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Journal of Chromatography A, 1305 (2013) 310– 319

Contents lists available at ScienceDirect

Journal of Chromatography A

j our nal homep age: www.elsev ier .com/ locate /chroma

he evaluation of 25 chiral stationary phases and the utilization ofub-2.0 �m coated polysaccharide chiral stationary phases viaupercritical fluid chromatography

hris Hamman ∗, Mengling Wong, Ignacio Aliagas, Daniel F. Ortwine,oseph Pease, Donald E. Schmidt Jr., Joseph Victorinoiscovery Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, United States

r t i c l e i n f o

rticle history:eceived 15 April 2013eceived in revised form 10 July 2013ccepted 10 July 2013vailable online 17 July 2013

a b s t r a c t

A rapid screening method to identify the best conditions for chiral separations is described. We analyzeda representative set of 80 racemic compounds against 25 different chiral stationary phases with threedifferent mobile phases to identify the combination of columns and mobile phases that will separate themost compounds on the initial screen. While the OD separated the largest number of compounds, wefound the best combination of six columns to be the AD, AS, AY, CC4, ID and Whelk-O1. The second team

eywords:hiralhiral screeningFCub-2.0 �m particlesolysaccharide chiral stationary phases

included the CCC, Cellulose-1, Cellulose-3 or OJ, IA, IE and IF. All 80 compounds were separated with aresolution range of 0.65–15.36. Screening the covalently bonded phases provided separation for 79 ofthe 80 compounds. We also found ethanol (0.1% NH4OH) separated more compounds than methanol(0.1% NH4OH) or isopropanol (0.1% NH4OH). As part of this study, we also compared the effectiveness ofstationary phases that have the same chiral selector. Finally, we demonstrated the effectiveness of usinga fast, 1.5-min screening method that utilizes a 1.7 �m coated polysaccharide chiral stationary phase.

. Introduction

Chiral chromatography is an important tool for pharmaceuticaliscovery and development. The ability to isolate pure enantiomers

s important to fully understanding the true potency of individualnantiomers. Supercritical fluid chromatography (SFC) paired withhiral stationary phases has established itself as the technique ofhoice for separating and purifying racemic mixtures [1–13]. SFCas advantages over conventional liquid chromatography, namelyhat supercritical or subcritical carbon dioxide has lower viscositynd much faster mass transfer properties than traditionally usedolvents which result in much faster analysis times without sac-ificing efficiency [4,6,14]. Additionally, SFC is a green techniqueue to its reduced organic solvent consumption [9]. Finally, it hasajor advantages on the preparative scale as a user only needs to

ry down the organic portion of the mobile phase.As mentioned previously, SFC paired with chiral stationary

hases (CSPs) is a powerful tool for resolving racemic mixtures.

ithin the last ten years, many new phases from a variety of man-

factures have been introduced, including new immobilized CSPsrom Chiral Technologies, cyclofructan selectors from AZYP, LLC and

∗ Corresponding author. Tel.: +1 650 467 3586.E-mail address: hamman.christopher@gene.com (C. Hamman).

021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2013.07.046

© 2013 Elsevier B.V. All rights reserved.

coated CSPs from a variety of other vendors. In this study we directlycompare 25 CSPs for their ability to separate a test set of 80 com-pounds using three different mobile phases: methanol, ethanol andisopropanol each with 0.1% NH4OH added. The bulk of this studyis dedicated to describing a screening hierarchy designed to givethe best possible chance of separation success with a six-columnscreen. We also compare the three mobile phases against each otherand compare phases with similar selectors.

As part of this study we also investigated the use of smallparticles (sub-2.0 �m) with the latest SFC instrumentation. Pair-ing small particles with SFC has gained recent attention and theresults are very compelling. Berger was able to generate over22,000 plates on a 3.0 mm × 100 mm, 1.8 �m silica column [15].He also demonstrated superior efficiencies using 2.6 �m core–shellparticles where he was able to generate over 29,000 plates ona peak retained less than a minute [16]. Guillarme et al. wereable to generate a peak capacity of greater than 250 compoundsin approximately 40 min [14]. However, the work by Berger andGuillarme focused on achiral applications. There has been somework done combining sub-2.0 �m CSPs with normal phase chro-matography. Kotoni et al. demonstrated the effectiveness of the

Whelk-O1 chiral selector bonded to a sub-2 �m particle with animpressive highlight of separating trans-stilbene oxide in under10 s [17]. While there have been a few studies investigating using3.0 �m particles coated with polysaccharide CSPs [2,18,19], very

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ittle has been researched on combining sub-2.0 �m particles withoated polysaccharide chiral phases with SFC to further decreaseun times. In this study we will explore the utility of using a.7 �m coated (3,5-dimethylphenylcarbonate) on both a cellulosend amylose phase from Orochem Technologies. A 1.5-min gra-ient screening method is described that takes advantage of theub-2.0 �m CSPs.

. Experimental

.1. Chemicals

Carbon dioxide was obtained from Praxair (Des Moines, IA).ethanol (MeOH) and isopropyl alcohol (IPA) were purchased from

MD Chemicals (Gibbstown, NJ, USA). Ethyl alcohol (EtOH, 100%))as purchased from Koptec (King of Prussia, USA). Ammoniumydroxide (28–30%) solution, Norphenylephrine HCl, acebutololCl, bupivacaine HCl, chlorpheniramine maleate salt, disopyra-ide phosphate salt, molindone HCl, warfarin, mianserin HCl,

rimipramine maleate salt, propanolol HCl, methoxamine HCl, clen-uterol HCl, mirtazapine, mebeverine HCl, butaclamol, pindolol,roger’s base, 1,1-bi-2-naphthol, nisoxetine HCl, mandelamide,enzoin, ibuprofen, bendroflumethazide, thalidomide, flurbipro-en, 1,1-binaphthyl-2,2-diamine, ketoprofen, sulindac, metoprolol,ndapamide, and miconazole were purchased from Sigma–AldrichSt. Louis, MO, USA). Trans-stilbene oxide and homatropine HBrere purchased from TCI (Tokyo, Japan). Atenolol was purchased

rom Spectrum Chemical (Gardena, NJ, USA). Fenoterol HBr wasurchased from MP-Biomedicals (Solon, OH, USA). Salbutamol sul-ate and flavanone were purchased from Alfa Aesar (Ward Hill, MA,SA). Napropamide was purchased from Ultra (Red Bank, NJ, USA).,2,2-trifluoro-1-9-anthryl was purchased from Oakwood Products

nc. (West Columbia, SC, USA). Ketoconazole was purchased fromoronto Research Chemicals (Toronto, ON, Canada).

