Combining multi-detector GPC measurements with …...Excellent repeatability › Only possible to...
Transcript of Combining multi-detector GPC measurements with …...Excellent repeatability › Only possible to...
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Combining multi-detector GPC measurements with UPLC separation
John D Stenson [email protected] Product Technical Specialist – SEC/GPC
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Overview › Why multi-detector UPLC?
› Challenges involved in multi-detection UPLC
› Optimisation of OMNISEC REVEAL for UPLC
› Early results
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Why are we interested in UPLC?
› Improved resolution › Less sample › Less solvent › Less time
UPLC GPC
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Why are we interested in UPLC? › Excellent repeatability
› Only possible to obtain
relative Mw of samples
› Additional detectors will allow the calculation of Absolute Mw, size and structure
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The multi-detection pyramid
δ Refractive Index
UV absorbance
Light scattering intensity δ viscosity
Concentration
Molecular weight (SLS)
Intrinsic viscosity
Hydrodynamic radius (Rh)
Mark-Houwink parameters
Branching
Composition
Radius of gyration (Rg)
Directly measure:
Directly calculate:
Indirectly calculate:
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Challenges involved with Multi-detection UPLC › Dispersion and band-broadening the biggest challenge
Detectors connected in series, signals must be aligned
Cell entry/exit Cell volume and shape Any other dead volumes
• Connectors and bulkheads • Filters
› These affects can be corrected for using algorithms for in GPC but due to the low volumes can be a problem in UPLC
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Malvern GPC/SEC systems
OMNISEC TDAmax
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Analytical GPC - TDA › In analytical GPC (columns = 8
x 300 mm), the issue is small
› Even a narrow polymer peak is likely to be 1 ml wide
› Dispersion contribution is small and easily corrected between first and fourth detectors (and others in between)
Raw data
BB corrected
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Hardware improvements – OMNISEC REVEAL › Move the RI › Reduce other inter-detector tubing volumes › All detectors are now closer together › OMNISEC REVEAL Optimised for UPLC
Ultraviolet Refractive index RALS/LALS Intrinsic viscosity
OMNISEC
Flow path
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Hardware improvements › Analytical GPC columns
› Raw data comparison
No band broadening corrections
› Dispersion between first and
fourth detectors (worst case) visibly improved
› OMNISEC includes: Reduced inter-detector tubing volume Different detector order (RI first)
TDA
Standard OMNISEC
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Hardware improvements › UPLC columns
› OMNISEC includes:
Reduced inter-detector tubing volume Different detector order (RI first)
› Dispersion between first and
fourth detectors (worst case) visibly improved
TDA
Optimised OMNISEC
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DATA COMPARISON UPLC TO GPC – EARLY RESULTS
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Polystyrene Standards › Series of standard mixes:
PS6000 – PS2500 PS65K – PS30K PS400k – 170K
› GPC mode LT4000L
• Two 8x300mm columns • Exclusion limit ~400K PS
› UPLC mode Waters APC XT columns – 450Å & 125Å
• Two 4.6x150mm columns • Exclusion limit ~400K PS
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PS65K – PS30K – Rv Comparison
› Run volume including permeation peak UPLC 5 mL Analytical GPC 24 mL
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PS65K – PS30K UPLC Raw data
› RI detector responses appear similar › 1st detector in REVEAL, last in APC
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PS65k – PS30K Multi-Detection results
GPC UPLC
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PS6000 – PS2500 Multi-Detection Results GPC UPLC
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PS400K – PS170K Multi-Detection Results GPC UPLC
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BSA standard– UPLC raw data
› Some resolution lost between the monomer and dimer.
› 1st detector in REVEAL, last in APC
› Waters Protein BEH column – 200Å
• One 4.6x150mm columns • Exclusion limit ~450K Protein
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BSA standard
› Hydrodynamic radius can be calculated for monomer and dimer
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Summary › Multi-detection UPLC creates unique band broadening issues
› OMNISEC REVEAL optimised for UPLC can eliminate many of
these issues
› Early results show good multi-detection results for polymers and proteins