Latest developments in multi-detector GPC/SEC
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This presentation was part of ISPAC 2015, the International Symposium on Polymer Analysis and Characterization. Mark Pothecary details the latest developments in multi-detector Gel Permeation Chromatography/Size Exclusion Chromatography (GPC/SEC). This includes an overview of the new OMNISEC system including the chromatography module, OMNISEC RESOLVE and the detector module, OMNISEC REVEAL and the software. It discusses the technical advances within the hardware as well as design aspects and considerations that have gone into the design. For more information please visit www.malvern.com/omnisec
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Latest developments in multi-detector GPC/SEC
- 1. Latest developments in multi-detector GPC/SEC Mark Pothecary Product Manager – GPC
- 2. 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:
- 3. Introduction to OMNISEC system
- 4. 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
- 5. OMNISEC Separations module OMNISEC RESOLVE Sample characterization OMNISEC REVEAL
- 6. 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
- 7. 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 100ng PS105 Retention volume (mL) 8.02 Mn (Da) 105,470 Mw(Da) 108,180 Mz (Da) 110,570 PD(Mw/Mn) 1.026
- 8. 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) 9.083 Mn (g/mol) 63,800 Mw (g/mol) 65,070 Mz (g/mol) 66,290 Mw/Mn 1.02
- 9. PEO 196 Da by Triple Detection ! › 4 mg/ml, 100 µl injection volume
- 10. 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
- 11. 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 position UV/Vis PDA Refractive index RALS/LALS Intrinsic viscosity Viscomet er signal 0
- 12. 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)
- 13. 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
- 15. Repeatability of 8 overlaid injections of NBS 1478
- 16. 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 Linear Branched Brominated
- 17. Branching vs bromination › The branched and brominated polystyrene samples can be distinguished using the PDA
- 18. 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
- 19. PLGA Mark-Houwink overlays 50:50, 65:35 and 75:25
- 20. 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 21 HPC 45,600 69,000 111,000 1.113 10.21 HPMC 98,300 306,000 692,000 7.085 29.85 HBMC 102,000 362,000 831,000 8.556 33.54
- 21. 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
- 22. 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
- 23. Find out more at: www.malvern.com/omnisec
Editor's Notes
- Latest developments in multi-detector GPC/SEC
- 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.
- This presentation provides an overview of the OMNISEC system including the chromatography module, OMNISEC RESOLVE and the detector module, OMNISEC REVEAL and the software. It discusses the technical advances within the hardware as well as design aspects and considerations that have gone into the design
- OMNISEC is a complete SEC system comprised of two modules, OMNISEC RESOLVE is the chromatography module, the equivalent of the GPCmax. OMNISEC REVEAL is the integrated detector module, the equivalent of the TDA.
- The two parts of the system are the separations module, which is called OMNISEC RESOLVE and the detector or sample characterisation module, OMNISEC REVEAL.
- OMNISEC REVEAL is based on the same philosophies that make the TDA a very powerful advanced SEC detector module. This includes the RALS/LALS detector for the measurement of molecular weight. We use the RALS detector because it has the sensitivity required to measure low molecular weight polymers and proteins. The LALS detector has the accuracy to measure the molecular weight of large molecules. [we do have MALS now in the SEC-MALS 20 but we still maintain that the RALS/LALS detector is as sensitive and accurate]. The inclusion of multiple detectors brings more information than just the sum of their parts. For instance, light scattering gives molecular weight and viscomster gives intrinisic viscosity ut the combination of them allows you to measure hydrynamic radius; to make structural comparisons using Mark-Houwink plots and to do branching analysis. The combination of two concentration detectors allow copolymer or conjugate analysis. [Branching and copolymer analysis will both be available in a future software release] It also offers the benefits of an integrated system. By including all the detectors in a single compartment it means better temperature control which means better chromatography. Integration also means reduced interdetector tubing and therefore reduced band broadening.
- The improvement in signal-to-noise means that the limit of detection has also been significantly improved. It is now possible to measure accurately as little as 100ng of PS 105k. Of course, the RI sensitivity improvements also contribute to this ability to measure at such low sample loadings
- The improvement in signal-to-noise means that the limit of detection has also been significantly improved. It is now possible to measure accurately as little as 100ng of BSA. Of course, the RI sensitivity improvements also contribute to this ability to measure at such low sample loadings
- Here is that 196 Da PEO in more detail. The RALS peak is small but clear.
- The viscometer in OMNISEC REVEAL includes an exchangeable bridge. [CLICK] All of the capillaries can be removed in a single block and replaced in about 10 minutes.
- As the capillary moves up and down the sleeve, it changes the signal in the viscometer. [CLICK] The baseline here starts negative as the mechanism if fully extended. As the capillary moves up and down, the balance changes. The software searches automatically to find the position closest to zeo. In this way, the viscometer can always balance itself. Viscometer measurements therefore always start from a balanced position.
- In summary, the detector module, OMNISEC REVEAL contains a redesigned RALS/LALS detector with significant sensitivity improvements; a new viscometer with an autobalancing mechanism and a user-exchangeable bridge; an improved RI detector enabling it to be the first detector in sequence and with some signal to noise improvements; a new UV/Vis absorbance detector with a wider wavelength range; and improved temperature control of the detectors from 20 to 65 degrees C. This includes the detectors themselves but also the inter-detector tubing.
- This slide summarizes the features in OMNISEC RESOLVE. Its autosampler’s injection reproducibility is excellent. Its zero overhead autosampler is capable of making injections iwthout any sample waste which is great for precious samples such as novel polymers. The temperature controlled autosampler can cool thermolabile samples such as proteins or maintain sample solubility as in xylene soluble applications. It is capable of making measurements from 96 well plates as well as vials. Its low volume degasser enables fast solvent exchange and rapid detector stabilization. Its pump contains an integrated seal washing mechanism to protect the pump seals from any salt precipitation. The large column oven has room for up to 8 analytical columns or a GE column such as a superdex columnn.
- Here is an overlaid mark-houwink plot of a 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
- PLGA is a polymer most commonly used in drug delivery applications. It is biodegradeable and biocompatible. It’s used in a number of applications like implants and stents, degradeable sutures and drug delivery. In drug delivery applications it is conjugated with some small molecule drug. As it degrades, the drug molecules are released in a timely fashion. Researchers are very interested in controlling the rate of drug release i.e. how much is released over how long a period of time. PLGA is made from two components, poly lactic acid and glycolic acid to form the PLGA. The molecular weight of the polymer and the composition of the two components will both affect the rate of drug release. Here is an overlay of 10 injections of a sample of PLGA which is 50% PLA and 50% glcyolide. We start with the RI CLICK - to the RALS CLICK – to the IV See the excellent overlay and repeatability
- Here is an overlaid Mark-Houwink plot for PLGA samples of different composition. As the glycolic acid content increases, the density of the molecule increases and it moves down the Mark-Houwink plot. The sensitivity of OMNISEC and its repeatability means that these measurements are of excellent quality are possible and these kind of samples can be well measured and distinguished.
- The different celluloses will all have different molecular weight distributions, structures, densities, effects on viscosity etc. etc. etc. The numbers demonstrate this, but again, the Mark-Houwink plot clearly visualises the differences. Here we can see HPC is the most dense, followed by HEC, HPMC, and HBMC. This of course will depend on molecular weight, structure/branching and level or derivatisation so in an uncontrolled environment will be quite unpredictable but using this tool allows users to compare the samples and relate their properties to those of the final products.
- Find out more at www.malvern.com/omnisec