DBPIA-NURIMEDIA

Korean Society for Biotechnology and Bioengineering Journal 29(3): 205-209 (2014) http://dx.doi.org/10.7841/ksbbj.2014.29.3.205 ISSN 1225-7117 / eissn 2288-8268 연구논문 고성능액체크로마토그래피의다당유도체를기초로한키랄고정상에서이동상첨가제가키랄아민의광학분리에미치는영향 백만정 1, 윤혜란 2, 이원재 * Effect of Mobile Phase Additive on Enantiomer Resolution for Chiral Amines on Polysaccharide-derived Chiral Stationary Phases by High Performance Liquid Chromatography Man-Jeong Paik 1, Hye-Ran Yoon 2, and Wonjae Lee* 접수 : 2014 년 4 월 8 일 / 게재승인 : 2014 년 6 월 24 일 2014 The Korean Society for Biotechnology and Bioengineering Abstract: Chromatographic enantiomer resolution of chiral amines was performed on several covalently immobilized and coated chiral stationary phases (CSPs) based on polysaccharide derivatives under the mobile phase conditions containing base or acid or acid/base additive. The chromatographic parameters including separation factors and capacity factors were greatly influenced by the nature of the mobile phase containing base or acid or salt additive as well as the used CSPs. When 0.05% triethylamine/0.05% trifluoroacetic acid as an additive in the mobile phase was used on all CSPs in this study, the greatest enantiomer resolution was observed except for Chiralpak AD. Also, it was shown that the change of base additive into acid or salt in the mobile phase may directly affect chiral recognition mechanisms between the 조선대학교약학대학약학과 College of Pharmacy, Chosun University, Gwangju, 501-759, Korea Tel: +82-62-230-6376, Fax: +82-62-222-5414 e-mail: wlee@chosun.ac.kr 1 순천대학교약학대학약학과 1 College of Pharmacy, Sunchon National University, Sunchon 540-950, Korea 2 덕성여자대학교약학대학약학과 2 College of Pharmacy, Duksung Women's University, Seoul 132-714, Korea chiral selectors and analytes occurring during enantiomer separation, resulting in the change of elution orders. Keywords: Chiral amine, Chiral stationary phase, Enantiomer resolution, Mobile phase additive 1. INTRODUNTION 키랄물질의광학분리를위해다당류셀룰로오스와아밀로오스유도체를키랄선택자 (chiral selector) 로이용하여만들어진키랄고정상 (chiral stationary phase) 으로많은거울상이성질체화합물을고성능액체크로마토그래피에서성공적으로분리해왔다 [1-3]. 