Foundation GPC Part 3 – Gel Permeation Chromatography Instrumentation.

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Foundation GPC Part 3 – Gel Permeation Chromatography Instrumentation
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Transcript of Foundation GPC Part 3 – Gel Permeation Chromatography Instrumentation.

  • Slide 1
  • Foundation GPC Part 3 Gel Permeation Chromatography Instrumentation
  • Slide 2
  • 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
  • Slide 3
  • 3 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
  • Slide 4
  • 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
  • Slide 5
  • 5 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
  • Slide 6
  • 6 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
  • Slide 7
  • 7 Eluent : THF Columns : PLgel 100 Test probe : ODCB Optimum flow rate for small molecule separations is around 1.0ml/min
  • Slide 8
  • 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
  • Slide 9
  • 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.
  • Slide 10
  • 10 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
  • Slide 11
  • 11 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
  • Slide 12
  • 12 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
  • Slide 13
  • 13 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
  • Slide 14
  • 14 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
  • Slide 15
  • 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
  • Slide 16
  • 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
  • Slide 17
  • 17 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. 8,500,0004. 34,500 2. 1,130,0005. 5,100 3. 170,0006. 580 0.05% 0.10% 0.15%0.20%
  • Slide 18
  • 18 Effect of Injector Loop Size on Resolution Column :PLgel 3m MIXED-E 300x7.5mm Eluent : THF Flow rate : 1.0 ml/min Sample : Epikote 1001 epoxy resin 20l loop 200l loop Injection loop is a major contribution to system dead volume, use reduced injection volume and increase concentration to maintain sensitivity
  • Slide 19
  • 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
  • Slide 20
  • 20 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
  • Slide 21
  • 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
  • Slide 22
  • 22 Effect of Length on Resolution Eluent:THF (stabilized) FlowRate:1.0ml/min Detector:UV Samples : PL EasiCal PS-1 calibrants, two injections 1 x PLgel Column Mp values Injection 1Injection 2 1. 75000006. 2560000 2. 8417007. 320000 3. 1480008. 59500 4. 285009. 10850 5. 293010. 580 3 x PLgel Column
  • Slide 23
  • 23 Resolution Rs =2(V 1 -V 2 ) (W 1 +W 2 ) Elution Volumes of peaks 1 and 2 are V 1 and V 2 Peak Widths of peaks 1 and 2 are W 1 and W 2 Specific Resolution per Molecular Weight Decade Rsp =0.25 D Where D = slope of calibration Sigma = peak variance (related to peak width) Resolution in GPC
  • Slide 24
  • 24 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
  • Slide 25
  • 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
  • Slide 26
  • 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
  • Slide 27
  • 27 Effect of Temperature on Separations in Polar Solvents Column : PLgel 5um MIXED-C 300x7.5mm Eluent : DMF Flow rate :1.0ml/min PEO/PEG standards 990,000252,000 86,00018,000 4,800200 Increased temperature : Reduced operating pressure Improved resolution, particularly at high MW
  • Slide 28
  • 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
  • Slide 29
  • 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
  • Slide 30
  • 30 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
  • Slide 31
  • 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
  • Slide 32
  • 32 Differential Refractive Index Detector R = reference cell (usually static) S = sample cell, eluent flowing through Response = K ri * (dn/dc) * concentration Where K is a constant, (dn/dc) is the refractive index increment and C is concentration
  • Slide 33
  • 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 (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 5m 104+ 500 Flow Rate:1.0ml/min Detector:DRI
  • Slide 34
  • 34 Linear Hydrocarbons PeakHCMWRI 1C 12 H 26 1701.4216 2C 16 H 34 2261.4340 3C 22 H 46 310 4C 32 H 66 4501.4550 Columns 2 x PLgel 5um 50 300x7.5mm Eluent THF Flow rate 1.0ml/min Solutes Linear hydrocarbons, all prepared at equal concentration Low MW dn/dc Effects 1 2 3 4 Refractive index of a homologous series changes rapidly below a MW of around 1000.