.2. Analytical instrumentation

All data was collected on a UPC2 from Waters Corporation (Mil-ord, MA, USA) equipped with a sample manager, a binary solventelivery pump, a SQD single quadrupole mass detector, a photoiode array detector, and a convergence manager (automated backressure regulator). The pump was equipped with a 50 �L mixerrom ASI (Richmond, CA, USA). The instrument was controlled by

aters EmpowerTM Pro 3 Software. The flow was split post UV, butre-back pressure regulator and some of the flow was diverted tohe mass spectrometer (it is very difficult to measure as the carbonioxide outgases when it hits ambient temperatures).

.3. Columns

The analytical chiral columns we used in our study includedhe Chiralpak AD-3 (AD), Chiralcel OD-3 (OD), Chiralcel OJ-3 (OJ),hiralcel OZ-3 (OZ), Chiralpak AS-3 (AS), Chiralpak AY (AY), Chiral-ak IA-3 (IA), Chiralpak IB-3 (IB), and Chiralpak IC-3 (IC), Chiralpak

D-3 (ID), Chiralpak IE-3 (IE), Chiralpak IF-3 (IF) from Chiral Tech-ologies (West Chester, PA, USA), the Lux Cellulose-1 (Cellulose-1),ux Cellulose-2 (Cellulose-2), Lux Cellulose-3 (Cellulose-3), Luxellulose-4 (Cellulose-4), and Lux Amylose-2 (Amylose-2) fromhenomenex (Torrance, CA, USA), the Whelk-O1 (S, S) from Regisechnologies (Morton Grove, IL, USA), the CCC and CC4 from ESndustries (West Berlin, NJ, USA), the Larihc CF6-RN (CF6-RN) andarihc CF6-P (CF6-P) from AZYP (Arlington, TX, USA), the Epitomize

A and Epitomize 1C from Orochem (Lombard, IL, USA), the Chiro-iotic T2 from Supelco (St. Louis, MO, USA) Most of the columnsere previously used. All column lengths were 50 mm and all col-mn diameters were 4.6 mm with the exception of the Epitomize

. A 1305 (2013) 310– 319 311

1A and Epitomize 1C which were 3.0 mm. The Whelk-O1, the Chi-robiotic T2, the CF6-RN, and the CF6-P had 5 �m particles, the twoEpitomize columns had 1.7 �m, and all other columns had 3 �mparticles. The injection volume was 2 �L for all columns except theEptiomize columns where it was 1 �L. All 40 commercially avail-able compounds and all 40 Genentech compounds were analyzedon every column with all three modifiers.

2.4. Analytical method and calculations

For the 4.6 mm × 50 mm, 3 or 5 �m columns, the gradient was5–60% over 1.8 min, hold at 60% until 2.4 min and brought back to5% at 2.45 min for a total of a 2.5 min method run. Since analyti-cal method will eventually be scaled, we decided not go above 60%organic as the solvent consumption and post-purification removalbecomes too burdensome. The flow rate was 4.0 mL/min, the back-pressure was 105 bar and the column temperature was 40 ◦C. Forthe 3.0 mm × 50 mm, 1.7 �m columns, the gradient was 5–60%in 1 min, held at 60% until 1.4 min and then brought back to 5%at 1.45 min. The flow rate was 1.5 mL/min, the backpressure was105 bar and the column temperature was 40 ◦C. For both methodsthe column equilibration was completed during the injection cycle(at least 60 s). The resolution was calculated via EmpowerTM 3 Prosoftware using the USP Resolution option, Rs = 2(t2 − t1)/(tw2 + tw1)[20]. The peak width was also calculated by EmpowerTM 3 Pro soft-ware. The peak capacity (Pc) was calculated using the equationPc = 1 + tg/Pw(5%) where tg is the time of the gradient and Pw(5%) isthe peak width at 5% of the height of the peak [21].

In Fig. 1, the pKa and log D were calculated using v1.1 of MoKa,available from Molecular Discovery Ltd. (www.moldiscovery.com)SFI is Solubility Forecast Index (equal to clog D + the number of aro-matic rings), a commonly used surrogate measure of solubility [22].

3. Results and discussion

Compounds were selected to provide a wide diversity of prop-erties that are encountered in medicinal chemistry programs. Fig. 1shows the distribution of commonly used physicochemical andstructural properties among the 80 compounds chosen. A widerange of property space coverage is apparent, consistent withthe properties of most marketed small molecule drugs [23]. Forexample, the basic pKa range was from 2 to 12. The Genentechcompounds were from 15 different projects and included final com-pounds, starting materials and intermediates.

As stated in the introduction, one of the major objectives of thisstudy was to rank CSPs and modifier combinations that give the bestchance of separation success. The columns were ranked accordingto two different parameters: partial (Rs value of ≥0.3) and baselineseparations (Rs value ≥1.50). Both parameters are important: par-tial separations can determine if there are enantiomers in a sample,where as baseline separations are more important for preparativechromatography. In addition to determining the optimal combi-nation of columns, we also wanted to compare various alcoholmodifiers, immobilized columns versus coated columns, success ofCSPs with the same chiral selector, and to demonstrate the poten-tial of for polysaccharide phases coated onto sub-2.0 �m particlesfor chiral screening purposes.

