Herbal drugs and fingerprints

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Herbal drugs and fingerprints

  1. 1. 49D.D. Joshi, Herbal Drugs and Fingerprints: Evidence Based Herbal Drugs,DOI 10.1007/978-81-322-0804-4_3, © Springer India 2012Plants contain thousands of constituents and arevaluable source of new therapeutic molecules.For new and effective herbal drug development,it is important to have a validated process to pre-pare plant extract and to isolate ingredients forfull structure elucidation and biological testing.The combination of biological and chemicalscreening leads to the important informationabout plant constituents. The chemical screen-ing by TLC analysis is illustrated in the form ofhi-tech art using high-performance thin-layerchromatography (HPTLC) for better separation,eliminating manual errors, and better repeatabilityas well as reproducibility of the test results. Itprovides a great deal of preliminary informationabout the content and nature of constituentsfound in the active fraction. Once the chemicalnature of a constituent is established via HPTLCanalysis, it is easier to develop validated processto prepare standardized extract and isolateingredient in pure form, structure elucidation,and biological testing with synergistic explanation[1]. HPTLC is a very simple and economicalanalytical method, useful for high-potentialqualitative characterization and quantitativedetermination of herbals and products. Its fieldof application covers virtually all classes ofsubstance with the exception of readily volatileand gaseous substances and can be extendedeasily to the preparative scale by using thickerlayers [preparative layer chromatography(PLC)]. The separated substances, dependingon their optical properties, can be detected,identified, and quantified in visible, infrared, orUV light, sometimes only after derivatizationwith a suitable reagent.Currently, quality evaluation is a main concernin herbal formulations due to variation in the con-tent of markers/active ingredients in the rawmaterials, due to different geo-climatic factorsand business reasons. A computerized densitom-eter is used for the fingerprinting, of concernspot, on its area and intensity, for true authentica-tion of test samples, against standard. Suchchemical fingerprinting is helpful for industries,research institutions, and regulatory authoritiesfor quality evaluation and to decipher the claimsmade for the products [2].Operational Summary of HPTLCThe whole analytical process for HPTLC may besummarized in the following steps [3]:1. Selection of stationary phase for HPTLCanalysis2. Sample preparation, clean up, and pre-chro-matographic derivatization, if any3. Application of sample on stationary phase4. Development of chromoplate5. Detection of spots including post-chromato-graphic derivatization6. Quantification7. DocumentationStationary phase selection for a new product isbased on the subject knowledge of the analystwhich is supported by the gained knowledge dur-ing experiments and TLC analysis for the same.3HPTLC: Herbal Drugsand Fingerprints
  2. 2. 50 3 HPTLC: Herbal Drugs and FingerprintsThe steps as spotting, evaluation, and documen-tation have been connected with computers andcameras respectively, which make the techniquemore hi-tech. HPTLC leads to difficulty in auto-mation, and because of its open character, it ishighly influenced by environmental factors. It istherefore essential that each step which mayrequire specific approach must be carefully vali-dated, much more than TLC analysis.HPTLC Pre-Coated PlatesThe uniformity and homogeneity of the station-ary phase during HPTLC analysis is directlylinked with reproducibility and versatility of theanalytical results. HPTLC uses the same type ofsilica gel 60 layers, as in traditional TLC, with athickness of 0.20–0.25 mm. However, the particlesize is much smaller, typically ranging from 4 to8 mm, with an optimum of 5–6 mm (Table 3.1).The commercial pre-coated HPTLC plates withpolymeric binders are sufficiently hard so as notto be easily damaged by the capillary tubes usedfor sample application. Use of smaller particlesof stationary phase, similar in size and quality toHPLC packing materials, gives a lower theoreti-cal plate height (H) and hence higher efficiencybut can be fully utilized if the plates are not over-loaded with too much sample, the spot size iskept small (about 1.0 mm), and the plate is devel-oped only to the extent necessary for completeresolution (often only 5 cm and rarely more than8 cm). A direct comparison of theoretical platesin HPTLC with HPLC serves little purpose as thenumber found is only valid for the spot