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/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 viscosityDirectly
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 <0.5% for maximum result quality › Zero overhead autosampler (vs 50µl in GPCmax)› Temperature controlled autosampler. Cooling to protect samples› Temperature controlled autosampler. Cooling to protect samples
from degradation (e.g proteins) or warming to maintain sample solubility (e.g. xylene solubles)
› Sampling from vials or from 96-well plates› Improved efficiency and smaller volume degasser (2ml vs 30 in
GPCmax – quicker solvent changeover/stabilization)› New pump with integral seal washing› Large capacity column oven
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…