We selected the 25 columns based on multiple factors. Thenumber one factor was to include every CSP for which we had acorresponding prep column. That included all of the phases fromPhenomenex (Cellulose 1, 2, 3, 4, and Amylose-1), the AD, AS, AY,

OD, OJ, IA, IB, IC, and ID from Chiral Technologies, and the Whelk-O1from Regis. As stated earlier, one of the objectives of this study wasto demonstrate the viability of using sub-2.0 �m particles coatedwith polysaccharide CSPs so we included the Epitomize 1A and the

312 C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319

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propanol relative to the other alcohols may be due to its lowerpolarity and thus was not able to elute some of the compoundsfrom the CSPs. Given the results from our data on the 25 CSPS and

Table 1A summary of the performance of methanol (0.1% NH4OH), ethanol (0.1% NH4OH),and isopropanol (0.1% NH4OH).

No. of cmpdsseparated,Rs > 0.3

No. of cmpdsseparated,Rs > 1.5

Median no. ofcmpds separatedacross all 25 CSPs,

ig. 1. Radar plot showing the wide distribution of molecular weight (MW), calcNH + OH), solubility forecast index (SFI), calculated most basic pKa (c pKa most bas

pitomize 1C CSPs. The remaining 8 phases were selected basedheir unique characteristics and their relative newness to the CSP

arket. This included the latest immobilized phases from Chiralechnologies, the IE and IF; the CCC column that uses an interest-ng blend of the cellulose tris 3-chloro-4-methylphenylcarbonatend the cellulose tris (3,5-dichlorophenylcarbonate) CSPs to sepa-ate compounds; and the cyclofructan CSPs, the CF6-RN and CF6-P.

e also wanted to compare similar chiral selectors for a couple ofhe chlorinated phases, so we added the OZ to compare with theellulose-2 and the CC4 to compare with the Cellulose-4. Finally,here has been very little published data using an antibiotic-basedSP with SFC, so we included the Chirobiotic T2 (Teicoplanin is thentibiotic).

.1. Solvent selection

One of the challenges we encounter in our lab is selectinghe first solvent to begin our chiral screen. We normally selected

ethanol based more on familiarity than any real data. Methanolas a few advantages over ethanol and isopropanol: it is less expen-ive, is the most polar of the alcohols, is the least viscous and isenerally the best of the three alcohols for dissolving our ana-ytes. The stronger polarity of methanol generally makes our soluteslute earlier, which has two positives: more compounds are eluteduring our screen and less organic solvent is consumed during an

socratic preparative run. One of our goals with this study is to findut if methanol offers the highest rate of separation success givents other benefits. Methanol has one downside in that it does notave as high of a safety profile as does ethanol or isopropanol.

In order to compare the modifier alcohols against each othere used two metrics: highest success rate of partial separations

Rs ≥ 0.3) and/or baseline separations (Rs ≥ 1.5) for the 80 com-ounds and the median number of partial separations across all the

SPs with each modifier. We selected a resolution value of greaterhan 0.3 for our partial separations because we felt that any resolu-ion greater than 0.3 clearly indicates chiral recognition and allowss to say definitively that there are two enantiomers. Given our

lipophilicity (clog D), total polar surface area (TPSA), number of NH + OH groupsd rotatable bonds (RotBonds) among the 80 compounds in this study.

goal is to purify molecules, having baseline resolution is clearlypreferred.

Table 1 summarizes the performance for each modifier. Sur-prisingly, ethanol outperformed methanol and isopropanol in bothmetrics. Ethanol at least partially separated 98% of the compoundscompared to 95% for methanol and 94% for IPA. Ethanol also base-line resolved 78% of the compounds versus 71% for methanol and61% for isopropanol. Ethanol also had the highest median number ofcompounds separated for partial separations across all 25 CSPs with33 versus methanol and isopropanol with 31 and 29, respectively.

Although ethanol and methanol were similar, ethanol clearlyis the better choice as the first solvent in the screening process.Although many of the columns performed better with ethanol,much of the improvement in the number of samples separatedcan be attributed to the coated 5-chloro-2-methylphenylcarbonate(AY and Amylose-2) CSPs (see Section 3.4.6 for more discussion onthis topic). These CSPs separated almost twice as many compoundswhen using ethanol as compared to methanol or isopropanol. Iso-propanol was significantly worse than the other two modifiers;however it was able to separate the two compounds that ethanolfailed to separate and three of the four that methanol failed to sep-arate. The two compounds that did not separate using ethanol alsodid not separate when using methanol. The poor results by iso-

Rs > 0.3

MeOH (0.1% NH4OH) 76 (95%) 57 (71%) 31EtOH (0.1% NH4OH) 78 (98%) 62 (78%) 33IPA (0.1% NH4OH) 75 (94%) 49 (61%) 29

C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319 313

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ontrary to our current practice, ethanol should be the first choicef solvent followed by isopropanol, as it was able to fill the gapsrom ethanol, and then methanol.

.2. Best combination of columns and solvent systems

Although all 80 compounds could be partially separated and 65f the 80 could be baseline separated if we screened all 25 CSPsgainst the three solvent systems, this approach is impractical dueo time constraints. Thus we worked to identify the best subset ofolumns to screen against one solvent system first and then movento other solvent systems for subsequent screening. The practicaloal is to be able to separate the maximum number of enantiomericairs using a single solvent and small set of chiral columns. Oururrent system is equipped with a six-column switcher, so we canasily accommodate up to six columns in a given screen.

There have been a many studies that “rank” the effectiveness ofndividual CSPs [2,9,24–29]. Most of the studies start with the CSPhat separates the largest number of compounds in their study andhen proceed onto the CSP that separates the second most and so on.ig. 2a–c shows the results of how each CSP fared when using eachodifier. The OD separated the most compounds and the Lirahc-

separated the fewest compounds, regardless of modifier. Sincee routinely screen six columns at a time, we are more concerned

bout the ability of those six columns to be able to separate as manyompounds as possible versus the success of an individual CSP. Theolumn that separates the largest number of compounds may note a member of the six if all of its separations can be completedsing a different CSP. Furthermore, the top six phases may separate

he same set of samples. For example, the OD and the Cellulose-1ere the two best columns when using isopropanol (see Fig. 2c);

ut, they had many redundant separations, so including both ofhem for a six-column screen would not be very effective. In short,

0.1% NH4OH. (b) Results from screening the 25 CSPs versus the 80 compounds innds in isopropanol with 0.1% NH4OH.

we are more concerned with the outcome of the “team” of columnsversus the individual column performance.

