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THIN-LAYER CHROMATOGRAPHY | Overview☆
THIN-LAYER CHROMATOGRAPHY | Overview☆ E Reich and V Maire-Widmer, CAMAG Laboratory, Muttenz, Switzerland ã 2013 Elsevier Inc. All rights reserved.
Introduction Definition and General Description Historical Development Basic Concepts Principles of Separation Describing the Result Influence of the Developing Chamber Practical Approaches Sample Application Chromatogram Development Visualization and Derivatization Densitometric Evaluation Documentation Special and Hyphenated Techniques High-Performance Thin-Layer Chromatography Automated Multiple Development Forced-Flow Techniques Hyphenated Techniques Applications
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Introduction This article serves as an overview covering definition, historical development, basic principles, and advantages of thin-layer chromatography (TLC) in comparison to column chromatography. It concludes with a summary of common applications. Details on theory, instrumentation, plate technology, and method development are covered in subsequent articles.
Definition and General Description TLC is a form of liquid chromatography. It is used for rapid separation of samples into components based on differences in their retention behavior. The stationary phase, usually an adsorbent such as silica gel, is coated onto a rectangular support such as a glass, aluminum, or plastic plate as a thin layer. Liquid samples are applied as spots or bands onto the dry stationary phase forming a line close to one edge of the plate. The TLC plate is placed vertically in a chromatographic chamber with lid, containing an amount of the liquid mobile phase sufficient to cover the bottom of the chamber, but not reaching up to the application position of samples on the plate (Figure 1). The mobile phase is drawn through the layer by capillary action starting separation when reaching the applied samples (start position). When the front of the mobile phase has moved to a certain predefined height (developing distance) the plate is removed from the chamber, appropriately dried, and, if necessary, derivatized for detection of samples. The resulting chromatogram is evaluated qualitatively by visual or densitometric comparison of the migration distance of the separated components to those of reference standards analyzed simultaneously on the same plate. Often, the behavior of substances during derivatization is also observed. Quantitative determination is possible by comparison of the intensity of the separated zones to those of known standard concentrations. TLC is a very popular chromatographic technique because it is experimentally simple, enormously flexible, and inexpensive. It can lead rapidly to reliable qualitative and quantitative results primarily based on visual impression. TLC is an offline technique (Figure 2). An analysis is performed as a sequence of individual steps, which are independent with respect to time and location. Compared to chromatography in columns, which is an online process, this design does not only offer great flexibility for technical solutions but also almost unlimited possibilities for combination of parameters to optimize the chromatographic result. In TLC, a large number of samples can be analyzed and compared to one another simultaneously on the same plate. The actual chromatographic separation provides exactly the same conditions for samples and references and there is no
☆
Change History: May 2013. E Reich made changes in section ‘Hyphenated Techniques’.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.00540-0
1
2
THIN-LAYER CHROMATOGRAPHY | Overview
Expected developing distance
Z0
Figure 1 Schematic description of the development of a TLC plate. Z0 is the distance between the application position and the level of the developing solvent. After the mobile phase has reached the desired distance, the plate is removed from the tank.
danger of permanently contaminating the system by components of the sample’s matrix because each plate is used only once. After separation, the TLC plate functions as a storage device for the chromatogram and thus enables multiple detection/evaluation. For all steps of the TLC process modern instruments are on the market, ensuring reproducible results at various levels of sophistication. As an open system the TLC plate is easily affected by environmental factors such as humidity, fumes, light, and mechanical stress during handling. To ensure reproducibility of results this must be taken into account when designing and performing a TLC experiment.
Historical Development At the end of the 1930s, adsorption chromatography in columns as introduced by Tswett had become a powerful separation technique for plant extracts and natural products. Simultaneously, the need for a more rapid alternative suitable for identification of separated substances led to the invention of an open chromatographic system. In 1938, Izmailov and Shraiber reported the separation of belladonna alkaloids on a thin adsorbent layer, coated onto microscopic slides. Development of circular chromatograms was achieved by placing small amounts of various solvents to the center of samples previously applied as spots onto the layer. This method was an extremely rapid microtechnique requiring only small amounts of stationary and mobile phases. In 1944, Consden, Gordon, and Martin took a different approach using the principle of partitioning. This resulted in the invention of paper chromatography, which soon became a universal chromatographic technique. TLC did not advance much further until the 1950s when several significant improvements were made. Kirchner, Miller, and Keller in 1951 incorporated a fluorescent indicator into the stationary phase. Silica gel was becoming the most widely used adsorbent. In 1956, Stahl introduced the term ‘thin-layer chromatography’. This, together with Merck developing standardized aluminum oxide, kieselguhr, and silica gel based on Stahl’s specifications and Desaga bringing a basic instrument kit to the market, manifested the birth of TLC as a recognized and broadly accepted analytical technique. In 1962, Stahl with his fundamental book, Thin Layer Chromatography – A
THIN-LAYER CHROMATOGRAPHY | Overview
3
Application
Development
Derivatization
Detection Documentation
Evaluation
Report
Figure 2 Steps of TLC as an offline technique.
Laboratory Handbook, gave a forum to the leading researchers in the field of TLC and summarized the knowledge of that time. As a standard analytical technique, TLC was soon widely adopted by pharmacopoeias and official method collections. The improvement of plate materials, culminating in precoated high-performance layers of reproducible quality in the late 1970s, the rapid development of the theoretical foundation, and the appearance of powerful densitometers became the basis for reliable quantitative analyses by TLC. At that time, high-performance liquid chromatography (HPLC) evolved and began to compete with TLC as a quantitative technique. The 1980s saw remarkable improvements particularly in instrumentation, automation of individual TLC steps, and theoretical concepts contributed by Snyder, Kaiser, Ebel, and Geiss. In 1987, Geiss published Fundamentals of Thin Layer Chromatography, which is regarded as one of the most influential books on the subject. The Journal of Planar Chromatography was founded in 1988 as a platform for discussion of all aspects of modern TLC. Aside from the traditional way of performing TLC, several other approaches have also been taken. They include forced-flow techniques using centrifugal force, a pump (over-pressured layer chromatography, OPLC), or an electrical field to move the mobile phase. Hyphenated techniques were introduced combining TLC separation with infrared, Raman, or mass spectrometric detection. A technique for analysis of analytes that can be evaporated (IATROSCAN) utilizes separation performed on the surface of reusable thin chromatographic rods combined with a flame ionization detector. Also, gradient techniques have been developed for TLC, the most powerful of which is AMD – automatic multiple development – patented by Burger. Today, TLC is a mature technique that meets all requirements of a modern analytical tool. Although it has lost its dominant role as a routine procedure to HPLC due to the lack of full automation, it is still indispensable for rapid qualitative analyses (identification), as a screening tool, and as a complementary technique in research and development.
