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.

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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

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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.

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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.

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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

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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

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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

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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 of a similar particle size to the sorbent, may be added to the layer to visualise separated samples by fluorescence quenching. TLC plates with a narrow preadsorbent zone located along one edge of the layer are available to aid manual sample application.

Silica gel is the most important stationary phase for TLC, with other inorganic oxide adsorbents, such as alumina, kieselguhr (a silica gel of low surface area) and Florisil (a synthetic magnesium silicate), of minor importance. Most silica gel sorbents have an

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average pore size of 6 nm and are designed for the separation of small molecules (relative molecular mass < 700). The chromatographic properties of the inorganic oxide adsorbents depend on their surface chemistry and specific surface area. For silica gel, silanol groups are the dominant adsorption sites. The complementary sample properties that govern retention are the number and type of functional groups and their spatial location (Fig 3). The influence of functional group properties on selectivity is illustrated in Fig 2 for the separation of ethynyl steroids. The steroids with phenolic groups are the most strongly retained, followed by hydroxyl groups, and then ketone and ester groups. Subtle separation differences through steric hindrance at a functional group and differences in ring conformations are also seen, which allow the separation of steroids with very similar chemical properties.

Figure 3. General adsorption scale for separations by silica gel TLC.

Chemically bonded layers are prepared from silica gel by reaction with various organosilane reagents to form siloxane bonds, with some of the silanol groups present on the silica surface (Table 3). Reversed–phase alkylsiloxane–bonded layers with a high level of surface bonding cannot be used with mobile phases that contain a significant amount of water (>30% v/v) because of the inadequate mobile–phase velocity generated by capillary forces. Water compatibility for alkylsiloxane–bonded layers is achieved by increasing the particle size, using a reproducible although lower degree of silanisation, and by using modified binders. These layers are referred to as water wettable and are used for all types of reversed–phase separations, while layers with a high degree of silanisation are used predominantly with non–aqueous mobile phases. Alkylsiloxane bonded phases are used primarily (but not exclusively) for the separation of water–soluble polar drugs and weak acids and bases after ion suppression (buffered mobile phase) or ion–pair formation. Water compatibility is not a problem for polar chemically bonded phases, which can be used for

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both normal- and reversed–phase separations. For separations that cannot be achieved on silica gel, the polar chemically bonded phases are the most widely used stationary phases. The 3–aminopropylsiloxane–bonded layers can function as a weak anion exchanger for the separation of polyanions with a buffered mobile phase. Cellulose layers provide only weak retention of common drug substances and are used primarily to separate very polar compounds in biochemistry.

Table 27.3.: Clarke’s Analysis of Drugs and Poisons

Type of modification Functional group Applicationa

For reversed phase separations generally, Alkylsiloxane Si–CH 3 but not exclusively

Separation of water–soluble polar organic Si–C H 2 5 compounds (RPC)

Weak acids and bases after ion suppression Si–C H 8 17 (RPC)

Strong acids and bases by ion–pair Si–C H 18 37 mechanism (RPC)

Phenylsiloxane Si–C6H5 Of limited use for drug analysis

Cyanopropylsiloxane Si–(CH2)3CN Useful for both RPC and NPC

In NPC it exhibits properties similar to a low–capacity silica gel.

In RPC it exhibits properties similar to short–chain alkylsiloxane–bonded layers (it has no selectivity for dipole–type interactions)

Used mainly in NPC and IEC; limited Aminopropylsiloxane Si–(CH ) NH 2 3 2 retention in RPC

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Type of modification Functional group Applicationa

Selectively retains compounds by hydrogen–bond interactions in NPC; separation order generally different to that in silica gel

Functions as a weak anion exchanger in acidic mobile phases (IEC)

Spacer bonded Si–(CH2)3OCH2 Si- Used in NPC and RPC, but more useful for

propanediol CH(OH)CH2OH NPC because of low retention in RPC

Polar drugs selectively retained by hydrogen bond and dipole–type interactions in NPC; more hydrogen–bond acidic and less hydrogen–bond basic than aminopropylsiloxane–bonded layers in NPC; more retentive than aminopropylsiloxane–bonded layers in RPC

Similar retention to short–chain alkylsiloxane–bonded layers, but different selectivity for hydrogen–bonding drugs

Table 3. Retention properties of silica based chemically bonded layers

* NPC, normal–phase chromatography; RPC, reversed–phase chromatography, IEC, ion–exchange chromatography

TLC has found limited use for the separation of enantiomers. The most widely used approach employs ligand–exchange chromatography on reversed–phase layers impregnated with a solution of copper acetate and (2S,4R,2RS)-N-(2–hydroxydodecyl)-4–hydroxyproline. Separations result from stability differences in diastereomeric complexes formed between the drug, copper and the proline selector. Suitable drugs for this application require an amino acid or α-hydroxycarboxylic acid group for complex formation. A more versatile approach to the separation of enantiomeric drug substances by reversed–phase TLC is the use of chiral selectors, such as cyclodextrins or bovine serum albumin, as mobile–phase additives.

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Technique

The technique of TLC involves a number of separate steps, namely preparing the layer, applying the sample, developing the plate and detecting the separated zones. These steps are described below.

Layer pretreatments

Prior to chromatography it is common practice to prepare the layers for use by any or all of the following steps: washing, conditioning and equilibration. Layers may also be cut to preferred sizes using scissors for plastic- or aluminium–backed plates and diamond or carbide glass–cutting tools for glass–backed plates. Newly consigned precoated layers are invariably contaminated, or quickly become so, because of residual contaminants from the manufacturing process, contact with packaging materials and adsorption of materials from the atmosphere. To remove contaminants, single or double immersion in a polar solvent, such as methanol or propan–2–ol, for about 5 min is generally superior to predevelopment with the mobile phase. For trace analysis, sequential immersion and predevelopment may be required to obtain the best results.

For inorganic oxide adsorbents the absolute RF (see later) value and the reproducibility of RF values depend on the layer activity. The latter is controlled by the adsorption of reagents, most notably water, through the gas phase. Physically adsorbed water can be removed from silica gel layers by heating at about 120° for 30 min. Afterwards, the plates are stored in a grease–free desiccator over blue silica gel. Heat activation is not normally required for chemically bonded layers. Equilibration of activated layers by exposure to the atmosphere is extremely rapid and layer activation is at times an unnecessary step. In modern air–conditioned laboratories, layers achieve a consistent level of activity that should provide sufficient reproducibility for most separations. Inorganic oxide layers can be adjusted to a defined activity by exposure to a defined gas phase in an enclosed chamber. Since manipulation in the atmosphere almost certainly readjusts this activity, it is best performed after application of the sample zones in a developing chamber that allows both layer conditioning and development in the same chamber (e.g. a twin–trough chamber), or in a separate conditioning chamber immediately before development. Atmospheres of different constant relative humidity can be obtained by using solutions of concentrated sulfuric acid or saturated solutions of various salts. Acid or base deactivation can be carried out in a similar manner by exposure to, for example, ammonia or hydrochloric acid fumes.

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Sample application

Drugs are applied to TLC plates as spots or bands of minimum size with a homogeneous distribution of material within the starting zone. For high–performance layers, with desirable starting spot diameters of about 1.0 to 2.0 mm, this corresponds to a sample volume of 100 to 200 nL if applied by a dosimeter (micropipette). For conventional TLC plates, sample volumes five- to ten–fold greater are acceptable. Desirable properties of the sample solution are summarised in Table 4. If scanning densitometry is used for detection, manual sample application with hand–held devices is inadequate. For densitometry, the starting position of each spot must be known accurately, which is achieved easily with mechanical devices that operate to a precise grid mechanism. Also, the sample must be applied to the layer without disturbing the surface, something that is nearly impossible to achieve using manual application.

Sample application devices for TLC encompass a wide range of sophistication and automation. The most popular devices for quantitative TLC use the spray–on technique. A controlled nitrogen–atomiser sprays the sample from a syringe or capillary, to form narrow, homogeneous bands on the plate surface. The plate is moved back and forth under the atomiser on a translational stage to apply bands of any length between zero (spots) and the maximum transit length of the spray head. Bands are typically 0.5 or 1.0 cm in length, with the longer bands used primarily for preparative–scale separations. The rate of sample deposition is also adjustable to accommodate sample solutions of different volatility and viscosity. An advantage of spray–on devices is that different volumes of a single standard solution can be applied for calibration purposes and the standard addition method of quantification is carried out easily by overspraying the sample already applied to the layer with a solution of the standard. Fully automated sample applicators can be programmed to select samples from a rack of vials and deposit fixed volumes of the sample, at a controlled rate, to selected positions on the plate. The applicator automatically rinses itself between sample applications and can spot or band a whole plate with different samples and standards without operator intervention.

Glass microcapillaries for conventional TLC and fixed–volume dosimeters (which consist of a 100 or 200 nL platinum–iridium capillary sealed into a glass–support capillary) for high–performance TLC are also commonly used for sample application and require less sophisticated instrumentation. The capillary tip is brought into contact with the plate surface using a mechanical device to discharge its volume. A click–stop grid mechanism is used to provide an even spacing of the samples on the layer and a frame of reference for sample location during scanning densitometry.

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Layers with a preadsorbent zone (a narrow zone prepared from a silica gel of low surface area with weak retention) simplify some aspects of sample application. This allows relatively large sample volumes or dirty samples to be applied to the preadsorption zone and their reconcentration to a narrow band at the interface between the preadsorbent and separation zones by a short development prior to chromatography. However, since the distribution of the sample may not be even within the band, the quantitative accuracy of densitometric measurements may be lowered using this approach.

Development

The principal development techniques in TLC are linear, circular and anticircular, with the velocity of the mobile phase controlled by capillary forces or forced–flow conditions. In any of these modes continuous or multiple development can be used to extend the application range. Radial development is used rarely for drug analysis and is not considered further. Forced–flow development requires sophisticated equipment not commonly found in analytical laboratories, and is not described here.

For linear (or normal) development, samples are applied along one edge of the plate and the separation developed for a fixed distance in the direction of the opposite edge. Viewed in the direction of development, the chromatogram consists of a series of compact symmetrical spots of increasing diameter or, if samples are applied as bands, in rectangular zones of increasing width.

In continuous development the mobile phase is allowed to traverse the layer under the influence of capillary forces until it reaches some predetermined position on the plate, at which point it is evaporated continuously. Evaporation of the mobile phase usually occurs at the plate atmospheric boundary using either natural or forced evaporation. Continuous development is used primarily to separate simple mixtures with a short development length and a weaker (more selective solvent) than employed for normal development.

In unidimensional multiple development, the TLC plate is developed for some selected distance, then either the layer or the mobile phase is withdrawn from the developing chamber, and adsorbed solvent evaporated from the layer before repeating the development process. The principal methods of unidimensional multiple development are summarised in Table 5. Multiple development provides a very versatile strategy for separating complex mixtures, since the primary experimental variables of development distance and composition of the mobile phase can be changed at any development step, and the number

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of steps varied to obtain the desired separation. Multiple development provides a higher resolution of complex mixtures than does normal or continuous development, can easily handle samples of a wide polarity range (stepwise gradient development) and, because the separated zones are usually more compact, leads to lower detection limits. Equipment for automated multiple development is commercially available.

For drug mixtures that span a wide retention range, some form of gradient development is required to separate all the components in either a single chromatogram or in separate chromatograms for successive developments. Continuous solvent–composition gradients, as commonly employed in HPLC, are used rarely in TLC. These require experimental conditions that are less convenient than those for step mobile–phase gradients. In addition, step gradients can be constructed easily to mimic a continuous linear gradient, with the added advantage that the zone refocusing effect can be employed to minimise zone broadening. Gradients of increasing solvent strength are used to fractionate complex mixtures by separating just a few components in each step. Individual drugs are usually identified and quantified at the intermediate steps at which the drugs of interest are separated. In this way, the zone capacity can be made much larger than predicted for a complete separation recorded as a single chromatogram. However, this approach is tedious when many components are of interest and it is difficult to automate. Alternatively, if incremental multiple development is used, the sample can be separated for the shortest distance in the strongest mobile phase, with each subsequent, longer development using mobile phases of decreasing solvent strength. This strategy is most useful when the final separation is to be recorded as a single chromatogram, but it is limited in zone capacity because all the components must be located between the sample origin and the final solvent front. The two approaches for exploiting solvent–strength gradients are thus complementary and selection is made based on the properties of the sample. The decreasing solvent–strength gradient approach is the operating basis of automated multiple–development chambers.

In two–dimensional TLC the sample is spotted at the corner of the layer and developed along one edge of the plate. The solvent is then evaporated, the plate rotated through 90° and redeveloped in the orthogonal direction. If the same solvent is used for both developments, the sample is redistributed along a line from the corner at which the plate was spotted to the corner diagonally opposite. In this case, only a small increase in resolution can be anticipated. The realisation of a more efficient separation system implies that the resolved sample should be distributed over the entire plate surface. This can be achieved only if the selectivity of the separation mechanism is complementary in the orthogonal directions. Using two solvent systems with complementary selectivity is the

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simplest approach to implement in practice, but it is often only partially successful. In many cases the two solvent systems differ only in their intensity for a given set of properties and are not truly orthogonal. Chemically bonded layers can be used in the reversed–phase and normal–phase modes, and they enable the use of additives and buffers as a further way to adjust selectivity. The acceptance of two–dimensional TLC for quantitative analysis, though, will depend on providing a convenient method for in situ detection and data analysis. It seems unlikely that two–dimensional development will be more widely used in TLC, except for qualitative analysis, until the problems of detection are solved.

Development chambers

The development process in TLC can be carried out in a variety of vessels that differ significantly in design and sophistication. For convenience these are often categorised under the headings of normal (N-chamber) and sandwich (S-chamber), and further subdivided according to whether the internal atmosphere is saturated (NS or SS) or unsaturated (NU or SU). Sandwich chambers have a depth of gas phase in front of the layer of less than 3 mm, with other chamber designs indicated as normal chambers. Saturation of the vapour phase is achieved by using solvent–saturated pads or filter papers as a chamber lining.

The twin–trough chamber is the most popular of the simplest TLC developing chambers. It consists of a standard rectangular developing tank with a raised, wedge–shaped bottom. The wedged bottom divides the tank into two compartments, so that it is possible to either develop two plates simultaneously or to use one compartment to condition the layer prior to development. The horizontal developing chamber (Fig 4) can be used in either the normal or sandwich configuration for either conventional edge–to–edge or simultaneous edge–to–centre development. Starting the development simultaneously from opposite edges allows the number of samples separated to be doubled in the same time. The sandwich configuration of the horizontal developing chamber is not suitable for mobile phases that contain volatile acids, bases or large amounts of volatile polar solvents, such as methanol or acetonitrile, because of the restricted access of the saturated vapour phase to the dry portion of the separation layer.

The automated developing chamber increases laboratory productivity and improves the reproducibility of separations by providing precise control of layer conditioning, mobile–phase composition, solvent–front migration distance and drying conditions. This chamber can be used in the normal or sandwich configuration with all the operational

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features preselected on a microprocessor–based control unit and monitored by sensor technology.

The automated multiple–development chamber (Fig 5) provides the necessary conditions and control for automated separations by incremental multiple development with a decreasing solvent–strength gradient. The operating parameters of layer conditioning, solvent–front migration distance, mobile–phase composition and drying time for each development, and the total number of developments for the separation, are entered into the computer–based control unit. The complete separation sequence is carried out without further intervention. Each development is typically 3 to 5 mm longer than the previous one and, depending on the complexity of the desired mobile phase gradient, a total of 10 to 30 developments are used, which requires 1.5 to 4.5 h for completion.