used forcalculation. The basic problem is that all analytesdo not travel the same distance and are notmeasured in retention time as in columnchromatography.As HPTLC have higher performance thanTLC, so it is possible to carry out separations onHPTLC that were not possible on TLC platesand, for those where it was possible, to shortenthe time of separation dramatically. HPTLC istherefore a more rapid, efficient, and sensitivetechnique than conventional TLC. For in situquantitative analysis using spectro-densitome-ters, it is essential that HPTLC layers are used forthe most reliable results.Detection and VisualizationLike TLC, HPTLC requires the visualization anddetection, and similar practices are used for thatbut at more precise level. These practices may becategorized as [3]:Nondestructive TechniquesIn this practice, the chromoplate remains intact,may be evaluated by:Table 3.1 Comparison between silica gel pre-coated HPTLC and TLC plates [3]Property HPTLC layer TLC layerParticle size 5–6 mm 10–12 mmPore diameter 60 Å 40, 60, 80, 100 ÅPlate dimensions 10×10 cm, 20×20 cm,10×20 cm5×10 cm, 5×20 cm,10×20 cm, 20×20 cmLayer thickness 0.20–0.25 mm 0.20–0.25 mmAnalysis per plate Up to 75 Up to 16Spot size recommended ~1 mm 2–5 mmSpot loading 50–200 nl 1–5 mlBand size recommended 5–10 mm 10–15 mmBand loading 1–4 ml 5–10 mlSensitivity limit Upper pg (fluorescence) ngNormal development time 2–30 min 15–20 min
  3. 3. 51Detection and VisualizationVisible DetectionTherearecompoundsthathavecolor,forexample,natural and synthetic dyes, chlorophyll, and nitro-phenols, to give an absorption in the visible partof the electromagnetic spectrum. These areclearly seen in visible light and do not requireany further treatment for visualization.Ultraviolet DetectionThere are many compounds that appear color-less in normal light but can absorb electromag-netic radiation at shorter wavelengths. These areoften detected in the UV range, normally at200–400 nm. Often exposure to UV light atshort-wave radiation (254 nm) or long-waveradiation (365 nm), with commercial UV lampsand cabinets, which function at either or both ofthese wavelengths. To aid visualization, manycommercial pre-coated HPTLC layers containan inorganic phosphorescent or an organicfluorescent indicator (Table 3.2). Detection byabsorbance in these cases relies on the phospho-rescence or fluorescence being quenched by thesample components. This process is commonlycalled “fluorescence quenching” in both cases,although more accurately for most indicatorsdesignated F254it is described as phosphores-cence quenching.Reversible ReactionsMany compounds do not absorb visible or UVlight, quench fluorescence, or fluoresce whenexcited by visible or UV light. In these cases,suitable detection reagents are used to givecolored chromatographic zones in visible light orat shorter wavelengths in the UV. Dependingupon the nature of analyte and developing reagent,it may be reversible reactions (i.e., nondestruc-tive techniques), for example, iodine vapor andammonia.Iodine is a universal reagent detecting thepresence of many organic species on thin layers,but some reactions with iodine are irreversible.The use of iodine as a vapor enables the detectionof separated substances rapidly and economicallybefore final characterization with a group-specificreagent. Where lipophilic zones are present on achromatographic layer, the iodine molecules con-centrate in the substance zones giving yellow–brown chromatographic zones on lighter yellowbackground. The preparation of the reagent sim-ply involves putting a few iodine crystals in a drychromatography tank, replacing the lid, andallowing the iodine vapor to fill the air space fora few hours. The developed chromatogram isthen introduced into the chamber, and as soon asthe chromatographic zones are recognized, thelayer is removed and the results recorded. Theadsorbed iodine is allowed to slowly evaporatefrom the layer surface under a dry stream of air atroom temperature; a fume cupboard facility is anideal location for this. These chromatograms canbe subjected to further treatment with other uni-versal or with more specific functional groupreagents. If more permanent results of the iodineimpregnation are required, then the chromato-graphic zones are sprayed or dipped in a starchsolution (0.5–1% w/v) to give blue starch–iodineinclusion complexes. However, it is important tocarry out this procedure after partial evaporationTable 3.2 Some fluorescence intensifier and their application areas [3]Intensifier Compounds detected Enhancement StabilizationTriton X-100 (1% v/v solution inhexane or heptane)Fatty acids asdansyl amides At least tenfold YesPolyethylene glycol 400 or 4,000(10% w/v in methanol)Compounds with alcoholic(-OH) functional groups.20- to 25-fold UnknownParaffin liquid (33% v/v in hexane) Aflatoxins threefold to fourfold UnknownParaffin liquid (33% v/v in hexane) Ketosteroids, cholesterol,cortisoltenfold UnknownParaffin liquid (33% v/v in hexane) Dansyl amides tenfold YesParaffin liquid (33% v/v in hexane) Gentamicins Yes, but level unknown Yes
