HPLC Detectors are key components in High-Performance Liquid Chromatography (HPLC) instruments. Herein we are introducing the types of Liquid Chromatography Detectors as guidance to the Industry.
HPLC Detectors introduction
HPLC detectors will respond to the “Physico-chemical” property of the analyte. This might include UV absorbance, mass-to-charge ratio, fluorescence, refractive index, etc. The invention of the ‘flow-cell’ (flow-through cell) by Tiselius in 1940, which allowed dynamic and continuous measurement of the refractive index of the column effluent marked a breakthrough in the development of modern liquid chromatography.
The detector measures the Physicochemical property (Ex: UV absorption) of the mobile phase as it elutes from the column. The response of the detector will change due to changes in the contents of the mobile phase. Most detectors will respond linearly to changes in the analyte concentration in the mobile phase.
The most common HPLC detectors are based on the absorbance of UV (or Visible) light by the analyte molecule. These detectors are popular because of their low cost, robustness, reasonably low detection limits, and ease of use. There are several common types of ‘UV’ detectors including; single wavelength, multiple wavelengths, or diode array configurations. The diode array detector also allows some limited qualitative functionality via the ability to dynamically collect UV spectra.
Applications of HPLC Detectors
Several other detector types are available that use other physicochemical properties of the analyte molecule and these include:
- Fluorescence
- Electrochemical
- Electrical Conductivity
These HPLC detectors tend to employ when high sensitivity is required and/or the analyte molecule does not absorb UV radiation.
Mass Spectrometric detectors are emerging as important and these are now widely used in many areas of analytical chemistry. These are used for the detection of low analyte concentrations, for qualitative analyses, or in situations where the separation of analyte components is difficult.
Refractive index detection is useful when analyte molecules do not respond to any other detector type or if the analyte shows large differences in refractive index from the mobile phase used.
Ideal HPLC detectors
The ideal HPLC detector should meet the below requirements.
- Either be equally sensitive to all eluted peaks or only record those of interest
- Do not affect by changes in temperature or in mobile phase composition
- Be able to monitor small amounts of compound (trace analysis)
- Not contribute to band broadening (small cell volume)
- React quickly to detect narrow peaks which pass through the cell
- Be easy to manipulate and robust
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General Definitions of HPLC Detectors
General Terms / Definitions and Concepts of HPLC Detectors are provided below.
HPLC Detector Selectivity
Non-selective detectors react to the bulk property of the solution passing into the detector. When a compound elutes from the column, this bulk property changes, and the change can measure and record (i.e. refractive index). Selective detectors do not react to the bulk solution passing through but measure a response due to a specific property of the solute molecule (i.e. UV absorbance).
HPLC Detectors Sensitivity
The smallest detectable signal has usually estimated the equivalent to three times the height of the average baseline noise – this would give a signal-to-noise ratio of 3:1 for the ‘Limit of Detection’ of the detector. If the amount of analyte injected is less than this, then the signal ceases to be distinguishable from noise. For quantitative analysis, a signal-to-noise (S/N) ratio of 10:1 is recommended for the ‘Limit of Quantitation.
HPLC Detector Drift and Noise
Noise is the amplitude of all random variations of the HPLC detector signal. Drift is the average slope of the noise envelope.
HPLC Detector Flow Cell
The flow cell of the HPLC has a solid quartz body. A slit is integrated into the quartz body.
The maximum back pressure for a standard FLD flow cell is lower than that for DAD/VWD flow cells and is in the region of about 20 bar. Excessive back pressure will result in flow cell destruction. The light is split in the flow cell to allow light to pass onto both the reference diode and photomultiplier.
Emission Monochromator Grating
Before the luminescence reaches the emission monochromator, a cut-off filter removes light below a certain wavelength, to reduce noise from 1st order scatter and 2nd order stray light. The selected wavelength of light is reflected onto the slit in the wall of the photo-multiplier compartment.
The grating is turned using a DC motor, the position of the grating determining the wavelength or wavelength range of the light falling onto the Photomultiplier. The grating can be programmed to change its position and therefore the wavelength during a run. For spectra acquisition and multi-wavelength detection, the grating rotates at about 4000 rpm.
Photomultiplier
Incident photons hit the photocathode and generate electrons. These electrons can accelerate by an electric field between several arc-shaped dynodes. Depending upon the voltage difference of any pair of dynodes, one photon hitting the photocathode will produce many electrons, thus multiplying the signal.
Photodiode (Reference Diode)
A reference diode is located behind the flow cell of the HPLC detector. The diode measures the excitation light transmitted by the flow cell and corrects flash lamp fluctuations and long-term intensity drift. The response of the reference diode is subtracted from that of the photomultiplier to give a flatter and less noisy baseline.
