Related Experiment Video
Updated: May 13, 2026

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Use of an internal reference for the quantitative HPLC-UV analysis of solid-phase reactions: a case study of
Jan Spengler1, Anna-Iris Fernandez-Llamazares, Fernando Albericio
1Institute for Research in Biomedicine , Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain.
Abstract:
Here we evaluated the use of internal reference compounds for the rapid assessment of reactions performed in solid-phase. An internal reference compound (commercially available) was bound to the resin, together with the substrate, and cleaved with the products after completion of the reaction. The peak area of the reference compound in the HPLC-UV chromatograms can be correlated directly with those of other compounds present in the reaction mixture, thereby allowing a quantitative interpretation of the chromatograms with respect to conversion and yield. The usefulness of this method was demonstrated by optimization of a protocol for the synthesis of proline-based tripeptides.
Related Concept Videos
High-Performance Liquid Chromatography: Introduction
In HPLC, two phases play a critical role in the separation process:
High-Performance Liquid Chromatography: Elution Process
Chemical Shift: Internal References and Solvent Effects
The internal reference compound generally used in NMR spectroscopy is tetramethylsilane (TMS). TMS is preferred because it is chemically inert, soluble in NMR solvents, and easily removable. Also, the highly shielded methyl protons in TMS yield an intense...
High-Performance Liquid Chromatography: Instrumentation
High-Performance Liquid Chromatography: Types of Detectors
