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Going for Greener LC–MS

Liquid chromatography (LC) is used in a great many analytical applications worldwide, and is commonly coupled with mass spectrometry (MS) to detect, identify and monitor compounds, proteins or peptides in environmental, biological or chemical samples. Many of these laboratories have large analytical LC instruments running continuously, on a daily basis. This results in considerable consumption, over time, of hazardous organic solvents and additives.

In recent years, scientists have become more concerned about the environmental impact of their research, and have started to seek “greener” instruments, methods and reagents. However, switching equipment or methods can introduce unwanted variation or cast doubt on comparability of data. Therefore

A look at the role of micro-LC–MS to reduce the environmental impact of chromatography laboratories.

Tina Settineri, Eksigent, Part of AB Sciex, Dublin, California, USA.

it is essential that any alternative method or instrumentation is at least as good as previous approaches in terms of performance, reliability, speed, throughput and sensitivity.

For LC–MS, greener approaches include reducing the use and generation of harmful solvents and additives; refinement of methods to reduce sample use; increasing throughput; and using cleaner, more reliable instrumentation that requires less maintenance and component replacement.

Reduction of Solvents and AdditivesLC–MS uses hazardous solvents and additives, particularly acetonitrile, which can be harmful to animals as well as contributing to undesirable effects on the environment. It was recently estimated that one conventional

LC instrument, equipped with a 15–25-cm column (4.6-mm inner diameter packed with 5 μm particles) running at 1 mL/min on continuous operation, generates approximately 500 L solvent per year.1 Typically, 50% of this solvent would be organic solvent, and in most cases this is acetonitrile.

Reducing chemical waste and the use of harmful solvents in analytical laboratories is a major target for greener LC–MS. This could be done through two different approaches: firstly, by replacing these with non-hazardous alternative solvents; and secondly, by reducing the volume of harmful solvents used.

Finding suitable alternatives to harmful solvents is not necessarily straightforward. Acetonitrile is favoured for its excellent combination of characteristics, including

minimal chemical reactivity; low acidity; the ability to dissolve a wide range of solutes; low UV cut-off; and compatibility with MS. In recent years, some researchers have successfully developed LC methods using pure water as the mobile phase solvent2,3 — possibly inspired by the acetonitrile shortage that resulted in 2008–2009 when China’s production stopped for the Olympic Games. While the use of pure water would substantially reduce the use of harmful solvents in certain applications, it would require considerable method development for alternative solvents to be proven in all LC applications.

Other alternatives include acetone4 and methanol, which are considered to be more “environmentally friendly” alternatives for LC

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applications5; although methanol does have some toxicity, its disposal costs are lower than those of acetonitrile. While some methods have been successfully developed using methanol for the mobile phase6,7, methanol’s characteristics as a solvent are sufficiently different from those of acetonitrile to mean significant method development would need to be performed before it could replace acetonitrile in existing, validated procedures. Therefore it seems doubtful that analytical laboratories would abandon acetonitrile altogether in the near future.

A new, more environmentally friendly alternative has become available using CO2 in standard, reverse phase systems, such as the Waters Acquity UPC2 System (Massachusetts, USA) and the Agilent 1260 Infinity system (Santa Clara, California, USA). These systems are suitable for separation and analysis of compounds that are often problematic for LC, such as hydrophobic and chiral compounds, lipids, thermally-labile samples and polymers. The compressed CO2 mobile phase is less toxic and less costly than liquid mobile phases or carrier gases, and is considered the perfect complement to MS because of its low solvent load, high resolution, narrow peaks and fast separations. These systems still provide high throughput, with short cycle times and variable volume injection.

The second approach, reducing the volume of solvents used and waste generated, has gained much more traction within the LC community. Over the past ten years, several instrumentation providers have developed a variety of miniaturized systems that are designed to work with lower flow rates — around 10–100 times lower than traditional, high flow analytical systems, thus reducing solvent volumes.

There are two broad classes of miniature LC systems: micro-LC, with flow rates in the region of 5–200 μL/min, including the eskpert micro LC 200 (Eksigent, part of AB Sciex, Dublin, California, USA) and nano LC with flow rates ranging from around 100 nL–10 μL/min such as the Waters nanoAcquity Ultra Performance (Massachusetts, USA) and the Eksigent Nano LC Ultra (Eksigent, part of AB Sciex, Dublin, California, USA). Nano LC has the advantage of being highly sensitive, while also using only very small sample volumes. Most nano LC users work at flow rates under 1 μL/min, but for many LC–MS applications, nano LC can be too slow, taking perhaps 60–90 min for a typical quantitative protein analysis using a trap and elute workflow, compared with 8–12 min when using micro-LC (see example data in Figure 1).

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Figure 1: Throughput comparison: Total ion chromatograms using nanoflow and microflow liquid chromatography for typical protein quantification method with the system.(a) Using nano liquid chromatography at 300 nL/min (75 μm × 15-cm column) with trap at 200 μm × 0.5 mm, the procedure takes 35 min with an additional 20-min trap.(b) Direct injection micro liquid chromatography at 10 μL/min (300 μm × 15-cm column), the run time is completed in 4 min. In this example, micro-LC offers more than eight times higher throughput. (Data courtesy of Xu Wang, AB SCIEX.)

