A Comprehensive Guide to the Types of HPLC Detectors: Applications, Advantages, and Disadvantages

Types of HPLC Detectors

There are different types of HPLC detectors available in market but the idea Characteristics of a HPLC detector, as above:

  1. They should be highly sensitive and respond in a predictable manner.
  2. Detector should be able to detect all solutes or have predictable specificity if not.
  3. Temperature and carrier flow should have no effect on it.
  4. Should be able to respond regardless of whether or not the mobility phase is active.
  5. The detector should not cause extra-column peak broadening and should be dependable and simple to use.
  6. They have a reaction that is proportional to the amount of solute present.
  7. The detector should not harm the solute in any way.
  8. It is expected to provide qualitative data on the identified peak.

There are the lists of types of HPLC Detectors present for Analysis:

1. UV-VISIBLE DETECTORS (UV/Visible Detector in HPLC)

  • In modern HPLC, Photometers based on ultraviolet (UV) and visible light absorption are the most useful detectors.
  • This three UV, VIS and PDA detectors are under category of absorbance detectors.
  • For many solutes, these detectors have a high sensitivity, but samples must absorb light in the UV – visible range (i.e, 190–600 nm).
  • During the analysis, the sample goes through a clear colour-less glass cell, called a flow cell.
  • When UV light is exposed on the flow cell, the sample absorbs a portion/part of UV light. Therefore, the intensity of UV light observed for the mobile phase (without sample) and the eluent-containing sample will differ.
  • During the analysis, the sample passes through a flow cell, which is a clear, colourless glass cell. 
  • When UV light irradiated on the flow cell, a portion of the UV light is absorbed by the sample. As a result, the UV light intensity detected in the mobile phase (without sample) and the eluent-containing sample will be different.
  • The amount of sample can be calculated by measuring this difference. Because UV absorbance varies depending on the wavelength employed, it is critical to select an appropriate wavelength based on the application.
  • A typical UV detector permits user to select a wavelength between 195 to 370 nm.
  • A VIS detector employs a longer wavelength than a UV detector (400 to 700 nm). UV/VIS detectors are detectors that cover both the UV and VIS wavelength ranges (195 to 700 nm) and provide a wider wavelength selection.
  • Further divided in three detectors as below,

Read: Difference between UV and PDA Detector in HPLC

A. Fixed-Wavelength Detectors:

  • This hplc uv detector works with the fixed wavelength (Only one nm).
  • The 254-nm line from the low-pressure mercury (Hg) lamp is the most popular wavelength for use with the fixed-wavelength UV detector.
  • 214, 220, 280, 313, 334, and 365 nm can be accomplished by use of other lamps (e.g., zinc), phosphors Lamp.

B. Variable-Wavelength Detectors (VWD):

  • It is also known as variable-wavelength and diode-array detectors. A broad-spectrum UV lamp (D2 -deuterium lamp) is directed through a slit and onto a diffraction grating.
  • The light is split into wavelengths by the grating, which is then turned to guide a single wavelength (or a narrow range of wavelengths) through the slit and detection cell and onto a photodetector.
  • For detection in the visible region, a tungsten lamp is used instead of D2 lamp(deuterium).
Types of HPLC Detectors- Variable Wavelength Detectors (VWD)

C. Diode-Array Detectors (DAD) or PDA Detector:

  • It is also called photodiode-array detector (PDA Detector). HPLC -PDA Detector is the most widely & most commonly used detector in hplc analysis. It’s a universal detector in liquid chromatography detectors range.
  • Photodiode-array detector (PDA Detector) has a similar optical path to the variable wavelength detector (VWD), except that the white light from the lamp passes through the flow cell prior to striking the diffraction grating.
  • The photodiode array (PDA) gets its name from the fact that the grating may spread the spectrum across an array of photodiodes.
  • The DAD can be used to gather data across a chromatogram at one or more wavelengths or to acquire complete spectra on one or more analytes in a run.
  • The most important thing is that the PDA detector in HPLC mainly used for Peak Purity of sample.
  • The most Common Benefits of UV-Vis Detectors are:
    • Capable of very high sensitivity (for samples that absorb in the UV)
    • Good linear range (>105)
    • Can be made with small cell volumes to minimize extra-column band broadening
    • Relatively insensitive to mobile-phase flow and temperature changes
    • Very reliable
    • Easy to operate
    • Non-destructive of sample
    • Widely varying response for different solutes
    • Compatible with gradient elution
    • Detection wavelength can be selected
    • Internal troubleshooting and calibration checks are common

