Journal of Chromatography A, 1323 (2014) 49–56
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
A general method for the separation of triphenylphosphine oxide and reaction products using high performance countercurrent chromatography Neil A. Edwards a,∗ , Gail Fisher b , Guy H. Harris c , Nikki Kellichan d a
Dynamic Extractions, Ltd., 890 Plymouth Road, Slough, Berkshire, UK GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, UK c Dynamic Extractions, Inc., 11 Deer Park Drive, Suite 200, Monmouth Junction, NJ 08852, USA d University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK b
a r t i c l e
i n f o
Article history: Received 7 February 2013 Received in revised form 28 October 2013 Accepted 29 October 2013 Available online 6 November 2013 Keywords: Countercurrent chromatography Preparative chromatography Reverse phase liquid chromatography Triphenylphosphine oxide Impurity removal Mitsunobu reaction
a b s t r a c t A standardised separation methodology was developed for the purification of crude reaction mixtures containing triphenylphosphine oxide (TPPO) using high performance countercurrent chromatography (HPCCC). A solvent system consisting of hexane/ethyl acetate/methanol/water (5:6:5:6) was used in 1 column volume elution–extrusion mode. The HPCCC methodology was compared with classical RP HPLC purification using a set of 12 representative Mitsunobu reaction mixtures. HPCCC was seen to yield a 65% increase in the average recovery of the target component whilst providing similar final target purities to those obtained by HPLC. By eliminating the need for method development for individual samples, the HPCCC methodology described within provides a simple and efficient means for the purification of the entire family of TPPO-containing reaction products. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Medicinal chemistry presents many challenges in relation to the purification of crude reaction mixtures. There are a huge variety of reaction conditions and reagents in the toolbox of the modern medicinal chemist that are used to generate a diverse range of target compounds. Some by-products are common to multiple, widely used reaction types and many can often prove problematic to remove via existing methods. One example of such an impurity is triphenylphosphine oxide (TPPO), which is generated as a by-product of several useful and commonly used reactions in organic synthesis, including the Mitsunobu, Wittig, and Staudinger reactions [1–3]. Removal of TPPO can often present purification problems when using chromatography that employs a solid stationary phase; recovery and purity of target material can be degraded due to streaking or tailing of the TPPO peak. Countercurrent chromatography (CCC) is a technique that relies on differential partitioning of analytes between biphasic solvent mixtures [4]. Immobilisation of one solvent phase on the column
∗ Corresponding author. Tel.: +44 01753 696979. E-mail address: [email protected] (N.A. Edwards). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.089
by use of a centrifugal force establishes a liquid stationary phase, the remaining phase is then pumped as the mobile phase in order to elute the components of an injected mixture. HPCCC is a high performance iteration of CCC with instruments generating a high centrifugal force field of 240 × g, facilitating mobile phase flow rates that allow run times that are comparable to preparative HPLC [5]. HPCCC achieves high resolution through the careful choice of an appropriate solvent system to control the highly tuneable selectivity. Solvent systems are formed from mixtures of two or more solvents that generate two immiscible phases. Many possible solvent systems and solvent system series have been developed and successfully applied to a wide range of separation problems [6]. There are a multitude of benefits to the use of HPCCC that make it a powerful chromatographic tool and the technique is increasingly being recognised as a solution with relevance and application to modern medicinal chemistry purifications [7]. The technique is orthogonal and complementary to other forms of chromatography such as HPLC, often providing straightforward resolution of otherwise difficult separations. The absence of a solid stationary phase generally provides a high tolerance of crude samples containing contaminants such as unspent reagents and heavy metal residues; this can often eliminate the need for sample workup and significantly reduce sample preparation time. An active stationary phase volume of 70–90% of the HPCCC column volume is typical and yields
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PPh 3
R3-OH DIAD
OPPh3
Fig. 1. General reaction scheme for the Mitsunobu coupling.
a high dynamic loading capacity which facilitates high chromatographic throughput (mass of sample processed per unit time) and high concentration of solute in the resulting fractions, minimising downstream processing and reducing solvent consumption for a given sample mass. An important operational mode, elution–extrusion countercurrent chromatography (EECCC), further exploits the liquid stationary phase; solutes not yet eluted, but separated within the column can be rapidly extruded without loss of resolution [8]. Solutes with distribution ratios (D, defined as concentration of a solute in the stationary phase divided by that in the mobile phase) greater than those already eluted are found in the stationary phase and are separated according to their individual distribution ratios. Furthermore, these retained solutes do not experience significant chromatographic band broadening [9]. Simple extrusion of the column contents (both mobile and stationary liquid phases) is used to exploit this separation thus shortening run times for solutes with larger distribution ratios, reducing solvent consumption, and providing complete recovery of applied sample. Despite advances in aspects of HPCCC method development, including automated screening techniques and rapid scouting [10], the time and expertise required to develop an appropriate solvent system for a given separation has often been seen as a barrier to the wider adoption of the technique. By adopting a novel approach and developing a portfolio of separation conditions that focus on the removal of common impurities, method development is eliminated and the benefits of HPCCC are significantly more accessible for exploitation within a drug discovery environment. For the example of TPPO removal from a reaction mixture; separation conditions eluting this component at optimum resolution with a D of 1 provides a high probability of resolving remaining components, including the target compound. Total sample recovery, high throughput, reduced sample preparation and the elimination of effects such as peak tailing, promise to make this a powerful application in medicinal chemistry. Techniques such as flash chromatography are commonly used for such separations but can exhibit issues with contamination of targets due to peak tailing of TPPO. Preparative HPLC provides a good existing solution to the resolution of such mixtures and, as such was used for a comparison to the HPCCC methodology described. 2. Experimental 2.1. Solvents All solvents used for analytical and preparative scale RP-HPLC and HPCCC were of HPLC grade. 2.2. Materials A set of 12 crude Mitsunobu reaction mixtures were supplied by GSK, Stevenage. The samples contained target components formed from the coupling of a common template of a commercially available functionalised pyridine alcohol with a range of 12 commercially available monomer alcohols including substituted benzene alcohols, phenethyl alcohols, aliphatic alcohols and heteroaromatic alcohols. A general Mitsunobu reaction scheme is shown in Fig. 1.
