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.

INTRODUCTION TO SPECTROSCOPY

Spectroscopy was originally the study of the interaction between radiation and matter as a function of wavelength (λ). In fact, historically , spectroscopy referred to the use of visible light dispersed according to its wavelength, e.g. By a prism. Later the concept was expanded greatly to comprise any measurement of a quantity as a function of either wavelength or frequency. Thus it also can refer to a response to an alternating field or the varying frequency (ν). Spectroscopy originated through the study of visible light dispersed according to its wavelength. Later the concept was expanded greatly to comprise any interaction with radiative energy as a function of its wavelength or frequency. A further extension of the scope of the definition added energy (E) as a variable, once the very close relationship E = hν for photons was realized (h is the Planck constant). A plot of the response as a function of wavelength—or more commonly frequency—is referred to as a spectrum.

Spectrometry is the spectroscopic technique used to assess the concentration or amount of a given species. In those cases, the instrument that performs such measurements is a spectrometer or spectrograph.
Spectroscopy/spectrometry is often used in physical and analytical chemistry for the identification of substances through the spectrum emitted from or absorbed by them. Spectroscopy/spectrometry is also heavily used in astronomy and remote sensing. Most large telescopes have spectrometers, which are used either to measure the chemical composition and physical properties of astronomical objects or to measure their velocities from the Doppler shift of their spectral lines.