Detection

About 1 to 10 μg of coloured substances with a quantitative reproducibility rarely better than 10–30% can be detected by visual inspection of a TLC plate. This may be adequate for qualitative methods, but for reliable quantification in situ spectrophotometric methods are preferred, as they are more accurate and far less tedious and time consuming than excising zones from the layer for determination by conventional solution spectrophotometry. The fluorescence–quenching technique enables visualisation of UV-absorbing drugs on TLC plates that incorporate a fluorescent indicator. The zones of UV-absorbing substance appear dark against the brightly fluorescing background of a lighter colour when the plate is exposed to UV light of short wavelength. The method is not universal, since it requires overlap between the absorption bands of the indicator (γmax ≈ 280 nm with virtually no absorption below 240 nm) and the drug, but in favourable cases it is a valuable and non–destructive method for zone location.

All optical methods for the quantitative in situ evaluation of TLC chromatograms are based upon measuring the difference in optical response between a sample–free region of the layer and regions of the layer in which separated substances are present. Reflectance measurements can be made at any wavelength from the UV to the near infrared (185 to 2500 nm). The relationship between signal and sample amount in the absorption mode is non–linear, and does not conform to any simple equation. The principal method of quantification in TLC is by calibration using a series of standards that span the concentration range of the drug to be determined. The calibration curve is usually based on a second–order polynomial fit for the calibration standards, with individual samples

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quantified by interpolation only.

The determination of drugs that fluoresce on TLC plates is fundamentally different to absorption measurements. At low sample concentrations the fluorescence signal F is described adequately by F = φI0εbC, where φ is the quantum yield, I0 the intensity of the excitation source, ε the molar absorption coefficient, b the thickness of the TLC layer and C the sample amount. With the exception of the sample amount all terms in this expression are constant, or fixed by the experiment, and therefore the fluorescence emission is linearly dependent on the sample amount over two or three orders of magnitude.

Derivatisation reactions

There is a long history of the use of derivatisation reactions in TLC to visualise colourless compounds. Many of these reactions are of a qualitative nature, which was not a problem when TLC was used rarely for quantification. Some of these reactions have been adapted to the demands of quantitative scanning densitometry, as either pre- or post–chromatographic treatments, and new reagents and methods have been added specifically for quantitative measurements in TLC.

In post–chromatographic reactions the reagents can be applied to the layer through the gas phase or by evenly coating the layer with a solution of the reagents. Gas–phase methods are fast and convenient, but restricted by the number of useful reagents. Examples include iodine, ammonia and hydrogen chloride, which are applied by inserting the layer into a tank that contains a saturated atmosphere of the reactive vapour. Spraying or dipping are used to apply reagents in solution to the layer. Spray techniques that use simple atomisers have long been used in TLC, but reagent application by this method is quite difficult to perform well. The homogeneity of the reagent distribution over the layer depends on many factors, such as the droplet size, distance between the spray device and layer, direction of spraying and discharge rate of the reagent. If ventilation of the workspace is inadequate, spray techniques can be a potential health hazard. For quantitative analysis, immersion of the layer into a solution of the reagents in a controlled manner, referred to as dipping, is the preferred technique, since it does not rely on manual dexterity and produces superior results in scanning densitometry. Some spray reagents do not make good dipping solutions because they contain solvents that are too aggressive or viscous for convenient application (aqueous concentrated acids and bases, for example). Dipping solutions are usually less concentrated than spray reagents and water is often replaced by an alcohol for adequate permeation of reversed–phase layers. In general, it is necessary to reformulate dipping solutions from earlier recipes for spray solutions and, possibly, to change the reaction

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conditions. Automated low–volume dipping chambers provide a uniform speed and dwell time for the immersion process, which typically requires only a few seconds, and is long enough to impregnate the layer with solution, but not long enough to wash sample components off the layer.

Post–chromatographic derivatisation reactions can be classified as reversible or destructive, depending on the type of interaction between the reagents and separated drugs, and as selective or universal, based on the specificity of the reaction. The most common reversible methods employ iodine vapour, water, fluorescein, or pH indicators as visualising reagents. In the iodine vapour method, the dried plate is enclosed in a chamber that contains a few crystals of iodine; components on the chromatogram are stained more rapidly than the background and appear as yellow–brown spots on a light yellow background. Simply removal of the plate from the visualisation chamber to allow the iodine to evaporate can reverse the reaction. Spraying a TLC plate with water reveals hydrophobic compounds as white spots on a translucent background when the water–moistened plate is held against the light. Solutions of pH indicators (e.g. bromocresol green, bromophenol blue) are widely used to detect acidic and basic drugs.

Irreversible methods are more common for quantification and comprise hundreds of reagents based on selective chemistries reduced to standard operations over several decades of use. Some typical examples used in drug identification are summarised in Table 6. Reagents that are specific to functional groups or selective for compound classes can be applied to determine low levels of substances in complex matrices such as biological fluids and plant extracts.

The fluorescence response for drugs and their derivatives on TLC layers is sometimes less than that expected from solution measurements, is observed at different excitation and emission wavelengths than in solution, and may decrease with time. Adsorption onto the sorbent layer provides additional nonradiative pathways for the dissipation of the excitation energy, which is most probably lost as heat to the surroundings and reduces the observed fluorescence signal. The extent of fluorescence quenching often depends on the sorbent used for the separation and is generally more severe for silica gel than for chemically bonded sorbents. In most cases, impregnating the layer with a viscous liquid, such as liquid paraffin or Triton X-100, before evaluating the separation enhances the emission signal (in favourable cases ten- to 200–fold). The general mechanism of fluorescence enhancement is assumed to be dissolution of the sorbed solute with enhancement in response due to the fraction of solute that is transferred to the liquid phase, where fluorescence quenching is less severe. Viscous solvents are employed to minimise zone broadening from diffusion in the liquid phase during the measurement process.

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Slit–scanning densitometers

Commercial instruments for scanning densitometry usually allow measurements in the reflectance mode by absorbance or fluorescence. Most instruments employ grating monochromators for wavelength selection and spectrum recording in the absorption mode. For fluorescence measurements a filter, which transmits the emission wavelength envelope but attenuates the excitation wavelength, is placed between the detector and the plate. The separations are scanned at selectable speeds up to about 10 cm/s by mounting the plate on a movable stage controlled by stepping motors. A fixed sample beam is shaped into a rectangular area on the plate surface, through which the plate is transported in the direction of development. Each scan, therefore, represents a lane of length defined by the solvent–front migration distance and width by the slit dimensions of the source. Distorted chromatograms can be corrected by track optimisation, in which the sample zones are integrated as if the slit had moved along an optimum track from peak maximum to peak maximum. In modern TLC the relative standard deviation from all errors, instrumental and chromatographic, can be maintained below 2 to 3%, which makes it a very reliable quantitative tool.

Image analysers

For image analysers, scanning takes place electronically using a combination of a computer with video digitiser, light source, monochromators and appropriate optics to illuminate the plate and focus the image onto a charged–coupled device video camera. The captured images are initialised, stored and transformed by the computer into chromatographic data. Background subtraction and thresholding are common data–transformation processes. Image analysers provide fast data acquisition, simple instrument design and convenient software tools that search and compare sample images. Technological limitations currently prevent image analysers from competing with mechanical scanners in terms of sensitivity, resolution and available wavelength–measuring range. They have proved popular for less–demanding tasks, for the development of field–portable instruments and as a replacement for photographic documentation of TLC separations.

Other instrumental detection methods

Radioisotope–labelled drugs and their metabolites can be detected selectively with good sensitivity by imaging detectors that use windowless gas–flow proportional counters as detectors. The proportional counter is filled with a mixture of argon and methane gas, which

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is ionised locally by collision with beta or gamma rays produced by radioactive decay in the sample zones that contain radioisotopes. The local bursts of ionised gas molecules are sensed by a position–sensitive detector and stored in computer memory. These signals are accumulated for quantitative measurements.

Flame ionisation has been used to detect samples of low volatility that lack a chromophore for optical detection. The separation is performed on specially prepared, thin, quartz rods with a surface coating of sorbent attached by sintering. The rods are developed in the normal way, usually held in a support frame that also serves as the scan stage after the rods have been removed from the developing chamber and dried. The rods are moved at a controlled speed through a hydrogen flame and the signal processed in a similar manner to the flame ionisation detector used in gas chromatography. The linear working range of the detector is about 3 to 30 μg for most substances. There are few reported applications in drug analysis.

General interfaces are available for the in situ measurement of mass, infrared and Raman spectra of separated zones on TLC plates. Individual results in terms of sensitivity and spectral quality are impressive, but none of these methods are used routinely in drug analysis laboratories. This is a possible area for development.

Method development

The development technique is selected based on the number of detectable components in the mixture and their polarity range (Table 7). A single development with capillary–controlled flow may be too difficult or impossible for mixtures that contain more than eight to ten components of interest. In addition, if the range of polarities is too wide, multiple development techniques using mobile phase gradients are necessary. It is only necessary to separate the components of major interest from each other and from the less important components, which need not be separated individually. Method development is easier if standards for the relevant compounds are available. Standards simplify zone tracking and enable detection characteristics and the possibility of spectroscopic resolution of incompletely separated zones to be established. Standards are also required for calibration, if quantification is required, and to construct spectral libraries for identification purposes. The expected concentration range of relevant compounds may indicate the need for derivatisation to obtain the required detection limits and to increase zone separation of neighbouring compounds if one compound is a minor component with similar migration properties to a major component.

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Development method Dimensions Zone capacity

Predictions from theory

Capillary–controlled flow 1 <25

<80 (up to 150 depending on Forced flow 1 pressure limit)

Capillary–controlled flow 2 <400

Forced flow 2 Several thousand

Based on experimental observations

Capillary–controlled flow 1 12–14

Forced flow 1 30–40

Capillary–controlled flow (AMD) 1 30–40

Capillary–controlled flow 2 About 100

Table 7. Zone capacity calculated or predicted for different conditions in TLC

A general guide to the selection of stationary phases for TLC separations is summarised in Fig 6. Silica gel is generally the first choice to separate drugs of low molecular weight that are soluble in moderately polar organic solvents. Reversed–phase chromatography on chemically bonded layers is generally used to separate drugs that are difficult to separate on silica gel because of inadequate retention, inadequate selectivity or zone asymmetry. Ionic compounds and easily ionised compounds are separated frequently by reversed–phase chromatography using buffered mobile phases (weak acids and bases) or ion–pair reagents (strong acids and bases). Only a limited number of stationary phases are available for ion–exchange chromatography, which is not a widely used separation mechanism in TLC.

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Figure 6. Mode selection guide for TLC (LSC, liquid–solid chromatography on an inorganic oxide layer; BPC, liquid–solid chromatography on a chemically bonded layer; RPC, reversed–phase chromatography with a chemically bonded layer and an aqueous organic mobile phase; IPC, ion–pair chromatography with reversed–phase separation conditions; PC, precipitation chromatography used to separate polymers based on solubility differences in a mobile–phase solvent gradient.

Since the solvent used for the separation is evaporated prior to detection, a wider range of UV-absorbing solvents are commonly used in TLC than is the case for HPLC. Solvents must be of high purity, since involatile impurities and stabilisers remain sorbed to the layer that causes problems in the detection step. Multicomponent mobile phases can produce a mobile–phase gradient in the direction of development through demixing. If demixing is complete, zones with sharp boundaries are formed, which separate the chromatogram into sections of different solvent composition and, therefore, selectivity. Demixing effects are less apparent when saturated developing chambers are used. These considerations hinder optimisation strategies based on the composition of the mobile phases as popularised in HPLC.

The selection of a mobile phase to separate simple mixtures need not be difficult and can be arrived at quickly by guided trial and error methods. A solvent of the correct strength for a unidimensional development migrates the sample components into the RF range 0.2 to 0.8,

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or thereabouts and, if of the correct selectivity, distributes the sample components evenly throughout this range. Solvent systems can be screened in parallel using several development chambers, as prescribed in the PRISMA model. To select suitable mobile phases, the first experiments are carried out on TLC plates in unsaturated chambers with ten solvents, chosen from the different selectivity groups, indicated by bold type in Table 8. After these screening experiments with single solvents, the solvent strength is either reduced or increased so that the substance zones are distributed in the RF range 0.2 to 0.8. If the substances migrate into the upper third of the plate, the solvent strength is reduced by dilution with hexane (the strength–adjusting solvent). If the substances remain in the lower third of the plate with the single solvents, the solvent strength is increased by the addition of a strong solvent, such as water or acetic acid. A similar procedure is followed in the reversed–phase mode, except that solvent selection is limited to water–miscible solvents and water is used as the strength–adjusting solvent. From these trial experiments, those solvents that show the best separation are selected for further optimisation in the second part of the model.

Selectivity group Solvent

I n-Butyl ether, diisopropyl ether, methyl t-butyl ether, diethyl ether

II n-Butanol, propan–2–ol, propanol, ethanol, methanol

III Tetrahydrofuran, pyridine, methoxyethanol, dimethylformamide

IV Acetic acid, formamide

V Dichloromethane, 1,1–dichloroethane

VI Ethyl acetate, methyl ethyl ketone, dioxane, acetone, acetonitrile

VII Toluene, benzene, nitrobenzene

VIII Chloroform, dodecafluoroheptanol, water

Table 8. Solvent–strength parameters and selectivity groups for solvents used for separations on silica gel

Between two and five solvents can be selected to construct the PRISMA model for solvent optimisation. Modifiers required to maintain an acceptable zone shape, such as acids and ion–pair reagents, can be added in a low and constant concentration, so that their influence

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on solvent strength can be neglected. The PRISMA model (Fig 7) is a three–dimensional geometrical design that correlates solvent strength with selectivity of the mobile phase. The model consists of three parts: the base or platform (represents the modifier), the regular part of the prism with congruent base and top surfaces, and the irregular truncated top prism (frustum). The lengths of the edges of the prism (SA, SB, SC) correspond to the solvent strengths of the neat solvents (A, B, C). Since the selected solvents usually have different solvent strengths, the lengths of the edges of the prism are generally unequal and the top plane of the prism is not parallel and congruous with its base. If the prism is cut parallel to its base at the height of the lowest edge (determined by the solvent strength of the weakest solvent, solvent C in Fig 7), the lower part gives a regular prism, and the top and any planes, which represent weaker solvents diluted with a strength–adjusting solvent, are parallel equilateral triangles. The upper frustum of the model is used for mobile–phase optimisation of polar drugs in normal phase TLC, while the regular part is used to separate moderately polar drugs in normal–phase TLC and all separations by reversed–phase TLC.

Figure 7. The PRISMA mobile–phase optimisation model, showing the construction of the prism and the arrangement of selectivity points on the top face or horizontal plane cut through the prism.

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For polar compounds, optimisation is always started on the top irregular triangle of the model, either within the triangle, when three solvents are selected, or along one side, for binary mobile phases. Any solvent composition on the face of the triangle can be represented by a three–co–ordinate selectivity point (PS), each co–ordinate corresponding to the volume fraction of the solvent at that position on the triangle (Fig 7). Optimisation is commenced by selecting solvent combinations that correspond to the centre point PS = 333 and three other points close to the apexes of the triangle PS = 811, 181 and 118. If the separation obtained is insufficient, other selectivity points are tested around the solvent combination that gave the best separation. On changing the selectivity points on the top triangle the solvent strength changes as well, especially when the solvent strengths of the solvents used to construct the prism are significantly different. The solvent strength should be adjusted with the strength–adjusting solvent as required to maintain the separation in the optimum RF range. Failure to obtain the beginning of a separation requires that a new prism be constructed, using a different solvent for at least one of the edges.

