Elsevier

Process Biochemistry

Volume 92, May 2020, Pages 69-77
Process Biochemistry

Electrochromatographic separation of hydrophobic amino acid enantiomers by molecularly imprinted capillary columns

https://doi.org/10.1016/j.procbio.2020.02.033Get rights and content

Highlights

  • Molecularly imprinted capillary columns (MICC) were synthesized.

  • Hydrophobic amino acids were separated enantiomerically by MICC.

  • MICC enabled high selectivity for l-tryptophan, l-tyrosine, and l-phenylalanine.

  • A short analysis time of 20 min was achieved in Capillary Electrochromatography.

Abstract

In the present study, the enantiomeric forms of hydrophobic amino acids (l-tryptophan, l-tyrosine, and l-phenylalanine) were separated by molecularly imprinted capillary columns (MICC) via Capillary Electrochromatography (CEC) for the first time. The monomer ratio, crosslinker ratio, template molecule ratio, the porogen ratio and type, polymerization time, and also the effect of temperature were examined to increase the permeability properties of MICC. FTIR, SEM and BET analyses were realized for the characterization of MICC. The effect of the electric field, organic solvent ratio, and pressure were carried out experimentally to determine the optimum conditions. The separation performances of MICC and the non-imprinted capillary columns (NICC) were compared electrochromatographically.

Introduction

Enantiomers may exhibit different physiological effects in biochemical processes through different recognition mechanisms as a result of their characteristic properties. Amino acids, the important part of the building blocks of living cells, play an important role in human health because of their numerous biological activities [1]. Amino acids of biochemical importance may exist in both enantiomeric forms with the advantage of chiral selection, and these properties make amino acids important for the chemical industry [2,3]. The needs of amino acids in enantiomeric purity, especially in the areas of biochemistry, are reported in the literature through studies carried out in many fields [4]. For example, l-tryptophan (L-TRP), one of the essential amino acids, is used as a pharmaceutical anti-depressant. On the other hand, d-tryptophan, which is not known for its significant contribution, can be a source of contamination for L-TRP. [5]. Chromatographic techniques are the most preferred methods for chiral separation due to their advantages, such as fast, efficient, and selective response in terms of analytical and required sensitivity [6]. CEC system is a combination of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) techniques. The background electrolyte (BGE) in the column interacts with the stationary phase by the support of the electroosmotic flow, which is the driving force for the analysis, providing a high selectivity, high efficiency, and high resolution with a low sample and solvent consumption [7]. So, CEC is one of the powerful methods frequently used in enantiomeric separation analyses in recent years [8,9]. Besides, due to its advantages of high selectivity, high resolution, high theoretical plate numbers, and fast migration time, CEC is now widely applied in many fields such as pharmaceutical, environmental, clinical, biological, and agricultural-food analyses [10]. The most commonly used CEC columns are generally classified into three types, named as open-tubular (OT), packed, and monolithic columns [11].

Monolithic columns have superior properties such as not having the disadvantages of the packed columns, including the preparation of frit that tend to break easily, which causes air bubbles as well as the need to pack the solid phase particles into the capillaries compactly [[12], [13], [14], [15]]. With the development of column technology, monolithic columns have been used as the application tools due to the increased need for HPLC and CEC [[16], [17], [18]].

Molecular imprinting is a very attractive method to be used for the preparation of specific recognition sites in the monolithic columns [[19], [20], [21]]. These molecularly imprinted polymers (MIPs) show selectivity for the target molecules [[22], [23], [24], [25], [26]]. Compared to natural receptors, MIPs offer very attractive advantages due to their low cost, chemical stability, and long shelf life [27,28]. The high-affinity property of MIPs can be considered as the strongest advantage for the CEC system [[29], [30], [31]]. There are many studies in the literature about the separation of hydrophobic amino acids by CEC [25,30,31].

