shutterstock_654920956Icebergs and chromatography?

Have you ever had the chance to witness the beauty of glaciers? I was lucky to visit Alaska’s Glacier Bay in 2012 and I even luckier to see and hear glacial calving to form a new baby iceberg!

While I was able to see the creation of a small iceberg, I did notice that only a small fraction of what was actually calved off the glacier was visible above the surface of the water. Doing a quick internet search taught me that for a typical iceberg close to 87% of the iceberg is below the surface of the water1. As a tourist on a boat I was only seeing a small fraction of that iceberg floating in the ocean, and I was missing so much.

In a lot of ways this is a great analogy to high pressure liquid chromatography (HPLC). In HPLC, we have a sample that we want to analyze so we separate the sample on a column (similar to calving off small icebergs) and detect the analytes of interest. As many of us know, there are a variety of detectors to choose from, each with their own set of advantages and disadvantages. One of the most common detection methods used for HPLC is UV/Vis detection. While UV/Vis detection is widely used it can only identify analytes that have an inherent chromophore. While not as dramatic, this can be likened to the iceberg – it’s possible that we’re only see a part of the entire picture (i.e. we might only see what is above the surface of the water, but miss all of the great beauty below the surface).

Luckily, there are plenty of solutions to allow you to see what you’ve been missing (and the best part is you don’t have jump into the icy water to see what else there is to see… in the chromatography sense at least), but we’re going to focus on a technology that is unique to Thermo Fisher Scientific; Charged Aerosol Detection (CAD).

What is Charged Aerosol Detection (CAD)?

Most detection options in liquid chromatography require certain physico-chemical properties to detect a substance, such as a chromophore for UV light absorption or ionizability for mass spectrometry.

CAD is a nebulizer-based detector that provides near universal detection of non-volatile and most semi-volatile analytes. It also features uniform response independent of the chemical structure of the analyte, excellent sensitivity, and a wide dynamic range and is compatible with gradient elution (more to come on that a little later).

Check out our charged aerosol detection resource page to dive deeper and learn:

  • What makes CAD powerful?
  • How does CAD work?
  • What applications are ideal for CAD?

Combine the unique capabilities of the Thermo Scientific™ Charged Aerosol Detector with the powerful separation of a Thermo Scientific™ Vanquish™ Duo UHPLC system

The Vanquish Duo system for Inverse Gradient provides a uniform response for all detectable analytes with charged aerosol detection (CAD). CAD is a powerful tool to detect all non-volatile and many semi-volatile analytes that are typically overlooked (like the part of the iceberg underwater) if they do not contain a chromophore with UV/Vis detection. CAD is a nebulizer-based detector, sensitive to changes in solvent composition, and while it is compatible with gradient conditions, it works optimally with isocratic methods. To minimize the effect of solvent composition changes during a gradient method, a second pump can be used to compensate for solvent composition changes to achieve isocratic flow to the detector.

The Vanquish Duo system for Inverse Gradient provides a full solution offering:

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  • Uniform response with CAD under gradient elution conditions
  • Reliable standard-free quantification of knowns and unknowns
  • Simplifying method setup with automatic Inverse Gradient calculation considering all system volumes
chromatography

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Solvent composition effects detection for nebulizer based detectors

Although compatible with gradient elution, the response of any nebulized-based detector, like CAD, depends on the composition of the mobile phase. Higher organic content affects the efficiency of nebulization positively, increasing the percentage of analyte reaching the detector, resulting in a higher response.

By using an inverse gradient for post-column compensation, the eluent entering the detector always has the same solvent composition, like in an isocratic elution. No matter where the compound elutes during the gradient, response is consistent. This consistent response allows for quantitative information with high confidence even if there is a lack of an individual standard.

chromatography-2

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  • In a reversed-phase gradient separation, the organic composition increases over time. As the organic content increases, the efficiency of nebulization increases, leading to a higher percentage of analyte reaching the detector seen later in the gradient (red box, conventional gradient elution).
  • A second inverse gradient that mirrors the analytical gradient can be applied post column to achieve isocratic flow to the CAD. The inverse gradient can be used to correct for solvent composition related variations in response for analytes (blue box, inverse gradient composition).

While you might be concerned that analyte dilution will occur with the addition of the inverse gradient, there is no reduction in signal response, as a CAD is a mass proportional detector, where all solvent is being removed during the evaporation stages leaving all the charged analyte particles to be detected.

Things to know about the Vanquish Duo system for Inverse Gradient:

  1. The Vanquish Duo for Inverse gradient is appropriate for all applications which use CAD under gradient elution conditions. An additional optical detector (from our HPLC and UHPLC Detector portfolio) can also be used to get additional information from your sample.
  1. There are 1000 and 1500 bar options for maximum flexibility.
    Build the system with a single autosampler and either the new Thermo Scientific™ Vanquish™ Dual Pump F (up to 1000 bar, two independent ternary-solvent blending flow streams in one housing) or two single Thermo Scientific™ Vanquish™ Binary Pump H (up to 1500 bar)/ Thermo Scientific™ Vanquish™ Binary Pump F (up to 1000 bar).
  2. There’s no extra work needed for method development with the easy-to-use tools built into the Thermo Scientific™ Chromeleon Chromatography Data Systems (CDS) Software.

You can use the automatic gradient conversion tool in Chromeleon CDS, which automatically identifies the Inverse Gradient instrument configuration, and starts the dedicated method wizard. The wizard calculates and applies the Inverse Gradient to the second pump in the Vanquish Duo System for Inverse Gradient taking into account all internal volumes. This way you get highest quantification accuracy and maximized ease-of-use.

Now that you know you about the benefits of CAD, give it a try and sit back and relax sipping on a hot cocoa knowing you are getting a better glimpse of the iceberg beneath the water.

Check out this app note on the Quantification of paclitaxel, its degradants, and related substances using UHPLC with charged aerosol detection

Learn more about Thermo Fisher Scientific’s solution for Inverse Gradient visit www.thermofisher.com/VanquishDuo

1US Coast Guard Navigation Center, US Department of Homeland Security, accessed 3/29/2018.