HPLC

HPLC’s continued popularity in the separations laboratory and dominance of the separations market in general is no secret to professionals in the field of chromatography. And with a total market size of $4.7 billion forecast by 2012 (SDi Global Assessment Report, 10th Ed.), that popularity shows no sign of waning.

Joining us for this discussion are Sergio Guazzotti of Thermo Fisher Scientific; Fraser McLeod of Dionex Corporation; Helmut Schulenberg-Schell of Agilent Technologies; and Steve Watts of Hitachi High Technologies America.

The acetonitrile shortage has had a major impact on the HPLC field. How has it affected the research and development of HPLC as a whole?

Guazzotti:In the face of the acetonitrile shortage, researchers have looked at ways of reducing its use, replacing it in their applications, or recycling it. In the case of reducing its use, there are a number of strategies that are being used. For example, by employing shorter, narrower columns packed with small particles chromatographic separations using UHPLC require less than 10% of the solvent used in conventional HPLC methods. This has resulted in a higher transfer of HPLC methods into UHPLC and in a more detailed cost/value evaluation when deciding on the purchase of an LC. It has caused many to consider not only the original capital investment (e.g., higher for a UHPLC than a HPLC), but also the solvent consumption associated with the chromatographic separations (lower employing UHPLC methods than HPLC methods).When evaluating replacement of acetonitrile by other solvents, researchers have considered alternative solvents such as methanol. This is interesting since in the early years of HPLC a lot of methanol was being used. Later, acetonitrile gained popularity due to its solvent properties, in particular, its close proximity of polarity index (P’=5.8) with that of methanol (P’=5.1), but a much reduced viscosity (resulting in lower system back pressures), in particular when working with mobile phases mixed with water. Since the acetonitrile shortage, we are seeing a larger number of methods being carried out with methanol. With the luxury of now having LC pumps that can sustain much higher pressures than those used in the early years, the difference in viscosity between methanol and acetonitrile is not playing such a big role as before.

The other alternative some people have been able to adopt has been to recycle the eluent when using an isocratic separation. There are now automatic means of recycling solvents that provide economic and environmental advantages.

Independently of the acetonitrile situation normalizing in the future, the shortage has left in all researchers a permanent impact, with everybody now being more conscious of solvent use and of ways of reducing solvent costs, as well as dependence on a particular solvent.

McLeod:Naturally some companies are looking to use solvents other than acetonitrile, but this is not easy. The major benefits of acetonitrile are the low UV cut-off point (190 nm) and its low viscosity (reduces system back pressure and increases separation speed). There is no other solvent that can do this, so many labs continue to use acetonitrile despite the high price. They mitigate the cost factor by using smaller volume columns, which typically reduce solvent consumption by 90% or more.

Schulenberg-Schell:Analytical labs have definitely become more cost-conscious with respect to solvent consumption. Research to use less expensive solvents or to reduce acetonitrile usage has increased. Using a lower column inner diameter, or moving to faster UHPLC methods are major trends as it allows for smaller column i.d. and shorter run times, yet still it can improve data quality. However, acetonitrile remains an essential tool due to its unique elution strength in liquid chromatography.

Watts:The acetonitrile shortage affected all aspects of the HPLC industry and market demands have shifted. Although the technologies utilized to address the acetonitrile shortage had already existed (UHPLC systems, narrow bore columns, smaller particle sizes, and shorter columns, etc.), it forced the industry to re-evaluate the current and future requirements of HPLC end-users. Every customer has identified unique challenges they are trying to overcome, and our job is to help them reach their goals.

What trends have you most intrigued in the HPLC field?

Guazzotti:One of the trends that was intriguing in the past was UHPLC. Now we see that UHPLC is here to stay since the advantages it provides are clearly demonstrable. I think the combination of UHPLC with ways of increasing laboratory productivity will result in new technologies that will mark new trends in the HPLC field.

McLeod:There is a definite trend to smaller-sized columns, but many users find these difficult to use as any extra column (volume) introduced on the HPLC can have a significant, detrimental effect on the quality of analysis. Therefore, the most intriguing trend I see is the use of smaller volume columns made much easier by technology such as the introduction of novel fitting systems or making it easier to modify systems to use these columns.

Schulenberg-Schell:I am impressed by the strong interest in new flexible LC instrumentation that enables customers to run both conventional and UHPLC methods on a single instrument. LC users have quickly figured out how easily they can manage conversion to better methods if they can get rid of their aging conventional instruments and run both on a single system.

