30 2 TLC: Herbal Drugs and FingerprintsThin-Layer ChromatogramThe quick screening of herbals/herbal productsfor ﬁngerprints and quality is possible, usingthin-layer chromatogram, even in least availablefacilities. The entire process may be summarizedas below:Preparation of TLC Plate in LaboratoryIn TLC, an adsorbent is applied to a supportingplate in a thin layer; generally, a binding agent isused to adhere the adsorbent to the support,although some work is done without a binderusing very ﬁnely divided adsorbent which clingsto the support and forms a rather soft layer. Thisis to be distinguished from the loose-layer chro-matograms in which the adsorbent does notadhere to the supporting plate and must thereforebe developed in a horizontal or near-horizontalposition. Mostly, a mixture of adsorbent andbinder is applied as thin slurry, and the excessmoisture is removed under varying conditionsdepending on the adsorbent, the binder, and thedesired degree of activity. TLC plates are madeby mixing the adsorbent, such as silica gel, witha small amount of inert binder like calcium sulfate(gypsum) and water. This mixture is spread asthin slurry on an unreactive carrier sheet, usuallyglass, thick aluminum foil, or plastic, and theresultant plate is dried and activated by heating inan oven for 30 min at 110°C. The thickness of theadsorbent layer is typically around 0.1–0.25 mmfor analytical purposes and around 1–2 mm forpreparative TLC. After the starting point ismarked about 1.0 cm from the bottom of theplate, the ﬁnish line is marked a convenientdistance from the starting point. This is done witha very soft lead pencil, and care is taken not todisturb the adsorbent layer at the point of sampleapplication since this leads to deformed spots.The solution of the compound is deposited at thestarting line by means of a micropipette, and theplates are then placed in a closed container con-taining a layer of solvent about 0.5 cm deep. Thesolvent ascends the plate by capillary attractionuntil it reaches the ﬁnish line, at which time theplate is removed and solvent allowed to evapo-rate. The locations of the various substances arethen determined by developing spots to the color-less compounds making visible.Ready-Made TLC PlatePresently, ready-made TLC plates with station-ary phase either of silica gel (SiO2) or alumina(Al2O3) are easily available in market. A TLCplate is a sheet of aluminum foil, glass, metal, orplastic which is coated with a thin layer of a solidadsorbent (usually silica, modiﬁed silica, or alu-mina) and may easily reduce to the desired size.The stationary phase on the plates is of uniformthickness and consists of ﬁne particle size. Thealuminum sheets are preferred, as can be cutwithout much more labor.Selection of Suitable Plate SizeThe ready-made TLC plates generally used are of20×20-cm sheets. Each large sheet is cut hori-zontally into small pieces which are 10 cm inlength and of desired widths; the more sampleswe can plan to run on a plate, the wider it needsto be. Skillful handling is required as coating ofadsorbent may disturb and/dirty (Fig. 2.1) duringreduction of size, of the TLC plate.SpottingSample to be analyzed is spotted by either capil-lary or syringe on the TLC plate; on the baseline(Fig. 2.2) the process is called “spotting.” If thesample is not already in solution, it is dissolve insolvent of volatile nature such as hexanes, ethylacetate, or methylene chloride to have 1% solu-tion. For concentrated sample, it is necessary todilute it to avoid a smear or streak; however, some-times only we have to go by trial and error to havewell-sized spot for easy reading. Microcaps withcapillary (Fig. 2.3) or syringe are used for volu-metric spotting with care that it does not disturb
31Thin-Layer ChromatogramFig. 2.1 Cutting suitablesize of TLC plateFig. 2.2 Baseline marks forspottingFig. 2.3 Microcaps withcapillary
32 2 TLC: Herbal Drugs and FingerprintsFig. 2.4 Sample applicationon the plateFig. 2.5 Mobile phase indeveloping chamberthe coating of adsorbent. A small spot of solutioncontaining the sample is applied on the plate,about one centimeter above from the base (Fig. 2.4).The plate is then dipped in to a suitable solvent(i.e., mobile phase) and placed in a sealed con-tainer. As solvent moves up in the plate by capil-lary action and meets the sample mixture, which isdissolved and is carried up the plate by the solvent.Different compounds in the sample mixture moveat different rates due to differences in solubility inthe solvent and due to differences in their polarityto the stationary phase. Results also vary depend-ing on the solvent used, for example, if the solventis 90:10 mixture of hexane to ethyl acetate, thenthe analyte would be mostly nonpolar. This meansthat during separation on TLC plate, the nonpolarparts will have move further up the plate.Preparation of Developing ChamberThe developing container for TLC may be aspecially designed chamber, a jar with a lid, or abeaker with a watch glass on the top/aluminumfoil/butter paper with rubber band; pour mobilephase into the beaker to a depth of just less than0.5 cm, cover the beaker, swirl it gently, and allowit to stand till dip TLC plate on it (Figs. 2.5 and 2.6).