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THIN-LAYER CHROMATOGRAPHY | Overview
Basic Concepts Principles of Separation The stationary phase in TLC is usually an adsorbent (Table 1) composed of very fine and highly porous particles coated as a thin layer onto a support. Today, the most widely used stationary phase is silica gel 60. With a solvent or solvent mixture as mobile phase, a liquid–solid chromatographic system is formed. Separation of sample components can be primarily described as adsorption chromatography. Sample molecules are retained due to specific interaction on the surface of the stationary phase while migration takes place when the molecules are dissolved in the mobile phase. A dynamic adsorption equilibrium is established for each sample component. The equilibrium is characterized by a partition coefficient KA representing the ratio of concentration of the sample component A in the stationary phase (cs) and in the mobile phase (cm); KA ¼ cs/cm. Separation of two samples A and B can only be achieved if their partition coefficients are different. Typical forces causing adsorption of molecules on the stationary phase include nonspecific dispersion forces, dipole–dipole interactions, acid–base interactions, hydrogen bonding, electrostatic interaction between ions, and others. There are two extreme cases. If the sample is strongly adsorbed and only sparingly soluble in the mobile phase it will remain at the application position. Weakly adsorbed and readily soluble samples will migrate with or close to the solvent front. A suitable combination of stationary and mobile phase will result in a separation of sample components. They will be located at different migration positions when the development of the TLC plate is interrupted as soon as the mobile phase has reached the specified developing distance. The ability of a solvent to displace an adsorbed sample molecule from the surface of a given stationary phase is called solvent strength. It can be measured as energy that is released when a solvent molecule is adsorbed. Although it is not simply a measure of dipole moment, solvent strength in adsorption chromatography is often expressed as solvent polarity. Adjusting the solvent strength of the mobile phase affects the position of the sample components in the chromatogram. The quality of separation, expressed as resolution between zones of the chromatogram, depends on the selectivity of the TLC system. Adjusting the composition of the mobile phase is the primary way of changing selectivity. Apart from adsorption phenomena, a variety of other separation mechanisms can be utilized. If chemically bonded phases or liquid stationary phases on an inert support are used as stationary phase, separation is predominantly based on partition equilibria. Examples include octadecyl, aminopropyl, diol, cyanopropyl modified silica gel, impregnated silica gel, and cellulose or kieselguhr. Separations based on ion exchange, complex formation, size exclusion, and even chiral recognition are also performed.
Describing the Result In column chromatography, the chromatographic system has a fixed length. Each sample component can be characterized by the time it requires to pass through the column and reach the detector. This is the retention time tR and it is measured at the peak maximum. In TLC, the analysis takes a fixed time (development time) during which sample components can migrate. Depending on its retention each sample component will reach a specific migration distance (MD) and remains there as a zone (spot) for detection when the TLC plate is dried (Figure 3). A compound with high MD would in comparison have a short retention time in column chromatography, provided the separation mechanism is the same. The MD of a zone is measured in millimeters from the application position to the point of highest concentration (intensity). When the chromatogram is evaluated densitometrically this point represents the peak maximum. As retention time is dependent on the mobile phase velocity, the migration distance is dependent on the position of the mobile phase front zf. Therefore, a relative measure has been introduced. The RF value (retardation factor) of a zone is the ratio of its migration distance to that of the mobile phase front. RF ¼ MD/zf. RF values are always <1. For convenience it is common to multiply the RF value by 100 and report hRF with two digits. It is possible to compare the migration distance of an unknown (A) to that of a reference compound (ref) to yield the Rrel value. Rrel ¼ MDA/MDref. Although the RF value is characteristic for a substance in a given TLC system, it must be treated with caution because it is affected by several parameters. In practice, it is often difficult to reproduce RF values exactly. The Rrel value has no physical meaning. The principle of qualitative analysis is a comparison of RF values obtained from samples and standards on the same TLC plate. If two substances are the same they will have the same RF. However, different substances may also migrate to the same position. For further confirmation of identity a specific derivatization or recording of the ultraviolet (UV) spectrum of the substances on the plate can be utilized. The size and intensity of the zone of the analyte is visually compared for estimation of quantity to that of standards at several known levels on the same plate. A precise quantitation is possible by scanning or video densitometry by which Table 1
Important inorganic and organic adsorbents
Inorganic
Organic
Bonded phases on silica gel
Silica gel Aluminia Kieselguhr (diatomaceous earth) Magnesium silicate
Cellulose Polyamide Sephadex
Octadecyl-, octyl-, dimethyl-, diphenylsilanized Aminopropylsilanized Cyanopropylsilanized Diol (propanediol)
THIN-LAYER CHROMATOGRAPHY | Overview
5
A
ref.
Zf
MDA MDref.
Figure 3 Schematic presentation of result in TLC. MD, migration distance; zf, distance between application and front; RF ¼ MD/zf.
WB1 DZ WB2
Figure 4 The resolution (Rs) between two separated peaks is calculated from the chromatogram: Rs ¼ 2DZ/WB1 þ WB2.
the absorption or fluorescence of separated zones of each chromatogram track is recorded. The resulting analog curves are integrated and evaluated based on peak height or area. The fundamental requirements for reliable quantitation are baseline resolved zones and symmetric peaks for the compounds in question. The resolution between two zones or peaks can be calculated from the chromatogram (Figure 4).
Influence of the Developing Chamber In TLC, the chromatographic system consists of a plate coated with the stationary phase and a developing chamber containing the mobile phase. As seen in Figure 5, a gas phase consisting of components of evaporated solvent molecules is also present. It is a matter of time, geometry, and whether fitting the chamber with filter paper enlarges the surface of the developing solvent, but as long as the chamber is tightly closed, saturation is eventually established.
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THIN-LAYER CHROMATOGRAPHY | Overview
Figure 5 The composition of the gas phase is affected by: (1) equilibrium of gas phase with developing solvent on the bottom of the chamber (saturation); (2) and (3) equilibrium between gas phase and stationary phase ((2) absorption of gaseous molecules, (3) evaporation of mobile phase).
