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Green chiral HPLC study of the stability of Chiralcel OD under high temperature liquid chromatography and subcritical water conditions夽 S. Droux a , M. Roy b , G. Félix b,∗ a b

KIRALYA, Parc Biocitech, 102 Avenue Gaston Roussel, 93230 Romainville, France CINaM (CNRS UMR 7325), Aix-Marseille Université, Campus de Luminy, 13288 Marseille Cedex 9, France

a r t i c l e

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Article history: Received 21 November 2013 Received in revised form 26 March 2014 Accepted 29 March 2014 Available online xxx Keywords: Subcritical water chromatography Super heated water Reversed phase and normal chromatography Chiral separations Substituted polysaccharide stationary phase

a b s t r a c t We report here the study of the stability under subcritical water conditions of one of the most popular polysaccharide chiral stationary phase (CSP): Chiralcel OD. This CSP was used under high temperature and reversed phase conditions with acetonitrile and 2-propanol as modifier, respectively. The evolution of selectivity and resolution was investigated both in normal and reversed mode conditions with five racemates after packing, heating at 150 ◦ C and separations of some racemic compounds under different high temperatures and mobile phase conditions. The results show that after using at high temperature and subcritical water conditions the selectivity was only moderately affected while the resolution fell dramatically especially in reversed mode due to the creation of a void at the head of the columns which reflects the dissolution of the silica matrix. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Environmental problems are becoming more and more significant worldwide and compelling local and federal governments to take increasingly constraining measures regarding pollution and solvents. Most chiral HPLC separations are carried out in organic solvents, but the choice of mobile phase components is often restricted for cost and safety reasons. To help overcome these problems, the use of subcritical water (SCW, also called superheated water) could be an effective solvent replacement, because water is cheap, noninflammable, safe, nontoxic, and easily recyclable. The stability of commercial stationary phases under high temperature and subcritical water conditions has been studied by several research groups and reviewed [1–4]. These works concluded to the break of the siloxane bond for example in the case of C18 phases but showed also that the polymeric C18 phases having a high coverage were more resistant towards this degradation. In addition, the advantages and the limitations of high-temperature in liquid chromatography have been also discussed [5]. We have

recently shown that the separation of enantiomers can be performed under high temperature chromatography and subcritical water conditions on Chiralpak AD and Chiralcel OD [6–8]. As our first published results showed some anomalies occurred in several separations like abnormal peaks asymmetry of with CelluCoat and AmyCoat or like rapid loss of the resolution with the bonded Chiralpak IA and IB [6], whereas this problem of asymmetry appeared only after three weeks of use with Chiralpak AD and Chiralcel OD [7]. At this time we had no explanation of this phenomenon except a hypothetic degradation of the polysaccharide that has not been proven yet. But to our knowledge, no study has been yet published on the stability of polysaccharide chiral stationary phases under high temperature and subcritical water conditions. So we report here the study of the behavior of Chiralcel OD in normal and reversed mobile phase conditions at room temperature after treatment under subcritical water and high temperature conditions.

2. Experimental 2.1. Reagents

夽 IG002158 This paper is part of the special issue “Chiral Separations 2013” edited by Ruin Moaddel. ∗ Corresponding author. Tel.: +330620459246; fax: +330491829301. E-mail address: [email protected] (G. Félix).

Racemate compounds (benzoin, benzoin methyl ether, flavanone, trans-stilbene oxide and Tröger base) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). HPLC grade

http://dx.doi.org/10.1016/j.jchromb.2014.03.034 1570-0232/© 2014 Elsevier B.V. All rights reserved.