33Thin-Layer ChromatogramNowadays, there are applicators aided withsoftware for mounting purpose, where in spite ofspots, narrow bands are produced. Such type ofdevices is recommended when manual contact,with samples due to safety reasons, is strictlyprohibited as extremely toxic solutions, microbi-ologically contaminated samples, and radioactivecompounds.Chromoplate GenerationWhen we place the spotted TLC plate in thedeveloping chamber, cover it and allow undis-turbed on bench top until the solvent is abouthalf a centimeter below the top of the plate.During the span when TLC plate is inside chamber,a partially competing process occurs as (1) inbetween the components of developing solventsand their vapor, an equilibrium is established,known as chamber saturation; (2) stationaryphase adsorb molecules from gas phase (adsorp-tive saturation) and loaded into the surface ofstationary phase; (3) the wet part of the layer withmobile phase also interacts with gas phase; and (4)during migration, the components of the mobilephase can be separated by the stationary phaseunder certain conditions, causing the formationof secondary fronts.Evaluation of TLC PlateIn TLC plates, often a small amount of aﬂuorescent compound, usually manganese-acti-vated zinc silicate, is added to the adsorbentthat allows the visualization of spots under UV254-nm wavelength. The adsorbent layer itselfhas ﬂuorescence, but spots of analyte quench it.Compounds separated may not be UV detectable,so several methods exist to visualize the spots byspraying reagents . Once visible, the Rfvalueof each spot is determined by dividing the dis-tance traveled by the product by the total distancetraveled by the solvent (the solvent front). Thesevalues depend on the solvent used and the typeof TLC plate and are not physical constants.Evaluation can be either visible or scanner is usedto measure the spot density, enabling the analystfor quantitative results by the density at a speciﬁc Rfvalue and then calculated the marker compoundin the sample with reference to area :Fig. 2.6 TLC plate indeveloping chamber
34 2 TLC: Herbal Drugs and FingerprintsRetention FactorThe retention factor (Rf) is deﬁned as the distancetraveled by the compound divided by the distancetraveled by the solvent, from the baseline, forexample, if a compound travels 2.1 cm and thesolvent front 2.8 cm, the Rfis 0.75:Presentation of TLC ResultsThe position of substance zone (spot) in theTLC can be described with the aid of the reten-tion factor Rf, which is deﬁned as quotientobtained by dividing distance between the sub-stance zone and the starting line by the distancebetween solvent front and starting line. Rfvaluedoes not give any information about the meth-odology used and other boundary parameters.When Rfvalue is multiplied by 100, it isreferred as hRfvalue. Before presenting theresults, selectivity reproducibility and robust-ness of the analytical method are necessary.The Rffor a compound is a constant from oneexperiment to the next, only if the chromato-graphic conditions are similar (i.e., solventsystem, adsorbent, thickness of the adsorbent,amount of analyte mounted, and temperature).If the identity of a compound is suspected butnot yet proven, an authentic sample of the com-pound, or standard, is spotted and run on a TLCplate side by side (or on top of each other, i.e.,co-spotting) with the compound in question. Iftwo substances have the same Rfvalue, they arelikely (but not necessarily) the same com-pound. If they have different Rfvalues, they aredeﬁnitely different compounds. Notable pointis that this identity check should be performedon a single plate because it is difﬁcult to dupli-cate all the factors which inﬂuence Rfexactlyfrom experiment to experiment.Procedure to Determine RfValueof UnknownOn a TLC plate, mark a spot with pencil approxi-mately 1 cm from the end of the slide, far enoughso that it will stay above the solvent in the devel-oping jar. Pens should never be used on TLCplate because the ink will also develop as spotsin the plate. Care is to be taken not to disturbthe surface of the silica while marking. On top ofthe plate, label the spots with pencil accordingto choice (i.e., A=anthracene, C=cholesterol,T=test sample). Notable point, never to touch aTLC plate on the face, only on the sides and backare used; otherwise, extra spots may appear. Theplate now looks like as below (Fig. 2.7).Now using volumetric capillary/clean cutoff-ﬂat syringe needle, dip it in the solution to bespotted. The liquid rises in the needle by capil-lary action. Now very brieﬂy touch the needle tothe TLC slide on the pencil mark to spot thematerial. The spot should be as small in diameterArea (sample) Volume (standard)%ingredientArea (standard) Volume (sample)Cone.of standard100Cone.of sample×=×× ×2.1 cmorigindistance travelled by the compounddistance travelled by the solventSolvent frontRf =Rf = =2.10.752.8new positionof compound2.8 cm
35Thin-Layer Chromatogramas possible to keep the spots sharp and must notrun into another spot. Let the ﬁrst spot dry, thentouch the needle down on top of it two or threemore times to ensure adequate sample. Betweenspotting new samples, the needle should becleaned with suitable solvent. Now let the spotdry, then carefully place the TLC plate in a devel-oping chamber/jar containing about ½cm of theappropriate mobile phase on the bottom. Coverthe chamber/jar to ensure saturation of the air inthe chamber with solvent. Two or three platesmay be developed at the same time, if desired.When the mobile phase has reached about threefourths of the way up the plate, take it out of thechamber and quickly mark the solvent front withpencil before it evaporates. Let the plate dry andvisualize the spots in the following way. First,hold the plate under an ultraviolet light and markthe spots that ﬂuoresce (never look directly at aUV light). Next step is to place the plate in iodinechamber and mark new spots, if any that becomevisible. Third, after removing the plate from theiodine chamber, dip it quickly into a 2% solutionof phosphomolybdic acid in 95% ethanol, wipeoff the back, and set it on a warm hot plate. Thehot plate should be on a setting low enough not tomelt the plastic plate but high enough to cause thespots to appear. If the plate curls, it may be neces-sary to hold it down with tongs or wire gauze.When the dark green, orange, or brown spotshave appeared, remove the plate from the hotplate. Measure the