  4. 4. 52 3 HPTLC: Herbal Drugs and Fingerprintsof iodine from the layer. Starch treatment hasthe best results when iodine is still retained in theseparated chromatographic zones but has gonefrom the background layer. Otherwise, it will bedifficult to distinguish the zones from a back-ground that will also be stained blue.Iodine detection works well on silica gel 60and aluminum oxide layers. However, results areusually poor on reversed-phase layers as the lipo-philicity of the layer does not differ appreciablyfrom the chromatographic zones. Iodine vaporreversible reactions occur with a wide range oforganic lipophilic molecules, for example, fats,waxes, some fatty acids and esters, steroids, anti-oxidants, detergents, emulsifiers, and many mis-cellaneous pharmaceuticals.Ammonia vapor is often used in conjunctionwith other reagents to improve the contrastbetween the separated chromatographic zonesand the layer background. The most commonusage is in the visualization of organic acids withpH indicators. Although indicators, such as bro-mocresol green and bromophenol blue, detect thepresence of a variety of organic acids, furthertreatment with ammonia vapor sharpens the con-trast between analytes and background layerresulting in greater sensitivity. On segregation ofammonia source, ammonia gradually evaporatesaway from the chromoplate, and the sensitivity ofdetection reverts to that prior to treatment.Exposure to ammonia vapor can be achieved bysimply holding the chromatographic plate face-down over a beaker of strong ammonia solution.However, more elegantly, it can be performed bypouring ammonia solution into one compartmentof a twin-trough developing tank and placing theTLC plate in the dry compartment. With the lid inplace, the TLC plate is exposed to an almost evenconcentration of vapor. The process is reversiblewith time as the ammonia soon evaporates fromthe sorbent surface.Nonreversible ReactionsA few techniques and practices used to visualizethe spots for HPTLC have chemical reactionsthat cannot be in original stage; such practices areknown as nonreversible reactions. Fluorescentdyes are commonly used for the nondestructivedetection of lipophilic substances, for example,fluorescein, dichlorofluorescein, eosin, rhodamineB and 6 G, berberine, and pinacryptol yellow.Reagents for dipping chromatograms are preparedasdye(10–100mg)inmethanolorethanol(100 ml).After air drying, the detected chromatographiczones appear brightly fluorescent on a lighterfluorescent background under UV light (254 nm).Although very effective on silica gel, cellulose, andkieselguhr layers (sensitivity from low micro-gram to low nanogram range), these dyes do notrespond on reversed-phase silica gels; sometimesexposure to ammonia vapor after dye treatmentimproves sensitivity.Destructive TechniquesOxidation and/or derivatization due to chemicalreactions occurring on the chromatographic layerbetween a reagent and separated analytes is adestructive technique. In this case, the visualizedcompounds are no longer the original one. Themajor techniques as destructive are charring andthermal activation. Charring techniques involvetreatment of the developed chromatogram with asuitable reagent, followed by heating the layer atrelatively high temperatures to degrade anyorganic species to carbon. As can be appreciated,the reaction is somewhat nonspecific, and hence,charring has been included in what is termed uni-versal reagents. The most popular charringreagent is sulfuric acid, applied to the chromato-graphic layer as a dilute solution (10–20% v/v inmethanol/water); however, orthophosphoric acidand chromosulfuric acid have proved successfulin more of the specific circumstances. The tem-perature and heating time depends on the natureof the compounds to be charred. This can varyfrom 5 to 20 min at 100–180°C. Dilute solutionof sulfuric acid in water/methanol ensures ade-quate wetting of the TLC/HPTLC layers. Onheating, the solvents evaporate steadily and acidconcentrates and finally chars the organic materialpresent. Although it is a very simple detectiontechnique, but sulfuric acid charring does havelimitations especially where commercially man-ufactured chromatography plates are concerned.
