Chemistry
Thin-Layer Chromatography
Thin-layer chromatography (TLC) is a chromatographic technique used to separate and analyze mixtures. It involves the use of a thin layer of adsorbent material on a flat support, where the sample is applied and then separated as it moves up the plate through capillary action. TLC is commonly used in chemical analysis for its simplicity, speed, and cost-effectiveness.
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12 Key excerpts on "Thin-Layer Chromatography"
eBook - PDF- Satinder Ahuja, Neil Jespersen(Authors)
- 2006(Publication Date)
- Elsevier Science(Publisher)
Chapter 13 Thin-Layer Chromatography Pamela M. Grillini 13.1 INTRODUCTION Thin-Layer Chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capa-city to analyze multiple samples simultaneously. It has been applied to many disciplines including biochemistry [1,2] , toxicology [3,4] , pharma-cology [5,6] , environmental science [7] , food science [8,9] , and chemi-stry [10,11] . TLC can be used for separation, isolation, identification, and quantification of components in a mixture. It can also be utilized on the preparative scale to isolate an individual component. A large variety of TLC equipment is available and discussed later in this chapter. High-performance Thin-Layer Chromatography (HPTLC) has become widely used and while it follows the same principles as TLC, it makes use of modern technology including automatic application devices and smaller plates, which allow for better sensitivity. TLC is related to paper chromatography (PC) as both use a station-ary phase and a liquid phase to move the sample [12] . A common ex-ample of PC is the separation of black ink into its individual colors. Because the individual molecules behave differently when exposed to a solvent such as water or isopropyl alcohol, they are retained on the paper at different intervals, creating a visible separation of the indi-vidual components. This helps to identify each component in a mixture. 13.2 THEORY AND BASIC PRINCIPLES In TLC, the sample is applied as a small spot or streak to the marked origin of stationary phase supported on a glass, plastic, or metal plate. The sample solvent is allowed to evaporate from the plate that is then placed in a closed chamber containing a shallow pool of mobile phase at Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47013-6 r 2006 Elsevier B.V.
eBook - PDF- Satinder Ahuja(Author)
- 2003(Publication Date)
- Academic Press(Publisher)
8 Thin-Layer Chromatography I. STATIONARY PHASES FOR TLC A. Adsorption B. Partition C. Ion Exchange D. Size Exclusion E. Miscellaneous II. TLC OF ENANTIOMERIC COMPOUNDS A. Achiral Stationary Phase and Achiral Mobile Phase B. Achiral Stationary Phase and Chiral Stationary Phase Additives C. Chiral Stationary Phase and Achiral Mobile Phase III. SAMPLE APPLICATION IV. MOBILE PHASES A. Commonly Used Mobile Phases V. DEVELOPMENT OF CHROMATOGRAMS A. Effect of Temperature B. Gradient Elution C. MiscellaneousTechniques VI. DETECTION AND QUANTITATION VII. APPLICATIONS A. Amino Acids B. Pharmaceuticals C. Vitamins D. Dyes REFERENCES QUESTIONS FOR REVIEW Thin-Layer Chromatography (TLC) is an open-bed chromatographic technique that is generally carried out on a thin layer of stationary phase coated on a glass plate. It is routinely used in many laboratories in the chemical/pharmaceutical and related industries, for both qualitative and semiquantitative work. Quantitative analyses, of course, can also be performed. Some laboratories find this technique extremely useful and claim that very good precision can be achieved, even at very low levels of analyses. 1 However, elaborate steps must be taken to assure precision. These are discussed at some length later in this chapter The origin of TLC can be traced back to 1938 when two Russian researchers, Izmailov and Shraiber, utilized a technique called drop chroma-tography on horizontal thin layers. It took another twenty years for this 113 technique to become a practical tool when Stahl described equipment and efficient sorbents for the preparation of plates (for more details, see Stahl’s book listed in Reference 2). The technique suffered from relatively low efficiency for another twenty years before making a great advance that resulted from introduction of smaller and more uniform particle sizes in the sorbents. Greater reproducibility in the preparation of layers has helped to achieve better reproducibility of results.