distance from the originalposition of spotting to the spot and to the solventfront. Calculate the Rfvalue and record it .Spot Development by Iodine VaporsIodine vapor is used for quantitative estimation oflipids on TLC plates is based on the fact that mostlipids can be stained by iodine vapor, in con-trolled conditions, and the intensity of staining isproportional to the actual amount of lipid in thespot. The method consists of (1) exposing thedeveloped plate to iodine vapor, (2) spraying itwith a suitable solvent to prevent halogen evapo-ration, (3) collecting the stained lipids by scrap-ing the spots off the plate, and (4) determining bya rate-sensing method the absorbed iodine. Themethod has been successfully applied to analysisof several common phospholipids, long chainfatty acids, cholesterol, etc. .Documentation of FingerprintsThe choice for ﬁngerprints depends on the natureof the constituents that is present in plant materialor on customer’s speciﬁcation. TLC is widelyemployed in herbal authentication, and the major-ity of pharmacopoeial monographs for herbsinclude a TLC identiﬁcation test. TLC separatesmixtures of compounds to leave a “ﬁngerprint”of separated compounds on a plate coated withsilica gel. This ﬁngerprint can be compared withthat of an authentic sample or pure referencecompounds. The chemical proﬁle (ﬁngerprint) ofraw material or of intermediate product (speciﬁcextracts) or of the ﬁnished products against refer-ence material deﬁnes claims made on certiﬁcateof analysis as routine methodology for qualitycontrol (Fig. 2.8) .Two-Dimensional TLCWhen the analyte to be studied is unknown, theremay be many components of very close polarity,and clear-cut separation of the components maynot be achieved. In such cases, two-dimensionalTLC (2D-TLC) separation has advantage(Fig. 2.9). In this case, a single spot of a mixtureis applied near to one of the corner of a 20×20-cmplate and developed in one direction as usual.(Sideways view of plate)Top of slide• ACT••Bottom of slideFig. 2.7 Pre-spotting planon the TLC plate
36 2 TLC: Herbal Drugs and FingerprintsFig.2.8 TLC ﬁngerprints (where 1=reference, 2=sample).TLC No. 1: Fingerprint of forskolin during puriﬁcationfrom Coleus forskohlii. TLC No. 2: Fingerprint of baco-sides during herb selection. TLC No. 3: Fingerprint ofsaponins during extract (20% asiaticoside) preparation.TLC No. 4: Fingerprints of withanolides in Withaniasomnifera for herb selectionFig. 2.9 2D-TLC of C. forskohlii root extract 
37Thin-Layer ChromatogramThe plate is then removed, dried, and redevelopedin a second mobile-phase system so that directionof solvent ﬂow is at right angle with respect to theﬁrst run. The spot location is detected as previouscase. Each spot will with new Rfvalues .TLC BioautographyUse of TLC for chemical and biological screeningtogether is known as “TLC bioautography,” amultidisciplinary approach, which provides aground for efﬁcient collaboration of differentdisciplines for a new drug discovery as well asanalysis of food and feed. It is based on the factthat toxins or other negatively acting agentsreduce the metabolic activity of the microbes,which is proportional to the luminescence andthe stability of compounds on the TLC platewhich can be veriﬁed easily. For a new drug dis-covery, isolation of single component, followedby assay for its biological activity, is a tediousand expensive process. Under such stress, TLCbioautography has a good compatibility withlarge number of samples which can be studiedfor various activities (e.g., acetylcholinesteraseinhibitor, antibacterial, antifungal, free radicalscavenger), whose biological properties have notbeen documented earlier. This technique is usedfor selection of herbals to study therapeuticproperties, combines TLC with in situ bioassay,and allows localization of active constituents incomplex mixture. Agar diffusion, direct TLCbioautography, and agar overlay bioautographyare in general practices. For discovering newantioxidants in herbals, TLC plate is sprayed with2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.Antioxidant reduces the radical, producing whitespots on the purple ground. The TLC bioautog-raphy is a future tool for the study of herbal for-mulation to understand/explain the synergisticphenomena in new drug discovery .Bioluminescence and TLC AnalysisBioluminescence is the production and emissionof light by a living organism as the result of achemical reaction during which chemical energyis converted to light energy. The bioluminexassay is a unique biosensor method that directlycouplesnaturalbioluminescencetoTLC(Fig.2.10).This rapid assay can be used to support materialidentity, detect toxins and chemical adultera-tions, identify potential bioactive compounds, andmonitor manufacturing processes. TLC has tradi-tionally been used as a reliable and economicalanalytical technique for the simultaneous separa-tion of multiple samples using a minimum of harshchemicals or solvents. A developed TLC plate isFig. 2.10 Toxicity proﬁles for 15 wastewater samples; decreased luminescence indicates toxic substance zones(Source: ChromaDex)
38 2 TLC: Herbal Drugs and Fingerprintscoated with the nonpathogenic, bioluminescentmarine bacteria Vibrio ﬁscheri. The compoundwhich interferes with the metabolic process of thebacteria inhibits bioluminescence and is, there-fore, detected as contrasting dark spots on theluminescent background of the TLC plate. Activityis measured by a reduction in light emission ofthe bacteria producing a toxicity pattern charac-teristic of each analyzed sample that can beviewed and quantiﬁed directly on the TLC plate.Results occur within minutes and can be docu-mented photographically (CCD camera, X-ray,Polaroid, or 35-mm ﬁlm). This technology hasbeen developed into a kit system that provides aneffective means of prescreening a variety ofcomplex mixtures in order to minimize the use ofenvironmentally costly conventional methods .Bioluminex has been tested with a wide varietyof toxins, from naturally occurring biotoxinssuch as biocides to heavy metals. Bioluminexsets the standard for consistent regulatory useand helps ensure the quality of products, frombiomass to bottle. Standard toxicity tests onlyestablish the overall toxicity (biological activity)for complex mixtures like wastewater or crudenatural product extracts. Identiﬁcation of theactive compounds requires the tedious isolation ofsingle components followed by assays of