combining multi-detector gpc measurements with …...excellent repeatability › only possible to...

Combining multi-detector GPC measurements with UPLC separation

John D Stenson [email protected] Product Technical Specialist – SEC/GPC

Overview › Why multi-detector UPLC?

› Challenges involved in multi-detection UPLC

› Optimisation of OMNISEC REVEAL for UPLC

› Early results

Why are we interested in UPLC?

› Improved resolution › Less sample › Less solvent › Less time

UPLC GPC

Presenter

Presentation Notes

These improvements lead to there own unique challenges.

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

Presenter

Presentation Notes

As we can see from the image on the right, UPLC offers excellent repeatability. In this case we are looking at 100 injections of an expoy resin run in THF. Fingerprinting of results is obvious before we look at the % RSD values obtained. The only draw back of this is that the results produced are relative Mw numbers, so related to the standards used to calibrate the system. If our samples have a very different chemical structure to those of our standards then the results we measure may be very far away from the actual Mw of our sample. If we are interested in know the absolute Mw values of our samples we need to look at adding additional detectors to our system.

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:

Presenter

Presentation Notes

This diagram shows how light scattering and the other detectors all fit together in a multi-detector system. In this way we can see that as well as molecular weight, the detectors combine to give even more information. At the bottom, we have what the detectors are actually measuring – light scattering intensity, a change in refractive index, UV absorbance, or a change in solution viscosity. [CLICK] These measurements allow us to calculate concentration, molecular weight and intrinsic viscosity (molecular density). [CLICK] When these measurements are combined we can also get more information such as hydrodynamic radius and structural parameters called Mark-Houwink parameters (although these are not so important for proteins). From the structural data we can then calculate Branching.

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

Presenter

Presentation Notes

The biggest challenge is dispersion and band broadening of the sample as it flows thorugh the system. As we know in a multi-detection system the detectors are connected in series, so the sample flows from one detector to the next etc. When using multi-detection GPC the detector channels must be aligned with each other. This ensures tat

Malvern GPC/SEC systems

OMNISEC TDAmax

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

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

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

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

Presenter

Presentation Notes

So big improvements already with the OMONISEC system when compared to the TDA. But we still need to take this further to get a UPLC multi detector system. The next section will look at an OMNISEC system with optimised for UPLC.

DATA COMPARISON UPLC TO GPC – EARLY RESULTS

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

PS65K – PS30K – Rv Comparison

› Run volume including permeation peak UPLC 5 mL Analytical GPC 24 mL

Presenter

Presentation Notes

HPLC data run with standard REVEAL set up. Same CC mixture but different concentrations and inj volumes due to being run a different times. Typical run time for RI. If including IV then add a couple of minutes.

PS65K – PS30K UPLC Raw data

› RI detector responses appear similar › 1st detector in REVEAL, last in APC

PS65k – PS30K Multi-Detection results

GPC UPLC

PS6000 – PS2500 Multi-Detection Results GPC UPLC

PS400K – PS170K Multi-Detection Results GPC UPLC

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

Presenter

Presentation Notes

The extreme example when looking for BB corrections.

BSA standard

› Hydrodynamic radius can be calculated for monomer and dimer

Presenter

Presentation Notes

We can clearly see we are able to calculate Mw’s for the Monomer, Dimer, Trimer and the higher Mw aggregate peak. Run through results. In this case it in possible to calculate the Rh for the Monomer and dimer where we have a stronger IV signal but not the higher Mw aggregates.

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

Thanks for listening…

John D Stenson [email protected]

combining multi-detector gpc measurements with …...excellent repeatability › only possible to...

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