Selecting a Detector for LC

Although a blog is not a good way to teach all there is to know about LC detectors, I have tried to put together information to give an overview of the most common detectors.  Restek does not sell HPLC instruments or detectors, but we think we might be able to help if you are very new to the world of LC. Before getting started, if you have a textbook on analytical chemistry, it would most likely be useful also to review the section on LC detectors. Since new techniques are developed frequently, to obtain the most current information, i.e., the “latest and greatest”, the information offered online from your instrument manufacturer would be helpful as well.

For convenience, I have summarized and rated some key attributes in the table below for the detection methods discussed in this blog. Please keep in mind that all of these will vary depending on the specific method and analytes, perhaps different detector models and vendors, and also the level of training for the analyst. The attributes listed are rated on a scale of 1 to 4, with 4 being the highest and best, 1 being the lowest.

 

Detector	Selectivity	Sensitivity	Ease of use	Linearity
UV	2	3	4	4
PDA/DAD	2	3	3	4
Fluorescence	3	4	3	2
ELSD	1	2	1	1
RI	1	1	2	1
MS	3	3	4	3
MS/MS	4	4	3	3

 

UV Detector

The UV Detector is by far the most common detector used for liquid chromatography. Sometimes these detectors are offered as UV-VIS detectors, which can also measure absorbance in the visible range of the electromagnetic spectrum.   Absorption of UV light occurs when the energy applied causes electrons to jump from their ground state to an excited state, an orbital at higher energy, to be exact. Functional groups that containing electrons that easily absorb UV are called chromophores. Often the best chromophores are functional groups that contain conjugated double bonds or delocalized pi electrons, such as in a benzene ring.

To determine the best wavelength to use, generate a wavelength scan and look for the wavelength(s) at which maximum absorbance occurs.   An alternate approach is to identify wavelengths that have the strongest absorbance for specific functional groups in the molecular structure.  Tables are available in literature for this, for example, the “Correlation Table for Ultraviolet Active Functionalities” (Bruno and Svoronos), found in the CRC Handbook of Chemistry and Physics (https://www.crcpress.com/CRC-Handbook-of-Chemistry-and-Physics-96th-Edition/Haynes/9781482260960)

Response is determined by Beer’s Law, which demonstrates that absorbance is directly proportional to concentration: A=ELc 

Where:

• A=absorbance, E= molar extinction coefficient (molar absorptivity), L=path length of detector flow cell, c=concentration of analyte (Molarity)
• E depends on structure and properties of the analyte, as well as wavelength.

 

Diode Array (DAD) or Photodiode Array (PDA) Detectors

DAD and PDA are one in the same; terminology just varies between different vendors. In a practical sense, these are a 3-dimensional version of a UV detector.   A diffraction grating enables the detector to continuously and rapidly monitor across all wavelengths simultaneously.  The result looks like a chromatogram for UV, done at multiple wavelengths.

A DAD or PDA may be particularly useful in method development or research projects where there is a need to analyze for unknown compounds, possibly with unknown functional groups attached. The advantage is the ability to collect data from any selected wavelength and to change that selection after the initial data workup if desired.

Fluorescence Detector

Fluorescence light is emitted when a higher energy, short wavelength of light is absorbed and as a result, a lower energy, longer wavelength light is emitted. Fluorescence detectors are very sensitive and also very selective. However, since very few compounds have natural fluorescence properties, often derivatization is needed to attach fluorescing chromophores. This may be done either pre or post-column, and usually requires an additional pump and a rotary switching valve. Post-column derivatization is always safer for the HPLC column, although the setup is sometimes more complicated.

Similar to UV, compounds that emit a strong fluorescence signal usually contain conjugated pi-electrons or aromatic rings. The intensity of the signal is determined by the excitation and emission wavelengths. Typically, the excitation wavelength is held constant and the emission wavelength is varied for maximum response.

ELSD

The ELSD or “Evaporative Light Scattering Detector” is a good alternative to UV if your analysis is for a set of compounds that lack any UV chromophores, when you wish to avoid derivatization, and when an LC/MS or LCMSMS is not available.  The ELSD works by nebulizing the sample and measuring the degree of light scattering, which is related to the mass of the analyte.  The ELSD is used very commonly for sugar and for carbohydrate analyses and also sometimes for lipid analyses.  It is somewhat limited in that no buffers can be used in the mobile phase and the detector often exhibits a nonlinear response, or at least it has a very limited range linear response.

Refractive Index (RI) Detector

RI detectors can be universally applied to a wide variety of analytes, since no chromophores are required.  The detector works by measuring the refraction of light through the sample as it passes through flow cell.  The refraction from the sample is compared against the background produced by measuring mobile phase alone in a reference cell.   Similar to ELSD, this technique might be used when mass spec is not available. It is also usually less expensive than an ELSD.  Drawbacks include possible issues with interferences, low sensitivity, and the inability to use with gradients or buffers.

Mass Spectrometry (LCMSD or LCMSMS)

Although the most costly technique in terms of capital expense, mass spec has quickly become one of the primary detection techniques for LC.  With LCMSD, the mass-to-charge ratio of ions, shown as m/z, is measured, which can be very selective.  LCMSMS is even more selective, since it measures the mass-to- charge ratio of parent (precursor) and daughter (product) ions after subsequent fragmentation.   One advantage of such selectivity is that analytes can usually be distinguished from one another, even if they coelute.  This allows for more compounds to be analyzed simultaneously, which particularly useful for screening methods.

LCMS works by treating the sample/analyte molecule as follows:

• IONIZE- Occurs at the source. In positive mode (most common), protons are attached. In negative mode, electrons are attached/ protons removed from precursor molecule. (precursor=parent). The most common sources are Electrospray and APCI (chemical ionization).
• FOCUS- Ions are drawn through an orifice and skimmers by vacuum, guided by the octopoles or ion bridge into mass analyzer, with assistance of nitrogen as both a drying gas and as curtain gas.
• SEPARATE- The separation step is done by a mass analyzer or mass filter. Quadrupole, Time of Flight, and Ion Trap are the most common.
• DETECT- Signal generated when ions reach the detector and trigger release of electrons. Example: electron multiplier.

LCMSMS works the same way, except the analyte ion undergoes fragmentation after the initial separation, usually in a something called a Collision Cell. The collision cell is located between the two mass analyzers.  The fragment ion, called a product or daughter ion, is separated in a 2^nd mass analyzer. The sequence of events for the overall process with LCMSM can be described as this:

IONIZE> FOCUS> SEPARATE> FRAGMENT> FOCUS>  SEPARATE>  DETECT

The greatest benefits from LCMSMS are realized by operating in MRM or SRM modes (Multiple Reaction Monitoring or Selected Reaction Monitoring, respectively.) Terminology differs by manufacturer. In this mode, MRM pairs are selectively monitored and reported. The pairs are represented as m/z of the parent ion followed by  “>” and then the m/z of the fragment or daughter ion. 

These are just a few details about LCMSMS that I have highlighted.  If interested in learning more, I suggest contacting an instrument manufacturer and/or enrolling in a course for training.

For more information about all of the detectors we have discussed here, below are a few links that you may find useful from various sources.

From Hitachi-High Tech:

http://www.hitachi-hightech.com/global/products/science/tech/ana/lc/basic/course8.html/

From Bioforum:

http://www.forumsci.co.il/HPLC/Detectors_handouts.pdf

From Chromatography online (LCGC magazine):

http://www.chromatographyonline.com/seeing-believing-detectors-hplc

 

I hope this overview of detection methods has been helpful. Thank you for reading.