theirbiological effects. In addition, there is the risk offalse results due to interference or interactionby any number of compounds in complex sub-stances. Bioluminex overcomes the limitations ofthis conventional approach by speciﬁcally assign-ing biological activity to single components ofmixtures. Because of parallel sample processing,the TLC bioluminescence technique represents aversatile and rugged method with high samplethroughput. Bioluminex has been successfullyvalidated in environmental applications forfood samples, natural products, and a multi-tude of toxicity-related problems. Furthermore,ChromaDex has customized this advanced TLC–bioassay technique for speciﬁc research needs byemploying genetically engineered microorgan-isms, as BioluminexTSKit, available in market.The technique has wide scope in the analysisof drinking water and wastewater/efﬂuent forscreening biocide, leaching, in food, beverage,dietary supplements screening (raw materials andproduct), in veterinary applications (testing feed,supplements, and urine), in testing for toxicresidues in soils, in testing for residues in manu-facturing equipment, in validation of organicproducts or certify soil, and in equipment andwater used in organic farms as well as in determi-nation of an unknown compound’s toxicity.Detection of Colorless CompoundsIn general, the detection of colored compoundscauses no problem, and the same is true for com-pounds which exhibit ﬂuorescence or phospho-rescence under ultraviolet light. In some caseswhere the color is not very intense, a sprayreagent may be used to increase the sensitivityby spraying the chromatogram. There are numer-ous spray reagents which can be used to makethe various colorless compounds visible on thechromatogram. These can be divided into twoclasses: (1) those which are general reagents andwill detect a large number of different types ofcompounds (e.g., 10% H2SO4,iodine vapor) and(2) those which are more speciﬁc in nature, indi-cating the type of compound or functional groupthat is present (e.g., Dragendorff’s reagent foralkaloids, potassium hydroxide for coumarinsand anthraquinones, ferric chloride for tanninsand phenolic compounds, ninhydrin for alphaamino acids) .Combination of TLC with OtherTechniquesTLC has been directly coupled with column chro-matography by using various splitters with a vari-able drive to control the application of a portion ofthe column elute to the TLC plate. Similarly, TLCis used for the selection of proper mobile phase toHPLC and optimizes the analytical conditions.The spots obtained from TLC may be eluted, con-centrated, and then subjected to either HPLC orGLC analysis. The high boiling substances arepreferred for TLC–GLC analysis. Mass spec-trometry (MS) and TLC are combined together to
39Thin-Layer Chromatogramknow the molecular weight of the compound andfragmentation patterns. Desired spot from TLCwith deﬁned Rfvalue can be scraped, dissolved insuitable solvent, ﬁltered, and may be studied byNMR or IR spectrometry for structure elucida-tion, for authentication of the target component.Criteria for Selectivity, Reproducibility,and RobustnessTLC analysis mainly concerned with determi-nation of identity, purity, and assay or togetherall, for which two- or multidimensional TLCare the options. The selection of stationeryphase with analyte is a part of experience andtheoretical knowledge to the chemical compo-sition of stationary phase. For satisfactory sep-aration efﬁciency, the mean particle size,particle size distribution, and morphology ofparticle are to be considered. It is advisable touse prepared TLC plate to secure better repro-ducibility of good fame for quality. Solventsystem up to six components is used, providedthat this must have the appearance of single-phase system with no sign of cloudiness. Theanalogy between solvent system and mobilephase becomes logical when we use mixture ofsolvents, as the solvent system placed in thedevelopment chamber releases some of itscomponents into the pores of stationery phase,where it forms a liquid stationary phase. In thisequilibrium, the mobile phase and solvent havedifferent meaning .Special HazardsLooking directly at an ultraviolet (UV) light tendsto cause eyes cataracts, and shining it on the skinpromotes skin cancer. For these reasons, the UVlamp should be kept in the hood and only heldover the TLC slides for a few seconds to visualizethem. The lamp should be pointed downward atall times and turned off immediately after use.UV rays are absorbed by glass, so a transparentglass is used in UV cabinet to observe the TLCplate. Cyclohexane and toluene are ﬂammableandarenarcoticinhighconcentrations.Chloroformis an anesthetic and a possible carcinogen underconditions of daily exposure for several years.All organic solvents and plate developing cham-bers are kept in the fume hood to avoid solventexposure .Troubleshooting in TLC AnalysisThe aforesaid steps seem that TLC is quite aneasy procedure, but what about the ﬁrst time werun a TLC, and see spots everywhere and blurred,streaked spots, etc., as with any technique, withpractice we get better. A few notable points duringprocess development for unknown compoundsmay be helpful as :1. When compound runs as a streak rather than aspot indicates that sample is overloaded. Tohave desired results, run the TLC again afterdiluting sample, or sample might just containmany components, creating many spots whichrun together and appear as a streak. Perhaps,the experiment did not go as well as expected,change mobile phase as per own analyticaljudgment.2. When sample runs as a smear or an upwardcrescent, compounds which possess stronglyacidic or basic groups (amines or carboxylicacids) sometimes show up on a TLC platewith this behavior. Addition of a few drops ofammonium hydroxide (amines) or acetic acid(carboxylic acids) to the eluting solvent helpsto obtain a clear plate.3. If sample runs as a downward crescent, likelythe adsorbent may be disturbed during thespotting, causing the crescent shape.4. If plate solvent front runs crookedly, either theadsorbent has ﬂaked off the sides of the plateor the sides of the plate are touching the sidesof the container (or the paper used to saturatethe container) as the plate develops. Crookedlyrun plates make it harder to measure Rfvalueaccurately.5. Many random spots are seen on the plate,indicating that during operation analyst haveaccidentally dropped any organic compoundon the plate.