Reaction conditions were as follows; to pyridinol template (0.43 mmol, 1 equiv.) and MgSO4 , was added a solution of monomer alcohol (0.65 mmol, 1.5 equiv.), triphenylphosphine (0.65 mmol, 1.5 equiv.) and diisopropyl azodicarboxylate (0.65 mmol, 1.5 equiv.) in 1 ml of 2-methyl THF. The mixtures were stirred overnight at room temperature. The resulting solutions were divided in to two aliquots of equal volume before concentration to dryness in a nitrogen gas blow down evaporator at 35 ◦ C.
2.3. Equipment 2.3.1. GSK (Stevenage, UK) 2.3.1.1. Analytical RP-HPLC MS (LC–MS). LC–MS analyses were conducted on a Waters Acquity UPLC system fitted with a Waters BEH C18 column (50 mm × 2.1 mm, 1.7 m packing). Mass detection was performed with a Waters ZQ mass spectrometer.
2.3.1.2. Preparative UV-directed RP-HPLC. Preparative RP-HPLC was performed on an Agilent 1200 system comprising two G1361A prep pump solvent delivery modules, G2258A autosampler, G1365A DAD detector, two G1364B fraction collection modules and ChemStation software (Rev. B.01.01.[317]) (Agilent Technologies UK Ltd., Edinburgh, UK). The purifications were performed on a Waters Xbridge C18 prep column (19 mm × 100 mm, 5 m packing) fitted with an Xbridge Prep C18 guard column (19 mm × 10 mm, 5 m packing).
2.3.2. Dynamic Extractions Ltd (Slough, UK) 2.3.2.1. Analytical RP-HPLC. Analytical RP-HPLC was performed on an Agilent 1100 system comprising a G1311A Quaternary Pump solvent delivery module, G1322A Degasser, G1313A ALS autosampler, G1315A DAD detector, G1316A column oven and ChemStation software (Rev. B.01.03.[204]) (Agilent Technologies UK Ltd., Edinburgh, UK). The analyses were performed on a Phenomenex GeminiTM C18 analytical column (4.6 mm × 50 mm, 3 m packing diameter). 2.3.2.2. HPCCC. HPCCC separations were performed on a Dynamic Extractions Spectrum instrument (Slough, UK) which was fitted with both an analytical scale column with a volume of 25 ml, an I.D. of 0.8 mm, a ˇ-value range from 0.64 to 0.81, and a revolution radius of 85 mm and a semi-preparative scale column with a volume of 130 ml, an I.D. of 1.6 mm a ˇ-value range of 0.52–0.86, and a revolution radius of 85 mm. Cooling was provided by a Neslab ThermoFlex 1400 chiller. Semi-preparative scale separations were performed on a system comprising SSI Q-grad pump, LabAlliance Model 500 UV detector, Agilent 35900E Dual channel interface and Agilent EZ Chrom Elite (version 3.3.1 SP1) software.
2.4. Preparative RP-HPLC purification 2.4.1. Analytical RP-HPLC method The following RP-HPLC method was used for the analysis of crude samples and for the analysis of purified target materials. Solvent A: 0.1% (v/v) formic acid (FA) in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 3–100% B over 1.5 min; hold at 100% B for 0.4 min then 100–3% B over 0.1 min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. Mass spectrometer conditions used were alternate-scan positive and negative electrospray with a scan range of 100–1000 AMU, a scan time of 0.27 s and an inter scan delay of 0.10 s.
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51
Table 1 Typical preparative RP-HPLC method for the purification of Mitsunobu reaction products. Time (min)
% Solvent A
% Solvent B
Flow rate (ml/min)
Comments
Equilibration 0–1 1–20 20–20.5 20.5–30
99 99 99 → 70 70 → 1 1
1 1 1 → 30 30 → 99 99
20 20 20 20 20
Isocratic Isocratic Linear gradient Linear gradient Isocratic
2.4.2. Sample preparation For preparative HPLC separation, each sample was dissolved in DMSO (1 ml) and divided into two aliquots of equal volume for injection across 2 identical runs. 2.4.3. Preparative RP-HPLC method High pH mobile phase conditions were applied to all samples, using Solvent A: 10 mmol aqueous ammonium bicarbonate (pH10) and Solvent B: acetonitrile + 0.1% ammonia. Focussed gradients of solvents A and B were individually determined for each of the 12 samples based upon the HPLC retention time of the target. Flow rates were 20 ml/min and cycle time was 40 min including a 10 min equilibration step. A typical HPLC gradient is provided in Table 1. Fractions were collected on the basis of UV absorption, using a summed signal from a wavelength of 210–350 nm and a threshold of 25 mAU. 2.5. Semi-preparative HPCCC purification 2.5.1. Analysis RP-HPLC method The following RP-HPLC method was used for the initial analysis of crude samples and for the subsequent analysis of preparative HPCCC derived fractions and purified target material. Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. Analysis of final rich cuts containing target compounds was performed at GSK by the method described in Section 2.4.1 to facilitate accurate comparison of results. 2.5.2. Sample preparation For semi-preparative HPCCC separation, each sample was dissolved in equal volumes of the upper and lower phases of HEMWat solvent system 16 (hexane/ethyl acetate/methanol/water, 5:6:5:6, by volume) [12] to a total sample volume of 6 ml for injection in a single run. 2.5.3. General conditions for all semi-preparative HPCCC separations Upper and lower phases of solvent systems phases were prepared by combining hexane, ethyl acetate, methanol and water in the appropriate ratios in a separating funnel. The solvent system was agitated to equilibrate before separation of the two resulting phases. Reverse phase, elution–extrusion HPCCC conditions were applied as follows: A Dynamic Extractions Spectrum HPCCC semipreparative column was filled with the stationary phase (upper phase) at a flow rate of 10 ml/min. The column was rotated at 1600 rpm in a clockwise direction to provide a 240 × g centrifugal force field. Mobile phase (lower phase) was pumped in the direction from the centre inlet of the column to the peripheral outlet at a flow rate of 6 ml/min. The sample solution was injected following equilibration of the column (the moment at which mobile phase elutes from the column outlet) and eluted with mobile phase
Fig. 2. Generalised representation of 1 column volume elution–extrusion method. (A) Variation of log D with elution volume; (B) elution volumes of solutes with D = 0.01, 0.2, 1, 5, and 100.
