Past Contributions and Future Visions

Photo credit: Stockbyte/Getty Images

A popular feature of The Chromatographic Society’s Golden Jubilee publication was an article by Waters describing their heritage and setting out their contributions to the development of chromatography. In this special feature, some of the companies who are regular exhibitors at Society events answer questions on the current and future status of chromatography.

Agilent Technologies

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

The developments in capillary GC and HPLC have seen some of the most important developments. Reference could be made to the development of the particle technology, with initially fully porous material and then the development of superficially porous materials for HPLC. However, the biggest developments really have been the coupling of separation technology with detection technology - in particular the coupling of mass spectrometry. Mass spectrometry coupled to separation devices has revolutionized many industries in terms of the detection limits that have been achieved, and also for the identification of compounds, particularly in the field of protein analysis.

What has been your company’s highest impact contribution to chromatography over the past 60 years? Why you think the development had such a high impact?

Agilent has been market leaders in both HPLC and also GC and so choosing between these is difficult. The key to the success has very much been based on state-of‑the‑art instrumentation that keeps on performing, with industry standards such as the Agilent 1200 series HPLC Systems and the Agilent 7890 GC system.

The biggest impact that this technology has had is in making the market more accessible to a wider range of scientists. The initial separation scientist was a highly trained academic who had the ability to do front-line maintenance, column manufacture, and complex data interpretation. The greater use of computer technology has vastly simplified the interface with the instrumentation and the advancements in the robustness of the hardware have ensured that the accessibility of the technology has reached a much wider audience. This does present challenges in ensuring that the technology is robust enough for customers.

How does your company see the current state-of-the-art of chromatography and, in this context, what are your company doing to address unmet needs?

Agilent prides itself on understanding what the customer really wants, and delivering solutions to support this. Customers have real samples to analyze and this can be forgotten in the drive to provide new exciting solutions. The sample types that are being analyzed are becoming more complex though, and this will drive the separation technology to greater separation power. Improving the separation through brute force in HPLC, by simply changing the particle size, has seen improvements in the separation performance for customers, however, this is not a step change and certainly with the developments of biopharmaceuticals and the incredibly complicated mixtures comprising many tens of thousands of individual components, the world of separation science does need to see a step change.

One of the approaches that is being investigated by Agilent is the use of multidimensional chromatography, an approach that can provide resolutions that will far exceed those of more conventional technology. The academic world is leading the way here, but they are being supported by manufacturers such as Agilent, who are keen to better understand the advantages of this approach to improving the analysis of complicated sample sets. The application of this technology to real-world analysis and in particular the application to complicated protein samples has a very exciting future. In order to get the best of this technology, it is important to have identified the types of stationary phases that need to be employed to drive the best separation and the technology required to support this.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look?

The future of chromatography is very difficult to predict, although if the last 10 years are anything to go by there have not been so many significant changes. The biggest users of chromatography are still the pharmaceutical industry, however, the focus is changing, moving from laboratories based solely in the West to a staggering growth in Asia Pacific countries. The growth of the biopharmaceutical industry also has the potential to change the types of chromatography that are being used. I think for certain there will be improvements in the sensitivity of detectors, and also a reduction in the footprint. I think in terms of the chromatography, the more complex samples that customers are needing to separate will result in more advanced forms of chromatography becoming more commonplace, and the use of 2D LC will start to become mainstream.

The world of GC has seen developments and incremental changes will continue to happen over the coming years. Looking back over the previous 10 years, it is a well-established industry with a large degree of inertia to change, driven primarily by the regulators of the industries where the technology is employed. Certainly the developments in mass spectrometry will have an effect on the capabilities of gas chromatography.

There is other technology that catches the headlines such as SFC and CE, however, these technologies, although they have their niche, have never really been employed within the mainstream analytical laboratories as front-line separation instruments. Inertia has driven this to a certain extent, but the technology in both areas has yet to demonstrate a quantum leap in performance over conventional liquid and gas chromatography.