For reversed–phase TLC, the solvation–parameter model provides a convenient computer–aided approach to method development. Suitable water–miscible solvents with a range of selectivity include methanol, propan–2–ol, 2,2,2–trifluoroethanol, acetonitrile (or dioxane), acetone (or tetrahydrofuran) and dimethylformamide (or pyridine). For optimisation of systems (stationary phases and binary mobile phases), preliminary results in the form of system maps (a continuous plot of the system constants against mobile–phase composition) are required. System maps are a permanent record of the system properties used in all calculations and are available for most common layers and indicated solvents for selectivity optimisation. For each computer–simulated separation a retention map is calculated from the system map and displays the computed RF values as a continuous function of the binary mobile–phase composition. A typical retention map for the computer–predicted separation of analgesics on an octadecylsiloxane–bonded layer with 2,2,2–trifluoroethanol–water mixtures as the mobile phase is shown in Fig 8. Solvent compositions that result in an acceptable zone separation are identified easily by visual inspection. Computer simulation of retention maps allows those systems (defined as a combination of stationary and mobile phase) likely to provide an acceptable separation to be identified before experimental work commences. The agreement between model predicted and experimental RF values is generally good, typically better than 0.05 RF units. A mixture–design approach is used to extend this method to ternary solvent mixtures.

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Figure 8. Retention map for the simulation of the separation of analgesics by reversed–phase TLC on an octadecylsiloxane–bonded layer with 2,2,2–trifluoroethanol–water as the mobile phase [1, chlorphenamine (chlorpheniramine); 2, ibuprofen; 3, naproxen; 4, phenacetin; 5, aspirin; 6, caffeine; 7, acetaminophen].

For drug mixtures of a wide polarity range, stepwise changes in solvent composition are required to achieve a satisfactory TLC separation. Models to calculate migration distances using incremental multiple development with increasing and decreasing solvent–strength gradients have been described, but are complicated and not widely used. Optimised gradients for automated multiple development are usually arrived at by more pragmatic means. Methods based on a universal gradient commence with methanol, end with hexane and use either dichloromethane or methyl t-butyl ether as the intermediate solvent for separations on silica gel. By scaling and superimposing the chromatogram of the separation above the theoretical gradient profile, those regions of the chromatogram that affect the separation are identified easily. The solvent composition for the initial and final development steps is adjusted to eliminate those portions of the gradient that do not contribute to the separation. The gradient shape is modified to enhance resolution in those regions of the chromatogram that are separated poorly or to minimise regions devoid of sample zones. For moderately complex mixtures this approach is often satisfactory. If, after the above adjustments, the separation is inadequate it is necessary to identify a more

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selective solvent for problem regions in the gradient. The PRISMA model can be used at this point to identify more selective solvents to incorporate into the gradient as a replacement for the initial, terminal or base solvent.

Preparative thin–layer chromatography

Preparative TLC is used mainly to purify drugs or to isolate drug metabolites and impurities in amounts of about 1 to 100 mg for subsequent use as reference materials, structural elucidation, biological activity evaluation and other purposes. Scale up from analytical TLC is achieved by increasing the thickness of the layer (loading capacity increases with the square root of the layer thickness) and by increasing the plate length used for sample application. Precoated TLC plates for preparative chromatography vary in size from 20 cm × 20 cm to 20 cm × 40 cm and are coated with 0.5 to 10 mm thick layers, with the most popular thickness being 1.0 to 2.0 mm. As the average particle size (≈ 25 μm) and size distribution (5 to 40 μm) are larger for preparative layers, and as sample overload conditions are used commonly in preparative chromatography, invariably inferior separations in a longer time (≈ 1 to 2 h) are obtained compared with analytical separations. Resolution can be increased significantly by using wedge–shaped, gradient–thickness layers. These layers have a uniform increase in thickness from 0.3 mm at the bottom to 1.7 mm at the top. Sample bands are focused during migration by the negative mobile–phase velocity gradient created by the layer geometry.

Sample application is a critical step in preparative TLC, and if performed improperly can destroy all or part of the separation. The sample, usually as a 5 to 10 % (w/v) solution in a volatile solvent, is applied as a band along one edge of the layer to give a maximum sample load of about 5 mg/cm for each millimetre of layer thickness. Sample loads are usually lower for difficult separations and for cellulose and chemically bonded layers. Any of the automated band applicators for analytical TLC are suitable for sample application in preparative TLC. Manual sample application by syringe or glass pipette must be performed carefully to avoid damaging the layer and producing irregularly shaped migrating zones. A short predevelopment, of about 1 cm with a strong solvent, is often useful to refocus manually applied bands. Preparative layers with a preadsorbent zone are useful for manual sample application, since the focusing mechanism can be used to correct for poor sample–application technique. In all cases, it is important that the sample solvent is evaporated fully from the layer prior to the start of the separation to avoid the formation of distorted separation zones. It is usual to leave a blank margin of 2 to 3 cm at each vertical

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edge of the layer to avoid uneven development.

Most of the changes in preparative TLC over the past decade have occurred in the method of development. Conventionally, ascending development in large–volume tanks that hold a number of preparative layers in a rack is used commonly. In laboratories that perform preparative TLC on a regular basis, higher resolution and shorter development times are achieved by using forced–flow development or rotation planar chromatography (accelerated development using centrifugal force). These methods allow conventional development and elution with on–line detection and automated fraction collection to be used.

After development, physical methods of zone detection are used to identify the sample bands of interest. Layers that contain a UV indicator for fluorescence quenching or the adsorption of iodine vapours are useful for this purpose. If a reactive spray reagent is used for visualisation, it should be sprayed on a small strip of the chromatogram only, so as not to contaminate the remainder of the material. Once the bands of interest are located, the zones are scraped off the plate carefully with a spatula or similar tool. A number of devices based on the vacuum–suction principle for removing the marked zones from the plate are available also. Soxhlet extraction, liquid extraction or solvent elution with a polar solvent is used to recover drugs from the sorbent. For solvent extraction, water is often added to dampen the silica gel prior to extraction with a water–immiscible organic solvent. Chloroform and ethanol (methanol is less suitable because of its higher silica solubility) are widely used for solvent elution. Colloidal silica can be removed by membrane filtration prior to vacuum stripping of the solvent.

Retardation factor

The retardation factor, or RF value, is the fundamental parameter used to characterise the position of a sample zone in a TLC chromatogram. For linear development it represents the ratio of the distance migrated by the sample compared to the distance travelled by the solvent front:

R F = ZX/(Zf − Z0)

where ZX is the distance travelled by the sample from its origin, (Zf − Z0) the distance travelled by the mobile phase from the sample origin, Zf the distance travelled by the mobile phase measured from the mobile phase level at the start of the separation, and Z0 the distance from the sample origin to the mobile phase level at the start of the separation. The

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boundary conditions for RF values are 1 ≥ RF ≥ 0. The RF value is generally calculated to two decimal places. Some authors prefer to tabulate values as whole numbers, as hRF values equivalent to 100RF.

Drug identification

The RF value is affected too adversely by measurement difficulties and by variations in experimental and environmental conditions to be a useful identification parameter on its own. When standard substances are available, it is common practice to run standards and samples in the same system for improved confidence in identification based on RF values. If scanning densitometry is used, an acceptable agreement in RF values is generally supported by the automated matching of specific absorbance ratios or full spectra for the samples and standards.

In drug–screening programmes, in which simultaneous separation of standards and samples is impractical, the certainty of drug identification is improved by simultaneous separation of a series of related standard substances that allow the experimental RF values to be corrected to standardised RF values from automated library searches:

c c c hR F(X) = hRF(A) + [Δ /Δ][hRF(X) − hRF(A)]

c c c Δ = hRF(B) − hRF(A)

Δ = hRF(B) − hRF(A)

where hRF(X) is the RF value for substance X, hRF(A) and hRF(B) are the RF values for the standard substances that bracket hRF(X), and the superscript c indicates the corrected value for X and the accepted values for A and B. Alternatively, a calibration curve of experimental

RF values against the accepted RF values for the standards can be prepared and used to transpose experimental RF values to corrected RF values. Typically, four evenly spaced standard substances with the sample origin (hRF = 0) and solvent front (hRF = 100) are included as additional reference points.

Database searches

Database searches are used in systematic toxicological analysis to identify suspect

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substances in biological fluids and post–mortem tissue samples. Extracted samples are separated in one or more standard TLC systems. The corrected RF values, often combined with the results of sequential post–chromatographic colour reactions, are then entered into the search program. The input data are automatically compared against a database of reference drugs, common metabolites, natural contaminants, etc., for identification. A number of chemometric procedures can be used for data analysis, but the most common approach is based on the mean list method.

It is assumed that the errors in individual measurements are random and can be described by a standard deviation. The precision of the separation system can then be described as the mean of the standard deviation of all substances separated in the system, called the system mean standard deviation. This allows a confidence interval or window to be assigned to the system as some multiple (typically three) of the system mean standard deviation. Each RF value in the system database that appears in the window could be confused with the original substance. The number of substances identified as above is called the list length. Repeating the process for all RF values in that system and averaging the individual list lengths provides the mean list length for that system. The mean list length indicates, on average, the number of substances in the database that qualify as candidates for the identification of a single drug. The shorter the mean list length, the greater the information potential of the system. Combining the results from additional retention parameters in complementary standard separation systems, colour reactions, spectroscopic data, etc., minimises the mean list length to the point that only a small number of candidate compounds for the unknown are indicated. More specific tests can then be used to identify the unknown from among the small number of indicated possibilities. For systematic drug identification in forensic toxicology, commonly two or more complementary TLC systems combined with the results from several in situ sequential colour reactions are used. For drugs of toxicological interest, a mean list length from two to ten is possible.

Systematic drug identification

Systematic toxicological analysis takes advantage of the separation of an unknown substance in standard TLC systems (or other chromatographic systems) to establish the probable identity of the substance by reference to a database of candidate compounds using a statistical comparison approach, such as the mean list method. Suitable chromatographic techniques for systematic toxicological analysis must meet the following criteria:

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1. the drugs must exhibit acceptable chromatographic properties in the separation system

2. the RF values for the drugs must be distributed evenly over the full RF range

3. the RF values are standardised in such a way that good interlaboratory reproducibility is obtained

4. when more than one separation system is used, there must be a low correlation of RF values in the selected systems.

TLC systems that meet these requirements are described below.

Since pH-dependent extractions are customarily used in drug extraction and work–up procedures, generally different TLC systems are used to separate acidic and basic drugs, with neutral drugs likely to occur in both fractions. The Committee for Systematic Toxicological Analysis of the International Association of Forensic Toxicologists (TIAFT) recommended 11 separation systems for drug identification (Table 9). Four systems (1 to 4a) are to separate neutral and acidic drugs and seven systems (4b to 10) are to separate neutral and basic drugs. Reference data are presented for about 1600 toxicologically relevant substances. For general drug screens, the use of two separation systems with a low correlation is recommended: systems 2 and 4(a) for neutral and acidic drugs and systems 5 and 8 for neutral and basic drugs (systems 7 and 8 are nearly as good). Combining colour reactions with the TLC data improves the certainty of identification significantly. Four colour reactions are carried out on the same plate in sequence. After each step the colour is noted and encoded by means of a colour chart (1, yellow; 2, orange; 3, brown; 4, red; 5, purple; 6, black; 7, blue; 8, green; 0, no spot observed). The sequence consists of formaldehyde vapour and Mandelin’s reagent, water, fluorescence under 366 nm irradiation and modified Dragendorff’s reagent. Other sequential colour reactions can be encoded and utilised in the same way. The Merck Tox Screening System (MTSS) contains the TIAFTTLC database and several other useful tools for searches using other chromatographic and spectroscopic databases and user–created databases.

TLC system

Chamber Stationary Reference c Error No Mobile phase hR type phase compounds* F window†

Chloroform–acetone (1) Saturated Silica gel Paracetamol 15 7 (4:1)

Clonazepam 35

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TLC system

Chamber Stationary Reference c Error No Mobile phase hR type phase compounds* F window†

Secobarbital 55

Methylphenobarbital 70

(2) Ethyl acetate Saturated Silica gel Sulfathiazole 20 8

Phenacetin 38

Salicylamide 55

Secobarbital 68

Chloroform–methanol (3) Saturated Silica gel Hydrochlorothiazide 11 8 (9:1)

Sulfafurazole 33

Phenacetin 52

Prazepam 72

Sulfadimidine 13 Ethyl Hydrochlorothiazide 34 (4a) acetate–methanol–25% Saturated Silica gel 11 Temazepam 63 ammonia (17:2:1) Prazepam 81

20 Ethyl Morphine Codeine 35 (4b) acetate–methanol–25% Saturated Silica gel Hydroxyzine 10 53 ammonia (17:2:1) Trimipramine 80

(5) Methanol Unsaturated Silica gel Codeine 20 8

Trimipramine 36

Hydroxyzine 56

Diazepam 82

22 Methanol–n-butanol Codeine 48 (6) (3:2) containing 0.1 Unsaturated Silica gel Diphenhydramine 9 65 mol/L sodium bromide Quinine Diazepam 85

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TLC system

Chamber Stationary Reference c Error No Mobile phase hR type phase compounds* F window†

Silica gel impregnated 18 Atropine Codeine Methanol–25% with 0.1 33 (7) Saturated Chlorprothixene 9 ammonia (100:1.5) mol/L KOH 56 Diazepam in methanol 75 and dried

Silica gel impregnated Codeine 6 20 Cyclohexane– toluene– with 0.1 Desipramine (8) Saturated 36 8 diethylamine (15:3:2) mol/L KOH Prazepam 62 in methanol Trimipramine and dried

Silica gel impregnated Desipramine 11 Chloroform–methanol with 0.1 Physostigmine 36 (9) Saturated 11 (9:1) mol/L KOH Trimipramine 54 in methanol Lidocaine 71 and dried

Silica gel impregnated 15 Amitriptyline with 0.1 30 (10) Acetone Saturated Procaine Papaverine 9 mol/L KOH 47 Cinnarizine in methanol 65 and dried

Table 9. TLC systems recommended by TIAFT for systematic toxicological analysis (drug database, de Zeeuw et al. 1992)

* Concentration of reference standards, 2 mg/mL of each substance. † Error window defined as three times the mean standard deviation

Romano et al. (1994) presented data for 443 drugs in four TLC systems using high–performance silica gel TLC plates (Table 10).

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TLC system

Chamber Stationary Reference c No Mobile phase hR * type phase compounds F

Morphine 25 Ethyl Strychnine 44 (1) acetate–methanol– 30% Saturated Silica gel Aminopyrine 70 ammonia (17:2:3) Cocaine 85

Clobazam 15 Cyclohexane– toluene– Aminopyrine 29 (2) Saturated Silica gel diethylamine (13:5:2) Mebeverine 47 Amitriptyline 60

Caffeine 09 Ethyl acetate– Ketamine 24 (3) Saturated Silica gel chloroform (1:1) Flunitrazepam 44 Prazepam 61

Silica gel impregnated Imipramine 20 with 0.1 mol/L Pericyazine 37 (4) Acetone Saturated KOH in Aminopyrine 62 methanol and Lidocaine 78 dried

Table 10. The separation systems recommended by Romano et al. (1994) for systematic toxicological analysis by TLC (drug database: Romano et al. 1994)

c *Error window estimated as 7–9% R F.

These systems use slight modifications of the mobile–phase compositions recommended by TIAFT. The UniTox system uses three TLC systems (Table 11). System 1 is designed to separate neutral and acidic drugs and systems 2 and 3 to separate basic, amphoteric and quaternary drugs. Two of the separation systems are based on reversed–phase separations designed to complement the more familiar silica gel separations. The database contains over 375 drugs of general toxicological interest, including a large number of amphetamines.

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TLC system

Chamber Reference c Error No Mobile phase Stationary phase hR type compounds F window

Diazepam 16 Octadecylsiloxane–bonded Secobarbital 35 (1) Methanol–water (13:7) Unsaturated 4 silica gel Phenobarbital 54 Paracetamol 74

Codeine 16 Toluene– acetone– Promazine 36 (2) Saturated Silica gel 5 ethanol–25% ammonia Clomipramine 49 Cocaine 66

Hydroxyzine 20 Methanol–water–concentrated Octadecylsiloxane–bonded Lidocaine 46 (3) Unsaturated 4 hydrochloric acid (50:50:1) silica gel Codeine 66 Morphine 81

Table 11. The UniTox system for systematic toxicological analysis by TLC [drug database, Ojanpera 1995; additional compounds (amfetamines) in Ojanpera et al. 1991]

The Toxi-Lab system is a TLC kit for toxicological drug screening; it contains equipment for extraction, development, detection and identification. Separations are performed on unsupported, particle–embedded glass–fibre sheets with holes punched in them to receive samples and standards as extraction or reference disks. A combination of silica gel and reversed–phase separations together with sequential colour reactions is used for identification and confirmation purposes. The database is designed for computer searches with results entered in a standard format.

