Foundation GPC Part 3 – Gel
Permeation Chromatography
Instrumentation
Introduction
 This
presentation introduces the equipment used in gel permeation
chromatography (GPC)
 The
role of each device shall be discussed, including troubleshooting
information
 The idea of integrated GPC systems shall be presented
2
Components of a GPC System
Pump: delivers flow down the column
Injection valve: Allows us to inject our samples
GPC column set: Performs the separation
Detector: detects the material leaving the columns
Optional extras: autosamplers, degassers, etc.
3
Pump
 Required to maintain a constant, steady liquid flow through the columns
 Isocratic pump (single channel)
 Pulseless or low pulse flow required to ensure good detector baselines
 Typically a reciprocating dual piston pump
 Fairly inexpensive, reliable and suitable for use in a number of solvents
 Service typically includes replacement of worn check valves and piston seals
4
Effect of Flow Rate on Resolution
 Flow
rate strongly
affects resolution
 Every
column has an
optimum flow rate, as in
all LC systems
 However
in GPC the
mass transfer effect is
much more prominent
5
Flow Rate and Efficiency
 A measure of the efficiency of
a chromatographic system is the
plate count
 Column
is divided into
number of theoretical plates
a
 Plates
are defined as the
smallest cross-sectional slice in
which the mobile and stationary
phases are in equilibrium
 The smaller the width (known
as height) of the plate, the
quicker the system comes to
equilibrium and the greater the
efficiency
 Plate counts controlled by the
Van Deemter relationship
6
Eluent :
Columns :
Test probe :
THF
PLgel 100Å
ODCB
Optimum flow rate for small
molecule
separations
is
around 1.0ml/min
7
Eluent :
Column :
THF
PLgel 10um MIXED-B
For high MW samples, high flow rate
should be avoided, reduced flow rate
may be required to improve resolution
8
Affect of Pump Flow rate in GPC
A small change in flow rate can have a large effect on MW. Flow rate
correction using an internal flow rate marker is commonly applied to
correct for small flow rate fluctuations.
9
Pump Issues - Variable Retention Time
Increasing retention times - Lab temperature changes may
result in retention time changes
 Overcome by thermostatting the columns
Insufficient equilibration time for the column may give unstable
retention behavior
 Allow at least 2 GPC column volumes through the column(s)
Decreasing retention times - Usually a result of the flow rate
speeding up
 Check the pump and reset the flow if necessary
10
Increasing Retention Times
Usually a result of the flow rate slowing down
 Check for the presence of bubbles in pump head
Retention beyond total permeation volume will be observed if there are specific
interactions between the sample and the with stationary phase
 Interactions can be Inhibited by adding modifiers to mobile phase
Adsorption of sample can occur if you are using a poor solvent, for instance
analysing polystyrenes in DMF
 Change eluent so that samples, standards and solvent are of similar polarity
11
Pump Pressure Variations
Pressure increasing – Can be caused by build-up of particulates in the sample
 can be avoided by filtering the samples and mobile phase
With certain solvents, solvent freezing in GPC tubing can cause pressure
problems
 For these solvents e.g. TCB elevate the temperature of the system
Pressure falling - Can be caused by pump cavitation
 Make sure you thoroughly degas solvents
If the pressure is low it could be due to insufficient flow to column
 Clear any blocked solvent lines
 Loosen cap of eluent reservoir to prevent pressure problems
12
High Pressure
A high pressure will result if the flow rate is too high
 Check pump flow rate independently by measuring with flow with stopwatch
High pressure will also result if the column has a blockage
 Filter samples to avoid this problem
 Use a guard column to improve the column lifetime
High pressure may be due to a blocked inlet frit on the column
 Reverse flow through column to clear any blockage
 Replace frit to repair the column
13
Pressure Fluctuation
Fluctuation will be caused by a leaking check-valve or pump
seal
 Replace or clean the check-valve
A bubble in pump head will also cause fluctuations
 Remove the bubble by purging the pump head
 Degas solvents thoroughly to avoid bubble build-up
Insufficient liquid flow to pump will cause pressure problems
 Mobile phase inlet may be blocked - remove and clean it!