for 30 min before extrusion with stationary phase at 10 ml/min for 16 min. A column chamber temperature of 30 ◦ C was maintained. Separations were monitored by UV absorbance at 254 nm. A constant stationary phase fraction (Sf ) of 0.8 was observed for all samples. 3. Results and discussion 3.1. HPCCC method development Separation using HPCCC is dependent upon the distribution ratio, D, which is defined as the ratio of the concentration of the solute in the stationary phase to that in the mobile phase. Thus a solute eluting at a D = 1, equivalent to one column volume, is equally distributed between the upper and lower phases of the solvent system. Note that there is symmetry in distribution ratios and peak width centred around the D = 1 position in the hypothetical chromatogram shown in Fig. 2A. Plotting EECCC data in this manner has been described as reciprocal symmetry plots [11]. The HPCCC operational mode for standardised separation was chosen to minimise the time of separation while ensuring complete recovery of sample. This was achieved using a one column volume elution–extrusion mode of operation [7]. In EECCC the column is eluted with a chosen volume of mobile phase after which, flow of mobile phase is stopped and the column contents are extruded from the column using fresh stationary phase. The eluted and extruded solutes from a separation vary continuously in polarity from polar to non-polar or non-polar to polar depending upon mobile and stationary phase selection (reverse phase or normal phase respectively). One column volume elution–extrusion mode HPCCC separations are symmetrical about the D = 1 position [11]. Compounds that are either more polar or non-polar than TPPO are eluted either before or after TPPO depending upon the choice of mobile and stationary phases. This is illustrated graphically for components with D = 0.01,
N.A. Edwards et al. / J. Chromatogr. A 1323 (2014) 49–56
3.2. Determination of HPCCC loading capacity for TPPO HPCCC separations were carried out in order to establish the maximum loading capacity for TPPO. Optimum loading is here defined as the highest loading at which there was no decrease in stationary phase retention, and thus, an associated loss of resolution. A series of semi-preparative scale separations were performed at loadings of 75, 150, 300 and 450 mg of triphenylphosphine oxide, the resulting chromatograms are provided in Fig. 3. Stationary phase retention was stable at injection sizes up to and including 300 mg, with expected broadening of the TPPO peak in proportion to loading. Bleeding of stationary phase was observed at higher loadings which would likely result in a proportional reduction in resolution. These results establish the loading capacity of TPPO to be up to 300 mg per injection on semi-preparative scale. Higher crude sample loadings may be tolerated provided the mass of the TPPO does not exceed 300 mg.
Absorbance 254nm
A
0
3.4. Preparative scale HPLC separation of Mitsunobu products Samples were purified using one of 4 generic mobile phase gradients. The method applied each individual sample was determined
20
30
40 Time (min)
10
20
30
40 Time (min)
10
20
30
40 Time (min)
10
20
30
40 Time (min)
B
0
C
3.3. Semi-preparative scale HPCCC separation of Mitsunobu products
0
D Absorbance 254nm
The standardised HPCCC method was applied to a set of 12 crude Mitsunobu reaction mixtures. The mixtures had masses in the range of 110–230 mg with reaction stoichiometry dictating a content of approximately 90 mg of TPPO. It was established above that a 300 mg loading of TPPO was possible with no loss of stationary phase. Therefore, the samples represented a modest single injection loading, thus negating the need for any further optimisation. For each sample, complete dissolution was achieved in 6 ml of upper and lower phase [1:1, v/v] with the resulting solution loaded onto the column in a single injection. The sample was then eluted and fractions were collected on a timed basis. Target fractions were identified by HPLC analysis and were combined on the basis of achieving maximum recovery. Final analysis of rich cuts containing target compound was performed by LC–MS. The resulting chromatograms are shown in Figs. 4 and 5 with the target peak shaded. It is evident that the TPPO peak elution is consistent, at 22.9 min, with the target peak and other components eluting earlier or later, broadly depending on their individual polarities. Distribution ratios of the target components were calculated and are provided in Table 2.
10
Absorbance 254nm
0.2, 1, 5, and 100 in Fig. 2B. Furthermore, it should be noted that the region of maximum resolution, the point with the least change in log D with respect to elution volume, using this method is at the D = 1 position as can be seen in Fig. 2A. The HEMWat solvent system series was chosen for this work due to its excellent and broad range of selectivity and solubility characteristics for small molecule chromatography [12,13]. This solvent system series consists of 22 combinations of hexane, ethyl acetate, methanol, and water which systematically change in polarity. Solvent system 16 (hexane/ethyl acetate/methanol/water, 5:6:5:6, v:v:v:v) provided a distribution ratio for TPPO of approximately 1. Acidic and basic modifiers were examined and had no significant effect on the retention volume of TPPO. However, pH control could be beneficial, particularly where target components have ionisable functionalities. Reverse phase elution mode, where the lower phase of the solvent system was used as the mobile phase and the upper phase as the stationary phase, was found to provide marginally superior resolution to that of the inverse, normal phase, operating mode.
Absorbance 254nm
52
0
Fig. 3. HPCCC chromatograms of the semi-preparative scale HPCCC loading study for TPPO. Sample Loading: (A) 75 mg, (B) 150 mg, (C) 300 mg, (D) 450 mg. Separation conditions: column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
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Fig. 4. HPCCC chromatograms from the semi-preparative HPCCC separation of Mitsunobu coupling reaction mixtures 1–6. Peaks corresponding to target reaction product are highlighted. Conditions: sample loading 110–230 mg; column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
from the analytical HPLC retention time of the target component. Generic loading and equilibration conditions were applied without further optimisation. Two identical injections were performed per sample with complete sample dissolution achieved
in DMSO (500 l per injection). Fractions were collected automatically, directed by UV absorbance, with collected material subsequently analysed by LC–MS. Analytical HPLC chromatograms for all samples are provided in Fig. 6A and B to allow direct
Table 2 Comparison of purities and recoveries from the HPCCC and HPLC purifications of Mitsunobu reaction products. Sample
Crude mass (mg)
1 2 3 4 5 6 7 8 9 10 11 12
110 122 131 149 229 143 166 174 172 219 182 224
11 26 53 48 41 34 73 22 31 51 37
Mean average
168.0
35.6
a
Crude target purity (UV-LCMS 210–350 nm)
a
Purity could not be accurately determined due to complexity of sample.