This means that the vapor in the gas phase is in equilibrium (1) with the developing solvent on the bottom of the chamber. When the dry TLC plate is placed in the chamber solvent vapor is readily adsorbed onto the stationary phase. If the stationary phase has no contact with the developing solvent, as it is possible in a twin trough chamber, over time another equilibrium (2) between the gas phase and the stationary phase is established. This is called preconditioning. It leads to adsorptive saturation of the stationary phase. When the mobile phase rises up the stationary phase a third equilibrium (3) is approached. While (1) is dependent on the vapor pressure of the solvent components and (2) is controlled by adsorptive forces, (3) is affected by both parameters. During chromatography all three equilibria are effectively influencing each other as well as the chromatographic result. Experimental details, type, and geometry of the chamber are therefore important parameters that can be used to optimize separation. Even in so-called saturated chambers chromatography is typically performed in nonequilibrium, which makes it difficult to mathematically describe all processes in exact detail. Generally, there are neither good nor bad chambers; however, results obtained in one may be quite different from those obtained in another chamber. In consequence, it is imperative for obtaining reproducible results that the chamber and all of its parameters are clearly defined and kept constant. As a rule of thumb saturated chambers tend to give lower RF values for the same separation than unsaturated chambers. The zones in saturated chambers are somewhat more diffuse, but reproducibility of the result is much higher than in unsaturated chambers. TLC can also be performed in sandwich chambers, where covering the stationary phase with a counterplate limits the gas phase. As a result of this the stationary phase remains active and cannot be preloaded with solvent molecules. The mobile phase is then separated into its components. So-called secondary fronts are formed and can interfere with the separation. In saturated chambers secondary fronts
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THIN-LAYER CHROMATOGRAPHY | Overview
are usually eliminated because the polar components of the mobile phase are preferably preadsorbed through the gas phase. Secondary fronts are one of the principal drawbacks of OPLC where the gas phase is completely eliminated.
Practical Approaches Sample Application During sample application two principal requirements must be met. The samples must be precisely and correctly positioned in order to identify separated components based on their migration distance/RF values and the sample volume must be controlled in order to allow quantitative evaluation of the chromatogram. The size of the plate determines the number of samples. Samples should not be applied too close to the edges of the plate and must be evenly and sufficiently spaced for best results. Typical application parameters for high-performance thin-layer chromatography (HPTLC) plates are summarized in Table 2. Application of samples can be performed manually or with the help of instruments. For manual application the positions are carefully marked on the TLC plate with soft pencil. Use of a spotting guide helps avoiding any damage to the layer. Samples are transferred with the help of disposable capillaries, graduated micropipettes, or microliter syringes and deposited onto the plate as spots. If larger volumes are applied this is done in portions with intermediate drying of the application position. The smaller the applied spot, the better is the obtainable resolution of the chromatogram. Best results in sample application are obtained with instruments, particularly those that use the spray-on technique. For maximum resolution and best precision in quantitative TLC, samples are sprayed on as narrow bands. Following sample application it is common to mark the desired developing distance on one edge of the plate.
Chromatogram Development Chromatogram development is performed in a chromatographic chamber, often called a tank. There are many different kinds of chambers, but flat bottom or twin trough chambers with a rectangular base are most common. The size of the chamber should be appropriate for the size of the plate. A saturated chamber is created, when one or more walls of the chamber are fitted with filter paper, which is thoroughly wetted with the developing solvent. For saturation to establish, the charged chamber is left closed for a defined period of time prior to introducing the TLC plate and starting development. Unsaturated chambers do not contain filter paper. Chromatography is started immediately after the chamber is charged with developing solvent. The level of solvent in the chamber is chosen in a way that the sample application position on the plate is clearly above it. Most plates are developed in a saturated chamber in vertical position, but horizontal developing chambers are also available (Figure 6). With special chambers circular and anticircular development can also be performed. For circular development samples are applied to a square plate around the center forming a circle. The mobile phase is applied to the center of the plate. Anticircular development requires delivery of mobile phase from a ring gap to the plate onto which samples have been applied forming a circle of large diameter. All developments discussed so far were single developments. Multiple developments are also possible. After intermediate drying of the plate a subsequent development can be performed (1) over the same, (2) over a longer, and (3) over a shorter developing distance. While in case (1) the goal is to improve separation by using the same mobile phase for development, in case (2) the first development over a few millimeters with a strong mobile phase is typically used to concentrate the sample into a narrow application zone. The second development with a suitable mobile phase is used for separation. In case (3), a nonpolar matrix can be removed from the sample and transported to the upper edge of the plate with a weak mobile phase leaving the compounds of interest at the application position. In the second development with a stronger mobile phase the actual separation of the sample is performed. When the mobile phase has reached the desired developing distance of typically 6 cm on HPTLC plates and 12 cm on TLC plates development is interrupted, the plate is removed from the chamber, and dried. Table 2
Typical parameters for sample application on HPTLC plates
Parameter
Band
Spot
Distance from lower edge of plate in mm Minimum distance from left and right edge of plate in mm Minimum space in mm between bands/spotsa in mm Band length in mm Maximum diameter of application spot in mm
8 10 2 8
10 10 2
a
4
Most instruments do not allow programming of distance between bands/spots. Therefore distance between tracks (center to center) and band length must be chosen in order to meet the minimum distance requirement. For spot application, volume and application speed have to be determined empirically.
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THIN-LAYER CHROMATOGRAPHY | Overview
Figure 6 Comparison of flat-bottom chamber (a), twin trough chamber (b), and horizontal developing chamber (c): (1) HPTLC plate, (2) glass plate for sandwich configuration, (3) reservoir for developing solvent, (4) glass strip, (5) cover plate, (6) conditioning tray. Examples: The flat-bottom chamber is lined with filter paper soaked with the developing solvent for saturation. Twin trough chambers can be filled with two different liquids and are less solvent-consuming. Horizontal developing chambers are easy to use in sandwich configuration.
Visualization and Derivatization One of the most striking features of TLC is the possibility of presenting the chromatographic result visually. Visual detection is possible when the separated sample components are colored, have native fluorescence, or absorb UV light so that they quench the fluorescence of the indicator, which can be built into the layer of the TLC plate. Derivatization can enhance visualization dramatically by improving the detectability of all or selected zones of the chromatogram. Numerous chemical reactions are known to convert the analyte into a colored or fluorescing derivative. Nonspecific reactions such as charring with acid are broadly applicable, whereas specific derivatization such as the reaction of amines with ninhydrin can be used to single out compounds of certain functionality from a separated mixture. The convenient availability of biochemical reactions and in situ biological tests add extra flexibility to the detection step in TLC. Examples include inhibition of an enzyme such as choline esterase or measurement of effects on the growth of bacteria or yeast cells. For chemical derivatization, the developed TLC plate is either sprayed with or immersed into a solution of the reagent. While spraying requires small amounts of reagent and provides great flexibility the advantage of immersion lies in ensuring a homogenous and reproducible coverage. In many cases derivatization is completed with a heating step using either an oven or a plate heater.
