Entering the Second Dimension - - Chromatography Online
Entering the Second Dimension


The Column
Volume 10, Issue 14, pp. 2–4

Michael Frank, Senior Director of Global Marketing, Liquid Phase Separation Business at Agilent Technologies, talks about the rise of two-dimensional liquid chromatography (2D LC) and its potential for routine analysis.

Q: Why has 2D LC become more popular?


Michael Frank
A. Firstly, when we are talking about "2D LC" here, we are referring to comprehensive two-dimensional liquid chromatography (2D LC) or heart-cutting 2D LC and not any other LC techniques involving two columns, such as pre-concentration or alternating column regeneration.

The need for peak capacity, that is, the ability to separate as many compounds as possible in a complex sample, has probably not changed dramatically. Scientists have always had to deal with complex natural products, polymers, and biopharmaceuticals. Most recently, ultrahigh-pressure liquid chromatography (UHPLC) with mass spectrometry (MS) became the preferred choice for complex sample analysis. MS is obviously a great tool to get very complex samples separated (in the m/z-domain) but it also has some weaknesses. MS cannot separate isobaric compounds. Peak suppression is also a serious issue, particularly for very complex samples. High-end mass spectrometers are also very expensive.


PHOTO CREDIT: KUTAY TANIR/GETTY IMAGES
UHPLC has definitely increased the peak capacity in liquid chromatography (LC) in terms of practical separation times. However, reducing particle sizes further and accepting more and more back pressure achieves only a relatively small improvement in peak capacity compared to a 2D LC approach.

For complex samples 2D LC offers more separation power compared to one-dimensional liquid chromatography (1D LC) and modern 2D LC systems can be combined with UHPLC and coupled to a mass spectrometer. Therefore, with this a new level of separation power, significant increases in peak-capacity can be achieved. This makes modern 2D LC very attractive for researchers.

Q: What are the drawbacks associated with 2D LC?

A. Historically, researchers had to build their own 2D LC systems based on standard LC-modules and had to be very creative in getting such a system controlled by the data system because these were designed to support 1D LC systems with one gradient being executed. For comprehensive 2D LC, in the second dimension the gradients have to be repeated up to 100 times. The interfacing valve between the first and second dimension has to be synchronized precisely and the gradient delivered from the second dimension pump. We have seen 2nd dimension gradients as short as 9 s, being repeated over 200 times. Without optimized hardware, drivers, and firmware this is difficult to achieve, but these challenges have been overcome with the availability of commercially available 2D LC systems.

Q: What specific advantages does 2D LC offer over existing techniques?

A. As mentioned previously for complex samples 2D LC will always deliver higher separation power compared to a 1D LC — even 1D UHPLC. The reason is pretty simple, in an optimized 1D (U)HPLC system, separation power can only be increased by increasing column length: For example, by connecting two columns.

In 2D LC the separation power nearly multiplies with the two columns and any advantages of UHPLC can also be utilized in 2D LC. For example, smaller particle columns can also be used in a 2D LC-configuration.

Turning now to MS: 2D LC will certainly not replace MS as a means to separate complex samples but it can significantly improve the power of an LC–MS system. For example, isobaric compounds that are indistinguishable in MS can be separated with a higher probability as with an 1D LC–MS system and, more importantly, the significantly increased separation power reduces the chance of having compounds co-eluting that might lead to ion-suppression of another species.

Q: 2D LC is often more associated with being a tool for researchers. Do you see it being more commonly used in routine analysis?

A. This is certainly true if we look to the systems that have been used in the past because programming capabilities were often required to set up a 2D LC system.

2D LC is currently used by analytical scientists involved in research and method development in pharmaceutical, biopharmaceutical, and chemical method development laboratories, quality assurance groups of food producers, and federal drug-control agencies. The technique has scope to become available to a much broader audience.

So, yes, 2D LC is on the way to becoming more commonly used in routine analysis, but only for complex samples.


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