Analytical and Semi-preparative Chromatography of Aromatic Compounds on a Single HPLC System

The Gilson PLC 2050 Purification System was evaluated in its capacity to scale up a separation of aromatic compounds, performing the separation using analytical HPLC, as well as semi-preparative HPLC. The results demonstrated the Gilson systems capability to achieve reproducible injections on analytical columns generating chromatograms comparable to a typical analytical HPLC system as well as scaling up to preparative columns.

Introduction
A simple mixture of four compounds was used to evaluate the capacity of the Gilson PLC 2050 to scale up a separation from analytical to semi-preparative HPLC. The analytical method was first developed on a LaChrom Elite™ analytical HPLC system and then transferred to the Gilson PLC 2050 (Figure 1). The method is then scaled up 50-fold using the PLC 2050 system to allow for higher throughput separations on a semi preparative HPLC column (25 mm I.D.).

Materials & Methods
LaChrom Elite (Hitachi) is equipped with a 10 mL/min quaternary gradient pumping system, Photodiode Array Detector (200-800 nm), and EZChrom Elite™ Chromatography Data Software.
PLC 2050 (Gilson) is equipped with a 50 ml/min quaternary gradient pumping system, UV/Vis detector (200-840nm), and Gilson Glider Prep Software.
Analytical Method is the same for all tests.

Experimental conditions used to generate the analytical and preparative chromatograms:
Column - 
Analytical: Merck Purospher® RP18e (250 mm x 4.6 mm I.D. 5μm with pre column)
Preparative: Merck LiChrospher® 100 RP-18 (250mm x 25 mm I.D. 5μm)
Sample - Mixture of hydroquinone, catechol and phenol at 0.5g/L and piperine at 0.01g/L.
Detection - UV/Vis detector at 280nm.
Eluent A - phosphate buffer (0.02M, pH = 5.5 adjusted with NaOH 6N).
Eluent B - Methanol
Method - 
20% B - 3 min
20% to 90% B - 7 min
90% B - 20 min.

Results & Discussion
In this test, the profile of analytical separation (20 uL injection) on the PLC 2050 was similar to the chromatogram obtained using the LaChrom EliteTM HPLC system. Furthermore, the use of a preparative column (1 ml injection) with the PLC 2050 demonstrates clear separation of the compounds.

Summary

• The Gilson PLC 2050 system allows for reproducible analytical injections on 4.6 mm I.D. columns with high efficiency.
• This application note also demonstrates scale up to a 25 mm I.D. column.
• It is also possible to use the Gilson PLC products to perform similar scale-up with preparative columns, up to 100 mm I.D. (results not shown).