Cyclodextrins in Analytical Chemistry: HostâGuest Type Molecular

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Cyclodextrins in Analytical Chemistry: Utility of Host-Guest Type Molecular Recognition Lajos Szente, and Julianna Szemán Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 20 Jun 2013 Downloaded from http://pubs.acs.org on June 20, 2013

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Analytical Chemistry 1

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Cyclodextrins in Analytical Chemistry: Host-Guest Type Molecular Recognition

Lajos Szente* and Julianna Szemán CYCLOLAB Cyclodextrin Research and Development Laboratory Ltd. H-1097 Budapest, Illatos út 7. Hungary e-mail: [email protected] Fax: +36-1-347-6068 Cyclodextrins are utilized in many diverse fields of analytical chemistry, due to their propensity to form reversible inclusion complexes and recognize analytes selectively. This feature article shows how these nano-cavities can serve analysts in sample preparation, sensitivity- and selectivity improvement, enantio-separation, creating single-molecule sensors and automatizing DNA sequencing.

*corresponding author

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Historical events In 1952, while investigating the structure and properties of cyclodextrins (CDs) Cramer predicted that the nanometer-sized, apolar and chiral cavities of CDs would have a great utility in analytical chemistry, in particular in separation science1. Some thirty years later, Armstrong gave strong evidence of Cramer’s prediction and laid down the fundamentals of cyclodextrin-assisted separation science 2,3,4. Between 1980 and 1988, Armstrong and co-workers pioneered the development and optimization of analytical methods suitable for CD-based isomer separation and even marketed CD-bonded chromatographic columns for selective and efficient enantiomer separation5. During the period of 1980 to 1990, various types of CDs became everyday commodities in separation science, and also as enabling additives in the enhancement for the sensitivity and selectivity of different analytical techniques6. Li and Purdy reviewed the application of cyclodextrins in diverse fields of analytical chemistry7. This insightful review covered the structural aspects of cyclodextrins that enabled the improvement of different chromatographic separations, enhancement of sensitivity and accuracy of analytical spectrometric methods including luminescence spectroscopy and electrochemical analyses. A similar survey of the non-chromatographic analytical application of CDs was published in 19988. Hinze et al summarized the possibilities of using CDs as reagents in analytical chemistry and diagnostics9. Some recent reviews deal with the versatile role of CDs as chiral selectors in different separation techniques 10,11. The goals of this paper are to establish the basic principles of molecular recognition- based CD-assisted analytical methods, to highlight recent advances, and to demonstrate the benefits and limits of their application in this field.

CYCLODEXTRIN BASICS The analytical applications of CDs are based on reversible, host-guest (cyclodextrin analyte) interactions Enzyme-modified starch derivatives, cyclodextrins have long been utilized in different industrial fields such as pharmaceuticals, food and personal care products, etc12. The nontoxic, environmentally-friendly sugar-based macro rings consist of D-glucopyranose units linked with α-1,4 glycosidic bonds. The three major, industrially manufactured and applied parent CDs are the six glucose-membered αCD, the seven glucose-membered βCD and the eight glucose containing γCD. (See Figure 1) ACS Paragon Plus Environment

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Figure 1. Structure of αCD (left), βCD (middle), and γCD (right)

These discrete toroids have a slightly lipophilic inner cavity (due to the presence of glycosidic oxygen bridge and hydrogen atoms) and hydrophilic outer surface (due to the presence of primary and secondary hydroxyl groups). Consequently, the parent CDs are water-soluble and capable of recruiting fat-soluble compounds to reside in their lipophilic cavities. One of their most characteristic functional properties is to form reversible, noncovalent inclusion complexes with compounds that geometrically fit inside the cavity and are less polar than water. The practical utilization of CDs in analytical chemistry is almost exclusively related to this host - guest type molecular recognition process also known as supramolecular complex formation,13 (see Figure 2). Since the first published results, the analytical applications of CDs have been continuously expanding, and today CDs are considered to be versatile additives of many kinds of molecular recognition-based analytical methods, such as separation science, spectroscopic methods, electroanalyses, single molecule sensing and clinical diagnostics.