  5. 5. 53Detection and VisualizationMost binder whether present in homemade orcommercial plates affected to a greater or lesserextent depending on the temperature and time ofheating. Overheating of plates with organic bind-ers may have a gray or even black background,rendering it useless.It has been observed that some developedzones on a TLC/HPTLC layer when heated athigh temperatures have fluoresced on exposure toUV light, for example, lysergol and lumilysergol(using mobile phase chloroform–methanol–ammonium hydroxide, 85:14.5:0.5, and heatingat 100°C). This process has been given the titlethermochemical activation. Separations on mod-erately polar aminopropyl-bonded silica gel lay-ers have been observed to give the most consistentand sensitive results for this process of detection.The reaction mechanism by which thermochemi-cal activation takes place is not fully elucidated,but the following has been suggested as a proba-ble sequence. The surface of the silica gel-bondedlayer acts as a catalyst. Under the influence of thecatalytic adsorbent surface, substances rich inp-electrons are formed that conjugate to formproducts having fluorescent at excited state. It hasbeen observed that compounds with possible het-eroatoms, such as nitrogen, oxygen, sulfur, orphosphorus, will more readily respond to thermalactivation than pure hydrocarbons. Changes inpH often alter the excitation and emission wave-lengths. The fluorescent compounds formed arequite stable. The fluorescence can frequently beintensified and stabilized by coating the chro-matogram with liquid paraffin or a polyethyleneglycol. The fluorescent enhancer is dissolved inhexane or heptane (5% w/v). If the aminopropyl-bonded layer contains a fluorescent indicator(F254), then appreciable fluorescence quenchingcan occur under UV light at 254 nm. A few com-pounds that have weak fluoresce, like vanillicacid and homovanillic acid, can exhibit strongfluorescent absorption after thermal activationand fluorescence enhancement. Thermal activa-tion is also effective for the detection of cate-cholamines, fruit acids, and some carbohydrates.Spots are also detected by derivatizationreactions either before or after development;however, the popularity of detection of thechromatographic zones after development withchemical reagents compared with chemicalderivatization before development is reflected inthe number of methods available in the scientificliterature. Many hundreds of reagents and reagentprocedures are available for the post-chromato-graphic visualization, whereas relatively fewdescribe pre-chromatographic detection. In casewhere visualization before chromatographicdevelopment has been recommended, the resultsare quite unique and specific.The post-chromatographic visualization issimilar to the TLC detection, which is achievedby spraying or dipping. Some reactions occurimmediately, and colored chromatographic zonesappear on contact with the reagent or more usu-ally after drying or heating at a defined tempera-ture (Table 3.3). The choice of whether thereagent is applied as a spray or by dippingdepends on a number of factors. Spraying usesless solvent, can be accomplished with simpleatomizer devices, and is completed in a shortperiod. However, spraying exposes the surround-ing atmosphere; uneven spray, etc. are drawbacksof the techniques. The salient features of spray-ing reagent are below:1. Sensitivity for detection2. Specificity of the reagent for the analyte ofinterest3. Background effects, more specific when platesare to be scanned spectrophotometrically4. Stability of detection reagent5. Stability of the chromatogram after chemicalor thermal treatment6. Ease of preparation of the spraying or dippingreagent7. Hazards associated with the preparation anduse of a particular detection reagentCommon Visualizing ReagentsA few common reagents for detection of non-UV–Vis. compounds in HPTLC are as follows.Iodine Vapor/SolutionIt is also called “iodine reaction” possibly resultsin an oxidative product. The reaction pathway isnormally irreversible (but sometimes reversiblealso, as previously discussed); in most instances,