Distribution ratio of target in HEMW at 16
6.21 1.35 8.18 11.25 9.00 89.95 2.95 3.21 0.31 1.27 0.49 0.44
Target recovery (mg)
% Target purity (UV-LCMS 210–350 nm)
HPCCC
HPLC
HPCCC
HPLC
7.1 7.5 10.1 21.1 26.5 31.6 12.5 25.7 9.5 17.6 24.1 24.4
7 5 5 16 9 21 7 24 7 18 5 8
100 95 97 100 100 92 100 100 94 98 92 83
100 100 93 98 93 99 17 100 100 100 100 100
18.1
11.0
96
92
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Fig. 5. HPCCC chromatograms from the semi-preparative HPCCC separation of Mitsunobu coupling reaction mixtures 7–12. Peaks corresponding to target reaction product are highlighted. Conditions: sample loading 110–230 mg; column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
comparison of sample complexity with that observed by HPCCC. The target peak is highlighted and TPPO can be seen to elute at 4.85 min in all samples. Example chromatograms of crude samples purified by each of the 4 generic preparative HPLC methods utilised are provided in Fig. 6C; the target peak is highlighted and the TPPO peak is indicated by an asterisk immediately to the left.
3.5. Comparison of RP-HPLC and HPCCC separation methods for the processing of crude Mitsunobu reaction mixtures The resulting purities and yields for each target isolated by both HPCCC and HPLC methodologies are given in Table 2. From the data provided in Table 2, it can be calculated that the mean recovery of target material is 65% greater by HPCCC than by HPLC. The preparative HPLC results were perhaps skewed by outliers in the recoveries of Sample 11 and 12; disregarding these results, average preparative HPLC recovery increases to 11.9 mg, with HPCCC providing a 52% greater average recovery. The greater recoveries obtained by HPCCC are not surprising, as quantitative recoveries are expected from this solvent only technique. However, due to the relatively small sample size and the large variability in HPLC recoveries, it is not possible to draw strong conclusions as to
whether the scale of HPCCC recovery gains observed for this sample set would be representative of a wider range of examples. In general, the purity of targets from both HPLC and HPCCC was comparable with average purity being greater by HPCCC for the complete sample set but with HPLC purities marginally superior
Table 3 Comparison of run parameters for the HPCCC and HPLC purification of Mitsunobu reaction products. Run parameter
HPCCC
HPLC
Runs per sample Sample loading/injection (mg) Typical sample Loading/injection (mg) Cycle time/injection (min) Mobile phase flow rate – elution including equilibration (ml/min) Mobile phase flow rate – extrusion (ml/min) Solvent use per run (ml) Solvent use per sample (ml) Average target recovery (mg) Average target % purity (UV-HPLC)
1 110–230 600
2 55–115 30–100
50 6
40 20
10
n/a
530 530 18.1 96
800 1600 11 92
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Fig. 6. (A) Analytical HPLC chromatograms of Mitsunobu coupling reaction mixtures 1–6. Peaks corresponding to target reaction product are highlighted. Conditions: Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. (B) Analytical HPLC chromatograms of Mitsunobu coupling reaction mixtures 7–12. Peaks corresponding to target reaction product are highlighted. Conditions: Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. (C) Preparative HPLC chromatograms of Mitsunobu coupling reaction mixtures 1, 6, 7 and 10. Peaks corresponding to target reaction product are highlighted, the TPPO peak is indicated by an asterisk to its immediate left. Conditions: Solvent A: 10 mmol aqueous ammonium bicarbonate (pH10) and Solvent B: acetonitrile + 0.1% ammonia, flow rate 20 ml/min; focussed gradients of solvents A and B were individually determined for each of the 12 samples based upon the HPLC retention time of the target, cycle time was 40 min including a 10 min equilibration step. Fractions were collected on the basis of UV absorption, using a summed signal from a wavelength of 210–350 nm and a threshold of 25 mAU.
if Sample 7 is disregarded. This result is of note due to the standardised HPCCC strategy applied to all samples, whereby greatest resolution is provided for TPPO, rather than the component of interest. The preparative HPLC method utilised focused gradients based
upon the HPLC elution time of target components for each on the individual samples. To facilitate comparison of the two techniques, various parameters from this set of separations are compared in Table 3.
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The higher potential loading capacity of HPCCC allowed the samples to be processed in a single run whereas two injections were necessary for the equivalent sample by HPLC. This yielded significant enhancements of throughput and solvent usage using HPCCC, as described in Table 3. When considering these comparisons it should be noted that there is room for further optimisation of both methodologies, in terms of both throughput and efficiency.
By eliminating the requirement for individual sample method development, HPCCC could become greatly more accessible for the high throughput processing of crude reaction mixtures; offering predictable, reliable chromatography with benefits including total sample recovery, high throughput and reduction in both sample preparation and solvent usage. References
4. Conclusions HPCCC was demonstrated to provide an efficient clean up method for reaction mixtures containing TPPO using a standardised methodology. For this sample set, target recoveries from HPCCC were greater than those processed via preparative HPLC, while purities were generally comparable. The same HPCCC strategy should have scope for application in a wide range of medicinal chemistry applications through the development of a portfolio of pre-determined methods for commonly generated reaction mixtures or chromatographically problematic impurities. However, due to the limited size of the sample set described here, further purification campaigns would be necessary to confirm the full extent of the applicability of this approach.