+

Figure 2. Schematic illustration of reversible host-guest complex formation (blue: cyclodextrin, green: guest compound or analyte) ACS Paragon Plus Environment

Analytical Chemistry

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Cyclodextrins can be viewed as “molecular shape sorters” that provide the ability to selectively trap and temporarily retain substances (analytes) of proper geometrical fitting in their chiral nano-cavities. Thus CDs are capable of recognizing certain analytes in a highly selective and sensitive manner. This molecular recognition manifests in practically useful effects such as the separation of isomeric mixtures, the enhancement of detection sensitivity, and can be used in the automation of diagnostic processes, etc. The versatility of CDs in analytical chemistry is related to their unique structures and to the fact that CDs retain their analyte-recognizing and entrapping properties under relatively broad range of experimental conditions (pH, medium polarity, ionic strength, temperature, etc.). The numbers speak for themselves: Current landscape of analytical applications Today, the total number of publications and patents on CDs exceeds 53,000. In the past two decades, the applications of CDs in analytical chemistry have increased dramatically, in particular in the field of separation science. To illustrate the main types of analytical CD applications, careful literature search was performed with targeted key words (see Table 1.)

Table 1. Total number of publications on the utilization of CDs in analytical chemistry by selected keywords (ending in March 2013)* Keywords

Number of hits

chiral separation

4,680

fluorescence detection

3,100

capillary electrophoresis

2,450

detection limit

1,400

sensitivity

420

sensing

450

voltammetry

368

*Based on CycloLab’ Cyclodextrin News literature database, where the total number of entries is over 53,000


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CYCLODEXTRINS IN SAMPLE PREPARATION Sample preparation is a crucial step in any analytical method, especially in chromatography where the samples have to be homogenous, free of interferences and safe for the column. Frequently, the component of interest is present in levels too low for detection. Sample preparation can concentrate the component to adequate levels for measurement. The selective molecular interactions provided by the different CD cavity dimensions offer the possibility for selective trapping and removing either the target analyte or the interfering matrix components from complex analytical samples. This process improves the accuracy and reliability of most common analytical procedures by decreasing or eliminating the matrix effects. Such CD-enabled extraction cartridges have been employed to isolate or selectively enrich analytes from food, pharmaceutical products, tissues- and environmental samples 14,15. How do cyclodextrin-enabled cartridges work? In the first step of sample preparation the samples interact with the sampler or CD-coated adsorber, which can selectively retain the target analytes from dilute complex samples. The retained analyte is then eluted (liberated) for analysis by adding a competitive complexforming analytically inert agent (see Figure 3). This competitive displacement principle has been successfully used and validated for different analytes such as steroidal compounds, bile acids, pharmaceuticals, etc. 16

Figure 3. Suggested mechanism of sample preparation with CD-enabled cartridges.



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Cyclodextrins can also be used as “sample traps” for volatile or gaseous analytes 17. For example, CD-enabled silica particles can be used as “traps” for the capture of volatile organic solvents from contaminated air. Both the efficiency of sample capture and the recovery of the retained target analytes were found acceptable and reproducible in the environmental analysis of airborne toxic organics, e.g., benzene, toluene, ethyl-benzene, oxylene, m-xylene and p-xylene. The results indicate good reproducibility with a coefficient of variation below 6% and high recovery rates.18 Selective precipitation by cyclodextrins The analysis of complex biological samples often requires careful sample preparation steps to remove interfering matrix components. CDs are suitable inert agents to remove lipids and complex lipoproteins in lipemic serum. Unlike the use of organic solvents or centrifugation, the CD-based serum clarification is simple, mild, non-hazardous, resulting in an effective means to remove the interfering lipid particles from biological specimens19. A similar serum clarification procedure uses alpha-CDs for the selective removal of fatty acids that cause interference during the automated colorimetric determination of serum calcium levels. For example, calcium levels were determined in pooled serum containing palmitic acid and in serums of patients with high glucose and elevated lipids or from hemodialysis patients by colorimetric methods. All methods gave lower values for the Ca concentrations in the presence of fatty acids. Pre-treatment of the samples with alpha-CD eliminated this interference.20 Early on enantiomeric enrichment via cyclodextrin precipitation/crystallization was shown effective 21.