40 2 TLC: Herbal Drugs and FingerprintsIdentiﬁcation of Marker Compoundsin Herbal DrugsTLC is used for the analysis of aliphatic mono-carboxylic acids, keto acids, hydroxy acids,dicarboxylic acids, aromatic carboxylic acids,phenolcarboxylic acids, alcohols (except methanoland ethanol) and glycols, their derivatives, alkaloids(purine, pyridine, phenyl alkylamine, dipyridine,pyridine–pyrrolidine, quinoline, isoquinoline,indole, and pyrrolizidine), using Dragendorffreagent, amino acids, proteins, peptides, antibiotics,carbohydrates, dyes (both oil soluble as well aswater soluble), hydrocarbons (using hexane asmobile phase in silica TLC plate), lipids, nucleicacids and nucleosides, pesticides (chlorinated,phosphorus, carbamates, pyrethrins, etc.), phenoliccompounds,screeningofpharmaceuticals(samplesfor drug of abuse, sulfa drugs, contraceptives,laxatives, etc.), ﬂavonoids, steroids, vitamins,antioxidants, inorganic ions, etc. The herbal rawmaterial as well as herbal drugs may have adul-teration; hence, the proper identiﬁcation ofmaterial is essential before use. There is simplequantitative test by TLC against the referencestandard, as the ﬁngerprints and TLC proﬁleclearly indicate about the adulteration. In thisway, substitution by exhausted drugs can beavoided as the dried exhausted cloves and umbel-liferous fruits after extraction of their volatile oilclosely resemble to the genuine drug. Similarly,addition of synthetic to fortify inferior products,such as adding citral to the oil of lemon or benzylbenzoate to balsam Peru, can be checked.In an attempt to establish a certiﬁed quality ofmarketed herbal drugs, crude, powdered, or incombination, TLC is used to decipher the activeprincipals, identiﬁcation of claimed herb, andpossible contamination/adulterants. The TLCmethod is efﬁcient, rapid, and combines the sen-sitivity and simplicity with low cost for the deter-mination of main active principles of medicinalplants (alkaloids, anthraquinones, coumarins,essential oils, ﬂavonoids, glycosides, saponins,tannins, etc.), which are the main ingredientsresponsible for potential pharmacological effects.The technique is so efﬁcient that it is being usedas primacy analytical tool in the new drug discov-ery for herbal drugs. Herbal drugs are obtainedfrom cultivated or wild plants. They vary more orless in composition and properties depending onthe habitat, climate zone, and annual variations.This causes problems in the identiﬁcation andpurity tests of herbal drugs.Case Study 1: There are many Equisetum sub-species and hybrids species described in litera-ture. These Equisetum species along with havingalkaloid (species like Equisetum palustre) have to bedetected by TLC analysis (Figs. 2.11 and 2.12) .Fig. 2.11 TLC of Equisetum species, where: 1=rutoside, 2=hyperoside, 3=caffeic acid, 4=E. arvense, 5=E. arvense(China), 6=E. palustre, 7=E. arvense 
41Identiﬁcation of Marker Compounds in Herbal DrugsCase Study 2: Passionﬂower (Passiﬂora incar-nata) is used in phytotherapy as a mild sedativeand anxiolytic agent and has considerable quali-tative and quantitative variability with respect toits content of C-glycosyl ﬂavones, some of whichare used as marker compounds for extracts.Analysis of plant material cultivated in Australiarevealed two chemically distinct groups; hence,an investigation was carried out to determinewhether distinct intraspeciﬁc chemotypes exist inthis species. Eleven P. incarnata samples wereanalyzed by HPLC, LC–MS, and two differentTLC methods. The samples fell into two distinctgroups with respect to their C-glycosyl ﬂavoneproﬁle, with little within-group variation. Onechemotype was dominated by isovitexin andschaftoside/isoschaftoside. The other chemotypewas characterized by a high level of swertisin,with low levels of schaftoside/isoschaftoside.The two chemotypes were readily identiﬁed byboth HPLC and TLC .TLC Method 1: This was the method forpassionﬂower in the British Pharmacopoeia(2007). Mobile phase was composed of water-anhydrous formic acid methyl ethyl ketone ethylacetate (10:10:30:50, v/v), and the plates weredeveloped to a distance of 150 mm at room tem-perature. Detection was by way of two sprayreagents, natural products reagent (2-aminoethyldiphenylborinate, sigma, 1% in methanol) fol-lowed by Macrogol 400 (polyethylene glycol,sigma, 5% in methanol) .TLC Method 2: This method was adopted fromWidmer, Meier, and Schaffner and was publishedby CAMAG, Switzerland, on their website (http://www.camag.com/index.php; n.d.). Mobile phasewas composed of tetrahydrofuran-toluene-formicacid-water (16:8:2:1, v/v), and the plates weredeveloped to a distance of 52 mm at room tem-perature. The heated plates were sprayed withnatural products reagent (0.5% in ethyl acetate)followed by polyethylene glycol 4000 (Fluka,5% in dichloromethane) .TLC Method 2 was superior to the Method 1(i.e.,the British Pharmacopoeial method) in terms ofclarity and resolution, and Method 2 allowed forthe differentiation between the two P. incarnatechemotypes (Fig. 2.13) .Similarly, qualitative analysis for differentchemical classes of therapeutics, using differentmobile phases (Table 2.1), can be performed byTLC as:Fig. 2.12 E. arvense and E. palustre in TLC analysis 