Types of HPLC Detectors
Different types of HPLC detectors and principles are provided below for easy reference.
Ultraviolet Detector (HPLC UV Detector)
The most commonly used HPLC detector is the UV-Visible detector. The detector fulfills many of the criteria for an ‘ideal detector’ including:
- Sensitive
- Wide Linear Range
- Unaffected by Temperature Fluctuation
- Selective and suitable for gradient elution
The schematic diagram shows the basic flow cell of a UV detection system. As the mobile phase leaves the analytical column it enters, and fills the flow cell. Light from the UV (Deuterium) or Visible (Tungsten) lamp shines through the flow cell and its content.
The light intensity of the emergent light from the flow cell is measured using photodiodes, which produce an electrical signal when exposed to light. The greater the intensity the greater, the greater the absorbance and the larger the resultant signal. Analytes that contain a UV chromophore(s) will cause a large absorbance difference when they elute into the flow cell and the light exiting the flow cell will reduce markedly generating a large response. By constantly measuring this electrical signal it is possible to produce a plot of Absorbance against Time in the Chromatogram.
Quantitation
Quantification using this UV-Visible detector is possible because of the linear relationship between absorbance and concentration of the analyte. This relationship is defined by Beer’s Law (avail which is shown next.
UV absorbance
In UV-Visible detection, the useful detection wavelength range is between 210 nm and 850 nm (with a tungsten lamp). Deuterium lamps are used for the excitation of analyte molecules in the UV region (~180 – 380nm), and Tungsten lamps are used for measurements in the visible region (380 – 800nm). Below 210nm is possible for the solvent uses in the mobile phase to interfere with the analyte absorbance measurement.
Electrons tightly bound in single carbon/ carbon or carbon-hydrogen bonds absorb electromagnetic energies corresponding to wavelengths less than 180 nm – below the useful operating range for a typical UV-Vis detector. Analyte molecules containing only CC or C-H bonds do not show high sensitivity in UV-Vis detectors. Unshared electrons in the outer orbital of constituent atoms may exhibit larger absorbance’s in the useful range.
This would include the unshared electrons of sulfur, bromine, and iodine. Electrons within the unsaturated systems such as double or triple bonds within organic molecules are relatively easily excited by UV radiation and generally show absorbance within the useful UV-Visible region of the spectrum. Therefore, compounds with unsaturated and aromatic characteristics generally exhibit useful absorbance spectra.
Variable Wavelength Detectors (VWD)
VWD is one of the popular HPLC detectors. The schematic opposite shows the optical path of a conventional variable wavelength detector. Polychromatic light from a deuterium or tungsten lamp is focused onto the entrance slit of a monochromator using spherical and/or planar mirrors.
The monochromator selectively transmits a narrow band of light to the exit slit. The wavelength of measurement is selected via the data system –this is achieved using a grating mounted on a turntable. The grating is positioned to allow the light of the correct wavelength to pass to the send mirror and so on through the flow cell or the reference diode.
The magnitude of analyte absorbance is determined by measuring the intensity of the light reaching the photodiode without the sample (reference photodiode). And comparing it with the intensity of light reaching the photodiode after passing through the sample (sample photodiode).
Many variable wavelength detectors are time programmable and can program to switch wavelengths during analysis in order to select a suitable wavelength for each analyte. It is not easily possible to dynamically record sample spectra with this type of detector –although it is possible using stopped-flow techniques. The wave length of the used light to measure sample absorbance can change by altering the position of the grating, the next diagram presents the main elements that are currently present in a variable wavelength detector.
Diode Array Detector (Photodiode Array Detector)
Photodiode Array Detector is also a widely used HPLC detector in the pharma industry. The diode array detector can provide detection at a single wavelength or multiple wavelengths. This detector also has the ability to dynamically acquire and store spectra for peak purity analysis, library searching, and/or the creation of extracted signals.
The combined tungsten and deuterium lamps emit radiation from 190-850 nm. The radiation is collimated through the flow cell, then a mechanically controlled slit. The radiation is dispersing at the holographic grating into individual wavelengths of light. Each photodiode receives a different narrow wavelength band. A complete spectrum is taken approximately every 12 ms and spectra and signals are created and stored. The array consists of over 1000 diodes, each of which measures a different narrow-band wavelength range.
Measuring the variation in light intensity over the entire wavelength range yields an absorption spectrum. The entrance slit can do the program. So, if high sensitivity is required, the slit opens too wide and if the maximum spectral resolution is desired, then the slit is narrow. Spectral resolution and detector sensitivity are inversely proportional.
