Chromatography

Chromatography lecture and my learning diaries

The chromatography is a collective terms for separation of mixtures techniques. The sample (Mixtures) is dissolved in the mobile phase, which carried it through the stationary phase. The separation was achieved by the difference of travel time/speeds. Several chromatography methods have been developed during the past time including paper chromatography, gas chromatography, and high performance liquid chromatography (HPLC). In this lecture, the teacher mainly introduced the principle of HPLC.
HPLC is a technique to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual component. The instruments consists of PUMP (moves the mobile phase and sample through the column), injector (add samples), column and detector.
Different liquid chromatography including gel filtration, ion exchange, affinity and reversed phase chromatography are utilized according to the protein/peptide properties including size, net surface charge, hydrophobicity, respectively. Different methods can be combined according to the purpose, but the most important is keep it simple because quite a lot of samples are lost during the process.

Introduction to MS

Mass spectrometry (MS) can identify chemical composition of a sample based on the mass-to-charge ratio of charged particles. The instrument mainly contains three parts including ion source, mass analyzer and detector. The samples are ionized in the ion source using chemical or electron modes. Ions from the ion source were separated according to the m/z ratios in the analyzer part.
Two biological mass spectrometries were introduced during the lecture: Matix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). Both of them belong to soft ionization methods. The irradiated substance is embedded in crystallized matrix in MALDI. In contrast of MALDI, the ionization in ESI is achieved by spraying a solution into an electrical field. MS has been applied in many fields of protein research including protein identification, molecular weight determination, characterisation of post-translation modifications, relative quantification and also protein complex, etc.
It is important to note that the protein needs to be in solution without salts and detergents and should be purified before the MW measurement. For protein identification, the protein needs to be digested into peptide before analyzing by MS. The identification can base on peptide mass finger print (PMF) and MS/MS data from one or more peptides.