42 2 TLC: Herbal Drugs and FingerprintsTLC Analysis for AlkaloidsThe systematic procedure for the analysis ofalkaloids by means of thin-layer chromatography,with the mixture of cyclohexane-chloroform-diethylamine (5:4:1) as screening the alkaloidson the basis of polarity and further analysis usingchloroform–acetone–diethylamine (5:4:1) andchloroform–diethylamine (9:1), is still a guidelinein analysis of new unknown herbo-combinationfor alkaloids by TLC, subsequently developingspots with Dragendorff’s reagent. The mostpreferred adsorbent TLC plate for alkaloids hasbeen silica gel 60 F254. Once detecting the alka-loid, the mobile phase is determined based onsatisfactoryandvalidableparameters.Theauthorswere beneﬁted by the above concept duringdeveloping in-house protocol for Gloriosa glabraFig. 2.13 TLC plate showing P. incarnata swertisin chemotype (1–6) and isovitexin chemotype (7, 8), where C=chlo-rogenic acid, H=hyperoside, R=rutin Table 2.1 Brief list of common solvent systems for TLC Chemical group Absorbent Solvent system frequently used DetectionAlkaloids Silica gel 1. Methanol–chloroform (85:15) 1. UV2. Toluene–ethyl acetate–diethylamine (70:20:10)2. DragendorffAnthocyanins Silica gel, cellulose n-butanol–acetic acid–water(40:10:20)1. UV2. Anisaldehyde- sulfuric acidCardiac glycosides Silica gel 1. Ethyl acetate–methanol–water(81:11:8)1. Kedde reagent2. Chloroform–methanol–water(65:35:10)2. Antimony chlorideFlavonoids Silica gel Chloroform–acetone–formic acid(75:16.5:8.5)UVIndoles Silica gel Chloroform–ethyl acetate–formicacid (5:4:1)p-DimethylaminocinnamaldehydeMonosaccharides Silica gel n-butanol–acetic acid–ether–water(9:6:3:1)1. Aniline hydrogen phthalate2. UVPhenols Silica gel Acetic acid–chloroform (1:9) Folin reagentPolyacetylenes Silica gel Chloroform–methanol (1:9) 10% H2SO4Saponins Silica gel 1. Chloroform–methanol–water(60:35:5)1. Vanillin/ sulfuric acid2. n-butanol–water (1:1) (upperphase)2. Anisaldehyde sulfuric acidTerpenes Silica gel 1. Chloroform–methanol (95:5) 1. 10% Sulfuric acid2. Ethyl acetate–cyclohexane (60:40) 2. Anisaldehyde- sulfuric acid
43Identiﬁcation of Marker Compounds in Herbal Drugsmethanolicextract,evaluatingforbothcolchicinesand colchicoside in a single TLC plate usingmobile-phase chloroform–methanol (8.5:1.5) andevaluating at 254 nm, previously as it was beendone in two steps: chloroform–methanol (9.5:0.5)for colchicines and chloroform–methanol (8:2)for colchicoside. The best industrial applicationof TLC is the determination of selected substance inpharmacopoeialproducts,whenitisbeingmeasuredat the level of less than 0.1% (e.g., related sub-stances in yohimbine, berberine hydrochloride,thiocolchicoside, reserpine as pharmacopoeialproduct especially in USP). Test solution: A50-ml separating funnel was charged with 10 mlof the preparation to be tested added 1.0 ml ofconcentrated aqueous ammonia and 10 ml ofchloroform, after which the mixture is shaken for3 min. Then the chloroform extract is separatedand, in the case of emulsiﬁcation, centrifuged for3 min at 2,500 rpm to complete phase separation.Reference solution: The reference solution isprepared by dissolving 0.01 mg of sanguiritrine(working sample) solution in 19.5 ml of methylalcohol and 0.5 ml of ammonia solution. Spottingon TLC plate: Sample of the test solution (30 ml)and of the reference solution (20 ml) was appliedon the start line of a TLC plate (silica gel 60 F254).The plate is dried in air for 3 min, placed into avertical cell with the diethyl ether–petroleumether–methanol mixture (35:15:1) and chromato-graphed in the ascending mode. When the solventfront reaches the end of the path, the plate isextracted from the cell, dried in air at roomtemperature until the solvent mixture is removed,and examined on exposure to UV radiation witha wavelength of 360 nm. The chromatogram ofsanguiritrine shows two spots: orange (sangui-narine) and yellow (chelerythrine). The chro-matogram of the test solution must display bandof the same color and mobility (i.e., Rfvalue) asthose on the reference pattern .TLC Analysis for PhenolsVolumes have been written about the TLC anal-ysis of phenolic compounds (derivatives of benzoicacid, cinnamic acid, coumarins, ﬂavonoids,tannins, and lignins). For simple phenols, thebestsolventsarepetroleumether(60–80°C)-carbontetrachloride-acetic acid (4:6:1) and chloroform–acetone–diethylamine (4:2:0.2). Polyhydricphenols are best separated by using chloroform-acetic acid (5:1), chloroform–acetone–aceticacid (10:2:1) and benzene–acetic acid (5:1).These spots are made visible by spraying with anacetone solution of p-nitrobenzene diazoniumﬂuoroborate. Phenolic compounds are ubiquitousin the plant kingdom, being the most abundantsecondary metabolites. Though bio-phenols arefound in all plants, their quantitative distributionvaries between different tissues of plant andwithin different populations of the same plantspecies. Bio-phenols of fruits, vegetables, herbs,spices, and cereals have high therapeutic poten-tial and are in use as herbal drug. TLC is theprimary one and best screening technique forbio-phenols analysis. Lignin, the second mostabundant compound in the nature, on hydrolysis,gives bio-phenols. Methanol has been reported asa suitable solvent for short-time extraction asphenolic glycosides degraded with longer span.The primary characterization of phenolics isusually done by TLC .TLC Analysis for SaponinsSaponins are glycosides which produce stablefoams when their aqueous solution shaken. Onacid hydrolysis, saponins split into sugars andthe corresponding sapogenins. Saponins basedon the carbon skeleton of sapogenin are termedas terpenoid saponins (e.g., Centella asiatica,Bacopa monnieri, Stevia rubidiana) and steroidalsaponins (e.g., Tribulus terrestris, Withaniasomnifera, Gymnema sylvestre) were analyzedby TLC, in authors lab, using mobile-phase chlo-roform – methanol and water in different compo-sitions and subsequently developing spots withanisaldehyde–sulfuric acid and drying plate at110°C for 10 min. The spots were evaluatedagainst reference preparation, either as qualitativeor quantitative determination (withanolides inWithania somnifera, have steroidal skeleton, butthese are steroidal lactones). Chromoplates were