- Tony Edge, R&D Manager, Agilent Technologies

Anatune

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

Anatune has been largely involved in GC since its formation and the most important developments here are:   the air-bath oven, a genius bit of design that enables different heated zones to be linked together without cold-spots; the fused silica capillary column, which made high-resolution GC a practical proposition; and the low-cost bench-top, quadrupole MS detector - How did we ever manage without it?

What has been your company’s highest impact contribution to chromatography over the past 60 years? Why you think the development had such a high impact?

Anatune’s existence flowed from my belief that insufficient attention was being given to sample preparation and introduction for GC–MS. This was (and still is) the area where many of the errors and much of the cost associated with GC analysis resides. Anatune, together with some other like-minded small companies around the world, has made the automation of sample preparation and introduction a practical proposition; lowering costs and improving data quality as a consequence.

How does your company see the current state-of-the-art of chromatography and, in this context, what are your company doing to address unmet needs?

When compared to other technologies, the truth is that analytical chemistry is slow to change. The drive for better quality data is good, but it does mean that nowadays, the costs of implementing innovations are considerable. As a result, for established applications there is significant resistance to change unless there are compelling economic reasons to move away from the status quo. This is an observation, not a criticism. For Anatune, 20 years on from when we started, our mission to make the automation of GC sample preparation and injection the norm is still work in progress.

We pay attention to emerging trends and, currently, issues broadly related to health, safety, and environmental considerations are a significant source of the new applications that we come across;  with these emerging applications there is much more scope for innovation.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look?

As we discussed earlier, there is a lot of inertia built into separation science - so in 10 years time, I think for established applications, things will look much the same as they do now - instrument (especially mass spectrometer) performance will be better and this will make the automation easier, but it will be evolution rather than revolution.

For emerging applications things will be different. GC–MS will still be a gold-standard reference technique, but we see more and more applications that require a much greater density of data than GC (or LC) can currently deliver. Whichever way you look at these applications, the fundamental problem is that chromatography is slow - it takes time to generate theoretical plates. Right now, this is chromatography’s biggest weakness. At this point I don’t see where the improvements needed are going to come from; but you never know - there are a lot of clever people in this business.

Anatune has always been a dyed‑in-the wool GC company, and for the first time we have to use non‑chromatographic technologies (such as selected-ion flow-tube [SIFT]-MS) to give us the speed of analysis some customers are demanding.

- Ray Perkins, Managing Director, Anatune

Crawford Scientific

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

Crawford Scientific has extensive expertise in column technology and applications, and so our focus on the important innovations naturally falls with the column technology, which includes the development of spherical silica with low surface activity post-bonding and high mechanical strength. These have been used for a host of applications including the plethora of reversed‑phase phases that are available as well as chiral and mixed mode ligands. Latterly of course, high efficiency phases have been produced using sub-2-µm particles as well as being adapted to produce core–shell morphologies that lend higher efficiency without the large increases in back pressure.

In gas chromatography (GC), the development of coated capillary columns was revolutionary and the improvements in efficiency and peak capacity propelled the technique into widespread use globally.

The development of polymeric substrates for liquid phase separations heralded techniques such as ion chromatography (IC) and size-exclusion chromatography (SEC), which are so useful in industrial applications.

Important developments in instrumentation include the development of (somewhat) affordable high resolution and high mass accuracy detectors for HPLC and GC and of course the robust interfacing of HPLC with mass spectrometric detection via the atmospheric pressure interface (API) in the early 1980s was worthy of the Nobel Prize, which the technology earned. Multistage mass spectrometry brought huge advancements in sensitivity and the introduction of the computing data system (CDS) combined with the automatic sampler delivered massive efficiency savings in data acquisition and reporting.

What has been your company’s highest impact contribution to chromatography over the past 60 years? Why you think the development had such a high impact?

Our biggest impact has been our ability to remain independent, and to offer our clients the best products and services to enable their science. We have assembled a team of very committed sales and technical staff, who work with a vast range of resources to find the right products and applications to help solve our clients’ problems and enable them to do better science. We can then help our clients to implement these solutions through a range of consultancy services.