키랄고정상을사용하여키랄 carboxylic acid 를광학분리하고자할때일반적분석방법으로소량의산을이동상에첨가제로사용하였다 [4-6]. 같은맥락으로키랄아민을키랄고정상에서광학분리하고자할경우, 일반적으로소량의아민을첨가제로이동상에사용하여효과적으로수행하여왔다 [3]. 키랄아민화합물을광학분리하는데있어서소량의아민을이동상에첨가하는이유는분석물질인아민과고정상의실리카와의비특이성상호작용을 (non-specific interaction) 최소화하여분석물질의머무름시간을줄여주거나또는피크가꼬리끌기 (tailing) 되어용리되는것을막아주는효과를가지고있기때문이다 [7]. 그런데몇연구그룹에서키랄아민을광학분리할때소량의아민

206 Korean Society for Biotechnology and Bioengineering Journal 29(3): 205-209 (2014) 을염기첨가제로이동상에가하여분석하는일반적인분석법대신에산을첨가제로사용한광학분리결과를보고하였다 [7-10]. 그연구에서, 키랄아민분석물질의종류와분석에사용된키랄컬럼에따라이동상에산첨가제를사용하는것이광학분리에가끔씩효과적이라는결과를보여주었다. 그러나선행연구에서키랄아민화합물을광학분리할때염기이동상첨가제로사용하는경우와비교해서산을이동상첨가제로얻은분석결과만을단순보고하는경우가대부분이었고이에대한체계적인연구가진행되지아니하였다. 그러므로본연구에서는다당유도체를모체로하는대표적인키랄컬럼인 Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak AD, Chiralcel OD 를사용하여산또는염기등의첨가제를각각함유한이동상에서키랄아민을광학분리하는크로마토그래피연구결과를비교하여이들이동상첨가제사용이광학분리에미치는영향을살펴보며그연구결과를보고하고자한다 [2]. 2. MATERIALS AND METHOD 2.1. 재료및시약 HPLC 용매로사용하는 hexane, ethanol (EtOH) 은 J. T Baker (Phillipsburg, NJ) 로부터구입하였고 trifluoroacetic acid (TFA), triethylamine 및분석물질인 α-methylbenzylamine, 1-(1-naphthyl)ethylamine, phenylglycine (PG) methyl ester, phenylalanine (Phe) methyl ester, 2-phenylglycinol 모두 Aldrich (Milwaukee, WI) 혹은 Sigma (St. Louis, Missouri) 회사로부터구입하였다. 2.2. 분석기기및방법고성능액체크로마토그래피실험은다음의기기들로구성된 HPLC 를사용하여상온에서수행하였다. HPLC 구성기기로 Waters model 1525 binary pump, 20 µl loop 를가진 Rheodyne model 7125 주입기, a dual absorbance detector (Waters 2487 detector) 를사용하였다. HPLC 용키랄컬럼으로 Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak AD, Chiralcel OD (250 mm L 4.6 mm I.D. 일본 Daicel Chemical 회사 ) 을사용하였다 [4-6,11]. Chiralpak IA, Chiralpak IB, Chiralpak IC 는다당유도체를키랄선택자로사용하여실리카에코팅시켜제조된키랄컬럼이고 Chiralpak AD, Chiralcel OD 는다당유도체를실리카에코팅시켜제조된키랄컬럼이다 [2,5,6]. HPLC 이동상용매로는 에 0.1% Et 3 N, 0.05% Et 3 N/0.05% TFA, 0.1% TFA 를각각첨가제로넣어사용하였고유속은 1 ml/min, UV 254 nm 에서검출하여분석하였다. 시료농도는 10 mg/ml 이고주입부피를 (injection volume) 1 µl 로실험하였다. 3. RESULTS AND DISCUSSION Table 1~5 에서다당유도체를키랄선택자로이용하는 5 개의키랄컬럼에서키랄아민의광학분리를위해이동상에산, 염기, 산 / 염기등을각각첨가하여얻은연구결과를보여주고있다. 전체적인결과에서보여주는것처럼광학분리의선택성과머무름시간은산또는염기등의첨가제가포함된이동상의성질에크게영향을받는것으로나타나는데이러한실험결과는사용하는키랄컬럼의종류에따라매우다르게나타났다. 