HPLC DAD Detectors (Bandwidth)
The Bandwidth parameter in Diode Array detection relates to the number of diode responses which are averages in order to obtain a signal at a particular wavelength. A wide bandwidth has the advantage of reducing noise by averaging over a greater diode range. Because Noise is random, averaging the response over a larger range of diodes will reduce noise.
As the bandwidth increases, the signal intensity (detector sensitivity) increases as some diodes will result in a lower absorbance, compared to a reading using only the single most intense wavelength (max). A wide bandwidth results in a larger range of wavelengths being averaged when producing a spectral data point –resulting in a loss in spectral resolution.
The next figure presents the effect of the bandwidth in the chromatogram. Note how the resultant chromatogram and peak spectra change. As with most detection systems in analytical chemistry detector sensitivity and resolution are inversely proportional.
DAD Detectors – Slit Width
Some diode array UV-Vis detectors have a variable slit at the entrance of the spectrograph. This is an effective tool for adapting the detector to the changing demands of different analytical problems. A narrow slit (width) provides improved spectral resolution for analytes that give UV spectra with enough fine detail to be useful for qualitative analysis. For example, the improved spectral resolution will increase the confidence of library-matching search results when attempting to identify unknown peaks within a chromatogram.
A wide slit (width) allows more of the light passing through the flow cell to reach the photodiode array, hence the signal intensity and detector sensitivity increase. Baseline noise is also reduced –again leading to an increase in signal-to-noise ratio. However, with a wider slit, the spectrograph’s optical resolution (its ability to distinguish between different wavelengths) diminishes.
The wavelength of light falling on each diode becomes less specific as the light becomes more diffuse. Any photodiode receives light within a range of wavelengths determined by the slit width, and so spectral resolution decreases. The next graph highlights what happens when altering the slit width. Note how the resultant baseline noise and the peak spectral change.
External Standard Quantitation
The external standard (ESTD) quantitation procedure is the basic quantification procedure in which both calibration and unknown samples are analyzed under the same conditions. The results (usually peak height or peak area measured using a data system) from the unknown sample are then related to those of a calibration sample, using a calibration curve, to calculate the amount in the unknown.
Fluorescence Detector Principles
Fluorescence HPLC Detector is widely used in the industry. The picture opposite is a representation of an energy level diagram of a molecule. A molecule in one of its singlet states (S) has all electron spins paired. A molecule in a triplet state (T) has a pair of electrons where their spins are unpaired. When a photon is absorbed by the molecule, the energy of the molecule is raised from the ground state (S 0 ) to one of its singlet excited states (S 1, S 2 ). Once in the excited state, the molecule can undergo several different processes in order to lose the acquired energy. These are:
- Luminescence
- Fluorescence
- Phosphorescence
Only a minority of molecules show Fluorescence, which makes the Fluorescence detector one of the most specific detectors available for HPLC analysis. The diagram below presents some features of selected transition energies.
Luminescence
Luminescence is the emission of light and occurs when molecules change from an excited state to their ground state. Molecules can be excited by different forms of energy, each with its own excitation process. For example, when the excitation energy is light, the process is called photoluminescence. In basic cases, the emission of light is the reverse of absorption.
Fluorescence and Phosphorescence
Fluorescence and Phosphorescence are both examples of photoluminescence and occur in more complex molecules.
Phosphorescence
Phosphorescence is a longer process than fluorescence because one of the electrons involved in the excitation changes its spin, (Inter System Crossing) during a collision with a molecule of solvent, for example. The excited molecule is now in a so-called triplet state T. The molecule must change its spin back again before it can return to its ground state.
Since the chance of colliding with another molecule with the necessary spin for change is slight, the molecule remains in its triplet state for some time. During the second spin change, the molecule loses more energy by relaxing without radiation. The light which is emitted during phosphorescence, therefore, has less energy and is at a longer wavelength than fluorescence. Phosphorescence typically takes 10-3 seconds.
Fluorescence
Fluorescence: When a more complex molecule transforms from its ground energy state into an excited state, the absorbed energy is distributed into various vibrational and rotational sub-levels. When this, same molecule returns to the ground state, this vibrational and rotational energy is first lost by relaxation without any radiation.
Then the molecule transforms from this energy level to one of the vibrational and rotational sublevels of its ground state, emitting light. Internal conversion, a transition from S2 to S1 is highly favored because there are typically excited levels in the next lowest singlet state that have the same energy as the higher energy singlet state. If a molecule emits light 10 –9 to 10-5 seconds after it was illuminated then the process is fluorescence.
Fluorescence Detectors – Excitation & Emission
The excitation and emission spectra of Quinidine are shown next. The excitation spectrum corresponds to the absorption of photons from the ground to the excited states of the molecule. It will be very similar to the absorbance spectrum obtained with a diode array detector. There will be some differences in the spectra due to variations in the optical components of the HPLC detectors.