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solvents were from Carlo Erba (Marseille, France). Water was deionized by passing through an Elgastat UHQ II system. Chiralcel OD silica gel having a particle size of 20 ␮m was purchased from Chiral Technologies Europe (Illkirch, France). The columns (150 × 4.6 mm) were packed using a Touzart & Matignon column packer (Vitry, France). 2.2. Instrumentation The HPLC subcritical water system consisted of two Shimadzu LC-6A pumps, a Shimadzu GC-14 oven, a Shimadzu SPA 6A spectrophotometric detector set at 254 nm equipped with a high pressure cell (∼30 bars), a Jasco CD-2095 plus chiral detector equipped with a Jasco HI-Press cell followed by a Tescom regulator maintained at a constant back pressure of 16-17 bars. Pumps and UV detector were piloted by a Shimadzu SCL-6A system controller. The injection valve was a Rheodyne model 7125 (20 ␮L sample loop). A preheating capillary was placed into the oven between the injection valve and the column. The length of the capillary was 2 m according to Rocca et al. [9]. All the capillaries used have an internal diameter of 0.13 mm. A cooling bath was placed between the oven and the detector to decrease the temperature of the mobile phase to room temperature. The data acquisitions were processed on a Borwin 1.5 version acquisition software (JMBS developments, Grenoble, France). The chromatographic studies in normal phase were performed on a Jasco system (Jasco France, Nantes, France) fitted with a PU 980 intelligent pump equipped with a LG 980-02 tertiary gradient unit, a DG 980-50 line degasser, a 7725i Rheodyne injection valve with a 20 ␮L loop, a UV 975 intelligent UV/vis detector and a Jasco CD2095 plus chiral detector. The data acquisitions were processed on a Borwin 1.21.60 version acquisition software (JMBS developments, Grenoble, France). 2.3. Chromatographic procedure The different steps of the procedure used to appreciate the evolution of Chiralcel OD are listed in Table 1. The test compounds used in normal and in reversed mode are: benzoin (Bz), benzoin methyl ether (BME), flavanone (Fla), trans-stilbene oxide (TSO) and Tröger base (TB). For the studies of the resolution of racemates in function of the temperature, three solutes (benzoin, benzoin methyl ether and trans-stilbene oxide) were used under different mobile phase compositions based on water/acetonitrile and water/2-propanol. 3. Results and discussion The procedure chosen allows to know the behavior of the column at the starting point (after packing), to measure the impact of the heating on the coating and finally to see what can happen after several hours of use at elevated temperature both on the selectivity and the resolution. In our previous paper, we have extracted the low molecular weight polysaccharides of the coating with water in three steps (50, 100 and 150 ◦ C) [8]. The time of heating can be favorably reduced by heating directly at 150 ◦ C. On another hand, we have also shown that in several cases the addition of modifier is necessary to obtain a resolution at 150 ◦ C [6–8] so we have heated the column at 150 ◦ C in presence of 20% of modifier knowing that the use of higher modifier percentage is without interest in sub-critical water chromatography. As the more popular modifier in reversed phase chiral chromatography is acetonitrile and 2-propanol we have focus our choice on these two solvents. One column was devoted to the use of acetonitrile (Chiralcel OD-1), the second to 2-propanol (Chiralcel OD-2). All the heating required about six hours for recovering the

Fig. 1. Evolution of the chromatogram of BME in normal mode. (A) After packing. (B) After heating. (C) After tests. Mobile phase: Heptane/2-PrOH-90/10. Flow rate: 1 mL/min. Detection UV: 254 nm.

baseline of lowest absorption. After cooling it is necessary to wash the column at room temperature with pure modifier until a stable baseline was obtained to extract the dissociated coating which remained in the column. Finally the two columns were used under high temperature and subcritical conditions with acetonitrile and 2-propanol for OD-1 and OD-2, respectively. After each principal operation (packing, heating, subcritical studies) the columns were tested in normal (steps 1, 8 and 14) and reversed (steps 3, 6 and 12) modes. The results are listed in Tables 2–4 (normal mode) and Table 3 (reversed mode). Examples of the evolution of chromatogram of two compounds in each mode are shown in Figs. 1 and 2. As we have a very old packer, the tests after packing show slight difference in selectivity and resolution in each mode and for example Tröger base is separated on OD-1 and not on OD-2 in normal mode. But these differences have no influence on the studies because to appreciate the evolution only relative values are necessary. The tests realized both in normal and reversed mode after heating show, in all cases, a less than 10% decrease of the selectivity. It appears that the extraction of the low molecular weight polysaccharide has a very slight influence on the selectivity. It is the same for the resolution that slightly decreases in the two modes except for BME (Fig. 1) and TB in normal mode and TSO and Bz in reversed mode. At this point we have no explanation to understand these anomalies. Only three compounds (Bz, BME, TSO) can be separated under heating and subcritical conditions. The separation of benzoin enantiomers was performed in pure water at 150 ◦ C but only BME racemate can be resolved in pure water at 150 ◦ C starting from 2-propanol in mobile phase. However, with acetonitrile, 5% of modifier is necessary to obtain a resolution at 150 ◦ C. With TSO the resolution of the enantiomers can be effective at 110 ◦ C with 10% of acetonitrile or 15% of 2-propanol. But the main problem is that the resolutions decrease dramatically in all cases and the baseline of the chromatograms also increases. At the beginning, we thought that it was due to the subcritical conditions.