특히사용한컬럼중에서, Chiralpak IA 와 Chiralpak AD 는동일한 amylose tris (3,5-dimethylphenylcarbamate) 키랄선택자를실리카에각각공유결합하거나코팅시켜제조된컬럼이며, 마찬가지로 Chiralpak IB 와 Chiralcel OD 는동일한 cellulose tris (3,5-dimethylphenylcarbamate) 키랄선택자를실리카에각각공유결합하거나코팅시켜제조된컬럼이다 [2,5,6]. 이동상첨가제로 0.1% Et 3 N 또는 0.05% Et 3 N/ 0.05% TFA 를사용할경우에는 Table 1 의키랄선택자인 amylose 유도체가공유결합된 Chiralpak IA 에서 Table 4 의키랄선택자가코팅된 Chiralpak AD 보다더좋은광학분리결과를보이나, 0.1% TFA 첨가제를사용할때에는 Table 4 의 Chiralpak AD 가더좋은광학분리를보여주었다. 반면에 Table 5 의 cellulose 유도체가코팅된 Chiralcel OD 에서는이동상첨가제종류에관계없이 Table 2 의 cellulose 유도체가공유결합된 Chiralpak IB 보다분명하게더좋은광학분리결과를보여주었다. Table 1, 4 의 Chiralpak IA 와 Chiralpak AD 그리고 Table 2, 5 의 Chiralpak IB 와 Chiralcel OD 는동일한키랄선택자를각각공유결합하거나코팅시켜제조한컬럼이기때문에제조과정 Table 1. Separation of the enantiomers of chiral amines using the mobile phase with additive on Chiralpak IA 1 α-methylbenzylamine 1.00 2.12-1.00 1.72-1.00 7.35-2 1-(1-Naphthyl)ethylamine 1.02 2.59 0.20 R 1.08 2.08 0.75 S 1.00 5.10-3 PG methyl ester 1.25 3.60 5.51 R 1.12 2.49 1.63 S 1.05 3.64 0.28 S 4 Phe methyl ester 1.17 4.72 4.50 R 1.63 2.34 5.81 S 1.56 3.65 1.54 S 5 2-Phenylglycinol 1.02 7.12 0.22 R 1.14 2.95 1.65 S 1.10 3.38 0.46 S c Resolution factor. d The absolute configuration of the second eluted enantiomer.

고성능액체크로마토그래피의다당유도체를기초로한키랄고정상에서이동상첨가제가키랄아민의광학분리에미치는영향 207 Table 2. Separation of the enantiomers of chiral amines using the mobile phase with additive on Chiralpak IB 1 α-methylbenzylamine 1.00 3.54-1.00 3.10-1.00 6.27-2 1-(1-Naphthyl)ethylamine 1.08 3.97 0.17 R 1.87 4.10 9.77 R 1.42 7.98 2.55 R 3 PG methyl ester 1.06 2.43 0.78 R 1.06 4.29 0.87 R 1.05 7.83 0.37 R 4 Phe methyl ester 1.00 1.93-1.00 3.46-1.00 6.68-5 2-Phenylglycinol 1.00 7.63-1.00 4.03-1.00 9.87 - c Resolution factor. d The absolute configuration of the second eluted enantiomer. Table 3. Separation of the enantiomers of chiral amines using the mobile phase with additive on Chiralpak IC 1 α-methylbenzylamine 1.11 3.15 1.41 R 1.19 4.44 3.04 R 1.19 2.09 1.25 R 2 1-(1-Naphthyl)ethylamine 1.39 3.17 4.99 R 1.89 4.81 9.91 R 1.70 3.05 2.90 R 3 PG methyl ester 1.00 5.48-1.48 10.26 6.91 R - NE e - 4 Phe methyl ester 1.07 4.89 1.60 R 2.46 7.34 