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I-RITE Statement Archive
About I-RITE

Drawing Chemical Maps of Complex Samples

My Moberg
Department of Chemistry
Uppsala University
March 2002


In my research I develop analytical methods to study the chemistry of complex samples, such as body fluids or samples from nature. The strategy I use is to hyphenate two techniques, one technique to separate the component in the sample by applying the chemical rule "like dissolve like" and one technique to characterize them according to their mass and electrical charge. This gives me a map of the sample components.

The reason for looking at a specific sample varies; sometimes only one component in the sample is of interest, in which case the method development becomes a "fishing out" procedure, and sometimes a set of components are of interest. In one of my projects I am developing an experimental method to study the peptide part of cerebrospinal fluid (CSF). This fluid surrounds the central nervous system (CNS), the brain and the spinal cord, and is isolated from the bloodstream by barriers. Fluctuations in CSF will thus reflect alterations in the function of the barriers or variations in production of substances in the brain. It has been shown that peptides play an important part in many neural diseases and hence analytical methods to study the peptide part of CSF are of significant interest to neural chemists. Traditionally very time consuming and/or non-specific techniques have been used for this purpose but the introduction of combined techniques offers new possibilities.

A common approach in all my projects is that I use reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) to analyze samples. Liquid chromatography is a separation technique utilized to separate different analytes in a liquid sample by applying the rule "like dissolve like". The separation is achieved by injection of the sample onto a column (a steel or glass cylinder) packed with small particles and through which a liquid is pumped continuously. The injection results in a plug of sample in the beginning of the column. As the sample plug is pushed through the column the components will interact with the surface of the particles to different extent according to their chemistry. It is like adding water to a mixture of vinegar and oil. The vinegar and oil are immiscible and will be divided in two layers and since the water is more similar to vinegar in its chemistry it will mix more readily with the vinegar than with the oil. In RPLC the oily part is analogous to the "stationary" particles while the vinegar part is the "mobile" liquid. The analytes with a chemistry more like the mobile phase will arrive at the end of the column first, while those preferring the stationary phase will travel much more slowly through the column. This leads to a separation of the analytes in the time frame.

Once the sample constituents have been separated they need to be identified. Identification, or at least characterization, of the sample components can be achieved with mass spectrometry. In the mass spectrometer gaseous ions (components with an electrical charge) are detected according to their mass-to-charge ratio. In a mass spectrum the intensities in counts per second of different mass-to-charge ratios are given. Sometimes a compound gives only one mass-to-charge ratio but most often a set of fragments and adducts are detected. This information characterizes the compound being analyzed, but the information is not always enough for direct identification. The combination of RPLC and MS renders separation both in the time and in the mass-to-charge domain. This relationship can be represented graphically by drawing the time on one axis and the mass-to-charge ratio on the other, which gives a map of the sample components.

The hyphenation of RPLC and MS is used in a number of research areas, such as pharmaceutical and environmental chemistry, but still a number of difficulties concerning analyzing complex samples exist, especially when sets of components are to be analyzed simultaneously. Not only do we need to develop new experimental methods, we also need to look over how we handle and interpret all the information we gain from an RPLC-MS run. Finding appropriate evaluation techniques also becomes a part of drawing maps of complex samples and in the future I will incorporate this into my research.