44 2 TLC: Herbal Drugs and Fingerprintsexamined before and after spraying under UVand day light. The individual optimization ofTLC techniques was helpful in the process devel-opment of puriﬁcation at higher purity levels ofphytochemicals (>98% purity of the ingredient)to detect the impurity proﬁle of the ingredient,using organic solvents with water (sometimes indifferent pH values, as buffer, etc.), as well as todevelop HPLC analysis methods, selection ofanalytical column, and to determine combinationof mobile phase and gradient parameters, as wellas regeneration of column prior to the nextanalysis.As plant saponins are sensitive to the struc-tural variation (e.g., saponins mostly are hydro-philic, but digoxin is also lipophilic), so these arestandardized with saponin mixtures isolated fromthe plant species in which the concentration ismeasured (gravimetric analysis). However, oneplant species may contain some saponins whichcan be determined with a biological test andothers cannot be. That is why biological andcolorimetric determinations do not provide accu-rate data and have to be recognized as approximate(hence gravimetric analysis is preferred). TLC (onnormal and reversed phases) and 2D TLC provideexcellent qualitative information and in combina-tion with online coupling of a computer withdual-wavelength ﬂying-spot scanner and two-dimensional analytical software which can beused for routine determination of saponins inplant material. The densitometry of saponins hasbeen very sensitive, however, to plate quality,spraying technique, and the heating time, andtherefore appropriate saponin standards have tobe run in parallel with the sample. Gas–liquidchromatography has limited application for deter-mination since saponins are quite big moleculesand are not volatile compounds. Thus, there areonly few applications of GC for determinationof intact saponins. The method has been usedfor determination of TMS, acetyl or methylderivatives of an aglycones released duringsaponin hydrolysis. However, structurally differ-ent saponins show different rates of hydrolysis,and precise optimization of hydrolysis conditionsis essential. Besides, during hydrolysis, a numberof artifacts can be formed which can inﬂuencethe ﬁnal results. High-performance liquidchromatography on reversed-phase columnsremains the best technique for saponin determi-nation and is the most widely used method forthis group of compounds. However, the lack ofchromophores allowing detection in UV limitsthe choice of gradient and detection method. Thepre-column derivatization with benzoyl chloride,coumarin, or 4-bromophenacyl bromide has beenused successfully in some cases, allowing UVdetection of separation. Standardization andidentiﬁcation of the peaks in HPLC chromato-grams have been based on comparison of theretention times with those observed for authenticstandards. But new hyphenated techniques,combining HPLC with mass spectrometry andnuclear magnetic resonance, are developingrapidly and allow online identiﬁcation of sepa-rated saponins. Capillary electrophoresis hasbeen applied for saponin determination only in alimited number of cases, and this method is stillbeing developed .TLC Analysis for TerpenoidsThe low polar nature of terpene and its deriva-tives, solvents of low polarity, are used for TLCseparation of these components; spots are mea-sured either UV-visible or developed using iodinevapors or 5% sulfuric acid after iodinization andheating at 110°C for 10 min in oven. Author inhis laboratory is evaluating forskolin from Coleusforskohlii, using cyclohexane-ethyl acetate (6:4)and developing spots with 7.5% H2SO4afteriodinization and subsequently heating it at 110°Cfor 10 min. The technique is too valuable to assaypurity of forskolin which matches with HPLCassay. The TLC ﬁngerprints for essential oilsOcimum sanctum (tulsi), Centella asiatica (gotukola), and Convolvulus pluricaulis (shankh-pushpi), in a combo-herbal drugs, were developedusingmobile-phasetoluene–ethylacetate(85:15),which was evaluated at 365 nm after drying inhot air, and ﬁnally spots were developed using7.5% H2SO4and heating plate at 110°C for10 min in oven, by the authors, where separatemarker appears for individual ingredient, using
45Identiﬁcation of Marker Compounds in Herbal Drugsagainst reference botanical material (RBM).While in another combo-herbal formulation, theTLC ﬁngerprints for O. sanctum were developedby ether-hexane (7:3), and spots were developedwith 2% H2SO4and drying it at 110°C for 10 min,reddish ﬂuorescence marker for O. sanctum, vis-ible at 365 nm. The polarity of compoundsincreases with increasing number of –OH groups,while the alkyl derivative of hydroxyl groupdecreases the polarity, and further increase in sizeof alkyl group again decreases the polarity.Hydrogenation of side chain had only slight effecton polarity; similarly, hydrogen band formationalso reduces the polarity in terpenoids. In order toselect the optimum mobile phase for TLC, mobilephase was toluene–ethyl acetate (4:1), n-hexane–ethyl acetate (9:1), hexane–chloroform (5:1), andbenzene–ethyl acetate (5:1). The best separationof components was achieved with the last solventmixture. The bands of terpenoids in the chro-moplate were revealed by treating the TLC plateswith an anisaldehyde–sulfuric solution, followedby heating to 100°C for 10 min.Test solution: A 50-ml separating funnel wascharged with 10 ml of the preparation to be tested10 ml of n-hexane; after which the mixture isshaken for 3 min, and hexane phase was transferredinto a 50-ml round-bottom ﬂask. The extractionwas repeated with the same amount of n-hexane.Then extracts are combined, and the solvent isdistilled off to dryness in a rotary evaporator at atemperature not exceeding 50°C. The residue wasdissolved in 1 ml of ethyl acetate. Reference solu-tion: The reference solution is prepared by dis-solving 10 mg of menthol in 2 ml of ethyl acetate.Spotting on TLC plate: Sample of the test solution(20 ml) and of the reference solution (5 ml) wereapplied, on the start line of a TLC plate [silica gel60 F254, (mobile-phase benzene: ethyl acetatemixture) (5:1)] and chromatographed in theascending mode until the solvent reaches a level of10 cm in the plate and dried the plate in air at roomtemperature until the solvent disappears. Then theplate is treated with anisaldehyde solution andheated at 105°C for 10 min. The developed chro-matogram is examined in daylight .TLC with DART–MSUsing TLC, to generate more versatile andspeciﬁc information on extract of herbal drugs,“direct analysis in real time (DART) ion source”Relativeintensity200 250cba300288.11301304.14758[MH]+[MH]+NHNHNNevodamineritaecarpineNNevodiamine[MH]+;304.1450rutaecarpine[MH]+;288.1107H2COO350 400 450 500m/zm/z200 250 300 350 400 450 500Relativeintensity00100100Fig. 2.14 Evodiae fructus extract in TLC DART–MS . (a) Evodiae fructus, (b) TLC chromatogram of the extract ofEvodiae fructus, under 365 nm, (c) DART–MS spectra of rutaecarpine and evodiamine
