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Evaluation of injection conditions for preparative supercritical fluid chromatography
Journal of Chromatography A, 1250 (2012) 256–263
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Evaluation of injection conditions for preparative supercritical fluid chromatography Larry Miller a,∗ , Ian Sebastian b a b
Amgen, 360 Binney Street, Cambridge, MA 02142, USA Merck, 126 E. Lincoln Ave, Rahway, NJ 07065, USA
a r t i c l e
i n f o
Article history: Available online 7 June 2012 Keywords: Supercritical fluid chromatography Preparative chromatography Chiral chromatography Enantioseparations
a b s t r a c t Preparative supercritical fluid chromatography (SFC) has become the preferred method for the rapid purification of drug candidates during the pharmaceutical discovery process. This paper will discuss the evaluation of injection techniques for preparative SFC. A thorough evaluation of mixed stream vs. modifier streamTM injection was performed. It was shown that for the majority of the compounds evaluated, modifier stream injection gave better resolution relative to mixed stream injection. The improvement in resolution with modifier stream injection increased as injection volume increased. In addition, a study evaluating the effect of dissolution solvent on chromatographic performance for the preparative resolution of enantiomers using SFC showed that dissolution solvent had minimal impact on preparative resolution for the preparative SFC separation of trans stilbene oxide. © 2012 Elsevier B.V. All rights reserved.
1. Introduction For over twenty years, preparative HPLC has been the most frequently used technique for purifications in support of pharmaceutical discovery. Recently, supercritical fluid chromatography (SFC) has become a viable alternative for the analysis and purification of small molecules during drug discovery [1–5]. This is mainly due to the increasing commercial availability of analytical and preparative equipment appropriate for discovery and early development activities. With SFC a majority of the solvent in the mobile phase, usually greater than 60% is supercritical CO2 . The critical point for CO2 is a temperature of 31 ◦ C and a pressure of 73 atm. Above this point CO2 exists as a supercritical fluid and has properties intermediate between a liquid and a gas. The low viscosity and high diffusivity of the SFC mobile phase allows higher flow rates relative to HPLC, resulting in shorter run times and increased efficiencies. Increasing mobile phase velocities in SFC have significantly less impact on efficiency compared to HPLC. An SFC system can flow at linear velocities at least twice those seen in HPLC and achieve approximately the same efficiencies. In addition, the lower pressure drop in SFC allows higher linear velocities than those possible with HPLC. This increase in flow rates often results in higher productivities (material purified per unit time) relative to HPLC. The increased productivity allows compounds to be purified in a shorter time frame, reducing the time required to generate pure
∗ Corresponding author. Tel.: +1 617 444 5008; fax: +1 617 577 9913. E-mail address: [email protected] (L. Miller). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.101
compounds for pharmaceutical testing and accelerating the drug discovery process. Most pharmaceutically applicable compounds are moderately polar and CO2 alone is insufficient for elution from a chromatographic column. In most cases a polar modifier such as methanol must be added. A major advantage of preparative SFC vs. preparative HPLC is lower solvent usage. The lower solvent usage in preparative SFC is achieved by replacing a majority of the mobile phase with CO2 . CO2 is removed post chromatography by decreasing pressure, leaving only the modifier. This results in higher product concentrations post chromatography, reducing the time required for post purification solvent removal and product isolation. Additionally, SFC is an environmentally conscious technology. CO2 used in SFC is generally recovered as a byproduct of manufacturing processes, resulting in no net increase in CO2 . Overall solvent volumes for preparative SFC are 2–10 times less than seen in preparative HPLC. The reduction in solvent volumes results in reduced time and cost to isolate the purified material. Preparative separations require the introduction of larger amounts of material onto the separation column. Sample dissolution in the same solvents and solvent polarity as used for elution is ideal to minimize peak broadening and distortion and maximize preparative productivities. Poor solubility in carbon dioxide (in addition to difficulty in handling carbon dioxide under liquid or supercritical conditions) makes this approach impractical for many of the compounds purified using preparative SFC. The investigation of injection techniques for packed column SFC has been occurring for greater than twenty years. Initial attempts to increase sample loading for preparative SFC used solventless
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
257
The preparative SFC system was modified for this study (Fig. 1). A high pressure injection valve, model E60-TD from Valco Instruments (Houston, TX) was installed for sample introduction. In mixed stream operation the injection valve was installed just after the mixer. In modifier stream injection the injection valve was installed in the modifier line just prior to the mixer. In normal operation of the SFC350, the CO2 and modifier stream are mixed and then passed through a heat exchanger. For this study only the CO2 stream passed through the heat exchanger. The modifier used for all studies was methanol w/0.2% (v/v) diethylamine. For all analytical and preparative experiments system temperature was set to 40 ◦ C. All analytical experiments were conducted with system back pressure regulator set at 120 bar. All preparative experiments were conducted with system back pressure regulator set at 100 bar. Under these conditions the system was operated in a subcritical state. 2.2. Materials
Fig. 1. Instrument schematic for mixed stream and modifier stream injection.
injections. This process involved trapping the sample to be purified onto a pre-column prior to injection onto the separation column [6–8]. This is accomplished by pumping sample solution onto a pre-column from which the dissolution solvent was then removed by passing warm gas (nitrogen) over the pre-column. SFC mobile phase is then pumped through the pre-column to solubilize the sample and sweep onto the separation column. This approach has been shown to increase preparative loadings over standard injection techniques. Another approach is to couple supercritical fluid extraction (SFE) and preparative SFC. This technique was shown to increase loading capacity ten times higher and allow injection of a few hundred milligrams onto a 10 mm × 250 mm column [9]. Both solventless injection and SFE/SFC allowed increased injections in preparative SFC but were time consuming and were never utilized heavily for preparative SFC. There are currently two approaches used for sample introduction in preparative SFC. The first, mixed stream injection, introduces the sample solution just prior to the column, after carbon dioxide and the modifier solvent are mixed. This approach injects the sample just prior to the column but has the issue of decompression of the injector loop contents prior to loading with sample solution. The second approach, modifier stream injection, introduces sample solution into the modifier flow stream, prior to mixing with carbon dioxide. Modifier stream injection has been shown to improve peak shape in preparative SFC under gradient conditions [10,11]. Modifier stream injection can be compared to at-column dilution (ACD) injection technique utilized for preparative reverse phase chromatography [12,13]. An extensive comparison of mixed stream and modifier stream injection has not been published. This paper compares mixed stream and modifier stream injection techniques for the analytical and preparative separation of a series of achiral and racemic compounds under isocratic SFC conditions.