eBook - PDFApplied Thin-Layer Chromatography
Best Practice and Avoidance of Mistakes
- Elke Hahn-Deinstrop(Author)
- 2007(Publication Date)
- Wiley-VCH(Publisher)
This book is mainly intended for the younger scientific generation. For teachers it tries to encourage a form of teaching close to practical “real-life” TLC analysis, and the many practical tips also offer invaluable support for the less experienced users in industrial and official laboratories. Last but not least, it can be used by the analyst in a pharmaceutical laboratory as a work of reference. 1.1 What Does TLC Mean? Chromatography means a method of analysis in which a mobile phase passes over a stationary phase in such a way that a mixture of substances is separated into its compo- nents. The term “Thin-Layer Chromatography”, introduced by E. Stahl in 1956, means a chromatographic separation process in which the stationary phase consists of a thin layer applied to a solid substrate or “support” [1, 2]. For some years, TLC has also been referred to as planar chromatography. However, apart from the fact that paper chromatography, which is also a planar method, is now hardly used, I do not think that this term will ever be widely accepted because the abbreviation PC could easily be confused with the abbreviation for personal computer. 1.2 When Is TLC Used? An essential precondition is that the substances or mixtures of substances to be ana- lyzed should be soluble in a solvent or mixture of solvents.
eBook - PDFExperimental Organic Chemistry
A Miniscale & Microscale Approach
- John Gilbert, Stephen Martin(Authors)
- 2015(Publication Date)
- Cengage Learning EMEA(Publisher)
180 Experimental Organic Chemistry ■ Gilbert and Martin phases with a different equilibrium constant or partition coefficient, the compo-nents divide into separate regions termed migratory bands (Fig. 6.1). The component that interacts with or binds more strongly to the stationary phase moves more slowly in the direction of the flow of the mobile phase. The attractive forces that are involved in this selective adsorption are the same forces that cause attractive interactions between any two molecules: electrostatic and dipole-dipole interactions, hydrogen bonding, complexation, and van der Waals forces. The chromatographic methods used by modern chemists to identify and/or purify components of a mixture are characterized by the nature of the mobile and stationary phases. For example, the techniques of thin-layer (TLC), column, and high-performance liquid chromatography (HPLC) each involve liquid-solid phase interactions. Gas-liquid partition chromatography (GLC), also known as gas chro-matography (GC), involves distributions between a mobile gas phase and a station-ary liquid phase coated on a solid support. These important techniques can be used as tools to analyze and identify the components in a mixture as well as to separate the mixture into its pure components for preparative purposes. Although there are other chromatographic techniques, such as ion exchange and paper chromatogra-phy, a review of those methods is beyond the scope of this discussion. 6.2 T H I N -L A Y E R C H R O M A T O G R A P H Y Thin-Layer Chromatography (TLC) is a form of solid-liquid adsorption chroma-tography and is an important technique in organic chemistry for rapid analysis of small quantities of samples, sometimes as little as 10 2 9 g. Thus, TLC is frequently used to monitor the progress of reactions and of preparative column chromato-graphic separations as well as to determine the optimal combinations of solvent and adsorbent for such separations (Sec. 6.3).
eBook - PDF- Haleem J. Issaq(Author)
- 2001(Publication Date)
- CRC Press(Publisher)
4 Thin Layer Chromatography Joseph Sherma Lafayette College, Easton, Pennsylvania The term thin layer chromatography will be used throughout this chapter rather than planar chromatography, a general classification that has been used recently to denote chromatography modes having a flat stationary phase. Paper chromatography is a type of planar chromatography, but the term has been used incorrectly to include electrochromatography or electrophoresis be-cause this method does not involve the elements necessary for designation as a chromatographic method as defined by Strain in 1942 [1], i.e., substances in a narrow initial zone are caused to undergo differential migration on a porous, sorptive stationary phase having a selective resistive action by the nonselective driving force of a liquid or gaseous mobile phase. I. EARLY HISTORY OF THIN LAYER CHROMATOGRAPHY (1889 TO EARLY 1960s) The history of thin layer chromatography (TLC) has been traced back to experiments performed by the Dutch biologist Beyerinck in 1889 [2]. These predate the early work on column chroma-tography reported by Tswett in 1906 [3] but are predated by the paper chromatography work of Runge, Schoenbein, and Goppelsroeder [4] during the period 1834-1888. Beyerinck allowed a drop of a mixture of hydrochloric and sulfuric acids to diffuse through a thin layer of gelatin on a glass plate. The hydrochloric acid traveled faster than the sulfuric acid and formed a ring around the latter. The hydrochloric acid zone was made visible by reaction with silver nitrate and the sulfuric acid with barium chloride. Tswett did not study TLC but discussed adsorption of compounds on strips of paper during capillary analysis [5]. Consden, Martin, and Gordon reintroduced paper chromatography in 1944 [6], and it grew into a universally used microanalyti-cal method during the next 10 years.
