Purification of chlorogenic acids by solid phase extraction

Abstract

Solid phase extraction (SPE) with usage of syringe cartridges DIAPAK C18 for purification and concentration of chlorogenic acids from green coffee beans or infusions was worked up. The problem is a consequence of low lipophilicity of chlorogenic acids that is resulted in low retention of the solutes at reversed-phase conditions. The method was adopted for the HPLC determination of the acids by reversed-phase special gradient mode with spectrophotometric detection (325 nm). In the mode the three chlorogenic acid isomers were eluted by isocratic mode for correct solutes quantification without regard to solvatochromic effects and a gradient elution for desorption of highly retained concomitant solutes. The acidification of the mobile phase was shown to be necessary to achieve the best reversed-phase sorption at SPE as well as for subsequent chromatographic performance. Utilization of two cartridges connected in series was proposed to control the breakthrough of the least retainable 3-caffeoylquinic (neochlorogenic) acid (3CQA) during SPE, while acidification of water extracts to pH 1 – 0 is highly desirable to achieve 2.5-fold concentration after solute reextraction by a proper solvent (30 vol. % of CH3CN and 3 vol. % of HCOOH in water) with subsequent dilution (1 : 1) with water. For plant sources without 3CQA extracts may be purified and other chlorogenic acids may be concentrated up to 5-fold due to better sorbtivities of 5-caffeoylquinic (chlorogenic) and 4-caffeoylquinic (cryptochlorogenic) acids compared to 3CQA. The attention was paid to influence of sample solvent composition and injected sample volume upon chromatographic performance to escape appearance of chromatographic artefacts leading to chromatographic peak distortion and splitting. Methanol excess in sample solution had a higher impact upon peak distortion compared to acetonitrile.