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. 2019 Mar 28;24(7):1215.
doi: 10.3390/molecules24071215.

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology

Diego Sebastián Insuasty Cepeda  1 , Héctor Manuel Pineda Castañeda  2 , Andrea Verónica Rodríguez Mayor  3 , Javier Eduardo García Castañeda  4 , Mauricio Maldonado Villamil  5 , Ricardo Fierro Medina  6 , Zuly Jenny Rivera Monroy  7
Affiliations

Synthetic Peptide Purification via Solid-Phase Extraction with Gradient Elution: A Simple, Economical, Fast, and Efficient Methodology

Diego Sebastián Insuasty Cepeda et al. Molecules. .

Abstract

A methodology was implemented for purifying peptides in one chromatographic run via solid-phase extraction (SPE), reverse phase mode (RP), and gradient elution, obtaining high-purity products with good yields. Crude peptides were analyzed by reverse phase high performance liquid chromatography and a new mathematical model based on its retention time was developed in order to predict the percentage of organic modifier in which the peptide will elute in RP-SPE. This information was used for designing the elution program of each molecule. It was possible to purify peptides with different physicochemical properties, showing that this method is versatile and requires low solvent consumption, making it the least polluting one. Reverse phase-SPE can easily be routinely implemented. It is an alternative to enrich and purified synthetic or natural molecules.

Keywords: gradient elution; peptide; preparative purification; solid phase extraction (SPE); solid phase peptide synthesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dwell time determination [26,27,28]. Programed elution gradient (red line), experimental gradient performed by the HPLC system (black line). Delay time (ti), gradient time (tG), dwell time (tD).
Figure 2
Figure 2
RP-HPLC analysis of crude 20–25LfcinB/32–35BFII. The chromatographic profile shows a main peak at 5.10 min, corresponding to the peptide with a purity of 60%. The chromatographic profile of fractions N° 3, 6, and 11 collected during reversed-phase solid phase extraction (RP-SPE) purification is also shown. Specifically, those fractions contained 11, 18, and 23% solvent B, respectively.
Figure 3
Figure 3
Purification of Fmoc-Asn(GlcAc4)-OtBu (2) by RP-SPE. (A) Crude product chromatographic profile. (B) Chromatograms of collected fractions (6 to 13) (left) and used elution program (right). (C) Purified product chromatographic profile (fractions 11–13).
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