eBook - PDF- Ivor Smith, J. W. T. Seakins, Ivor Smith, J. W. T. Seakins(Authors)
- 2013(Publication Date)
- Butterworth-Heinemann(Publisher)
CHAPTER 3 THIN LAYER CHROMATOGRAPHY Ivor Smith and R. S. Ersser ALTHOUGH a number of workers had previously described procedures for thin layer chromatography, TLC, the first practical description of apparatus and technique was that of Stahl. (1) His contribution was not limited to this, however, as it was equally important to make avail-able suitable materials for preparing the layer and his investigation of the factors concerning the manufacture of such materials was of at least equal importance. Indeed the first suitable materials were commercially described as after Stahl and many still carry that label. TLC is, in principle, a variant of paper chromatography, PC. Where-as PC came into general use rather slowly because the apparatus was usually home-made and somewhat temperamental, TLC was adopted rapidly. This was to be expected as the wealth of practical information gained over years of use with paper could be applied almost unchanged to the new technique. The technique of TLC can be divided into two discrete techniques, namely the preparation of suitable TL plates and chromatography on such plates. Once the plates are prepared, the technique of chromatography is identical with that used for paper. PC, i.e. chromatography on a sheet (or layer) of cellulose, was highly successful in the field of ionic and polar molecules. It was highly unsuccessful in the field of non-polar or lipid molecules. TLC on silica gel brought the chromatography of lipids into use as a simple routine technique comparable with the paper technique for aminoacids. Not only were the separations as simple to perform but they occurred in minutes instead of hours, the technique required smaller quantities of materials as the spots were smaller and reagents appeared more sensitive but, also, more corrosive location reagents could be used on the inert silica gel layer.
- Satinder Ahuja, Karen Mills Alsante(Authors)
- 2003(Publication Date)
- Academic Press(Publisher)
Currently in the pharmaceutical industry, commercially precoated high-performance TLC (HPTLC) plates with fine particle layers are commonly used for fast, efficient, and reproducible separations. The choices of mobile phase range from single component solvent systems to multiple-component solvent systems with the latter being most common. The majority of TLC applications are normal phase, which is also a complementary feature to HPLC that uses mostly reverse-phase columns. The migration of each component in a mixture during TLC is a result of two opposing forces: capillary action of the mobile phase and retardation action of the stationary phase. Both forces contribute to achieve differential migration of each component. Developed TLC plates can be detected by various means, based on the nature of the sample. They could be non-destructive (UV/densitometer), destructive (derivatizing agents), or the combination of both. The results can be documented by photography and saved electronically for archiving and future reference. 204 P. M. GORMAN AND H. JIANG C. Why Use TLC? Today, while HPLC is widely used for separation and quantification, TLC remains a valuable and commonly used separation technique because of its features complementary to HPLC. The majority of TLC applications use normal-phase methods for separation, whereas reversed-phase methods dominate in HPLC. Some of the most important features of TLC compared to HPLC are briefly discussed here. 1. Open format of stationary phase and evaluation of the whole sample —In TLC separation, a mixture is applied to the stationary phase followed by development. It is an open system from separation to detection. In contrast to TLC, HPLC is a closed-column system in which a mixture is introduced into the mobile phase and solutes are eluted and detected in a time-dependent manner.