Pesticides are a further class of toxic substances of interest to systematic toxicological analysis because of their general availability, toxicity and potential confusion with drugs. Erdmann et al. (1990) developed a database for 170 commonly used pesticides separated in three standardised TLC systems (Table 27.12). The systems in Table 27.12 supplement those in Table 27.9, in which many common pesticides migrate with the solvent front. Systems 1 and 2 are recommended for general screening and system 3 for the identification of special compounds not distinguished in the first two systems.

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TLC system

Chamber Stationary Reference c No Mobile phase hR type phase compounds F

Hexane–acetone (1) Saturated Silica gel Triazophos 21 (4:1)

Parathion–methyl 30

Pirimiphos–methyl 49

Quintozene 84

Toluene–acetone (2) Saturated Silica gel Carbofuran 20 (19:1)

Azinophos–methyl 42

Methidathion 56

Parathion–ethyl 85

Chloroform–acetone (3) Saturated Silica gel Nicotine 11 (1:1)

Ioxynil 39

PCP 60

Methabenzthiazuron 85

Table 12. Standardised TLC systems for the screening of pesticides (pesticide database, Erdmann et al. 1990)

General applications

Thousands of general and validated methods are available for the determination of drugs as pharmaceutical products and in biological fluids. Since the zone capacity of TLC systems is small, there are no general methods for drugs as a class, but there are a large number of methods for individual drugs defined by therapeutic or chemical categories. These still

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represent substantial diversity driven by the need to optimise selectivity for each group of substances taken for analysis. This information can provide a useful starting point for system selection, but is no general substitute for systematic method development. For these reasons universal methods for general drug analysis do not exist and earlier attempts at systematised approaches for different drug categories have failed to keep pace with the growth in number of drugs in those categories. In addition, systems recommended for the separation of individual drug categories rarely prove optimal for the separation of individual drugs and their impurities or metabolites.

Systems for thin-layer chromatography

The TLC systems given below are general screening methods for nitrogenous bases (Systems TA, TB, TC, TL, TAE and TAF), for acids and neutral compounds (Systems TD, TE, TF and TAD) with a further three general screening methods (Systems TAJ, TAK and TAL). Furthermore, seven systems specific for pesticides (Systems TW, TX, TY, TZ, TAA, TAB and TAC) and another 19 systems covering specific groups of drugs are also listed. The drugs are divided into chemical or pharmacological groups, but some other drugs are included with certain groups if they are chemically similar and would be extracted with that group.

There may not be one best system for a particular separation and a number of systems can be applied from those suggested. However, each of the systems described has been selected because it gives a good spread of Rf values, has high reproducibility, and has low correlation with the other systems selected for that group of drugs. These systems have proved useful for a large number of groups of drugs over the years and are robust and dependable. At least three systems are given for each group, where possible. The Rf values of the reference compounds suggested for the general screening systems have been derived using solutions of approximately 2 mg/mL of each substance.

Fluorescent plates should always be used and the absorption or fluorescence of the drug under ultraviolet light (both 254 and 350 nm) should be used as a location procedure. The suggested locating agents include general ones to visualise any drug that might be present as well as more specific ones to pick out individual classes of drugs.

Note

In the tables of Rf values, a dash indicates that no value is available for the compound.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 38 Thin–layer Chromatography (TLC)

Systems described in Table 27.9 have been assigned the following codes:

Code Number in Table 27.9

TA 7

TB 8

TC 9

TD 1

TE 4

TF 2

TL 10

TAD 3

TAE 5

TAF 6

Screening systems

Basic nitrogenous drugs

A. H. Stead et al., Analyst, 1982, 107, 1106–1168 and R. A. de Zeeuw et al., Thin-layer Chromatographic Rf Values of Toxicologically Relevant Substances on Standardized Systems: Report XVII of the DFG Commission for Clinical-Toxicological Analysis, 2nd Edn, VCH, Weinheim, 1992.

System TA

Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Methanol:strong ammonia solution (100:1.5). Reference compounds: Atropine Rf 18, Codeine Rf 33, Chlorprothixene Rf 56, Diazepam Rf 75.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 39 Thin–layer Chromatography (TLC)

System TB

Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Cyclohexane:toluene:diethylamine (75:15:10). Reference compounds: Codeine Rf 06, Desipramine Rf 20, Prazepam Rf 36, Trimipramine Rf 62.

System TC

Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Chloroform:methanol (90:10). Reference compounds: Desipramine Rf 11, Physostigmine Rf 36, Trimipramine Rf 54, Lidocaine Rf 71.

System TL

Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, 0.1 M potassium hydroxide in methanol, and dried. Mobile phase: Acetone. Reference compounds: Amitriptyline Rf 15, Procaine Rf 30, Papaverine Rf 47, Cinnarizine Rf 65.

System TAE

Plates: Silica gel G, 250 μm thick. Mobile phase: Methanol. Reference compounds: Codeine Rf 20, Trimipramine Rf 36, Hydroxyzine Rf 56, Diazepam Rf 82.

System TAF

Plates: Silica gel G, 250 μm thick.

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Mobile phase: Methanol:n-butanol (60:40) and 0.1 mol/L NaBr. Reference compounds: Codeine Rf 22, Diphenhydramine Rf 48, Quinine Rf 65, Diazepam Rf 85.

Location reagents for systems TA, TB and TC

Ninhydrin spray Spray the plate with the reagent and then heat in an oven at 100° for 5 min. Violet or pink spots are given by primary amines and yellow colours by secondary amines. FPN reagent Red or brown-red spots are given by phenothiazines and blue spots by dibenzazepines. This reagent may be used to overspray a plate which has been previously sprayed with ninhydrin spray. Dragendorff spray Yellow, orange, red-orange, or brown-orange spots are given by tertiary alkaloids. This reagent may be used to overspray a plate which has been previously sprayed with ninhydrin spray and FPN spray. Acidified iodoplatinate solution Violet, blue-violet, grey-violet, or brown-violet spots on a pink background are given by tertiary amines and quaternary ammonium compounds. Primary and secondary amines give dirtier colours. This solution may be used to overspray a plate which has previously been sprayed with ninhydrin spray, FPN reagent and Dragendorff spray. Mandelin’s reagent This reagent is preferably poured onto the plate because of the danger of spraying concentrated acid. Many different colours are given with a variety of drugs (see Chapter 19 and the Index of Colour Tests). Marquis reagent This reagent is preferably poured onto the plate because of the danger of spraying concentrated acid. Black or violet spots are given by alkaloids related to morphine. Many different colours are given with a variety of drugs (see Chapter 19). Acidified potassium permanganate solution Yellow-brown spots on a violet background are given by drugs with unsaturated aliphatic bonds. Rf values Rf values for drugs in these systems will be found in drug monographs and in the Indexes of Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 41 Thin–layer Chromatography (TLC)

Acidic and neutral drugs

A. H. Stead et al., Analyst, 1982, 107, 1106–1168 and R. A. de Zeeuw et al., Thin-layer Chromatographic Rf Values of Toxicologically Relevant Substances on Standardized Systems: Report XVII of the DFG Commission for Clinical-Toxicological Analysis, 2nd Edn, VCH, Weinheim, 1992.

System TD

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:acetone (80:20). Reference compounds: Paracetamol Rf 15, Clonazepam Rf 35, Secobarbital Rf 55, Methylphenobarbital Rf 70.

System TE

Plates: Silica gel G, 250 μm thick. Mobile phase: Ethyl acetate:methanol:strong ammonia solution (85:10:5). Reference compounds: Sulfadimidine Rf 13, Hydrochlorothiazide Rf 34, Temazepam Rf 63, Prazepam Rf 81.

System TF

Plates: Silica gel G, 250 μm thick. Mobile phase: Ethyl acetate. Reference compounds: Sulfathiazole Rf 20, Phenacetin Rf 38, Salicylamide Rf 55, Secobarbital Rf 68.

System TAD

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:methanol (90:10). Reference compounds: Hydrochlorothiazide Rf 11, Sulfafurazole Rf 33, Phenacetin Rf 52, Prazepam Rf 72.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 42 Thin–layer Chromatography (TLC)

Location reagents for systems TD, TE and TF

Acidic drugs

Van Urk reagent Spray the plate with the reagent and then heat in an oven at 100° for 5 min. Yellow spots are given by sulfonamides and by meprobamate, blue spots are given by ergot alkaloids, and pink or violet spots are given by some other compounds, e.g. phenazone. Ferric chloride solution Blue or violet spots are given by phenols. This solution may be used to overspray a plate which has been previously sprayed with Van Urk reagent. Mercurous nitrate spray Barbiturates give dark spots which fade slowly; with some dilute solutions the spots fade rapidly. Acidified potassium permanganate solution Yellow-brown spots on a violet background are given by drugs with unsaturated aliphatic bonds, e.g. secobarbital. This solution may be used to overspray a plate which has been previously sprayed with mercurous nitrate spray.

Neutral drugs

Furfuraldehyde reagent Violet to blue-black spots are given by some neutral compounds, e.g. carbamates. Acidified iodoplatinate solution This solution may be used to overspray a plate which has been previously sprayed with furfuraldehyde reagent. Rf values Rf values for drugs in these systems will be found in drug monographs and in the Indexes of Analytical Data in Volume 2; they are also included in the systems for specific groups of drugs which follow. Note It is worth noting that system TE can be used for acidic, neutral and basic drugs. Furthermore, systems TC and TAD use the same mobile phase so that acidic, neutral and basic drugs can be run in the same tank, although on separate plates. It should also be noted that the above systems for basic nitrogenous drugs are also able to separate neutral drugs if the latter are present in the sample or in the basic extract thereof.Finally, the Index of Colour Tests lists colour reactions with TLC spray reagents for approximately 250 compounds and may therefore serve as an indication of colour reactions specific to certain

Analytical Toxicology - Thin–layer Chromatography (TLC) | 43 Thin–layer Chromatography (TLC)

classes of compounds.

General screening systems

The TLC systems listed below (Systems TAJ, TAK and TAL) were developed primarily by Professor George Maylin, New York State Racing Wagering Board, Drug Testing Programme, as well as System TAM listed under steroids.

System TAJ

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:ethanol (90:10).

System TAK

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:cyclohexane:acetic acid (4:4:2).

System TAL

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:methanol:propionic acid (72:18:10).

Location reagents for systems TAJ, TAK, TAL and TAM

Cupric chloride Dragendorff spray Fearon’s reagent Ferric chloride, ethanol and sulfuric acid Fluorescamine Gibb’s reagent Conc. hydrochloric and ethanol Iodine Mandelin’s reagent Modified Ehrlich’s reagent

Analytical Toxicology - Thin–layer Chromatography (TLC) | 44 Thin–layer Chromatography (TLC)

Sodium nitrite Ninhydrin See Index of Colour Tests for colour reactions with TLC spray reagents.

Amfetamines, other stimulants and anorectics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Amfetamine 43 20 9 43 18 12 75 – – –

Bemegride – – – 68 – – 88 – – –

Benzfetamine 73 67 70 87 70 60 – – – –

Brucine 16 – 17 – 1 5 7 4 – 64

Cathine 42 25 5 – – – – – – –

Chlorphentermine 44 18 17 48 8 14 77 – – –

Diethylpropion 76 62 63 85 64 55 56 44 2 35

Fenbutrazate 72 47 78 86 67 88 – – – –

Fencamfamin 54 62 34 77 30 21 – – – –

Fenfluramine 48 42 16 61 11 20 – 7 25 68

Fenproporex – – – 77 – – – – – –

Mazindol 63 7 13 53 13 46 65 4 – 24

Meclofenoxate 77 26 42 67 22 46 – – – –

Metamfetamine 31 28 13 42 5 9 63 – 3 45

Methylenedioxymethamfetamine 33 24 – 39 – 8 – 3 17 57

Methylphenidate 57 35 41 66 23 40 70 11 4 70

Analytical Toxicology - Thin–layer Chromatography (TLC) | 45 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Pemoline 60 – 23 36 40 81 81 12 14 60

Phendimetrazine 57 36 51 62 24 49 41. – – –

Phenmetrazine 50 14 27 46 14 34 45 20 8 60

Phentermine 46 26 24 48 12 11 78 2 5 36

Pipradrol 54 59 38 81 39 19 79 – – –

Prolintane 50 67 32 79 25 22 – – – –

Tacrine 43 5 4 – 10 – – – – –

Anaesthetics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Benzyl Alcohol – – – – – 86 – – – –

Benzocaine 67 6 57 77 66 84 87 – – –

Bupivacaine 69 42 73 80 65 69 79 59 5 40

Butacaine 71 7 30 83 64 44 76 27 5 20

Butanilicaine 76 14 54 75 61 65 – – – –

Butyl Aminobenzoate 75 6 63 – 70 83 90 – – –

Chloroprocaine 59 5 23 – 37 – – 9 – 32

Cinchocaine 63 25 34 67 35 42 – – – –

Cocaine 65 45 47 77 54 35 30 13 – 2

Cyclomethycaine 58 55 36 – 25 – – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 46 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Diperodon 70 15 58 – 66 – – – – –

Dyclonine 60 49 40 – 25 – – – – –

Etidocaine 91 – – 75 – 80 – 66 19 75

Hexylcaine 1 – – 80 – – – 29 15 70

Lidocaine 70 35 71 80 63 72 69 55 – 28

Mepivacaine 65 31 62 66 48 63 60 28 2 40

Methohexital – – – 58 – 85 – – – –

Oxetacaine 52 10 7 38 15 61 – – – –

Oxybuprocaine 62 23 41 83 36 54 – – – –

Piperocaine 55 53 37 76 27 24 56 – – –

Pramocaine 70 43 55 73 41 62 60 59 20 90

Prilocaine 77 29 64 75 60 62 79 50 22 69

Procaine 54 5 31 71 30 36 42 6 – 22

Propoxycaine 58 3 33 – 28 – – 11 – 45

Proxymetacaine 62 26 41 – 35 – – – – –

Quinisocaine 61 55 46 – 28 – – 25 13 66

Tetracaine 57 15 32 64 16 43 39 12 – 25

Thialbarbital – – – 43 – – – – – –

Analgesics, NSAIDs

The tabulated systems, previously described, may be used or System TG, below, which gives good separations.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 47 Thin–layer Chromatography (TLC)

System TG

Plates: Silica gel G, 250 μm thick. Mobile phase: Ethyl acetate:methanol:strong ammonia solution (80:10:10).

Location reagents The reagents for systems TD, TE and TF can be used as well as those given below. Chromic acid solution A variety of colours are given by certain substances, e.g. diclofenac, red; diflunisal, blue- grey; feprazone, yellow; flufenamic acid, blue; indometacin, grey-brown; meclofenamic acid, violet; mefenamic acid, green; oxyphenbutazone, yellow; phenylbutazone, brown; salsalate, brown; sulindac, white. Ludy Tenger reagent Orange or orange-brown spots are given by certain substances.