  6. 6. 54 3 HPTLC: Herbal Drugs and Fingerprintsitisobservedwithorganicunsaturatedcompoundspresent in the separated chromatographic zones.Electrophilic substitutions, addition reactions,and the formation of charge-transfer complexesoccur with iodine. An added feature is that iodinealso possesses fluorescence-quenching proper-ties; the chromatographic zones that have iodineappear as dark zones on a TLC layer containingfluorescent indicators (Table 3.4) [3].Nitric Acid VaporMany compounds such as ephedrine, sugars, tes-tosterone, and xanthine derivatives have yellowor blue fluoresce after nitration, at 365 nm. Mostaromatic compounds can be nitrated with thefumes from concentrated fuming nitric acid. Thedeveloped chromatogram is heated to about160°C for 10 min and kept while still hot into achambercontainingthenitricacidvapor.Nitrationproceeds at a reasonable rate, and generally thechromatographic zones are rendered yellow orbrown.Redox ReactionOxidation and reduction reactions are frequentlyused for visualization techniques as reactions aregroup specific, depending on the particularreagent used. The main redox reactions forTable 3.3 Some popular visualization reagents for TLC/ HPTLC [3]Visualization reagent Reagent conditions Groups detectedEhrlich’s reagent 4-Dimethylaminobenzaldehyde (2%, w/v) in25% (w/w) hydrochloric acid/ethanol (50:50,v/v). After treatment, heat at 110°C for 2 minAmines, indolesFolin and Ciocalteu’sreagentAs per literature PhenolsGibb’s reagent 2, 6-Dibromoquinone-4-chloroimide(0.5%, w/v) in methanol. After treatment,heat at 110°C for 5 minPhenols, indoles, thiols,barbituratesBlue tetrazolium reagent Blue tetrazolium (0.25%, w/v) in sodiumhydroxide solution (6%, w/v in water)/methanol (25:75, v/v)Corticosteroids,carbohydratesTillman’s reagent 2, 6-Dichlorophenolindophenol sodium salt(0.1%, w/v) in ethanol. After treatment, heatat 100°C for 5 minOrganic acids includingvitamin CIron (III) chloride reagent Iron (III) chloride (1%, w/v) in ethanol/water(95:5, v/v). After treatment, heat at 100°Cfor5 minPhenols, ergot alkaloids,inorganic anions, enols,hydroxamic acids,cholesteryl estersEP reagent 4-Dimethylaminobenzaldehyde (0.2%, w/v)and orthophosphoric acid (3%, v/v) in aceticacid/water (50:50, v/v). After treatment, heatat 80°C for 10 minTerpenes, sesquiterpeneestersJensen’s reagent Chloramine T (10%, w/v) and trichloroaceticacid (0.4%, w/v) in chloroform–methanol–water (80:18:2, v/v). After treatment, heatat 120°C for 10 minDigitalis glycosidesN-Bromosuccinimidereagent0.5%, w/v solution in acetone. Aftertreatment, heat at 120°C for 20 minAmino acids, Z-protectedamino acids, hydroxylflavones, hydroxylquinonesO-Phthalaldehyse-sulfuric acid reagentO-Phthalaldehyde (1%, w/v) in methanol/sulfuric acid (90:10, v /v). After treatment,heat at 80°C for 3 minErgot alkaloids,b-blockers, indolederivatives, histidylpeptides
  7. 7. 55Detection and Visualizationdeveloping visible spots are as follows: Emerson’sreagent [4-aminoantipyrine-potassium hexacy-anoferrate (III)] for detection of arylamines andphenols; chlorine-o-toluidine reagent for vita-mins B1, B2, and B6and triazines; chloramine Tfor steroids and purine derivatives; and chlorine–potassium iodide–starch reagent for amino,imino, and amido groups and triazine herbicides.By contrast, reduction reactions include phosph-omolybdic acid for lipids, phospholipids, andsome steroids; tin(II) chloride-4-dimethylamin-obenzaldehyde reagent for the detection of aro-matic nitrophenols; blue tetrazolium reagent forcorticosteroids; Tillman’s reagent (2,6-dichloro-phenolindophenol) for organic acids, includingvitamin C; and silver nitrate–sodium hydroxidereagent for reducing sugars and sugar alcohols.Iodoplatinate ReagentThis is an effective reagent for a wide rangeof nitrogen containing compounds, includingalkaloids, ketosteroids, quaternary ammoniumcompounds, thiols, thioethers, opiates, sulfoxides,tricyclic antidepressants, and vitamins D3, K1,and B1. A range of colors are produced on thechromatogram depending on the analyte. The limitof sensitivity for detection is often in the lownanogram range. Iodoplatinate reagent, a typicaldipping reagent, consists of the following: 10%(w/v) hexachloroplatinic acid aqueous solution(3 ml), 6% (w/v) potassium iodide aqueous solu-tion (100 ml), and 10% (v/v) methanol aqueoussolution (97 ml). After dipping, the TLC platesare dried at 80°C for 5 min. Further heating at115°C for 5 min can improve sensitivity for someanalyte.Group-Specific ReactionMany reagents are functional group specificmeaning that they give specific reactions withcertain organic and inorganic chemical groups. Inmost cases, the reaction mechanism has beenfully elucidated. As general rule, these reagentsare very sensitive with detection limits usually inmiddle to low nanogram range, for example [3].Hydrazone FormationA hydrazone is a class of organic compounds withthe structure R1R2C=NNH2. They are related toketones and aldehydes by the replacement of theoxygen with the=NNH2functional group. Theyare formed usually by the action of hydrazine onketones or aldehydes. The reagent employed forhydrazone formation is2,4-dinitrophenylhydra-zine in acidic solution [100 mg in 100-ml ethanol/phosphoric acid (50:50)]. After dipping or spray-ing the chromoplate with the reagent, the reactionis completed by heating at 110°C for 10 min.This is a specific reagent for aldehydes, ketones,and carbohydrates. Yellow or orange-yellowhydrazones, or osazones in the case of carbohy-drates, are formed on the chromoplate. Ascorbicacid and dehydroascorbic acid are