Combating Challenging Separations With Advanced 2D Liquid Chromatography Technologies

Liquid chromatography (LC) systems are the backbone of laboratories within the pharmaceutical and biopharmaceutical industries. As an essential piece of equipment for bio-therapeutic analysis, they provide in-depth characterization and purity confirmation of single peaks.

However, as the scientific and medical fields advance, samples are becoming increasingly complex to analyze and scientists are seeking more confidence in their results. Furthermore, laboratories are under growing pressure to operate as efficiently as possible, by simplifying workflows and optimizing time, while minimizing bench space. Can innovative two-dimensional LC (2D-LC) systems provide the answer?  

Challenges with 1D-LC technology

Conventional LC separations are referred to as one-dimensional (1D) as the sample is subjected to a single separation process. However, despite considerable improvements in the commercially available LC instrumentation and column technologies, 1D-LC sometimes struggles to meet the separation and resolution demands of the analytical task.¹

Interfering matrix and samples of high complexity, such as metabolites, fermentation broth or polymers including multiple structurally similar compounds, present an analytical challenge to 1D-LC in terms of resolution power, efficiency and selectivity. This is also true for samples with difficult-to-separate analytes, including those with a large number of structural isomers.

A further issue relates to mass spectrometry (MS) detector compatibility. Often, conventional ultraviolet (UV)-based LC methods are not compatible with MS and time-consuming re-development or off-line sample processing is required. Where compatibility is not an issue, compound disturbances may lead to ion suppression and decrease the sensitivity, or even worse result in analytes not being detected.

Achieving in-depth analysis with 2D-LC

2D-LC systems present a practical solution to achieve full characterization of substances or peak purity confirmation on more complex samples. This technology connects two different chromatographic columns in sequence, with the effluent from the first system (first dimension) being transferred onto the second column (second dimension). In essence, one compound, or one fraction, is analyzed by two different separation modes to provide additional selectivity to resolve difficult analytes or deplete interfering matrices.

There are many types of 2D-LC techniques that vary in automation degree, as well as the number of fractions utilized and the way they are stored. First, 2D-LC systems can either be classified as offline or online. In offline techniques, fractions are collected externally into vials and then re-injected onto a second column.² Conversely, online 2D-LC systems involve transfer of the fractions of the first-dimension separation to a second-dimension column in a single flow path inside the system. The interface connecting both dimensions usually consists of a combination of fluidic valves and storage loops or trap columns.

Then, there are two different types of online 2D-LC regarding the number of transferred fractions: comprehensive and heart-cutting. In comprehensive 2D-LC systems, the whole first dimension eluate is transferred in a large number of fractions. This method is a powerful tool in achieving separation of highly complex samples with 100–1000 components. However, comprehensive 2D-LC is often over-qualified for most analytical tasks, when only one or a few fractions are of greater interest.

This is where heart-cut 2D-LC systems are better suited. Single or multiple heart-cutting 2D-LC is used for analyses where only a single or a few peaks are transferred from the first-dimension eluate to the second dimension for further separation. In single heart-cut 2D-LC, a single fraction is transferred, whereas several fractions are transferred in multi-heart-cut 2D-LC. The fractions are stored in loops or traps until the second dimension is available for analysis.

Higher separation power with added flexibility

Modern heart-cut 2D-LC methods have many advantages compared to 1D-LC methods and are delivering higher separation power, while enabling straightforward peak purity confirmation. Furthermore, they are valuable tools in overcoming the MS incompatibility of traditional UV-based LC methods. As a result, this gives scientists confidence that they have the highest quality data from their analysis.

Moreover, 2D-LC methods can be advanced further, thanks to the latest developments in high performance liquid chromatography (HPLC) technology. Here, dual split sampler systems have been designed that combine both 1D-LC and heart-cut 2D-LC systems in one piece of equipment.

These innovative systems can be used either as one heart-cut 2D-LC system or as two independent 1D-LC systems to give scientists the utmost flexibility in their laboratory. When 1D-LC analysis is sufficient, the system can be used in 1D mode for high sample throughput, or for more difficult samples and separations, the heart-cut 2D-LC mode can be specified.

Dual split sampler systems create a flexible chromatography system, enabling scientists to switch easily between applications. They allow fast switching between online 2D-LC and 1D-LC operation without manual replumbing, thus saving time and increasing capacity further. Using the latest systems, scientists can achieve the confidence delivered by a 2D-LC system with the productivity gain from two 1D-LC systems.

Gaining confidence in product purity

As scientists work with evermore challenging samples and difficult-to-resolve analytes, such as those found in biopharmaceutical formulations, polymers, omics, and natural medicines, systems that deliver confident, accurate and robust results are essential.

By removing interfering matrices or overlaying peaks, or allowing the connection between UV and MS methods, 2D-LC methods are enabling full peak characterization and purity confirmation of complex substances. Heart-cut 2D-LC is used in pharmacokinetics and therapeutic drug monitoring of biological samples (for example plasma, blood and urine),³ and there are wider uses such as in food safety and environmental testing.

In particular, scientists using innovative heart-cut 2D-LC systems with a dual split sampler can accelerate sample analysis time to enhance productivity. These innovative HPLC systems provide complete, automated and flexible workflows, allowing pharmaceutical manufacturers to expand their analytical capabilities to meet the operational efficiency demands of quality control laboratories.

References

1. Guiochon, G. The limits of the separation power of unidimensional column liquid chromatography, J. Chrom. A 2006, 1126, 6–49. doi: 10.1016/j.chroma.2006.07.032

2. Stoll, D. R.; Carr, P. W., Two-dimensional liquid chromatography: A state of the art tutorial, Anal. Chem. 2017, 89, 519–531. doi: 10.1021/acs.analchem.6b03506