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O. Mitsunobu, Y. Yamada, Bull. Chem. Soc. Jpn. 40 (1967) 2380. G. Wittig, U. Schöllkopf, Chem. Ber. 87 (1954) 1318. H. Staudinger, J. Meyer, Helv. Chim. Acta 2 (1919) 612. Y. Ito, R.L. Bowman, Science 167 (1970) 281. H. Guzlek, P.L. Wood, L. Janaway, J. Chromatogr. A 1216 (2009) 1086. Y. Ito, J. Chromatogr. A 1065 (2005) 145. A. Berthod, in: A. Berthod (Ed.), Countercurrent Chromatography, The Liquid Stationary Phase, Elsevier, Amsterdam, 2002. A. Berthod, J.B. Friesen, T. Inui, G.F. Pauli, Anal. Chem. 79 (2007) 3371. A. Berthod, M. Hassoun, G. Harris, J. Liq. Chromatogr. Relat. Technol. 28 (2005) 1851. I. Sutherland, C. Thickitt, N. Douillet, K. Freebairn, D. Johns, C. Mountain, P. Wood, N. Edwards, D. Rooke, G. Harris, D. Keay, B. Mathews, R. Brown, I. Garrard, P. Hewitson, S. Ignatova, J. Chromatogr. A 1282 (2013) 84. B. Friesen, G.F. Pauli, Anal. Chem. 29 (2007) 2320. I. Garrard, L. Janaway, D. Fisher, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 151. F. Oka, H. Oka, Y. Ito, J. Chromatogr. 538 (1991) 99.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
A general method for the separation of triphenylphosphine oxide and reaction products using high performance countercurrent chromatography Neil A. Edwards a,∗ , Gail Fisher b , Guy H. Harris c , Nikki Kellichan d a
Dynamic Extractions, Ltd., 890 Plymouth Road, Slough, Berkshire, UK GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, UK c Dynamic Extractions, Inc., 11 Deer Park Drive, Suite 200, Monmouth Junction, NJ 08852, USA d University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK b
a r t i c l e
i n f o
Article history: Received 7 February 2013 Received in revised form 28 October 2013 Accepted 29 October 2013 Available online 6 November 2013 Keywords: Countercurrent chromatography Preparative chromatography Reverse phase liquid chromatography Triphenylphosphine oxide Impurity removal Mitsunobu reaction
a b s t r a c t A standardised separation methodology was developed for the purification of crude reaction mixtures containing triphenylphosphine oxide (TPPO) using high performance countercurrent chromatography (HPCCC). A solvent system consisting of hexane/ethyl acetate/methanol/water (5:6:5:6) was used in 1 column volume elution–extrusion mode. The HPCCC methodology was compared with classical RP HPLC purification using a set of 12 representative Mitsunobu reaction mixtures. HPCCC was seen to yield a 65% increase in the average recovery of the target component whilst providing similar final target purities to those obtained by HPLC. By eliminating the need for method development for individual samples, the HPCCC methodology described within provides a simple and efficient means for the purification of the entire family of TPPO-containing reaction products. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Medicinal chemistry presents many challenges in relation to the purification of crude reaction mixtures. There are a huge variety of reaction conditions and reagents in the toolbox of the modern medicinal chemist that are used to generate a diverse range of target compounds. Some by-products are common to multiple, widely used reaction types and many can often prove problematic to remove via existing methods. One example of such an impurity is triphenylphosphine oxide (TPPO), which is generated as a by-product of several useful and commonly used reactions in organic synthesis, including the Mitsunobu, Wittig, and Staudinger reactions [1–3]. Removal of TPPO can often present purification problems when using chromatography that employs a solid stationary phase; recovery and purity of target material can be degraded due to streaking or tailing of the TPPO peak. Countercurrent chromatography (CCC) is a technique that relies on differential partitioning of analytes between biphasic solvent mixtures [4]. Immobilisation of one solvent phase on the column
∗ Corresponding author. Tel.: +44 01753 696979. E-mail address: [email protected] (N.A. Edwards). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.089
by use of a centrifugal force establishes a liquid stationary phase, the remaining phase is then pumped as the mobile phase in order to elute the components of an injected mixture. HPCCC is a high performance iteration of CCC with instruments generating a high centrifugal force field of 240 × g, facilitating mobile phase flow rates that allow run times that are comparable to preparative HPLC [5]. HPCCC achieves high resolution through the careful choice of an appropriate solvent system to control the highly tuneable selectivity. Solvent systems are formed from mixtures of two or more solvents that generate two immiscible phases. Many possible solvent systems and solvent system series have been developed and successfully applied to a wide range of separation problems [6]. There are a multitude of benefits to the use of HPCCC that make it a powerful chromatographic tool and the technique is increasingly being recognised as a solution with relevance and application to modern medicinal chemistry purifications [7]. The technique is orthogonal and complementary to other forms of chromatography such as HPLC, often providing straightforward resolution of otherwise difficult separations. The absence of a solid stationary phase generally provides a high tolerance of crude samples containing contaminants such as unspent reagents and heavy metal residues; this can often eliminate the need for sample workup and significantly reduce sample preparation time. An active stationary phase volume of 70–90% of the HPCCC column volume is typical and yields
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PPh 3
R3-OH DIAD
OPPh3
Fig. 1. General reaction scheme for the Mitsunobu coupling.
a high dynamic loading capacity which facilitates high chromatographic throughput (mass of sample processed per unit time) and high concentration of solute in the resulting fractions, minimising downstream processing and reducing solvent consumption for a given sample mass. An important operational mode, elution–extrusion countercurrent chromatography (EECCC), further exploits the liquid stationary phase; solutes not yet eluted, but separated within the column can be rapidly extruded without loss of resolution [8]. Solutes with distribution ratios (D, defined as concentration of a solute in the stationary phase divided by that in the mobile phase) greater than those already eluted are found in the stationary phase and are separated according to their individual distribution ratios. Furthermore, these retained solutes do not experience significant chromatographic band broadening [9]. Simple extrusion of the column contents (both mobile and stationary liquid phases) is used to exploit this separation thus shortening run times for solutes with larger distribution ratios, reducing solvent consumption, and providing complete recovery of applied sample. Despite advances in aspects of HPCCC method development, including automated screening techniques and rapid scouting [10], the time and expertise required to develop an appropriate solvent system for a given separation has often been seen as a barrier to the wider adoption of the technique. By adopting a novel approach and developing a portfolio of separation conditions that focus on the removal of common impurities, method development is eliminated and the benefits of HPCCC are significantly more accessible for exploitation within a drug discovery environment. For the example of TPPO removal from a reaction mixture; separation conditions eluting this component at optimum resolution with a D of 1 provides a high probability of resolving remaining components, including the target compound. Total sample recovery, high throughput, reduced sample preparation and the elimination of effects such as peak tailing, promise to make this a powerful application in medicinal chemistry. Techniques such as flash chromatography are commonly used for such separations but can exhibit issues with contamination of targets due to peak tailing of TPPO. Preparative HPLC provides a good existing solution to the resolution of such mixtures and, as such was used for a comparison to the HPCCC methodology described. 2. Experimental 2.1. Solvents All solvents used for analytical and preparative scale RP-HPLC and HPCCC were of HPLC grade. 2.2. Materials A set of 12 crude Mitsunobu reaction mixtures were supplied by GSK, Stevenage. The samples contained target components formed from the coupling of a common template of a commercially available functionalised pyridine alcohol with a range of 12 commercially available monomer alcohols including substituted benzene alcohols, phenethyl alcohols, aliphatic alcohols and heteroaromatic alcohols. A general Mitsunobu reaction scheme is shown in Fig. 1.