The emission spectra occur at higher wavelengths (lower energy) and correspond to the photon emission from the lowest level singlet state to the ground state of the molecule. Spectra such as these can be collected for each component in the chromatogram to optimize the analysis for each individual component. The principles of determining optimum wavelengths are explained in more detail next.
HPLC Detectors – Refractive Index Detectors Instrumentation
Refractive Index HPLC Detectors are widely used in the Pharma industry. Initially, both the sample and reference cell are flushed with the mobile phase. The reference cell is then closed and solvent flows only through the sample cell. The refractive index of the mobile phase in both cells is the same and the position of the zero glass can adjust so that the detector is in optical balance with an equal amount of light falling on each diode. When the sample elutes from the column into the sample cell the refractive index of the cell contents changes.
The change in RI deflects the light beam as it passes through the flow cell resulting in an unequal amount of light falling on each diode. The change in current from the diodes that this causes is amplified and used to produce the calibrated detector signal. This signal expressed, as nano Refractive Index Units (nRIU), corresponds to the difference between the refractive index of the sample in the sample cell and the mobile phase in the reference cell. where:
RI Detector instrument components
1. Mobile Phase Container: Mobile phase reservoir also collects mobile phase when in recycling mode.
2. Heat Exchanger: RI changes with temperature –it is desirable to keep the temperature of the mobile phase in the detector constant though out the analysis.
3. Sample Cell: Mobile phase continuously flows though the sample cell. The RI of the Sample Cell is compared with the RI of the Reference cell. The greater the concentration of analyte in the sample cell the greater the difference in RI.
4. Reference Cell: This is purged with a ‘blank’ phase before the analysis/injection of the sample. The RI of the Sample Cell is compared with the RI of the Reference cell. The greater the concentration of analyte in the sample cell the greater the difference in the RI HPLC detector.
5. Sample Photomultiplier: Light intensity is measured using a photomultiplier tube. This converts light intensity into an electrical signal that can measure and recorded. The difference in response between the reference photodiode and sample photodiode records.
6. Beam Splitter: Split the beam of light allowing light from the same source to pass onto the reference and sample photodiodes
7. Lens and Slit: Focuses the Beam of light onto the Beam Splitter
8. Reference Photomultiplier: A reference photodiode is located prior to the flow cell. The diode measures the light transmitted by the lamp prior to refraction. The difference in response between the reference photodiode and sample photodiode records.
9. Waste Container: Collects mobile phase eluent when not in recycling mode
10. Recycle Valve: As most RI detectors are unsuited to gradient analysis (the composition of the mobile phase in the reference cell remains constant though out the analytical run) it is possible to recycle the mobile phase
11. Purge Valve: Enables filling of the reference cell with ‘blank’ eluent before the analysis/injection of the sample. The RI of the Sample Cell is compared with the RI of the Reference cell. The greater the concentration of analyte in the sample cell the greater this difference in RI
12. Detector Lamp: Light Source.
RI Detector, important notes
When the Purge Valve is ‘ON’, the mobile phase flows into the reference cell via the heat changer, fills the reference cell flows back out via the same heat exchanger through the purge valve and either to waste or to the solvent bottle depending on the position of the recycle valve. Once the reference cell is full the purge valve closes to stop the flow to the reference cell.
When the Purge Valve is ‘OFF’, the mobile phase flows in though the entrance port, through the heat exchanger into the sample cell, back out through the heat exchanger, into the purge valve, and either to waste or to the solvent bottle depending upon the position of the recycle valve.
Refractive Index (RI) Detector Principles
This is one of the best HPLC detectors. When a beam of light passes from one medium into another, both the wave velocity and direction change. The change in direction is called refraction. The refractive index (R.I.) detector is a nonselective or universal detector. This means that it can detect almost every compound, but not at low levels.
The detection principle is based on the comparison of the refractive index of the eluent and sample with a reference solution of the eluent only. Because the composition of the eluents must remain fixed throughout the analysis, this detector is not suitable for gradient analysis. The basics of Refractive Index detection are described in the schematic diagrams next.
The reference section of the flow cell is filled with eluent only prior to analysis. In this way a ‘baseline’ response due to the eluent can be determined. The reference cell remains filled throughout the analysis.
The mobile phase flows continually through the sample section of the flow cell during the analysis. As the sample passes into the flow cell the refractive index will change and the resultant change will cause the light beam to deviate away from the photomultiplier point detector.
The change in light intensity is proportional to the change in refractive index. Higher sample concentrations will give greater refraction and therefore a greater response change on the photomultiplier. The relationship between the angle of incidence and the angle of refraction is expressed in Snell’s Law of refraction.
Hope, you enjoyed the above article on HPLC Detectors.
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