High performance liquid chromatographs (HPLCs) use a liquid mobile phase to separate the components of a mixture. The components are dissolved in a solvent and forced to flow through a chromatographic column under high pressure. In the column, the mixture is resolved into its components. The components flow through a detector and a chromatogram is generated. Most HPLCs use high-pressure pumps to force solvents through packed stationary beds. Isocratic pumps use constant mobile phase composition to elute compounds. By contrast, gradient pumps vary the strength of the organic solvent to elute different compounds. Pumps with flow control are well-suited for HPLCs that deliver gradients automatically. Specifications, such as flow rate, maximum pump pressure, and pump accuracy vary among devices. In simple systems, the sample is introduced with an injection valve. More complex HPLCs incorporate an autosampler with a microprocessor. 

Applications

HPLCs use several detection methods. Ultraviolet (UV) detectors measure the ability of a sample to absorb light at one or more wavelengths. Light scattering detectors nebulize the column effluent, vaporize the solvent, and then detect droplets in a light scattering cell. Electrochemical detectors measure the current from the oxidation/reduction reaction of an analyte at a suitable electrode. Radiochemical detectors use tritium or carbon-14 to detect the fluorescence associated with beta-particle ionization. Mass spectroscopy detectors ionize a sample and use a mass analyzer to detect the ion current. Nuclear magnetic resonance detectors irradiate nuclei that are placed between the poles of a strong magnet. The radiation is absorbed, the parallel nuclei enter a higher energy state, and each atom produces a spectra specific to its location and chemical composition. Some HPLCs measure the change in the refractive index of the column effluent passing through the flow cell. Others detect the fluorescence that occurs when compounds are excited by shorter wavelength energy and emit higher wavelength radiation.

Features

HPLCs vary in terms of general features, display options, and interface options.

General features include:

• self-calibration
• self-test diagnostics
• data storage
• programmability.

Devices with a built-in chromatograph and application software are commonly available. Some HPLCs are suitable for extreme temperatures. Others output an analog voltage, analog current, frequency, or pulse.

Examples include:

• amplitude modulation (AM)
• frequency modulation (FM)
• pulse width modulation (PWN)
• sine waves,
• pulse trains

Analog devices usually include an analog meter and a front panel with switches and knobs. Digital devices often provide a serial or parallel computer interface and a video display. Serial protocols include universal serial bus (UBS), RS232, RS422, and RS485.

Related Information

Engineering360—HPLC Columns Information

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