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Chromatography

Chromatography is a group of techniques that enables the separation of mixtures. This separation is based on differences between the unique affinity of the mixture’s components to the two phases of chromatography: mobile and stationary.

In the mobile phase, the sample moves through a chromatographic column (where the stationary bed is within a tube).

In the stationary phase, the sample interacts with the stationary bed and undergoes separation.

While in the column stage, elution (washing of the stationary phase containing the mixture with the mobile phase) is carried out by a pre-defined methodology. The eluent (mobile phase) is collected in fractions, which are put on a thin-layer chromatography (TLC) plate. After running the plate, analysis takes place and the desired material is collected by the TLC plate. Analytical high-performance liquid chromatography (HPLC) procedures can also be performed.

While there are many chromatographic techniques, TAPI primarily uses two methods:

  • Low-pressure flash chromatography uses big particle size silica of fixed length to obtain fast flow of the mobile phase. TAPI uses glass columns with diameters ranging from 3 to 45 cm, with standard silica 60. This method is effective for vitamins and prostaglandins (such as calcitriol, calcipotriene, paricalcitol and latanoprost).
  • High-pressure preparative chromatography uses small particle size silica, with silica length varying according to the pressure developed in the column. TAPI uses stainless steel columns with diameters ranging from 5 to 15 cm. This method is well suited for three types of sorbents:
    • regular silica for vitamins
    • cyano silica for prostaglandins
    • reverse-phase silica (C18) for peptides and similar materials (e.g., caspofungin). It is effective for some vitamins and prostaglandins (such as calcipotriene, paricalcitol, latanoprost and travoprost).