2. Experimental 2.1. Equipment The preparative SFC was a Prep 350 from Thar. (Pittsburgh, PA, USA).
The preparative column was obtained from Chiral Technologies, Inc. (Exton, PA, USA) or Princeton Chromatography (Cranbury, NJ) as pre-packed columns. All chemicals were purchased from Sigma–Aldrich or through VWR. The solvents were reagent grade or better and obtained from a variety of sources. 3. Results and discussion 3.1. Analytical injection evaluation 3.1.1. Racemic compounds An initial study was designed to evaluate two injection conditions using analytical scale loadings. The racemates used for all studies are summarized in Table 1. These racemates were chosen to provide a range of modifier percentages for elution. Using a Chiralpak AD-H (5 m, 3 cm × 25 cm), 1 ml of a 1 mg/ml methanol solution of each of the racemates was injected. Total flowrate (CO2 and modifier) was kept constant at 126 ml/min. The co-solvent percentage (methanol w/0.2% diethylamine) was modified to achieve a consistent retention (3–4 min, average k of 2.5–3.75) for each racemate. Plate count was measured for both enantiomers (plate counts were calculated using Advanced Chemistry Development ChromManager software). The plate counts are lower than expected for a five micron stationary phase due to the preparative SFC system having larger dwell/dead volumes than seen with an analytical SFC system, resulting in band broadening and lower plate counts. In addition many of the test racemates are not ideal probe molecules, containing functional groups that give non-specific interactions and broader peaks. While use of a preparative SFC system with reduced dwell/dead volumes would have resulted in more accurate data, as data for both injection techniques was collected on the same system, any inefficiencies were identical for both injection techniques. These results are summarized in Table 2. A graphical comparison of these results is shown in Fig. 2. Comparison of plate counts showed four racemates that showed no difference (<25% difference) between injection techniques. Six racemates showed higher (>25%) plate counts with modifier stream injection and one racemate showed higher plate counts with mixed stream injection. Evaluation of the data in Table 2 for the peak 1 shows that the largest increase in plate count for modifier stream vs. mixed stream injection are seen with peaks with k < 1. Once retention increases to k > 2 (with the exception of racemates 1 and 11) there is little difference in plate count. The plate count data for the mixed stream injection of chiral compound 3 is unusual. The plate count for peak 2 is higher than for peak 1. This was not seen for modifier stream injection. The racemate has k of 0.93 and 5.42 for the
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Chiral test compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Disopyramide 4-Benzoloxy-2-azetidinone ␣-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol Fenoterol 1-(4-Chlorobenzylhydryl)piperazine Sulconazole Warfarin 1,5-Dimethyl-4-phenyl-2-imidazolidinone Propranolol Pantothenol Benzyl mandelate Sulfinpyrazone Mianserin HCl Trans stilbene oxide
Achiral test compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Tetracaine HCl Metoprolol tartrate Doxepin HCl Nabumetone Theophylline Tetramisole HCl Naproxen sodium Dibucaine HCl Gemfibrozil Metronidazole Tolbutamide n-Propyl-4-hydroxybenzoate p-Hydroxybenzoic acid Antipyrine Procainamide HCl Salicylanilide Labetalol HCl Chloramphenicol Dipyridamole Sulfamethoxazole Trichlormethiazide Furosemide 5-(4-Hydroxyphenyl)-5-phenylhydantoin Naringenin
Table 2 Plate count and k for analytical modifier stream and mixed stream injection. Chiral Percent Modifier stream compounds modifier injection
Mixed stream injection
N, k (peak 1) N, k (peak 2) N, k (peak 1) N, k (peak 2) 25 25 35 30 25 25 25 15 20 25 20
200
150
100
50
0 1
2
3
4
746, 1.18 5251, 2.87 3823, 0.98 1474, 0.70 1063, 3.35 2215, 3.89 1317, 2.42 770, 3.31 891, 1.76 1634, 1.69 1927, 2.43
141, 2.12 4934, 3.43 3391, 5.26 257, 2.65 765, 4.23 2127, 4.75 887, 4.37 618, 4.37 600, 2.57 1202, 3.62 1358, 4.20
601, 1.08 4751, 2.81 1886, 0.93 450, 0.57 795, 3.40 2023, 3.76 1383, 2.31 656, 3.27 463, 1.63 842, 1.57 4385, 2.29
101, 2.12 4415, 3.26 3038, 5.42 128, 2.50 557, 4.27 1781, 4.63 816, 5.13 496, 4.33 279, 2.43 685, 3.60 1664, 4.12
5
6
7
8
9
10
11
-50
-100
enantiomers. The poor plate count for peak 1 is directly related to the impact of mixed stream injection on less retained peaks. The slug of methanol from injection causes a localized severe disturbance in the SFC mobile phase causing peak shape distortion. Maximum analytical efficiency is obtained when analytes are dissolved in mobile phase. With SFC this is not practical and standard practice is to dissolve in modifier (methanol) only. Methanol has a higher polarity than the CO2 /methanol mobile phase. The higher polarity of the methanol can cause some of the analyte not to be adsorbed on the stationary phase, resulting in peak breakthrough or distortion. As retention is decreased, the distortion becomes more severe.
1 2 3 4 5 6 7 8 9 10 11
Percent Increase in Plate Count for Modifer Stream Relave to Mixed Stream Injecon
Table 1 Test compounds used for evaluation.
Compound Number
Fig. 2. Comparison of plate count for modifier stream vs. mixed stream for analytical injection of racemates. Blue: peak 1; red: peak 2. Positive number indicates modifier stream injection giving higher resolution, negative number indicates mixed stream injection giving higher resolution. See text for experimental conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3.1.2. Achiral compounds The analytical study described above for racemic compounds was duplicated using achiral compounds. A total of 24 compounds (listed in Table 1) were used for this study. The test compounds were chosen to provide a wide range of modifier percentages that maintained an approximately constant elution time (3–5 min, k of 2.5–5). Flowrate was kept constant at 126 ml/min. Using a Princeton 2-ethylpyridine column (5 m, 3 cm × 25 cm), 1 ml injections of a 1 mg/ml methanol solution of each of the test compounds were made and plate count measured. Results are summarized in Table 3. It is interesting that all plate counts at 30% modifier are low. Plate counts for chiral solutes at 30% modifier were as expected (Table 2), seeing to eliminate instrumentation as a source of the low plate counts. Fig. 3 compares the difference in plate count for each injection technique at different modifier percentages. At low modifier conditions (5 and 10%), modifier stream injection affords higher Table 3 Plate count for analytical modifier stream and mixed stream injection. Achiral compounds
k a
Modifer percentage
Modifier stream injection (N)
Mixed stream injection (N)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
3.23 4.09 2.43 1.18 3.34 1.70 4.59 1.36 1.66 1.67 4.40 2.57 2.75 1.10 2.00 2.63 1.58 2.42 2.65 3.75 3.11 3.05 1.22 2.15
5 5 5 5 5 10 10 10 10 10 10 10 10 10 15 15 20 20 20 20 30 30 30 30
3848 3448 2699 1614 3323 3224 3064 3015 3032 2391 3763 1971 2163 2410 1013 1933 1459 1963 899 1233 213 478 479 248
1486 659 1639 2437 2552 2064 2909 1222 3036 2554 3270 2157 2372 1440 891 2791 1066 2518 1155 2310 223 524 524 335
a
k measured from analytical analysis.
80
3500
60
3000 Average Plate Number
Percent Change Plate Count Modifier Stream vs. Mixed Stream Injection
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
40 20 0 -20 -40 0
5
10
15
20
25
30
35
2500 2000 1500 1000 500
Percent Modifier
plate counts relative to mixed stream injection. At higher modifier conditions (>10%), the difference between the two injection techniques was less pronounced. At 15 and 20% modifier mixed stream injection was slightly better than modifier stream injection. While this data is interesting it may not be representative due to the smaller number of compounds tested at 15 and 20% (2 and 4 respectively) compared to 5 and 10% (5 and 9 compounds). There appears to be a trend toward the choice of injection technique being less critical as the modifier percentage increases. At low modifier percentages and using modifier stream injection, the time to apply the sample to the head of the column is increased due to the low flow rate of the modifier pump. For example with a system flow rate of 126 ml/min and 5% modifier, the modifier flow rate is ∼6 ml/min. At this flow rate a 1 ml injection would take ∼10 s to be applied to the head of the column using modifier stream injection. This compares to ∼½ second with mixed stream injection. One would predict that the increased injection time would result in broader peaks and a lower plate count for modifier stream relative to mixed stream injection. For the low modifier percentage studies (5 and 10%) the opposite was observed, with modifier stream giving higher plate counts. This is due to the larger disturbance in mobile phase polarity when methanol is injected using mixed stream injection into a low modifier percent mobile phase. One milliliter of methanol was injected into an SFC mobile phase of 5 or 10% modifier causes a larger localized disturbance in mobile phase polarity, and ultimately peak distortion relative to a higher modifier percentage. This disturbance in peak shape more than offsets the increase in peak width seen with increased injection time using mixed stream injection. A series of experiments were performed to determine if the modifier percentage used for separation had an impact on the best analytical injection technique. Fig. 4 plots the average plate count for the compound analyzed at each modifier percentage for the two injection techniques. As modifier percentage increases there is a general trend toward lower average plate counts for both injection techniques. At low modifier percentages (5 and 10%) modifier stream injection is significantly better. Above 15% modifier there is less difference between the two injection techniques. Most of the analytical SFC systems on the market utilize mixed stream injection, choosing to inject sample immediately prior to the column. This eliminates injection delays that would be observed using modifier stream injection and isocratic elution at low modifier percentages. A more detailed study of injection techniques using an analytical SFC system would be of interest and may provide guidance on better equipment design.
