chemistry

Reversed phase liquid chromatography LC/HPLC What is reverse phase liquid chromatography (LC) / HPLC? Reverse phase chromatography is the most commonly used LC or HPLC separation mode . It is used to separate nonpolar molecules in solution . In reverse phase the stationary phase is nonpolar and the mobile phase is polar . The name “ reversed phase ” is derived from the opposite technique of “ normal phase ” chromatography which involves the separation of molecules based upon their interaction with a polar matrix (silica beads) in the presence of a nonpolar mobile phase (nonpolar solvent). This chromatographic technique is far superior to the other modes of separation in the variety of target compounds it can analyze. The mechanism in reverse phase separation is shown in Fig. I.1 and  has to do with the binding of a solute - having both hydrophobic and hydrophilic parts (groups) -  to a stationary hydrophobic molecule (station...

The object of chromatography is to separate a sample into a series of chromatographic peaks , within a reasonable time, each representing a single component of the sample. The resolution (R) between two peaks (Fig. 1) is given by equation 1: R = (t R2 – t R1 ) / [(w 2 + w 1 )/2]  = 2 Δ t / (w 2 + w 1 )          (1) Where, t R2 και t R1 are the retention times of the retained components 1 και 2 measured from injection to peak maximum and w 2 and w 1 are peak widths in units of time measured at the base of the peaks 1 and 2. Fig 1: Peak resolution in chromatography. Where, t R2 και t R1 are the retention times of the retained components 1 και 2 measured from injection to peak maximum and w 2 and w 1 are peak widths in units of time measured at the base of the peaks 1 and 2. For two peaks that are close w 2 = w 1 and equation (1) gives: R =  Δ t / w 1      (2) Resolution depends upon two factors: Nar...