We are small enough to remain very flexible and responsive to our customers’ needs and have worked hard to maintain our independence whilst continuing to work with the world’s leading suppliers who trust us to deliver a best-in-class service on their behalf.

We have served analytical science for 31 years and in those years the company has built a huge amount of knowledge across the whole spectrum of analytical techniques and application areas. It is this experience, combined with the best columns and consumables available, which we bring to bear when supplying products and services to our clients. In recent years we have begun to build our specialist analytical laboratory services with the acquisition of Hall Analytical Laboratories in Manchester, UK, which allows us to support our clients’ requirements within our own facilities as well as at the client site.

We are a small, independent, chromatography products and services provider based in the United Kingdom, however, through the vision of our Managing Director, Sam Crawford, we have been able to make a global impact in learning and development within analytical science. We have been providing classroom and on-site training in analytical science for 25 years, however, our global presence has grown through the provision of our on-line professional development website CHROMacademy (www.chromacademy.com). This has been in development for over 15 years, and in the early days, on-line and computer-based learning were not as well accepted as they are now. However, the continuation of this programme was never in question and today we are proud to have many tens of thousands of members worldwide, who all receive much‑needed training and development from the learning resources within CHROMacademy. This global presence was in no small part made possible through our partnership with LCGC magazine, who helped us to market and sell the product on a worldwide basis.

How does your company see the current state-of-the-art of chromatography and, in this context, what is your company doing to address unmet needs?

We continue to work with our customers to provide knowledge and expertise.

As any science matures, the spirit of innovation and necessity of invention decline in favour of higher throughput and generic ways of working. Of course this is not to say that we lack high quality, innovative, and inquisitive people - absolutely not. Our role is to ensure that our clients have the right products to enable them to do the best science they can, and where they need some assistance to really push the envelope using new products or applications, then our large technical team is able to bring their own wide-ranging experience and expertise into play.

Many challenges still remain in the industrial application of analytical science:

The growth of biopharmaceutics has seen rapid change in the way we characterize and measure these substances. We continue to work with clients to improve their capabilities for understanding entities such as monoclonal antibodies and other protein-based therapeutic agents.

We will continue to assist clients to take advantage of the latest column technologies to improve the quality and timeliness of their data and to meet evolving regulatory requirements.

Our experts at Hall Analytical Laboratories are specialists in mass spectrometry and we continue to work with clients to provide advanced data and help them to understand the information available from spectrometric techniques.

We will also continue to research into new and effective ways of delivering the right information at the right time via the right platform to our clients globally.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look?

For the most part, chromatography relies on chemistry to provide individual components for detection. Whilst this remains the case, a “column” of some sorts will always be necessary, no matter what form it takes (miniature, narrow, encased, etched onto a chip, etc.). So, we will continue to support separation science through the provision of the best chemistries to enable our clients to perform the separations they need.

This being said, we can see systems becoming much more integrated, with lower dead volumes and a more “plug and play” principle as we continue to see the industrialization of instrumentation. Combined with improvements in the specificity of detector systems, this will lead to lower detection limits and improved sample throughput.

We believe that mass spectrometric and spectroscopic techniques will continue to flourish and provide increasingly less ambiguous data through improved accuracy and sensitivity. If we combine these advances with improved data manipulation techniques, we believe that the ability to elucidate even the most complex samples will become increasingly simple.

These improvements in instrumentation and software will mean that the operator becomes increasingly less likely to intervene between loading up the autosampler and reporting the result, which of course takes us further away from the scientific processes that occur within the instrument. We will continue to develop services and resources that enable analytical scientists to understand the principles which underpin instrument operation, software design, and separation science in order to better understand what to do when things go wrong, to spot when insidious problems are occurring, and to develop ever better methods to serve their businesses.

- Anthony Taylor, Technical Director, Crawford Scientific Limited

Shimadzu

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

Firstly, the realization of benchtop instrumentation for routine laboratory use was the starting point for current chromatographs and forced a lot of related developments.