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The reduced flow rate of micro-LC can still provide equal, or better, chromatographic resolution and sensitivity to traditional UHPLC, yet with sub-2 μL injection volumes typically. To maintain equal or better chromatographic resolution at these lower flow rates, however, micro-LC systems need to have very low delay (void) volumes (1–3 μL). Ideally, delay volumes should be significantly less than a typical column volume to enable ultrafast gradients for micro-LC–MS, resulting in high throughput without compromising chromatography.8 Significantly, some of the latest micro-LC systems can save up to 95% of solvent consumption compared with traditional systems, yet also provide UHPLC at high pressure (see Table 1).

The success of these systems relies on design and optimization of all their elements for miniaturized LC, rather than

simply scaling down a traditional system. Optimization of all components includes specially designed pumping systems to reduce mixing times and the delay (void) volume; smaller internal diameter (i.d.) columns that require minimal volumes of solvent and sample injection; and even the connection with a coupled mass spectrometer must be optimized. Scaling down a traditional UHPLC system does not achieve the same benefits.

Method RefinementA number of researchers have demonstrated that even “greener” micro-LC–MS can be achieved by reducing the number of sample preparation steps required for an analysis, thereby limiting the use of reagents and organic solvents.9,10 Pre-concentration procedures, such as solid-phase extraction (SPE) are often required when there are low

concentration sensitivity issues, but such procedures add considerable time, labour and solvent consumption to the workflow. Development of automated, on-line SPE coupled directly with LC–MS–MS has been shown to considerably reduce reagent consumption and generation of hazardous chemical waste, as well as increasing throughput and laboratory efficiency.

Reducing Sample VolumeMicro-LC also offers the advantage of requiring less sample, which is particularly beneficial for analysts in clinical research and proteomics, for example, where biological samples may be of limited availability, and/or expensive to collect and store. By acquiring data from smaller sample volumes, scientists can reduce their tissue use and also limit their impact on the environment by using fewer subjects, taking smaller samples from subjects, and reducing sample transport and storage requirements.

Micro-LC is growing in popularity with LC–MS users, particularly in food and environmental analysis laboratories and quantitative proteomics research, where the technique’s improved sensitivity from reduced limits of quantitation (LOQ) can significantly improve data quality and help to reduce the need for repetitions

of experiments because of inconclusive findings.

Improving Up-timeThe eskpert micro LC 200 system mentioned previously relies on a specially designed pump that only strokes once for each run, compared with traditional UHPLC reciprocating pumps that stroke several times. Such single-stroking pumps are usually more robust, so require less maintenance. Furthermore, these pumps have fewer components that last longer than reciprocating pumps, whose seals and check valves have to be replaced every three to six months on average.

In addition, using micro-LC for LC–MS means at least ten times less solvent goes into the mass spectrometer. Consequently, micro-LC often reduces the frequency that a coupled mass spectrometer needs to be cleaned. This not only improves the system’s up-time and throughput, but also reduces the consumption of cleaning fluids for the system. Finally, the vast reduction in solvent consumption using micro-LC has extra benefits because laboratories can significantly reduce their storage volumes of solvents and reagents, as well as reducing the costs, space and paperwork associated with transporting waste products.

Table 1: Micro liquid chromatography offers improved throughput with low delay times*.

Traditional UHPLC Microflow UHPLC

Flow rate 1.5 mL/min 0.060 mL/min

Column diameter 2.1 mm 0.50 mm

Mobile phase solvent waste volume (per run) 1,925 μL 66 μL

Solvent used per 1,200 injections 2.31 L 2 mL

*Data courtesy of Eksigent, part of AB SCIEX, acquired using ekspert microLC 200 system.

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E-mail: support@absciex.comWebsite: www.eksigent.com

ConclusionsThere are several approaches that analytical scientists can take to develop more “environmentally friendly” laboratory methods. Nano flow LC enables the greatest elimination from the workflow of harmful solvents, but can be too slow for many LC–MS applications.

Micro-LC offers many key advantages for scientists who wish to reduce the negative effects of their research on the environment, without compromising method sensitivity, throughput, efficiency or data quality. The technique is gaining popularity particularly among LC–MS and LC–MS–MS users working in analytical drug discovery applications (for example, drug metabolism and pharmacokinetics, [DMPK]), environmental analysis, clinical research and quantitative proteomics where sample volumes may be limited but laboratories need to achieve higher throughput than is possible with nano flow LC.

Today’s micro-LC systems have been shown to reduce solvent consumption by as much as

95% compared with traditional UHPLC systems, while offering improved sensitivity, reliability and up-time. Until suitable alternatives to acetonitrile and methanol are widely proven across all LC applications, micro-LC offers mumerous advantages for greener, high-throughput LC.

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Tina Settineri, PhD, is director of HPLC products and assistant general manager at Eksigent, part of AB Sciex, where she leads product marketing and product management for HPLC products. Tina has been working in LC–MS since the

Article reprinted from 22nd August 2012 issue of

1990s, when she started as an application scientist in protein mass spectrometry. She has worked in various product management and marketing roles for both instrumentation and consumables over the past 14 years. She has previously held positions with Applied Biosystems and Life Technologies from 1997–2011, including roles as director of proteomics mass spectrometry, director of pharma programmes and director of TaqMan protein assays. Tina rejoined AB Sciex through Eksigent in 2011.

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