CYCLODEXTRINS TO IMPROVE SENSITIVITY The molecular basis of sensitivity improvements by CDs in analytical processes can be explained by a significant and temporary change of the analyte micro-environment (from aqueous hydrophilic to the lipophilic phase) in the CD cavity. This results in an enhanced spectral response. Moreover, the CD cavity is a unique micro-environment similar to that of organic solvents and can cause spectral shifts, changes in excited state lifetimes, etc. The first observation of such CD-assisted sensitivity improvements was presented by Friedrich Cramer in 1951, reporting on the spectral changes of colored substrates recorded in the presence of CDs 1, 22.


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Cyclodextrins in luminescence spectroscopy Most organic compounds show enhanced fluorescence or phosphorescence in ordered organic systems, e.g., in lipophilic solvents, whereas the intensity of luminescence of the same compounds can be poor in hydrophilic environments, e.g. in water. In an aqueous solution, the relatively lipophilic nano-cavities of CDs offer such a local micro-environment for fluorescent analytes resulting in useful enhancement of the fluorescence or phosphorescence signals of the complexed analyte. The CD cavity behaves like an organic solvent, providing an apolar surrounding for the entrapped chromophores. This altered lipophilic microenvironment can provide favored polarity and acid/base equilibria for enhanced quantum efficiencies and increase the luminescence intensity. The polarity of the actual microenvironment inside the CD cavity is similar to that of some oxygen-containing organic solvents such as 1,4-dioxane, tert-amyl alcohol, or 1-octanol. Thus, the CDcomplexed analyte experiences a significantly lower polarity in the local cavity environment as well as reduced freedom of movement in the cavity resulting in hindered intramolecular rotations23. Moreover, the postulated mechanism behind the fluorescence enhancement for fluorophores by inclusion complexation is that CDs protect the fluorescing singlet state or the phosphorescing triplet state of the analyte from external quenchers and, as a consequence of inclusion, the rotation of the guest molecule becomes hindered. Both of these effects may result in a decrease in the vibrational deactivation. Kinoshita and co-workers were the first who reported on the usefulness of CDs in advanced luminescence spectroscopy by reporting on the improved fluorimetric determination of dansyl-amino acid derivatives 24,25. Cyclodextrin-assisted fluorescence spectroscopy for mycotoxin analysis Mycotoxins are considered one of the most significant chronic dietary risk factors besides synthetic food contaminants, plant toxins, food additives and agrochemical residues. While traditional mycotoxin analysis has been performed using mainly chromatographic techniques, which provide high sensitivity and accuracy, emerging techniques, such as fast screening assays suitable for simultaneous in situ multi-toxin determination are of growing importance. One of these novel approaches is based on the fact that mycotoxin fluorescence can be significantly enhanced in the presence of cyclodextrins26. A portable fluorometer for the rapid and highly sensitive screening of aflatoxins in realworld food samples was constructed using cyclodextrin-enhanced fluorescence detection. The compact and easy-to-handle device enables simple and rapid monitoring of aflatoxins in milk, with an enhanced detection limit. The cyclodextrin-enabled aflatoxin sensor was ACS Paragon Plus Environment


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found suitable for preliminary screening of non-contaminated and contaminated farm milk products at early stages of the production chain with improved sensitivity at the 25 ppt analyte level 27. Certain types of CDs such as dimethyl-βCD enhance the fluorescence of a number of mycotoxins. Such specific mycotoxins include those with native fluorescence, such as the aflatoxins, ochratoxin-A and zearalenone as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The CD-based analysis by CE and HPLC methods all using dimethyl-βCD in the mobile phase, allowed the reliable identification of mycotoxins by laser induced fluorescence detection at levels as low as several 10 ng/g sample 28. Cyclodextrin-enabled sensors: toward single molecule sensing The sensitivity of sensors working on fluorescence, phosphorescence, infrared absorption, surface acoustic wave or electric (piezo-electric or voltammetric) principle can be improved by CDs. The chromophore- or fluorophore-tagged cyclodextrins are particularly selective and sensitive thus useful to improve sensor performance. The molecular mechanism behind this phenomenon is the change of fluorescence spectra in the presence of a competitive guest substance (i.e., target analyte). These sensors are selective to the compounds

which

have higher affinity toward cyclodextrin than the

appended

chromophore/fluorophore group occupying the CD cavity (see Figure 4).

