Thin–layer Chromatography (TLC) Thin–layer chromatography (TLC) is a widely used technique for the separation and identification of drugs. It is equally applicable to drugs in their pure state, to those extracted from pharmaceutical formulations, to illicitly manufactured materials and to biological samples. TLC as we know it today (see Fig 1) was established in the 1950s with the introduction of standardised procedures that lead to improved separation performance and reproducibility, and paved the way for its commercialisation and an increase in the number of published applications. The 1970s saw the introduction of fine–particle layers and associated instrumentation required for their correct use. In this form, TLC became known as high–performance TLC, instrumental TLC or modern TLC to distinguish it from its parent, now generally referred to as conventional TLC. High–performance TLC has not displaced conventional TLC from laboratory studies and the two approaches coexist today because of their complementary features (Table 1). Conventional TLC provides a quick, inexpensive and portable method for qualitative analysis. It requires minimal and readily available instrumentation and uses easily learned experimental techniques. High–performance TLC is characterised by the use of kinetically optimised layers for faster and more efficient separations, takes advantage of a wider range of sorbent chemistries to optimise selectivity and requires the use of instrumentation for convenient (automated) sample application, development and detection. High–performance TLC provides accurate and precise quantitative results based on in situ measurements and a record of the separation in the form of a chromatogram, such as the example in Fig 2. While all modern laboratories are capable of drug analysis by conventional TLC, only those laboratories equipped with the necessary instrumentation for high–performance TLC have this option. Analytical Toxicology - Thin–layer Chromatography (TLC) | 1 Thin–layer Chromatography (TLC) Figure 1. Time line depicting the evolution of modern TLC. Parameter High–performance TLC TLC Plate dimensions (cm2) 10 × 10 20 × 20 Layer thickness (mm) 0.1 or 0.2 0.1–0.25 Starting spot diameter (mm) 1–2 3–6 Diameter of separated spots (mm) 2–6 6–15 Solvent front migration distance (cm) 3–6 10–15 Analytical Toxicology - Thin–layer Chromatography (TLC) | 2 Thin–layer Chromatography (TLC) Parameter High–performance TLC TLC Time for development (capillary flow) (min) 3–20 20–200 Detection limitsa Absorption (ng) 0.1–0.5 1–5 Detection limits Fluorescence (pg) 5–10 50–100 Nominal particle size range (μm) 3–7 5–20 Apparent particle size (μm)b 5–7 8–10 Minimum plate height (μm) 22–25 35–45 Optimum velocity (mm/s) 0.3–0.5 0.2–0.5 Porosity Total 0.65–0.70 0.65–0.75 Porosity Interparticle 0.35–0.45 0.35–0.45 Porosity Intraparticle 0.28 0.28 Table 1. Characteristic properties of silica gel precoated TLC layers a For drugs with favourable detection properties. b Determined by chromatographic measurements. Precoated TLC layers are prepared from silica gel with a narrower particle size range than typical bulk materials available for self–made layers. Analytical Toxicology - Thin–layer Chromatography (TLC) | 3 Thin–layer Chromatography (TLC) Figure 2. Separation of ethynyl steroids (birth–control pill components) by high–performance TLC. Two 15 min developments with the mobile phase hexane–chloroform–carbon tetrachloride–ethanol (7:18:22:1) on a silica gel 60 high–performance TLC plate. Chromatogram was recorded by scanning densitometry at 220 nm. In the basic TLC experiment, the sample is applied to the layer as a spot or band near to the bottom edge of the layer. The separation is carried out in a closed chamber by either contacting the bottom edge of the layer with the mobile phase, which advances through the layer by capillary forces, or the mobile phase is forced to move through the layer at a controlled velocity by an external pressure source or centrifugal force. A separation of the sample results from the different rates of migration of the sample components in the direction travelled by the mobile phase. After development and evaporation of the mobile phase, the sample components are separated in space, their position and quantity being determined by visual evaluation or in situ scanning densitometry aided by the formation of easily detected derivatives by post–chromatographic chemical reactions, as required. Analytical Toxicology - Thin–layer Chromatography (TLC) | 4 Thin–layer Chromatography (TLC) Separations by column liquid chromatography (HPLC) and TLC occur by essentially the same physical process. The two techniques are frequently considered as competitors, when it would be more realistic to consider them