Latest developments in multi-detector GPC/SEC · Latest developments in multi-detector GPC/SEC Mark...

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Latest developments in multi-detector GPC/SEC GPC/SEC Mark Pothecary PhD Product Manager GPC Product Manager GPC Malvern Instruments International Symposium on GPC/SEC and Related Techniques - 2015
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Transcript of Latest developments in multi-detector GPC/SEC · Latest developments in multi-detector GPC/SEC Mark...

  • Latest developments in multi-detector GPC/SECGPC/SECMark Pothecary PhDProduct Manager – GPCProduct Manager – GPCMalvern Instruments

    International Symposium on GPC/SEC and Related Techniques - 2015

  • The multi-detection pyramid

    Hydrodynamic radius (Rh)

    Mark-Houwinkparameters

    Branching

    Indirectly calculate:

    Molecular weight (SLS)

    Intrinsic viscosity

    radius (Rh) parameters

    Radius of

    calculate:

    Concentration

    (SLS) viscosityRadius of gyration (Rg)

    Directly calculate:

    Composition

    δ Refractive Index

    UV absorbance

    Light scattering intensity δ viscosity

    Directly measure:

  • Introduction to OMNISEC system

  • OMNISECSeparations moduleOMNISEC RESOLVE

    Sample characterizationOMNISEC REVEAL

  • Evolved integrated multi-detection› RALS/LALS detector measures molecular weight and has the

    sensitivity for small scatterers and is independent of anisotropic scattering for large scatterersscattering for large scatterers

    › Triple detection offers additional measurements of: Hydrodynamic radius Mark-Houwink plotsp

    › Integration benefits Better control of temperature = better chromatography Integrated detectors mean reduced tubing length and reduced band broadening g g g g

    = better results (significantly improved in OMNISEC) Robust detector cells and all in series means better sensitivity

  • Light ScatteringRefractive index

    › Light Scattering signal-to-noise approx. 10 times improvedRefractive index

    TDA

    UV/Vis PDA

    TDA

    RALS/LALS

    OMNISEC

    Intrinsic viscosity

  • Light ScatteringRefractive index

    › Molecular weight quantification at 100 ng!› Polystyrene 105 KDa in THF, 0.01 mg/mL, 10 µL injection

    Refractive index

    UV/Vis PDA

    Parameter 100 ng PS105K

    RALS/LALS

    RV (mL) 8.737Mn (g/mol) 97,750Mw (g/mol) 103,100Mz (g/mol) 110,700(g/ o ) 0, 00Mw/Mn 1.055

    Intrinsic viscosity

  • Viscometer technologyRefractive index

    › The exchangeable bridge can be removed and replaced in just 10 minutes

    Refractive index

    UV/Vis PDAIP+ -

    RALS/LALSDP-+SEC IN OUT

    Intrinsic viscosity

  • ViscometerRefractive index

    › Automated bridge balancing mechanism› As the capillary moves within its sleeve, the resistance changes

    altering the bridge balance

    Refractive index

    altering the bridge balance

    UV/Vis PDA

    RALS/LALS

    Intrinsic viscosity

  • Viscometer technologyRefractive index

    › Automated capillary bridge balancing: Easier installation of a new bridge Prolong the life of an old bridge

    Refractive index

    Prolong the life of an old bridge› The software automatically searches for the zero position UV/Vis PDA

    RALS/LALS

    Viscometer signal

    00

    Intrinsic viscosity

  • Detector module – OMNISEC REVEAL› Redesigned RALS/LALS with significant (10x)

    sensitivity improvements› New viscometer design (self-balancing bridge with› New viscometer design (self-balancing bridge with

    user-exchangeable capillaries)› Improved RI (8x improvement in S/N), including change

    of position in detector moduleof position in detector module› Completely new UV-diode array (full UV/Vis range)› Temperature control of all detectors and inter-detector

    t bi (20 t 65°C)tubing (20 to 65°C)

