Thin Layer Chromatography in Chiral Separations and Analysis

Teresa Kowalska, Joseph Sherma

• Publisher: CRC Press
• Year: 2007
• eBook ISBN: 9781000654318
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Thin layer chromatography (TLC) is well suited for performing enantioseparations for research as well as larger-scale applications. A fast, inexpensive, and versatile separation technique, there are many practical considerations that contribute to its effectiveness. Thin Layer Chromatography in Chiral Separations and Analysis is the first bo

1 Overview of the Field of Chiral TLC and Organization of the Book

Teresa Kowalska and Joseph Sherma

CONTENTS

• 1.1 Chirality Background
• 1.2 Basic Strategies of Chromatographic Enantioseparations
• 1.3 The Areas of Primary Demand for Enantioseparations
• 1.4 The Role of TLC
• 1.5 Organization of the Book
• 1.6 Epilogue
• References

1.1 Chirality Background

Chirality is a form of stereoisomerism that embraces the relationship between two or more isomers having the same structure (i.e., the same linkages between atoms) but with different configurations (spatial arrangements). Chiral compounds are further classified into enantiomers, that is, molecules that are mirror images of each other, and diastereoisomers, which are stereoisomers that are not mirror images.

An enantiomer is one of a pair of nonsuperimposable mirror image molecules. Two molecules are enantiomers if they are mirror images of each other that cannot be superimposed by any rotation or translation. Physical and chemical properties of the two enantiomers making a pair (also known as two antimers or antipodes) are almost identical, except for their optical property of rotating polarized light in opposite directions. The almost identical physical and chemical properties of the two antimers pose a very difficult task for all who, for one reason or another, need to separate them as two different species. In other words, enantioseparation is among the most difficult tasks in analytical chemistry.

Diastereoisomers are stereoisomers that have more than one center of asymmetry in their structure and are not enantiomers or mirror images of each other. Contrary to enantiomers, diastereoisomers can have different physical properties and reactivity. Because of greater differences in physical and chemical properties among diastereomers, separation and isolation of these isomeric compounds having the same structure but different spatial arrangement are relatively easier than with the enantiomers, but still a difficult task.

The phenomenon of chirality is omnipresent in nature, and its presence in humans, animals, and plants determines their chemical structure and also the majority of their living functions. One of the greatest, and so far unexplained, mysteries of biophysics is the predominance of homochirality among living organisms. According to the definition adopted by the International Union of Pure and Applied Chemistry (IUPAC), only a sample that contains all molecules of the same chirality type can be considered as homochiral (of course, within the limits of the available detection sensitivity). The most striking manifestation of homochirality in nature is that human and animal organisms are built exclusively of the L-amino acids (i.e., the left-handed form) and of the D-carbohydrates (i.e., the right-handed form).

This biophysical puzzle spans many different areas of the natural and life sciences, but in a certain sense it also poses an important question for philosophy and even religion. Why do we not encounter on earth “the life reflected in the mirror,” and why do we not encounter our own antimeric “quasi-twins?” The simplest scientific and also philosophical question related to homochirality can be formulated in this way: Is homochirality an inevitable precursor of the organic life on our planet, or, to the contrary, can we consider homochirality as a free play of the forces of nature and a purely random phenomenon? At this point, one starts raising questions about the origin of life on earth and comes very close not only to theoretical organic geochemistry and astrobiology but also to the domain of religion.

Due to the omnipresence of chirality in nature, the majority of metabolic processes occurring in living organisms are stereospecific. It is a well-recognized fact that the biological catalysts known as enzymes have asymmetric active centers to properly fit in biological receptors that are also asymmetric. In this sense, homochirality, even if we do not understand its origin, seems to reflect the wisdom of nature and its great economy. It undoubtedly leads to a massive gain in time and energy, because living organisms do not need to select the properly handed substrates from a pool of the left- and right-handed ones at each individual metabolic step.

1.2 Basic Strategies of Chromatographic Enantioseparations

Chromatographic separation of two antimers, most often referred to as enantioseparation, can be carried out following either a direct or an indirect strategy. It is perhaps noteworthy that this duality of alternative options is characteristic not only of enantioseparations by means of thin layer chromatography (TLC), which is the primary subject of this book, but also of high-performance liquid chromatography (HPLC) and gas chromatography (GC).