Secondly, improvements in the manufacturing and synthesis of stationary phases, for example, from 10–20 µm and larger particles down to sub-2-µm particles in LC and the fused silica capillary columns for GC contributed a lot to the acceptance and spread of chromatographic techniques.

Last but not least, big improvements on the detection side extended the application range and offered new chances to use such systems: photo diode array detectors in LC, a variety of LC–MS and
GC–MS detectors, and a breakthrough in ionization techniques and practical implementation, especially for LC–MS. For many years, GC–MS was the “gold standard” in terms of high-sensitivity systems, whereas nowadays
LC–MS and GC–MS are seen more as complementary techniques. Of course, each laboratory and each analytical task has its own preferences. A special instrument to highlight in the field of detectors is certainly the Orbitrap‑MS.

Major impacts on the market were the development of the capillary GC columns and the release of ultrahigh‑pressure liquid chromatography (UHPLC) systems (>100 MPa). Both issues underlined the necessity of renewing the complete system design by the manufacturers and forced users to reconsider existing “standard” methods in terms of analytical time, sensitivity, and suitability for state‑of‑the-art analytical instruments.

As well as the developments in chromatography itself, thanks to the development in PC technology and modern user interfaces for dedicated software, such systems have become more suitable for the operators and surely supported the wide spread of these techniques in modern laboratories.

What has been your company’s highest impact contribution to chromatography over the past 60 years?

Instrumentation wise, Shimadzu started with GC 60 years ago and we have produced LC instruments for 40 years; we are proud of the release of the photo diode array detector in 1982 as one of the first suppliers. Many customers remember our integrators, which offered an easier and more reliable processing of obtained data.

As a manufacturer of chromatographs, our major impact was to offer instrumentation that makes the most of the latest developments in separation science. It was always our intention to make these instruments more reliable and applicable for routine use.

Supporting special developments, such as comprehensive LC×LC and GC×GC, in terms of hardware and software is just one example where we see our role having a big impact on chromatography.

Recently, Shimadzu introduced a supercritical fluid extraction/supercritical fluid chromatography (SFE/SFC) system. We see this as an interesting combination of sample pretreatment and separation. Even without the use of toxic organic solvents during the extraction process, difficult and time-consuming extractions can be performed faster and more efficiently.

How does your company see the current state-of-the-art of chromatography and, in this context, what is the company doing to address unmet needs?

State-of-the-art chromatography is very much driven by developments in mass spectrometry. Standard chromatography matters, such as stationary phase development and technical aspects, seem to be less important.

Users in many application areas seem to be bound to existing methods and techniques, which will probably remain the case for quite a while, even though this was predicted to change more quickly. This also means that current developments are focused on just a small group of users - which may be growing - while a larger group of analysts in routine laboratories gain less from the advantages of those developments. For example, UHPLC was introduced years ago, but we still see a need to optimize the instrumentation in terms of robustness and system design. We also see that current instrumentation has to be further improved to meet all requirements for ease of use, robustness, and flexibility. Improvements in current instrument control and data processing, and if needed, on the concept of the software, for example, would be necessary to enable truly “intuitive” operation for software users.

From an end-user’s perspective, interchangeability of instruments and data becomes more and more important. Instrument control by another vendor’s software and data compatibility are also important issues.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look like?

Following the trends towards ease of use, faster separation, and high(er) sensitivity detection, we see several aspects and challenges.

We will see advances in miniaturization of the system’s hardware in parallel with developments in using reduced flow rates and faster ways to achieve separations, including shorter columns, smaller particles, new stationary phases, and coated chips. Multidimensional systems will become easy to use systems for routine analysis. Sample preparation will be integrated into the analytical process.

Right now standard LC detectors are still UV-based (classic UV and PDA) and in some areas still based on refractive index detection. In the future, MS techniques will definitely play a more important role and new MS detectors may take over business from the “standard” detectors, UV and PDA.

There will also be an increase in ready-to-use systems for specific analytical tasks. The appearance, size of such systems, software control, and operation will be different from current ones. We can increasingly expect MS as standard detection in such systems, either as the only one, or as complementary technique to highly specific detectors.