Densitometric Evaluation If a separated substance absorbs UV or white light or can be excited to fluoresce, densitometric evaluation of the chromatogram is possible without derivatization. With a densitometer, also called scanner, the tracks of the TLC plate are evaluated by registering the reflected/emitted light when a narrow beam of light of a single wavelength is moved across the plate. A photomultiplier converts
THIN-LAYER CHROMATOGRAPHY | Overview
9
the light into an electrical signal, which as a function of migration distance presents the analog curve of the chromatogram. Following integration of the raw data the peak height and/or area can be quantitated. Densitometry is a very sensitive measurement. Typically less than 100 ng of substance per zone can be detected. In some cases, such as aflatoxins, detection limits are in the picogram range. Most modern densitometers allow recording of in situ UV spectra, which can be used to confirm identity or purity of separated compounds. Densitometry is performed after derivatization, if that improves the detectability of the analyte. Examples include introduction of fluorophoric groups, charring of nonabsorbing compounds, specific color reactions.
Documentation Documentation of a TLC chromatogram as an image is conveniently possible. Traditionally photographs have been taken but the availability of electronic devices such as flatbed scanners or digital cameras have simplified the process. The principal advantage of electronic images is their durability. Added benefit is the possibility of qualitative and quantitative densitometric evaluation.
Special and Hyphenated Techniques High-Performance Thin-Layer Chromatography HPTLC is based on the use of special layers made from narrowly distributed fine particles of 5 mm. It is actually not a special technique because all fundamental parameters, theoretical considerations, and practical aspects of classical TLC still apply. However, due to the consequent use of specialized instrumentation HPTLC does not only achieve miniaturization of the chromatogram, but also and more importantly a significant improvement of sensitivity, reproducibility, and separation power. A modern HPTLC workstation including devices for each individual chromatographic step is software controlled and provides good manufacturing or good laboratory practice compliant results similar to that of HPLC and GC instruments.
Automated Multiple Development Automated multiple development (AMD) is a gradient technique consisting of a variable number of developments with intermediate drying steps, which are achieved by application of vacuum to the special developing chamber. In a computer-controlled method, each subsequent development proceeds to a higher developing distance using increments of 2–3 mm than the previous step. Furthermore, each run uses a weaker (in adsorption chromatography less polar) mobile phase. As a consequence, the separated zones are repeatedly focused. In combination with the mobile phase gradient this results in a significant increase of the separation power. Results obtained with AMD are predictable and very reproducible.
Forced-Flow Techniques The separation efficiency of TLC is limited due to the fact that capillary forces move the mobile phase. Forced-flow techniques are an attempt to solve this problem. One approach, rotational planar chromatography, utilizes centrifugal forces and has become a widely used preparative technique. A circular plate is mounted on a centrifuge and the sample followed by the mobile phase is applied close to the center of the plate. Separated sample components can be collected when they elute from the rotating plate. Another very flexible forced-flow technique is OPLC introduced by Tyihak and Mincsovics. The plate is completely sealed on all four edges. Following sample application the plate is covered with a Teflon foil, which is part of a cassette. In a special pressure chamber the cover foil seals the plate in vertical direction under external pressure. With the help of a programmable pump the mobile phase is forced with constant velocity through an inlet in the coversheet of the cassette to travel in a laminar flow through the chromatographic layer toward an outlet located at the opposite edge of the plate. The two principal drawbacks of the technique, which can considerably interfere with the separation and must therefore be minimized in the experimental setup, are solvent demixing resulting in secondary fronts when multicomponent mobile phases are employed and a so-called front of total wetness. This is a disturbance at the region where gas bubbles, which are trapped in the pores of the adsorbent when the mobile phase first enters the layer, are finally displaced by solvent molecules. OPLC has found various analytical, semipreparative, and preparative applications. The system can be operated in an online mode like HPLC, offline mode like regular TLC, or in a mixed mode.
Hyphenated Techniques Several so-called hyphenated techniques have been developed, where the developed TLC plate is transferred to a modified spectrometer to record in situ the Fourier transform infrared, surface enhanced Raman, or mass spectra of the separated zones. This way more detailed structural information can be obtained to complement the data from densitometric evaluation. A true hyphenation is the direct application of the eluate from a microbore HPLC column onto an HPTLC plate, which is then developed by AMD.
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THIN-LAYER CHROMATOGRAPHY | Overview
Table 3
Major applications of TLC
Sample type
Application
Pharmaceutical
Impurities in synthetic drugs Stability tests of synthetic drugs Content uniformity Pharmacokinetic studies and drug monitoring Assay Enantiomeric purity Product uniformity Impurity determination Surfactants, dyes Cleaning validation Identity, purity, and stability tests of plant drugs Quantitation of marker compounds Organic acids, lipids Carbohydrates Porphyrins and bile pigments Amino acids, peptides Steroids, doping Mycotoxins (incl. aflatoxins) Drug and pesticide residues Antioxidants, preservatives Pigments, dyes, spices, flavors, vitamins Pesticides residues Water and soil analysis Poisons Drugs of abuse Inks
Industrial
Herbal Biomedical
Nutritional
Environmental Forensic
For further investigation by mass spectrometry (MS) separated sample components can either be eluted by a commercially available TLC-MS interface or analyzed directly on the plate by DART (direct analysis in real time) or DESI (desorption electrospray ionization) techniques. Also commercially available is an instrument for MALDI (matrix assisted laser desorption/ionization) MS.
Applications Typical applications of TLC are listed in Table 3. In many areas including herbal, pharmaceutical, and environmental analyses the flexibility of the technique as a research tool is a great asset. Other applications such as content uniformity, residue, and doping test as well as cleaning validation take advantage of short analysis times due to parallel chromatography of many samples. Qualitatively, TLC is primarily used in general research and in quality control of raw materials for rapid and cost-efficient identification. Quantitative applications include assays, determination of impurities and process monitoring.
Further Reading 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Frey, H. P.; Zieloff, K. Qualitative und Quantitative Du¨nnschicht-Chromatographie; VCH Verlagsgesellschaft mbH: Weinheim, 1993. Geiss, F. Fundamentals of Thin Layer Chromatography; Alfred Hu¨thig Verlag: Heidelberg, 1987. Hahn-Deinstrop, E. Applied Thin-Layer Chromatography; Wiley-VCH: Weinheim, 1998. Pachaly, P. DC-Atlas. Wissenschaftliche Verlagsgesellschaft: Stuttgart, 1991. Poole, C. F.; Poole, S. K. The Essence of Chromatography; Elsevier: Amsterdam, 2003. Sherma, J.; Fried, B., Eds.; Thin-Layer Chromatography, Techniques and Applications; 3rd ed.; Dekker: New York, 1994. Sherma, J.; Fried, B., Eds.; Handbook of Thin-Layer Chromatography; 3rd ed.; Dekker: New York, 2003. Stahl, E. Thin-Layer Chromatography, A Laboratory Handbook; 2nd ed.; Springer: Berlin, 1969. Touchstone, J. C. Practice of Thin-Layer Chromatography; Wiley: New York, 1992. Wagner, H.; Bladt, S. Plant Drug Analysis, A Thin Layer Chromatography Atlas; 2nd ed.; Springer: Berlin, 1995.