0 5
0
10
15 20 % Modifer (MeOH)
25
30
35
Fig. 4. Average plate count vs. modifier percentage for achiral compounds using modifier stream and mixed stream injection technique (blue diamond: modifier stream injection, magenta square: mixed stream injection). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3.2. Preparative injection evaluation For the preparative injection study a total of 13 racemates were evaluated. Each racemate was dissolved in 20 mg/ml in methanol. 1, 2, 3, 4 and 5 ml injections were made on a Chiralpak AD-H (5 m, 3 cm × 25 cm). Flowrate was kept constant at 126 ml/min. Co-solvent percentage was varied to achieve approximately consistent retention for each racemate (3–4 min, average k for peak 1 and peak 2 of 2.5–3.75). From each injection, resolution was measured for both enantiomers (resolution was calculated using Rs = (t2 − t1 )/(1/2(tw1 + tw2 )) where t1 and t2 are retention times for peaks 1 and 2 respectively and tw1 and tw2 are peak width at baseline for peaks 1 and 2). The results from these analyses are summarized in Table 4. Of the 14 racemates, modifier stream injection was better for five racemates (3, 4, 6, 13 and 14), mixed stream injection was better for one racemate (7) and seven racemates (2, 5, 8, 9, 10, 11 and 12) showed no difference between injection techniques. Fig. 5 shows the average percentage increase in resolution (±one standard deviation) for modifier stream injection vs. mixed stream injection. On average modifier stream injection giving higher resolutions relative to mixed stream injection. This advantage increased
35.0
Percent Increase in Resolution for Modifer Stream Injection compared to Mixed Stream Injection
Fig. 3. Comparison of plate count for modifier stream vs. mixed stream for analytical injection of achiral compounds at various modifier percentages. Each data point is average of compounds analyzed under those conditions. Positive number indicates modifier stream injection giving higher resolution, negative number indicates mixed stream injection giving higher resolution. See text for experimental conditions.
259
30.0 25.0 20.0 15.0 10.0 5.0 0.0 0
1
2
3
4
5
6
(5.0)
Injection Volume (mL) Fig. 5. Average percent increase in resolution for modifier stream injection vs. mixed stream injection vs. injection volume for preparative separation of chiral compounds.
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
Table 4 Resolution for preparative injection of racemates using modifier stream and mixed stream injection. Racemate
Volume (ml)
Rs modifier stream injection
Rs mixed stream injection
2
1 2 3 4 5
0.86 0.59 0.45 0.31 0.28
0.85 0.68 0.54 0.33 0.28
1 2 3 4 5
7.62 5.82 4.88 4.30 3.71
7.03 5.24 4.04 3.20 3.09
4
1 2 3 4 5
2.60 2.46 1.75 1.84 1.53
1.81 1.21 0.74 0.64 0.64
5
1 2 3 4 5
0.66 0.49 0.38 0.28 0.24
0.69 0.54 0.40 0.25 0.21
6
1 2 3 4 5
1.13 0.98 0.80 0.75 0.68
0.95 0.66 0.48 0.43 0.32
7
1 2 3 4 5
2.68 2.10 1.88 1.75 1.48
2.68 2.33 2.05 1.72 1.59
8
1 2 3 4 5
0.95 0.81 0.66 0.64 0.53
0.87 0.86 0.82 0.75 0.72
9
1 2 3 4 5
1.00 0.89 0.66 0.64 0.64
1.11 0.84 0.57 0.35 0.12
10
1 2 3 4 5
2.99 2.33 2.04 1.86 1.55
2.97 2.54 2.07 1.67 1.34
11
1 2 3 4 5
2.41 1.88 1.37 1.21 1.09
1.49 1.78 1.53 1.27 1.19
12
1 2 3 4 5
2.30 2.27 2.24 1.96 1.93
2.45 2.34 2.16 2.00 1.86
13
1 2 3 4 5
2.55 1.83 1.52 1.22 1.05
2.45 1.70 1.39 1.12 0.88
1 2 3 4 5
3.24 2.40 1.92 1.61 1.49
3.03 2.24 2.04 1.62 1.47
3
14
1 Resolution
260
0.8 0.6 0.4 0.2 0 0
2 4 Injection Volume (mL)
6
Fig. 6. Effect of injection volume on preparative resolution for mixed stream injection of racemate 9. Injection quantity kept constant at 100 mg.
as the injection volume increased. When a preparative injection is made using mixed stream injection, a plug of methanol enters the column. This injection technique results in a temporary, localized increase in mobile phase polarity. This polarity increase can cause peak distortion, resulting in reductions in resolution. As the volume of the methanol plug increases there is a more pronounced impact on peak shape and thus resolution decreases. To illustrate this point further, racemate 9 was prepared at 20, 40, 80 and 160 mg/ml in methanol. Injection volume was adjusted to maintain a constant load of 100 mg (5, 2.5, 1.25, 0.63 ml respectively). Resolutions for these separations were 0.244, 0.629, 0.882 and 0.893. The results are summarized in Fig. 6. The chromatograms are shown in Fig. 7. As the injection volume increased there is a drastic impact on peak shape, resulting in reduced resolution. As a benchmark the resolution is 0.64 for a 5 ml (100 mg @ 20 mg/ml) injection using modifier stream injection conditions (chromatogram shown in Fig. 7). The data from Table 4 was evaluated to determine if retention time had an impact on preparative resolution. Fig. 8 shows the increase in resolution observed for modifier stream injection vs. mixed stream injection with retention time for peak 1. Fig. 9 shows the same data for peak 2. The data shows that for shorter retention (<3 min on 3 cm × 25 cm column @ 126 ml/min) that modifier stream injection affords higher resolution. Above 3 min retention the two injection methods are approximately equivalent. Another variable that may impact choice of injection method is modifier percentage in the mobile phase. One would predict at low modifier percentages that modifier stream injection would prevail and as the modifier percentage increased the differences would be less. Unfortunately the number of racemates that eluted at higher modifier percentages (>25%) was too small to draw any definite conclusions. In summary it appears that the most important variable on which injection technique will provide best resolution is injection volume. With increasing injection volume, modifier stream injection affords increased resolution relative to mixed stream injection. Retention time also has an impact; with a retention factor of less than approximately 3.5, modifier stream injection is the better choice, above a retention factor of 3.5 the difference between the methods is reduced. Modifier percentage may also play a role but limited data at higher modifier percentages did not allow a meaningful comparison of the injection techniques. 3.3. Effect of dissolution solvent on preparative resolution Ideally for chromatographic purifications the sample should be dissolved in the same solvent as the mobile phase. This is not always possible due to limited solubility of the compounds in the chromatographic mobile phase. In that case it is standard practice in preparative chromatography to utilize dissolution solvents different from the chromatographic mobile phase. This practice allows purification of compounds that are poorly soluble in the mobile
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
261
Fig. 7. Preparative separation of racemate 9 using mixed stream injection. Load kept constant at 100 mg. Injection volumes: (A) 0.63 ml, (B) 1.25 ml, (C) 2.5 ml, and (D) 5 ml. The injection of 100 mg (5 ml injection volume) using modifier stream injection is shown in chromatogram E.
