HPLC Detectors

The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column. The bandwidth and height of the peaks may usually be adjusted using the coarse and fine tuning controls, and the detection and sensitivity parameters may also be controlled (in most cases). There are many types of detectors that can be used with HPLC. Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS).

Refractive Index (RI) detectors measure the ability of sample molecules to bend or refract light. This property for each molecule or compound is called its refractive index. For most RI detectors, light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell directs the mobile phase passing through the column while the other directs only the mobile phase. Detection occurs when the light is bent due to samples eluting from the column, and this is read as a disparity between the two channels.
Ultra-Violet (UV) detectors measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths:
A)Fixed Wavelength measures at one wavelength, usually 254 nm
B)Variable Wavelength measures at one wavelength at a time, but can detect over a wide range of wavelenths
C)Diode Array measures a spectrum of wavelengths simulateneously.

UV detectors have a sensitivity to approximately 10-8 or 10 -9 gm/ml.

Fluorescent detectors measure the ability of a compound to absorb then re-emit light at given wavelengths. Each compound has a characteristic fluorescence. The excitation source passes through the flow-cell to a photodetector while a monochromator measures the emission wavelengths. Has sensitivity limit of 10-9 to 10-11 gm/ml.

Radiochemical detection involves the use of radiolabeled material, usually tritium (3H) or carbon-14 (14C). It operates by detection of fluorescence associated with beta-particle ionization, and it is most popular in metabolite research. Two detector types:
A)Homogeneous- Where addition of scintillation fluid to column effluent causes fluorescence.
B)Heterogeneous- Where lithium silicate and fluorescence caused by beta-particle emission interact with the detector cell.
Has sensitivity limit up to 10-9 to 10-10 gm/ml.

Electrochemical detectors measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential.
Has sensitivity of 10-12 to 10-13 gm/ml

Mass Spectroscopy (MS) Detectors- The sample compound or molecule is ionized, it is passed through a mass analyzer, and the ion current is detected. There are various methods for ionization:
A) Electron Impact (EI)- An electron current or beam created under high electric potential is used to ionize the sample migrating off the column.
B)Chemical Ionization- A less aggresive method which utilizes ionized gas to remove electrons from the compounds eluting from the column.
C)Fast Atom Bombarbment (FAB)- Xenon atoms are propelled at high speed in order to ionize the eluents from the column.
Has detection limit of 10-8 to 10-10 gm/ml.

Nuclear Magnetic Resonance (NMR) Detectors- Certain nuclei with odd- numbered masses, including H and 13C, spin about an axis in a random fashion. However, when placed between poles of a strong magnet, the spins are aligned either parallel or anti-parallel to the magnetic field, with the parallel orientation favored since it is slightly lower in energy. The nuclei are then irradiated with electromagnetic radiation which is absorbed and places the parallel nuclei into a higher energy state; consequently, they are now in "resonance" with the radiation. Each H or C will produce different spectra depending on their location and adjacent molecules, or elements in the compound, because all nuclei in molecules are surrounded by electron clouds which change the encompassing magnetic field and thereby alter the absorption frequency.

Light-Scattering (LS) Detectors- When a source emits a parallel beam of light which strikes particles in solution, some light is reflected, absorbed, transmitted, or scattered. Two forms of LS detection may be used to measure the two latter occurrences:
A) Nephelometry- This is defined as the measurement of light scattered by a particulate solution. This method enables the detection of the portion of light scattered at a multitude of angles. The sensitivity depends on the absence of background light or scatter since the detection occurs at a black or null background.
B) Turbidimetry- This is defined as the measure of the reduction of light transmitted due to particles in solution. It measures the light scatter as a decrease in the light that is transmitted through the particulate solution. Therefore, it quantifies the residual light transmitted. Sensitivity of this method depends on the sensitivity of the machine employed, which can range from a simple spectrophotometer to a sophisticated discrete analyzer. Thus, the measurement of a decrease in transmitted light from a large signal of transmitted light is limited to the photometric accuracy and limitations of the instrument employed.

Near-Infrared Detectors- Operates by scanning compounds in a spectrum from 700 to 1100 nm. Stretching and bending vibrations of particular chemical bonds in each molecule are detected at certain wavelengths. This is a fast growing method which offers several advantages: speed (sometimes less than 1 second), simplicity of preparation of sample, multiple analyses from single spectrum, and nonconsumption of the sample