In effort to analyze the 531,300 possible combinations and selectthe best team of six columns with the best solvent system, wedeveloped a fast algorithm via Pipeline Pilot software (Accelrys,PipelinePilot v7.5). This algorithm, called a set-covering problem[30], was used to determine the 6 best CSPs to use with each solvent.Unfortunately, the authors have not published this algorithm at thetime of publication. Rather than starting with the CSP that separatesthe largest number of compounds, the algorithm started with thesamples that were separated by only one CSP. A snap shot of thealgorithm output when using EtOH (0.1% NH4OH) as co-solvent isdisplayed in Table 2 for the ten most difficult compounds to sepa-rate. The first two compounds listed were not separated (Rs ≥ 0.3)by any CSP, as indicated by “none” listed in the “Columns with Suc-cess” column. Genentech compound #3 was separated by only theAD. The AD CSP was then added to the team of the 6 best. In total,the AD separated 37 compounds with a Rs ≥ 0.3, so all 37 of thosecompounds were “checked” off as separated. There were two othercompounds (Genentech compound #4 and ketoprofen) that alsoseparated on one CSP (those CSPs being the AY and the ID), so thosetwo CSPs were added to the team 6 and all of the compounds theyseparated from the remaining 43 were “checked” off as separated.

The algorithm then continued onto compounds that were sepa-rated by only two CSPs. If any of those compounds were separatedon the AD, AY or ID the algorithm understood those samples werealready separated and moved on to samples that had two CSPsthat were not the AY, AD or ID. Continuing with our example, nor-phenylephrine HCl was only separated on the CC4 and IE whenusing ethanol (0.1% NH4OH)—both of which were not on our team

of 6 columns. To determine which column was added, the algo-rithm chose based on how many total separations the CSP offered.In this example, the CC4 separated 36 samples and the IE sepa-rated only 29 samples. So the CC4 is added to the team of 6. On

314 C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319

Table 2A snap shot of the algorithm output for generating resolutions greater than 0.3 for ethanol (0.1% NH4OH) as a modifier.

Compound Columns with success Column choice Columns on team

Genentech compound #1 None None NoneGenentech compound #2 None None NoneGenentech compound #3 AD AD ADGenentech compound #4 ID ID AD, IDKetoprofen AY AY AD, ID, AYNorphenylephrine HCl CC4, IE IE, CC4 AD, ID, AY, CC4Acebutolol HCl AY, IC AD, ID, AY, CC4

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he other hand, acebutolol was only separated when using ethanol0.1% NH4OH) on the AY and IC CSPs. Since the AY was already onur team of 6, the algorithm continued on to the next sample thatas only separated by two CSPs. The algorithm continues this pro-

ess until all of the compounds that had an Rs ≥ 0.3 have a CSP thateparated them on the team. With this unique approach, we wereble to separated 78 of the 80 compounds using only six CSPs (AD,S, AY, CC4, Whelk-O1, ID). Interestingly, out of the top 7 columns

OD, Cellulose-2, AY, Cellulose-4, IA, OZ, and CCC) that offered theargest number of partial separations with ethanol (0.1% NH4OH)see Fig. 2a), only the AY made it onto the team.

The results from the algorithm are summarized in Table 3.ne will notice that the list of CSPs providing partial separations

or each modifier is quite different, with two exceptions: the ADnd Whelk-O1 were needed in all three modifiers. One will alsootice the algorithm needed more than the allotted six columns

n order to partially separate the maximum number of compoundsor methanol and isopropanol, so one column will need to be elim-nated for each modifier. For methanol, eliminating the AD or OZeduces the total number of samples separated by 2, whereas elim-nating any of the remaining five CSPs reduces the number ofamples separated by more than 2. So either the AD or OZ cane eliminated. For isopropanol, eliminating either the AD, IA orC would all reduce the total number of samples separated by 2,

hereas eliminating any of the remaining five CSPs reduces theumber of samples separated by more than 2. So either the AD, IAr IC can be eliminated.

Interestingly, while the amylose-based phases offered manyovel baseline separated compounds with methanol and ethanol,hey did not add any novel baseline separations when usingsopropanol as a modifier. This result suggests that the threeimensional shape of the amylose phases is not very amenable toeparations when in a carbon dioxide/isopropanol medium. Furthertudies would need to be done to confirm this conclusion.

Given our results, we found that the best team of six columns tose for our 80 compound set were (in alphabetical order) the AD, AS,Y, CC4, ID and Whelk-O1 CSPs. This combination of CSPs partiallyeparated 78 of 80 compounds and baseline separated 51 of 80 com-ounds with ethanol (0.1% NH4OH) alone. Although the number ofartially separated compounds does not increase, when the team

as screened with methanol (0.1% NH4OH) and isopropanol (0.1%H4OH) the number of baseline separations went up to 58 (seeable 4). After screening the first team we only need to partiallyeparate 2 more compounds and baseline separate an additional 7

able 3he best combination of columns as determined by the Pipeline Pilot algorithm to separaactor.

Rs > 0.3 (partial)

Ethanol (0.1% NH4OH) AD, AS, AY, CC4, Whelk-O1, ID

Methanol (0.1% NH4OH) AD, Cellulose-4, IF, OD, OJ, OZ, Whelk-OIsopropanol (0.1% NH4OH) AD, IA, IC, ID, IE, OD, Whelk-O1

AD, ID, AY, CC4AS, IE AD, ID, AY, CC4, AS

AD, ID, AY, CC4, AS

more compounds to reach the maximum of 80 partial resolved and65 of 80 baseline resolved using all 26 CSPs. With that in mind, wesuggest team two include (in alphabetical order) the CCC, Cellulose-1, Cellulose-3/OJ, IA, IE and IF. With the addition of team two to theteam one results, all 80 compounds were partially separated and64 of the 65 possible were baseline separated. Since the Cellulose-3 and OJ were closely correlated (Section 3.4.3), it does not matterwhich of the pair is included in team two, the number of partial andbaseline separations does not change. Although the Cellulose-1 andOD are very closely correlated, the Cellulose-1 provided one extrabaseline separation that the OD did not, so that is why we includedit in team two. The last remaining baseline separation required theCellulose-4 with methanol (0.1% NH4OH).