2. FLUORESCENCE DETECTORS:

  • Fluorescence detectors are very sensitive and selective for solutes that fluoresce when excited by UV radiation.
  • Analyte atoms are stimulated by employing a specified wavelength and subsequently emit a light signal (fluorescence). To determine the analyte concentration, the intensity of the emitted light is measured.
  • Because of its high sensitivity, the fluorescence detector is particularly useful for trace analysis, or when either the sample size is small or the solute concentration is extremely low.
  • Most clinical samples, pharmaceuticals, petroleum products, and natural products have fluorescent absorbance.
  • For a few compounds which do not have fluorescence absorbance or low absorbance, they treated with fluorescence derivatives such as dansyl chloride.
Types of HPLC Detectors-FLUORESCENCE DETECTORS (FID)

3. Electrochemical (Amperometric) Detectors (EC Detector)

  • Selective electrochemical (EC) HPLC Detectors measurements can identify several chemicals that can be oxidised or reduced in the presence of electric potential at very low concentrations. The current between the polarizable and reference electrodes is measured as a function of applied voltage using this method.
  • Catecholamine and other neurotransmitters are commonly determined by EC detection.
  • In an electrochemical cell, a glassy carbon electrode is most typically utilised.
  • There are several different types of ECs. The detection is grounded on amperometry, coulometry, polarography, and conductometry method.

4. Radioactivity Detectors

  • As radio-labeled solutes elute from the HPLC column, radioactivity detectors are utilised to keep track of them. The emission of light in the flow cell as a result of radioactive decay of the solute, followed by emission of -, -, or -radiation, is used for detection.

5. Conductivity Detectors (Electrical conductivity HPLC detectors)

  • Conductivity detectors measure a change in the conductivity of the column effluent as it flows through the cell using low-volume detector cells.
  • Ion chromatography and ion exchange applications in which the analyte does not have a UV chromophore are the most common uses for conductivity detectors.
  • Sample as a solution containing ionic components will conduct electricity. The conductivity detector calculates electronic resistance and the measured value is directly proportional to the concentration of ions present in the sample solution. Thus, it is mostly useful in ion chromatography.  
  • The conductivity detector is appropriate for detecting inorganic ions (e.g., lithium, sodium, ammonium, potassium) in water samples, plating baths, power plant cooling fluids, and other similar applications.
  • Also known as Bulk Property HPLC Detectors as it measure the conductivity of Mobile Phase.
  • Conductivity also used to identify organic acids like acetate, formate, and citrate.

6. Chemiluminescent Nitrogen Detector (CLND Detector)

  • The HPLC column effluent is nebulized with oxygen and a carrier gas of argon (Ar) or helium (He) and pyrolyzed at 1050 °C in the furnace.
  • Nitrogen-containing mixes (excluding N2) are oxidized to nitric oxide (NO), which is subsequently combined with ozone to generate nitrogen dioxide (NO2*).
  • When NO2* decays to its ground state, a photon is released, which can be detected by a photometer. The signal is proportional to the nitrogen content of the original sample.
Types of HPLC Detectors-Chemiluminescent Nitrogen Detector (CLND Detector)

7. Chiral Detectors

Based on the application chiral HPLC detectors divided as below,

  1. Polarimeters Detector (PLD): It determines the degree of rotation of polarized light (ideally in the 400 to 700 nm range) as it travels through the sample. The degree of rotation is determined by the chiral compound’s concentration as well as its molecular structure.
  2. Optical rotary Dispersion Detectors (ORD): It perform on a same principle to polarimeters (PLD), but it uses lower wavelengths.
  3. Circular Dichroism Detectors (CD) : When an analyte flows through a flow cell, they measure the differential in absorption of right and left circularly polarised light. The analyte should have a chromophore with absorption in the 200 to 420 nm range for strong CD results.

