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…