Analgesics, NSAIDs

Molecule TA TB TC TD TE TF TG TL TAD TAE TAJ TAK TAL

Acetanilide – – – 45 70 45 – – 52 80 – – –

Alclofenac – – – 18 4 28 12 – 33 – 17 70 90

Aletamine 59 37 40 – – – – 42 – – – – –

Aloxiprin – – – 4 9 10 – – 22 – – – –

Aminophenazone 66 21 – 25 62 10 – – 58 70 53 – 76

Aspirin 90 – – 18 9 30 – – 31 78 40 65 90

salicylic acid – – – 7 10 1 – – 24 86 – – –

salicyluric acid – – – – – – – – – – – – –

Azapropazone 68 53 5 – 8 – – 67 – 88 11 6 61

Benorilate 67 – 51 – – – – 62 – 86 – – –

Benoxaprofen – – – – – – 14 – – – 56 80 99

Benzydamine 44 36 22 – – – – 9 – 16 – – –

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Molecule TA TB TC TD TE TF TG TL TAD TAE TAJ TAK TAL

Bufexamac – – – 11 18 19 36 – 31 – – – –

Cinchophen 75 – 2 – 8 – – – 7 82 – – –

Clonixin – – – – – – 30 – – – 40 14 80

Diclofenac 90 – – 25 12 27 29 – 47 90 40 64 84

Diflunisal – – – 8 16 5 37 – 18 89 6 69 69

Dipyrone 84 – 1 – 2 – – 2 2 85 – – 24

Etenzamide 64 3 59 – 76 – – 55 – 87 – – –

Etofenamate – – – – 78 – – – – 89 – – –

Etoxazene 65 – 56 – – – – 67 – – 57 2 70

Famprofazone 72 37 74 – 87 – – 67 – 90 – – –

Fenbufen 92 – – 18 4 30 9 – 39 – 43 68 91

Fenclofenac – – – – – – 20 – – – – – –

Fendosal 95 – – – 22 – – – – – 5 68 83

Fenoprofen 96 – – 42 6 38 16 – 50 – 58 78 76

Feprazone – – – – 19 – 45 – – 92 – – –

Floctafenine – – – – 85 – – – – 85 – – –

Flufenamic Acid 96 – – – 18 – 37 – – 84 55 78 95

Flunixin 96 – – – 12 – 33 – – – 37 20 83

Flurbiprofen – – – 30 6 30 16 – 45 – 47 69 91

Glafenine 67 1 38 – 46 3 – 40 – 81 – – –

Ibuprofen – – – 46 6 57 18 – 54 75 59 76 93

Indometacin 94 – – 16 5 13 20 – 38 83 46 90 90

Analytical Toxicology - Thin–layer Chromatography (TLC) | 49 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TG TL TAD TAE TAJ TAK TAL

Indoprofen – – – – – – 08 – – – – – –

Ketoprofen – – – 27 6 25 14 – 41 85 54 82 98

Meclofenamic – – – – 12 43 38 – – – 59 77 92 Acid

Mefenamic Acid 96 – – 41 11 48 32 – 54 87 68 86 95

Methyl Salicylate 96 – – – 84 68 – – – – 95 86 –

Morazone 58 8 46 – 58 – – 31 – 61 – – –

Naproxen – – – 33 6 38 14 – 44 82 60 75 93

Nefopam 50 33 32 – 59 – – 17 – 30 23 17 71

Nifenazone 57 – – – 36 – – – – 58 – – –

Niflumic Acid – – – 3 11 3 28 – 15 88 27 56 90

Oxyphenbutazone 77 – – 52 9 62 25 – 57 90 56 41 92

Paracetamol 95 – – 15 45 32 – – 26 77 30 5 73

Phenacetin – – – 38 68 37 – – 52 83 58 41 89

Phenazone 65 4 – 18 45 14 – – 50 66 51 18 83

Phenazopyridine 59 1 50 – 70 – – 53 – 80 46 56 91

Phenylbutazone 79 – – 78 65 68 23 – 76 87 90 76 97

M (5-hydroxy) – – – 8 3 18 – – – 88 – – –

Piroxicam – – – 51 17 38 – – 71 88 69 45 94

Propyphenazone 71 32 – 61 74 49 – – 65 81 – – –

Salicylamide – – – 38 50 55 – – 43 83 – – –

Salsalate – – – – – – 23 – – – – – –

Sulindac – – – 14 4 10 13 – 34 87 39 40 92

Analytical Toxicology - Thin–layer Chromatography (TLC) | 50 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TG TL TAD TAE TAJ TAK TAL

Tenoxicam – – – – 14 6 – – – 87 – – –

Tiaprofenic Acid – – – – 4 5 – – – 86 30 80 98

Tolfenamic Acid – – – – 14 31 – – – – – – –

Tolmetin – – – 13 5 20 10 – 30 85 20 59 83

Zomepirac – – – 12 4 12 – – – 88 19 65 88

Anti-emetics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAJ TAK TAL

Benzquinamide 65 7 69 – 36 – 53 6 91

Cyclizine 57 48 41 68 16 39 23 11 72

Difenidol 61 56 45 91 51 – 24 13 67

Granisetron – 18 – 51 – 14 – – –

Metoclopramide 47 1 7 51 13 17 – – –

Metopimazine 56 – 11 – 12 – – – –

Thiethylperazine 51 30 41 52 8 27 22 11 83

Anti-fungals

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TAE

Buclosamide 90 2 67 – 90

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Molecule TA TB TC TE TAE

Chlorphenesin 82 – – 62 –

Clotrimazole – – – 76 80

Diamthazole 52 30 30 – –

Econazole 80 9 61 75 78

Fenticlor – – – – 91

Fluconazole – – – 35 67

Flucytosine – – – 9 57

Griseofulvin – – – 69 78

Hydroxystilbamidine 1 – – – –

Itraconazole – 1 – 79 87

Ketoconazole – – – 50 68

Miconazole 73 11 67 80 77

Thioacetazone 78 – – – –

Antibacterials

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TAE

Amikacin – – 00

Aminosalicylic Acid 70 – –

Cefaloridine 42 – –

Chloramphenicol 69 00 86

Analytical Toxicology - Thin–layer Chromatography (TLC) | 52 Thin–layer Chromatography (TLC)

Molecule TA TB TAE

Cycloserine 44 1 –

Dequalinium Chloride 3 – –

Dibrompropamidine 1 – –

Ethambutol 30 03 12

Ethionamide 65 00 –

Furazolidone 44 00 56

Hexetidine 70 48 30

Isoniazid 47 1 55

Methenamine 30 4 12

Metronidazole 58 2 75

Minocycline – – 88

Morinamide 54 8 –

Nalidixic Acid – – 63

Nitrofurantoin – 00 84

Noxytiolin 74 – –

Oleandomycin 45 – –

Propamidine 1 1 –

Protionamide 66 1 77

Pyrazinamide 63 3 71

Rifamycin SV 84 – –

Salinazid 84 1 –

Thioacetazone 78 – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 53 Thin–layer Chromatography (TLC)

Molecule TA TB TAE

Tiocarlide 80 7 –

Tobramycin – – –

Trimethoprim 55 00 45

Troleandomycin 65 – –

Vancomycin 22 – –

Anticholinergics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Adiphenine 64 56 60 83 51 49 – – – –

Atropine 18 5 3 24 1 5 28 – – 14

Atropine Methonitrate 2 – – – – – – – – –

Benzatropine 13 26 6 – 2 6 – – – –

Benzilonium Bromide 3 – – – – – – – – 5

Biperiden 64 68 64 83 64 45 – 37 12 73

Chlorphenoxamine 53 47 36 70 17 29 – 11 5 54

Clidinium Bromide 2 – – 1 – 3 – – – –

Cyclopentolate 57 32 39 64 26 46 – 23 2 42

Cycrimine 66 67 61 – 60 – – – – –

Dicycloverine 68 67 64 84 54 55 – 42 25 84

Diethazine 58 57 51 77 39 33 54 – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 54 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Emepronium Bromide 5 – – 2 – 3 – – – –

Eucatropine 46 18 13 60 12 – – 3 2 33

Glycopyrronium Bromide 3 – – 1 – 3 – – – –

Hexocyclium Metilsulfate 2 – – 1 – 3 – – – –

Homatropine 1 5 1 23 1 7 27 – 2 34

Homatropine Methylbromide – – – – – – 12 – – –

Hyoscine 55 6 37 48 18 49 47 61 49 93

Hyoscyamine 18 – – 26 – – – – 1 34

Isopropamide Iodide 5 – 5 3 – 3 41 – – 19

Mepenzolate Bromide 1 – – – – – – 42 4 52

Methanthelinium Bromide 2 – – 76 – 3 – – – –

Metixene 50 45 25 61 12 21 – 16 22 73

Orphenadrine 55 48 33 68 16 25 49 14 2 47

Oxyphencyclimine 2 1 3 6 – 2 18 – 1 24

Oxyphenonium Bromide 3 – 1 1 – 2 36 – – –

Pentapiperide Metilsulfate 1 – 1 – – – – – – –

Penthienate Methobromide 2 – – 3 – 9 – – – –

Piperidolate 69 55 81 82 55 54 52 49 11 64

Procyclidine 48 62 31 74 23 20 68 – – 36

Profenamine 67 64 47 83 66 31 55 22 10 57

Propantheline Bromide 4 – 4 4 – 3 31 – – 20

Tigloidine 42 39 21 – 7 – – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 55 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Tricyclamol Chloride 6 – – – – – – – – –

Trihexyphenidyl 68 66 61 83 59 43 75 38 22 80

Tropicamide 65 – – 51 – – – – – –

Anticonvulsants and barbiturates

The tabulated systems, previously described, together with the associated location reagents may be used or System TH, below, which gives good separations.

Systems TH

Plates: Silica gel G, 250 μm thick. Mobile phase: Isopropyl alcohol:chloroform:strong ammonia solution (90:90:20).

Location reagents for systems TD, TE and TH

Mercuric chloride-diphenylcarbazone reagent White spots on a violet background are given in neutral systems, and violet spots on a pink background are given if the plate is alkaline. Acidified potassium permanganate solution Yellow-brown spots on a violet background are given by drugs with unsaturated aliphatic bonds. Zwikker’s reagent Pink spots are given by 5,5-disubstituted barbiturates, green spots are given by thiobarbiturates, and faint pink spots are given by bromobarbiturates and by 1,5,5- trisubstituted barbiturates. The test is not very sensitive. Fluorescein solution Spray the plates with a 10% solution of sodium hydroxide and heat at 100° in an oven for 5 min before applying the reagent. Pink spots are given by bromobarbiturates. Mercurous nitrate spray Barbiturates give dark spots which fade slowly; with some dilute solutions the spots fade rapidly.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 56 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TH TL TAD TAE TAJ TAK TAL

Allobarbital – – – 50 34 66 53 – 56 87 – – –

Alverine 66 65 39 – – – – 38 – – – – –

Ambucetamide 73 5 68 – 76 – – 61 – 76 – – –

Amobarbital – – – 52 44 66 74 – 58 88 – – –

Aprobarbital – – – 48 40 65 66 – 57 86 – – –

Barbital – – – 41 32 61 51 – 57 84 – – –

Beclamide 65 8 65 – – – – 64 – 90 – – –

Benactyzine 66 40 53 – – – – 53 – 52 34 3 48

Brallobarbital – – – 52 30 68 47 – 57 87 – – –

Butalbital – 1 – 54 44 67 67 – 57 87 – – –

Butetamate 69 59 57 – 81 – – 47 – 48 – – –

Butobarbital – – – 50 41 65 68 – 58 86 – – –

Carbamazepine 60 2 56 – 56 – – 47 – 79 44 64 94

Clonazepam 72 – 53 35 67 45 – 61 56 85 50 53 91

Cyclobarbital – – – 50 40 64 59 – 58 88 – – –

Cyclopentobarbital – – – 50 39 65 62 – 59 90 66 63 90

Dimoxyline 68 16 75 – 87 – – 58 – – 64 10 93

Enallylpropymal – – – 71 58 71 87 – 70 – – – –

Ethosuximide 70 5 – 50 66 53 – – 59 84 – – –

Ethotoin 88 – – 53 71 54 – – 60 – 61 66 91

Flavoxate 62 36 67 – 77 – – 45 – 48 52 71 92

Heptabarb – – – 50 38 64 62 – 59 88 – – –

Hexethal – – – 53 44 67 74 – 60 – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 57 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TH TL TAD TAE TAJ TAK TAL

Hexobarbital – – – 65 53 65 85 – 69 85 – – –

Ibomal – – – 50 32 66 61 – 56 91 – – –

Idobutal – – – 55 41 69 71 – 59 – – – –

Mebeverine 63 40 53 – 86 – – 49 – 32 – – –

Mephenytoin – – – 62 74 58 – – 66 – 64 70 91

Mesuximide 76 – – – 86 – – – – 90 85 70 98

Metharbital – – – 66 54 65 86 – 69 87 – – –

Methylphenobarbital – – – 70 41 67 72 – 70 86 – – –

Nealbarbital – – – 58 44 68 78 – 60 92 – – –

Octamylamine 22 28 11 – – – – 25 – – – – –

Oxcarbazepine – – – – 54 20 – – – 78 – – –

Papaverine 61 8 65 – 69 – – 47 – 74 66 8 93

Paramethadione 86 – – – 7 60 – – 56 – 87 70 94

Pentobarbital – – – 55 45 66 76 – 59 90 – – –

Phenacemide – – – 22 65 40 – – 50 – – – –

Pheneturide 76 – – 38 71 53 – – 59 – – – –

Phenobarbital – – – 47 28 65 38 – 53 85 – – –

Phensuximide 75 – – 71 77 59 – – 72 – 81 71 96

Phenytoin – – – 33 41 55 – – 53 86 48 84 96

Pipoxolan 77 53 68 – – – – 56 – – – – –

Primidone 88 – – 8 41 23 – – 28 76 29 60 86

Secbutabarbital – – – 50 48 64 69 – 57 88 – – –

Secobarbital – – – 55 45 68 78 – 62 88. – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 58 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TH TL TAD TAE TAJ TAK TAL

Sultiame – – – 23 57 43 – – 42 81 – – –

Talbutal – – – 53 46 67 71 – 60 92 – – –

Thiamylal – – – – 55 75 – – – – – – –

Thiopental – – – 77 49 74 80 – 68 – 73 71 92

Trimethadione – – – – – – – – – – – – –

Valproic Acid – – – – – 52 – – – – – – –

Vinbarbital – – – 50 34 65 56 – 57 89 56 62 91

Vinylbital – – – 38 39 64 – – 66 89 – – –

Antidepressants and antipsychotics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Acetophenazine 53 3 25 38 3 34 32 5 – 33

Amitriptyline 51 50 32 69 15 27 51 13 5 56

Benperidol – – – – 32 62 69 – – –

Butriptyline 59 61 48 – 38 – – 22 8 61

Carfenazine 54 5 27 39 7 39 – 8 – 51

Clomipramine 51 53 34 72 18 26 54 18 16 75

Clorgiline 67 42 70 – 59 – – – – –

Clozapine 57 4 38 55 17 42 – – – –

Desipramine 26 19 11 40 3 7 71 7 23 72

Dibenzepin 54 22 35 55 14 38 22 – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 59 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Dosulepin 51 49 42 65 16 27 41 – – –

Doxepin 51 48 37 63 13 24 45 23 14 71

Droperidol 67 2 48 58 36 71 73 37 2 46

Fluoxetine – 13 – 47 – 11 – – – –

Fluvoxamine – 12 – 46 – 18 – – – –

Imipramine 48 48 23 67 13 21 47 7 2 52

Iprindole 47 49 34 – 16 – – – – –

Iproniazid 69 1 23 41 17 70 69 – – –

Isocarboxazid 71 20 74 75 61 84 86 67 67 92

Lofepramine – – – 90 – 82 – – – –

Maprotiline 15 18 5 36 2 6 71 – – 50

Mebanazine 70 48 69 – 63 – – – – –

Mianserin 58 39 58 68 23 48 50 – – –

Moclobemide – 1 – 52 – 65 – – – –

Nialamide 70 2 25 – 4 68 64 – – –

Nomifensine 56 9 29 64 31 53 52 25 6 50

Nortriptyline 34 27 16 – – – – 1 9 68

Noxiptiline 53 43 35 66 18 29 – – – –

Opipramol 54 6 22 38 7 35 39 6 5 59

Paroxetine – 4 – 40 – 8 – 4 4 54

Phenelzine 77 37 12 83 63 29 82 74 74 93

Protriptyline 19 18 7 38 2 6 69 4 28 76

Analytical Toxicology - Thin–layer Chromatography (TLC) | 60 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Remoxipride – 14 – 54 – 26 – – – –