At the same time, established methods and approaches from today will remain, which means that compatibility in terms of instruments and methods will play an important role and force vendors to consider instrumentation compatible to the ones we offer today.

- Björn-Thoralf Erxleben, Senior Manager, Marketing Europe, Shimadzu

Thermo Fisher Scientific

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

The developments in chromatography over the past 60 years have been immense. We have gone from primitive paper chromatography, which was manual, time-consuming, and only a visually qualitative technique to fully quantitated, automated, high-resolution, high-throughput chromatography in several forms. The basic principles of paper chromatography have gone on to spawn a range of chromatography techniques, each of which has seen huge developments over the years.

The development of UHPLC is an example of where gradual improvements in chromatography both created and then overcame limitations. Column chemistries have also advanced. The high resolution form of HPLC came first with an improvement in the column chemistry, only to be limited by the dispersion inherent in traditional HPLC systems. The low-dispersion UHPLC systems available now are essential to see the benefits of high‑resolution chemistries.

In line with very narrow peak widths from gas chromatography (GC) and UHPLC, the detector response rates had to be fast enough to collect enough data points over a peak with a much decreased transit time in the detector. Electronics are now fast enough to produce data collection rates of up to 200 Hz. The ability to analyze thousands of samples each day produced the need for the automated high-throughput data handling workstations we now see in laboratories.

Ion chromatography (IC) has also improved. The single biggest advance in IC was the idea of using a suppressor to serve two functions: the first to remove the eluent by a neutralization reaction to limit background and reduce noise, and the second to convert analytes into a more conductive form. This combination increases the sensitivity of IC, and today, all suppressors are based on this fundamental concept.

There have also been advances in GC; specifically the development of fused silica capillaries in the late 1970s dramatically improved the performance and efficiency of GC. 

The shift from single workstations to enterprise deployments has simplified the chromatography workflow and management of data.

What has been your company’s highest impact contribution to chromatography over the past 60 years?

Thermo Fisher has made a number of contributions over the past 60 years to the different chromatography techniques.

In HPLC, there has been a significant industry shift in the last 15 years to using higher pressure instrumentation for analyses. Thermo Fisher has played a large part in this transition and currently produces an instrument with the highest pressure rating on the market, enabling rapid analysis and improved resolution for complex samples. In addition, our zero-volume, fingertight fittings have radically advanced ease-of-use in standard and nanoflow HPLC. 

In IC, the biggest contribution by Thermo Fisher has been the development of the eluent generator. This development has truly revolutionized IC, as the automatic production of eluents provides incredible ease of use, better detection limits, and the cleanest possible eluent.

Software, and the development of the Thermo Scientific Chromeleon Chromatography Data System (CDS), is a marked contribution in the chromatography software and data management field. The system has now developed into a multivendor software control for laboratories, the first to allow an analytical site to control all the instruments. When integrated with our enterprise-level informatics portfolio, including Thermo Scientific SampleManager LIMS, SDMS, and LES, it increases laboratory efficiency and productivity and connects the laboratory to our customer’s enterprise.

In GC, the incorporation of high‑performance MS directly into the gas chromatograph has been a huge advantage to using high resolution accurate mass spectrometry (HRAMS) because it can help determine samples that haven’t yet been characterized.

Detection of compounds of interest is another aspect where Thermo Fisher has excelled. Universal detection with universal response was realized with the introduction of the charged aerosol detector in 2004. However, Thermo Fisher will always be remembered for the introduction of Orbitrap MS, first commercially introduced in 2005, which maintains a position of prominence as a technique for MS detection.

In the area of sample preparation, breakthrough techniques such as accelerated solvent extraction (ASE) and autotrace automated SPE have significantly enhanced sample preparation workflows for solid samples and liquid samples, respectively. More recently, the Thermo Scientific SMART Digest kit has enabled significant improvements over conventional in-solution protein digestion technologies. The kit reduces preparation times and associated errors, enabling fast, sensitive, and reproducible results for high‑throughput applications.

How does your company see the current state-of-the-art of chromatography and, in this context, what is the company doing to address unmet needs?