phase or to reduce the injection volume for compounds with minimal solubility. It is well known in HPLC that injection from stronger solvents than the mobile phase can lead to poor peak shape, sample breakthrough and reduced loadings [13]. A series of experiments were performed to study the impact of varied polarity dissolution solvents on the preparative separation of racemate 14, trans stilbene oxide (TSO). TSO was dissolved in 20 mg/ml in numerous solvents and solvent mixtures. Four milliliter injections (80 mg total) were injected on a 3 cm × 25 cm Chiralpak AD-H preparative column using mixed stream injection. The results are summarized in Table 5. These experiments showed the dissolution solvent had
minimal impact on plate count, retention time or resolution regardless of the dissolution solvent strength. The same type of study was performed for preparative HPLC. 50 mg of TSO in 20 mg/ml in different solvents was injected on a 3 cm × 15 cm Chiralpak ADH column. The mobile phase was 10/90 (v/v) isopropanol/heptane. Due to solvent immiscibility, fewer dissolution solvents were investigated for HPLC. The results from this study are summarized in Table 6. This work shows that dissolution solvent had a large impact on plate count for the first eluting enantiomer (k = 1.14). The impact was less pronounced for the second eluting enantiomer (k = 4.44). The larger the difference between the polarity of the sample
262
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263 500
Dissolution solvent
Rt1 (min)
Rt2 (min)
N1
N2
Rs
Methanol Ethanol Acetonitrile Isopropanol 75/25 isopropanol/heptane 50/50 isopropanol/heptane 25/75 isopropanol/heptane 10/90 isopropanol/heptane Heptane 50/50 methanol/dimethylether 75/25 methanol/dichloromethane
2.55 2.56 2.55 2.59 2.60 2.60 2.59 2.59 2.57 2.59 2.62
3.96 3.99 3.94 4.08 4.06 4.07 4.05 4.02 4.00 4.01 4.06
620 522 750 507 548 642 691 722 762 421 690
358 334 418 300 329 355 388 399 399 436 420
1.88 1.87 1.97 1.86 1.88 2.02 1.96 2.05 2.03 1.85 1.96
a Chiralpak AD-H, 3 cm × 25 cm, 126 ml/min, 25% methanol w/0.2% diethylamine, 80 mg injection (4 ml @ 20 mg/ml), mixed stream injection.
Percent Incrase in Resoluon for Modifer Stream Relave to Mixed Stream Injecon
Table 5 Effect of dissolution solvent on preparative SFC separation of racemate 14.a
400
300
200
100
0 2.00
0.00
4.00
6.00
8.00
10.00
12.00
-100
Table 6 Effect of dissolution solvent on preparative HPLC separation of racemate 14.a Dissolution Solvent
Rt1
Rt2
N1
N2
Rs
Heptane 10/90 isopropanol/heptane 25/75 isopropanol/heptane 50/50 isopropanol/heptane 75/25 isopropanol/heptane Isopropanol Ethanol
3.22 3.22 3.19 3.17 3.10 3.06 3.22
8.27 8.22 8.13 8.18 8.11 8.16 8.37
3229 2740 2691 2192 1980 391 1600
718 826 885 834 840 718 613
6.83 6.97 7.37 7.32 7.16 6.19 6.19
Chiralpak AD-H, 3 cm × 15 cm, 42 ml/min, 10/90 (v/v) isopropanol/heptane, 50 mg injection (2.5 ml @ 20 mg/ml).
Percent Increase in Resoluon for Modifier Stream relave to Mixed Stream Injecon
a
500
Fig. 9. Percent increase in resolution for modifier stream/mixed stream vs. retention time for second eluting peak of racemates. Positive number indicates modifier stream injection giving higher resolution; negative number indicates mixed stream injection giving higher resolution.
70 50
30
10
10
-10
70
100
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4:48
-10
10
10
4.00
3:36
4:48
-10
3:36
4:48
D
50 30
2:24
2:24
70
30
-10
0
3:36
C
50
200
-100
2:24
B
50
30
400 300
70
A
2:24
3:36
4:48
Peak 1 Retenon Time (minutes)
Fig. 8. Percent increase in resolution for modifier stream/mixed stream vs. retention time for first eluting peak of racemates. Positive number indicates modifier stream injection giving higher resolution; negative number indicates mixed stream injection giving higher resolution.
dissolution solvent and the polarity of the mobile phase, the larger the impact on plate count. The plate count for peak 1 using isopropanol as dissolution solvent was much lower than expected. These results were duplicated to ensure they were accurate. The reduction in plate count for isopropanol was much larger than with ethanol, indicating that the polarity of the dissolution solvent is not the only factor. It is possible that the increased viscosity of isopropanol and resulting temperature increase may have played a role in the low plate count. In addition the increased viscosity of the solute plug may cause viscous fingering, resulting in the poor chromatography results observed [14]. The impact of dissolution solvent is best illustrated for the separation of peak 1 and an achiral impurity that elutes just after peak 1. Chromatograms of this region are shown in Fig. 10. As the polarity of the dissolution solvent increases the separation between peak 1 and the impurity decreases. With a dissolution solvent of 50% isopropanol in heptane, and more polar, the separation is lost.
E
70 50
50
30
30
10
10
-10
-10
2:24
3:36
F
70
4:48
2:24
3:36
4:48
G
70 50 30 10 -10
2:24
3:36
4:48
Fig. 10. HPLC chromatograms of preparative separation of trans stilbene oxide with various dissolution solvents. (A) Heptane, (B) 10/90 isopropanol/heptane, (C) 25/75 isopropanol/heptane, (D) 50/50 isopropanol/heptane, (E) 75/25 isopropanol/heptane, (F) isopropanol, and (G) ethanol. X axis is time in minutes.
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
Unfortunately this achiral impurity is not resolved from peak 1 under SFC conditions. As this study was only performed with one compound, and mobile phase polarities were not identical for the HPLC and SFC separations, additional studies should be performed to determine if this is a general phenomenon of preparative SFC.
263
Acknowledgements The authors thank Wolfgang Goetzinger, Manny Ventura and Kyung Gahm for assistance with the technical review of this article. References
4. Conclusions The choice of injection technique for preparative SFC can have a large impact on peak shape, resolution and preparative productivity. For the majority of compounds studied, modifier stream injection was a better choice for injection relative to mixed stream injection. In mixed stream injection there is a temporary localized increase in mobile phase strength due to the methanol used for injection which can lead to peak distortion and reduced resolution. The difference is more pronounced as the injection volume increases and as retention time decreases. The polarity of the dissolution solvent has little impact on efficiency, retention or resolution for the preparative SFC separation of trans stilbene oxide. This data was in contrast to the preparative HPLC separation where dissolution solvent polarity had a large impact on separation efficiency.