 Elevate reservoir above pump head to help siphoning
14
Injection Valve
 Required
to allow introduction of the
sample into the flowing eluent stream
 Usually
a 6 port Rheodyne or Valco
manual valve is used, with automatic
triggering
 Service
usually involves changing the
valve seal in the case of a leak
 Leaks are sometimes seen from worn
rotor seal in the injection valve
 Injection valve siphoning can draw
solution from the waste - lower waste bottle
15
Injection Volume




16
GPC columns have a relatively large volume (typically 300x7.5mm)
Injection volumes for GPC can therefore be higher than for HPLC
As a rule of thumb, 50ul per 300x7.5mm column length will have little
effect on band broadening
Minimise injection volume for high efficiency separations (e.g. 3um
columns) to avoid band broadening which will decrease resolution
Effect of Concentration on Peak
Shape and Resolution
0.05%
0.10%
Column :
Eluent :
Flow rate :
Detector :
PLgel 10um MIXED-B
300x7.5mm
THF
1.0ml/min
UV
Polystyrene standards
0.15%
17
0.20%
1. 8,500,000
4. 34,500
2. 1,130,000
5. 5,100
3. 170,000
6. 580
Effect of Injector Loop Size on Resolution
20µl loop
Column :
Eluent :
Flow rate :
Sample :
PLgel 3µm MIXED-E
300x7.5mm
THF
1.0 ml/min
Epikote 1001
epoxy resin
200µl loop
Injection loop is a major
contribution to system dead
volume, use reduced injection
volume
and
increase
concentration
to
maintain
sensitivity
18
Columns
 The columns perform the separation
 The choice and care of columns is critical to good chromatography
 Columns will be the focus of the next presentation
19
Effect of Particle Size on Resolution
 Smaller
particle size leads
to greater efficiency and
resolution
 Smaller
particle size also
leads to shear degradation
 Therefore
only use 3um
particle sizes for very low
molecular weight separations
 High
molecular weight
separations require large
particle sizes
20
On-column Shear Degradation in GPC
Sample of cellulose carbanilate was analysed in THF eluent at
1.0ml/min with DRI and PD2020 dual angle light scattering detector to
measure bulk weight average molecular weight (Mw) of the polymer
as it eluted from the column.
Effect of column characteristics on measured Mw
21
Effect of Length on Resolution
Eluent:
FlowRate:
Detector:
Samples :
calibrants,
THF (stabilized)
1.0ml/min
UV
PL EasiCal PS-1
two injections
1 x PLgel Column
22
Mp values
Injection 1
Injection 2
1. 7500000
6. 2560000
2. 841700
7. 320000
3. 148000
8. 59500
4. 28500
9. 10850
5. 2930
10. 580
3 x PLgel Column
Resolution in GPC




Resolution Rs
=
2(V1-V2)
(W1+W2)
Elution Volumes of peaks 1 and 2
are V1 and V2
Peak Widths of peaks 1 and 2 are
W1 and W2
Specific Resolution per Molecular
Weight Decade
Rsp
=
0.25
sD
Where D = slope of calibration
Sigma = peak variance (related to
peak width)
23
Poor Column Lifetime
Columns can be degraded by attack of polymeric materials by mobile phase
 Use THF & TCB stabilised with antioxidant.
Shorter lifetime are observed with high temperature using small particle
columns
 Switch to larger particle size to reduce problem
Deterioration can also occur due to contaminant build-up on the column
 This can be avoided by using guard column which can be discarded
24
Column Ovens
 Ovens
are used to heat and maintain the temperature in a GPC
separation
 They come in a range of specifications, from low temperature all the way
up to very high temperatures
 Temperature can be important in GPC
 Some GPC experiments are impossible
temperature
25
without working at elevated
Why use Elevated Temperature?