The requests of the analytical community for improvements in chromatography have been fairly consistent over the last few years: higher resolution, improved sensitivity, faster analysis, and all with ease of use. The evolution of chromatography from a research tool used in a central facility to a widely used instrument across many applications is a key driving force behind these demands.

Instruments now need to be essentially push button: easy‑to‑use and as user-friendly as a smartphone, with the ability to produce data at a high throughput without compromising sensitivity and robustness. The incorporation of easy-to-use tools such as tablets, as seen on instruments such as the Thermo Scientific Dionex Integrion HPIC system, enables users to intuitively interact with the instrument to quickly obtain the data they need.

The introduction of automation and the smart monitoring of chromatography systems help laboratories reduce maintenance and instrument downtime by flagging when consumables need to be changed and when the instrument is conducting experiments under conditions that are not ideal to produce the best results, resulting in optimal performance.

To look at resolution, a state‑of‑the‑art chromatography system must have low dispersion and high pressure capabilities to achieve and maintain the resolution provided by the modern chromatography media. The chromatography system and column are integral to each other and must both be up to the task. To achieve the goal of high‑resolution, chromatographic media have become smaller in particle size and columns have become longer, both of which increase the demands on the pressure capabilities of the system. The new Vanquish Horizon UHPLC system increases the industry standard upper pressure limit of UHPLC to 1500 bar.

Sensitivity can come through various approaches. The detector of choice often reflects this, which is why we provide a wide range of detection capabilities from fluorescence, electrochemistry, CAD, UV with light pipe technology, all the way to Orbitrap mass detection. To achieve higher sensitivity, one option is to reduce the scale of the chromatography. Nanoscale systems have proven essential in the field of proteomics. The advances that allow these systems to be used with the ease of use expected from standard‑flow systems include nanoViper connections, which are fingertight with near zero‑dead‑volume, allowing inexperienced users to set up nano flow rates. Developments in software will further improve the ease of use for chromatography systems, particularly in industries where compliance with regulations is crucial.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look like?

Chromatography will continue to develop in many ways over the next few years, becoming an increasingly powerful analytical tool. In line with changing manufacturing and laboratory needs, reducing environmental impact and lowering costs, the future will bring change and adaptability to accommodate new and more efficient ways of working. A need for more information at higher throughput in Quality by Design (QbD) pushes the QC and production divisions into the use of simple LC coupled to mass spectrometry (MS) assays to maintain control of ongoing manufacturing processes. Manufacturers will need to make these techniques amenable to less‑skilled operators, which will involve simplified workflows and intuitive analysis software.

Another significant move that is underway is the adoption of a more holistic approach with greater support and collaboration in solving analytical problems rather than simply just supplying the equipment. This has already been seen in clinical applications but will soon move to other markets too, such as the biopharmaceutical industry, which has traditionally adopted very complex workflows. For example, peptide mapping involves a complex sample preparation step, for which there is now a simple digest kit available. There are also several opportunities to remove sample preparation altogether and increase the information accessible with high-resolution intact MS using Orbitrap technology. The use of these global methods in analysis will reduce the amount of time taken in method development and increased automation of traditionally manual and often error-prone techniques will also be a driver for increased productivity. Software will continue to play a critical role by turning data into knowledge and will adopt new technology and delivery models, like the cloud, to accelerate processing and simplify deployment. 

With regards to specific chromatography techniques, some great developments will be seen in IC. The pressure rating is likely to continue to increase, bringing with it many performance gains. While currently in the 10–15-min range, in the future we could see analysis times drop to the 30–60 s range, providing an instant information stream. This will be particularly advantageous in many areas of manufacturing that require a near continuous data output.

Though the industry is prone to change over the next 10 years, Thermo Fisher will continue to support the needs of industry and research, keeping aligned with future developments and listening to the driving forces behind them.

- Jakob Gudbrand, President, chromatography and mass spectrometry, Thermo Fisher Scientific

Waters Corporation

Overall, what have been the most important developments in chromatography over the past 60 years (other than those made by your company)?