In summary, we suggest screening the six columns in teamone with ethanol (0.1% NH4OH) against unknown compounds. Forany compounds not separated with the first screen, screen thosewith team one with methanol (0.1% NH4OH) and isopropanol (0.1%NH4OH). If necessary, then proceed to screen team two against allthree solvent systems. Using this streamlined approach, we haveachieved a high degree of success, both partial and baseline, in theminimum amount of time.

3.3. Immobilized column success

Covalently bonded chiral stationary phases offer advantagesover coated phases as they are able to tolerate a wide variety ofsolvents. The added solvent tolerability frees the user to dissolvetheir samples in stronger solvents such as DMSO and DMF withoutthe fear of dissolving the CSP. Furthermore, the chiral selectivity ofthe columns can be further optimized by using solvents other thanCO2 with methanol, ethanol, isopropanol and/or acetonitrile. Milleret al. demonstrated that dichloromethane and or tetrahydrofuranmixed with methanol as a modifier provided very different selectiv-ity than a methanol only modifier for some molecules [31]. Zhanget al. also showed interesting results using various percentages ofTHF or ethyl acetate under normal phase conditions [32]. However,some studies have demonstrated that under normal conditions (i.e.CO2 with an alcohol or acetonitrile) the immobilized CSPs separatefewer compounds than their coated CSP counterparts [2].

There were ten covalently bonded chiral stationary phases ana-

lyzed in this study: Chirobiotic T2, CF6-P, CF6-RN, IA, IB, IC, ID, IE, IFand the Whelk-O1. We decided to remove the Chirobiotic T2, CF6-Pand CF6-RN from this analysis due to the inability of these phases toseparate most of the compounds under SFC conditions. We ran the

te the highest percentage of compounds for each solvent system at each resolution

Rs > 1.5 (baseline)

AD, AS, AY, Cellulose-1, Cellulose-4, IA, ID, IE, IF, Whelk-O11 AD, AS, Cellulose-3, Cellulose-4, IA, ID, IF, OD, Whelk-O1

OD, CCC, Cellulose-4, ID, IE, OD, OJ, Whelk-O1

C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319 315

Table 4The breakdown of separation success by resolution.

Team 1 (AD, AS, AY, ID, CC4, Whelk-O1) Separated with Rs ≥ 0.3 (out of 80) Separated with Rs ≥ 1.5 (out of 80)

EtOH (0.1% NH4OH) 78 51Then IPA (0.1% NH4OH) 78 55Then MeOH (0.1% NH4OH) 78 58

Team 1 + Team 2 (CCC, Cellulose-1, Cellulose-3/OJ, IA, IE, IF) Separated with Rs ≥ 0.3 (out of 80) Separated with Rs ≥ 1.5 (out of 80)

EtOH (0.1% NH4OH) 78 64Then IPA (0.1% NH OH) 80 64

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3.4.3. Cellulose tris (4-methylbenzoate), Cellulose-3 vs. OJThere were two cellulose tris (4-methylbenzoate) chiral selec-

tors in our study, the Cellulose-3 and the OJ. The resolution R2 value

4

Then MeOH (0.1% NH4OH) 80

lgorithm using the data from the remaining 7 stationary phases toee if the success rate of using only immobilized phases comparesith that of all of the stationary phases. We were very surprised

y the results. The immobilized CSPs separated 74 of the 80 (93%)ompounds with an Rs ≥ 0.3 when using methanol, 72 of the 8090%) with ethanol and 71 of the 80 (89%) with isopropanol. If oneere to screen all seven of the immobilized phases against the threeodifiers, the success rate jumps to 79 of the 80 compounds suc-

essfully separated with a resolution greater than 0.3. If a user wererying to get a quick identification if enantiomers are present, themmobilized columns would be a great start.

With such an excellent success rate with just screening sevenmmobilized and very rugged CSPs, one might be tempted to relyolely upon the covalently bonded phases. However, partial sep-ration of the samples is only part of the story, particularly if theoal is to scale up to the preparative scale. The success rate of themmobilized CSPs with a resolution greater than 1.5 is 54 out of 80,

arkedly lower than the success rate of 65 out of 80 for when all5 of the CSPs are screened. Further investigation needs to be doneo see if the number of baseline resolutions would go up if somef the solvent combinations Miller or Zhang used in their studiesere employed for these 80 compounds [31,32].

.4. Comparing similar chiral selectors

There are a few CSPs featured in this study that utilize the samehiral selector, as shown in Table 5, and we evaluated the per-ormance of each group of CSP. We carried out this evaluation bylotting the resolution values from all three mobile phases for eachhiral selector. This provided us with two pieces of data: (A) howimilar two columns were in terms of selectivity by an R2 value andB) the tendency for one phase to provide a higher resolution valuever the other as determined by the slope of a linear regressionine. If the slope is greater than one, the CSP on the y-axis providesarger resolutions on average than the CSP on the x-axis. Conversely,f the slope is less than one, the x-axis CSP provides larger reso-utions on average. For example, an R2 value of .99 and a slopealue of 1.4 for two CSPs indicates they offer very similar selectiv-ty but the CSP on the y-axis offers much higher average resolutionalues. Table 6 summarizes the results from the chiral selectorvaluations.

.4.1. Cellulose tris (3,5-dimethylphenylcarbamate), Cellulose-1s. IB vs. OD vs. Epitomize 1C

There were four cellulose tris (3,5-dimethylphenylcarbamate)hiral selectors in our study, three coated CSPs, the Cellulose-1, theD and the Epitomize 1C, and the one immobilized CSP, the IB. Weill discuss the Epitomize 1C in Section 3.6 with the sub 2.0 �m

articles. As one can see from Fig. 2a–c, the OD separated the mostamples out of any phase we tested. While the OD and Cellulose-1re highly correlated (R2 for the resolutions was 0.93, see Fig. 3),he resolution slope value for the Cellulose-1 (x-axis) vs. the OD

64

(y-axis) was 1.36 indicating the OD averages much larger resolutionvalues versus the Cellulose-1. The IB was worse than the OD andCellulose-1 in its ability to separate the 80 compounds selected forthis study. The R2 value for the resolutions for the OD and IB was0.72 demonstrating the two are related, but the resolution slopevalue for the OD (x-axis) vs. the IB (y-axis) was 0.44 indicating theOD was far superior.