- Klara Valko(Author)
- 2020(Publication Date)
- Elsevier Science(Publisher)
Particle-loaded membranes are used as stationary phase in the Toxi-Lab system for toxicological drug screening by TLC [ 41 ]. Monolithic films of silica gel [ 42 ]and organic polymers [ 43, 44 ] can be used in ultra-TLC. These structures do not require a binder to form a stable layer. Silica monoliths have been optimized for separation of small molecules and polymers for peptides and nucleotides. 10.3. Mobile phases for Thin-Layer Chromatography Mobile phases used for TLC have to fulfil various requirements. Some of these do not differ from the characteristics of mobile phases used for HPLC; however, others are specific for TLC. The UV absorbance of mobile phases for TLC may be neglected, as the mobile phase is evaporated between the separation and the detection. For this reason, acetone can be easily used for TLC, while this solvent has only minor use in HPLC as a consequence of its strong absorbance at 254 nm. The solvents have to be easy to purify, must be inexpensive, must have low viscosity, and must be compatible with the stationary phase and binder being used. Relative strengths of various solvents are given in Table 10.10. 10.3.1. Optimization of solvent systems Solvent system optimization can be done on the basis of trial and error, according to literature data, or the intuition and experience of the chromatographer [ 50 ]. The mobile-phase optimization procedure is based on Snyder's solvent characterization [ 51 ] and is called the PRISMA system [ 50 ], which uses a three-step optimization procedure
eBook - PDF- C.F. Poole, S.K. Poole(Authors)
- 2012(Publication Date)
- Elsevier Science(Publisher)
As an analogy, the separation number envisages the chromatogram as being similar to a string of beads, each bead touching its neighbor with no unoccupied space between the beads. The separation number is thus an inflated estimate of the real 671 separation capacity, since real chromatograms do not usually consist of an array of equally spaced peaks. In general, unless the separation number exceeds the number of components in the sample by a significant amount the separation will normally be difficult to achieve. 7-3 STATIONARY PHASES FOR TLC TLC plates may be prepared in the laboratory by standardized procedures [1-3], although the exacting experimental conditions required for their reproducible preparation are more easily obtained in a manufacturing setting. Consequently, most laboratories, today, use commercially available precoated plates. Precoated plates for high performance, conventional and preparative TLC are available in thicknesses from 0.1 to 2.0 mm supported by either glass, aluminum or plastic backing sheets. Most plates also contain a binder such as gypsum, starch or salts of poly (acrylic acid), in amounts of 0.1 to 10% (w/w) , to impart the desired mechanical strength, durability and abrasion resistance to the sorbent layer. A UV-indicator, such as manganese-activated zinc silicate of a similar particle size to the sorbent, may be added to the layer for visual evaluation of separated samples by fluorescence quenching. TLC plates with a binary layer of two different, separated sorbents, forming a narrow interface parallel to one edge, are available for two-dimensional TLC. If one of the layers is a form of silica with very weak retention properties, it can be used as a concentrating zone, to aid sample application.
eBook - PDFThin-Layer Chromatography
A Modern Practical Approach
- Peter E Wall, Roger M Smith(Authors)
- 2007(Publication Date)
- Royal Society of Chemistry(Publisher)
3.3 Partition Separations In partition TLC, 3,4 a liquid stationary phase is adsorbed or chemically bound to the silica gel support. The stationary phase should ideally be insoluble in the mobile phase. On application of the sample to the sorbent layer, the components of the sample equilibrate between the mobile and stationary phases. The migration of components is dependent upon their relative solubility or preference for the mobile or stationary phases. On this basis, a component that has greater affinity for the mobile phase will be eluted before one that has more affinity for the stationary phase. To illustrate how this works consider a small portion of solid support containing a unit of stationary phase, (see Figure 3, unit (i)). A mixture of two substances, (A) and (B) is applied to the solid support. As the mobile phase flows through this unit, equilibration occurs and 80% of (A) and 40% of (B) prefers the mobile phase. As the mobile phase passes on to the next unit as shown in Figure 3, unit (ii), the sample remaining in the stationary phase will re-equilibrate itself. This process will continue with further equilibrium stages and substance (A) will migrate faster than substance (B). On a TLC or HPTLC plate there are many such equilibrium stages that are known as theoretical plates. Resolution obviously improves with the number of theoretical plates. As a general rule for TLC, the number of theoretical plates is 4 600 and for HPTLC typically 4 5000. 3.4 Ion-exchange Separations The ion-exchange process is dependent on the sorbent containing ions that are capable of exchanging with ions of like charge in the sample or mobile phase. The exchange of ions is dependent on the affinity of the support for the various ionic Figure 3 Unit (i) is the first portion of stationary phase under the effect of flowing mobile phase where separation can begin. Unit (ii) is the second portion of stationary phase.
