When it comes to liquid chromatography there are numerous choices available for the detection of your analyte, but which of these offers the greatest sensitivity, is most suitable for your particular application(s) and most affordable? In this article I will briefly outline the choices of (U)HPLC detectors available, their pros and cons, and provide some sources for further information.
UV-Vis Absorption Detectors – These are the most commonly used detectors in liquid chromatography and can be used to detect a broad range of analytes across many application areas. There are also different types of UV-Vis absorption detectors: Diode Array Detectors (DAD) offer high levels of sensitivity and a near-uniform response whilst Variable Wavelength Detectors (VWD) deliver great linearity and robustness for routine use. For example, if you are analysing trace impurities I would suggest a DAD, whilst for routine QC procedures a VWD would potentially be a better option.
Pros: Simple to use, broad applications and cost effective
Cons: Analyte must contain a chromophore
This application note is a good example of the sensitivity afforded by a DAD using LightPipe™ technology to further enhance sensitivity.
Flourescence Detection – Such detectors are one of the most sensitive, offering sensitivities three to six orders of magnitude higher than UV-Vis. Modern fluorescent detectors also allow the monitoring of multiple excitation/emission wavelength pairs simultaneously.
Pros: High sensitivity and selectivity
Cons: Analyte must contain a fluorophore or be derivatized or labelled with a fluorescent label
You can watch an overview of fluorescence detection for HPLC in this whiteboard video.
Electrochemical Detectors – These detectors are typically used for the sensitive and selective detection of compounds in complex matrices that undergo oxidation or reduction, such as neurotransmitters. Used with amperometric cells for high sensitivity, low sample volumes, and coulometric sensors to maximize both selectivity and sensitivity.
Pros: High sensitivity and selectivity
Cons: Compound must exhibit electrochemical activity
Refractive Index Detectors – The often forgotten detector! 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 chromophores and in sugar and polymer detection.
Pros: Cost effective and analytes do not need to contain a chromophore
Cons: Not suitable for gradient methods and also relatively low sensitivity
Charged Aerosol Detection – This detector, frequently abbreviated to CAD, offers universal detection with a near-uniform response, meaning that it can be used for a wide range of analytes that do not contain a chromophore or fluorophore, and can be used for relative quantitation without the use of reference standards.
Pros: Universal detector, good for analytes lacking a chromophore or that ionise poorly, and provides relative quantitation
Cons: Poor detection of volatile and some semi-volatile compounds
For a complete list of applications that can utilise a charged aerosol detector, please view this CAD bibliography.
Mass Spectrometry – Not always considered a detector but more of a separate instrument, the mass spectrometer offers both detection and identification with the ability to use different liquid chromatography flow rates (nano, micro, capillary and analytical).
Pros: High sensitivity, wide compound detection range and identification
Cons: Expensive and generally not as intuitive as other detectors
Conductivity and pH Monitors – These are not for the detection of analytes but for monitoring pH and conductivity as the name suggests. These are useful for monitoring salt and pH gradients and are mostly used in biopharmaceutical separations.
As you can see, there are many options when it comes to (U)HPLC detectors, with each having their unique capabilities and applications. Which one is most suited to your needs is dependent on a number of factors such as application, analyte(s), budget, and ease-of-use. In many instances it may be appropriate to combine different detectors to get a comprehensive understanding of the sample.
We recently conducted a five-part webinar series dedicated to HPLC detectors, overviewing the various types of detectors, mechanisms of operation, which detector to choose for particular applications and tips and tricks. Please visit thermofisher.com/lc-webinars to view these on-demand.
Learn more about the various types of detectors on this informative webpage, with links to the specific detector types.