HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography), HPLC, is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture.

Basic block diagram

HPLC is accomplished by injection of a small amount of liquid sample into a moving stream of liquid (called the mobile phase) that passes through a column packed with particles of stationary phase. Separation of a mixture into its components depends on different degrees of retention of each component in the column. The extent to which a component is retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phase. In HPLC this partitioning is affected by the relative solute/stationary phase and solute/mobile phase interactions. Thus, unlike GC, changes in mobile phase composition can have an enormous impact on your separation. Since the compounds have different mobilities, they exit the column at different times; i.e., they have different retention times, tR. The retention time is the time between injection and detection. There are numerous detectors which can be used in liquid chromatography. It is a device that senses the presence of components different from the liquid mobile phase and converts that information to an electrical signal. For qualitative identification one must rely on matching retention times of known compounds with the retention times of components in the unknown mixture.Quantitative analysis is often accomplished with HPLC. An automatic injector providing reproducible injection volumes is extremely beneficial, and are standard on modern commercial systems.

HPLC is just one type of liquid chromatography, meaning the mobile phase is a liquid. In this lab you will use what is called reversed phase HPLC. Reversed phase HPLC is the most common type of HPLC. What reversed phase means is that the mobile phase is relatively polar, and the stationary phase is relatively non-polar. Thus non-polar compounds will be more retained (i.e. Have longer retention times) than a polar compound. In normal phase HPLC, the mobile phase is relatively non-polar and the stationary phase is relatively polar. Other more general types of HPLC include partition, adsorption, ion-exchange, size-exclusion, and thin-layer chromatography.

Application :

• Quantitative/qualitative analyses of amino acids, nucleic acids, proteins in physiological samples.
• Measuring levels of active drugs, synthetic by-products, degradation products in pharmaceuticals.
• Measuring levels of hazardous compounds such as pesticides and insecticides.
• Monitoring environmental samples.
• Purifying compounds from mixtures.

LASER BASED LIQUID PARTICLE COUNTER

A particle counter is an instrument that detects and counts particles. By its very nature a particle counter is a single particle counter, meaning it detects and counts particles one at a time. The nature of particle counting is based upon either light scattering, light obscuration, or direct imaging. A high energy light source is used to illuminate the particle as it passes through the detection chamber. The particle passes through the light source (typically a laser or halogen light) and if light scattering is used, then the redirected light is detected by a photo detector. If direct imaging is used, a halogen light illuminates particles from the back within a cell while a high definition, high magnification camera records passing particles. Recorded video is then analyzed by computer software to measure particle attributes. If light blocking (obscuration) is used the loss of light is detected. The amplitude of the light scattered or light blocked is measured and the particle is counted and tabulated into standardized counting bins.

  The laser beam passes through the walls of a glass container or a flow-thru cell

Particle counter uses as its basic light source a laser diode (650nm wavelength). The beam from this laser is spatially filtered and focused by a lens assembly to form a small and well-defined illuminated volume within the liquid being inspected. A scanning mechanism provides a circular displacement of this illuminated volume at a constant rate of speed. As the illuminated volume moves across a particle suspended in the liquid, some light from the beam will be scattered. This is known as Fraunhofer diffraction. Most of this scattered light is in the near-forward direction and is collected by the optical system of the photodetector assembly. The flash of light striking the photodetector will cause an electrical pulse in the preamplifier connected to the photodetector. The amplitude and width of this pulse are a function of the size of the particles, The analog signals generated by the light pulses are routed to a computer and digitized.

Applications :

• Quality control of hydraulic fluids and oils.
• De-ionized water and acid testing for semiconductor manufacturing.
• Vial and ampule inspection for pharmaceuticals.
• Silt and sediment sizing.
• Oceanographic particles.
• Sizing for corrosive chemicals and solvents.
• Cell counting where physical force would damage particles.
• Particle agglomeration studies.
• Water treatment plants.
• Filter efficiency control.
• Powdered solids manufacturing.