F

F A CD

CD

Figure 4. Analyte-induced conformational change of chromophore-tagged CDs (Cavity replacement-based analyte sensing) 29 Legend: A: analyte, CD: cyclodextrin, F: appended fluorophore ACS Paragon Plus Environment

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Cyclodextrin-enabled protein nano-pores: rings in the pores Engineered transmembrane protein pores have been considered as promising sensor elements for stochastic analyte detection. Analyte compounds alter the ionic current flowing through a protein pore causing altered transmembrane electric current. The analyte-created current signature and the residence time of the analyte-caused blockade can be used to identify and quantify analytes. The sequencing of human genome may cost today as much as about 10,000 dollars. There were numerous attempts to reduce the price and analysis time of DNA sequencing to practical levels, ultimately to about a thousand dollars/analysis and to less than an hour time frame. One promising approach uses the idea of threading a single DNA molecule through a nano-sized protein pore anchored in a lipid membrane and identifying each DNAbase by changes in the ion current flowing across the pore 30. The structure and geometry of protein nanopores enable the entrapment and manipulation of single analyte molecules. Most of the currently used engineered nanopores are bacterial (Staphylococcal) toxins, like alpha-hemolysin, a heptameric assembly of the protein. These protein pores are fixed into a lipid bilayer, which is a powerful insulator, so the electrical conductance through the membrane will be due to interactions between the salt ions and the heptamer protein pore. The lumen of protein pores can be functionalized with CDs resulting in a “sugar-ring in protein-pore” supramolecular arrangement. The molecular adapter CD will fulfill the analyte sensing function by recognizing analytes that move through the cyclodextrin cavity. During the action of these single-molecule detectors, the reversible interaction of the analyte with a modified protein nano-pore causes change in the ion-current flow across the pore. The change in the ion-current amplitude and the mean dwell time of the analyte enable the identification of the analyte, whereas the frequency of those conductivity change events during sensing correlates with the amount (concentration) of the analyte. The discovery that cyclodextrin-enabled protein nanopores can be used for stochastic sensing and DNA sequencing was proposed by Hagan Bayley’s group31. Bayley and colleagues later covalently attached a beta-cyclodextrin derivative inside the protein nanopore. This chemically fixed CD adapter cannot dissociate from the nanopore ensuring that no gaps occur during stochastic detection. The fixed nature of the CD molecular adapter results in distinctive current flow through the pore, whenever analytes interact with it . This is particularly important in DNA sequencing, where the fixed molecular adapter is needed for the continuous reading of DNA fragment nucleotides32.



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CYCLODEXTRIN-MEDIATED SEPARATIONS Cyclodextrins can be utilized in a wide variety of separation techniques, such as chromatographic methods, electrophoresis, isotachophoresis, isoelectric focusing, microdialysis, separation on hollow fibers, foam flotation enrichment, solid- and liquid-phase extractions, separation through liquid and composite membranes, applications in molecularly imprinted polymers etc. 33 Since the first recognition that CDs have enantio-discriminative properties published in 1959 34, more than 4000 papers have dealt with CD-assisted enantiomer separation. There is no doubt that one of the most valuable applications of CDs as analytical tools, is their utilization in resolving enantiomer compounds. There are a number of criteria for successful chiral recognitions by CDs. A general mechanism of chiral recognition cannot be given, as enantioselective retention mechanisms depend on the reaction conditions and the chemical microenvironment of CD’s cavity entrance. In most cases it is proved that no inclusion complex between analyte and CDs govern enantiomer separations. The enantio-recognition is due to the combination of different molecular interactions such as H-bonding, dipolar and steric interactions between hydroxyl groups of CDs and analytes35. Armstrong explained the enantio-recognizing properties of CDs by referring to the structure and geometry of these nano rings. He postulated that the highly ordered and stereochemically unique arrangement of the D-glucose units forming the macrocycle is responsible for enantiorecognition. The facts that each glucose of the CD cylinder is of chiral structure (with five chiral centers), that the glucose units are found in chair conformation, that all secondary OH-groups of glucose units are pointed in clock-wise direction, whereas all primary OH-groups at the opposite rim of the CDs are pointed in counter-clock-wise direction, alone or in their combination play a role in the chiral recognition36. Two types of practical applications of CDs are known in enantiomer separations: •