[1] D.R. Brocks, F.M. Pasutto, F. Jamali, J. Chromatogr. 581 (1992) 83. [2] E.R. Francotte, Switz. Chimia 51 (10) (1997) 717. [3] E. Francotte, Preparative Enantioselective Chromatography, Blackwell Publishing, 2005. [4] L. Miller, D. Honda, R. Fronek, K. Howe, J. Chromatogr. A 658 (1994) 429. [5] E.R. Francotte, in: S. Ahuga (Ed.), Switz. Chiral sep., American Chemical Society, 1997, p. 271. [6] G. Cretier, R. Majdalani, J. Neffati, J.L. Rocca, Chromatographia 38 (March (5/6)) (1994) 330. [7] Y. Hirata, Y. Kawaguchi, K. Kitano, Chromatographia 40 (January (1/2)) (1995) 42. [8] J.Y. Gao, C.E. Kibbey, Chromatographia 51 (June (11/12)) (2000) 747. [9] Y. Yamauchi, M. Kuwajima, M. Saito, J. Chromatogr. 515 (1990) 285. [10] T.A. Berger, K.D. Fogelman, US2003/0034307 A1. [11] K. Fogelman, HPLC 2004 Poster, Philadelphia, PA, June, 2004. [12] U.D. Neue, C.B. Mazza, J.Y. Cavanaugh, Z. Lu, T.E. Wheat, Chromatographia Supplement 57 (2003) S-121. [13] K.F. Blom, J. Combust. Chem. 4 (2002) 295. [14] R.A. Shalliker, H.J. Catchpoole, G.R. Dennis, G. Guiochon, J. Chromatogr. A 1142 (2007) 48.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Evaluation of injection conditions for preparative supercritical fluid chromatography Larry Miller a,∗ , Ian Sebastian b a b
Amgen, 360 Binney Street, Cambridge, MA 02142, USA Merck, 126 E. Lincoln Ave, Rahway, NJ 07065, USA
a r t i c l e
i n f o
Article history: Available online 7 June 2012 Keywords: Supercritical fluid chromatography Preparative chromatography Chiral chromatography Enantioseparations
a b s t r a c t Preparative supercritical fluid chromatography (SFC) has become the preferred method for the rapid purification of drug candidates during the pharmaceutical discovery process. This paper will discuss the evaluation of injection techniques for preparative SFC. A thorough evaluation of mixed stream vs. modifier streamTM injection was performed. It was shown that for the majority of the compounds evaluated, modifier stream injection gave better resolution relative to mixed stream injection. The improvement in resolution with modifier stream injection increased as injection volume increased. In addition, a study evaluating the effect of dissolution solvent on chromatographic performance for the preparative resolution of enantiomers using SFC showed that dissolution solvent had minimal impact on preparative resolution for the preparative SFC separation of trans stilbene oxide. © 2012 Elsevier B.V. All rights reserved.
1. Introduction For over twenty years, preparative HPLC has been the most frequently used technique for purifications in support of pharmaceutical discovery. Recently, supercritical fluid chromatography (SFC) has become a viable alternative for the analysis and purification of small molecules during drug discovery [1–5]. This is mainly due to the increasing commercial availability of analytical and preparative equipment appropriate for discovery and early development activities. With SFC a majority of the solvent in the mobile phase, usually greater than 60% is supercritical CO2 . The critical point for CO2 is a temperature of 31 ◦ C and a pressure of 73 atm. Above this point CO2 exists as a supercritical fluid and has properties intermediate between a liquid and a gas. The low viscosity and high diffusivity of the SFC mobile phase allows higher flow rates relative to HPLC, resulting in shorter run times and increased efficiencies. Increasing mobile phase velocities in SFC have significantly less impact on efficiency compared to HPLC. An SFC system can flow at linear velocities at least twice those seen in HPLC and achieve approximately the same efficiencies. In addition, the lower pressure drop in SFC allows higher linear velocities than those possible with HPLC. This increase in flow rates often results in higher productivities (material purified per unit time) relative to HPLC. The increased productivity allows compounds to be purified in a shorter time frame, reducing the time required to generate pure
∗ Corresponding author. Tel.: +1 617 444 5008; fax: +1 617 577 9913. E-mail address: [email protected] (L. Miller). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.101
compounds for pharmaceutical testing and accelerating the drug discovery process. Most pharmaceutically applicable compounds are moderately polar and CO2 alone is insufficient for elution from a chromatographic column. In most cases a polar modifier such as methanol must be added. A major advantage of preparative SFC vs. preparative HPLC is lower solvent usage. The lower solvent usage in preparative SFC is achieved by replacing a majority of the mobile phase with CO2 . CO2 is removed post chromatography by decreasing pressure, leaving only the modifier. This results in higher product concentrations post chromatography, reducing the time required for post purification solvent removal and product isolation. Additionally, SFC is an environmentally conscious technology. CO2 used in SFC is generally recovered as a byproduct of manufacturing processes, resulting in no net increase in CO2 . Overall solvent volumes for preparative SFC are 2–10 times less than seen in preparative HPLC. The reduction in solvent volumes results in reduced time and cost to isolate the purified material. Preparative separations require the introduction of larger amounts of material onto the separation column. Sample dissolution in the same solvents and solvent polarity as used for elution is ideal to minimize peak broadening and distortion and maximize preparative productivities. Poor solubility in carbon dioxide (in addition to difficulty in handling carbon dioxide under liquid or supercritical conditions) makes this approach impractical for many of the compounds purified using preparative SFC. The investigation of injection techniques for packed column SFC has been occurring for greater than twenty years. Initial attempts to increase sample loading for preparative SFC used solventless
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
257
The preparative SFC system was modified for this study (Fig. 1). A high pressure injection valve, model E60-TD from Valco Instruments (Houston, TX) was installed for sample introduction. In mixed stream operation the injection valve was installed just after the mixer. In modifier stream injection the injection valve was installed in the modifier line just prior to the mixer. In normal operation of the SFC350, the CO2 and modifier stream are mixed and then passed through a heat exchanger. For this study only the CO2 stream passed through the heat exchanger. The modifier used for all studies was methanol w/0.2% (v/v) diethylamine. For all analytical and preparative experiments system temperature was set to 40 ◦ C. All analytical experiments were conducted with system back pressure regulator set at 120 bar. All preparative experiments were conducted with system back pressure regulator set at 100 bar. Under these conditions the system was operated in a subcritical state. 2.2. Materials
Fig. 1. Instrument schematic for mixed stream and modifier stream injection.
injections. This process involved trapping the sample to be purified onto a pre-column prior to injection onto the separation column [6–8]. This is accomplished by pumping sample solution onto a pre-column from which the dissolution solvent was then removed by passing warm gas (nitrogen) over the pre-column. SFC mobile phase is then pumped through the pre-column to solubilize the sample and sweep onto the separation column. This approach has been shown to increase preparative loadings over standard injection techniques. Another approach is to couple supercritical fluid extraction (SFE) and preparative SFC. This technique was shown to increase loading capacity ten times higher and allow injection of a few hundred milligrams onto a 10 mm × 250 mm column [9]. Both solventless injection and SFE/SFC allowed increased injections in preparative SFC but were time consuming and were never utilized heavily for preparative SFC. There are currently two approaches used for sample introduction in preparative SFC. The first, mixed stream injection, introduces the sample solution just prior to the column, after carbon dioxide and the modifier solvent are mixed. This approach injects the sample just prior to the column but has the issue of decompression of the injector loop contents prior to loading with sample solution. The second approach, modifier stream injection, introduces sample solution into the modifier flow stream, prior to mixing with carbon dioxide. Modifier stream injection has been shown to improve peak shape in preparative SFC under gradient conditions [10,11]. Modifier stream injection can be compared to at-column dilution (ACD) injection technique utilized for preparative reverse phase chromatography [12,13]. An extensive comparison of mixed stream and modifier stream injection has not been published. This paper compares mixed stream and modifier stream injection techniques for the analytical and preparative separation of a series of achiral and racemic compounds under isocratic SFC conditions.