Introduction Definition and General Description Historical Development Basic Concepts Principles of Separation Describing the Result Influence of the Developing Chamber Practical Approaches Sample Application Chromatogram Development Visualization and Derivatization Densitometric Evaluation Documentation Special and Hyphenated Techniques High-Performance Thin-Layer Chromatography Automated Multiple Development Forced-Flow Techniques Hyphenated Techniques Applications
1 1 2 4 4 4 5 7 7 7 8 8 9 9 9 9 9 9 10
Introduction This article serves as an overview covering definition, historical development, basic principles, and advantages of thin-layer chromatography (TLC) in comparison to column chromatography. It concludes with a summary of common applications. Details on theory, instrumentation, plate technology, and method development are covered in subsequent articles.
Definition and General Description TLC is a form of liquid chromatography. It is used for rapid separation of samples into components based on differences in their retention behavior. The stationary phase, usually an adsorbent such as silica gel, is coated onto a rectangular support such as a glass, aluminum, or plastic plate as a thin layer. Liquid samples are applied as spots or bands onto the dry stationary phase forming a line close to one edge of the plate. The TLC plate is placed vertically in a chromatographic chamber with lid, containing an amount of the liquid mobile phase sufficient to cover the bottom of the chamber, but not reaching up to the application position of samples on the plate (Figure 1). The mobile phase is drawn through the layer by capillary action starting separation when reaching the applied samples (start position). When the front of the mobile phase has moved to a certain predefined height (developing distance) the plate is removed from the chamber, appropriately dried, and, if necessary, derivatized for detection of samples. The resulting chromatogram is evaluated qualitatively by visual or densitometric comparison of the migration distance of the separated components to those of reference standards analyzed simultaneously on the same plate. Often, the behavior of substances during derivatization is also observed. Quantitative determination is possible by comparison of the intensity of the separated zones to those of known standard concentrations. TLC is a very popular chromatographic technique because it is experimentally simple, enormously flexible, and inexpensive. It can lead rapidly to reliable qualitative and quantitative results primarily based on visual impression. TLC is an offline technique (Figure 2). An analysis is performed as a sequence of individual steps, which are independent with respect to time and location. Compared to chromatography in columns, which is an online process, this design does not only offer great flexibility for technical solutions but also almost unlimited possibilities for combination of parameters to optimize the chromatographic result. In TLC, a large number of samples can be analyzed and compared to one another simultaneously on the same plate. The actual chromatographic separation provides exactly the same conditions for samples and references and there is no
☆
Change History: May 2013. E Reich made changes in section ‘Hyphenated Techniques’.
Reference Module in Chemistry, Molecular Sciences and Chemical Engineering
http://dx.doi.org/10.1016/B978-0-12-409547-2.00540-0
1
2
THIN-LAYER CHROMATOGRAPHY | Overview
Expected developing distance
Z0
Figure 1 Schematic description of the development of a TLC plate. Z0 is the distance between the application position and the level of the developing solvent. After the mobile phase has reached the desired distance, the plate is removed from the tank.
danger of permanently contaminating the system by components of the sample’s matrix because each plate is used only once. After separation, the TLC plate functions as a storage device for the chromatogram and thus enables multiple detection/evaluation. For all steps of the TLC process modern instruments are on the market, ensuring reproducible results at various levels of sophistication. As an open system the TLC plate is easily affected by environmental factors such as humidity, fumes, light, and mechanical stress during handling. To ensure reproducibility of results this must be taken into account when designing and performing a TLC experiment.
Historical Development At the end of the 1930s, adsorption chromatography in columns as introduced by Tswett had become a powerful separation technique for plant extracts and natural products. Simultaneously, the need for a more rapid alternative suitable for identification of separated substances led to the invention of an open chromatographic system. In 1938, Izmailov and Shraiber reported the separation of belladonna alkaloids on a thin adsorbent layer, coated onto microscopic slides. Development of circular chromatograms was achieved by placing small amounts of various solvents to the center of samples previously applied as spots onto the layer. This method was an extremely rapid microtechnique requiring only small amounts of stationary and mobile phases. In 1944, Consden, Gordon, and Martin took a different approach using the principle of partitioning. This resulted in the invention of paper chromatography, which soon became a universal chromatographic technique. TLC did not advance much further until the 1950s when several significant improvements were made. Kirchner, Miller, and Keller in 1951 incorporated a fluorescent indicator into the stationary phase. Silica gel was becoming the most widely used adsorbent. In 1956, Stahl introduced the term ‘thin-layer chromatography’. This, together with Merck developing standardized aluminum oxide, kieselguhr, and silica gel based on Stahl’s specifications and Desaga bringing a basic instrument kit to the market, manifested the birth of TLC as a recognized and broadly accepted analytical technique. In 1962, Stahl with his fundamental book, Thin Layer Chromatography – A
THIN-LAYER CHROMATOGRAPHY | Overview
3
Application
Development
Derivatization
Detection Documentation
Evaluation
Report
Figure 2 Steps of TLC as an offline technique.
Laboratory Handbook, gave a forum to the leading researchers in the field of TLC and summarized the knowledge of that time. As a standard analytical technique, TLC was soon widely adopted by pharmacopoeias and official method collections. The improvement of plate materials, culminating in precoated high-performance layers of reproducible quality in the late 1970s, the rapid development of the theoretical foundation, and the appearance of powerful densitometers became the basis for reliable quantitative analyses by TLC. At that time, high-performance liquid chromatography (HPLC) evolved and began to compete with TLC as a quantitative technique. The 1980s saw remarkable improvements particularly in instrumentation, automation of individual TLC steps, and theoretical concepts contributed by Snyder, Kaiser, Ebel, and Geiss. In 1987, Geiss published Fundamentals of Thin Layer Chromatography, which is regarded as one of the most influential books on the subject. The Journal of Planar Chromatography was founded in 1988 as a platform for discussion of all aspects of modern TLC. Aside from the traditional way of performing TLC, several other approaches have also been taken. They include forced-flow techniques using centrifugal force, a pump (over-pressured layer chromatography, OPLC), or an electrical field to move the mobile phase. Hyphenated techniques were introduced combining TLC separation with infrared, Raman, or mass spectrometric detection. A technique for analysis of analytes that can be evaporated (IATROSCAN) utilizes separation performed on the surface of reusable thin chromatographic rods combined with a flame ionization detector. Also, gradient techniques have been developed for TLC, the most powerful of which is AMD – automatic multiple development – patented by Burger. Today, TLC is a mature technique that meets all requirements of a modern analytical tool. Although it has lost its dominant role as a routine procedure to HPLC due to the lack of full automation, it is still indispensable for rapid qualitative analyses (identification), as a screening tool, and as a complementary technique in research and development.