9.51 R - NE e - 5 2-Phenylglycinol 1.07 8.43 1.00 R 1.59 8.52 6.53 R 1.40 3.65 6.14 R c Resolution factor. d The absolute configuration of the second eluted enantiomer. e No elution for 1 hr. Table 4. Separation of the enantiomers of chiral amines using the mobile phase with additive on Chiralpak AD 1 α-methylbenzylamine 1.00 1.80-1.00 1.25-1.12 4.43 0.68 S 2 1-(1-Naphthyl)ethylamine 1.00 2.40-1.05 1.65 0.53 S 1.29 8.38 3.12 S 3 PG methyl ester 1.18 3.00 1.62 R 1.07 2.10 0.38 S 1.05 6.53 0.30 S 4 Phe methyl ester 1.23 3.73 2.52 R 1.62 1.88 4.53 S 1.77 4.30 3.16 S 5 2-Phenylglycinol 1.00 6.64-1.10 2.18 1.05 S 1.13 12.41 0.68 S c Resolution factor. d The absolute configuration of the second eluted enantiomer. Table 5. Separation of the enantiomers of chiral amines using the mobile phase with additive on Chiracel OD with 0.05% Et 3 N/0.05% TFA 1 α-methylbenzylamine 1.00 4.18-1.08 2.39 0.57 S 1.07 2.98 0.19 S 2 1-(1-Naphthyl)ethylamine 1.29 4.22 0.88 R 3.05 6.05 11.48 R 2.52 5.86 3.52 R 3 PG methyl ester 1.20 3.57 1.47 R 1.21 4.14 1.80 R 1.28 4.11 1.67 R 4 Phe methyl ester 1.00 2.63-1.12 2.81 0.92 S 1.11 3.40 0.73 S 5 2-Phenylglycinol - NE e - 1.07 3.40 0.51 R 1.06 4.34 0.13 R c Resolution factor. d The absolute configuration of the second eluted enantiomer. e No elution for 1 hr. 에서키랄선택자로사용된키랄선택자의키랄성을얼마나유지하고있느냐가광학분리에영향을줄수있다. 키랄선택자를공유결합하거나코팅시킨것에따른차이가광학분리에약간의영향을줄수있다고하더라도, 키랄선택자와분석물질간의키랄인지매커니즘 (chiral recognition mechanism) 에서는전체적으로유사할것이라예상한다 [5,6]. 실제, 동일한키랄선택자가사용되었다고볼수있는 Table 1, 4 의 Chiralpak IA 와 Chiralpak AD 그리고 Table 2, 5 의 Chiralpak IB 와 Chiralcel OD 에서동일한첨가제의이동상을사용하였을경우에광학분리되어용출된용리순서 (elution order) 가동일하게나타나는것이이에대한하나의증거라말할수있다. 한예로, 0.05% Et 3 N/0.05% TFA 를첨가제로얻은분석결

208 Korean Society for Biotechnology and Bioengineering Journal 29(3): 205-209 (2014) 과에서 Table 1 의 Chiralpak IA 와 Table 4 의 Chiralpak AD 에서광학분리된 4 개의분석물질의 S-isomer 가모두두번째로용출되나, Table 2 의 Chiralpak IB 와 Table 5 의 Chiralcel OD 에서는광학분리된 2 개의분석물질의 (entry 2 와 3) R-isomer 가모두두번째로용출되어나타난다. 그러나이러한결과보다도, 앞에서도언급한바와같이동일키랄컬럼상에서이동상에산또는염기, 산 / 염기등의첨가제가각각다르게사용되었을때키랄아민의광학분리가전체적으로상당히달라지고있음을실험결과에서보여주고있다 [8]. 