2. Experimental 2.1. Equipment The preparative SFC was a Prep 350 from Thar. (Pittsburgh, PA, USA).
The preparative column was obtained from Chiral Technologies, Inc. (Exton, PA, USA) or Princeton Chromatography (Cranbury, NJ) as pre-packed columns. All chemicals were purchased from Sigma–Aldrich or through VWR. The solvents were reagent grade or better and obtained from a variety of sources. 3. Results and discussion 3.1. Analytical injection evaluation 3.1.1. Racemic compounds An initial study was designed to evaluate two injection conditions using analytical scale loadings. The racemates used for all studies are summarized in Table 1. These racemates were chosen to provide a range of modifier percentages for elution. Using a Chiralpak AD-H (5 m, 3 cm × 25 cm), 1 ml of a 1 mg/ml methanol solution of each of the racemates was injected. Total flowrate (CO2 and modifier) was kept constant at 126 ml/min. The co-solvent percentage (methanol w/0.2% diethylamine) was modified to achieve a consistent retention (3–4 min, average k of 2.5–3.75) for each racemate. Plate count was measured for both enantiomers (plate counts were calculated using Advanced Chemistry Development ChromManager software). The plate counts are lower than expected for a five micron stationary phase due to the preparative SFC system having larger dwell/dead volumes than seen with an analytical SFC system, resulting in band broadening and lower plate counts. In addition many of the test racemates are not ideal probe molecules, containing functional groups that give non-specific interactions and broader peaks. While use of a preparative SFC system with reduced dwell/dead volumes would have resulted in more accurate data, as data for both injection techniques was collected on the same system, any inefficiencies were identical for both injection techniques. These results are summarized in Table 2. A graphical comparison of these results is shown in Fig. 2. Comparison of plate counts showed four racemates that showed no difference (<25% difference) between injection techniques. Six racemates showed higher (>25%) plate counts with modifier stream injection and one racemate showed higher plate counts with mixed stream injection. Evaluation of the data in Table 2 for the peak 1 shows that the largest increase in plate count for modifier stream vs. mixed stream injection are seen with peaks with k < 1. Once retention increases to k > 2 (with the exception of racemates 1 and 11) there is little difference in plate count. The plate count data for the mixed stream injection of chiral compound 3 is unusual. The plate count for peak 2 is higher than for peak 1. This was not seen for modifier stream injection. The racemate has k of 0.93 and 5.42 for the
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Chiral test compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Disopyramide 4-Benzoloxy-2-azetidinone ␣-(2,4-Dichlorophenyl)-1H-imidazole-1-ethanol Fenoterol 1-(4-Chlorobenzylhydryl)piperazine Sulconazole Warfarin 1,5-Dimethyl-4-phenyl-2-imidazolidinone Propranolol Pantothenol Benzyl mandelate Sulfinpyrazone Mianserin HCl Trans stilbene oxide
Achiral test compounds 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Tetracaine HCl Metoprolol tartrate Doxepin HCl Nabumetone Theophylline Tetramisole HCl Naproxen sodium Dibucaine HCl Gemfibrozil Metronidazole Tolbutamide n-Propyl-4-hydroxybenzoate p-Hydroxybenzoic acid Antipyrine Procainamide HCl Salicylanilide Labetalol HCl Chloramphenicol Dipyridamole Sulfamethoxazole Trichlormethiazide Furosemide 5-(4-Hydroxyphenyl)-5-phenylhydantoin Naringenin
Table 2 Plate count and k for analytical modifier stream and mixed stream injection. Chiral Percent Modifier stream compounds modifier injection
Mixed stream injection
N, k (peak 1) N, k (peak 2) N, k (peak 1) N, k (peak 2) 25 25 35 30 25 25 25 15 20 25 20
200
150
100
50
0 1
2
3
4
746, 1.18 5251, 2.87 3823, 0.98 1474, 0.70 1063, 3.35 2215, 3.89 1317, 2.42 770, 3.31 891, 1.76 1634, 1.69 1927, 2.43
141, 2.12 4934, 3.43 3391, 5.26 257, 2.65 765, 4.23 2127, 4.75 887, 4.37 618, 4.37 600, 2.57 1202, 3.62 1358, 4.20
601, 1.08 4751, 2.81 1886, 0.93 450, 0.57 795, 3.40 2023, 3.76 1383, 2.31 656, 3.27 463, 1.63 842, 1.57 4385, 2.29
101, 2.12 4415, 3.26 3038, 5.42 128, 2.50 557, 4.27 1781, 4.63 816, 5.13 496, 4.33 279, 2.43 685, 3.60 1664, 4.12
5
6
7
8
9
10
11
-50
-100
enantiomers. The poor plate count for peak 1 is directly related to the impact of mixed stream injection on less retained peaks. The slug of methanol from injection causes a localized severe disturbance in the SFC mobile phase causing peak shape distortion. Maximum analytical efficiency is obtained when analytes are dissolved in mobile phase. With SFC this is not practical and standard practice is to dissolve in modifier (methanol) only. Methanol has a higher polarity than the CO2 /methanol mobile phase. The higher polarity of the methanol can cause some of the analyte not to be adsorbed on the stationary phase, resulting in peak breakthrough or distortion. As retention is decreased, the distortion becomes more severe.
1 2 3 4 5 6 7 8 9 10 11
Percent Increase in Plate Count for Modifer Stream Relave to Mixed Stream Injecon
Table 1 Test compounds used for evaluation.
Compound Number
Fig. 2. Comparison of plate count for modifier stream vs. mixed stream for analytical injection of racemates. Blue: peak 1; red: peak 2. Positive number indicates modifier stream injection giving higher resolution, negative number indicates mixed stream injection giving higher resolution. See text for experimental conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3.1.2. Achiral compounds The analytical study described above for racemic compounds was duplicated using achiral compounds. A total of 24 compounds (listed in Table 1) were used for this study. The test compounds were chosen to provide a wide range of modifier percentages that maintained an approximately constant elution time (3–5 min, k of 2.5–5). Flowrate was kept constant at 126 ml/min. Using a Princeton 2-ethylpyridine column (5 m, 3 cm × 25 cm), 1 ml injections of a 1 mg/ml methanol solution of each of the test compounds were made and plate count measured. Results are summarized in Table 3. It is interesting that all plate counts at 30% modifier are low. Plate counts for chiral solutes at 30% modifier were as expected (Table 2), seeing to eliminate instrumentation as a source of the low plate counts. Fig. 3 compares the difference in plate count for each injection technique at different modifier percentages. At low modifier conditions (5 and 10%), modifier stream injection affords higher Table 3 Plate count for analytical modifier stream and mixed stream injection. Achiral compounds
k a
Modifer percentage
Modifier stream injection (N)
Mixed stream injection (N)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
3.23 4.09 2.43 1.18 3.34 1.70 4.59 1.36 1.66 1.67 4.40 2.57 2.75 1.10 2.00 2.63 1.58 2.42 2.65 3.75 3.11 3.05 1.22 2.15
5 5 5 5 5 10 10 10 10 10 10 10 10 10 15 15 20 20 20 20 30 30 30 30
3848 3448 2699 1614 3323 3224 3064 3015 3032 2391 3763 1971 2163 2410 1013 1933 1459 1963 899 1233 213 478 479 248
1486 659 1639 2437 2552 2064 2909 1222 3036 2554 3270 2157 2372 1440 891 2791 1066 2518 1155 2310 223 524 524 335
a
k measured from analytical analysis.