also detectedby this reagent giving yellow zones on a whitebackground. The sensitivity limit is in the order of10 ng per chromatographic zone.Table 3.4 Iodine reactions on the TLC layer [3]Compounds ReactionPolycyclic aromatic hydrocarbons, indole,and quinoline derivativesFormation of oxidation productsQuinine alkaloids, barbiturates, unsaturatedlipids, capsaicins, and calciferolAddition of iodine to the double bondsOpiates, brucine, ketazone, and trimethazone Iodine addition to the tertiary nitrogen for the opiates. Additionreaction withthe -OCH3group of the brucine. Ring-openingreaction for the ketazone and trimethazoneThiols and thioethers Oxidation of sulfur and addition across the double bondin the thiazole ringAlkaloids, phenothiazines, and sulfonamides Complex formation
  8. 8. 56 3 HPTLC: Herbal Drugs and FingerprintsDansylationDansyl [5-(dimethylamino)-1-naphthalenesulfo-nyl] chloride and other derivatives are used toproduce fluorescent dansyl derivatives of aminoacid, primary and secondary amines, fatty acid, andphenols. The dansylation of carboxylic acid is indi-rect as the acid amides must first be formed. Thisconversion is readily achieved with the reagent.The detection limit is 1–2 ng for fatty acids; how-ever, one of the problems with post-chromato-graphic dansylation is the background fluorescenceit produces. Unfortunately, the fluorescent contrastbetween the chromatographic zones and back-ground results in reduced sensitivity.DiazotizationAzo dyes are strongly colored and can be pro-duced readily from aromatic nitro- and primaryamines and phenols present in the separated chro-matographic zones. This can be achieved in twobasic ways. Nitro compounds are reduced to pri-mary arylamines. These are diazotized withsodium nitrite and then coupled with phenols toform the azo dyes. Conversely, phenols can bedetected by reaction with sulfanilic acid in thepresence of sodium nitrite. The resulting azo dyesare often stable for a period of months. A novelapproach to the detection of phenols is to impreg-nate the layer with sulfanilic acid hydrochloride(2.5% w/v in water) before chromatography andapplication of the sample. After drying the plate120°C for 30 min, the phenolic samples areapplied in the usual way. Following developmentand drying, the layer is sprayed with fresh sodiumnitrite solution (5% w/v). The azo dyes formedhave a high stability, immediately appearing ascolored zones that maintain their color for weeksafter first visualization.Metal ComplexesA number of transition metals act as electronacceptors to form complexes with organic com-pounds that are rich in electrons. Colored metalcomplexes are formed by electron movement todifferent energy states in the transition metal ion.Copper (Cu2+) readily forms such complexes orchelates with carboxylic acids including thiogly-colicanddithioglycolicacids.Asuitabledetectionreagent is copper(II) sulfate 5-hydrate (1.5% w/v,water/methanol). Most acids appear as blue zoneson a pale blue background. The limit of sensitiv-ity is 5 mg/zone. Copper is also used in the biuretreaction with proteins, resulting in the formationof a reddish-violet complex, and with aromaticethanolamines to form blue-colored chelates.Iron (Fe3+) and cobalt (Co2+) can also be used in asimilar way with the formation of reddish-violetzones for phenolic compounds and blue zones inthe presence of ammonia vapor for barbiturates,respectively.Ninhydrin TestNinhydrin is a well-known detection reagent forthe visualization of amino acids, peptides, amines,and amino sugars. The limit of sensitivity rangesfrom 0.2 to 2 mg per chromatographic zonedepending on the amino acid. The colored zonescan vary from yellow and brown to pink and vio-let, depending on the sorbent layer and pH. Thecolors fade quickly unless stabilized by the addi-tion of metal salts of tin, copper, or cobalt.Copper(II) nitrate or acetate is the usual saltschosen as additives. A typical formulation forsuch a ninhydrin dipping reagent is 0.3% (w/v) inpropan-2-ol with the addition of 6 ml/100 ml ofaqueous copper(II) acetate (1% w/v). After dip-ping, the TLC layer is heated at 105°C for 5 min.For better resolution between glycine and serine,collidine is added to the ninhydrin at conc. of5-ml/100-ml reagent.Natural Product ReagentNatural product reagent (NPR), as diphenyl boricacid-2-aminoethyl ester, readily forms complexeswith 3-hydroxyflavones via a condensation reac-tion and is used extensively for visualization ofcomponentsinherbalpreparationsinTLC/HPTLCanalysis. A suitable dipping reagent consists ofdiphenyl boric acid-2-aminoethyl ester (1 g) dis-solved in methanol (100 ml). This solution shouldbe freshly prepared when needed, especially wherequantitative results are required. The chromoplateis thoroughly dried, dipped in the reagent for a fewseconds, dried again in a stream of warm air, andthen dipped in a polyethylene glycol (PEG) 4000(5% w/v) solution in ethanol. The reagent is
