Mass Spectrometry and HPLC of peptides Lab Report

Mass Spectrometry and HPLC of peptides - Lab Report Example The mass of the protonated and solvated molecules pass through the mass analyzer that establishes the mass of the analyte. Reversed phase high pressure liquid chromatography relies on the reversible water-hating associations between the molecules of the solute present in a polar mobile phase and the immobilised hydrophobic ligand in the solid support to effect separation (David 2001). The dispersal of the solute between the two phases is a function of the binding attributes of the medium, the polarity of the solute and the constituents of the mobile phase. The polarity of the mobile phase is lowered by the addition of organic solvents, which minimize the hydrophobic associations between the solute and the stationary phase hence causing desorption. Hydrophobic molecules end up spending more time on the solid support hence requiring a high solvent concentration to promote desorption. The mass to charge ratio (m/z) is the proportion of the mass of a substance to the number of ions present in a substance. This value is obtained sing the formula m/z = (MW + nH+)/n where m/z = the mass-to-charge ratio, which is indicated on the axis of the spectrum; MW is the molecular mass of the sample; n is the integer number of charges on the ions and H is the mass of a proton, which is equivalent to 1.008 Da. The above equation can be used to find the molecular weight of a sample provided that the sum of charges on an ion is established. The m/z value is read from the spectrum and substituted into the equation. Usually, during the calculation of charge, it is assumed that any two adjacent members in the sequence of multiplied charged ions differ by one charge. The mass of charge ratio (m/z) of large molecules such as ionised proteins, for example, lysozyme and bovine serum albumin is obtained in the same manner as described. The charge of the ion is found by monitoring adjacent charge states and substituted into the above equation alongside the m/z ratio that