80
3500
60
3000 Average Plate Number
Percent Change Plate Count Modifier Stream vs. Mixed Stream Injection
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
40 20 0 -20 -40 0
5
10
15
20
25
30
35
2500 2000 1500 1000 500
Percent Modifier
plate counts relative to mixed stream injection. At higher modifier conditions (>10%), the difference between the two injection techniques was less pronounced. At 15 and 20% modifier mixed stream injection was slightly better than modifier stream injection. While this data is interesting it may not be representative due to the smaller number of compounds tested at 15 and 20% (2 and 4 respectively) compared to 5 and 10% (5 and 9 compounds). There appears to be a trend toward the choice of injection technique being less critical as the modifier percentage increases. At low modifier percentages and using modifier stream injection, the time to apply the sample to the head of the column is increased due to the low flow rate of the modifier pump. For example with a system flow rate of 126 ml/min and 5% modifier, the modifier flow rate is ∼6 ml/min. At this flow rate a 1 ml injection would take ∼10 s to be applied to the head of the column using modifier stream injection. This compares to ∼½ second with mixed stream injection. One would predict that the increased injection time would result in broader peaks and a lower plate count for modifier stream relative to mixed stream injection. For the low modifier percentage studies (5 and 10%) the opposite was observed, with modifier stream giving higher plate counts. This is due to the larger disturbance in mobile phase polarity when methanol is injected using mixed stream injection into a low modifier percent mobile phase. One milliliter of methanol was injected into an SFC mobile phase of 5 or 10% modifier causes a larger localized disturbance in mobile phase polarity, and ultimately peak distortion relative to a higher modifier percentage. This disturbance in peak shape more than offsets the increase in peak width seen with increased injection time using mixed stream injection. A series of experiments were performed to determine if the modifier percentage used for separation had an impact on the best analytical injection technique. Fig. 4 plots the average plate count for the compound analyzed at each modifier percentage for the two injection techniques. As modifier percentage increases there is a general trend toward lower average plate counts for both injection techniques. At low modifier percentages (5 and 10%) modifier stream injection is significantly better. Above 15% modifier there is less difference between the two injection techniques. Most of the analytical SFC systems on the market utilize mixed stream injection, choosing to inject sample immediately prior to the column. This eliminates injection delays that would be observed using modifier stream injection and isocratic elution at low modifier percentages. A more detailed study of injection techniques using an analytical SFC system would be of interest and may provide guidance on better equipment design.
0 5
0
10
15 20 % Modifer (MeOH)
25
30
35
Fig. 4. Average plate count vs. modifier percentage for achiral compounds using modifier stream and mixed stream injection technique (blue diamond: modifier stream injection, magenta square: mixed stream injection). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3.2. Preparative injection evaluation For the preparative injection study a total of 13 racemates were evaluated. Each racemate was dissolved in 20 mg/ml in methanol. 1, 2, 3, 4 and 5 ml injections were made on a Chiralpak AD-H (5 m, 3 cm × 25 cm). Flowrate was kept constant at 126 ml/min. Co-solvent percentage was varied to achieve approximately consistent retention for each racemate (3–4 min, average k for peak 1 and peak 2 of 2.5–3.75). From each injection, resolution was measured for both enantiomers (resolution was calculated using Rs = (t2 − t1 )/(1/2(tw1 + tw2 )) where t1 and t2 are retention times for peaks 1 and 2 respectively and tw1 and tw2 are peak width at baseline for peaks 1 and 2). The results from these analyses are summarized in Table 4. Of the 14 racemates, modifier stream injection was better for five racemates (3, 4, 6, 13 and 14), mixed stream injection was better for one racemate (7) and seven racemates (2, 5, 8, 9, 10, 11 and 12) showed no difference between injection techniques. Fig. 5 shows the average percentage increase in resolution (±one standard deviation) for modifier stream injection vs. mixed stream injection. On average modifier stream injection giving higher resolutions relative to mixed stream injection. This advantage increased
35.0
Percent Increase in Resolution for Modifer Stream Injection compared to Mixed Stream Injection
Fig. 3. Comparison of plate count for modifier stream vs. mixed stream for analytical injection of achiral compounds at various modifier percentages. Each data point is average of compounds analyzed under those conditions. Positive number indicates modifier stream injection giving higher resolution, negative number indicates mixed stream injection giving higher resolution. See text for experimental conditions.
259
30.0 25.0 20.0 15.0 10.0 5.0 0.0 0
1
2
3
4
5
6
(5.0)
Injection Volume (mL) Fig. 5. Average percent increase in resolution for modifier stream injection vs. mixed stream injection vs. injection volume for preparative separation of chiral compounds.
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
Table 4 Resolution for preparative injection of racemates using modifier stream and mixed stream injection. Racemate
Volume (ml)
Rs modifier stream injection
Rs mixed stream injection
2
1 2 3 4 5
0.86 0.59 0.45 0.31 0.28
0.85 0.68 0.54 0.33 0.28
1 2 3 4 5
7.62 5.82 4.88 4.30 3.71
7.03 5.24 4.04 3.20 3.09
4
1 2 3 4 5
2.60 2.46 1.75 1.84 1.53
1.81 1.21 0.74 0.64 0.64
5
1 2 3 4 5
0.66 0.49 0.38 0.28 0.24
0.69 0.54 0.40 0.25 0.21
6
1 2 3 4 5
1.13 0.98 0.80 0.75 0.68
0.95 0.66 0.48 0.43 0.32
7
1 2 3 4 5
2.68 2.10 1.88 1.75 1.48
2.68 2.33 2.05 1.72 1.59
8
1 2 3 4 5
0.95 0.81 0.66 0.64 0.53
0.87 0.86 0.82 0.75 0.72
9
1 2 3 4 5
1.00 0.89 0.66 0.64 0.64
1.11 0.84 0.57 0.35 0.12
10
1 2 3 4 5
2.99 2.33 2.04 1.86 1.55
2.97 2.54 2.07 1.67 1.34
11
1 2 3 4 5
2.41 1.88 1.37 1.21 1.09
1.49 1.78 1.53 1.27 1.19
12
1 2 3 4 5
2.30 2.27 2.24 1.96 1.93
2.45 2.34 2.16 2.00 1.86
13
1 2 3 4 5
2.55 1.83 1.52 1.22 1.05
2.45 1.70 1.39 1.12 0.88
1 2 3 4 5
3.24 2.40 1.92 1.61 1.49
3.03 2.24 2.04 1.62 1.47
3
14
1 Resolution
260
0.8 0.6 0.4 0.2 0 0
2 4 Injection Volume (mL)
6
Fig. 6. Effect of injection volume on preparative resolution for mixed stream injection of racemate 9. Injection quantity kept constant at 100 mg.
as the injection volume increased. When a preparative injection is made using mixed stream injection, a plug of methanol enters the column. This injection technique results in a temporary, localized increase in mobile phase polarity. This polarity increase can cause peak distortion, resulting in reductions in resolution. As the volume of the methanol plug increases there is a more pronounced impact on peak shape and thus resolution decreases. To illustrate this point further, racemate 9 was prepared at 20, 40, 80 and 160 mg/ml in methanol. Injection volume was adjusted to maintain a constant load of 100 mg (5, 2.5, 1.25, 0.63 ml respectively). Resolutions for these separations were 0.244, 0.629, 0.882 and 0.893. The results are summarized in Fig. 6. The chromatograms are shown in Fig. 7. As the injection volume increased there is a drastic impact on peak shape, resulting in reduced resolution. As a benchmark the resolution is 0.64 for a 5 ml (100 mg @ 20 mg/ml) injection using modifier stream injection conditions (chromatogram shown in Fig. 7). The data from Table 4 was evaluated to determine if retention time had an impact on preparative resolution. Fig. 8 shows the increase in resolution observed for modifier stream injection vs. mixed stream injection with retention time for peak 1. Fig. 9 shows the same data for peak 2. The data shows that for shorter retention (<3 min on 3 cm × 25 cm column @ 126 ml/min) that modifier stream injection affords higher resolution. Above 3 min retention the two injection methods are approximately equivalent. Another variable that may impact choice of injection method is modifier percentage in the mobile phase. One would predict at low modifier percentages that modifier stream injection would prevail and as the modifier percentage increased the differences would be less. Unfortunately the number of racemates that eluted at higher modifier percentages (>25%) was too small to draw any definite conclusions. In summary it appears that the most important variable on which injection technique will provide best resolution is injection volume. With increasing injection volume, modifier stream injection affords increased resolution relative to mixed stream injection. Retention time also has an impact; with a retention factor of less than approximately 3.5, modifier stream injection is the better choice, above a retention factor of 3.5 the difference between the methods is reduced. Modifier percentage may also play a role but limited data at higher modifier percentages did not allow a meaningful comparison of the injection techniques. 3.3. Effect of dissolution solvent on preparative resolution Ideally for chromatographic purifications the sample should be dissolved in the same solvent as the mobile phase. This is not always possible due to limited solubility of the compounds in the chromatographic mobile phase. In that case it is standard practice in preparative chromatography to utilize dissolution solvents different from the chromatographic mobile phase. This practice allows purification of compounds that are poorly soluble in the mobile
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
261
Fig. 7. Preparative separation of racemate 9 using mixed stream injection. Load kept constant at 100 mg. Injection volumes: (A) 0.63 ml, (B) 1.25 ml, (C) 2.5 ml, and (D) 5 ml. The injection of 100 mg (5 ml injection volume) using modifier stream injection is shown in chromatogram E.
