Macromolecule and Nanoparticle Characterization

Geofrey Wyatt, President at Wyatt Technology talks to AZoM about macromolecule and nanoparticle characterization.

How has Wyatt become one of the world’s leading providers of solutions for macromolecular and nanoparticle characterization tools?

Our customers come first, and the corollary to that: the customer is always right. Customers are our passion and the entire reason we exist and thrive at Wyatt Technology. In order to become a leader, you have to have a vision that others share. We’re fortunate to be able to share a simple one: that we are here to delight the customer, by whatever means necessary.

Do we always succeed? I’m afraid not, but we keep trying again, and again, and again. From the time of our company’s start in 1982 to the present, we’ve insisted upon the highest technological pedigree from our employees as well as our Scientific Advisory Board, which has on it one member of the National Academy of Sciences and another scientist who became a Nobel Laureate.

If our science is impeccable from the top down, great instruments are going to be built. And our track record bears this out.

We’ve got instruments in more than fifty countries, we’re still family-owned and operated, and we’re the world’s leading manufacturer of instruments for absolute macro-molecular characterization. We are the only company in the world focused exclusively on such systems, their design, and their application.

What are the key theoretical principles of your products?

Without getting too specific, the underlying theory of many of our products involves the scattering of light from dilute solutions of molecules or nanoparticles.

By observing the intensity of light scattered from a sample as a function of angle, our instruments can determine the molecular weights, sizes and distributions of a wide range of molecules.

Whether you’re looking at the aggregation phenomena of proteins, or the massive molar masses of starch molecules, or the random-coiled brilliance of ophthalmic grade hyaluronic acid, or just the scattering properties of quantum dots, the chances are very good that we have an instrument ideally-suited to it.

The entire product line is focused on direct measurement of the primary physical parameters of macromolecules and nanoparticles in solution: molar mass, size, charge and interactions.

The majority of the instruments employ optical technology, especially light scattering in one form or another, affording a suite of non-destructive, solution-based, first-principles techniques rooted firmly in rigorous scientific principles. These main principles are: multi-angle, static light scattering (MALS) determines molar mass, M_w, and size, r.m.s. radius r_g; dynamic light scattering (DLS) measures the diffusion constant, which relates to another type of size - hydrodynamic radius, r_h; and phase-analysis light scattering (PALS) measures electrophoretic mobility which, in combination with DLS, determines net molecular charge.

Label-free, immobilization-free interaction analysis is a straightforward derivative of MALS: the binding affinity, K_D, and absolute molecular stoichiometry of complexes in dynamic equilibrium are determined via the composition/concentration dependence of molar mass (CG-MALS).

While CG-MALS affords a strict thermodynamic interpretation of data, some users prefer the estimation of interaction parameters via our high-throughput, low-volume DLS microwell plate reader (CG-DLS).

Customers choose these techniques because they are not willing to settle for relative measurements and dubious calibrations. And they choose Wyatt instruments because they will not settle for anything but the best quality and support.

Earlier this year, you announced the availability of the Optilab UT-rEX (UHPLC refractometer with EXtended range), for use with UHPLC systems using columns packed with small beads. How does this technology work?

The Optilab, which is the first RI detector of its kind, specifically designed for use with UHPLC systems uses a combination of miniaturized components and cutting-edge semiconductor photodiode technology as well as proprietary computer algorithms in order to make measurements—with very little band-broadening—of UHPLC samples.

Until now you could use UHPLC with a UV detector, an evaporative light scattering detector (no relation to our own light scattering instruments), or mass spectrometry. But there was never a non-destructive universal detector that didn’t depend on UV chromophores that had been available. Now there is.

What are the main benefits of your technology?

There are numerous benefits to the Optilab UT-rEX including the instrument being compatible with all UHPLC systems and the ability to be operated below ambient temperatures as easily as above ambient.

The temperature control can be programmed down to 4°C or as high as 50°C. Also, due to the extremely small volumes of UHPLC, the dead volume for the instrument is <1.5µl as compared to 10-20µl for other RI detectors.

As a result, the UT-rEX offers superior resolution. In addition to the instrument having no range or gain settings, the full range of instrument detection is always present and the full sensitivity exists over the entire range.

Optilab UT-rEX Refractive Index Detector by Wyatt.

How does your technology compare and contrast to competing products in the market?

As I mentioned before, the sun never sets on the Wyatt’s instruments because they may be found in more than fifty countries.