CDs are chemically bound to the stationary phases mainly in gas chromatography (GC) and high-performance liquid chromatography (HPLC),

•

CDs are used in the mobile phase as chiral additives, mostly in HPLC and capillary electrophoresis (CE) techniques

Cyclodextrins in gas chromatographic separations Since the first successful chiral GC separation made by Gil-Av and co-workers 37 in 1966, chiral GC has witnessed a dramatic explosion of CD-mediated chiral separations in many ACS Paragon Plus Environment

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fields including natural products, asymmetric synthesis, biological studies, environmental, agriculture, food, flavor and fragrance evaluations. CDs soon found their practical utility in enantiomer separation by GC. The first CD-based chiral GC separation was published in 1982 by Smolkova-Keulemansova, et al. 38. More than fifty CD derivatives have been so far designed and employed successfully in GC separations. Nearly 500 papers reported on the benefits of short chain per-alkylated CDs. Results of peralkyl CD-based chiral GC separations indicate that substitution of primary OH-groups by methyl moiety, and the substitution of secondary OH-groups with longer alkyl groups (e.g. n-butyl-, n-pentyl- groups) greatly improve enantioselectivity39.The first broadly effective chiral separations on chemically modified CDs were reported by Konig et al.40 Two of the most frequently applied CD derivatives in capillary GC separations are the heptakis-2,3-di-O-methyl-6-O-tert-butyl-dimethyl-silyl-βCD (TBDMS) and 2,6-di-O-pentyl-3O-trifluoroacetyl-βCD (DPTABCD).

41

These versatile chiral selectors in capillary GC have

replaced the previously used 2,3,6-tri-O-methyl βCD (TRIMEB). The reason for the superior applicability of TBDMS and DPTABCD over TRIMEB is that the tert-butyl- and n-pentyl groups allow improved distribution and orientation of the CDs in the polysiloxane carrier matrix. Another reason is that these CD derivatives are oils, thus can exhibit a homogeneous distribution throughout the polysiloxane phase, whereas TRIMEB is a crystalline solid and in a gaseous environment has poor mass transfer properties resulting in broad peaks and high retention times42. DPTABCD is directly bonded to the dimethylpoly-siloxane matrix and this chemical bond prevents the CD chiral selector from migrating to different locations on the film surface, ensuring homogeneous enantio-selectivity throughout the GC capillary coating43. The new Mars mission, the Curiosity Lab of NASA carries an analytical unit called Sample Analysis at Mars (SAM). This miniature analytical lab is designed to measure abundances of gases associated with life, such as hydrogen, nitrogen oxygen, methane, water vapor and carbon dioxide. The analysis unit consists of several spectrometers and a GC-MS unit, where six different GC capillary columns are employed. One of these columns is a 2,6-di-Opentyl-3-O-trifluoroacetyl-βCD based Chira-Sil-DexTM capillary GC column which is supposed to determine enantiomer ratios of chiral compounds isolated from Martian samples. The relative abundances of gases and eventual chirality of compounds in the samples will be an essential piece of information in the evaluation whether the Martian conditions could have supported life in the past or at present. The suitability and robustness of CD-based chiral GC columns employed in the NASA SAM ACS Paragon Plus Environment


