Latest developments in multi-detector GPC/SEC

Mark PothecaryProduct Manager – GPC

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:

Introduction to OMNISEC system

OMNISEC

› An OMNISEC system is an out of the box solution for SEC

› Comprising a complete system for mobile phase and sample delivery and separation, and an integrated detector module for detailed molecular characterization

› Two parts OMNISEC RESOLVE OMNISEC REVEAL

OMNISEC

Separations 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 scatterers

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

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

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

Light Scattering

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

UV/Vis PDA

Refractive index

RALS/LALS

Intrinsic viscosity

Parameter 100 ng PS105Retention volume (mL) 8.02Mn (Da) 105,470Mw (Da) 108,180Mz (Da) 110,570PD (Mw/Mn) 1.026

Light Scattering

› Molecular weight quantification at 100 ng!› BSA 66 KDa in PBS 0.05 mg/ml, 2 µL

injection› Accurate calculation of molecular weight

UV/Vis PDA

Refractive index

RALS/LALS

Intrinsic viscosity

RV (mL) 9.083Mn (g/mol) 63,800Mw (g/mol) 65,070Mz (g/mol) 66,290Mw/Mn 1.02

PEO 196 Da by Triple Detection !

› 4 mg/ml, 100 µl injection volume

Viscometer technology

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

UV/Vis PDA

Refractive index

RALS/LALS

Intrinsic viscosity

IP+ -

DP-+

SEC IN OUT

Viscometer technology

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

› The software automatically searches for the zero positionUV/Vis PDA

Refractive index

RALS/LALS

Intrinsic viscosity

Viscometer signal

0

Detector module – OMNISEC REVEAL

› Redesigned RALS/LALS with significant sensitivity improvements

› New viscometer design (self-balancing bridge with user-exchangeable capillaries)

› Improved RI, including change of position in detector module

› Completely new UV-diode array (full UV/Vis range)

› Temperature control of all detectors and inter-detector 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 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 with highly effective

temperature control

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 the structural changes are quite clear

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

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

LinearBranchedBrominated

Branching vs bromination

› The branched and brominated polystyrene samples can be distinguished using the PDA

PLGA 50:50 - 10 repeat injections

RIRALSIV

› At just 2 mg/ml of this 13 KDa PLGA, the RSD for 10 repeat injections is less than 1% for Mw, IV, and Rh measurements

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

Cellulose derivatives

› Different cellulose derivatives can be compared by number and on the Mark-Houwink plot

› These derivatives will have different effect on (e.g.) formulation viscosity

› This will depend on molecular weight, structure/branching and level of derivatisation

Sample Mn (g/mol)

Mw (g/mol)

Mz (g/mol) [η] (dL/g) Rh (nm)

HEC 62,600 223,000 712,000 3.572 21HPC 45,600 69,000 111,000 1.113 10.21HPMC 98,300 306,000 692,000 7.085 29.85HBMC 102,000 362,000 831,000 8.556 33.54

What does advanced 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

sample structure and size› An advanced system can measure

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

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

tubing to improve separation and result accuracy Reduced band-broadening (dispersion) between detectors for

improved accuracy

In short, advanced SEC means more and better information about a sample

Why is OMNISEC better?› Better light scattering sensitivity

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

polymers and proteins› Self-balancing viscometer with replaceable bridge

A better balanced viscometer means better sensitivity to small changes

More robust viscometer means less down-time and reduced service costs

› OMNISEC RESOLVE 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 protein research

In short, power, versatility and robustness

Find out more at: www.malvern.com/omnisec