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THIN-LAYER CHROMATOGRAPHY | Overview
Basic Concepts Principles of Separation The stationary phase in TLC is usually an adsorbent (Table 1) composed of very fine and highly porous particles coated as a thin layer onto a support. Today, the most widely used stationary phase is silica gel 60. With a solvent or solvent mixture as mobile phase, a liquid–solid chromatographic system is formed. Separation of sample components can be primarily described as adsorption chromatography. Sample molecules are retained due to specific interaction on the surface of the stationary phase while migration takes place when the molecules are dissolved in the mobile phase. A dynamic adsorption equilibrium is established for each sample component. The equilibrium is characterized by a partition coefficient KA representing the ratio of concentration of the sample component A in the stationary phase (cs) and in the mobile phase (cm); KA ¼ cs/cm. Separation of two samples A and B can only be achieved if their partition coefficients are different. Typical forces causing adsorption of molecules on the stationary phase include nonspecific dispersion forces, dipole–dipole interactions, acid–base interactions, hydrogen bonding, electrostatic interaction between ions, and others. There are two extreme cases. If the sample is strongly adsorbed and only sparingly soluble in the mobile phase it will remain at the application position. Weakly adsorbed and readily soluble samples will migrate with or close to the solvent front. A suitable combination of stationary and mobile phase will result in a separation of sample components. They will be located at different migration positions when the development of the TLC plate is interrupted as soon as the mobile phase has reached the specified developing distance. The ability of a solvent to displace an adsorbed sample molecule from the surface of a given stationary phase is called solvent strength. It can be measured as energy that is released when a solvent molecule is adsorbed. Although it is not simply a measure of dipole moment, solvent strength in adsorption chromatography is often expressed as solvent polarity. Adjusting the solvent strength of the mobile phase affects the position of the sample components in the chromatogram. The quality of separation, expressed as resolution between zones of the chromatogram, depends on the selectivity of the TLC system. Adjusting the composition of the mobile phase is the primary way of changing selectivity. Apart from adsorption phenomena, a variety of other separation mechanisms can be utilized. If chemically bonded phases or liquid stationary phases on an inert support are used as stationary phase, separation is predominantly based on partition equilibria. Examples include octadecyl, aminopropyl, diol, cyanopropyl modified silica gel, impregnated silica gel, and cellulose or kieselguhr. Separations based on ion exchange, complex formation, size exclusion, and even chiral recognition are also performed.
Describing the Result In column chromatography, the chromatographic system has a fixed length. Each sample component can be characterized by the time it requires to pass through the column and reach the detector. This is the retention time tR and it is measured at the peak maximum. In TLC, the analysis takes a fixed time (development time) during which sample components can migrate. Depending on its retention each sample component will reach a specific migration distance (MD) and remains there as a zone (spot) for detection when the TLC plate is dried (Figure 3). A compound with high MD would in comparison have a short retention time in column chromatography, provided the separation mechanism is the same. The MD of a zone is measured in millimeters from the application position to the point of highest concentration (intensity). When the chromatogram is evaluated densitometrically this point represents the peak maximum. As retention time is dependent on the mobile phase velocity, the migration distance is dependent on the position of the mobile phase front zf. Therefore, a relative measure has been introduced. The RF value (retardation factor) of a zone is the ratio of its migration distance to that of the mobile phase front. RF ¼ MD/zf. RF values are always <1. For convenience it is common to multiply the RF value by 100 and report hRF with two digits. It is possible to compare the migration distance of an unknown (A) to that of a reference compound (ref) to yield the Rrel value. Rrel ¼ MDA/MDref. Although the RF value is characteristic for a substance in a given TLC system, it must be treated with caution because it is affected by several parameters. In practice, it is often difficult to reproduce RF values exactly. The Rrel value has no physical meaning. The principle of qualitative analysis is a comparison of RF values obtained from samples and standards on the same TLC plate. If two substances are the same they will have the same RF. However, different substances may also migrate to the same position. For further confirmation of identity a specific derivatization or recording of the ultraviolet (UV) spectrum of the substances on the plate can be utilized. The size and intensity of the zone of the analyte is visually compared for estimation of quantity to that of standards at several known levels on the same plate. A precise quantitation is possible by scanning or video densitometry by which Table 1
Important inorganic and organic adsorbents
Inorganic
Organic
Bonded phases on silica gel
Silica gel Aluminia Kieselguhr (diatomaceous earth) Magnesium silicate
Cellulose Polyamide Sephadex
Octadecyl-, octyl-, dimethyl-, diphenylsilanized Aminopropylsilanized Cyanopropylsilanized Diol (propanediol)
THIN-LAYER CHROMATOGRAPHY | Overview
5
A
ref.
Zf
MDA MDref.
Figure 3 Schematic presentation of result in TLC. MD, migration distance; zf, distance between application and front; RF ¼ MD/zf.
WB1 DZ WB2
Figure 4 The resolution (Rs) between two separated peaks is calculated from the chromatogram: Rs ¼ 2DZ/WB1 þ WB2.
the absorption or fluorescence of separated zones of each chromatogram track is recorded. The resulting analog curves are integrated and evaluated based on peak height or area. The fundamental requirements for reliable quantitation are baseline resolved zones and symmetric peaks for the compounds in question. The resolution between two zones or peaks can be calculated from the chromatogram (Figure 4).
Influence of the Developing Chamber In TLC, the chromatographic system consists of a plate coated with the stationary phase and a developing chamber containing the mobile phase. As seen in Figure 5, a gas phase consisting of components of evaporated solvent molecules is also present. It is a matter of time, geometry, and whether fitting the chamber with filter paper enlarges the surface of the developing solvent, but as long as the chamber is tightly closed, saturation is eventually established.
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THIN-LAYER CHROMATOGRAPHY | Overview
Figure 5 The composition of the gas phase is affected by: (1) equilibrium of gas phase with developing solvent on the bottom of the chamber (saturation); (2) and (3) equilibrium between gas phase and stationary phase ((2) absorption of gaseous molecules, (3) evaporation of mobile phase).