  9. 9. 57Coupling of HPTLC with Spectrometryespecially good for the detection of rutin, chloro-genic acids, hypericum, and other flavonoids. Itcan also be used on most sorbent layers includingboth the normal and reversed-phase silica gels.The limit of sensitivity is about 1–5 ng/chromato-graphic zone. The purpose of the PEG 4000 is toenhance the fluorescence and to stabilize the emis-sion of light.Case StudyThe root, stem bark, and fruits of various Berberisspecies in the Himalayan region are well recog-nized for their alkaloid contents. Due to globaldemand for berberine alkaloids and their deriva-tives, various analytical tools such as HPLC, GC,and GC-MS have been used for berberine estima-tion. HPTLC, a technique for quality control andstandardization of traditional herbs like Berberisfor berberine content in root and stem bark ofthree Berberis (i e., B. asiatica, B. aristata, B.lycium), was used, and comparative analyticalassessment revealed that the berberine contentvaried both in root and stem bark samples. Moreberberine content observed in root samples ascompared to bark of all the investigated species.Among the species, Berberis asiatica containsmore berberine as compared B.lycium andB. aristata (Figs. 3.1 and 3.2) [4].Coupling of HPTLC with SpectrometryHPTLC is coupled with ultraviolet–visible, infra-red spectrometry, Raman spectrometry, photoa-coustic spectrometry, and mass spectrometry.FTIR has a high potential for the elucidation ofmolecular structures, and the characteristicabsorption bands are the clue for specific detec-tion, as it indicates the presence/absence ofspecific functional group. Almost all chemicalcompounds yield good FTIR spectra that aremore useful for identification of unknown sub-stances and discrimination between closelyrelated substances. The HPTLC–FTIR spectramake possible the detection and quantification ofeven non-UV-absorbing substances on HPTLCplates. These reasons make this hyphenated tech-nique more universally applicable. The HPTLCand FTIR coupling can be divided into twogroups, that is, indirect and direct methods [5].For indirect coupling there is transfer of thesubstance from a TLC spot to a non-absorbing IRmaterial (KBr or KCl) or in situ measurement ofexcised HPTLC spots when the spectra arerecorded directly from the plate. The directonline-coupled HPTLC–FTIR offers some advan-tages relative to other hyphenated techniquesB asiatica(root)B aristata(root)B lycium(root)B. asiatica(bark)B. aristata(bark)B. lycium(bark)Berberine2 µLBerberine4 µLBerberine6 µLFig. 3.1 HPTLC at 254-nm root and stem bark samples of various Berberis species [4]
  10. 10. 58 3 HPTLC: Herbal Drugs and Fingerprints(HPTLC–Raman spectroscopy, HPTLC–PA, andHPTLC–MS), such as the ease of operation andthe optimized operational aspects of online cou-pling. In direct-coupling HPTLC–FTIR method,a major difficulty is the absorption by conven-tional stationary phases, for example, silica gel,which absorb strongly in the IR range. It is verydifficult to obtain reliable spectra in the regionswhere the layer shows strong IR absorption. Thesilica gel, the most widely used adsorbent inHPTLC, presents absorption bands between1,350 and 1,000 cm−1and above 3,550 cm−1whichare superimposed on the spectra of compounds,and only the region between 3,550 and 1,350 cm−1can be evaluated. Therefore, measurements in thisregion are not possible, but it is possible to makemeasurements up to 1,000 cm−1on cellulose. Thebest results are obtained when the mixture of silicagel 60 and magnesium tungstate (1:1) is used asstationary phase. This adsorbent improves signal-to-noise ratios and enhances the performance ofthe diffuse reflectance of the matrix. Anotherproblem is due to the particle size, particle-sizedistribution, and the layer thickness, which affectthe scattering, remitting, and absorbing of theradiation by the matrix. A stationary phase with aparticle diameter of 10 mm, a narrow particle-sizedistribution, and a layer thickness of 200 mm onglass is found to be ideal in the mid-IR range.Finally, the binder or the fluorescence indicatoradded to the adsorbent and the mobile phase couldlead to altered HPTLC–FTIR spectra [5].The identification can be realized by fitting thereference spectra to sample spectra and visual com-parison. The compounds separated by HPTLC canbe also identified using an HPTLC–FTIR library.The band position, width, and intensity are automati-cally compared, and the reliability of the results isdescribed in terms of hit quality. Quantitative analy-sis with the HPTLC–FTIR technique is generallyapplied for the substances that do not absorb in theUV–Vis. range and when the precision required isnot too high. The lack of precision is due to theincrease of sample spot broadening with increasedmigrationdistanceandtothemeasurementnotbeingexactly at the peak maximum. These problems aredue to the circular infrared beam with small diame-ter. The determination of compounds is made on thebasis of evaluation of the peak areas in the Gram-Schmidt trace or in the window diagram, or by theevaluation of Kubelka-Munk spectra with integra-tion of their strongest bands. The method using theGram-Schmidt traces indicates the changes in absor-bance over the whole spectral region, and therefore,B asiatica(root)B aristata(root)B lycium(root)B. asiatica(bark)B. aristata(bark)B. lycium(bark)Berberine2 µLBerberine4 µLBerberine6 µLFig. 3.2 HPTLC at 366-nm root and stem bark samples of various Berberis species [4]