8. Refractive Index Detectors (RI Detector)

  • Differential refractometer (DRI) or refractive index detector (RI or RID) is another name if RI Detector.
  • RID responds to the difference in the refractive index of the column effluent as it passes through the detector flow cell.
  • Differential refractometers (DRI) contain a flow cell with two parts: one for the sample and one for the reference solvent. The detector measures the refractive index of both components.
  • When only solvent is passing through the sample component the measured refractive index of both components is the same, but when an analyte passes through the flow cell the two measured refractive index are different.
  • The difference appears as a peak in the chromatogram.
  • RID is most effective to detect substances with limited or no UV absorption.
  • Example of this materials are alcohols, sugars, fatty acids, polymers, carbohydrates, and polymer analysis.
  • Refractive Index Detector (RID) Know as universal detector.

Que. What is a universal detector for HPLC?

Ans. Differential refractometer (DRI) or refractive index detector (RI or RID) is known as universal Detector in HPLC Analysis.

Limitation of HPLC RI detectors:

  • Less sensitivity, Temperature-dependent on detection and they are not greatly suitable for gradient elution method.
Types of HPLC Detectors- Refractive Index Detectors (RI Detector)

9. Light-Scattering Detectors

A. Evaporative Light-Scattering Detector (ELSD) :

  • Evaporative light-scattering detectors or evaporative light scattering detector (ELSD) are based on evaporation of the mobile phase, followed by measurement of light scattered by particles of nonvolatile analyte.
  • The column effluent is nebulized in a stream of nitrogen or air and evaporated in a heated drift tube, leaving non-volatile particles suspended in the carrier gas stream. Light scattered by the particles is detected by a photodetector mounted at a fixed angle from the incident beam.
  • The ELSD is also considered a universal detector same as  the refractive index (RI) detector. Because it has the potential to be used for ‘‘any’’ analyte.
Evaporative Light-Scattering Detector (ELSD)

B. Condensation Nucleation Light Scattering Detector (CNLSD) :

  • The condensation nucleation light-scattering detector (CNLSD) improves the sensitivity and linear range of the traditional ELSD.
  • A saturated stream of solvent is added to the particles in the carrier gas after the mobile phase has evaporated. The particles serve as condensation nuclei, forcing the solvent to condense onto the particles, leading them to grow to a size where light-scattering detection can detect them more easily.

C. Laser Light-Scattering Detectors (LLSD)

  • It is also called as Multi Angle Light Scattering (MALS) or multiple angle laser light scattering detector.
  • As the material passes through the flow cell in the mobile phase, LLSD uses a laser light source focused at the flow cell. In the absence of reference standards, scattered light can be measured at several angles or plurality of angles (3 to 18) and utilized to determine the mass of the analyte with the correct mathematical modifications.

10. Mass Spectrometer (LC-MS Detector)-Mass Spectroscopic HPLC Detectors  

  • The MW (Molecular weight) of the analytes is used to detect them. The information obtained is particularly important for determining the structure of compounds.
  • It can also be used to measure very low detection limits of elemental and molecular components, which is not limited to structure identification.
  • It is Specific HPLC Detectors  as Mass spectroscopy detectors are renowned for their high selectivity and sensitivity, as they detect molecules by fragmenting them using electric fields and separating the fragments based on their mass-to-charge ratio. This makes them incredibly useful tools in a variety of industries.

11. Corona-Discharge Detector (CAD)

  • The mobile phase is evaporated and the column effluent is nebulized, just like in an evaporative light-scattering detector or a mass spectrometer.
  • The gas phase analytes are then combined with a stream of nitrogen gas that has been positively charged using a corona discharge device.
  • To increase signal quality, the charge is transferred to the analyte particles, and high-mobility charged species are removed in an ion trap. An electrometer reads the signal generated by the residual charged analyte ions.
Corona-Discharge Detector (CAD)

Here are the summary of all types of HPLC detectors useful in Pharmaceutical industries.

Reference:

  • Book-Lloyd R. Snyder, Joseph J. Kirkland, John W. Dolan(auth.) – Introduction to Modern Liquid Chromatography, Third Edition (2010)
  • Type of HPLC Detectors

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