phase or to reduce the injection volume for compounds with minimal solubility. It is well known in HPLC that injection from stronger solvents than the mobile phase can lead to poor peak shape, sample breakthrough and reduced loadings [13]. A series of experiments were performed to study the impact of varied polarity dissolution solvents on the preparative separation of racemate 14, trans stilbene oxide (TSO). TSO was dissolved in 20 mg/ml in numerous solvents and solvent mixtures. Four milliliter injections (80 mg total) were injected on a 3 cm × 25 cm Chiralpak AD-H preparative column using mixed stream injection. The results are summarized in Table 5. These experiments showed the dissolution solvent had
minimal impact on plate count, retention time or resolution regardless of the dissolution solvent strength. The same type of study was performed for preparative HPLC. 50 mg of TSO in 20 mg/ml in different solvents was injected on a 3 cm × 15 cm Chiralpak ADH column. The mobile phase was 10/90 (v/v) isopropanol/heptane. Due to solvent immiscibility, fewer dissolution solvents were investigated for HPLC. The results from this study are summarized in Table 6. This work shows that dissolution solvent had a large impact on plate count for the first eluting enantiomer (k = 1.14). The impact was less pronounced for the second eluting enantiomer (k = 4.44). The larger the difference between the polarity of the sample
262
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263 500
Dissolution solvent
Rt1 (min)
Rt2 (min)
N1
N2
Rs
Methanol Ethanol Acetonitrile Isopropanol 75/25 isopropanol/heptane 50/50 isopropanol/heptane 25/75 isopropanol/heptane 10/90 isopropanol/heptane Heptane 50/50 methanol/dimethylether 75/25 methanol/dichloromethane
2.55 2.56 2.55 2.59 2.60 2.60 2.59 2.59 2.57 2.59 2.62
3.96 3.99 3.94 4.08 4.06 4.07 4.05 4.02 4.00 4.01 4.06
620 522 750 507 548 642 691 722 762 421 690
358 334 418 300 329 355 388 399 399 436 420
1.88 1.87 1.97 1.86 1.88 2.02 1.96 2.05 2.03 1.85 1.96
a Chiralpak AD-H, 3 cm × 25 cm, 126 ml/min, 25% methanol w/0.2% diethylamine, 80 mg injection (4 ml @ 20 mg/ml), mixed stream injection.
Percent Incrase in Resoluon for Modifer Stream Relave to Mixed Stream Injecon
Table 5 Effect of dissolution solvent on preparative SFC separation of racemate 14.a
400
300
200
100
0 2.00
0.00
4.00
6.00
8.00
10.00
12.00
-100
Table 6 Effect of dissolution solvent on preparative HPLC separation of racemate 14.a Dissolution Solvent
Rt1
Rt2
N1
N2
Rs
Heptane 10/90 isopropanol/heptane 25/75 isopropanol/heptane 50/50 isopropanol/heptane 75/25 isopropanol/heptane Isopropanol Ethanol
3.22 3.22 3.19 3.17 3.10 3.06 3.22
8.27 8.22 8.13 8.18 8.11 8.16 8.37
3229 2740 2691 2192 1980 391 1600
718 826 885 834 840 718 613
6.83 6.97 7.37 7.32 7.16 6.19 6.19
Chiralpak AD-H, 3 cm × 15 cm, 42 ml/min, 10/90 (v/v) isopropanol/heptane, 50 mg injection (2.5 ml @ 20 mg/ml).
Percent Increase in Resoluon for Modifier Stream relave to Mixed Stream Injecon
a
500
Fig. 9. Percent increase in resolution for modifier stream/mixed stream vs. retention time for second eluting peak of racemates. Positive number indicates modifier stream injection giving higher resolution; negative number indicates mixed stream injection giving higher resolution.
70 50
30
10
10
-10
70
100
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4:48
-10
10
10
4.00
3:36
4:48
-10
3:36
4:48
D
50 30
2:24
2:24
70
30
-10
0
3:36
C
50
200
-100
2:24
B
50
30
400 300
70
A
2:24
3:36
4:48
Peak 1 Retenon Time (minutes)
Fig. 8. Percent increase in resolution for modifier stream/mixed stream vs. retention time for first eluting peak of racemates. Positive number indicates modifier stream injection giving higher resolution; negative number indicates mixed stream injection giving higher resolution.
dissolution solvent and the polarity of the mobile phase, the larger the impact on plate count. The plate count for peak 1 using isopropanol as dissolution solvent was much lower than expected. These results were duplicated to ensure they were accurate. The reduction in plate count for isopropanol was much larger than with ethanol, indicating that the polarity of the dissolution solvent is not the only factor. It is possible that the increased viscosity of isopropanol and resulting temperature increase may have played a role in the low plate count. In addition the increased viscosity of the solute plug may cause viscous fingering, resulting in the poor chromatography results observed [14]. The impact of dissolution solvent is best illustrated for the separation of peak 1 and an achiral impurity that elutes just after peak 1. Chromatograms of this region are shown in Fig. 10. As the polarity of the dissolution solvent increases the separation between peak 1 and the impurity decreases. With a dissolution solvent of 50% isopropanol in heptane, and more polar, the separation is lost.
E
70 50
50
30
30
10
10
-10
-10
2:24
3:36
F
70
4:48
2:24
3:36
4:48
G
70 50 30 10 -10
2:24
3:36
4:48
Fig. 10. HPLC chromatograms of preparative separation of trans stilbene oxide with various dissolution solvents. (A) Heptane, (B) 10/90 isopropanol/heptane, (C) 25/75 isopropanol/heptane, (D) 50/50 isopropanol/heptane, (E) 75/25 isopropanol/heptane, (F) isopropanol, and (G) ethanol. X axis is time in minutes.
L. Miller, I. Sebastian / J. Chromatogr. A 1250 (2012) 256–263
Unfortunately this achiral impurity is not resolved from peak 1 under SFC conditions. As this study was only performed with one compound, and mobile phase polarities were not identical for the HPLC and SFC separations, additional studies should be performed to determine if this is a general phenomenon of preparative SFC.
263
Acknowledgements The authors thank Wolfgang Goetzinger, Manny Ventura and Kyung Gahm for assistance with the technical review of this article. References
4. Conclusions The choice of injection technique for preparative SFC can have a large impact on peak shape, resolution and preparative productivity. For the majority of compounds studied, modifier stream injection was a better choice for injection relative to mixed stream injection. In mixed stream injection there is a temporary localized increase in mobile phase strength due to the methanol used for injection which can lead to peak distortion and reduced resolution. The difference is more pronounced as the injection volume increases and as retention time decreases. The polarity of the dissolution solvent has little impact on efficiency, retention or resolution for the preparative SFC separation of trans stilbene oxide. This data was in contrast to the preparative HPLC separation where dissolution solvent polarity had a large impact on separation efficiency.
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