The first strategy, known as direct separation, consists of introducing to the given chromatographic system a mixture of the two enantiomers without any preprocessing, that is, without derivatization, prior to the chromatographic run. It is a widely assumed conviction (and in most cases a true one) that direct separation can only be obtained in a chiral chromatographic system. Each chromatographic system is composed of a stationary phase and a mobile phase. Chiral chromatographic systems are most frequently composed of either a chiral stationary phase (CSP) with a nonchiral mobile phase or vice versa. Chromatographic systems composed of two chiral phases are avoided, one reason being that the optically pure stereoisomers are costly and especially because one chiral phase is sufficient. In HPLC, chromatographic systems with CSPs are more frequently applied than those with chiral mobile phases. An important reason is that chiral columns can be reused for many consecutive analyses, whereas the expensive chiral mobile phase modifiers often cannot be purified and reused. Chiral separations by means of TLC in many cases follow in the conceptual footsteps of successful, practical solutions elaborated earlier for HPLC, and this is one reason why applications of CSPs are more frequent than those of mobile phases containing chiral modifiers, even though both TLC plates and mobile phases are used only once.

The second strategy for separating enantiomer pairs is indirect separation. In this case, a mixture of the antipodes is derivatized with a chiral agent prior to chromatography in an appropriate system to give a respective mixture of diastereoisomers. Chemical structures of diastereoisomers and their resulting physical and chemical properties are much more differentiated than in the case of a pair of original enantiomers, and the separation of diastereoisomers is, therefore, a considerably easier experimental task than the separation of the corresponding enantiomers. In fact, chromatographic separation of diastereoisomers can be obtained in a chromatographic system composed of an achiral stationary phase and an achiral mobile phase. From a purely chromatographic point of view, indirect separation of the enantiomers can be obtained in much simpler and also less expensive chromatographic systems than those required for direct separation. On the other hand, preliminary derivatization of an enantiomer mixture can often prove to be a relatively complex and time-consuming step that many analysts would rather prefer to avoid. This book covers all the important aspects of both direct and indirect enantioseparations by means of TLC.

1.3 The Areas of Primary Demand for Enantioseparations

The first strong impulse to seriously consider the development of small- and large-scale separations of enantiomers as an important and urgent research task came from the pharmaceutical field, initiated by the infamous case of introducing to the market the racemic sedative drug known by the name thalidomide. Thalidomide was first introduced in Germany (and later in other countries, mostly Western European) on October 1, 1957, by the German pharmaceutical firm, Chemie Grünenthal. The drug was particularly advertised as an effective and safe sedative to pregnant women. At that time, nobody was aware of the fact that one drug enantiomer from a given pair can exert a positive therapeutic effect and its antipode can prove to be ineffective or even dangerous and harmful. This was precisely the case with thalidomide. One enantiomer acted as a very efficient sedative, whereas its antipode was strongly teratogenic. As a result, thousands of seriously crippled babies with underdeveloped limbs were born in many countries, since then known as generation of “thalidomide babies” or “flipper babies.”

The dramatic effect of thalidomide on human fetuses is still considered as one of the greatest mistakes ever committed in the history of modern pharmacy. However, it also served as the starting point of a massive research effort in the field of chromatographic enantioseparations. Perhaps, it is also worth mentioning that this once feared drug is now being rediscovered and is again in use [1]. In 1964, a physician treating leprosy patients in Israel for a painful condition known as erythema nodosum leprosum (ENL) prescribed thalidomide as a sedative. Surprisingly, the drug alleviated the symptoms of this painful condition. From that point onward, thalidomide was the therapy of choice for this application, including designation by the World Health Organization (WHO).

Moreover, thalidomide’s effects on the rapidly dividing cells of embryos suggested that it might destroy cancer cells. What was unique about thalidomide was its powerful teratogenicity that could be related to anticancer effects. Now, it is considered that thalidomide’s potential applications are essentially limitless. It can be used, under an emergency U.S. Food and Drug Administration (FDA) approval, to treat more than 70 forms of cancer and various skin, digestive, and immunological diseases. It may also be useful in treating the autoimmune deficiency syndrome (AIDS)-associated cachexia (wasting) and tuberculosis.