Fig. 2. Evolution of the chromatogram of BME in reversed mode. (A) After packing. (B) After heating. (C) After tests mobile phase: water/MeCN-60/40. Flow rate: 1 mL/min. Detection UV: 254 nm.

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Table 1 Steps of the procedure used to evaluate the column stabilities.

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Tests-1 Flushing Tests-2 Heating Washing Tests-3 Flushing Tests-4 Flushing Separation in temperature Washing Tests-5 Flushing Tests-6

Chiralcel OD 1

Chiralcel OD 2

Heptane/2-PrOH 90/10 2-PrOH Water/MeCN 60/40 150 ◦ C in water/MeCN-80/20 MeCN 30 ◦ C Water/MeCN 60/40 2-PrOH Heptane/2-PrOH 90/10 2-PrOH Water/MeCN 90 to 150 ◦ C MeCN 30 ◦ C Water/MeCN 60/40 2-PrOH Heptane/2-PrOH 90/10

Heptane/2-PrOH 90/10 2-PrOH Water/2-PrOH 50/50 150 ◦ C in water/2-PrOH 80/20 2-PrOH 30 ◦ C Water/2-PrOH 50/50 2-PrOH Heptane/2-PrOH 90/10 2-PrOH Water/2-PrOH 90 to 150 ◦ C 2-PrOH 30 ◦ C Water/2-PrOH 50/50 2-PrOH Heptane/2-PrOH 90/10

Table 2 Separation of the five racemates on Chiralcel OD-1. Mobile phase: heptane/2-PrOH-90/10. Flow rate: 1 mL/min. Detection UV: 254 nm.

Tests-1 after packing

Tests-4 after heating in water/MeCN

Tests-6 after use at high temperature

k1 k2 ˛ Rs k1 k2 ˛ Rs k1 k2 ˛ Rs

Bz

BME

Fla

TSO

TB

(+) 3.18 (−) 5.38 1.69 0.48 (+) 2.65 (−) 4.36 1.65 0.44 (+) 2.84 (−) 4.33 1.52 0.31

(+) 0.97 (−) 1.77 1.82 0.51 (+) 0.82 (−) 1.40 1.71 0.40 (+) 1.22 (−) 1.39 1.69 0.35

(−) 1.72 (+) 2.47 1.41 0.40 (−) 1.42 (+) 1.96 1.38 0.39 (−) 1.20 (+) 1.56 1.30 0.28

(−) 0.87 (+) 1.43 1.64 0.48 (−) 0.81 (+) 1.30 1.60 0.38 (−) 0.98 (+) 1.45 1.48 0.29

(+) 1.5 (−) 1.74 1.16 – (+) 1.24 (−) 1.29 1.04 – (+) 1.40 (−) 1.46 1.04 –

Table 3 Separation of the five racemates on Chiralcel OD-1. Mobile phase: water/MeCN-60/40. Flow rate: 1 mL/min. Detection UV: 254 nm.

Tests-2 after packing

Tests-3 after heating in water/MeCN

Tests-5 after use at high temperature

k1 k2 ˛ Rs k1 k2 ˛ Rs k1 k2 ˛ Rs

Bz

BME

Fla

TSO

TB

(+) 2.80 (−) 3.59 1.28 0.30 (+) 2.29 (−) 2.84 1.24 0.18 (+) 2.56 (−) 2.58 1.01 –

(+) 3.93 (−) 4.64 1.18 0.22 (+) 2.91 (−) 3.40 1.17 0.14 (+) 2.63 (−) 3.04 1.16 0.12