The longevity of the people who come to work for us is legendary which means that we create relationships with our customers that last decades. We take our business personally, and I mean that both metaphorically and literally.

We’re still family-owned and operated, we have no long- or short-term debt, no outside investors, and our destiny is in our own hands. Because we’re relatively small, compared to the multi-billion dollar conglomerates, we have people who can identify new technologies and bring them to bear in our instruments in relatively short periods of time.

We’re using such sophisticated solid state electronics and computers in our instruments that they make other products in the market look positively out-dated.

Wyatt’s Calypso system was used in a recent study to determine the binding affinity and stoichiometry of unknown proteins. What were the main findings of this study and did this study help highlight any benefits or weaknesses of this solution compared to other solution-based measurements?

Yes that’s right. The conclusions of the 2012 Molecular Interactions Research Group (MIRG) study were revealed at the Association of Biomolecular Research Facilities (ABRF) annual meeting in March.

In this study, a pair of unknown proteins, prepared and characterized by the ABRF, was sent out to multiple labs that volunteered to measure binding affinity and stoichiometry with their instruments. In a single afternoon consisting of 2 CG-MALS runs, the Wyatt Calypso system correctly determined that Protein Y (26.3kDa) contains two binding sites for Protein X (11.9kDa).

The first X binds with KD of 10nM and the second X binds with 14µM. This analysis agreed perfectly with extensive analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC) measurements performed by ABRF prior to distributing the samples.

CG-MALS was the only solution-based technique utilized among the participating labs. Other participants used surface plasmon resonance (SPR) instruments which require immobilization of one of the binding partners.

Interestingly, the affinities determined by SPR were significantly weaker than those found by any of the solution-based measurements (AUC, ITC, CG-MALS), possibly indicating that immobilization modifies this interaction.

Calypso II Instrument by Wyatt for the characterization of macromolecular interactions. Image courtesy of Wyatt.

What are the main challenges ahead for your products?

The biggest challenges lie in continuing to popularize our technologies and the benefits they bring to the laboratory.

As a small voice among the screams of the industry giants, we sometimes need to remember that a whisper will get more attention than a message that is yelled. But our Bibliography—the collection of peer-reviewed publications in which our equipment has been used—is now more than 8,500 articles strong.

When scientists want to see how certain measurements can best be accomplished, a literature search will turn up scores of papers every time. We need to continue to encourage our customers to publish prolifically.

Since we don’t rest on our laurels, we’re constantly pushing to make our existing instruments obsolete. Technology doesn’t stand still and neither do we.

There are always new detectors, computers, software, and materials that can be incorporated into our products to improve them. Andy Grove, the former Chairman of Intel said “Only the paranoid survive.” We’re always glancing in the rear-view mirror just to make sure no one is gaining on us.

How will your solutions advance the existing areas of application?

Scientists characterizing novel macromolecules and nanoparticles need pioneering, innovative and cutting-edge technological solutions to meet their demanding expectations. But they must also be sure that they can rely on their analytical instruments to give them the right answers.

Our products and software are designed by colleagues – expert scientists who appreciate the value of thoroughly validated measurements without fudges, so that our customers can proceed confidently in their own fields of expertise.

What’s more, we go beyond just technological innovation – we provide a powerful suite of instruments that work together to attack analytical problems from multiple angles. For example, in protein formulation you need to understand both propensity for aggregation – which we do with CG-MALS and CG-DLS, as well as the final aggregation state – for which we use MALS coupled to size exclusion chromatography or field-flow fractionation. Our instruments also work together as orthogonal methods to validate conclusions drawn from any one technique.

Where can we find further information?

To learn more about Wyatt’s innovative product range and their capabilities you can visit our website or our Twitter page @WyattTechnology.

About Geofrey K Wyatt

Mr. Wyatt graduated from Yale University. At Yale, he was the Editor-in-Chief of an on-campus journal, captain of his college’s soccer team, and full partner in an on-going entrepreneurial venture for three years.

He joined Wyatt Technology in 1983 as its second full-time employee. He has been with the company ever since, with the exception of two years during which he received his Master's degree from the Harvard Business School.

Geof is responsible for marketing and its coordination with sales. His extensive travel puts him in close contact with Wyatt’s direct and dealer networks overseas. He also works closely with the Company’s research scientists and the ways in which they interact with Wyatt’s manufacturing.