46 2 TLC: Herbal Drugs and Fingerprintsis being in use. This hyphenation system of TLCand DART–MS provides unique and speciﬁcinformation on the major constituents of crudeplant drug on TLC through uncovering high-resolution mass number of each band on the TLCplate directly in real time.Case Study-1: The three well-known KoreanPharmacopoeial herbal drugs were extractedand developed on a silica-coated TLC platewith preestablished conditions, as per KoreanPharmacopoeia IX, and developed TLC platewere placed between the DART ion source andTOF-MS analyzer to get real-time mass spectrafrom the bands on the plate directly. The markercoumarin compounds, decursin and decursinol,were successfully identiﬁed from the TLC platedeveloped with Angelicae gigantis radix, alongwith alkaloid compounds of rutaecarpine andevodiamine from Evodiae fructus and lignanmolecules of gomisin A, N, and schisandrin fromSchisandrae fructus (Fig. 2.14) .Case Study-2: The curcuminoids (mixture ofcurcumin, demethoxy curcumin, and bis-demethoxy curcumin) were successfully detecteddirectly from the raw rhizome of Curcuma longawhen a turmeric extract was separated on a TLCplate, studied with DART–MS, each band pro-duced molecular ion peaks corresponding to cur-cumin, demethoxy-curcumin, and bis-demethoxycurcumin. Molecular ions of curcuminoids inturmeric-containing beverages and curry powderwere also efﬁciently detected at the range of5–100 mg/ml. To establish the validity of analyticalmethods for curcuminoids (curcumin and itsderivatives) from various types of samples withDART–MS, data were compared with the resultsof HPLC and were strong proofs for speciﬁcity ofthe method. As DART–MS produces [M+H]+molecular ions of most compounds, so relativelysimple and clear mass spectra are obtained evenof multicomponent samples . In order to takeadvantage of the capacity of DART–MS for thereal-time analysis of individual compounds innatural raw materials, the technology has a hugescope in herbal drug industries .TLC Fingerprints for Batch to BatchConsistencyA study for TLC ﬁngerprints of an Unani formu-lation, named “Majoon-e-Sandal,” which iswidely used in the ailments of stomachic, antibil-ious, psychoneurosis, vomiting, and nausea, andprepared by mixing the powders of the Santalumalbum (110 g), Bambusa bambos (15 g), andStyrax benzoin (15 g) for the formulation compo-sition and kept separately. Tamarindus indicawas soaked in water for 2 h, crushed with hand,and ﬁltered through muslin cloth and kept sepa-rately. Punica granatum seeds were crushed withhand and ﬁltered it through muslin cloth and keptseparately. Crocus sativus was grinded by addingrose water and kept separately. Dissolve 750 g ofsugar in 500 ml of water; at the boiling stage,0.1% citric acid was added, mixed thoroughly,and ﬁltered through muslin cloth. Then, boil theﬁltrate on slow heat and add the mixed extractof Tamarindus indica and Punica granatumfollowed by Crocus sativus. Then, with the contentmixed thoroughly, prepare the 79% consistencyof quiwam. Remove the vessel from the ﬁre,while hot condition is added to the mixed powdersof the Santalum album, Bambusa bambos, andStyrax benzoin, followed by 0.1% of sodium ben-zoate; mix thoroughly to prepare the homogenousproduct. Allow to cool to room temperature, andpacked it in tightly closed container to protectfrom light and moisture. Majoon-e-Sandal is asemisolid, brown-colored characteristic of itsown odor and in sweet taste .Preparation of extracts: From three differentbatches, 2 g of sample, each separately, wassoaked in chloroform and alcohol, respectively,for 18 h, reﬂuxed for 10 min on water bath, andﬁltered. The ﬁltrates were concentrated on waterbath and made up to 5 ml in a standard ﬂask sepa-rately. Development of TLC ﬁngerprints: Thechloroform and alcohol extracts were applied onprecoated silica gel 60 F254TLC plate (E. Merck)as absorbent and developed the plate using sol-vent systems, toluene : ethyl acetate 9:1 and 1: 1,
47Referencesrespectively. After developing, the plates weredried and observed the color spots at UV-254,UV-366 nm, and spots were developed using van-illin–sulfuric acid spraying reagent. TLC studiesof chloroform and alcoholic extract of all the threebatch samples had identical spots in UV (254,366 nm), similar Rfvalues, even on developmentwith vanillin sulfuric acid reagent, after heating at105º for 10 min (Figs. 2.15 and 2.16) .TLC is a widely applied technique in herbalauthentication and used in majority of pharmaco-poeia’s monograph for correct identiﬁcation. UsingCo-TLC, unknown compounds can be easilyidentiﬁed with an authentic compound, so it iswidely adopted because of less time-consum-ing, semiquantitative, and a cheap technique.Furthermore, it also provides ﬁngerprints of thematerial under consideration; if the marker com-pound is known, then it becomes more precise, so itis an important tool for monitoring the identity andpurity of the plant material under consideration; inaddition, it also provides information about substi-tution and adulteration.References1. Abu-Hamdah S, Aﬁﬁ FU, Shehadeh M, Khalid S. Simplequality control procedures for selected medicinal plantscommonly used in Jordan. Pharm Biol. 2005;43(1):1–7.2. Sachan AK, Sachan NK, Kumar S, Sachan A, GangwarSS. Evaluation and standardization of essential oils fordevelopment of alternative dosage forms. Eur J SciRes. 2010;46(2):194–203.3. Joshi DD. Thin layer chromatography and biotechnol-ogy at molecular level. In: Textbook of molecular bio-technology. New Delhi: I.K. International Pub. House;2009. p. 1181–96. Sample Chapter 53.4. Klier B. Current problems with identiﬁcation ofherbal drugs. PhytoLab GmbH & Co. KG 91487Vestenbergsgreuth Germany. http://www.ga-online.org/ﬁles/Graz/WS-4_Klier.pdf. Accessed 29 Mar 2012.5. Wohlmuth H, Penman KG, Pearson T, Lehmann RP.Pharmacognosy and chemotypes of passionﬂowerFig. 2.15 TLC for chloroform extracts Fig. 2.16 TLC for alcohol extracts 
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