David Hawes

Stationary Phase Retention in CCC: Modelling the J‐Type Centrifuge as a Constant Pressure Drop Pump

Abstract To be able to design a J‐type centrifuge for a given need, a method of being able to predict peak elution is required. Predicting peak elution will also allow the user to optimise the process parameters for his or her needs. Such predictions require an accurate knowledge of the volume of the stationary phase retained in the coil for a given set of operating conditions. This paper builds upon an experimental relationship in that the stationary phase retention decreases proportionally to the square root of the mobile phase flow rate. Combining this experimental relationship with the hypothesis that the pressure drop across a coil is independent of mobile phase flow rate, and assuming that the mobile phase flow is laminar, the equation below is derived: Experimental evidence is presented supporting the above equation. The experimental evidence was gained using, a heptane–ethyl acetate–methanol–water (1.4:0.1:0.5:1) v/v phase system, in normal phase mode using three helical stainless steel coils. These stationary phase retention studies allowed the above equation to be tested under conditions of different rotational speeds and tubing internal diameter. The derived stationary phase retention characteristics from each retention study allowed pressure drop and Reynolds number data to be calculated. The pressure drop data shows that the pressure drop across a coil is constant and independent of the mobile phase flow rate.

Countercurrent Chromatography (CCC) and its Versatile Application as an Industrial Purification & Production Process

Abstract This paper describes the versatile operation of CCC, its potential for scale up and compares its operational performance with HPLC as a generic preparative purification process. In the words of the UK Biology & Biotechnology Science Research Council (BBSRC), there is a need for a “generation of new, robust, and usable techniques for bioprocess intensification and simplification” and, in particular, technology that can be scaled from laboratory to process scale easily and cheaply without any fundamental change to the principle of separation. CCC offers the potential to do this.

Review of Progress Toward the Industrial Scale‐Up of CCC

Abstract Considerable advances have been made in the last two years on the industrial scale‐up of countercurrent chromatography. This paper briefly reviews the scale‐up progress being made by three groups, two in France and one in the UK before giving details of advances being made at Brunel Institute for Bioengineering, Brunel University in the UK on the scale‐up of their J‐type centrifuges.

INDUSTRIAL SCALE-UP OF COUNTERCURRENT CHROMATOGRAPHY

The hydrodynamic, engineering, and chromatographic variables affecting scale-up of countercurrent chromatography (CCC) are examined. The predictable and linear scale-up from the current laboratory scale technology to industrial process scale, capable of kgm/month in the first phase, is demonstrated. Continued research will prepare the way for a new generation of tonne/annum capacity high throughput, high resolution CCC machines for pilot and plant scale separations of a range of bioprocess products.

A new small coil-volume CCC instrument for direct interfacing with MS

Abstract One of the major factors restricting the use of CCC as an analytical tool is the speed at which a separation may be conducted. This paper describes the phased development of a new low volume capacity Milli‐CCC device, which is as rapid as HPLC, achieving high resolutions in minutes as opposed to hours, with the capability of linking with a mass spectrometer (CCC/MS). The Milli‐CCC J‐type apparatus has gears enclosed in a lubricated case to minimize noise. Its volume with one coil mounted in a cantilever style is 4.6 mL with 2.5 m of 0.76 mm bore tubing. It can rotate at a maximum speed of 2100 rpm. Stationary phase retention factor higher than 60% could be obtained with 1500 rpm and 1 mL/min producing separation of compounds with K D distribution coefficient of 1 in less than 5 min. The connection to MS was straightforward.

Resolution in CCC: The Effect of Operating Conditions and Phase System Properties on Scale‐Up

Abstract Being able to accurately predict the eluted volume of a substance with a known distribution ratio will depend on accurately knowing the retention volume of the stationary phase. Sample resolution depends on a number of factors that are not so easy to predict: the properties of the phase systems, the number of mixing and settling cycles per unit time, the rate of mass transfer during mixing, and the quality of mixing between the phase systems. The extent of mixing between the phases will, in turn, depend on the flow rate of the mobile phase and the “g” field acting across the stratified phases within the coiled tubing. A systematic study is made of how sample resolution changes with the key operating variables associated with scale‐up: the mobile phase flow, the bore of the tubing, and the rotational speed. It shows how the commonly accepted characteristic, good resolution at low flow and poor resolution at high flow, slowly changes as tubing bore increases to one of poor resolution at low flow rising to optimum resolution at high flow and a slow decline in resolution at very high flow. Furthermore, it goes on to show that, as phase system physical properties change when moving from hydrophobic phase systems to more polar hydrophilic ones, the optimum resolution remains in a similar speed and flow range. It also shows that the key variable for scale‐up, the throughput of sample in kg/hour, increases significantly as mobile phase flow increases, provided the rotational speeds are high enough. Optimum throughput has not yet been reached, which is extremely promising for realising process scale CCC.

Determination of J‐Type Centrifuge Extra‐Coil Volume Using Stationary Phase Retentions at Differing Flow Rates

Abstract An alternative method for determining the extra‐coil volume (V ext) of a J‐type centrifuge and its ancillary equipment is given based upon displaced volumes of stationary phase. The extra‐coil volume (V ext) is determined by plotting the volume of stationary phase displaced from the coil and extra‐coil volume combined (V E ), against the square root of the mobile phase flow rate. The extra‐coil volume is the intercept on the V E axis for a zero flow rate of mobile phase. This extra‐coil volume is then used to produce a stationary phase retention characteristic where the intercept on the retention axis is 100% for a zero flow rate of mobile phase. Experimental evidence was gained for a heptane–ethyl acetate–methanol–water (1.4:0.1:0.5:1) v/v phase system, in normal phase mode using three helical stainless steel coils. The internal diameters of these coils were 3.73 mm, 5.33 mm, and 7.73 mm. Retention studies were conducted at rotational speeds between 600 and 1200 rpm. The same phase system was also tested in a multi‐layer PTFE coil in reverse phase mode at a rotational speed of 800 rpm. The internal diameter of the tubing for the PTFE coil was 1.6 mm. All of the coils used were fitted to a J‐type centrifuge with a rotor radius of 110 mm. These retention studies allowed the extra‐coil volume to be tested under conditions of different rotational speeds and coil tubing internal diameters. It was also tested using different tubing material, and in both normal and reverse phase modes.