  11. 11. 59Bibliographyit is suitable and practical for rapid determinations.The evaluation of the peak areas in the window chro-matogram is appropriate for the quantification ofindividual substances. An advantage of this methodis a better signal-to-noise ratio, but the disadvantageis the poorer precision. More precise results areobtained using the evaluation of Kubelka-Munkspectra. The limit of identification and determinationis 10 times higher than those obtained by densitom-etry. This method has the disadvantages of the mea-surement only of the fraction of the substance in thepeak maxima and the additional processing step. Inconclusion, none of these methods is perfect andappropriate for all samples. The choice of a methoddepends on the goals of the analysis [5].TLC and HPTLC are valuable tools for quali-tative determination of small amounts of impuri-ties. Lack of chemical markers is a major problemfor the quality control of herbal medicines. Inmany cases, we do not have sufficient chemicaland pharmacological data of chemical markers.Furthermore, there are many technical challengesin the production of chemical markers, for exam-ple, temperature, light, and solvents often causedegradation and/or transformation of purifiedcomponents; isomers and conformations mayalso cause confusions of chemical. Under suchconditions, HPTLC fingerprints have its values,using reference botanical standard for compari-sons and quality management policies [ISO 9000certification, good laboratory practices (GLP),good manufacturing practices (GMP), total qual-ity management (TQM) and validated instrumentsand services, etc.] in pharmaceuticals to have abetter quality of drugs.References1. Giri L, Andola HC, Purohit VK, Rawat MSM, Rawal RS,Bhatta ID. Chromatographic and spectral fingerprintingstandardization of traditional medicines: an overview asmodern tools. Res J Phytochem. 2010;4:234–41.2. Rajkumar T, Sinha BN. Chromatographic fingerprintanalysisofbudmunchiaminesinAlbiziaamarabyHPTLCtechnique. Int J Res Pharm Sci. 2010;1(3):313–6.3. Wall PE. Thin layer chromatography: a modernpractical approach, RCS chromatography monograph.Cambridge: Royal Society of Chemistry; 2005. ISBN0-85404-535-X.4. Andola HC, Rawal RS, Rawat MSM, Bhatta ID, PurohitVK. Analysis of berberine content using HPTLCfingerprinting of root and bark of three Himalayanberberis species. Asian J Biotechnol. 2010;2(4):239–45.5. Cimpoiu C. Qualitative and quantitative analysis byhyphenated(HP)TLC-FTIRtechnique.JLiqChromatogrRelat Technol. 2005;28:1203–13.BibliographyAhmad I, Aqil F, Owais M. Turning medicinal plants intodrugs. Modern phytomedicine, vol. 384. Weinheim:Wiley; 2006. p. 67–72.Bhutani KK. Fingerprinting of Ayurvedic drugs. EastPharm. 2000;507:21–6.Bobby N, Wesely EG, Johnson M. HPTLC profile studieson the alkaloids of Albizia lebbeck. Asian Pac J TropBiomed. 2012;2:1–3.Dhandapani A, Kadarkarai M. HPTLC quantification offlavonoids, larvicidal and smoke repellent activities ofCassia occidentalis L. (Caesalpiniaceae) againstmalarial vectore Anopheles Stephensi Lis (Diptera:Culicidae). J Phytol. 2011;3(2):60–71.Liang YZ, Xie P, Chan K. Quality control of herbal medi-cines. J Chromatogr B. 2004;812:53–70.Long F. Bio-pharmaceutical characterization of herbalmedicinal products. Drugs. 2001;44(4):102–8.Sagar BPS, Zafar R, Panwar R. Herbal drug standardiza-tion. Indian Pharm. 2005;4(35):19–22.Shahare MD, Mello PM. Standardization of Bacopa mon-nieri and its formulations with reference to BacosideA, by high performance thin layer chromatography.Int J Pharmacogn Phytochem Res. 2010;2(4):8–12.Shanbhag DA, Khandagale NA. Application of HPTLC inthe standardization of a homoeopathic mother tinctureof Syzygium jambolanum. J Chem Pharm Res.2011;3(1):395–401.Soni K, Naved T. HPTLC–its applications in herbal drugindustry. Pharma Rev. 2010 (July-August);112–7.WHO. Quality control methods for medicinal plant mate-rials. Geneva: WHO; 1998.YadavD,TiwariN,GuptaMM.Simultaneousquantificationof diterpenoids in Premna integrifolia using a validatedHPTLC method. J Sep Sci. 2011;34(3):286–91.

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