Sertraline – 46 – 72 – 25 – – – –

Tofenacin 45 26 21 48 7 14 – – – –

Trazodone 63 10 58 66 37 64 61 55 – 66

Trifluoperazine 53 33 30 55 8 30 29 – – –

Trifluperidol 73 13 – 77 – 55 76 – – –

Trimipramine 59 62 54 80 37 36 56 – – –

Viloxazine 42 6 23 36 6 25 – – – –

Zimeldine 47 27 25 48 – 20 – – – –

Zuclopenthixol 56 7 32 44 11 45 – – – –

Antihistamines

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Alimemazine 58 54 39 77 31 32 46 – – –

Antazoline 31 6 7 47 3 5 66 00 00 15

Bamipine 49 40 43 – 13 24 – – – –

Bromazine 54 44 43 – 13 27 48 – – –

Brompheniramine 45 33 16 – 6 12 – 1 – 28

Buclizine 75 61 83 – 72 – – – – –

Carbinoxamine 48 26 19 50 4 13 16 4 – 27

Analytical Toxicology - Thin–layer Chromatography (TLC) | 61 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Chlorcyclizine 57 42 46 67 14 35 52 21 10 70

Chloropyramine 52 41 28 63 17 22 – – – –

Chlorphenamine 45 35 18 46 2 12 21 – – 25

Cinnarizine 76 54 78 86 65 79 87 – – –

Clemastine 46 49 25 58 9 88 49 – – –

Clemizole 78 33 69 78 52 76 73 – – –

Cyclizine 57 48 41 68 16 39 52 23 11 72

Cyproheptadine 51 45 44 64 13 30 50 18 16 –

Deptropine 13 26 4 36 1 1 – – – –

45 33 68 28 48 Dimenhydrinate 55 and 88 and and and – and and – – – 00 10 02 87 46

Dimetindene 42 36 13 47 6 10 – 4 – 40

Dimetotiazine 56 13 48 – 28 43 – – – –

Diphenhydramine 55 44 33 65 15 27 48 – – –

Diphenylpyraline 46 42 28 68 8 23 49 61 50 92

Doxylamine 48 41 10 60 9 12 – – – 8

Histapyrrodine 60 – – 75 – 32 – – – –

Hydroxyzine 68 10 54 54 19 57 65 26 – 34

Isothipendyl 52 41 30 64 14 22 35 – – –

Levocabastine – – – 12 – 76 – – – –

Loratadine – 20 – 78 – 86 – – – –

Mebhydrolin 57 27 45 65 20 36 46 – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 62 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Meclozine 76 61 79 87 70 80 88 65 24 95

Mepyramine 51 39 25 58 14 22 33 – – –

Mequitazine 10 6 6 27 – 3 – – – –

Methapyrilene 52 41 26 66 13 21 24 20 – 48

Methdilazine 29 32 15 63 6 – – 12 19 72

Phenindamine 63 45 57 68 21 – 49 37 2 82

Pheniramine 45 35 13 46 3 14 26 – – –

Phenyltoloxamine 53 38 48 67 15 32 – 27 15 69

Pizotifen 48 45 – 64 – 28 – – – –

Promethazine 50 36 35 65 17 30 44 – – –

Propiomazine 55 34 42 68 26 30 52 34 16 81

Pyrrobutamine 54 54 37 71 18 25 66 24 25 86

Thenalidine 50 38 44 52 12 20 – – – –

Thenyldiamine 53 42 25 65 12 21 36 – – –

Thiazinamium 2 – – – – 1 25 – – – Metilsulfate

Thonzylamine 55 38 28 65 14 22 31 – – –

Tolpropamine 51 52 32 68 15 26 – – – –

Trimethobenzamide 42 2 – 47 – 24 – – – –

Tripelennamine 55 44 27 68 15 22 34 2 – 18

Triprolidine 51 11 20 55 6 19 30 95 3 69

Antimalarials

Analytical Toxicology - Thin–layer Chromatography (TLC) | 63 Thin–layer Chromatography (TLC)

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAL

Amodiaquine 62 8 40 74 37 38 – 9

Chloroquine 38 14 4 46 2 4 14 4

Chlorproguanil 3 – 1 – 1 – – –

Cinchonidine 49 6 8 44 6 24 55 70

Cinchonine 49 6 12 44 5 19 61 –

Halofantrine – 50 – 88 – 56 – –

Desbutylhalofantrine – 12 – 61 – 19 – –

Hydroxychloroquine 45 2 2 37 3 7 – 4

Primaquine 19 13 5 – 15 – – –

Proguanil 3 – 1 18 1 7 79 –

Pyrimethamine 61 2 31 58 21 66 – –

Quinine 51 2 11 45 4 26 65 –

Antineoplastics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TF TL TAD TAE

Aminoglutethimide – – – 65 47 – 53 –

Chlorambucil – – – 6 40 – 50 84

Cytarabine 5 – 1 – – 1 – 69

Diethylstilbestrol – 3 – 73 68 – – 92

Analytical Toxicology - Thin–layer Chromatography (TLC) | 64 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TF TL TAD TAE

Doxorubicin 12 – – – 00 – – –

Epirubicin – – – – – – – –

Fluorouracil – – – 4 20 – – –

Idarubicin – – – 6 – – – –

Mercaptopurine – – – 2 – – – 77

Pipobroman 66 2 58 – – 41 – –

Procarbazine 49 2 10 80 – 4 68 88

Vinblastine 60 1 60 57 – 29 – 46

Antiparkinsonians

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF

Amantadine 23 19 7 35 4 7 77

Benserazide 1 – 1 – 3 7 –

Bromocriptine 72 – 69 – 61 84 88

Levodopa – – – – – 11 –

Selegiline 74 57 69 – – – –

Antiprotozoals

The tabulated systems, previously described, may be used together with the associated location reagents.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 65 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAD TAE

Broxaldine 74 52 79 – 71 – 78

Broxyquinoline 51 – 6 – 3 – –

Dehydroemetine 43 6 21 – 2 – –

Hydroxystilbamidine 1 – – – – – –

Metronidazole 58 2 36 46 40 32 75

Nifuratel 73 – – 67 – – 77

Nimorazole – 3 44 58 33 – 60

Pentamidine 1 1 – – – – –

Antitussives

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Benzonatate 61 – – – – – – 56 23 90

Bromhexine 75 67 79 – 71 84 – 98 28 78

Carbetapentane 48 48 22 – – – – – – –

Cephaëline 53 1 19 – 8 – – – – –

Clofedanol 52 41 37 – 29 32 – – – –

Dextromethorphan 33 42 18 47 6 10 42 – – –

Dextrorphan 35 14 4 42 3 10 49 – – –

Dimethoxanate 39 18 24 49 6 21 38 10 84 49

Dropropizine 65 1 – 34 – 59 – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 66 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Guaifenesin – 2 – 39 – 81 – 42 27 74

Isoaminile 68 58 54 81 55 45 – – – –

Noscapine 64 21 74 78 64 72 75 65 18 93

Oxeladin 50 51 22 67 19 19 – – – –

Pholcodine 36 3 18 25 2 15 – – – –

Pipazetate 47 17 13 48 6 12 – – – –

Benzodiazepines and hypnotics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TD TE TF TL TAD TAE TAF TAJ TAK TAL

Acecarbromal – – – 49 57 48 – 60 22 84 61 58 90

Apronal – – – 33 67 52 – – 125 – – – –

Bromazepam 61 6 41 13 63 – 53 47 73 69 34 4 63

Brotizolam 72 5 52 15 52 5 27 53 231 71 – – –

M-6-hydroxy 68 1 35 5 28 4 13 37 – 76 – – –

M-α-hydroxy 72 2 46 7 45 6 31 41 – 78 – – –

Camazepam 76 12 73 55 75 – 65 69 82 83 – – –

Carbromal – 12 – 53 75 55 – 64 294 87 – – –

Chlordiazepoxide 62 2 50 10 52 – 22 53 76 77 48 2 79

Clobazam 62 8 70 53 75 – 62 70 84 85 – – –

Clomethiazole 64 44 69 – 76 – 58 – 333 85 – – –

Clonazepam 72 00 53 35 67 45 61 56 403 87 50 53 91

Clorazepic Acid 84 3 56 34 68 – 60 57 83 87 – – –

Ethchlorvynol – – – 81 – 74 – 82 657 – 73 77 96

Analytical Toxicology - Thin–layer Chromatography (TLC) | 67 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TL TAD TAE TAF TAJ TAK TAL

Ethinamate 76 5 – 49 76 59 – 58 661 87 58 69 91

Demoxepam 63 – 35 15 41 – 51 42 81 83 42 38 89

Diazepam 75 27 73 58 76 – 59 72 82 85 67 48 96

Flumazenil 71 3 63 30 61 14 44 61 738 72 – – –

Flunitrazepam 63 10 72 54 74 47 63 72 740 82 69 59 95

Flurazepam 62 30 48 3 71 3 40 41 756 45 32 8 73

Glutethimide 75 31 – 63 80 62 – 70 797 89 – – –

Ketazolam 66 14 64 45 74 – 66 62 83 80 – – –

Loprazolam 40 1 48 3 40 1 5 36 956 15 – – –

Lorazepam 52 1 36 23 43 – 28 42 82 82 46 42 86

Lormetazepam 52 6 61 46 59 45 50 60 960 82 – – –

Mecloqualone – 25 – – 76 – – – 985 – 77 68 96

Medazepam 67 41 74 54 78 – 62 73 79 83 70 12 95

Methaqualone 70 36 80 63 78 – 56 – 1032 84 – – –

Methylpentynol – – – 49 74 62 – 57 1072 – – – –

Methyprylon 58 – – 31 63 25 – 55 1080 – – – –

Midazolam 72 6 60 13 60 5 19 53 1099 70 – – –

Nitrazepam 68 – 36 35 64 46 55 53 1180 86 53 52 92

Nordazepam 62 3 55 34 67 – 60 57 82 83 53 60 92

Oxazepam 56 – 40 22 45 – 51 42 82 91 47 47 89

Prazepam 65 36 74 64 81 – 63 72 84 89 75 69 94

Quazepam 74 27 75 78 83 71 76 78 1434 96 – – –

M(2-oxo) 78 16 89 59 80 57 71 70 – 90 – – –

M(3-hydroxy-2-oxo) 71 2 55 42 69 52 59 58 – 90 – – –

M(3-hydroxy-N-dealkyl-2-oxo) 67 – 30 15 49 28 38 35 – 89 – – –

M(N-dealkyl-2-oxo) 75 2 56 34 72 45 58 60 – 88 – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 68 Thin–layer Chromatography (TLC)

Molecule TA TB TC TD TE TF TL TAD TAE TAF TAJ TAK TAL

Temazepam 53 8 59 51 62 47 53 65 1563 82 65 54 92

Triazolam 60 1 40 5 44 2 16 41 1647 65 – – –

Zopiclone – 4 – – 47 – – – 1732 – – – –

Bronchodilators

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF

Bambuterol – 2 – 37 – 18 –

Bambuterol monocarbamate – – – 21 – 19 –

Bamifylline 65 – 54 – 34 71 –

Butetamate 69 59 57 81 47 48 56

Protokylol 65 1 3 – 6 – –

Rimiterol – – – 6 – 7 –

Salbutamol 46 1 1 20 4 16 74

Cannabinoids

The tabulated systems, previously described, may be used together with the associated location reagents or Systems TI and TJ, below. These systems may be used for extracts of both cannabis and cannabis resin.

System TI

Plates: Silica gel G, 250 μm thick, dipped in, or sprayed with, a 10% solution of silver nitrate, and dried. Mobile phase: Toluene, using unsaturated (open tank) conditions.

Analytical Toxicology - Thin–layer Chromatography (TLC) | 69 Thin–layer Chromatography (TLC)

System TJ

Plates: Silica gel G, 250 μm thick, sprayed with diethylamine immediately before use. Mobile phase: Xylene:hexane:diethylamine (25:10:1).

Location reagents for systems TI and TJ

Fast blue B solution Cannabidiol gives an orange colour, cannabinol gives a violet colour, and Δ9- tetrahydrocannabinol gives a red colour. The colours may be intensified by overspraying with 1 M sodium hydroxide or by exposing the plate to ammonia fumes. Duquenois reagent After spraying with the reagent, overspray the plate with hydrochloric acid. Blue to violet colours are given by cannabinoids. Molecule TA TE TI TJ TAH TAJ TAK TAL

Δ9-THC 11 31 30 29 50 00 1 31

CBN 94 95 52 20 45 90 77 97

CBD 94 95 5 36 60 88 76 97

Cardioactive drugs

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Ajmaline 62 7 – 56 – – 22 – – – –

Amiodarone 72 62 68 82 – 55 54 64 – – –

Aprindine – 63 – 76 – – 20 – – – –

Azapetine 70 57 67 78 – 56 14 – 59 26 90

Bamethan 55 4 6 – – 00 23 – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 70 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Benziodarone – – – 23 58 – – – – – –

Benzthiazide – – – 9 51 – – – 31 6 71

Bethanidine 1 – – – – – – – – – –

Bretylium Tosilate 1 – – – – – – – – – –

Buphenine 74 3 14 62 – 50 33 83 – – –

Butalamine 68 – – 86 29 – – – – – –

Captopril – – – – 1 – – – – – –

Carbocromen 48 17 24 62 – 12 18 – – – –

Clonidine 62 8 31 70 – 53 44 76 9 2 51

Clopamide – – – 55 38 – – – – – –

Co–dergocrine Mesilate 66 1 48 – – 29 – – – – –

Cyclandelate – 37 – 80 77 – 87 95 81 71 95

Debrisoquine 1 – – – – 00 – – – 3 36

Deserpidine 72 3 77 81 – 66 73 – – – –

Digitoxin – – – 36 10 – 88 – 35 1 78

Digoxin – – – 33 05 – 85 – – – –

Dihydralazine 55 34 2 18 – 01 – – – – –

Disopyramide 45 7 8 60 – – 9 7 – – –

Doxazosin – – – 73 – – 71 – – – –

Enalapril – – – – – – 85 – – – –

(enalapril) – – – – 00 – – – – – –

(enalaprilat) – – – – 00 – – – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 71 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Encainide – 28 – 54 – – 16 – – – –

Felodipine – 2 – 77 60 – 87 – – – –

Flecainide – 6 – 49 – – 28 – – – –

Glyceryl Trinitrate – – – 86 72 – – – – – –

Guanethidine 01 – 2 1 – 00 3 30 – – 16

Guanoclor 03 – – – – 00 – – – – –

Guanoxan 01 – – – – 00 3 76 – – –

Heptaminol 23 1 2 22 – 05 14 – – – –

Hexamethonium Bromide 00 – – – – – – – – – –

Hexobendine 47 10 44 16 – 06 12 – – – –

Hydralazine 51 41 11 80 – 64 73 – 1 1 25

Hydroquinidine 45 3 8 43 – 05 20 70 – – –

Indoramin 84 – – 74 – – – – 13 10 77

Inositol Nicotinate 57 1 43 – – 16 – – – – –

Isoxsuprine 78 3 32 62 – 53 62 81 13 5 60

Labetalol – – – 29 01 – – 32 – – –

Lanatoside C – – – 6 – – – 89 – – –

Lidoflazine 70 11 63 70 – 36 – 70 77 – –

Lofexidine – – – 53 – – – 17 – – –

Lorcainide – 48 – 80 – – – 41 – – –

Mecamylamine 16 51 2 – – 04 – – – – 16

Methoserpidine 72 4 77 – – 64 – – – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 72 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Methyldopa 49 1 1 2 – 01 – 60 75 – –