Even from this single example of thalidomide and the diverse biological effects and curing potentials of its two antimers, it can easily be deduced that enantioseparations are crucial in human (and also veterinary) pharmacy and medicine. In the present period of dynamic development of advanced computer-aided strategies for molecular drug design, the number of newly devised molecular structures with the anticipated curing potential is growing quickly. Many of these compounds have an asymmetric structure and, hence, they can appear in both left- and right-handed forms. From the pharmacokinetic point of view, each of the two antipodes has to be treated as a completely different compound that is able to exert a unique effect on living organisms, which has to be assessed at a very early stage of investigation of the prospective drug. To carry out the preliminary biological and other tests with each individual enantiomer, efficient working tools that enable the separation and evaluation of the quantitative proportions between the two antimers in a reaction mixture, followed by their ultimate preparative isolation, are needed.

The search for drugs that are safe from a toxicological point of view makes investigations of the analysis of enantiomeric antipodes and their biological activity among the most important tasks in a long and meticulous sequence of steps leading from the computer-aided molecular drug design to the implementation of a successful pharmaceutical product on the drug market. However, there is one field of scientific activity, in a superficial way rather similar to computer-aided molecular drug design, which is focused on selection of the compounds with a properly tailored toxic potential. The chemometric strategies elaborated for drug design have been widely and successfully adopted in the search for efficient candidate pesticide compounds [2,3]. Also, in this case, many chemometrically devised and then synthesized substances are asymmetric, and biological activity of each individual antimer from a given pair has to be carefully examined. The principal objective is selection of the enantiomers with well-balanced biochemical properties, combining a possibly low-toxic potential toward humans with a possibly high-toxic potential toward the target organisms to be destroyed. It is a well-known fact that such target organisms (plants, when we consider herbicides, insects for insecticides, etc.) most often play a significant role in the interspecies nutrition chain, and through this chain they can relatively easily be transported by ingestion to humans. Another route for the dangerous migration of pesticides to humans is through natural precipitation (i.e., rain and snow) that is able to carry them into reservoirs of potable water (i.e., underground water, rivers, lakes, artificial reservoirs, etc.).

Chirality studies also proved very important for the dating of the organic fossils, and, hence, fortified archeology, geoarcheology, paleontobiology, and the related fields of knowledge with a very well-performing diagnostic tool based on naturally occurring homochirality. The principle is that all amino acids except glycine (the simplest amino acid that lacks an asymmetric carbon atom in its structure) are chiral, and in living human and animal bodies, they appear exclusively in the left-handed form. At the moment of biological death of the organism, the spontaneous racemization of the L-amino acids commences, with continuously growing content of the right-handed antimer and corresponding diminishing content of the left-handed one. It has not been difficult to experimentally determine the kinetic parameters (i.e., half-times and rate constants) of the spontaneous racemization with selected amino acids, depending on the external physical parameters of running this process (e.g., temperature, pH, pressure, reaction medium, etc.). This knowledge enabled elaboration of the method of dating the archeological findings, based on the degree of the spontaneous racemization of the amino acids contained in the organic fossils. The first amino acid selected to serve the purpose of a natural age marker was L-aspartic acid, and the first article on this subject was published in 1980 in the journal Nature [4]. It originated from the leading research group investigation on chirality and homochirality, and it is still considered as a momentous breakthrough in the field of dating archeological findings. The article [4] deals with the dating of the Dead Sea ...

Table of contents

• Cover
• Half Title
• Series Page
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Editors
• Contributors
• Chapter 1 Overview of the Field of Chiral TLC and Organization of the Book
• Chapter 2 Chirality
• Chapter 3 Commercial Precoated Layers for Enantiomer Separations and Analysis
• Chapter 4 Planar Chromatographic Enantioseparations on Noncommercial CSPs
• Chapter 5 Planar Chromatography Enantioseparations on Noncommercial CCSPs
• Chapter 6 Chiral Mobile Phase Additives
• Chapter 7 An Overview of the Chiral Separation Mechanisms
• Chapter 8 Separation of Diastereoisomers by Means of TLC
• Chapter 9 Selected Bottlenecks of Densitometric Detection with Chiral Analytes
• Chapter 10 Chirality of Pharmaceutical Products
• Chapter 11 Chiral Separation of β-Adrenergic Antagonists
• Chapter 12 Chiral Separation of Amino Acid Enantiomers
• Chapter 13 Chiral Separation of Nonsteroidal Anti-Inflammatory Drugs
• Chapter 14 Determination of Components in Selected Chiral Drugs
• Chapter 15 Chiral Separations Using Marfey’s Reagent
• Index