standards
APC Conditions:
Column set: 3 APC columns (150x4.6 mm ID) packed with BEH TMS particles,
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
Detector: RI, TD = 40 oC
~200 – 1M
Page 46
Run time: 6 min
1.8 5.6
RI d
ete
cto
r re
sp
on
se
, [m
V]
3 4 5
1 1
30K
560K
310K
200K
120K
98K
66K
44K
30K
22K
11.6
K
7000
3250
1700
580
220 (
BH
T)
Literature
1. Mori, S & Barth, H (1999) Size Exclusion Chromatography,
Springer Verlag, Berlin, Germany
2. Wu, C-S (2003) Handbook of Size Exclusion Chromatography
and Related Techniques. Marcel Dekker, New York, NY, U.S.A.
3. Striegel, AM, Yau, WW, Kirkland, JJ & Bly, DD (2009) Modern
Size-Exclusion Chromatography. John Wiley & Sons, Chichester,
UK
4. Polymer Handbook, 4th edition; Editors: Brandrup, E. H.
Immergut, and E. A. Grulke, Willey-Interscience, 1999
5. GPC/SEC/APC Vendor’s webpages:
www.agilent.com
www.jordilabs.com
www.malvern.com
www.phenomenex.com
www.pss-polymer.com
www.shodex.com
www.tosohbioscience.com
www.waters.com
www.wyatt.com
Page 47
Safety Considerations
Safety should be the highest priority
while working in the lab
Wear appropriate PPEs (lab coat,
safety glasses, gloves, goggles, face
shield, apron, etc.)
Sample prep steps/handling should be
performed in the hood or in a well
ventilated enclosure to minimize
exposure of operator and lab personnel
to chemical vapors
Spills – know the spill kit location,
handle by institution policy
All waste generated should be
disposed off in the proper waste stream
according to institution’s policy
Know eye wash and safety shower
locations
Ergonomics
Page 48
Summary
Page 49
Sample preparation is viewed as a routine task rather than as an integral
component in the analytical process, and as a result, it has long been
undervalued as a science and underdeveloped as a technology.
There is no universal solvent nor GPC/SEC/APC method
Solvent /Eluent selection is driven by
Sample Solubility
Column Packing Compatibility
GPC/SEC/APC method development / improvement is driven by
The analysis goal which defines the selection of:
Column(s)
Eluent
Flow rate
Injected mass (injection volume and concentration)
Temperature
Calibration Standards
Detection Options
THANK YOU FOR ATTENDING!
Page 50
ANY QUESTIONS?