Mexiletine 40 17 4 55 – 09 – 25 78 – –

Minoxidil 51 – 3 18 – 00 – 44 – – –

Moxisylyte 52 31 44 – – 19 – – – – –

Naftidrofuryl Oxalate 64 52 41 78 – 35 – 43 – – –

Nicametate 56 41 35 68 – 20 – 35 – – –

Nicergoline 64 – – 73 – – – 43 – – –

Nicofuranose 61 42 70 – – – – – – – –

Nicotinyl Alcohol 56 4 17 45 – 22 – 74 69 – –

Nifedipine 68 1 65 71 – 68 – 79 – – –

Pargyline 70 – – 60 – – – 77 – 71 20

Pempidine 24 68 3 – – 10 – – – – –

Pentaerithrityl – – – 72 – – – 92 – – – Tetranitrate

Pentifylline 55 6 66 66 – 46 – 72 – – –

Pentolonium Tartrate 00 – – – – 00 – – 1 – –

Pentoxifylline – – – 55 – – – 64 – 49 12

Perhexiline 41 57 8 59 – 06 – 8 – – –

(perindopril) – – – 6 00 – – – – – –

(perindoprilat) – – – 3 00 – – – – – –

Phenoxybenzamine 73 63 76 87 – 68 – 84 97 65 4

Phentolamine 32 1 3 33 – 02 – 6 – – 1

Analytical Toxicology - Thin–layer Chromatography (TLC) | 73 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Prajmalium Bitartrate 59 – – – – – – 8 – – –

Prazosin 60 1 47 59 – 49 – 68 74 39 –

Prenylamine 68 55 68 84 – 56 – 43 85 47 63

Procainamide 49 1 5 39 – 09 – 17 33 – –

Quinidine 51 4 12 49 – 06 – 30 63 – 2

Rescinnamine 73 1 75 81 – 64 – 77 79 – –

Reserpine 69 2 74 77 – 63 – 76 80 56 6

Sotalol 53 1 3 30 – 05 – 19 – – –

Strophanthin-K – – – – – – – 81 – – –

Tocainide 60 2 23 44 – – – 42 74 – –

Tolazoline 13 2 2 25 – 02 – 3 55 – –

Trimetaphan Camsilate 02 – – – 00 – – – – – 1

Trimetazidine 22 5 4 – – – – – – – –

Verapamil 59 23 70 73 – 42 – 43 61 – –

Xamoterol – – – 15 00 – – 18 – – –

Xantinol Nicotinate 41 – – 21 00 – – 26 – – –

Coumarins and other anticoagulants

The tabulated systems, previously described, may be used together with the associated location reagents.

TD TE TF TAD TAE TAJ TAK TAL

Acenocoumarol 52 16 48 60 92 68 51 92

Analytical Toxicology - Thin–layer Chromatography (TLC) | 74 Thin–layer Chromatography (TLC)

TD TE TF TAD TAE TAJ TAK TAL

Anisindione – 15 – – – 82 70 95

Dicoumarol 18 30 32 33 88 60 80 96

Diphenadione 11 46 33 53 – 51 74 91

Ethyl Biscoumacetate 4 24 32 21 – – – –

Phenindione 65 21 56 70 – – – –

Phenprocoumon 62 19 58 61 93 – – –

Warfarin 64 18 62 64 – – – –

Diuretics

The tabulated systems, previously described, may be used together with the associated location reagents.

Location reagents The reagents given for systems TD, TE and TF can be used as well as that given below. N-(1-Naphthyl)ethylenediamine solution Spray the plate with dilute sulfuric acid, expose it to nitrogen dioxide vapour for 15 min and then spray with the reagent. Molecule TA TD TE TF TAD TAE TAJ TAK TAL

Acetazolamide 85 4 3 31 18 84 18 1 60

Amiloride 24 – 24 – – 6 – – 29

Bendroflumethiazide – 25 52 71 30 – 38 11 72

Benzthiazide – 14 9 51 30 – 31 6 71

Bumetanide – 1 4 10 6 87 18 42 80

Chlorothiazide – 2 2 16 11 – 11 – 41

Chlortalidone – 4 42 40 23 88 17 10 63

Analytical Toxicology - Thin–layer Chromatography (TLC) | 75 Thin–layer Chromatography (TLC)

Molecule TA TD TE TF TAD TAE TAJ TAK TAL

Clopamide 79 19 55 38 39 – – – –

Clorexolone 76 31 60 51 47 79 – – –

Cyclopenthiazide – 21 66 62 27 – – – –

Cyclothiazide 77 18 59 60 26 – – – –

Epithiazide – 13 44 62 25 88 22 5 63

Etacrynic Acid 96 3 5 2 5 71 5 42 57

Ethiazide – 11 50 50 – – – – –

Ethoxzolamide 76 43 43 65 51 – 51 48 91

Furosemide – 1 6 7 7 86 10 25 70

Hydrochlorothiazide – 4 34 34 11 78 9 – 40

Hydroflumethiazide 86 7 36 47 13 87 9 – 43

Indapamide – 38 66 61 46 89 56 22 92

Mefruside – 45 67 58 55 – – – –

Methyclothiazide 87 19 53 50 27 – 30 8 69

Metolazone – 23 57 51 33 – 34 8 75

Polythiazide – 22 63 60 32 – 35 8 70

Quinethazone 75 4 40 21 15 – 11 6 56

Spironolactone – 66 78 51 75 84 73 64 96

Triamterene 51 – 30 – 13 50 4 – 40

Trichlormethiazide 80 15 14 60 23 88 24 5 61

Urea 55 – 15 – – – 5 15 30

Xipamide – 38 13 64 36 93 – – –

Analytical Toxicology - Thin–layer Chromatography (TLC) | 76 Thin–layer Chromatography (TLC)

Drugs of abuse

The tabulated systems, previously described, may be used together with the associated location reagents. A further three systems (TAH, TAI and TAN), described below may be used for drugs of abuse. Please refer to Chapter 2 for Rf values.

System TAH

Plates: Silica gel G, 250 μm thick. Mobile phase: Hexane:diethyl ether (80:20).

System TAI

Plates: Silica gel G, 250 μm thick. Mobile phase: Acetone.

System TAN

Plates: Silica gel G, 250 μm thick. Mobile phase: Butanol:acetic acid:water (2:1:1).

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

5-Methyltryptamine 56 – – – – – – – – –

Amfetamine 43 20 9 43 18 12 75 – – –

Benzfetamine 73 67 70 87 70 60 – – – –

Benzoylecgonine 21 0 1 – – – – – – –

Bufotenine 35 0 1 33 1 10 34 – – –

Cannabidiol 94 – – 95 – – – 88 76 97

Cannabinol 94 – – 95 – – – 90 77 97

Cocaine 65 45 47 77 54 35 30 13 0 2

Analytical Toxicology - Thin–layer Chromatography (TLC) | 77 Thin–layer Chromatography (TLC)

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Δ9-THC 11 – – 31 – – – 0 1 31

Diamorphine 47 15 38 49 4 26 33 25 5 64

Diethyltryptamine 46 15 10 63 11 14 56 2 3 41

Dimethyltryptamine 40 9 9 50 6 14 39 – – –

DOM 51 15 17 41 16 9 76 – – –

Ketamine 63 37 63 79 64 68 72 47 4 43

Lysergic acid 58 0 0 0 0 70 16 48 7 79

Lysergide 60 3 39 56 24 60 59 33 2 59

Mescaline 20 3 10 24 12 6 63 2 9 51

Metamfetamine 31 28 13 42 5 9 63 0 3 45

Methadone 48 59 20 77 27 16 60 8 0 45

Methylenedioxyamfetamine 39 18 12 42 17 10 76 – – –

Methylenedioxymethamfetamine 33 24 – 39 – 8 – 3 17 57

N-Methyltryptamine 18 – – – – – – – – –

p-Methoxyamfetamine 73 23 77 43 69 9 74 4 18 58

Monoacetylmorphine 46 6 19 – – – – 13 2 51

Morphine 37 0 9 20 1 18 23 0 0 15

Psilocin 39 5 9 47 9 14 48 – – –

Psilocybine 5 0 – 0 0 80 1 – – –

Ergot alkaloids

R. E. Ardrey and A. C.Moffat,J. Forens. Sci. Soc. 1979, 19, 253–282

Analytical Toxicology - Thin–layer Chromatography (TLC) | 78 Thin–layer Chromatography (TLC)

The tabulated systems, previously described, may be used together with the associated location reagents.

System TL, previously described, may be used or system TM below. Note that these systems can be run in a single tank as they use the same mobile phase and have low correlation of Rf values.

System TM

Plates: Aluminium oxide, 250 μm thick. Mobile phase: Acetone. Reference compounds: Lysergide Rf 70, Ergotamine Rf 48, Ergometrine Rf 26.

Location reagents for systems TL and TM

Naphthoquinone sulfonate solution Spray the plate with the reagent, then spray with a 10% v/v solution of hydrochloric acid and heat at 110° for 20 min. Red-violet spots on a light pink background are given by ergot alkaloids. Nitroso-naphthol solution Spray the plate with the reagent, then spray with a 10% v/v solution of hydrochloric acid and heat at 110° for 20 min. Blue-black spots on a yellow background are given by ergot alkaloids. Van Urk reagent After spraying the plate, heat in an oven at 100° for 5 min. Blue spots are given by ergot alkaloids.

Ergot alkaloids

Molecule TA TB TC TE TL TM TAE TAF TAJ TAK TAL

Co-dergocrine 66 1 48 – 29 64 – – – – –

Dihydroergotamine 60 1 28 42 14 40 58 – 33 3 84

Ergometrine 57 00 12 33 08 26 62 60 17 00 37

Ergotamine 63 1 34 44 23 48 68 64 – – –

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Molecule TA TB TC TE TL TM TAE TAF TAJ TAK TAL

Ergotoxine 66 1 62 – 48 67 – – – – –

Lysergamide 60 00 19 36 6 27 57 51 – – –

Lysergic acid 58 – – – 00 00 70 16 48 7 79

Lysergide 60 3 39 3 24 70 60 59 33 2 59

Methylergometrine 62 00 14 41 12 31 69 – – – –

Methysergide 65 1 21 45 12 33 – – 23 4 66

Narcotic analgesics and narcotic antagonists

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Alphaprodine 50 30 35 62 11 28 – 23 5 60

Amiphenazole 61 2 33 62 57 – – – – –

Anileridine 73 12 56 79 51 60 66 20 – 68.

Bezitramide 71 41 79 – 70 92 96 – – –

Buprenorphine 76 9 68 80 69 80 – 62 4 77

Butorphanol – – – – – – – 16 – 36

Codeine 33 6 18 35 3 21 22 10 – 26

Cyclazocine 53 15 13 65 25 24 74 6 5 60

Dextromoramide 73 42 71 79 60 72 78 – – –

Dextropropoxyphene 68 59 55 – – – – 33 4 51

Diamorphine 47 15 38 49 4 26 33 25 5 64

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Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Dihydrocodeine 26 8 13 29 2 11 19 6 – 38

Dihydromorphine 25 2 3 18 1 12 – – – 25

Dipipanone 66 67 33 87 70 27 72 – – –

Embutramide 72 – – 59 – – – 53 46 –

Ethoheptazine 40 45 19 55 4 12 41 8 12 65

Ethylmorphine 40 7 22 36 6 21 26 13 4 54

Fentanyl 70 43 74 78 58 70 77 59 8 84

Hydromorphone 23 3 9 18 2 12 14 – – –

Ketobemidone 47 2 9 37 6 26 – 3 1 40

Levallorphan 67 19 24 74 45 42 73 10 6 66

Levorphanol 35 13 7 – – – – – – –

Methadone 48 59 20 77 27 16 60 8 – 45

Morphine 37 – 9 20 1 18 23 – – 15

Nalbuphine – – – 34 – 58 – 19 – 20

Nalorphine 59 1 23 32 29 57 59 18 1 46

Naloxone 65 9 66 47 63 74 – 58 1 45

Naltrexone – – – – – – – 49 1 40

Norcodeine 13 – 5 – – – – – – –

Normethadone 56 40 34 – – – – 19 20 78

Normorphine 17 – – – – – – – – 28

Norpipanone 68 58 50 80 38 43 – – – –

Oxycodone 50 25 51 62 39 30 33 27 1 36

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Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Oxymorphone 48 10 37 33 30 27 36 13 – 13

Pentazocine 61 16 12 70 28 34 72 2 3 57

Pethidine 52 37 34 60 11 34 40 14 6 72

Phenazocine 68 16 39 74 49 50 81 26 20 90

Phenoperidine 71 26 64 76 58 70 82 – – –

Piminodine 67 36 64 88 59 63 77 51 24 90

Piritramide 70 1 45 61 42 73 74 – – –

Profadol 42 8 6 – 8 – – – – –

Racemorphan 34 14 9 – 2 – – – – –

Thebacon 45 20 34 49 11 24 25 – – –

Tilidate – – – 84 – 61 – 47 3 38

Tramadol – – – 78 – 30 – – – –

Trimeperidine 58 41 41 – 17 – – – – –

Oral hypoglycemics and antidiabetics

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TD TE TF TAD TAE TAK TAL

Acetohexamide – 39 12 43 53 – 66 91

Buformin 2 – – – – – – –

Carbutamide 78 – – – – 87 – –

Chlorpropamide 72 38 10 43 49 87 78 6

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Molecule TA TD TE TF TAD TAE TAK TAL

Glibenclamide 80 30 11 30 57 90 – –

Glibornuride – 40 5 60 54 92 – –

Gliclazide – – 9 – – 84 – –

Glipizide 87 – 7 – – 86 – –

Glymidine Sodium 76 – 5 – – – – –

Metformin 1 – 00 and 80 – – 03 and 93 – –

Phenformin 3 – – – – – 3 29

Tolazamide – 52 7 50 66 86 71 95

Tolbutamide 76 51 12 55 62 88 74 93

Pesticides

System TW M. E. Getz and H. G.Wheeler,J. Ass. Off. Analyt. Chem. 1968, 51, 1101–1107

Plates: Silica gel, 250 μm thick. Mobile phase: Cyclohexane:acetone:chloroform (70:25:5).

Location reagent Allow the plate to dry in air, heat at 110° for 2 h, allow to cool, spray with molybdate–antimony reagent, and then lightly overspray with ascorbic acid reagent.

System TX

Plates: Silica gel G, 250 μm thick. Mobile phase: N-hexane:acetone (80:20). Reference compounds: Triazophos Rf 20, Parathion-methyl Rf 30, Pirimiphos-methyl Rf 49, Quintozen Rf 84.

System TY

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Plates: Silica gel G, 250 μm thick. Mobile phase: Toluene:acetone (95:5). Reference compounds: Carbofuran Rf 20, Azinphos-methyl Rf 46, Methidathion Rf 60, Parathion-ethyl Rf 85.

System TZ

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform:acetone (90:10).

System TAA

Plates: Silica gel G, 250 μm thick. Mobile phase: Chloroform.

System TAB

Plates: Silica gel G, 250 μm thick. Mobile phase: Dichloromethane.

System TAC

Plates: Silica gel G, 250 μm thick. Mobile phase: Ethyl acetate:isooctane (85:15).

Please refer to the pesticides chapter (Chapter 14, Table 14.1) for Rf values for systems TX, TY, TZ, TAA, TAB and TAC.