GPC applications employing elevated temperature generally
fall into two categories :
1. To reduce solvent viscosity for improved chromatography
2. To achieve and maintain sample solubility
26
Effect of Temperature on
Separations in Polar Solvents
Column :
Eluent :
Flow rate :
PLgel 5um MIXED-C
300x7.5mm
DMF
1.0ml/min
Increased temperature :
 Reduced operating
pressure
 Improved resolution,
particularly at high MW
PEO/PEG standards
990,000 252,000
86,000 18,000
4,800
200
27
Effect of Temperature on Column Pressure
Column
Eluent
Flow rate
28
PLgel 5um MIXED-D
300x7.5mm
Toluene
1.0ml/min
Column pressure falls as
temperature increases due to
reduced viscosity
Typical Range of Solvents used in GPC
 A wide
range of solvents are
used in GPC with very varied
viscosities
 Elevated temperature helps to
reduce the viscosity of these
solvents
improving
column
lifetime
29
Leaks in a GPC System
Most common caused by loose connections between columns and detectors
 Check all the connectors and tighten if necessary
 If the leak persists, disassemble and replace the leaking connector
Internal Detector Leak can be seen in the detector, injection valve or pump
 Often due to solvent spillage near the instruments solvent sensor
 Can be due to failed detector seal or cracked cell – these must be replaced
 Leaks are sometimes seen from worn rotor seal in the injection valve
 Injection valve siphoning can draw solution from the waste - lower waste bottle
 Pump purge valve failure will cause leaks – tighten the valve or replace
 Pump seal and gasket failure will result in leaks - these must be replaced
Leaking can be seen in from the column end-fittings
 The end-fitting may be loose - tighten as necessary
 The frit & spreader in the column may need to be replaced
30
Concentration Detectors for GPC
 There are several concentration detectors that are used in conventional
GPC
 Differential refractive index (DRI)
 UV
 Infra-red
 Evaporative light scattering (ELSD)
 We will look at these in turn
31
Differential Refractive Index Detector
R = reference cell (usually static)
S = sample cell, eluent flowing through
Response = Kri * (dn/dc) * concentration
Where K is a constant, (dn/dc) is the refractive index increment and C is
concentration
32
Eluent Selection with DRI Detectors
Polydimethylsiloxane (PDMS) is soluble in
several common GPC solvents.
PDMS has a refractive index of 1.407 and
therefore it is isorefractive with THF and no
DRI signal is recorded.
Toluene
(n=1.496)
and
chloroform
(n=1.444) give good DRI signals and are
therefore preferred solvents for GPC of
PDMS polymers when DRI is the detector
of choice.
Columns:
Flow Rate:
Detector:
33
PLgel 5µm 104Å+ 500Å
1.0ml/min
DRI
Low MW dn/dc Effects
Columns
Eluent
Flow rate
Solutes
2 x PLgel 5um 50Å 300x7.5mm
THF
1.0ml/min
Linear hydrocarbons, all prepared at equal concentration
Linear Hydrocarbons
4
3
2
1
34
Peak
HC
MW
RI
1
C12H26
170
1.4216
2
C16H34
226
1.4340
3
C22H46
310
4
C32H66
450
1.4550
Refractive index of a
homologous
series
changes rapidly below a
MW of around 1000.