Reaction conditions were as follows; to pyridinol template (0.43 mmol, 1 equiv.) and MgSO4 , was added a solution of monomer alcohol (0.65 mmol, 1.5 equiv.), triphenylphosphine (0.65 mmol, 1.5 equiv.) and diisopropyl azodicarboxylate (0.65 mmol, 1.5 equiv.) in 1 ml of 2-methyl THF. The mixtures were stirred overnight at room temperature. The resulting solutions were divided in to two aliquots of equal volume before concentration to dryness in a nitrogen gas blow down evaporator at 35 ◦ C.
2.3. Equipment 2.3.1. GSK (Stevenage, UK) 2.3.1.1. Analytical RP-HPLC MS (LC–MS). LC–MS analyses were conducted on a Waters Acquity UPLC system fitted with a Waters BEH C18 column (50 mm × 2.1 mm, 1.7 m packing). Mass detection was performed with a Waters ZQ mass spectrometer.
2.3.1.2. Preparative UV-directed RP-HPLC. Preparative RP-HPLC was performed on an Agilent 1200 system comprising two G1361A prep pump solvent delivery modules, G2258A autosampler, G1365A DAD detector, two G1364B fraction collection modules and ChemStation software (Rev. B.01.01.[317]) (Agilent Technologies UK Ltd., Edinburgh, UK). The purifications were performed on a Waters Xbridge C18 prep column (19 mm × 100 mm, 5 m packing) fitted with an Xbridge Prep C18 guard column (19 mm × 10 mm, 5 m packing).
2.3.2. Dynamic Extractions Ltd (Slough, UK) 2.3.2.1. Analytical RP-HPLC. Analytical RP-HPLC was performed on an Agilent 1100 system comprising a G1311A Quaternary Pump solvent delivery module, G1322A Degasser, G1313A ALS autosampler, G1315A DAD detector, G1316A column oven and ChemStation software (Rev. B.01.03.[204]) (Agilent Technologies UK Ltd., Edinburgh, UK). The analyses were performed on a Phenomenex GeminiTM C18 analytical column (4.6 mm × 50 mm, 3 m packing diameter). 2.3.2.2. HPCCC. HPCCC separations were performed on a Dynamic Extractions Spectrum instrument (Slough, UK) which was fitted with both an analytical scale column with a volume of 25 ml, an I.D. of 0.8 mm, a ˇ-value range from 0.64 to 0.81, and a revolution radius of 85 mm and a semi-preparative scale column with a volume of 130 ml, an I.D. of 1.6 mm a ˇ-value range of 0.52–0.86, and a revolution radius of 85 mm. Cooling was provided by a Neslab ThermoFlex 1400 chiller. Semi-preparative scale separations were performed on a system comprising SSI Q-grad pump, LabAlliance Model 500 UV detector, Agilent 35900E Dual channel interface and Agilent EZ Chrom Elite (version 3.3.1 SP1) software.
2.4. Preparative RP-HPLC purification 2.4.1. Analytical RP-HPLC method The following RP-HPLC method was used for the analysis of crude samples and for the analysis of purified target materials. Solvent A: 0.1% (v/v) formic acid (FA) in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 3–100% B over 1.5 min; hold at 100% B for 0.4 min then 100–3% B over 0.1 min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. Mass spectrometer conditions used were alternate-scan positive and negative electrospray with a scan range of 100–1000 AMU, a scan time of 0.27 s and an inter scan delay of 0.10 s.
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51
Table 1 Typical preparative RP-HPLC method for the purification of Mitsunobu reaction products. Time (min)
% Solvent A
% Solvent B
Flow rate (ml/min)
Comments
Equilibration 0–1 1–20 20–20.5 20.5–30
99 99 99 → 70 70 → 1 1
1 1 1 → 30 30 → 99 99
20 20 20 20 20
Isocratic Isocratic Linear gradient Linear gradient Isocratic
2.4.2. Sample preparation For preparative HPLC separation, each sample was dissolved in DMSO (1 ml) and divided into two aliquots of equal volume for injection across 2 identical runs. 2.4.3. Preparative RP-HPLC method High pH mobile phase conditions were applied to all samples, using Solvent A: 10 mmol aqueous ammonium bicarbonate (pH10) and Solvent B: acetonitrile + 0.1% ammonia. Focussed gradients of solvents A and B were individually determined for each of the 12 samples based upon the HPLC retention time of the target. Flow rates were 20 ml/min and cycle time was 40 min including a 10 min equilibration step. A typical HPLC gradient is provided in Table 1. Fractions were collected on the basis of UV absorption, using a summed signal from a wavelength of 210–350 nm and a threshold of 25 mAU. 2.5. Semi-preparative HPCCC purification 2.5.1. Analysis RP-HPLC method The following RP-HPLC method was used for the initial analysis of crude samples and for the subsequent analysis of preparative HPCCC derived fractions and purified target material. Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. Analysis of final rich cuts containing target compounds was performed at GSK by the method described in Section 2.4.1 to facilitate accurate comparison of results. 2.5.2. Sample preparation For semi-preparative HPCCC separation, each sample was dissolved in equal volumes of the upper and lower phases of HEMWat solvent system 16 (hexane/ethyl acetate/methanol/water, 5:6:5:6, by volume) [12] to a total sample volume of 6 ml for injection in a single run. 2.5.3. General conditions for all semi-preparative HPCCC separations Upper and lower phases of solvent systems phases were prepared by combining hexane, ethyl acetate, methanol and water in the appropriate ratios in a separating funnel. The solvent system was agitated to equilibrate before separation of the two resulting phases. Reverse phase, elution–extrusion HPCCC conditions were applied as follows: A Dynamic Extractions Spectrum HPCCC semipreparative column was filled with the stationary phase (upper phase) at a flow rate of 10 ml/min. The column was rotated at 1600 rpm in a clockwise direction to provide a 240 × g centrifugal force field. Mobile phase (lower phase) was pumped in the direction from the centre inlet of the column to the peripheral outlet at a flow rate of 6 ml/min. The sample solution was injected following equilibration of the column (the moment at which mobile phase elutes from the column outlet) and eluted with mobile phase
Fig. 2. Generalised representation of 1 column volume elution–extrusion method. (A) Variation of log D with elution volume; (B) elution volumes of solutes with D = 0.01, 0.2, 1, 5, and 100.