It is also outstanding how UV sensitivity has improved most recently. We thought only mass spectrometry would allow us to obtain lower limits of detection. But customers have seen up to 10x improvement in UV sensitivity recently. That was considered impossible a while ago.

Watts:A major trend observed at my company has been a shift in the market toward application-specific HPLC systems. Many customers are in search of turn-key solutions that include optimized methodologies and application support, as well as the HPLC system hardware. For example, my company offers three different systems and technologies in performing amino acid analysis, and each allows the customer to begin analyzing samples immediately out-of-the-box. The result is an increase in productivity for the customer. In addition, some customers require our expertise in performing the method development for their application. Customers want assurance that the HPLC solution provided to them will successfully perform the application on their samples, and thus the customer can immediately produce useful data in support of their research activities.

What obstacles, other than acetonitrile, stand in the way of HPLC development?

Guazzotti:In order to improve method development it is crucial to utilize flexible systems. One of the obstacles for method development, in particular when working with UHPLC in order to minimize the method development time, has been the limitation of traditional UHPLC pumps to operate only in binary mode. Recent technological advances have permitted the development of UHPLC pumps with quaternary capabilities that provide performances that match or surpass those of traditional binary UHPLC pumps. This resulted in much higher flexibility in method development. The ability of coupling multiple systems or carrying out some type of automatic method development has also helped tremendously in this field.

McLeod:One of the major obstacles until now has been the lack of a standardized detection system that is universal, quantitative, and easy to use. Typically, analysts have to work with multiple detector types to identify and quantitate their analytes. Even MS has not been able to solve these issues as it is more complex to use, cannot easily ionize all analytes, and often cannot provide quantitative results.

Schulenberg-Schell:As much as every analytical lab would like to change to new methodology, giving more confidence, the regulations and operating procedures are still to be followed. Fortunately, regulations are changing to favor performance-based method definitions and add certain flexibility.

Watts:The primary obstacle of HPLC development is meeting customer requirements. We learn from our customers by asking the right questions and understanding their pains. With this information, we predict future market demands and design systems and solutions accordingly. It’s a challenging task, but one that we enjoy.

Do you expect any major developments to be presented at Pittcon 2010, or other major shows?

Guazzotti:Liquid chromatography continues to be a field where technological advances will continue to improve instrumental performance. We will continue to see advances that keep pushing the efficiency of chromatographic separations as well as the ability to carry out shorter and shorter runs and/or provide alternative ways to increase productivity. I am sure we will see some developments introduced at Pittcon 2010 as well as during HPLC 2010. Some of the key developments to be introduced might be related to pump technology, detector technology, and column technology.

Schulenberg-Schell:I am sure customers will report on their practical experiences exploiting routinely the new power range available for better quality results. And achieving this with the new type of method development has just gotten so much easier and faster.

Watts:New technologies are continually being developed, and UHPLC systems will continue to evolve. I would expect several developments to be presented for increasing system capabilities and performance and increasing laboratory productivity. Many of these developments will be in the form of turn-key HPLC solutions, and some of this will be a result of collaborations with third-party vendors.

What is one change/improvement you hope to see in the HPLC industry over the next few years?

Guazzotti:One improvement that I hope to see in the HPLC instrumentation over the next few years is the development of smart systems that can monitor changes in system performance in real time based on actual physicochemical measurements and adjust the system behavior accordingly. We are doing some of this now but I am hoping to see major developments in this field in the near future.

McLeod:I think we can do a lot to improve ease-of-use of HPLC systems. The goal should be to enable any analyst, independent of experience or training, to be able to immediately use an HPLC and to take full advantage of all the functionality.

Schulenberg-Schell:Many labs would like to move to a greener future. The new instruments most recently introduced for UHPLC and HPLC allow for the usage of green solvents, (e.g., ethanol) with higher viscosity. That definitely has taken an obstacle away to move to a greener chemistry in the lab and help save the environment.

Watts:A change that we hope to see continue is the customer’s appreciation for high-value, high-performance HPLC instrumentation. In light of recent economic conditions, most companies that utilize HPLC systems state that they have either entered a cost-saving initiative or they need more from their investments. They are being held accountable for every dollar they spend, and they are seeking high-value alternatives to their HPLC solutions.

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