eBook - ePubQuality Control and Evaluation of Herbal Drugs
Evaluating Natural Products and Traditional Medicine
- Pulok K. Mukherjee(Author)
- 2019(Publication Date)
- Elsevier(Publisher)
Detailed information of the specific extract producing plant should be accumulated. This will help in the selection of the mobile phase. A full literature search on the following aspects should be considered:- (i) Whether the species has been considered previously.
- (ii) What metabolites were detached if considered previously.
- (iii) Whether standard TLC techniques are accessible.
- (iv) In the event that the science of a species has not been examined, whether there are any data at the nonspecific level.
- (v) Chemotaxonomy, or the arrangement of a lifeform according to its chemical constituents, may be helpful in identifying chemically unknown genera, which may be helpful in finding related species, yielding related secondary metabolites (Gibbons and Gray, 1998 ).
- (i) Acidic compounds tend to “tail” on silica because of bonding between acidic groups (e.g., –CO2 H, –OH) of the compound and silanol group of the stationary phase. This can be corrected by adding a small amount of acid (e.g., 1% trifluoracetic acid or acetic acid) to the solvent system, which will keep the compound in nonionized form, thus reducing tailing.
- (ii) In the case of a basic compound, tailing can be avoided by the addition of a weak base, such as diethylamine or triethylamine.
- (iii) Nonpolar compounds, including fatty acids, glycerides, alkanes, and some lower terpenoids, need simple nonpolar solvent systems (e.g., cyclohexane, hexane, pentane, diethyl ether, hexane mixtures) and are not easily detected by UV (due to lack of chromophore) or by spray detection (due to charring of plates caused by reagents, such as vanillin-sulfuric acid).
- (iv) Polar compounds, including sugars, glycosides, tannins, polyphenolics, and certain alkaloids, need to be developed in polar solvent systems. During such development, compounds may be adsorbed onto the stationary phase, which is difficult to remove. Mobile phase selection should start with the use of a mono or binary system, for example, 100% CHCl3
- Thomas J. Bruno, Paris D.N. Svoronos(Authors)
- 2010(Publication Date)
- CRC Press(Publisher)
209 3 CHAPTER Thin Layer Chromatography CONTENTS Strength of Common TLC Solvents .............................................................................................. 211 Modification of the Activity of Alumina by Addition of Water .................................................... 213 Stationary and Mobile Phases........................................................................................................ 214 Typical Stationary and Mobile Phase Systems Used in the Separation of Various Inorganic Ions ....................................................................................................................... 225 Spray Reagents in Thin Layer Chromatography ........................................................................... 226 Protocol for Reagent Preparation ................................................................................................... 243 THIN LAYER CHROMATOGRAPHY 211 STRENGTH OF COMMON TLC SOLVENTS The following table contains the common solvents used in thin layer chromatography, with a measure of their “strengths” on silica gel and alumina. The solvent strength parameter, ε° , is defined as a relative energy of adsorption per unit area of standard adsorbent [1–3]. It is defined as zero on alumina when pentane is used as the solvent. This series is what was called the eluotropic series in the older literature. For convenience, the solvent viscosity is also provided. Note that the viscosity is tabulated in cP for the convenience of most users. This is equivalent to mPa⋅s in the SI convention. Additional data on these solvents may be found in the tables on high performance liquid chromatography. REFERENCES 1. Snyder, L. R. Principles of Adsorption Chromatography. New York: Marcel Dekker, 1968. 2. Willard, H. H., L. L. Merritt, J. A. Dean, and F. A. Settle. Instrumental Methods of Analysis. 7th ed. New York, Belmont: Van Nostrand, 1988. 3. Hamilton, R., and S.
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