3.4.2. Amylose tris (3,5-dimethylphenylcarbonate), AD vs. IA vs.Epitomize 1A

There were three amylose tri (3,5-dimethylphenylcarbonate)chiral selectors in our study, two coated CSPs, the AD and the Epit-omize 1A, and one immobilized CSP, the IA. We will discuss theEpitomize 1A in Section 3.6. Although the AD and IA are very similarin the number of compounds they separate with each mobile phase,their low R2 value of 0.3 demonstrates they are not well correlated.The AD faired much better than the IA for separating compoundswith a resolution greater than 1.5. The resolution slope value on theAD (x-axis) vs. the IA (y-axis) was 0.66, indicating the AD averagedmuch larger resolution values with respect to the IA. Our previousstudy [2] showed the AD was clearly better than the IA, so we werea little surprised by the two phases separating almost the samenumber of samples. Part of this inconsistency can be attributed tothe minimal sample set used in the first study. We often obtain lessretention on the IA compared with the AD and there were at leastsix samples that only had the first peak elute on the AD (the secondpeak appeared in the next run).

Fig. 3. A scatter plot of all the resolution values from the three mobile phasesgenerated on the Cellulose-1 and OD.

316 C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319

Table 5Summary of similar chiral selectors.

Column name Column chemistry Structure

Chiralcel OD, Lux Cellulose-1, Chiralpak IB, Epitomize 1C Cellulose tris (3,5-dimethylphenylcarbamate)

Chiralpak AD, Chiralpak IA, Epitomize 1A Amylose tris (3,5-dimethylphenylcarbamate)

Chiralcel OJ, Cellulose-3 Cellulose tris (4-methylbenzoate)

Cellulose-2, Chiralcel OZ, CCCa Cellulose tris (3-chloro-4-methyl phenylcarbamate)

Cellulose-4, CC4 Cellulose (4-chloro-3-methyl phenylcarbamate)

Amylose-2, Chiralpak AY, Chiralpak IF Amylose (5-chloro-2-methyl phenylcarbamate)

Chrialcel IC, CCCa Cellulose tris (3,5-dichlorophenylcarbamate)

cellul

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a The CCC contains both the cellulose tris (3,5-dichlorophenylcarbamate and the

or the two columns was 0.77, but the Cellulose-3 separated moreamples and provided more baseline separations than the OJ whensing methanol (0.1% NH4OH) and ethanol (0.1% NH4OH). The reso-

ution slope value for the Cellulose-3 (x-axis) vs. the OJ (y-axis) was.61 demonstrating the Cellulose-3 outperformed the OJ. In our ear-

ier study [2], the OJ performed slightly better than the Cellulose-3.his may arise because we have used a more extensive set of com-

ounds in this study with a greater range of physical propertieshan was used in our first study. The OJ did separate slightly moreamples than the Cellulose-3 when isopropanol (0.1% NH4OH) wassed as a modifier.

ose tris (3-chloro-4-methyl phenylcarbamate).

3.4.4. Cellulose tris (3-chloro-4-methylphenylcarbonate),Cellulose-2 vs. OZ

There were two cellulose tris (3-chloro-4-methylphenylcarbonate) chiral selectors in our study, theCellulose-2 and the OZ. These two chiral selectors were verysimilar to each other with a resolution R2 value of 0.80. The twophases were very similar with ethanol (0.1% NH4OH) and methanol

(0.1% NH4OH) mobile phase conditions. The OZ has a slight edgeover the Cellulose-2 in terms of number of samples separated,based mostly on that it separated 6 more samples with a resolutionequal or over 0.3 with isopropanol (0.1% NH4OH) was the modifier.

C. Hamman et al. / J. Chromatogr. A 1305 (2013) 310– 319 317

Table 6A comparison of the various similar chiral selectors. The R2 value is a measure of how well correlated the phases are and the resolution slope value determines which phaseprovides larger resolution values. A slope value <1 means the x-axis provides column a higher average resolution than the y-axis column and vice versa.

Chiral selector X-axis Y-axis R2 value Resolution slope

Cellulose tris (3,5-dimethylphenylcarbamate) Cellulose-1 OD 0.93 1.36OD IB 0.72 0.44Cellulose-1 IB 0.76 0.63

Amylose tris (3,5-dimethylphenylcarbonate) AD IA 0.30 0.66

Cellulose tris (4-methylbenzoate) Cellulose-3 OJ 0.77 0.61

Cellulose tris (3-chloro-4-methylphenylcarbonate) Cellulose-2 OZ 0.80 0.84Cellulose-2 CCC 0.20 0.52CCC OZ 0.15 0.47

Cellulose tris (4-chloro-3-methylphenylcarbonate) Cellulose-4 CC4 0.63 0.78

Amylose tris(5-chloro-2-methylphenylcarbonate)

Amylose-2 AY 0.40 1.27AY IF −0.02 0.70

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selector. There is one dichloro selector in this study, the IC, but itis immobilized. The resolution R2 value for the CCC versus the IC is−0.22 suggesting the two CSPs provide very dissimilar separations.The CCC versus the Cellulose-2 R2 is 0.20 demonstrating that the

Amylose-2

Cellulose tris (3,5-dichlorophenylcarbonate) IC

owever, the resolution slope value for the Cellulose-2 (x-axis) vs.he OZ (y-axis) was 0.84 indicating the Cellulose-2 averaged higheresolution values. Given their similarities, it seems as though theyan be used interchangeably.