for 30 min before extrusion with stationary phase at 10 ml/min for 16 min. A column chamber temperature of 30 ◦ C was maintained. Separations were monitored by UV absorbance at 254 nm. A constant stationary phase fraction (Sf ) of 0.8 was observed for all samples. 3. Results and discussion 3.1. HPCCC method development Separation using HPCCC is dependent upon the distribution ratio, D, which is defined as the ratio of the concentration of the solute in the stationary phase to that in the mobile phase. Thus a solute eluting at a D = 1, equivalent to one column volume, is equally distributed between the upper and lower phases of the solvent system. Note that there is symmetry in distribution ratios and peak width centred around the D = 1 position in the hypothetical chromatogram shown in Fig. 2A. Plotting EECCC data in this manner has been described as reciprocal symmetry plots [11]. The HPCCC operational mode for standardised separation was chosen to minimise the time of separation while ensuring complete recovery of sample. This was achieved using a one column volume elution–extrusion mode of operation [7]. In EECCC the column is eluted with a chosen volume of mobile phase after which, flow of mobile phase is stopped and the column contents are extruded from the column using fresh stationary phase. The eluted and extruded solutes from a separation vary continuously in polarity from polar to non-polar or non-polar to polar depending upon mobile and stationary phase selection (reverse phase or normal phase respectively). One column volume elution–extrusion mode HPCCC separations are symmetrical about the D = 1 position [11]. Compounds that are either more polar or non-polar than TPPO are eluted either before or after TPPO depending upon the choice of mobile and stationary phases. This is illustrated graphically for components with D = 0.01,
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3.2. Determination of HPCCC loading capacity for TPPO HPCCC separations were carried out in order to establish the maximum loading capacity for TPPO. Optimum loading is here defined as the highest loading at which there was no decrease in stationary phase retention, and thus, an associated loss of resolution. A series of semi-preparative scale separations were performed at loadings of 75, 150, 300 and 450 mg of triphenylphosphine oxide, the resulting chromatograms are provided in Fig. 3. Stationary phase retention was stable at injection sizes up to and including 300 mg, with expected broadening of the TPPO peak in proportion to loading. Bleeding of stationary phase was observed at higher loadings which would likely result in a proportional reduction in resolution. These results establish the loading capacity of TPPO to be up to 300 mg per injection on semi-preparative scale. Higher crude sample loadings may be tolerated provided the mass of the TPPO does not exceed 300 mg.
Absorbance 254nm
A
0
3.4. Preparative scale HPLC separation of Mitsunobu products Samples were purified using one of 4 generic mobile phase gradients. The method applied each individual sample was determined
20
30
40 Time (min)
10
20
30
40 Time (min)
10
20
30
40 Time (min)
10
20
30
40 Time (min)
B
0
C
3.3. Semi-preparative scale HPCCC separation of Mitsunobu products
0
D Absorbance 254nm
The standardised HPCCC method was applied to a set of 12 crude Mitsunobu reaction mixtures. The mixtures had masses in the range of 110–230 mg with reaction stoichiometry dictating a content of approximately 90 mg of TPPO. It was established above that a 300 mg loading of TPPO was possible with no loss of stationary phase. Therefore, the samples represented a modest single injection loading, thus negating the need for any further optimisation. For each sample, complete dissolution was achieved in 6 ml of upper and lower phase [1:1, v/v] with the resulting solution loaded onto the column in a single injection. The sample was then eluted and fractions were collected on a timed basis. Target fractions were identified by HPLC analysis and were combined on the basis of achieving maximum recovery. Final analysis of rich cuts containing target compound was performed by LC–MS. The resulting chromatograms are shown in Figs. 4 and 5 with the target peak shaded. It is evident that the TPPO peak elution is consistent, at 22.9 min, with the target peak and other components eluting earlier or later, broadly depending on their individual polarities. Distribution ratios of the target components were calculated and are provided in Table 2.
10
Absorbance 254nm
0.2, 1, 5, and 100 in Fig. 2B. Furthermore, it should be noted that the region of maximum resolution, the point with the least change in log D with respect to elution volume, using this method is at the D = 1 position as can be seen in Fig. 2A. The HEMWat solvent system series was chosen for this work due to its excellent and broad range of selectivity and solubility characteristics for small molecule chromatography [12,13]. This solvent system series consists of 22 combinations of hexane, ethyl acetate, methanol, and water which systematically change in polarity. Solvent system 16 (hexane/ethyl acetate/methanol/water, 5:6:5:6, v:v:v:v) provided a distribution ratio for TPPO of approximately 1. Acidic and basic modifiers were examined and had no significant effect on the retention volume of TPPO. However, pH control could be beneficial, particularly where target components have ionisable functionalities. Reverse phase elution mode, where the lower phase of the solvent system was used as the mobile phase and the upper phase as the stationary phase, was found to provide marginally superior resolution to that of the inverse, normal phase, operating mode.
Absorbance 254nm
52
0
Fig. 3. HPCCC chromatograms of the semi-preparative scale HPCCC loading study for TPPO. Sample Loading: (A) 75 mg, (B) 150 mg, (C) 300 mg, (D) 450 mg. Separation conditions: column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
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Fig. 4. HPCCC chromatograms from the semi-preparative HPCCC separation of Mitsunobu coupling reaction mixtures 1–6. Peaks corresponding to target reaction product are highlighted. Conditions: sample loading 110–230 mg; column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
from the analytical HPLC retention time of the target component. Generic loading and equilibration conditions were applied without further optimisation. Two identical injections were performed per sample with complete sample dissolution achieved
in DMSO (500 l per injection). Fractions were collected automatically, directed by UV absorbance, with collected material subsequently analysed by LC–MS. Analytical HPLC chromatograms for all samples are provided in Fig. 6A and B to allow direct
Table 2 Comparison of purities and recoveries from the HPCCC and HPLC purifications of Mitsunobu reaction products. Sample
Crude mass (mg)
1 2 3 4 5 6 7 8 9 10 11 12
110 122 131 149 229 143 166 174 172 219 182 224
11 26 53 48 41 34 73 22 31 51 37
Mean average
168.0
35.6
a
Crude target purity (UV-LCMS 210–350 nm)
a
Purity could not be accurately determined due to complexity of sample.