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lab unit, seem a valuable proof of the practical utility of CD-based enantiomer separation even under extreme conditions44. Cyclodextrin-mediated separations by HPLC Cyclodextrins bound to the stationary phase: Two major enantio-selective retention mechanisms are proposed by Armstrong.35 In the “polar-organic eluent mode” the analyte is separated by a combination of H-bonding and dipolar interactions. While in the “reversedphase mode” the analyte retention is due to hydrophobic interactions and the enantiorecognition is related to H-bonding and steric interaction at “mouth” of CD cavity. Direct enantiomeric separation by chiral stationary phases in HPLC remains one of the most important techniques for the analysis of enantiomeric purity as well as – convenient way to produce enantiopure compounds. Although there are a number of CD-containing HPLC columns on the market (e.g. CyclobondTM, ChiraDexTM, NucleodexTM), improvements in the preparation chemistry of stationary phases may result in more efficient enantio-recognitions. The CDs are introduced by covalently linking them to the silica surface. The quality of such CD-“decorated” HPLC stationary phases depends on the particle size of the silica, on the nature of the chemical linkage between the silica and the CD, on the amount of free silanol groups and on the structure of the chemically modified CDs bound to the silica. The most suitable types of CDs useful in stationary phases are the mono-substituted CDs, and the single isomer, pure CD-derivatives having different substituents. The high isomeric purity enables homogeneous surface coating and even monomeric surface modification. CD stationary phases have been successfully used for the separation of a number of drug molecules in reversed-phase, normal-phase and polar organic phase mode45. Cyclodextrins applied in the mobile phase: Due to the limited aqueous solubility of parent CDs, in this HPLC separation mode mainly highly water-soluble CD derivatives are used. Single isomeric or statistically substituted hydrophilic CD-derivatives, however, have long been used as efficient selectors in the mobile phase of enantio-selective HPLC . There are two major types of analyte-CD interactions in chiral separations: inclusion complex formation and analyte CD surface interaction based “outer sphere” complexation.45 The complex formation-based separation is typical for reversed-phase HPLC separations in aqueous mobile phase, where the analyte isomer actually enters the chiral CD cavity and interacts with hydroxyls or substituents located at the cavity entrances (NH2-, -CH2COOH, etc). The surface analyte interaction mode occurs mainly in HPLC techniques relying on polar organic mobile phases. In this case, the analyte preferably interacts with the CD functional groups located on the rim, (e.g., hydroxyl-, hydroxylalkyl-, carboxymethyl-, etc.) groups. Advantages of having the CDs in the mobile phase include that the selector can be ACS Paragon Plus Environment

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easily varied and inexpensive HPLC columns can be applied.46 The disadvantage of HPLC methods with CD-enabled mobile phase is that the selector additives may disturb the detection (with UV and MS detectors), as relatively large amount of selectors are needed.

Cyclodextrins in Capillary Electrophoresis (CE) One of the most thoroughly studied and published field of CD-mediated separations is the capillary electrophoresis. In this quick and efficient separation method CDs are almost exclusively employed in the mobile phase (running buffers) 47,48,49. The most suitable types of CDs in CE are the neutral parent CDs and the methyl-, ethyl- and 2-hydroxypropylderivatives of α-, β- and γCD. Naturally, electrically charged CDs such as single-isomer (the family of sulfato-CDs, peralkyl-mono-amino-CDs, etc.) and statistically derivatized carboxyalkyl-CDs, phosphated and sulfated-CD derivatives, are even more efficient chiral separation agents 50 (see Figure 5). The highly efficient chiral selector CD-sulfates were introduced in the CE separations by Stalcup.51

O CH3 OH O

*

mAU 14

N CH3

NH

S

12

O 10 8 6 4 2 0 12

14

16

18

20

22

24

Time (min)

Figure 5. CE separation of first eluting D-isomer and second eluting L-isomer of dansylphenylalanine with permethyl-mono-amino-βCD-containing running buffer. 50

Combined, dual selector-containing running buffers have been reported to exhibit great enantio-selectivities in CE52. In these mobile phases electro-neutral CD methyl-ethers were combined with charged carboxyalkyl-CDs and permethyl-monoamino-CDs.53 Attempts have been made to further miniaturize and simplify the CE instruments for easy and quick on site analyses, such as forensic and environmental studies done by portable microfluidic CE devices. Some onsite detection and quantitation of illicit drugs (e.g. methyl-amphetamines, heroin, etc.) were made possible – by a CD-enabled portable CE setup54. Separation and ACS Paragon Plus Environment