.4.5. Cellulose tris (4-chloro-3-methylphenylcarbonate), CC4 vs.ellulose-4

There were two cellulose tris (4-chloro-3-ethylphenylcarbonate) chiral selectors in this study, the CC4 and

he Cellulose-4. In this case, the modifier had a huge impact onhe performance of these two CSPs. With methanol (0.1% NH4OH)s the modifier, the Cellulose-4 clearly outperformed the CC4 byeparating 41 compounds to the CC4’s 26. The Cellulose-4 alsoearly doubled the number of baseline separations when usingethanol (0.1% NH4OH). However, with ethanol (0.1% NH4OH)

s the modifier, the two CSPs were very similar, baseline sepa-ating the same number of compounds (13) and the Cellulose-4eparating a mere two more compounds than the CC4. Finally,ith isopropanol (0.1% NH4OH) as the modifier, the CC4 clearly

utperformed the Cellulose-4. The CC4 separated 6 more com-ounds with a resolution greater than or equal to 0.3 and baselineeparated 4 more compounds than the Cellulose-4. We do notave a good explanation for this flip-flopping in performance and

t definitely deserves further study. The resolution R2 for the twohases was 0.63 and the resolution slope value for the Cellulose-4x-axis) vs. the CC4 (y-axis) was 0.78 indicating the Cellulose-4erforms better than the CC4 in terms of average resolution.

.4.6. Amylose tris (5-chloro-2-methylphenylcarbonate),mylose-2 vs. AY vs. IF

There are three amylose tris (5-chloro-2-ethylphenylcarbonate) chiral selectors in this study, two

oated phases, the Amylose-2 and the AY and one immobilizedhase, the IF. It is striking how differently these three phasesehaved despite all being the same selector. Between the twooated phases, the AY clearly outperformed the Amylose-2 inoth total separations and baseline separations, regardless ofodifier. The resolution slope value for the Amylose-2 (x-axis)

s. the AY (y-axis) is 1.27. As was mentioned in Section 3.1, theY and Amylose-2 experienced much better success in ethanolersus methanol (0.1% NH4OH) or isopropanol (0.1% NH4OH).or example, the AY separated only 21 compounds when using

ethanol (0.1% NH4OH), but separated 40 of the 80 compoundshen using ethanol. This was one of the more surprising results

n this study. The increase in separations is most likely due to thehree-dimensional shape of the selector when dissolved in the

IF 0.07 1.49

CCC −0.22 0.61

ethanol (0.1% NH4OH)/CO2 mobile phase [33]. The selector’s shapeis much more conducive to separating enantiomers in ethanolthan its shape in either methanol or isopropanol.

Since the IF is covalently bonded, it probably has a differentshape than its coated counterparts in the various mobile phasesand thus produces very different results. Fig. 4 shows the resolutionscatter plot of the AY and IF. The R2 value is −0.02. This suggests thatthe two phases are very complimentary to each other as they do nothave many redundant separations. With methanol (0.1% NH4OH)as the modifier, the IF separated 10 and 17 more compounds thanthe AY and Amylose-2, respectively. However, with ethanol (0.1%NH4OH), both the AY and Amylose-2 separated more compoundsthan the IF. With isopropanol (0.1% NH4OH), the AY separates onemore compound than the IF, but the IF provides 7 more baselineseparations than the AY. The Amylose-2 is much less effective thanthe IF when using isopropanol (0.1% NH4OH).

3.4.7. Cellulose tris (3,5-dichlorophenylcarbonate) CCC vs. IC andthe CCC vs. OZ/Cellulose-2

The CCC has a coated mixture of the cellulose tris (3,5-dichlorophenylcarbonate) and the cellulose tris (3-chloro-4-methylphenylcarbonate) selector in this study. The addition of the3-chloro-4-methyl selector decreases the solubility of the dichloro

Fig. 4. A scatter plot of all the resolution values from the three mobile phasesgenerated on the AY and IF.

3 atog

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18 C. Hamman et al. / J. Chrom

CC is very dissimilar from the Cellulose-2. The two low R2 valuesor the CCC with the IC and Cellulose-2 suggest the selectivity ofhe CCC is very different from the IC and Cellulose-2 even thoughhey share a selector.

.5. Evaluation of the new chiral phases

There were a number of new CSPs evaluated in this study that,o our knowledge, had not been previously studied with SFC: theF6-P, the CF6-RN, the Chirobiotic T2, the ID, the IE, the IF, the CC4nd the CCC. From Figs. 2a–c, one can get an idea of the successates of each of these columns. The CF6-P separated only 2 of the0 compounds. The CF6-RN also fared poorly, separating only 5f the 80 compounds. The Chirobiotic T2 was marginally betterhan the cyclofructan columns and separated 10 of 80 compoundsith methanol (0.1% NH4OH), 8 of 80 compounds with ethanol

0.1% NH4OH) and only 3 of 80 compounds with isopropanol (0.1%H4OH). Furthermore, none of these phases separated very chal-

enging molecules (molecules that separate on 3 or fewer CSPs). Its clear that these three phases are not very amenable to SFC con-itions. One recent study by Vozka et al. did report success using

different type of cyclofructan selector, the CF7-DMP paired withFC with some success [34].

The three new immobilized columns from Chiral Technolo-ies worked particularly well. The ID was the most consistent ofhe group and is a member of the primary team of six columnsescribed in Section 3.2 due to its unique separations. The IE did

ot separate many compounds with methanol (0.1% NH4OH) rela-ive to the competition, but did better with ethanol (0.1% NH4OH)nd was one of the top five columns in terms of separations whensopropanol (0.1% NH4OH) was the modifier. The IF performed

ig. 5. A chromatogram showing the separation of TSO on the 3.0 mm × 50 mm, 1.7 �m Epitomize column, the gradient was 5–60% over 1 min of MeOH (0.1% NH4OH), hold at 60radient was 5–60% over 1.8 min of MeOH (0.1% NH4OH), hold at 60% for 0.6 min, 4.0 mL/

r. A 1305 (2013) 310– 319

inversely from that of the IE, it was best when using methanol (0.1%NH4OH) and separated the fewest compounds under isopropanol(0.1% NH4OH). Perhaps most importantly, is that when these threecolumns were paired with the other three immobilized columnsfrom Chiral Technologies and the Whelk-O1, they partially sepa-rated an impressive 79 of the 80 compounds. The IE and IF werealso members of team two described in Section 3.2.