In the present study, to our knowledge, the hydrophobic amino acids (l-tryptophan (L-TRP), l-tyrosine (l-TYR) and l-phenylalanine (l-PHE)) were separated enantiomerically for the first time by molecularly imprinted capillary columns (MICC) via the CEC method. Capillary columns were characterized by FTIR-ATR, SEM, and BET analyses. Electrochromatographic separation of the alkylbenzenes mixture, which was applied as test compounds to evaluate the separation performance of monolithic columns, was performed. To determine optimum conditions, enantiomeric separations were carried out under different conditions such as electric field strength, pressure, and the organic solvent content of the background electrolyte. Detection of the imprinted enantiomeric species was performed under the determined optimum conditions. To examine the effectiveness of the molecular imprinting technique, chromatographic separations of MICC and NICC were compared.

The main objective of this work is to find out separation mechanism shown by MICC based on the functional N-methacryloyl-(L)-phenylalanine (MAPA) monomer, which has dual ability to form both hydrophobic matrix and functional group supplier in one mode without needing any other spacer arm or functional monomer and also a EOF supplier. In this study, we aimed to produce CEC columns which provide excellent instrumentation for enantiomeric separations of hydrophobic d,l-TRP, d,l-TYR and d,l-PHE amino acids with high selectivity and specificity by MIPs.

Section snippets

Instrumentation

Enantiomeric studies were performed with a CEC system (Prince CEC-760, Netherlands). The device includes a diode array detector (DAD), UV lamp, and electrical power supply. The μHPLC pump (Dionex) was used for the treatment of capillary columns. Besides, long capillary mode system was used for analysis, and this method was chosen as the analysis method. The polymerization of MICC and NICC was achieved in the water bath (Julabo, Germany) at 70°C.

Reagents and materials

The functional monomer MAPA was purchased from

Preparation of amino acid imprinted capillary columns

Vinyl groups of the monolithic polymer were covalently bound to the inner wall of the capillary column by silanization process to increase the stability of the column. Following the silanization process of the inner capillary wall, preparation of the MICC was performed by using the MAPA monomer, which was chosen as a functional monomer and as the electroosmotic flow enabler.

In this study, the effects of functional monomer MAPA ratio, crosslinker ratio, template molecule ratio, the porogen

Concluding remarks

Enantiomeric separation of amino acids was performed extensively by electromigration techniques such as CE and CEC because enantiomers are diagnostic biomarkers for different diseases. So, many applications in the amino acid analysis have been made in biochemical, pharmaceutical, and clinical fields [[42], [43], [44]]. In this study, The l-PHE, l-TRP, l-TYR-imprinted MICC having specific recognition capability was prepared and applied to the CEC system for highly selective separation of d,l

Authorship contributions

Category 1

Conception and design of study: Handan Yavuz, Fatma Yılmaz, Adil Denizli

acquisition of data: Koray Şarkaya

analysis and/or interpretation of data: Koray Şarkaya, Süleyman Aşır

Category 2

Drafting the manuscript: Ilgım Göktürk, Fatma Yılmaz, Handan Yavuz

revising the manuscript critically for important intellectual content: Adil Denizli

Category 3

Approval of the version of the manuscript to be published (the names of all authors must be listed):

Koray Şarkaya, Süleyman Aşır, Ilgım Göktürk,

Declaration of Competing Interest

The authors declare that they have no competing interests.

Acknowledgements

All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are named in the Acknowledgements and have given us their written permission to be named. If we have not included an Acknowledgements, then that indicates that we have not received substantial contributions from non-authors.

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      However, all methods follow the same basic steps; (i) producing a polymer in the presence of a template or target molecule covalently or non-covalently bonded to the functional monomer, (ii) the template molecule is removed from the polymer matrix (desorption step) to form specific binding sites for the target molecule, (iii) when the MIP interacts with the target-containing sample, the target molecule is specifically rebinding to the specific cavity in the complex medium [17]. MIPs as a mimic of the specific biological recognition element is well combined in analytical technologies such as chromatography [18,19], assays [20] and sensors [21]. Sensors are optical [22], mass-sensitive [23], electrical [24] or chemical [25] devices with the capability to selectively sensing biological types [26].

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