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detection of acidic and neutral impurities in heroin samples were peformed by using an anionic sulfobutylether-βCD additive in the background electrolyte. Improved selectivity and sensitivity in detection was achieved by using photodiode array UV absorption and laserinduced fluorescence detection. The limit of detection by laser-induced fluorescence for some heroin impurities (e.g. acetyl-thebaol) was 500-times lower than by traditional UV detection, due to the presence of the CD additive55. The suitability of the CD-enabled electrophoresis method for miniaturization was one of the most important reasons why NASA engineers selected the CD-assisted CE instrument on designing the lab-on-a-chip type chirality detectors. When hunting for signs of life on Mars, the chirality of Martian soil samples was an important aspect of getting to know if there has ever been life on Mars. The Mars Organic Analyzer is a microchip CE device, where chiral recognition is provided by a γCD in CE background electrolyte56. Any sign of a detectable enantiomer excess in the Martian samples would indicate the possibility of enzymatic (lifeoriginated) formation of substances, whereas the detection of 1:1 enantiomer ratio would point to compounds not associated with life. The practical value of the CD-based CE separation micro-device was tested by conducting analyses under simulated Martian conditions in the Atacama desert in Chile. The Mars Organic Detector was found to be robust and suitable for the separation and quantitation of a number of polyaromatic hydrocarbons (such as 9,10-diphenylanthracene, anthracene, anthanthrene, fluoranthene, perylene, etc.) at ppm levels using running buffers with added selectors such as sulfobutylether-βCD and methylether-βCD.57

FUTURE APPLICATIONS After about 30 years of intense and successful research and development using CDs in analytical chiral separations, the translation of these analytical scale methods to the semipreparative and preparative scale remains a real challenge. The technical feasibility of a pragmatic and efficient enantiomer separation by CD-enabled chromatographic techniques is low due to the low capacity of CD-based columns and mobile phase. Instead, more and more emphasis has been put on the application of CD-enabled multiple extraction methods for g and kg- scales enantiomer separation. It is thought, that there are many opportunities for analytical chemists collaborating with extraction technologists to solve this challenge. In addition to the utilization of cyclodextrins in separation methods, there is a number of emerging applications aiming at the improvement of sensitivity of analyte detection based ACS Paragon Plus Environment

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on molecular recognition. Further miniaturization and automation of robust analytical instruments for in situ and continuous analyses of complex environmental, food, pharmaceutical and biological samples will certainly require the development of novel labon- a-chip type analytical instruments. The most recent direction of CD-enabled analytical applications is their use in stochastic sensing. A good example of such a future direction is the development of a continuous DNA sequencer/reader for individual human genome analysis using protein nanopore with covalently tagged cyclodextrin adaptor as analyterecognizing sensor.58 Another CD-enabled protein nanopore for the detection of trace amounts of organophosphorous nerve agents can be envisioned as a rapid and sensitive technique for on-site analysis in environmental monitoring at the single molecule level.59

ACKNOWLEDGEMENT The authors acknowledge the assistance of Mr. Milo Malanga in the graphical rendering of figures and Dr. Eva Fenyvesi in reviewing the manuscript. We thank professor Akos Vertes for valuable discussions and comments.

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Biography Lajos Szente is a founding member of CycloLab Cyclodextrin Research and Development Laboratories, Ltd. in Budapest, Hungary. He received his Ph.D. in bio-organic chemistry from the Eotvos Lorand University, in Budapest, Hungary. He has a 35-year experience in the field of supramolecular chemistry and cyclodextrin technology. His current research focuses on the utilization of cyclodextrin nano-cavities for therapeutic-, analyticaland diagnostic purposes. Julianna Szemán is the head of the HPLC analytical laboratory at CycloLab. Dr. Szemán received her Ph.D. from the Technical University of Budapest. She studies the analytical applications of cyclodextrins, the development of chromatographic methods for cyclodextrins, and cyclodextrin inclusion complexes and chiral separations.


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