Molecule TW

Azinphos-methyl 57

Diazinon 82

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Molecule TW

Dichlorvos 42

Dimethoate 19

Disulfoton 100

Malathion 74

Mevinphos 23

Oxydemeton-methyl 00

Parathion 81

Parathion-methyl 77

Phorate 100

Trichlorphon 9

Phenothiazines and other tranquilisers

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Acepromazine 48 26 24 63 12 28 – 4 – 39

Acetophenazine 53 3 25 38 3 34 32 5 – 33

Azacyclonol 10 – 3 14 1 3 – – – 40

Benzoctamine 59 57 52 – 43 38 – 31 14 65

Butaperazine 53 28 37 5 5 26 – 10 – 42

Captodiame 66 49 – 77 – 47 – – – –

Chlormezanone 66 1 63 68 57 84 80 55 45 94

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Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Chlorpromazine 49 45 35 70 17 25 45 11 2 47

Chlorprothixene 56 51 51 74 25 34 51 20 8 65

Clopenthixol 56 7 32 44 11 45 – – – –

Clotiapine 59 41 59 – 23 – – – – –

Dixyrazine – – – 49 – 47 – – – –

Ethomoxane 60 34 47 – 36 – – – – –

Fluanisone 73 39 68 82 60 67 75 – – –

Flupentixol 62 6 33 46 – 50 – – – –

Fluphenazine 63 5 23 45 10 45 49 6 – 41

Fluspirilene 69 4 59 71 49 63 78 – – –

Haloperidol 67 11 27 76 33 51 75 6 2 61

Levomepromazine 57 47 38 76 46 32 49 27 19 81

Loxapine – 36 – 54 – 49 – 45 9 78

Mebutamate – – 33 60 56 82 85 35 47 87

Meprobamate 75 – 32 56 58 63 87 35 29 78

Mesoridazine 38 3 6 30 1 11 – 2 2 52

Molindone – – – – – – – 24 – 37

Oxypertine 68 4 65 78 58 74 – – – –

Pecazine 53 47 44 65 16 27 – – – –

Penfluridol 76 17 60 84 60 72 89 – – –

Perazine 48 25 37 47 3 21 23 – – –

Pericyazine 58 4 16 51 18 46 61 – – –

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Molecule TA TB TC TE TL TAE TAF TAJ TAK TAL

Perphenazine 55 7 29 42 9 40 40 – 3 56

Phenaglycodol – – – 71 – 84 – – – –

Pimozide 71 3 60 71 40 73 82 – – –

Pipamperone 56 1 12 43 8 33 61 – – –

Piperacetazine 56 6 19 – 17 – – 4 – 30

Pipotiazine 66 3 32 53 21 40 59 – – –

Prochlorperazine 49 34 37 55 7 26 26 18 9 74

Promazine 44 38 30 62 11 18 35 6 2 41

Prothipendyl 47 43 23 59 9 15 29 – – –

Sulforidazine – – – 54 – – – – – –

Sulpiride 38 – – 34 – 17 – – – –

Tetrabenazine 69 41 78 79 67 80 – 83 33 97

Thiopropazate 61 35 53 74 42 62 59 52 36 91

Thioproperazine 46 7 34 43 6 22 22 – – –

Thioridazine 48 42 30 67 13 20 55 9 2 51

Tiotixene 49 10 40 44 7 26 24 19 5 71

Triflupromazine 54 47 35 75 22 32 49 – – –

Trimetozine 61 11 72 68 52 80 – – – –

Tybamate 77 – – 65 – – – – – –

Psychomimetics and sympathomimetics

The tabulated systems, previously described, may be used together with the associated

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location reagents.

Molecule TA TB TC TE TL TAE TAF TAK TAL

Acetylcholine Chloride 2 – – – – – – – –

Adrenaline – – 1 13 – 3 – – –

Amidefrine 15 – 1 – 2 1 – – –

Carbachol – – – – – 4 23 – –

Clorprenaline 57 18 15 – 20 – – – –

Cyclopentamine 20 32 10 66 2 6 68 – –

Dobutamine 52 – 1 49 3 87 – – –

Dopamine 18 – – 43 – 14 59 – 7

Ephedrine 30 5 5 25 1 10 64 1 29

Etafedrine 44 35 9 56 15 14 – 6 49

Ethylnoradrenaline 42 1 2 15 24 – – – 15

Etilefrine 41 2 2 22 3 15 74 – –

Fenoterol 76 – 1 25 4 38 81 – –

Hexoprenaline 3 1 – 71 1 12 – – –

Hordenine 40 5 6 52 5 – – – 20

Hydroxyamfetamine 35 2 2 – 11 – – – 19

Isoetarine 59 – – 36 – 73 – – 26

Isometheptene 24 – – – – – – 4 43

Isoprenaline 40 – 1 21 3 14 69 – –

Mephentermine 25 34 8 40 2 6 – 3 36

Metaraminol 42 1 1 18 24 13 76 – 20

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Molecule TA TB TC TE TL TAE TAF TAK TAL

Methoxamine 55 24 4 11 38 12 73 12 50

Methoxyphenamine 23 26 4 32 2 7 – 20 64

Methylephedrine 32 – – 35 – 12 – – –

Naphazoline 14 3 6 27 – 3 52 – –

Orciprenaline 48 1 3 18 6 21 77 – –

Oxedrine 25 4 1 – – – – – –

Oxymetazoline 9 1 1 34 1 80 – 1 25

Phenylephrine 33 1 1 12 – 8 67 – 22

Phenylpropanolamine 44 4 4 – – – – 2 29

Pholedrine 29 3 3 27 3 9 – – –

Pilocarpine 53 – 32 44 12 52 45 – 44

Prenalterol 47 1 9 25 – – – – –

Pseudoephedrine 33 54 4 17 63 9 – 1 30

Salbutamol 46 1 1 20 4 16 74 – –

Terbutaline 47 1 1 21 5 18 77 – 29

Tetryzoline 13 7 2 26 2 5 60 – –

Tramazoline 6 4 2 30 2 4 – – –

Tuaminoheptane 33 1 7 – 24 – – – –

Xylometazoline 13 7 5 30 3 5 64 – –

Quaternary ammonium compounds

Systems TN and TO H. M. Stevens and A. C.Moffat,J. Forens. Sci. Soc. 1974, 14, 141–148

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System TN

Plates: Cellulose, 250 μm thick. Mobile phase: Ammonium formate:formic acid:water:tetra-hydrofuran (1:5:95:233).

System TO

Plates: Silica gel (without gypsum), 250 μm thick. Mobile phase: Methanol:0.2 M hydrochloric acid (80:20).

Location reagents for systems TN and TO

Acidified iodoplatinate solution Violet, blue-violet, grey-violet, or brown-violet spots on a pink background are given by quaternary ammonium compounds. Cobalt thiocyanate solution Blue spots are given by quaternary ammonium compounds. Molecule TN TO

Acetylcholine chloride 70 60

Atropine methonitrate 95 35

Bretylium tosilate 94 40

Cetrimide 100 50

Choline 60 60

Decamethonium bromide 56 16

Gallamine triethiodide 34 5

Guanethidine 56 50

Hexamethonium bromide 36 10

Pancuronium bromide 80 –

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Molecule TN TO

Paraquat dichloride 22 10

Suxamethonium chloride 35 10

Suxethonium bromide 40 23

Tubocurarine chloride 85 40

Steroids

The tabulated systems, previously described, may be used or Systems TP, TQ, TR, TS and TAM. Systems TP, TQ, TR and TS W. Lund, ed., Pharmaceutical Codex, 11th Edn, London, Pharmaceutical Press, 1979, 940.

System TP

Plates: Silica gel G, 250 μm thick. Mobile phase: Methylene chloride:ether:methanol:water (77:15:8:1.2).

System TQ

Plates: Silica gel G, 250 μm thick. Mobile phase: Dichloroethane:methanol:water (95:5:0.2).

System TR

Plates: Kieselguhr, 250 μm thick, impregnated with a mixture of acetone:formamide (9:1). Mobile phase: Toluene:chloroform (3:1).

System TS

Plates: Kieselguhr, 250 μm thick, impregnated with a mixture of acetone:propylene

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glycol (9:1). Mobile phase: Cyclohexane:toluene (1:1).

System TAM Professor George Maylin: personal communication.

Plates: Silica gel G, 250 μm thick. Mobile phase: The plate is run to 5 cm in a TLC system of chloroform:ethyl acetate:methanol (50:45:5), dried and then re-run to 7 cm in the solvent composition of system TE, ethyl acetate:methanol:strong ammonia solution (85:10:5).

Location reagents for systems TP, TQ, TR, TS and TAM

DPST solution Sulfuric acid–ethanol reagent Spray the plate and then heat at 105° for 10 min. p-Toluenesulphonic acid solution Heat the plate at 120° for 15 min, cool, spray with the reagent, heat again at 120° for 10 min, and respray. Molecule TA TB TE TF TP TQ TR TS TAE TAJ TAK TAL TAM

Androstanolone – – – – 78 11 90 72 – – – – –

Androsterone – 16 72 52 – – – – 90 – – – –

Beclometasone – – – – 75 38 89 42 – – – – –

Betamethasone – – – – 30 00 00 00 – 38 08 80 70

Betamethasone – – – – 58 27 20 02 – – – – – valerate

Cortisone 90 3 68 – 72 28 55 – 87 51 9 83 91

Desoxycortone – – – – 86 52 98 95 – 78 71 96 91

Dexamethasone – – – – 32 8 – – – 38 7 75 66

Dimethisterone – – – – 80 42 91 95 – – – – –

Dydrogesterone – – – – 86 53 96 98 – – – – –

Ethisterone – – – – 78 39 80 – – – – – –

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Molecule TA TB TE TF TP TQ TR TS TAE TAJ TAK TAL TAM

Ethylestrenol – – – – 79 50 94 99 – – – – –

Etynodiol Diacetate – 11 71 57 83 61 95 99 89 – – – –

Fludrocortisone – – – – – – – – – 55 35 91 90

Fludrocortisone 90 – 86 – 58 12 30 00 – – – – – acetate

Fluocinolone – – – – 42 8 10 1 – – – – 68 Acetonide

Fluocortolone – – 50 28 – – – – – – – – –

Fluocortolone – – – – 79 39 88 00 – – – – – hexanoate

Fluocortolone – – – – 78 35 89 58 – – – – – pivalate

Fluorometholone – – 68 52 – – – – 91 46 26 90 86

Fluoxymesterone – – – – 51 9 38 16 – 41 35 91 74

Gestonorone – 31 83 59 – – – – – – – – – Caproate

Halcinonide – – – – – – – – – 62 58 91 –

Hydrocortisone 96 00 45 28 27 02 08 00 86 36 05 74 58

Hydrocortisone – – – – 51 11 38 00 – – – – – acetate

Hydrocortisone – – – – 08 00 00 00 – – – – – hydrogen succinate

Hydrocortisone – – – – 00 00 00 00 – – – – – sodium phosphate

Hydroxyprogesterone – 38 85 63 – – – – 86 – – – –

Hydroxyprogesterone – – – – 81 55 99 90 – – – – – caproate

Lynestrenol – – – – 77 55 99 97 – – – – –

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Molecule TA TB TE TF TP TQ TR TS TAE TAJ TAK TAL TAM

Medroxyprogesterone – – – – 80 50 98 85 – – – – – Acetate

Megestrol Acetate – – – – 80 50 98 85 – – – – –

Metenolone – – 87 62 – – – – 92 – – – –

Methandienone 86 – 80 – 65 10 87 61 – 44 61 92 88

Methylprednisolone 87 – 41 27 23 80 3 – 87 31 13 78 56

Methyltestosterone 89 17 73 47 70 16 91 71 86 60 65 92 92

Nandrolone – – – – 88 49 97 95 – – – – –

Norethandrolone – – – – 71 20 95 78 – – – – –

Norethisterone – 20 76 57 71 22 87 63 86 – – – –

Norethisterone – – – – 87 39 98 90 – – – – – acetate

Noretynodrel – – – – 79 32 91 71 – – – – –

Oxymetholone 95 – 9 – 69 23 85 82 – 70 74 94 86

Paramethasone 91 – 88 – – – – – – 54 39 91 91

Prednisolone – – 41 24 20 – 2 – 86 19 3 65 54

Prednisone – – 45 28 41 – 10 – 84 33 4 74 60

Progesterone – 36 79 56 81 20 99 95 83 76 68 95 97

Stanozolol 78 – – – – – – – – 43 56 91 78

Testosterone: – 14 70 45 60 07 90 63 85 59 63 92 92

Testosterone – – – – 86 28 99 98 – – – – – phenylpropionate

Testosterone – – – – 78 12 99 98 – – – – – propionate

Triamcinolone: 79 – 27 – 09 00 00 00 – 14 6 65 33

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Molecule TA TB TE TF TP TQ TR TS TAE TAJ TAK TAL TAM

Triamcinolone – – – – 32 00 20 06 – – – – – acetonide:

Sulfonamides

Systems TT, TU and TV H. De Clercq et al. ,J. Pharm. Sci. 1977, 66, 1269–1275Sulfonamides are difficult to separate, but these systems are effective and may be used in combination. System TF, previously described, may also be used.

System TT

Plates: Silica gel G, 250 μm thick. Mobile phase: Hexanol.

System TU

Plates: Aluminium oxide, 250 μm thick. Mobile phase: Acetone:ammonia solution 25% (80:15).

System TV

Plates: Aluminium oxide, 250 μm thick. Mobile phase: Chloroform:methanol (70:30).

Location reagents for systems TF, TT, TU and TV

Acidified potassium permanganate solution Yellow-brown spots on a violet background are given by sulfonamides. Copper sulfate solution This detects N-substituted sulfonamides. Mercuric chloride–diphenylcarbazone reagent Blue spots are given by sulfonamides. Van Urk reagent

Analytical Toxicology - Thin–layer Chromatography (TLC) | 95 Thin–layer Chromatography (TLC)

After spraying, heat the plates in an oven at 100° for 5 min. Yellow spots are given by sulfonamides. Molecule TF TT TU TV

Carbutamide – 90 27 7

Chlorpropamide 43 84 43 3

Mafenide 1 – – –

Phthalylsulfacetamide 00 – – –

Phthalylsulfathiazole 00 2 4 4

Succinylsulfathiazole 00 2 1 1

Sulfamerazine 41 33 18 7

Sulfametopyrazine 50 – – –

Sulfacetamide 42 53 37 4

Sulfadiazine 39 24 22 3

Sulfadimethoxine 51 85 52 34

Sulfadimidine 45 50 27 62

Sulfaethidole 35 – – –

Sulfafurazole 52 74 48 4

Sulfaguanidine 6 21 90 48

Sulfamethizole 23 46 36 2

Sulfamethoxazole 54 88 33 2

Sulfamethoxydiazine 43 55 17 15

Sulfamethoxypyridazine 39 53 26 50

Sulfanilamide 46 61 96 66

Sulfaphenazole 51 89 70 13

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Molecule TF TT TU TV

Sulfapyridine 42 47 43 73

Sulfasalazine 00 – – –

Sulfasomidine 16 11 49 20

Sulfathiazole 20 53 40 5

Tolbutamide 55 98 35 4

Vitamins

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TC TE TL TAE TAF

Nicotinamide 54 21 40 27 68 66

Nicotinic Acid 58 17 – – 72 –

Pyridoxine 59 8 15 5 75 67

Thiamine 1 – 1 – 2 18

Xanthine stimulants

The tabulated systems, previously described, may be used together with the associated location reagents.

Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Acefylline Piperazine 4 1 1 – – 1 – – – – –

Caffeine 52 3 58 52 10 25 59 55 54 18 81

Diprophylline 48 – 12 25 – 12 70 59 – – –

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Molecule TA TB TC TE TF TL TAE TAF TAJ TAK TAL

Etamiphylline 54 12 39 74 – 17 – – 28 2 44

Etofylline – – – 38 6 – 66 – – – –

Fenetylline 55 3 45 54 – 14 44 – – – –

Proxyphylline 58 2 33 49 – 29 71 – – – –

Theobromine 53 1 31 34 4 21 59 54 32 8 65

Theophylline 75 1 30 11 9 11 74 66 40 21 78

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