as complementary. The attributes of TLC that provide for its co–existence as a complementary technique to HPLC are summarised in Table 2. Based on these attributes, TLC methods are most effective for the low–cost analysis of a large number of samples (e.g. drug screening in biological fluids and tissues, determination of the botanical origin and potency of traditional herbal medicines, stability testing and content uniformity testing), for the rapid analysis of samples that require minimum sample clean up or where TLC allows a reduction in the number of sample preparation steps (e.g. analysis of samples containing components that remain sorbed to the stationary phase or contain suspended microparticles). TLC is also preferred for the analysis of substances with poor detection characteristics that require post–chromatographic chemical treatment for detection. In other cases, HPLC methods are generally preferred, particularly if a large number of theoretical plates are necessary for a separation, for separations by size–exclusion and ion–exchange chromatography, and for trace analysis using selective detectors unavailable for TLC. Attribute Application Low–cost analysis and high–throughput Separation of samples in parallel screening of samples requiring minimal sample preparation Analysis of crude samples (minimising Disposable stationary phase sample preparation requirements) Analysis of a single or small number of samples when their composition and/or matrix properties are unknown Analysis of samples containing components that remain sorbed to the separation medium or contain suspended microparticles Samples that require Static detection post–chromatographic treatment for detection Samples that require sequential detection techniques (free of time constraints) for identification or confirmation Analytical Toxicology - Thin–layer Chromatography (TLC) | 5 Thin–layer Chromatography (TLC) Attribute Application Storage device Separations can be archived Separations can be evaluated in different locations or at different times Convenient fraction collection for coupled column–layer chromatography Total sample occupies the Sample integrity chromatogram, not just that portion of the sample that elutes from the column Table 2. Attributes of TLC providing the link to contemporary applications in drug analysis. Sommaire 1 Stationary phases 2 Technique 2.1 Layer pretreatments 2.2 Sample application 2.3 Development 2.4 Development chambers 3 Detection 3.1 Derivatisation reactions 3.2 Slit–scanning densitometers 3.3 Image analysers 3.4 Other instrumental detection methods 4 Method development 5 Preparative thin–layer chromatography 6 Retardation factor 6.1 Drug identification 6.2 Database searches Analytical Toxicology - Thin–layer Chromatography (TLC) | 6 Thin–layer Chromatography (TLC) 7 Systematic drug identification 8 General applications 9 Systems for thin-layer chromatography 9.0.1 Note 10 Screening systems 10.1 Basic nitrogenous drugs 10.2 Acidic and neutral drugs 10.3 General screening systems 10.4 Amfetamines, other stimulants and anorectics 10.5 Anaesthetics 10.6 Analgesics, NSAIDs 10.7 Analgesics, NSAIDs 10.8 Anti-emetics 10.9 Anti-fungals 10.10 Antibacterials 10.11 Anticholinergics 10.12 Anticonvulsants and barbiturates 10.13 Antidepressants and antipsychotics 10.14 Antihistamines 10.15 Antimalarials 10.16 Antineoplastics 10.17 Antiparkinsonians 10.18 Antiprotozoals 10.19 Antitussives 10.20 Benzodiazepines and hypnotics 10.21 Bronchodilators 10.22 Cannabinoids 10.23 Cardioactive drugs 10.24 Coumarins and other anticoagulants Analytical Toxicology - Thin–layer Chromatography (TLC) | 7 Thin–layer Chromatography (TLC) 10.25 Diuretics 10.26 Drugs of abuse 10.27 Ergot alkaloids 10.28 Ergot alkaloids 10.29 Narcotic analgesics and narcotic antagonists 10.30 Oral hypoglycemics and antidiabetics 10.31 Pesticides 10.32 Phenothiazines and other tranquilisers 10.33 Psychomimetics and sympathomimetics 10.34 Quaternary ammonium compounds 10.35 Steroids 10.36 Sulfonamides 10.37 Vitamins 10.38 Xanthine stimulants Stationary phases Conventional TLC plates can be prepared in the laboratory by standardised methods, but reproducible layer preparation is easier to achieve in a manufacturing setting and few laboratories prepare their own plates today. Precoated plates for high performance, conventional and preparative TLC are available in a range of sizes and different layer thickness, supported on glass, aluminium or plastic backing sheets. To impart the desired mechanical stability and abrasion resistance to the layer a binder, such as poly(vinyl alcohol), poly(vinyl pyrrolidone), gypsum or starch in amounts from 0.1 to 10% (w/w) is incorporated into the layer. An ultraviolet (UV)-indicator, such as manganese–activated zinc silicate