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?