Differential Refractive Index Detector









35
The most commonly used detector in GPC, "Universal" detector
Monitors difference in refractive index of eluent stream as solutes emerge
from column with respect to a static reference cell filled with the pure
solvent
Can give positive and negative peaks
Must have sizeable difference in refractive index of solvent and solutes
Extremely sensitive to pressure and temperature fluctuations
Modest sensitivity, unsuitable for low solute concentrations
Non-destructive to sample
Easy to use
Approximately linear response with concentration
Baseline Noise and Drift
Random noise is usually a result of the build-up of contamination in the
column or in the detector cell, steady baseline drift usually results from the
build up of contaminations
 Flush the column and the detector cell to waste
 Make sure the samples are clean – filter with 0.45µm filters
 Use high quality solvents for HPLC or GPC
Spikes are usually due to bubbles in detector
 Make sure you have degassed mobile phase before use
Random drift can also be cause by temperature changes
 If thermostatting, make sure you insulate the column and tubing
36
Baseline Drift at Start of Operation
Usually caused by the column settling down
 Make sure you allow sufficient time for column to equilibrate
Can be caused by the detector equilibrating
 Allow time to reach stability - very common for RI detectors
 Ensure detector is not in a draught or direct sunlight
Baseline variations can also be cause by RI Reference cell
contents decaying or degrading, especially at temperature
 Regularly flush the reference cell with mobile phase
37
Ghost and Negative Peaks
Ghost peaks are often peaks which come from the previous injection
 Make sure you do not inject next sample until previous one has fully eluted!
 If there is absorption, some material may elutes after the total permeation limit
 If there is absorption, make sure you flush the column completely
 During injection, ensure that injection loop is completely filled and flushed
 On RI detectors can occur is the dn/dc is less than the solvent
 Reversing signal polarity gives a positive peak
 On UV detectors can occur is the solute absorbs less than the eluent
 Need to change eluents to get a positive peak
Negative peaks and baseline disturbance at total permeation due to differences in
refractive indices of injection solvent and eluent
 Cannot be avoided, but it helps if the samples are prepared in the mobile phase
38
UV Detectors







39
Relies on UV absorbing groups being present in solute
Very sensitive detector with small cell volumes and therefore low system
dispersion
Good linearity
Insensitive to temperature and pressure fluctuations
Many polymers do not have chromaphores
Many solvents or solvent additives absorb UV and either prevent use or cause
decrease in sensitivity.
Sometimes used in conjunction with RI for copolymer analysis when only one
of the monomers has UV chromaphore.
Infrared Detectors







Relies on infrared absorbing groups in solute
Sensitivity low to moderate
Cell volumes tend to be much larger than other detectors and time constants
longer
Many solvents absorb IR and either prevent use or decrease sensitivity
Insensitive to temperature fluctuations
Niche market for polyolefin analysis at high temperature but with moderate
sensitivity
Can be used with RI for copolymer analysis
Note : GPC-FTIR using special flow cell (e.g. the PL-HTGPC/FTIR
interface) or eluent collection device (e.g. Lab Connections) has great
potential for identification of solutes by measuring complete FTIR
spectrum as a function of elution time.
40
The PL-HTGPC/FTIR Interface



41
Consists of heated cell, transfer line and temperature control box
Can be heated to 175°C
Designed for use with Varian, Perkin Elmer, Nicolet and Bruker spectrometers
Evaporative Light Scattering Detector







42
Monitors changes in eluent stream by evaporation of solvent and using
simple light scattering mechanism to detect solute particles
Economical detector with high temperature capability
Insensitive to temperature and compositional changes