The history of chromatography cannot be written without a nod to outside influences including the development of the personal computer and the internet. In a nutshell, the most important developments to occur with chromatography over the past 60 years include the commercialization of the first gas chromatograph in 1954 (okay 62 years); the first gel permeation chromatography system in 1963; the first high performance liquid chromatography (HPLC) system in 1967; the launch of rigid, fully-porous particles and bonded phases; the first ion chromatograph in 1975; the first computer-driven robotic sample preparation devices in 1984; the invention of the electrospray source for mass spectrometry in 1984; the development of the personal computer and the first chromatography data software for the PC in 1986; first sub‑2‑µm particles in the 1990s; the creation of the internet and on-line commerce in the 1990s; first commercially‑available LC systems in 2004 built around sub-2-µm particles; and the emergence of reliable and stable supercritical fluid chromatography (SFC) systems and columns employing 2.5-µm particle technology in 2012.

What has been your company’s highest impact contribution to chromatography over the past 60 years?

Scientists may not have known it then, but they were witnessing a revolution in separations science in the 1960s. The history and the growth of chromatography parallels the growth of Waters Corporation. From 1963 until Waters acquired Micromass in 1997, chromatography was the company’s central focus.

In 1963, Waters Associates introduced the GPC-100, an instrument that used the novel technique of gel permeation chromatography (GPC), also known as size-exclusion chromatography (SEC), to measure the molecular weight distribution of the most common polymers in use at the time - thermoplastics. Many consider this instrument to be the first high performance liquid chromatography system to reach the market. Others, consider the first HPLC system to be the Waters Associates ALC-100 system introduced in 1967.

During this time, Waters Associates’ signature contribution to chromatography was to take it out of academic and research laboratories - where scientists tinkered with homemade columns and instrumentation - and put it into widespread commercial use. This took entrepreneurship and investment capital, and the operational expertise to reliably manufacture instruments and columns and deliver them on time, at an affordable price, and make a profit. Perhaps, most importantly, Waters figured out how to educate and support customers in the new technique. This strategy became evident with the first GPC Symposium in 1965. The strategy was simple: get those who have been successful with GPC together in the same room with those who weren’t but who were motivated to learn, and watch the magic take place.

The early success with the GPC‑100 and GPC-200 led Waters Associates to introduce the ALC‑100, considered to be the first commercial HPLC system, in 1967.

From 1967, HPLC improved incrementally until 2004, which is the year Waters introduced its Acquity UltraPerformance LC (UPLC) System at Pittcon 2004 in Chicago, USA. This system became the first commercially available instrument built around sub-2-µm particles, for which it earned a Pittcon Editors Gold Award. Completely engineered from the ground up around the column, the Acquity System proved the potential of sub-2-µm particle column chemistries to increase separation speed, sensitivity, and resolution. Within five years, all of the major instrument providers introduced systems of their own.

How does your company see the current state-of-the-art of chromatography and, in this context, what is the company doing to address unmet needs?

Let me give you three good examples of how Waters is meeting the unmet needs of the chromatography laboratory. Among them is the need to get chromatographic data on exceedingly small sample volumes, the need to detect more potent therapeutics dosed a lower levels, and, when doing biomarker research, the need to find and identify metabolites, peptides, and proteins at ultralow concentrations. This calls for innovation in the area of microscale chromatography. However, microscale chromatography hasn’t been easy to pull off: It requires incredible patience, dexterity, and time. Very few chromatographers have been able to put it to good use.

The Waters ionKey-MS system was designed to make scientists much more successful with microscale chromatography. Incorporating a ceramic substrate inscribed with a 150 μm (i.d.) channel packed with 1.7-μm particles, the product was developed to provide reproducible and robust ultrahigh-pressure LC (UPLC/UHPLC) separations, provide up to 40× sensitivity improvement over 2.1 m i.d. columns, and provide plug-and-play ease-of-use, making it accessible to laboratory personnel at all skill levels.

