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Foundation GPC Part 3

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Title: Foundation GPC Part 3


1
Foundation GPC Part 3 Gel Permeation
Chromatography Instrumentation
2
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

3
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.
4
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

5
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

6
Flow Rate and Efficiency
  • A measure of the efficiency of a chromatographic
    system is the plate count
  • Column is divided into a number of theoretical
    plates
  • 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

7
Eluent THF Columns PLgel 100Å Test probe
ODCB
Optimum flow rate for small molecule separations
is around 1.0ml/min
8
Eluent THF Column PLgel 10um MIXED-B
For high MW samples, high flow rate should be
avoided, reduced flow rate may be required to
improve resolution
9
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.
10
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

11
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

12
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

13
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

14
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

15
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

16
Injection Volume
  • 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

17
Effect of Concentration on Peak Shape and
Resolution
0.10
0.05
Column PLgel 10um MIXED-B 300x7.5mm Eluent
THF Flow rate 1.0ml/min Detector UV
Polystyrene standards 1. 8,500,000 4. 34,500 2.
1,130,000 5. 5,100 3. 170,000 6. 580
0.15
0.20
18
Effect of Injector Loop Size on Resolution
20µl loop
Column PLgel 3µm MIXED-E 300x7.5mm Eluent
THF Flow rate 1.0 ml/min Sample 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
19
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

20
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

21
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
22
Effect of Length on Resolution
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
Eluent THF (stabilized) FlowRate 1.0ml/min Det
ector UV Samples PL EasiCal PS-1 calibrants,
two injections
1 x PLgel Column
3 x PLgel Column
23
Resolution in GPC
  • Resolution Rs 2(V1-V2) (W1W2)
  • 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 s D
  • Where D slope of calibration
  • Sigma peak variance (related to peak width)

24
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

25
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 without
    working at elevated temperature

26
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
27
Effect of Temperature on Separations in Polar
Solvents
Column PLgel 5um MIXED-C 300x7.5mm Eluent
DMF Flow rate 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
28
Effect of Temperature on Column Pressure
Column PLgel 5um MIXED-D 300x7.5mm Eluent
Toluene Flow rate 1.0ml/min
Column pressure falls as temperature increases
due to reduced viscosity
29
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

30
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

31
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

32
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
33
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
(n1.496) and chloroform (n1.444) give good DRI
signals and are therefore preferred solvents for
GPC of PDMS polymers when DRI is the detector of
choice. Columns PLgel 5µm 104Å 500Å Flow
Rate 1.0ml/min Detector DRI
34
Low MW dn/dc Effects
Columns 2 x PLgel 5um 50Å 300x7.5mm Eluent
THF Flow rate 1.0ml/min Solutes Linear
hydrocarbons, all prepared at equal concentration
Linear Hydrocarbons Peak HC MW RI 1 C12H26 170 1.4
216 2 C16H34 226 1.4340 3 C22H46 310 4 C32H66 450
1.4550
4
3
2
1
Refractive index of a homologous series changes
rapidly below a MW of around 1000.
35
Differential Refractive Index Detector
  • 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

36
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

37
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

38
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

39
UV Detectors
  • 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.

40
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.
41
The PL-HTGPC/FTIR Interface
  • Consists of heated cell, transfer line and
    temperature control box
  • Can be heated to 175C
  • Designed for use with Varian, Perkin Elmer,
    Nicolet and Bruker spectrometers

42
Evaporative Light Scattering Detector
  • 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

43
Varian 380-LC Evaporative Light Scattering
Detector
44
ELS Instrument Concept
45
Light Scattering Detection
  • Response dependent on particle size
  • Mechanism principally reflection/refraction
  • Ideally nebulisation should form uniform droplet
    size

46
Linearity of Response
  • GPC analysis using THF at 1ml/min
  • Lowest column loading 1.0µg on column, or 100µl
    of 0.01mg/ml solution

47
Sensitivity of DRI Versus ELS
Columns 2 x PLgel 5um MIXED-C 300x7.5mm Eluent
THF Flow rate 1.0ml/min Loading 0.1, 20ul
ELS is essentially independent of dn/dc,
improvement in sensitivity will depend on a
number of solute parameters
3
Mp values 1. 7,500,000 2. 841,700 3. 148,000 4.
28,500 5. 2,930
4
2
1
5
48
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

49
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
50
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
51
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
52
Styrene Butadiene Rubber (SBR) Analysis
Columns 2 x PLgel 20um MiniMIX-A
250x4.6mm Eluent THF Flow rate
0.3ml/min Loading 1mg/ml, 100µl
Oil extended SBR General grade SBR
This application illustrates the high sensitivity
of the PL-ELS1000, permitting the polymers to be
analysed at low loadings using narrow bore SEC
columns.
53
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 2 x PLgel 5um 50Å Eluent THF 0.1
diethanolamine
54
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 Mw404,000
Pullulan Mw22,800
55
High Temperature GPC
Columns 2 x PLgel 10um MIXED-B 300x7.5mm Eluent
TCB Flow rate 1.0ml/min Temperature
160C Detectors PL-ELS 1000
MISS OUT SLIDE No lONGER SOLD
NBS 1475 polyethylene
56
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
57
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

58
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

59
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

60
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

61
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.
62
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

63
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.

64
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

65
Example - The PL-GPC 50 Plus
  • Integrated system for GPC analysis up to 50C
  • Standard instrument fitted with a DRI detector
  • Can accommodate other detector options
  • Fully software controlled

66
Reproducibility on the PL-GPC 50 Plus
Raw data chromatograms
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
Molecular weight distributions
67
Example - PL-GPC220 Integrated GPC
68
Components of the PL-GPC220 Integrated GPC System
PUMP AND DEGASSER
69
HTGPC Analysis of Crystalline Polymers
Additional system requirements for these
difficult applications
  • Adequate temperature capability (30-220C)
  • Consistency of solvent delivery in continuous
    use
  • High temperature autosampler/injection system
  • DRI performance (sensitivity and stability)

70
PL-GPC220 Autosampler
  • 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

71
PL-GPC220 DRI Sensitivity
Columns 3 x PLgel 10um MIXED-B 300x7.5mm Flow
rate 1.0 ml/min Injection 200ul Test
probes Polystyrene standards
Many polymer/solvent combinations in HTGPC offer
very low dn/dc so DRI sensitivity is an important
issue
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