Always gives positive signal response
Requires difference in volatility of solute and solvent
Generally higher sensitivity than RI
Loss of volatile low molecular weight solutes can occur
Varian 380-LC Evaporative
Light Scattering Detector
43
ELS Instrument Concept
44
Light Scattering Detection
 Response dependent on particle size
 Mechanism principally reflection/refraction
 Ideally nebulisation should form uniform droplet size
45
Linearity of Response


46
GPC analysis using THF at 1ml/min
Lowest column loading 1.0µg on column, or 100µl of 0.01mg/ml solution
Sensitivity of DRI Versus ELS
Columns
Eluent
Flow rate
Loading
Mp values
2 x PLgel 5um MIXED-C 300x7.5mm
THF
1.0ml/min
ELS is essentially independent
of dn/dc, improvement in
0.1%, 20ul
sensitivity will depend on a
number of solute parameters
3
1. 7,500,000
4
2. 841,700
3. 148,000
4. 28,500
5. 2,930
47
2
1
5
Consequence of Non-linearity
 Non-linearity
results in
loss of response for low
concentration peak tails
 Distribution
narrower
than that calculated by
DRI, polydispersity low
48
Polymer Blends in THF, DRI Versus ELS
DRI
Columns 2 x PLgel 5um MIXED-C
300x7.5mm
Eluent
THF
Loading
0.2%, 20ul
Detectors DRI at 1V FSD
ELS1000 at 10V FSD
ELS 1000
Samples
Polystyrene
Polydimethylsiloxane
Blend
49
Polymer Blends in Toluene, DRI Versus ELS
Columns 2 x PLgel 5um MIXED-C
300x7.5mm
Eluent
Toluene
Loading
0.2%, 20ul
Detectors DRI at 1V FSD
ELS1000 at 10V FSD
Samples
Polystyrene
Polydimethylsiloxane
Blend
50
Analysis of Natural Rubber, DRI Versus ELS
Columns 3 x PLgel 10um MIXED-B
300x7.5mm
Eluent
Toluene
Loading ~0.2%, 200ul
Detectors DRI at 1V FSD
ELS1000 at 10V FSD
Zoom on additive region ELS
51
Styrene Butadiene Rubber (SBR) Analysis
Columns
Eluent
Flow rate
Loading
2 x PLgel 20um MiniMIX-A 250x4.6mm
THF
0.3ml/min
1mg/ml, 100µl
Oil extended SBR
General grade SBR
This application illustrates the
high sensitivity of the PLELS1000, permitting the
polymers to be analysed at low
loadings using narrow bore SEC
columns.
52
Polymer Additive Analysis Using the ELS
These additives are used as stabilisers and antioxidants in polymer
formulations. Not all of them have a UV chromophore and when extracted from
polymers they are usually present in very small quantities. The universality and
high sensitivity of the ELS makes it ideal for this type of application.
Columns
Eluent
2 x PLgel 5um 50Å
THF + 0.1% diethanolamine
Chimasorb 944
Irgafos 168
Irganox 1010
Tinuvin 622
Tinuvin 770
Tinuvin 327
53
GPC Using Polar Organic Solvents
Columns
PLgel 10um MIXED-B 300x7.5mm
Eluent
DMSO
Detectors
PL ELS 1000
Must use volatile salts as modifiers
for polar organic eluents (e.g.
ammonium acetate)
Pullulan Mw=404,000
Pullulan Mw=22,800
54
High Temperature GPC
Columns
2 x PLgel 10um MIXED-B 300x7.5mm
Eluent
TCB
Flow rate
1.0ml/min
Temperature
160°C
Detectors
PL-ELS 1000
MISS OUT SLIDE
No lONGER SOLD
NBS 1475
polyethylene
55
PVP/PVA Copolymer (Kollidon VA64)
Columns
2 x PL aquagel-OH MIXED 8um 300x7.5mm
Eluent
1. 70% 0.2M NaNO3, 0.01M NaH2PO4, pH7, 30% methanol
2. 70% 0.1M ammonium formate, 30% methanol
Flow rate
1.0 ml/min
Detector
1. DRI
2. ELS 1000
Volatile salts must be used
with
evaporative
light
scattering detection
56
Summary of ELS
Evaporative light scattering detection can offer some significant advantages in
GPC applications when compared to the more widely used differential
refractometer or alternative UV detector :
 Responds to compounds with no UV chromaphore
 Positive response for all non-volatile solutes
 Stable baseline, no drift with eluent or ambient temperature changes
 High sensitivity, ideal for low dn/dc polymer/solvent combinations
 No interference from spurious peaks around total permeation
 Fast setup and equilibration
57
Split Peaks
Often seen if the sample loading on the column is too large
 Reduce the size of the injection loop or the concentration
Can also be caused by a blocked or partially blocked frit
 Need to replace the frit in the column
 Stop the frit clogging by using an in-line solvent filter of about 2µm
A void or channel in the column will also cause split peaks
 Unfortunately you will need to replace column!