The ceramic substrate is then encased in an injection-moulded housing containing a column heater, electronic and fluidic connections, and an electrospray emitter. When the iKey device is locked into position in the source, all of the electronic and fluidic connections to the mass spectrometer are made automatically, thus eliminating any potential variability.

The sample is then introduced to and separated in the iKey and transported directly to the integrated emitter, which converts the eluent into an aerosol. The plume of fine droplets in the aerosol is ionized, giving the droplets a positive charge at which point they enter the vacuum of the MS where they are further separated. Because mass spectrometers are flow‑sensitive detectors where signal response is proportional to the amount of sample reaching the detector per unit of time, the lower solvent flow generates a finer, less disperse electrospray plume and smaller droplets. These small droplets go through fewer Coulomb Fission events and have a greater surface‑to-volume ratio compared to larger droplets. A higher percentage of analytes is ionized, resulting in a greater sampling efficiency by the mass spectrometer.

Another driving force is the desire for ease of use, more information per chromatographic separation, accurate quantification of sample analytes, and quality of results. This is where modular mass detectors designed for chromatography fit in. These detectors are about the size of an ordinary optical detector and are designed with scan rates that are compatible with today’s UPLC/UHPLC instruments. They use less energy: they can run effectively at 464W, and generate substantially less heat and use 85% less electricity than legacy products. Moreover, they operate on 100–240 V, so that, no matter where they are used, little in the way of modifications are required to fit it into an existing laboratory infrastructure. Since they don’t require a roughing pump for normal operation, they can be turned on or off at the push of a button unlike conventional mass spectrometers. These detectors also operate without the sample‑specific or user adjustments of typical mass spectrometers. Any chromatographer can consistently generate the highest quality mass spectral data routinely, without the need for any special training or expertise. Waters Acquity QDa Detector is an excellent example.

Finally, if one takes a hard look at the sample preparation protocols in use today, they’ll see a number of slow, multistep workflows in need of improvement. Until recently that was true of released N-glycan analysis. It can take 24 h or more to profile the glycans released from a monoclonal antibody. With a new chemistry and labelling technique, Waters RapiFluor-MS reagent and kit, labs can now release and tag the glycans and profile them in a little more than 1 h.

Has your company thought about where chromatography might be in 10 years’ time? If so, how might the future look?

Separations science continues to be at the core of everything we do. The demand for information that only chromatography can provide has never been greater. Also, the need for greater separations fidelity and sensitivity continues to push investment into instrument and chemistry R&D.

Resolving and characterizing more analytes per unit of time - increased peak capacity - is the driving force behind two-dimensional LC, microscale chromatography, supercritical fluid chromatography (SFC), solid core particle technology, and new software algorithms for processing and interrogating data in new ways. In the future we can expect to see chromatography become even more useful.

From an economical and environmental sustainability point of view, we can expect to see more down-scaling of chromatography. For example, microscale chromatography greatly decreases solvent consumption, which reduces solvent storage and disposal costs and lessens the impact on the environment. Microscale chromatography, by virtue of its reduced injection volumes, also has the potential of increasing the ROI of investments in mass spectrometry, given its ability to improve the sensitivity of mass spectrometry and lower the limits of detection.

The possibilities are always there for more simplified operation and increased automation of the entire sample preparation and sample analysis workflow and we think we’ll continue to see improvements there along with further improvements in separation technology allowing higher speeds and/or resolution for the analysis of complex samples, such as mixtures of intact proteins (such as top-down proteomics). This could potentially be achieved by further reducing particle size and by developing new particle morphologies and surface chemistries.

Waters continues to support the need of industry for process analytics, a growing market that is still young. Driving innovation in this regard is the need for just-in‑time manufacturing and for product release data obtained at-line or on-line, rather than in the laboratory. Some day in the near future, we can expect to see chromatography in one form or another make its way out of the laboratory and onto the production floor and play a central role in fine-tuning and checking the quality and purity of drugs, chemicals, foods, and beverages by providing real-time information for batch or for continuous manufacturing (flow chemistry) environments.

- Michael Yelle, Vice President, Separations Technology, Waters Corporation