I don’t think this is a good omen, just as I sat down to write this my own source of UV has just disappeared behind a cloud, perhaps it is trying to tell me something.
In HPLC, and many other areas of science, UV is a common method for detecting compounds and molecules. In fact, in a HPLC survey we conducted last year, nearly all respondents indicated that they used UV as a detection method in their HPLC. Those detectors measure the absorption of compounds in the ultraviolet range (190 – 360 nm) or visible (360 – 800 nm) range and are popular as they can detect a broad range of compounds, are easy to use, sensitive and offer reasonable specificity. In HPLC these detectors usually come in two forms, either a variable wavelength detector or a diode array detector. The variable wavelength detector is typically used for monitoring one wavelength; the diode array detector can easily acquire multiple wavelengths and also absorb spectra. However, what are the alternatives if UV detection just doesn’t offer you enough from a specificity, sensitivity or application perspective or your compound simply lacks a chromophore? Thankfully, with HPLC, there are quite a few good alternatives:
Alternatives to UV
Fluorescence Detection – This requires your compound to contain innate or natural fluorescence properties or it will require labeling with a fluorescent tag such as occurs with 2-AB labeling of glycans. The advantages of fluorescence are that it is very sensitive and highly selective.
Electrochemical Detection (ECD) – An electrochemical detector can be used with substances that can be oxidised or reduced with the chemical reaction generating an electrical signal that can be measured. As with fluorescence the advantages of ECD are that it is very sensitive and selective, however it does require your compound to exhibit electrochemical activity.
Refractive Index (RI) – The RI detector I feel is one that is often overlooked in exchange for more glamorous detectors. It works by measuring the difference between the refractive index of the mobile phase and the deflection of the sample using Snell’s Law. Its application areas include where compounds lack chomophores and in sugar and polymer detection. Its main advantage is that it offers universal detection, however its disadvantages include it being relatively insensitive, not suitable for gradient elution chromatography and also temperature and flow sensitive.
Charged Aerosol Detector (CAD) – Charged Aerosol Detectors really are a magic box in that they offer near universal detection of any non-volatile and semi-volatile analytes that lack chomophores and/or are poorly ionised. In addition the CAD provides relative quantification without the need for reference standards. To see how charged aerosol detection works, check out the short whiteboard video below:
The advantages of CAD are that it does offer near-universal detection, is relatively sensitive, works with gradient conditions and offers over four orders of dynamic range. It has applications in many areas and is constantly finding new applications, such as detection of glycans, without the need for fluorescence labelling as outlined in this poster note video.
Mass Spectrometry – While mass spectrometry is an HPLC detector, I feel it offers more than just simple analyte detection so I don’t perceive mass spectrometry in the same bracket as the HPLC detectors above. For more information on mass spectrometers and specifically Orbitrap mass spectrometers, I would encourage you to read this blog by my colleague Martin Hornshaw.
What to Use When UV Is Not Enough
That really depends on your application and the physio-chemical properties of your analyte(s) of interest and matching this to the appropriate detector. The detectors, though, are complimentary so UV can be combined with another detector, such as a charged aerosol detector, to ensure that no peak is missed, such as shown in this poster note.
In conclusion, UV is a popular and cost-effective detection mechanism in HPLC, however in some instances alternative detection methods are required to see all analytes and the addition of a complementary detector to UV can allow this universal detection of all analytes.
Learn more about the complete range of HPLC detector on our resource page.
Download the Charged Aerosol Detector Bibliography.