How HPLC Detectors Work

Types of UV-Vis detectors

For UV-Vis detection, chromatographers can choose between three types of detectors: a variable wavelength detector (VWD), diode array detector (DAD), or multiple wavelength detector (MWD). In variable wavelength detection, a single chosen wavelength from the UV-Vis spectrum illuminates the sample. In contrast, diode array and multiple wavelength detectors exposes the sample to the entire spectrum instead of a single chosen wavelength. The application needs or optical properties of the analyte(s) and sample matrix often determine the detector choice.

Fluorescence detectors are the most sensitive optical detectors and an excellent alternative to standard, absorption-based UV-Vis detectors for analytes with fluorescent properties or analytes tagged with fluorophores.

The sensitivity can be 10-1000 times higher for fluorescence versus UV-Vis absorption detectors but is limited to fluorescent molecules. For this reason, FLD is less common than UV-Vis.

Fluorescence detectors are exceptionally selective for fluorogenic compounds, and excitation and emission are tunable for a particular class of fluorophore.

Refractive index detectors typically measure the deflection of a light beam due to the difference between the refractive indices of the pure mobile phase and the mobile phase containing the analyte. Refractive index detectors are universal detectors, requiring only that the analyte be soluble in the mobile phase.

The downside of RID is sensitivity towards temperature and flow rate as well as eluent composition, which prevents its use with gradient separations. A well-defined thermostatting of the detector and precise flow control is required to maintain sensitivity.

The detection limit for a refractive index detector is considerably lower than UV-Vis and FLD, but there are applications where RIDs should be your first choice.

Electrochemical detectors are selective and extremely sensitive and often used to measure low-level, redox-active analytes in complex biological matrices. Although ECD performance often compares to the sensitivity in FLD, an advantage of electrochemical detection is a direct measurement of an analyte without using complex, time-consuming derivatization procedures.

How ECD works

An electrochemical detector measures the current produced when an electrochemically active compound undergoes oxidation or reduction at the surface of the electrode caused by an applied potential. According to Faraday's law, the resulting current is directly proportional to the concentration of the analyte experiencing the electrochemical reaction.

Charged aerosol detectors are near-universal. The high sensitivity, wide dynamic range, and uniform response make CAD excellent for application versatility. The three significant benefits of using CAD are:

1. Sensitive detection
   Independent of whether a chromophore is present or the compound forms gas-phase ions

2. Uniform response
   As in the ability to obtain the same response for all components regardless of the analyte structure

3. Standard-free quantitation for unknowns
   This functionality is essential when reference standards are unavailable for impurities and degradants

Evaporative light scattering detection (ELSD)

Like charged aerosol detectors, evaporative light scattering detectors are non-linear and near-universal. These detectors measure many semi- and non-volatile analytes and do not require an analyte to possess a chromophore or ionizable group.

How ESLD works

In the ELSD, aerosol detection depends on the light-scattering properties of the analyte, and light intensity is related to the quantity of analyte present. All evaporative light scattering detectors work the same way:

1. Nebulization via droplet formation from an eluent stream

2. Evaporation of the eluent leaving behind dried analyte particles

3. Exposure of dried analyte particles to a beam of light

4. Scattering of light by dried particles

5. Measurement of the scattered light by a photomultiplier

6. Quantification of analyte by photo multiplier tube

Liquid chromatography systems often pair with mass spectrometers. In combination with the retention time from the LC separation, MS detection provides an additional level of information by determining the mass-to-charge ratio of analytes contained in the sample.

Mass spectra contain information regarding the elemental and isotopic composition of analytes, which yields high detection specificity and is helpful for structural elucidation. These detectors are compatible with many analytes capable of forming gas-phase ions, from small inorganic salts to large macromolecules like proteins.

MS detection is more sensitive than other detection methods such as UV-Vis, does not require a chromophore or redox group, and enables the identification and structure elucidation of various molecules.  

How mass spectrometry works

Most HPLC detectors work by converting a physiochemical property of an analyte into an electrical signal.

There are many varieties of HPLC detectors. The common ones used are UV-Vis, fluorescence, mass spectrometry, charged aerosol, evaporative light scattering, and refractive index detectors.