Distribution ratio of target in HEMW at 16
6.21 1.35 8.18 11.25 9.00 89.95 2.95 3.21 0.31 1.27 0.49 0.44
Target recovery (mg)
% Target purity (UV-LCMS 210–350 nm)
HPCCC
HPLC
HPCCC
HPLC
7.1 7.5 10.1 21.1 26.5 31.6 12.5 25.7 9.5 17.6 24.1 24.4
7 5 5 16 9 21 7 24 7 18 5 8
100 95 97 100 100 92 100 100 94 98 92 83
100 100 93 98 93 99 17 100 100 100 100 100
18.1
11.0
96
92
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Fig. 5. HPCCC chromatograms from the semi-preparative HPCCC separation of Mitsunobu coupling reaction mixtures 7–12. Peaks corresponding to target reaction product are highlighted. Conditions: sample loading 110–230 mg; column volume 130 ml; upper phase stationary; flow rate 0–30 min = 6.0 ml/min of lower phase, 30–46 min = 10 ml/min of upper phase; Sf of 0.8; 1600 rpm; 30 ◦ C; UV detection at 254 nm.
comparison of sample complexity with that observed by HPCCC. The target peak is highlighted and TPPO can be seen to elute at 4.85 min in all samples. Example chromatograms of crude samples purified by each of the 4 generic preparative HPLC methods utilised are provided in Fig. 6C; the target peak is highlighted and the TPPO peak is indicated by an asterisk immediately to the left.
3.5. Comparison of RP-HPLC and HPCCC separation methods for the processing of crude Mitsunobu reaction mixtures The resulting purities and yields for each target isolated by both HPCCC and HPLC methodologies are given in Table 2. From the data provided in Table 2, it can be calculated that the mean recovery of target material is 65% greater by HPCCC than by HPLC. The preparative HPLC results were perhaps skewed by outliers in the recoveries of Sample 11 and 12; disregarding these results, average preparative HPLC recovery increases to 11.9 mg, with HPCCC providing a 52% greater average recovery. The greater recoveries obtained by HPCCC are not surprising, as quantitative recoveries are expected from this solvent only technique. However, due to the relatively small sample size and the large variability in HPLC recoveries, it is not possible to draw strong conclusions as to
whether the scale of HPCCC recovery gains observed for this sample set would be representative of a wider range of examples. In general, the purity of targets from both HPLC and HPCCC was comparable with average purity being greater by HPCCC for the complete sample set but with HPLC purities marginally superior
Table 3 Comparison of run parameters for the HPCCC and HPLC purification of Mitsunobu reaction products. Run parameter
HPCCC
HPLC
Runs per sample Sample loading/injection (mg) Typical sample Loading/injection (mg) Cycle time/injection (min) Mobile phase flow rate – elution including equilibration (ml/min) Mobile phase flow rate – extrusion (ml/min) Solvent use per run (ml) Solvent use per sample (ml) Average target recovery (mg) Average target % purity (UV-HPLC)
1 110–230 600
2 55–115 30–100
50 6
40 20
10
n/a
530 530 18.1 96
800 1600 11 92
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Fig. 6. (A) Analytical HPLC chromatograms of Mitsunobu coupling reaction mixtures 1–6. Peaks corresponding to target reaction product are highlighted. Conditions: Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. (B) Analytical HPLC chromatograms of Mitsunobu coupling reaction mixtures 7–12. Peaks corresponding to target reaction product are highlighted. Conditions: Solvent A: 0.1% (v/v) FA in water; Solvent B: 0.1% (v/v) FA in acetonitrile, flow rate 1 ml/min; linear gradient 5–95% B over 6 min; hold at 95% B for 2 min then 2 min at 5% B at an increased flow rate of 1.5 ml/min. Analysis was performed at 40 ◦ C. UV detection was a summed signal from a wavelength of 210–350 nm. (C) Preparative HPLC chromatograms of Mitsunobu coupling reaction mixtures 1, 6, 7 and 10. Peaks corresponding to target reaction product are highlighted, the TPPO peak is indicated by an asterisk to its immediate left. Conditions: Solvent A: 10 mmol aqueous ammonium bicarbonate (pH10) and Solvent B: acetonitrile + 0.1% ammonia, flow rate 20 ml/min; focussed gradients of solvents A and B were individually determined for each of the 12 samples based upon the HPLC retention time of the target, cycle time was 40 min including a 10 min equilibration step. Fractions were collected on the basis of UV absorption, using a summed signal from a wavelength of 210–350 nm and a threshold of 25 mAU.
if Sample 7 is disregarded. This result is of note due to the standardised HPCCC strategy applied to all samples, whereby greatest resolution is provided for TPPO, rather than the component of interest. The preparative HPLC method utilised focused gradients based
upon the HPLC elution time of target components for each on the individual samples. To facilitate comparison of the two techniques, various parameters from this set of separations are compared in Table 3.
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The higher potential loading capacity of HPCCC allowed the samples to be processed in a single run whereas two injections were necessary for the equivalent sample by HPLC. This yielded significant enhancements of throughput and solvent usage using HPCCC, as described in Table 3. When considering these comparisons it should be noted that there is room for further optimisation of both methodologies, in terms of both throughput and efficiency.
By eliminating the requirement for individual sample method development, HPCCC could become greatly more accessible for the high throughput processing of crude reaction mixtures; offering predictable, reliable chromatography with benefits including total sample recovery, high throughput and reduction in both sample preparation and solvent usage. References
4. Conclusions HPCCC was demonstrated to provide an efficient clean up method for reaction mixtures containing TPPO using a standardised methodology. For this sample set, target recoveries from HPCCC were greater than those processed via preparative HPLC, while purities were generally comparable. The same HPCCC strategy should have scope for application in a wide range of medicinal chemistry applications through the development of a portfolio of pre-determined methods for commonly generated reaction mixtures or chromatographically problematic impurities. However, due to the limited size of the sample set described here, further purification campaigns would be necessary to confirm the full extent of the applicability of this approach.
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