  • Separation module – OMINSEC RESOLVE› Injection reproducibility

  • OMNISEC system applications

  • Repeatability of 8 overlaid injections of NBS 1478

  • Comparing branched and brominated PS› Overlay of linear, branched and

    brominated polystyrene› The differences in magnitude of

    LinearBranched› The differences in magnitude of

    the structural changes are quite clear

    › Linear to branched is only a

    Brominated

    › Linear to branched is only a small change in the IV of the sample

    › Linear to brominated in this› Linear to brominated in this case is a much more significant change

  • Branching vs bromination› The branched and brominated

    polystyrene samples can be distinguished using the PDAdistinguished using the PDA

  • PLGA 50:50 - 8 repeat injections

    RIRALSLALSIV › At just 2 mg/ml of this 50 kDa PLGA, the RSD for 8 repeat injections is less

    than 2% for Mw, IV, and Rh measurements

  • PLGA Mark-Houwink overlays 50:50, 65:35 and 75:25

  • Example Triple Chromatogram of Polypropylene Glycols

    › Mobile Phase: THF Columns: 2 X T2500 Flow Rate: 1 mL/min› Mobile Phase: THF Columns: 2 X T2500 Flow Rate: 1 mL/min› Temperature: 30C Injections: 100 µL of 6 mg/mL dn/dc: 0.0454

  • Summary of Molecular values of Polypropylene Glycols

    Samples Mw Mn PDI IV dn/dc % Recovery

    PPG-1A 2,806 2,487 1.13 0.062 0.0454 100.1PPG 1A 2,806 2,487 1.13 0.062 0.0454 100.1PPG-1B 2,644 2,061 1.28 0.061 0.0454 101.6

    AVE 2,725 2,274 1.21 0.061 0.0454 100.9

    PPG-2A 2,831 2,691 1.05 0.067 0.0454 99.2G ,83 ,69 05 0 06 0 0 5 99PPG-2B 2,753 2,394 1.15 0.061 0.0454 100.1

    AVE 2,792 2,543 1.10 0.064 0.0454 99.6

    PPG-3A 2,789 2,717 1.03 0.062 0.0454 99.5

    › Mobile Phase: THF Columns: 2 X T2500 Flow Rate: 1 mL/min

    , ,PPG-3B 3,217 2,897 1.11 0.071 0.0454 99.4

    AVE 3,003 2,807 1.07 0.067 0.0454 99.4

    › Mobile Phase: THF Columns: 2 X T2500 Flow Rate: 1 mL/min› Temperature: 30C Injections: 100 µL of 6 mg/mL dn/dc: 0.0454

  • UV absorbing additive› Oligomer resolution and MW measurement over

    the different populations

    › THF, SVB columns, 35°C

  • Hyaluronic Acid – Linear and Crosslinked

    › Small but clear structural differences between the Linear and Cross-linked HA

  • What does multi-detector SEC offer?› Light scattering overcomes the basic flaw in conventional calibration (it

    is a relative technique) and measures absolute molecular weight› The addition of intrinsic viscosity adds extra information about the› The addition of intrinsic viscosity adds extra information about the

    sample structure and size› An advanced system can measure

    Concentration› In short, advanced SEC

    Concentration Molecular weight (distribution) Structure (IV), Mark Houwink parameters & size (Rh & Rg) Dn/dc

    means more and better information about a sample

    › An integrated advanced system has: Temperature control across columns, detectors and inter-detector tubing to

    improve separation and result accuracy

    p

    Reduced band-broadening (dispersion) between detectors for improved accuracy

  • Why is OMNISEC better?› Better light scattering sensitivity

    Accurately and sensitively measure lower molecular weight samples Measure novel polymers with low dn/dc (PLA)p y ( ) Measure lower concentrations and sample volumes of novel polymers and

    proteins

    › Self-balancing viscometer with replaceable bridge› In short power versatility A better balanced viscometer means better sensitivity to small changes More robust viscometer means less down-time and reduced service costs

    › OMNISEC RESOLVE

    › In short, power, versatility and robustness

    Temperature controlled autosampler protects samples in the queue from aggregation

    Zero injection volume overhead reduces sample waste of precious proteins Injections from 96-well microtiter plates, the standard sample format in proteinInjections from 96 well microtiter plates, the standard sample format in protein

    research

  • Thanks to our colleagues back in Houston and to you for listeningfor listening…