This means that the vapor in the gas phase is in equilibrium (1) with the developing solvent on the bottom of the chamber. When the dry TLC plate is placed in the chamber solvent vapor is readily adsorbed onto the stationary phase. If the stationary phase has no contact with the developing solvent, as it is possible in a twin trough chamber, over time another equilibrium (2) between the gas phase and the stationary phase is established. This is called preconditioning. It leads to adsorptive saturation of the stationary phase. When the mobile phase rises up the stationary phase a third equilibrium (3) is approached. While (1) is dependent on the vapor pressure of the solvent components and (2) is controlled by adsorptive forces, (3) is affected by both parameters. During chromatography all three equilibria are effectively influencing each other as well as the chromatographic result. Experimental details, type, and geometry of the chamber are therefore important parameters that can be used to optimize separation. Even in so-called saturated chambers chromatography is typically performed in nonequilibrium, which makes it difficult to mathematically describe all processes in exact detail. Generally, there are neither good nor bad chambers; however, results obtained in one may be quite different from those obtained in another chamber. In consequence, it is imperative for obtaining reproducible results that the chamber and all of its parameters are clearly defined and kept constant. As a rule of thumb saturated chambers tend to give lower RF values for the same separation than unsaturated chambers. The zones in saturated chambers are somewhat more diffuse, but reproducibility of the result is much higher than in unsaturated chambers. TLC can also be performed in sandwich chambers, where covering the stationary phase with a counterplate limits the gas phase. As a result of this the stationary phase remains active and cannot be preloaded with solvent molecules. The mobile phase is then separated into its components. So-called secondary fronts are formed and can interfere with the separation. In saturated chambers secondary fronts
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THIN-LAYER CHROMATOGRAPHY | Overview
are usually eliminated because the polar components of the mobile phase are preferably preadsorbed through the gas phase. Secondary fronts are one of the principal drawbacks of OPLC where the gas phase is completely eliminated.
Practical Approaches Sample Application During sample application two principal requirements must be met. The samples must be precisely and correctly positioned in order to identify separated components based on their migration distance/RF values and the sample volume must be controlled in order to allow quantitative evaluation of the chromatogram. The size of the plate determines the number of samples. Samples should not be applied too close to the edges of the plate and must be evenly and sufficiently spaced for best results. Typical application parameters for high-performance thin-layer chromatography (HPTLC) plates are summarized in Table 2. Application of samples can be performed manually or with the help of instruments. For manual application the positions are carefully marked on the TLC plate with soft pencil. Use of a spotting guide helps avoiding any damage to the layer. Samples are transferred with the help of disposable capillaries, graduated micropipettes, or microliter syringes and deposited onto the plate as spots. If larger volumes are applied this is done in portions with intermediate drying of the application position. The smaller the applied spot, the better is the obtainable resolution of the chromatogram. Best results in sample application are obtained with instruments, particularly those that use the spray-on technique. For maximum resolution and best precision in quantitative TLC, samples are sprayed on as narrow bands. Following sample application it is common to mark the desired developing distance on one edge of the plate.
Chromatogram Development Chromatogram development is performed in a chromatographic chamber, often called a tank. There are many different kinds of chambers, but flat bottom or twin trough chambers with a rectangular base are most common. The size of the chamber should be appropriate for the size of the plate. A saturated chamber is created, when one or more walls of the chamber are fitted with filter paper, which is thoroughly wetted with the developing solvent. For saturation to establish, the charged chamber is left closed for a defined period of time prior to introducing the TLC plate and starting development. Unsaturated chambers do not contain filter paper. Chromatography is started immediately after the chamber is charged with developing solvent. The level of solvent in the chamber is chosen in a way that the sample application position on the plate is clearly above it. Most plates are developed in a saturated chamber in vertical position, but horizontal developing chambers are also available (Figure 6). With special chambers circular and anticircular development can also be performed. For circular development samples are applied to a square plate around the center forming a circle. The mobile phase is applied to the center of the plate. Anticircular development requires delivery of mobile phase from a ring gap to the plate onto which samples have been applied forming a circle of large diameter. All developments discussed so far were single developments. Multiple developments are also possible. After intermediate drying of the plate a subsequent development can be performed (1) over the same, (2) over a longer, and (3) over a shorter developing distance. While in case (1) the goal is to improve separation by using the same mobile phase for development, in case (2) the first development over a few millimeters with a strong mobile phase is typically used to concentrate the sample into a narrow application zone. The second development with a suitable mobile phase is used for separation. In case (3), a nonpolar matrix can be removed from the sample and transported to the upper edge of the plate with a weak mobile phase leaving the compounds of interest at the application position. In the second development with a stronger mobile phase the actual separation of the sample is performed. When the mobile phase has reached the desired developing distance of typically 6 cm on HPTLC plates and 12 cm on TLC plates development is interrupted, the plate is removed from the chamber, and dried. Table 2
Typical parameters for sample application on HPTLC plates
Parameter
Band
Spot
Distance from lower edge of plate in mm Minimum distance from left and right edge of plate in mm Minimum space in mm between bands/spotsa in mm Band length in mm Maximum diameter of application spot in mm
8 10 2 8
10 10 2
a
4
Most instruments do not allow programming of distance between bands/spots. Therefore distance between tracks (center to center) and band length must be chosen in order to meet the minimum distance requirement. For spot application, volume and application speed have to be determined empirically.
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THIN-LAYER CHROMATOGRAPHY | Overview
Figure 6 Comparison of flat-bottom chamber (a), twin trough chamber (b), and horizontal developing chamber (c): (1) HPTLC plate, (2) glass plate for sandwich configuration, (3) reservoir for developing solvent, (4) glass strip, (5) cover plate, (6) conditioning tray. Examples: The flat-bottom chamber is lined with filter paper soaked with the developing solvent for saturation. Twin trough chambers can be filled with two different liquids and are less solvent-consuming. Horizontal developing chambers are easy to use in sandwich configuration.
Visualization and Derivatization One of the most striking features of TLC is the possibility of presenting the chromatographic result visually. Visual detection is possible when the separated sample components are colored, have native fluorescence, or absorb UV light so that they quench the fluorescence of the indicator, which can be built into the layer of the TLC plate. Derivatization can enhance visualization dramatically by improving the detectability of all or selected zones of the chromatogram. Numerous chemical reactions are known to convert the analyte into a colored or fluorescing derivative. Nonspecific reactions such as charring with acid are broadly applicable, whereas specific derivatization such as the reaction of amines with ninhydrin can be used to single out compounds of certain functionality from a separated mixture. The convenient availability of biochemical reactions and in situ biological tests add extra flexibility to the detection step in TLC. Examples include inhibition of an enzyme such as choline esterase or measurement of effects on the growth of bacteria or yeast cells. For chemical derivatization, the developed TLC plate is either sprayed with or immersed into a solution of the reagent. While spraying requires small amounts of reagent and provides great flexibility the advantage of immersion lies in ensuring a homogenous and reproducible coverage. In many cases derivatization is completed with a heating step using either an oven or a plate heater.
