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3. Centrifugal Partition Chromatography (CPC – this work)
2.5. Centrifugal Partition Chromatography [183]
Countercurrent separation and liquid/liquid chromatography are the two basic types of liquid/liquid separation methods. Centrifugal partition chromatography (CPC) is a method belonging to the countercurrent separation techniques wherein the stationary and mobile phases used in the separation are both liquids. The principle to retain the stationary phase inside the rotor by means of centrifugal force (thus making the phase suitable as stationary phase) was first described by Yoichiro Ito.[184] Coil planet centrifuge[185] and the first multistage mixer-settler centrifuge[186] are each of his realised devices.
The basis of the CPC device and its differentiation from other liquid chromatographic methods is also formed by such a rotor, comprising the column,[187] which rotates around its central axis – with a typical rotation speed range between 500 to 2000 rpm – producing such centrifugal force field which retains the liquid phase chosen to be stationary phase in stationary state. Inside the rotor, there are specially formed extraction cells in a geometric way connected by ducts in series with each other. The development of the basic equipment was implemented by Sanki Engineering Ltd during the 1980s.[188]
The operating principle of CPC (the driving force of the separation) lies in that different components of the mixture (sample) have a different partition coefficient (K) in the separated phases. Due to the centrifugal force arising from the rotation around the central axis, the liquid chosen to be stationary phase retained in the extraction cells remains stationary and the liquid chosen to be the mobile phase is pumped through these extraction cells. As a result of the mixing and settling operations, extraction occurs in each of these extraction cells and ducts. Depending on the relative density of the stationary and mobile phase, the direction of the pumping is chosen in a way that
(i) if the stationary phase is the denser phase (ascending mode or AM) then the flow of the mobile phase progresses from the rotational circumference towards the rotational central point, in other words towards the main axle of the CPC rotor;
(ii) if the stationary phase is the less dense phase (descending mode or DM), then the flow of the mobile phase progresses from the rotational central point towards the rotational circumference.
When feeding the sample in the form of short liquid sections – i.e. as plugs – the components are separated depending on not just the mixing and settling operations due to the
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(i) and (ii) circulating modes but those are separated from each other according to their differing partition coefficients in the different phases.
Figure 2.31. Working principle of CPC device and the elution order in different modes.
Continuous development of the centrifugal partition chromatographic devices results numerous realization over the past decades.[189–194] Starting from the 1990s, the method and the device have evolved primarily through French and British developments to its current forms. WO 00/58722[190] discloses a modified centrifugal partition chromatographic device providing more efficient separations than the prior solutions.
WO 2005/011835[191] discloses a centrifugal partition chromatographic device comprising at least one column and a separation method wherein the continuous injection of the feed occurs at an intermediate point of the series of extraction cells and the separation itself realised by alternating the sections of ascending and descending modes in a defined frequency in a way wherein according to the mode used the lighter and heavier phases are injected at different ends of the rotors. The implementation of this method has been reported by R. van den Heuvel et al.[195] or Hopmann et al.[196]
WO 2005/011835.,[191]discloses an improved device in which having at least one rotor wherein the continuous injection of the feed occurs at an intermediate point of the series of
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extraction cells, one or the other phase injected at different ends of the rotors and the fractions also collected at different ends of the rotors but the extraction cells have two-two inlets and outlets instead of having one-one as previously. The latter makes the device capable of applying the ascending and descending modes simultaneously compared to their prior progression with predetermined frequency. In this way, the continuously operating device realises the principle of a true moving bed. It is mentioned that on the basis of the disclosed comparative table the method of WO 2005/011835[191] on account its semi-continuous operation is indicated to be a simulated moving bed (SMB) type method.
In contrast, according to the principle of SMB countercurrent liquid chromatography the solid phase is stationary and its countercurrent movement relative to the mobile phase is simulated in a way that the feed inlets and collection exits are moved in a direction same as the mobile phase flows. If the solid phase is fill-ed into columns and one or more columns are used for each zone, the flow of the solid phase is simulated by moving the feed inlets and collection exits in identical direction as the mobile phase flows. Usage of more columns approaches more the principle of true moving bed.[197]
Figure 2.32. General scheme of a continuously operated simulated moving bed (SMB) chromatography system.[3]
O’Brien et al.[172] was first to describe a method, wherein SMB comprising six HPLC columns was used as in-line purification of a flow reaction. Figure 2 of the article shows that the method provides continuous operation.
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Applying SMB with the aim of increasing productivity for the purification of the products resulting from flow reactions is not recommended by Anderson et al.,[198] since solid state itself means the physical limitations of continuous procedures, thus the vapour and liquid states seem to be ideal and the process steps contacting solid state are limitative, i.e. adversely affect the productivity. However, the latter method would be difficult to realise also in accordance with the requirements of the current good manufacturing practice (cGMP).
Furthermore, SMB is known to be a cumbersome technique to develop and has a major drawback that the compound of interest must elute either as the first or the last component;[172]
consequently, it cannot be used generally. In contrast, the recently developed trapping multiple dual-mode[199] resolves this issue and enables the quasi-continuous purification of components where SMB cannot be employed.