Can be caused by a partially blocked or damaged flowpath in the injector
 Need to replace the rotor seal in the injector
Split peak may be due to a single peak with interfering components
58
 Need to prepare a fresh solution!
Peak Tailing
Tailing can result from excessive dead volumes
 Make sure the tubing length is minimised,
 Make sure the injection seal is tight and there are no leaks
 Ensure that the connector fittings are properly seated
Tailing can result from degradation of column
 Repair or replace the column!
Interaction of sample with surface of stationary phase can cause
tailing
 Overcome with using mobile phase additives
 Amines or salts to can be used in organic GPC
59
Peak Broadening
Large dead volumes will contribute significantly to peak broadening
 Always use LDV end fittings and connectors
 Minimise lengths and diameters of tubing wherever possible
Broadening will result if the eluent is too viscous
 May need to increase operational temperature
Broadening may result if the detector cell volume is too large
 If possible, use a smaller cell volume
Broadening will result if the column is not performing
 Repair or replace the column
60
Effects of Band Broadening
Modern high performance GPC columns
have minimised the effect of band
broadening in the separation. However poor
system design with large amounts of dead
volume can still cause loss of resolution.
System dead volume should be minimised,
especially when using very high efficiency
columns.
61
Poor Detector Sensitivity
The sample will not be observed if it is injected at a concentration below the
minimum detectable level
 Increase concentration or sample volume to get a good response
Sometimes a small peak will be observed for the first few sample injections
due to adsorption of sample onto the column
 Condition column with concentrated sample will reduce effect
Injecting an underfilled injection loop will give small peaks
 Ensure at least 3 times the sample loop volume is injected
62
Other System Components
 Other
components can be added to a modular GPC system as
required
 The most common additions are…
 Degassers used to removed dissolved
air from solvents,
preventing pumping issues
 Autosamplers
can be used to inject samples and automatically
trigger data collection.
63
Integrated GPC Systems
 Integrated
GPC systems include pump, injection valve, oven and
detectors in a single system, often with additional systems
 They have several advantages over a modular (‘separates') system
 They often provide an adequate temperature range for GPC
applications
 They
reduce system dead volume by minimising connecting tubing
between system components
 The presence of a controlled temperature environment that contains
all components leads to no localised variations in temperature
 The
systems have improved communications between components,
system intelligence provides high performance, high degree of
automation and comprehensive safety features
64
Example - The PL-GPC 50 Plus
 Integrated
system for GPC
analysis up to 50°C
 Standard
instrument fitted
with a DRI detector
 Can
accommodate
detector options
other
 Fully software controlled
65
Reproducibility on the PL-GPC 50 Plus
Raw data chromatograms
Molecular weight distributions
66
Inj no.
Mn
Mw
Peak area
1
17,289
76,818
333851
2
16,988
77,434
335496
3
17,248
77,514
332616
4
17,251
77,052
335635
5
17,348
76,520
334212
6
17,487
77,728
333511
7
16,898
77,578
335642
8
17,457
77,288
334923
Mean
17,302
77,241
334485
s.d.
220
687
1048
% var
1.3
0.5
0.3
Example - PL-GPC220 Integrated GPC
67
Components of the PL-GPC220
Integrated GPC System
PUMP AND
DEGASSER
68
HTGPC Analysis of Crystalline Polymers
Additional system requirements for these difficult applications
 Adequate temperature capability (30-220°C)
 Consistency of solvent delivery in continuous use
 High temperature autosampler/injection system
 DRI performance (sensitivity and stability)
69
PL-GPC220 Autosampler




70
40 vial position carousel
2ml glass vials with crimped aluminium caps
Sample maybe slowly stirred prior to injection
Two zone heating, minimised risk of sample degradation
PL-GPC220 DRI Sensitivity
Columns
Flow rate
Injection
Test probes
3 x PLgel 10um MIXED-B
300x7.5mm
1.0 ml/min
200ul
Polystyrene standards
Many polymer/solvent
combinations
in
HTGPC offer very low
dn/dc
so
DRI
sensitivity
is
an
important issue
71