32
May/June 2011
Chromatography…….. but not as we know it
by Keith Freebairn1 Guy Harris3
*, David Johns1 , Peter Hewitson4
, Nathalie Douillet1 , Ian Sutherland4
, Chris Thickitt1 , Svetlana Ignatova4
1 GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts., SG1 2NY, UK 2 Pfizer UK Group Ltd., Sandwich, Kent , CT13 9NW, UK
3 Dynamic Extractions Ltd, 890 Plymouth Road, Slough, SL1 4LP, UK 4 Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK
*Corresponding author. Tel: +44 (0) 1438 763705; Fax: +44 (0) 1438 764414 E-mail address:
keith.w.freebairn@
gsk.com
, Elsa Vilminot1 , Ben Mathews2 ,
The use of countercurrent chromatography (CCC) as a preparative technique is seen to occupy a niche area of separation science and is largely used to isolate natural products. The technique has however considerable untapped potential both at the laboratory preparative scale and also at larger scale………….
Introduction
This article gives a chromatographer’s perspective of the potential of countercurrent chromatography (CCC) in the pharmaceutical industry, specifically relating to high- performance countercurrent chromatography (HPCCC) instruments. The work described has been performed by a consortium consisting of GlaxoSmithKline, Pfizer, Dynamic Extractions and Brunel University and is part of a three year project sponsored by the UK Government’s Technology Strategy Board as part of its high value manufacturing programme.
Although CCC has always had a relatively low profile in separation science we believe that it has considerable unrealised potential to improve both laboratory and manufacturing efficiency. In the laboratory for example the opportunity is to enhance overall separation capability. This will require the integration of CCC technology to a similar extent to that achieved in HPLC giving the same degree of instrumental control and automated method development. This will allow the instrumentation to be integrated into a broader strategy for preparative separations. As a manufacturing tool, CCC promises lower costs compared to other large scale chromatographic separation technology with potential applications such as reclamation of waste streams for high value products.
CCC is applicable to preparative separations covering a range of scales from a few milligrams through to kilograms and can be operated in both batch and semi-continuous
modes. Relatively large scale chromatographic separations can be achieved using CCC and in batch mode for example throughputs of the order of 10kg/day have been projected[1]
.
Potential loading in a semi-continuous mode has not been established but initial research indicates that it will be about 5 to 6 times higher than for batch mode.
An overview of counter-current
– the basic principle involves subjecting two immiscible liquids to an external acceleration field generated by centripetal motion. Therefore unlike solid phase chromatography both stationary and mobile phase are liquids. The technique has been variously described as a multi-stage liquid-liquid extraction and a continuous countercurrent chromatography process. The column in CCC is open tubing which is initially filled with the liquid phase that becomes the stationary phase and the sample is injected with the mobile phase. Separation is based on the distribution of the sample between two immiscible liquid phases (e.g. heptane/ethyl acetate: methanol/water) and is characterised by the distribution ratio (KD) defined as the concentration in the stationary phase divided by the concentration in the mobile phase. This is also known as liquid-liquid partition chromatography.
chromatography CCC was first introduced by Yoichiro Ito in 1966 [2]
The liquid nature of the stationary phase leads to many unique features – high injected sample loading, high yields of purified
compounds, high reliability of retention and a number of different processing methods that can be used for this liquid–liquid extraction/chromatography process. Method transfer from one instrument to another, or one scale to another, is simple and predictable. From a quality angle, the reliability of retention that is available with CCC is seen to offer particular advantage, especially at manufacturing scale.
Stationary phase is retained in the column as a result of complex centripetal acceleration. Either liquid phase can be the mobile phase as illustrated in Figure 1. The advantage of having two liquid phases is that other operating modes rather than just standard elution are available. These can either save time or solvent usage and can also ultimately allow semi-continuous liquid chromatography to be performed.
Normal Phase
Organic Phase mobile Aqueous phase stationary
Reverse Phase
Organic phase stationary Aqueous Phase mobile Figure 1: Isocratic operating schemes for CCC
The latest high-performance countercurrent chromatography (HPCCC) instruments run at a higher rotational speed (typically “g” fields up to 240g) compared to conventional high
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