Although SMB and CPC methods resemble in that both of them are a separation method with the aim of increasing productivity, the latter has more advantages over SMB. Such advantages are inter alia that there is no need to replace the column and to recycle silica, solvent consumption is lower, flow rate is higher (hence lower run time), higher performances can be achieved (purity > 99%, recovery > 90%), there is no sample loss, the sample does not denature (it could not get irreversibly absorbed on the stationary phase) and last but not least its application field ranges from petrochemistry to the processing of peptides.[200]
Huang et al.[201] reports on the recent major developments of countercurrent liquid chromatographic methods. Advances in the elaboration of elution modes has resulted in improved techniques such as countercurrent liquid chromatographic gradient elution, dual- mode and multiple dual-mode (MDM) elution, recycling elution, extrusion elution, co-current elution or pH zone refining. The different application possibilities of these new techniques are summarised in the table below, wherein K is the partition coefficient (the ratio of the equilibrium concentrations of a compound in the stationary and mobile phase):
51 Table 2.11. Comparison of elution modes in CPC.
As an improved alternative method of CPC, the above referenced article also mentions the so-called multiple dual-mode (MDM) elution described by Delannay et al.,[202] which is primarily used in the separation of extracts of natural products.[203,204] Using this method two racemic mixtures, (±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5- dinitrobenzamide and N-(3,5-dinitrobenzoyl)-(±)-leucine were successfully separated using (S)-naproxen N,N-diethylamide as the chiral selector.[204]
Compared to the method disclosed in WO 2005/011835[191] which requires at least two rotors during pilot plant conditions, the MDM elution is capable of realising semi-continuous operating using only one rotor in a way that the already known dual-mode[187] is performed multiple timeswith or without sample re-injection between each of them.
Entry Elution mode Characteristics
Recommended for separation
target
Principal merit
1 Gradient elution
Change in flow rate, pH value, composition and salt concentration of
mobile phase
Solutes in a proper range of
K value
Reduce separation time and solvent
consumption 2 Dual-mode
elution
Change in the phase role and circulation direction
during the separation
Solutes in a very large range of K
value
Reduce separation time and solvent
consumption 3 Multiple dual-
mode elution
Change in the phase role and circulation direction several times during the
separation
Solutes with extremely different or similar K values
Achieve semicontinuous process; improve
resolution factor 4 Recycling
elution
The mobile phase recycle in the column
Solutes with similar K values
Improve resolution factor without increasing the consumption of
solvent 5 Extrusion
elution
Extrude stationary phase after classical elution step
Solutes in a very large range of K
values
Reduce separation time and solvent
consumption 6 Cocurrent
elution
Both phases in the column move in the same
direction with different speeds
Solutes in a very large range of K
values
Reduce separation time and solvent
consumption
7 pH zone
refining
Use acid and base in both phases as retainer and
eluter
Solutes whose electric charge depends on the
pH value
Increase sample loading capacity;
concentrate solutes in fractions
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According to the principle of dual-mode, the ascending and descending modes are alternately applied with a determined frequency in a manner that the samples are injected at the feed inlet of the currently applied mode. During these alternately applied modes both the change of the direction of mobile and stationary phases and change of the direction of the elution occur at certain points. In the case of ascending mode, component(s) having higher partition coefficient are collected, while in the case of descending mode those with lower partition coefficients, since the pseudo-partition coefficient is by definition the ratio of the concentrations of a particular compound in the stationary and mobile phase. K=1 means that the equilibrium concentrations measured in both phases are equal, while K>1 indicates that the equilibrium concentration is higher in the upper phase. When K<1, the equilibrium concentration is higher in the lower phase (Fig 2.31.).
Agnely and Thiébaut[205] extensively studied the dual-mode elution in theory and their comparison with classical elution concluded that in case of its application the resolution could be increased depending on the solute, sample volume and the biphasic solvent system. These findings were later confirmed by Mekaoui and Berthod;[206] however, considering the importance of the latter a vast number of in-depth analysis has been published.[207–209]
According to Delannay et al.[202] performing the above multiple times improves the resolution, since each flow inversion increases the number of theoretical plates, while the steady-state is achieved along with the periodical reinjection of a given mixture when the elution profile remains approximately constant. It is also mentioned, that determining the appropriate composition of the mobile and stationary phases in order to optimise the separation selectivity belongs to the general knowledge of the person skilled in the art.
In a later work, Mekaoui and Berthod[206] also confirmed the conclusion of both Delannay et al.[202] and Yang et al.,[210] that the resolution improves with the increasing numbers of flow inversions, but on the grounds of their modelling comprising a separation of two solutes, it was proposed that the total column volume plays a more important role in the resolution increase than the number of switching steps.
WO 2010/010366[211] discloses a feasible method and apparatus according to the principles set out in Delannay et al.[202]
The above cited documents report on the realised and possible applications of CPC in the neighbouring fields, such as the separation of monoclonal antibodies from host cell
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protein pool,[212] separation of myoglobin and lysozyme containing protein mixture,[213]
purification of a mycosporine from a crude methanolic extract of Lichina pygmaea using multiple dual-mode centrifugal partition chromatography[214] or the method enabling the separation of enantiomers connected to (S)-naproxen.[204]
As evident from the latter article, the CPC method is the subject of great interest in the field of chemistry, as well.[215,216] Just in 2017 numerous of CPC application was described in connection with purification of natural products,[217–243] computational solvent system screening for finding the appropriate biphasic liquid system and assessing partition coefficients.[244–248] Moreover, scaling up[249–253] of CPC purification and other interesting features of CPC technique[238,254–282]
were described.
Therefore, it is not surprising that in the field of analytical chemistry several in-line couplings of CPC with well-known instrumental analytical devices have been published. Such solutions are, for example, the direct coupling of CPC and mass spectrometry,[283] the monitoring of CPC by nuclear magnetic resonance,[284] but further couplings are also published.[285–289]
Besides coupling with analytical chemistry purposes, the CPC is not proposed in connection with the field of synthetic chemistry and flow chemistry, respectively. Solely one application known that by some means connected to the synthetic chemistry is the use of CPC as a continuous reactor by immobilizing whole-cells and enzymes in enzymatic synthesis.[290–296]
Consequently, the present discovery, that relates to the use of centrifugal partition chromatography coupled in-line to a multistep flow reaction for the separation or final product purification of the products, is conceptually novel.
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