The CC4 and CCC phases from ES Industries also worked well.The two phases are very complimentary of each other with an R2

of 0.15. The CC4 works particularly well with isopropanol (0.1%NH4OH) where it separated 36 of 80 compounds, 16 of whichwere baseline separated. The CC4 is also a member of the teamone discussed in Section 3.2. The CCC excelled with ethanol (0.1%NH4OH) where it separated 39 of 80 compounds, 20 of which werebaseline separated. The CCC is also a member of our team twocolumns.

3.6. 1.5-min chiral screening method

Chiral method development is still very much a trial anderror process [2,9,29,35]. As such, there exists a need for fastermethod development. There have been a few strategies employedto decrease the method development time for chiral screening withSFC. Barnhart et al. and Zhao et al. used a multiple sample per-injection method that utilized a system whereby the user wouldinjection multiple samples with different molecular weights ina single run [26,36]. By using the mass spectra, the user is able

to determine chiral separations. This “sample pooling” method isvery effective, however, requires a mass spectrometer. Anotherapproach has been to use the Waters X5 Method Station systemthat uses five columns and five UV detectors per injection. The flow

pitomize 1C column and on a 4.6 mm × 50 mm, 3.0 �m Cellulose-1 column. For the% for 0.4 min, 1.5 mL/min, 1 �L inj of 1 mg/mL TSO. For the Cellulose-1 column themin, 1 �L inj of 1 mg/mL TSO. UV = 220 nm.

atogr

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4

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[34] J. Vozka, K. Kalikova, C. Roussel, D.W. Armstrong, E. Tersarova, J. Sep. Sci. 36

C. Hamman et al. / J. Chrom

rom the injector is split five ways to each column and a UV detec-or is placed at the end of each column. With this methodology,ne can screen up to five columns per injection, providing a 5-old improvement in throughput. The problem with the Waters X5

ethod Station setup is that it requires the coordination of 5 differ-nt detectors and the instrument itself has a very large footprint.

Our approach is to use shorter columns packed with 3.0 �m par-icles with high flow rates to reduce the screening method run timeo 2.5 min [2]. We have employed this strategy for over 1500 com-ounds and experienced excellent results. However, there is always

better way. Given the excellent results we obtained with shortolumns and sub 2.0 �m particles with our UPLC system and ourchiral analytical SFC methods (results not shown), we wanted tonvestigate using columns packed with sub 2.0 �m particles thatave a polysaccharide chiral selector for chiral separations. Theope was that we would be able to quickly determine if there isnantioselectivity or not with a very fast run time while consumingess solvent.

Fig. 5 shows chromatograms of TSO separated using the 1.5 minethod on the 3.0 mm × 50 mm, 1.7 �m Orochem Epitomize 1C

olumn at 1.5 mL/min and using the 2.5 min method on the.6 mm × 50 mm, 3 �m Cellulose-1 at 4.0 mL/min. The peak widthst 5% of the peak height generated on the Epitomize 1C wereoughly double that of the Cellulose-1. The peak capacities are 41nd 36 for the Epitomize 1C and Cellulose-1, respectively, usinghe 5% peak width values. Using the 1.5 min method reduces the

ethod by 40%, consumes ¼ of the solvent and provides very sim-lar peak capacities. Notice that both peaks eluted in less than 36 s

ith peak widths less than 2 s and a resolution of 1.9 on the Epito-ize 1C.Although the methods were fast for the Epitomize columns, the

verall results did not fair as well as their large-particle, similarhiral selector peers. The Epitomize 1C has the same coated cel-ulose tris (3,5-dimethylphenylcarbamate) selector as the OD andellulose-1 but it failed to separate as many compounds. At best,he Epitomize 1C partially separated 29 of the 80 compounds with

ethanol (0.1% NH4OH) compared with 41 for the OD and 36 forhe Cellulose-1. The Epitomize 1A has the same coated amylose tris3,5-dimethylphenylcarbamate) as the AD but it also failed to sep-rate as many compounds. At it is best, the Epitomize 1A separatednly 21 compounds with EtOH (0.1% NH4OH) compared with 37 forhe AD. We are not sure why the Epitomize selectors are so poorlyorrelated and further studies need to be done. Although these sub-.0 �m phases have limited separation ability, they demonstratehat shorter methods are practical.

. Conclusions

The 6000 chromatograms we generated for this study providedreat insights into columns and solvent systems that should becreened first in order to provide the highest chance of separationuccess. We found ethanol (0.1% NH4OH) to be a more effec-ive co-solvent for separating enantiomers than methanol (0.1%H4OH) or isopropanol (0.1% NH4OH). Through the use of an algo-

ithm, we found the best primary team of six columns is AD,S, AY, ID, CC4, and Whelk-O1. This combination separated 78f the 80 compounds and baseline separated 58 of the 80 com-ounds. With a secondary team of columns, the CCC, Cellulose-1,ellulose-3 or OJ, IA, IE and IF, we were able to increase the num-er of partial separations to 80 out of 80 and baseline separationso 64.

Screening the seven most effective immobilized columns the

A, IB, IC, ID, IE, IF and Whelk-O1 provided separation for 79 ofhe 80 compounds used in this study—a truly remarkable number.nd that was only with the three solvent combinations used in this

[[

. A 1305 (2013) 310– 319 319

study. The fact that the immobilized columns are more rugged andoffer such great success rates makes them a very attractive option.

Finally, we demonstrated the potential of pairing sub 2.0 �mparticles coated with polysaccharide chiral stationary phases forrapid chiral screening. A 1.5 min chiral screening method was pro-posed that can provides a fast readout on whether a given phaseis able to separate enantiomers. We are hopeful that the resultspublished in this paper will inspire more vendors involved to cre-ate sub-2.0 �m CSPs that are optimized to work with the latestinstrumentation, such as the Waters UPC2. As we like to say inour lab, there is always a better way and we feel strongly that the1.5 min chiral screening method holds promise of improving thechiral screening process.

Acknowledgements

The authors wish to thank Peter Yehl, Mohammad Al-Sayah, andJason Gruenhagen for their helpful comments.

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