이러한이동상의첨가제가광학분리에미치는효과는광학분리된정도와머무름정도그리고광학분리된물질의용출된용리순서를통해서그러한현상이발견된다. 일반적으로 Table 1, 2, 4 에서의광학분리에서는산첨가제를사용할때가염기첨가제를사용하는것에비해용량인자값이상당히증가하게되지만, Table 3 의광학분리에서는첨가제로 0.05% Et 3 N/0.05% TFA 사용했을때가산또는염기첨가제를사용하는것에비해용량인자값이가장크게나타났으며, Table 1 과 4 에서의광학분리에서는첨가제로 0.05% Et 3 N/0.05% TFA 를사용했을때가용량인자값이가장작게나타났다. 또한 Table 4 에서약간예외적인부분이나타나긴하지만, 전체적으로 Table 1~3 과 Table 5 에서이동상첨가제로 0.05% Et 3 N/0.05% TFA 를사용하는경우가 0.1% Et 3 N 또는 0.1% TFA 를사용하는경우보다더좋은광학분리를나타내었다. 특히여러키랄컬럼중 Table 3 의 Chiralpak IC 에서 0.05% Et 3 N/0.05% TFA 를첨가제로얻은광학분리결과가전반적으로가장좋게나타났고분석물질에서모두기준분리를보여주었다. 대표적인예로, Chiralpak IC 에서 phenylglycine methyl ester 를광학분리할때, 이동상첨가제로 0.1% Et 3 N 을사용하는경우에전혀분리되지않았고, 0.1% TFA 를사용하는경우에도 1 시간동안에전혀용리되지않았다 (Table 3, entry 3). 그런데이동상첨가제로 0.05% Et 3 N/0.05% TFA 를사용하였을때는매우좋은광학분리결과를보인것처럼 이동상의산, 염기, 산 / 염기첨가제종류의변화만주어도광학분리결과가매우크게달라지는현상을발견할수있었다. 또한이렇게이동상의첨가제가광학분리에전반적으로미치는영향은키랄선택자와분석물질간의키랄인지매커니즘에직접적인영향을주기때문이고이는광학분리의용리순서에도매우큰영향을주는것으로관찰된다. 예를들어, Table 2, 3, 5 에서사용된컬럼들의경우이동상의첨가제변화에따른용리순서변화는없는것으로나타나지만, Table 1 의 Chiralpak IA 와 Table 4 의 Chiralpak AD 경우에는첨가제로 0.1% Et 3 N 을사용한용리순서가첨가제로 0.05% Et 3 N/ 0.05% TFA 또는 0.1% TFA 를사용한용리순서와는정반대로나타난다. 그래서전자의경우광학분리된분석물질의 R- isomer 가모두두번째로용출되어나타나지만후자의경우에는 S-isomer 가모두두번째로용출되어나타난다. 이는전자에서의키랄인지매커니즘이후자의경우와다르다는것을의미하고있다. 이동상에 0.1% TFA 나 0.05% Et 3 N/0.05% TFA 의첨가제를사용하게될경우, 분석물질인키랄아민은키랄 ammonium salt 를형성한후키랄선택자와의키랄인지를통한광학분리가진행될것이라예상된다 [ 여기서 0.05% TFA (6.7 mm) 와 0.05% triethylamine (3.6 mm) 를동시에사용할경우, 화학량적인 (stoichiometric) 관점에서 acidic 한조건을유지하게된다 ]. 그에비해이동상에 0.1% Et 3 N 의첨가제를사용하게될경우, 이동상이염기성을띄기때문에분석물질인키랄 free amine 자체가키랄선택자와의키랄인지를할것이라보아진다. 그러한광학분리용리순서의역전현상이산성을띠는이동상의경우에 Chiralpak IA 와 Chiralpak AD 에서만일어난다는것은이들컬럼에서는키랄선택자와의키랄 ammonium salt 와의 hydrogen bonding 으로인한 interaction 이존재하기때문이라추정할수있다. 이와는달리, Table 2, 5 의 Chiralpak IB 와 Chiralcel OD 뿐만아니라 Table 3 의 Chiralpak IC 에서는앞에서기술했던 Chiralpak IA 와 Chiralpak AD 에서일어나는첨가제의산, 염기, Fig. 1. Typical chromatograms showing additive effect in the mobile phase on enantiomer resolution of 1-(1-naphthyl)ethylamine on Chiralcel OD using (a) 1, (b) 0.05% Et 3 N/0.05% TFA and (c) 0.1% TFA, respectively; Flow rate= 1.0 ml/min; Detection UV 254 nm; Injection amount 10 µg.