Densitometric Evaluation If a separated substance absorbs UV or white light or can be excited to fluoresce, densitometric evaluation of the chromatogram is possible without derivatization. With a densitometer, also called scanner, the tracks of the TLC plate are evaluated by registering the reflected/emitted light when a narrow beam of light of a single wavelength is moved across the plate. A photomultiplier converts
THIN-LAYER CHROMATOGRAPHY | Overview
9
the light into an electrical signal, which as a function of migration distance presents the analog curve of the chromatogram. Following integration of the raw data the peak height and/or area can be quantitated. Densitometry is a very sensitive measurement. Typically less than 100 ng of substance per zone can be detected. In some cases, such as aflatoxins, detection limits are in the picogram range. Most modern densitometers allow recording of in situ UV spectra, which can be used to confirm identity or purity of separated compounds. Densitometry is performed after derivatization, if that improves the detectability of the analyte. Examples include introduction of fluorophoric groups, charring of nonabsorbing compounds, specific color reactions.
Documentation Documentation of a TLC chromatogram as an image is conveniently possible. Traditionally photographs have been taken but the availability of electronic devices such as flatbed scanners or digital cameras have simplified the process. The principal advantage of electronic images is their durability. Added benefit is the possibility of qualitative and quantitative densitometric evaluation.
Special and Hyphenated Techniques High-Performance Thin-Layer Chromatography HPTLC is based on the use of special layers made from narrowly distributed fine particles of 5 mm. It is actually not a special technique because all fundamental parameters, theoretical considerations, and practical aspects of classical TLC still apply. However, due to the consequent use of specialized instrumentation HPTLC does not only achieve miniaturization of the chromatogram, but also and more importantly a significant improvement of sensitivity, reproducibility, and separation power. A modern HPTLC workstation including devices for each individual chromatographic step is software controlled and provides good manufacturing or good laboratory practice compliant results similar to that of HPLC and GC instruments.
Automated Multiple Development Automated multiple development (AMD) is a gradient technique consisting of a variable number of developments with intermediate drying steps, which are achieved by application of vacuum to the special developing chamber. In a computer-controlled method, each subsequent development proceeds to a higher developing distance using increments of 2–3 mm than the previous step. Furthermore, each run uses a weaker (in adsorption chromatography less polar) mobile phase. As a consequence, the separated zones are repeatedly focused. In combination with the mobile phase gradient this results in a significant increase of the separation power. Results obtained with AMD are predictable and very reproducible.
Forced-Flow Techniques The separation efficiency of TLC is limited due to the fact that capillary forces move the mobile phase. Forced-flow techniques are an attempt to solve this problem. One approach, rotational planar chromatography, utilizes centrifugal forces and has become a widely used preparative technique. A circular plate is mounted on a centrifuge and the sample followed by the mobile phase is applied close to the center of the plate. Separated sample components can be collected when they elute from the rotating plate. Another very flexible forced-flow technique is OPLC introduced by Tyihak and Mincsovics. The plate is completely sealed on all four edges. Following sample application the plate is covered with a Teflon foil, which is part of a cassette. In a special pressure chamber the cover foil seals the plate in vertical direction under external pressure. With the help of a programmable pump the mobile phase is forced with constant velocity through an inlet in the coversheet of the cassette to travel in a laminar flow through the chromatographic layer toward an outlet located at the opposite edge of the plate. The two principal drawbacks of the technique, which can considerably interfere with the separation and must therefore be minimized in the experimental setup, are solvent demixing resulting in secondary fronts when multicomponent mobile phases are employed and a so-called front of total wetness. This is a disturbance at the region where gas bubbles, which are trapped in the pores of the adsorbent when the mobile phase first enters the layer, are finally displaced by solvent molecules. OPLC has found various analytical, semipreparative, and preparative applications. The system can be operated in an online mode like HPLC, offline mode like regular TLC, or in a mixed mode.
Hyphenated Techniques Several so-called hyphenated techniques have been developed, where the developed TLC plate is transferred to a modified spectrometer to record in situ the Fourier transform infrared, surface enhanced Raman, or mass spectra of the separated zones. This way more detailed structural information can be obtained to complement the data from densitometric evaluation. A true hyphenation is the direct application of the eluate from a microbore HPLC column onto an HPTLC plate, which is then developed by AMD.
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THIN-LAYER CHROMATOGRAPHY | Overview
Table 3
Major applications of TLC
Sample type
Application
Pharmaceutical
Impurities in synthetic drugs Stability tests of synthetic drugs Content uniformity Pharmacokinetic studies and drug monitoring Assay Enantiomeric purity Product uniformity Impurity determination Surfactants, dyes Cleaning validation Identity, purity, and stability tests of plant drugs Quantitation of marker compounds Organic acids, lipids Carbohydrates Porphyrins and bile pigments Amino acids, peptides Steroids, doping Mycotoxins (incl. aflatoxins) Drug and pesticide residues Antioxidants, preservatives Pigments, dyes, spices, flavors, vitamins Pesticides residues Water and soil analysis Poisons Drugs of abuse Inks
Industrial
Herbal Biomedical
Nutritional
Environmental Forensic
For further investigation by mass spectrometry (MS) separated sample components can either be eluted by a commercially available TLC-MS interface or analyzed directly on the plate by DART (direct analysis in real time) or DESI (desorption electrospray ionization) techniques. Also commercially available is an instrument for MALDI (matrix assisted laser desorption/ionization) MS.
Applications Typical applications of TLC are listed in Table 3. In many areas including herbal, pharmaceutical, and environmental analyses the flexibility of the technique as a research tool is a great asset. Other applications such as content uniformity, residue, and doping test as well as cleaning validation take advantage of short analysis times due to parallel chromatography of many samples. Qualitatively, TLC is primarily used in general research and in quality control of raw materials for rapid and cost-efficient identification. Quantitative applications include assays, determination of impurities and process monitoring.
Further Reading 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Frey, H. P.; Zieloff, K. Qualitative und Quantitative Du¨nnschicht-Chromatographie; VCH Verlagsgesellschaft mbH: Weinheim, 1993. Geiss, F. Fundamentals of Thin Layer Chromatography; Alfred Hu¨thig Verlag: Heidelberg, 1987. Hahn-Deinstrop, E. Applied Thin-Layer Chromatography; Wiley-VCH: Weinheim, 1998. Pachaly, P. DC-Atlas. Wissenschaftliche Verlagsgesellschaft: Stuttgart, 1991. Poole, C. F.; Poole, S. K. The Essence of Chromatography; Elsevier: Amsterdam, 2003. Sherma, J.; Fried, B., Eds.; Thin-Layer Chromatography, Techniques and Applications; 3rd ed.; Dekker: New York, 1994. Sherma, J.; Fried, B., Eds.; Handbook of Thin-Layer Chromatography; 3rd ed.; Dekker: New York, 2003. Stahl, E. Thin-Layer Chromatography, A Laboratory Handbook; 2nd ed.; Springer: Berlin, 1969. Touchstone, J. C. Practice of Thin-Layer Chromatography; Wiley: New York, 1992. Wagner, H.; Bladt, S. Plant Drug Analysis, A Thin Layer Chromatography Atlas; 2nd ed.; Springer: Berlin, 1995.