HPLC Laboratory Experiments

The sixth item is a series of three HPLC laboratory experiments. The first familiarizes the student with getting a system up and running and calibrating a column with standards. The second experiment shows how to clean a column and pacify a system. The last is a first, quick look at methods development. 

Strom, S.L. (1993) Production of phaeopigments by marine protozoa results of laboratory experiments analyzed by HPLC. Deep-Sea Res. 40, 57-80. 

In the early days of TLC, before the advent of HPLC, researchers experimented in their laboratories with developing chambers of glass, sometimes V4A steel or, for less aggressive solvent systems, plastics. Various chambers were used for the following purposes ... 

Usually, to analyze trichothecenes in foods and feeds, solvent extraction is essential. Aqueous methanol and acetonitrile are conunonly used for extraction. For applying GC-MS, HPLC, and LC-MS/MS, a sample needs to be cleaned up with a charcoal-alumina-celite, florisil, silica gel, or solid-phase extraction column. For laboratory experiments, TL is very useful because of its low cost and simplicity. 

M Munari, M Miurin, G Goi. Didactic application to riboflavin HPLC analysis. A laboratory experiment. J Chem Educ 68 78-79, 1991. 

The high cost of the HPLC-NMR system is of course a factor which must be taken into consideration. Since most laboratories have seen the value of NMR in its traditional form, it is only necessary to add an HPLC system, an appropriate flow probe and a flow-control unit to an existing spectrometer to enable HPLC-NMR experiments to be performed. The cost of these accessories, or even the cost of a complete system dedicated to HPLC-NMR, is offset by the efficiency of the method. The laborious extraction of minor components from complex mixtures followed by off-line analysis or in the case of synthetic drugs the synthesis of radiochemically... 

Eor the future, the reduction in cost of LC-MS systems will increase their accessibility and, in combination with GC-MS, will greatly improve the efficiency and accuracy with which the analysis of antidegradants, cure systems, plasticisers and other additives can be carried out. The combination of these two instruments will enable the full spectrum of polarity and molecular weight to be covered and enable a quantum leap in capability to be achievable by all analytical laboratories. Experiments which for years have had to be carried out by TLC because of the limitations of conventional HPLC instruments will now be carried out by LC-MS. 

Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. 

Much of the recent impetus for temperature control has focused on exploiting the effects of elevated temperature on viscosity and diffusion coefficients [2], These lead to faster separations and also allow smaller particle diameters to be employed with conventional HPLC hardware. As the viscosity of solvents decreases, the column pressure drops. This can be exploited by using faster flow rates and smaller particle diameters. All of this leads to faster separations. In one experiment in this laboratory, a separation which required 8 min at room temperature was reduced to 2 min at 50°C without changing the column. Speed enhancements of as much as 50-I00-fold have been reported [13] as shown in Figure 9.1. 

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