Foundation GPC Part 3 – Gel Permeation Chromatography

Instrumentation

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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

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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.

Components of a GPC System

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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

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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

Effect of Flow Rate on Resolution

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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

Flow Rate and Efficiency

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Eluent : THF

Columns : PLgel 100Å

Test probe : ODCB

Optimum flow rate for small molecule separations is around 1.0ml/min

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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

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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.

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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

Pump Issues - Variable Retention Time

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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

Increasing Retention Times

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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

Pump Pressure Variations

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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

High Pressure

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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

Pressure Fluctuation

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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

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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

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Effect of Concentration on Peak Shape and Resolution

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

0.05% 0.10%

0.15% 0.20%

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Effect of Injector Loop Size on Resolution

Column : PLgel 3µm MIXED-E

300x7.5mm

Eluent : THF

Flow rate : 1.0 ml/min

Sample : Epikote 1001

epoxy resin

20µl loop

200µl loop

Injection loop is a major contribution to system dead volume, use reduced injection volume and increase concentration to maintain sensitivity

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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

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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

Effect of Particle Size on Resolution

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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

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Effect of Length on Resolution

Eluent: THF (stabilized)FlowRate: 1.0ml/minDetector: UVSamples : PL EasiCal PS-1 calibrants, two injections

1 x PLgel Column

Mp valuesInjection 1 Injection 21. 7500000 6. 25600002. 841700 7. 3200003. 148000 8. 595004. 28500 9. 108505. 293010. 580

3 x PLgel Column

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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 D

Where D = slope of calibrationSigma = peak variance (related

to peak width)

Resolution in GPC

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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

Poor Column Lifetime

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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

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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

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Effect of Temperature on Separations in Polar Solvents

Column : PLgel 5um MIXED-C300x7.5mm

Eluent : DMFFlow rate : 1.0ml/min

PEO/PEG standards

990,000 252,000

86,000 18,000

4,800 200

Increased temperature :

Reduced operating pressure

Improved resolution, particularly at high MW

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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

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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

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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

Leaks in a GPC System

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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

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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

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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: PLgel 5µm 104Å+ 500ÅFlow Rate: 1.0ml/minDetector: DRI

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Linear Hydrocarbons

Peak HC MW RI

1 C12H26 170 1.4216

2 C16H34 226 1.4340

3 C22H46 310

4 C32H66 450 1.4550

Columns 2 x PLgel 5um 50Å 300x7.5mmEluent THFFlow rate 1.0ml/minSolutes Linear hydrocarbons, all prepared at equal concentration

Low MW dn/dc Effects

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2

3

4

Refractive index of a homologous series changes rapidly below a MW of around 1000.

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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

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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

Baseline Noise and Drift

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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

Baseline Drift at Start of Operation

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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

Ghost and Negative Peaks

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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.

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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.

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The PL-HTGPC/FTIR Interface

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

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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

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Varian 380-LC Evaporative Light Scattering Detector

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ELS Instrument Concept

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Light Scattering Detection

Response dependent on particle size

Mechanism principally reflection/refraction

Ideally nebulisation should form uniform droplet size

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Linearity of Response

GPC analysis using THF at 1ml/min

Lowest column loading 1.0µg on column, or 100µl of 0.01mg/ml solution

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3

4

5

Columns 2 x PLgel 5um MIXED-C 300x7.5mm

Eluent THF

Flow rate 1.0ml/min

Loading 0.1%, 20ul

Mp values

1. 7,500,000

2. 841,700

3. 148,000

4. 28,500

5. 2,930

Sensitivity of DRI Versus ELS

ELS is essentially independent of dn/dc, improvement in sensitivity will depend on a number of solute parameters

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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

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DRI

ELS 1000

Polymer Blends in THF, DRI Versus ELS

Samples

Polystyrene

Polydimethylsiloxane

Blend

Columns 2 x PLgel 5um MIXED-C

300x7.5mm

Eluent THF

Loading 0.2%, 20ul

Detectors DRI at 1V FSD

ELS1000 at 10V FSD

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Polymer Blends in Toluene, DRI Versus ELS

Samples

Polystyrene

Polydimethylsiloxane

Blend

Columns 2 x PLgel 5um MIXED-C

300x7.5mm

Eluent Toluene

Loading 0.2%, 20ul

Detectors DRI at 1V FSD

ELS1000 at 10V FSD

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Analysis of Natural Rubber, DRI Versus ELS

Zoom on additive region 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

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Styrene Butadiene Rubber (SBR) Analysis

This application illustrates the high sensitivity of the PL-ELS1000, permitting the polymers to be analysed at low loadings using narrow bore SEC columns.

Columns 2 x PLgel 20um MiniMIX-A 250x4.6mm

Eluent THF

Flow rate 0.3ml/min

Loading 1mg/ml, 100µlOil extended SBR

General grade SBR

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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

Chimasorb 944

Irgafos 168

Irganox 1010

Tinuvin 622

Tinuvin 770

Tinuvin 327

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GPC Using Polar Organic Solvents

Columns PLgel 10um MIXED-B 300x7.5mm

Eluent DMSO

Detectors PL ELS 1000Must use volatile salts as modifiers for polar organic eluents (e.g. ammonium acetate)

Pullulan Mw=404,000

Pullulan Mw=22,800

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Columns 2 x PLgel 10um MIXED-B 300x7.5mm

Eluent TCB

Flow rate 1.0ml/min

Temperature 160°C

Detectors PL-ELS 1000

High Temperature GPC

NBS 1475 polyethylene

MISS OUT SLIDENo lONGER SOLD

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PVP/PVA Copolymer (Kollidon VA64)

Volatile salts must be used with evaporative light scattering detection

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

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Summary of ELS

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

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 :

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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

Need to prepare a fresh solution!

Split Peaks

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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

Peak Tailing

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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

Peak Broadening

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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.

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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

Poor Detector Sensitivity

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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.

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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

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Example - The PL-GPC 50 Plus

Integrated system for GPC analysis up to 50°C

Standard instrument fitted with a DRI detector

Can accommodate other detector options

Fully software controlled

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Raw data chromatograms

Molecular weight distributions

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

Reproducibility on the PL-GPC 50 Plus

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Example - PL-GPC220 Integrated GPC

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Components of the PL-GPC220 Integrated GPC System

PUMP AND DEGASSER

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HTGPC Analysis of Crystalline Polymers

Adequate temperature capability (30-220°C)

Consistency of solvent delivery in continuous use

High temperature autosampler/injection system

DRI performance (sensitivity and stability)

Additional system requirements for these difficult applications

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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

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PL-GPC220 DRI Sensitivity

Columns 3 x PLgel 10um MIXED-B 300x7.5mm

Flow rate 1.0 ml/minInjection 200ulTest probes Polystyrene standards

Many polymer/solvent combinations in HTGPC offer very low dn/dc so DRI sensitivity is an important issue