(−) 10.76 (+) 11.14 1.04 – (−) 8.56 (+) 8.56 1.00 – (−) 7.75 (+) 7.75 1.00 –

(−) 16.48 (+) 18.56 1.13 0.20 (−) 13.89 (+) 15.50 1.12 0.11 (−) 12.84 (+) 13.48 1.05 –

(+) 8.22 (−) 8.22 1.00 – (+) 6.47 (−) 6.47 1.00 – (+) 5.35 (−) 5.35 1.00 –

As the Chiralcel OD is cellulose tris-(3,5-dimethylphenylcarbamate) coated on macroporous amino silica gel [10], only dissolution of silica can explain the dramatic fall of resolution. The opening of the column shows a void at the head of the

column, between 1 and 2 cm long, confirming the dissolution of silica. That also explains the decrease of the UV absorbance of the racemates especially in reversed mode due to the silica in the UV cell (Fig. 2). It is well known that over 60 ◦ C the

Table 4 Separation of the five racemates on Chiralcel OD-2. Mobile phase: heptane/2-PrOH-90/10. Flow rate: 1 mL/min. Detection UV: 254 nm.

Tests-1 after packing

Tests-4 after heating in water/2-PrOH

Tests-6 after use at high temperature

k1 k2 ˛ Rs k1 k2 ˛ Rs k1 k2 ˛ Rs

Bz

BME

Fla

TSO

TB

(+) 2.52 (−) 4.08 1.62 0.51 (+) 2.59 (−) 3.84 1.48 0.47 (+) 2.24 (−) 3.07 1.37 0.39

(+) 0.90 (−) 1.71 1.90 0.37 (+) 0.83 (−) 1.44 1.73 0.30 (+) 0.80 (−) 0.80 1.00 –

(−) 1.56 (+) 2.28 1.46 0.40 (−) 1.36 (+) 1.96 1.44 0.36 (−) 1.23 (+) 1.76 1.43 0.30

(−) 0.84 (+) 1.88 2.24 0.74 (−) 0.71 (+) 1.56 2.20 0.70 (−) 0.76 (+) 1.23 1.62 0.34

(+) 1.40 (−) 1.40 1.00 – (+) 1.40 (−) 1.40 1.00 – (+) 1.18 (−) 1.18 1.00 –

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stability of the silica column is very bad. Chiralcel OD does not make an exception to this rule. To confirm these observation, we opened our old columns (Chiralcel OD and Chiralpak AD) used for our previous works [6–8]. All have a hole at the head of the column, the hole is more marked when the column has been used with diethylamine that contribute to accelerate the dissolution. On the other hand, the decrease of the selectivity and resolutions after the columns heating at 150 ◦ C is not due to the extraction of oligosaccharides but more probably to the beginning of the dissolution of silica. The column failure begins during the extraction of the low molecular weight polysaccharides and increases gradually under subcritical water conditions and high temperatures what returns unusable columns after approximately three weeks as previously noticed [7]. Furthermore, chiral separations are generally more efficient at low temperature since most chiral recognition processes are enthapically driven. On the other hand, higher temperatures would help enantioselectivity for entropically-controlled chiral separations. The comparison of the elution orders whether under normal HPLC operation conditions and under high temperature liquid chromatography or subcritical water conditions shows no change in the elution order for all the racemates (Tables 3 and 4). It appears that high temperatures have no influence on the recognition processes and that cellulose tris(3,5-dimethyl-phenylcarbamate) keep its recognition power at elevated temperatures.

dramatically due to the dissolution of silica which breaks up the silica bed that seems to spell death for this technology. As some racemates can be separated with pure water at elevate temperature, one solution to overcome the problem generated by silica dissolution is to use other supports that can well resist to high temperature under subcritical condition like zirconia [11–14] or titania [14]. As cellulose tris(3,5-dimethylphenyl carbamate)coated zirconia has been successfully studied in chiral HPLC [15,16], the use of substituted polysaccharides coated on zirconia under high temperature liquid chromatography and subcritical water conditions appears to be a new challenge in green chromatography. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

4. Conclusion and remarks

[14]

These studies show that the selectivity of Chiralcel OD is moderately affected by the use of the column at high temperature under subcritical water conditions. However, the resolutions fall

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