  • Slide 35
  • 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
  • Slide 36
  • 36 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 0.45m 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
  • Slide 37
  • 37 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
  • Slide 38
  • 38 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
  • Slide 39
  • 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.
  • Slide 40
  • 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.
  • Slide 41
  • 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
  • Slide 42
  • 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
  • Slide 43
  • 43 Varian 380-LC Evaporative Light Scattering Detector
  • Slide 44
  • 44 ELS Instrument Concept
  • Slide 45
  • 45 Light Scattering Detection Response dependent on particle size Mechanism principally reflection/refraction Ideally nebulisation should form uniform droplet size
  • Slide 46
  • 46 Linearity of Response GPC analysis using THF at 1ml/min Lowest column loading 1.0g on column, or 100l of 0.01mg/ml solution
  • Slide 47
  • 47 1 2 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
  • Slide 48
  • 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
  • Slide 49
  • 49 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
  • Slide 50
  • 50 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
  • Slide 51
  • 51 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
  • Slide 52
  • 52 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, 100l Oil extended SBR General grade SBR
  • Slide 53
  • 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. Columns2 x PLgel 5um 50 Eluent THF + 0.1% diethanolamine Chimasorb 944 Irgafos 168 Irganox 1010 Tinuvin 622 Tinuvin 770 Tinuvin 327
  • Slide 54
  • 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 Mw=404,000 Pullulan Mw=22,800
  • Slide 55
  • 55 Columns 2 x PLgel 10um MIXED-B 300x7.5mm Eluent TCB Flow rate 1.0ml/min Temperature 160C Detectors PL-ELS 1000 High Temperature GPC NBS 1475 polyethylene MISS OUT SLIDE No lONGER SOLD
  • Slide 56
  • 56 PVP/PVA Copolymer (Kollidon VA64) Volatile salts must be used with evaporative light scattering detection Columns2 x PL aquagel-OH MIXED 8um 300x7.5mm Eluent 1. 70% 0.2M NaNO 3, 0.01M NaH 2 PO 4, pH7, 30% methanol 2. 70% 0.1M ammonium formate, 30% methanol Flow rate1.0 ml/min Detector1. DRI 2. ELS 1000
  • Slide 57
  • 57 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 :
  • Slide 58
  • 58 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 2m 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
  • Slide 59
  • 59 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
  • Slide 60
  • 60 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
  • Slide 61
  • 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.
  • Slide 62
  • 62 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
  • Slide 63
  • 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.
  • Slide 64
  • 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
  • Slide 65
  • 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
  • Slide 66
  • 66 Raw data chromatograms Molecular weight distributions Inj no.MnMwPeak area 117,28976,818333851 216,98877,434335496 317,24877,514332616 417,25177,052335635 517,34876,520334212 617,48777,728333511 716,89877,578335642 817,45777,288334923 Mean17,30277,241334485 s.d.2206871048 % var1.30.50.3 Reproducibility on the PL-GPC 50 Plus
  • Slide 67
  • 67 Example - PL-GPC220 Integrated GPC
  • Slide 68
  • 68 Components of the PL-GPC220 Integrated GPC System PUMP AND DEGASSER
  • Slide 69
  • 69 HTGPC Analysis of Crystalline Polymers Adequate temperature capability (30-220C) Consistency of solvent delivery in continuous use High temperature autosampler/injection system DRI performance (sensitivity and stability) Additional system requirements for these difficult applications
  • Slide 70
  • 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
  • Slide 71
  • 71 PL-GPC220 DRI Sensitivity Columns3 x PLgel 10um MIXED-B 300x7.5mm Flow rate1.0 ml/min Injection200ul Test probesPolystyrene standards Many polymer/solvent combinations in HTGPC offer very low dn/dc so DRI sensitivity is an important issue