고성능액체크로마토그래피의다당유도체를기초로한키랄고정상에서이동상첨가제가키랄아민의광학분리에미치는영향 209 염기에따라분석물질의용리순서의역전현상이일어나지않는다. 이는첨가제로이동상이염기성이아닌산성성질을띄어아민분석물질이키랄 ammonium salt 를형성하더라도키랄선택자와 ammonium salt 사이의키랄인지에서 hydrogen bonding interaction 이존재하지않기때문에위와같은용리순서의역전현상이나타나지않는것이라보인다. Fig. 1 는 Chiralcel OD 을사용하여 1-(1-naphthyl)ethylamine 을광학분리할때염기, 산또는산 / 염기각각첨가제를사용한이동상에서의전형적인크로마토그램을비교하며보여주고있다. Chiralcel OD 를사용할때광학분리가이동상의첨가제에따라매우큰영향을받게되는데 0.05% Et 3 N/0.05% TFA 첨가제를가했을때가장좋은광학분리를보여주고있으며 0.1% Et 3 N 염기첨가제를가했을때가장좋지않는광학분리를보여주고있다. 4. CONCLUSION 고성능액체크로마토그래피에서다당유도체를기초로하는키랄고정상들을사용하여산또는염기등의첨가제가포함된각각의이동상에서키랄아민의광학분리를수행하였다. 산또는염기등의첨가제가각각포함된이동상뿐만아니라사용한키랄고정상에따라광학분리된선택성과분리인자, 용량인자가크게영향을받았다. Chiralpak AD 를제외한모든컬럼에서이동상에 0.05% Et 3 N/0.05% TFA 를첨가제로사용했을경우가 0.1% Et 3 N 또는 0.1% TFA 를첨가제로사용했을경우보다도훨씬좋은광학분리를보였다. 이동상의산, 염기등의첨가제의변화만으로도광학분리결과에큰영향을주면서, 때로는용리순서에도상당히큰영향을주는것을볼때이러한첨가제변화는광학분리중에일어나는키랄인지매커니즘의상호작용에직접적으로연계되어있음을보여주고있는것이라보아진다. REFERENCES 1. G. Subramanian, (Ed.) (2001) Chiral Separation Techniques: A practical approach. 2nd revised ed., VCH, Weinheim. 2. W. Lee (2009) The application of chiral HPLC columns for enantiomer separation of chiral drugs. Yakhak Hoeji 53: 60-68. 3. Application Guide for Chiral HPLC selection, 4th ed., (2008) Daicel Chemical Industries, Ltd. 4. Li, Y. H. and W. Lee (2005) Liquid chromatographic enantiomer resolution of N-fluorenylmethoxycarbonyl α-amino acids and their ester derivatives on polysaccharide-derived chiral stationary phases. J. Sep. Sci. 30: 2057-2060. 5. Jin, J. Y., W. Lee, and C. S. Baek (2008) Enantiomer resolution of non-steroidal anti-inflammatory drugs on chiral stationary phases derived from polysaccharide derivatives. Chin. J. Anal. Chem. 36: 1207-1211. 6. Jin, J. Y., S. K, Bae, and W. Lee (2009) Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N-protected α-amino acids and their ester derivatives. Chirality 21: 871-877. 7. Strigham, R. W. and Y. K. Ye (2006) Chiral separation of amines by high-performance liquid chromatography using polysaccharide stationary phases and acidic additives. J. Chromatogr. A 1101: 86-93. 8. Ye, Y. K. and R. W. Strigham (2006) The effect of acidic and basic additives on the enantioseparation of basic drugs using polysaccharide-based chiarl stationary phases. Chirality 18: 519-530. 9. Tang, Y., W. L. Zielinski, and H. M. Bigott (1998) Separation of nicotine and nornicotine enantiomers via normal phase HPLC on derivatized cellulose chiarl stationary phases. Chirality 10: 364-369. 10. S. Caccamese, S. S. Bianca, and G. T. Carter (2007) Direct highperformance liquid chromatographic separation of the enantiomers of an aromatic amine and four aminoalchols using polysaccharide chiral stationary phases and acidic additive. Chirality 21: 647-653. 11. Xu, W. J., J. Y. Jin, and W. Lee (2011) Enantiomer separation of chiral amino alcohols as 9-anthraldimine derivatives on coated and covalently bonded chiral stationary phases based on polysaccharide derivatives by high performance liquid chromatography. Kor. Soc. Biotech. Bioeng. J. 26: 323-327.