Dyeing Reagents for Thin-Layer and Paper Chromatography

Contents
Introduction Index of compounds and compound classes and appropriate detection reagents Reagents in alphabetical order

5n most cases the chromatogram is specially treated at this stage. 0or details please refer to the directions ?uoted for the particular reagent concerned. Anless otherwise stated, subse?uent treatment should be ta"en as meaning drying at room temperature. Dipping: 5n case of ?uantitati'e e'aluation dipping of the chromatogram into the staining solution is becoming e'er more usual with respect to precision and repeatability. 5n general less concentrated reagent solutions prepared with less polar sol'ents are common for dipping purposes. %are should be ta"en in the choice of sol'ent to ensure that neither the chromatographically separated substances nor their reaction products are soluble in the sol'ent of the dipping reagent. Ready-to-use Spray Solutions (erc" sells a number of ready#to#use spray solutions in ),, ml glass bottles which can directly be connected to the rechargeable electro#pneumatically operated T$% sprayer 4Brd. 7o. ).,+9;,>C -niline phthalate Brd. 7o. ).,).<*.,),, 6romocresol green Brd. 7o. ).,)**;.,),, . ,: #Dichlorofluorescein Brd. 7o. ).,*.)*.,),, ;#4Dimethylamino>#benDaldehyde Brd. 7o. ).,8:...,),, Dragendorff#reagent Brd. 7o. ).,.,89.,),, (olybdatophosphoric acid Brd. 7o. ).,,;+,.,),, 7inhydrin Brd. 7o. ).,<:,9.,),, 2hodamine 6 Brd. 7o. ).,:<,..,),, fter-treatment 0re?uently, optimum color de'elopment after reagent application by spraying or dipping is only obtained by heating. - plate heater or an adEustable drying o'en is generally used for this purpose. Bccasionally, in the case of fluorescing chromatogram Dones an additional treatment with solutions of li?uid paraffin, polyethylene glycols, and other 'iscous li?uids can lead to a stabiliDation and also to a tremendous enhancement of the fluorescence intensity of compounds. Paper Chromatograms 0or the detection of paper chromatograms seeC 5.(. Hais, K. (ace", @aper %hromatography, @ublishing House %Dechoslo'a" -cademy of Sciences, @rague, and -cademic @ress, 7ew For" and $ondon, )*<8. 0. %ramer, @apier#%hromatographie, 3erlag %hemie, Weinheim, 9th Ed., )*<.. See also the rele'ant chapters in handboo"s on the subEect, e.g.C E. Stahl, Thin#$ayer %hromatography, Springer and -cademic @ress, 7ew For" and $ondon, .nd Ed., )*<*.

Introduction
The first collection of instructions for the preparation of staining reagents was published by K.G. Krebs, D. Heusser, and H. Wimmer in Egon Stahl s !Handboo" of Thin#$ayer %hromatography! in the si&ties and lateron in a re'ised form repeatedly as a brochure by E. (erc" Darmstadt, Germany until )*+, when the last unchanged edition was released. -bout ., years later two comprehensi'e boo"s on staining reagents were published by H. /or", W. 0un", W. 0ischer, and H. Wimmer !Thin#$ayer %hromatography 1 2eagents and Detection (ethods, 3ols. )a and )b! 43%H Weinheim, 5S67 8#9.:# .:+8;#< and 5S67 8#9.:#.+.,9#=> # somehow as a replacement for the anti?uated staining reagents brochure. 6oth 'olumes are recommended to any thin#layer chromatographer because they show for each reagent at least one appro'ed e&ample and furthermore they offer plenty of information not only on instructions for preparation and handling but also on reaction mechanisms, coloration of deri'ates, limits of detection etc. etc. -s still numerous chromatographers ha'e been fre?uently as"ing for the cancelled dyeing reagents boo"let its te&t was completely re'ised # mainly concerning ordering numbers, misprintings and other errors. 0or easy accessibility the resulting new list below is now being presented in the internet. -n alphabetical inde& of compounds and compound classes for which a detection reagent is being sought is gi'en at the beginning followed by the staining reagents listed in alphabetical order. 2eagents for paper chromatography are additionally mar"ed with !@%!. Staining of Thin-layer Chromatograms Spraying: Dry the chromatogram to remo'e the sol'ent, then cool. @lace it 'ertically into a spraying bo& or into a fume cupboard and protect the surroundings by co'ering with filter paper or the li"e. -pply the spray solution from about 8, cm until the layer is e'enly wetted but not for so long that the chromatogram begins to run with li?uid.

K. 2anderath, Thin#$ayer %hromatography, 3erlag %hemie and -cademic @ress, 7ew For" and $ondon, .nd Ed., )*<<.

Index of Compounds and Compound Classes and ppropriate Detection Reagents

-cetylene compounds -cids, aromatic -cids, organic -drenaline and deri'ati'es -lcohols, higher -ldehydes -l"ali chlorides -l"ali ions -l"aline earth metal ions -l"aloids -luminium ions -mides -mines -mines, aromatic -mines, ?uaternary -mino acids -mino acids, sulfur containing -minosugars -mmonium ions -mylase -nhydrides -nthro?uinone glycosides -ntimony ions -ntio&idants -rginine -rsenic ions -Dulenes 6arbiturates 6arium ions 6ile acids 6ismuth ions 7o. *< 7o. )98,.;* 7o. ;.,;8, <;, <<,::,:+, *),*8, ).., );+ 7o. *9,)89,.8+,.;) 7o. 8.* 7o. +<,))9 7o. *. 7o. 88, 7o. 88, 7o. 9<, :<, )8), )8., )88, )<., .,9, .)), .;< ,.;: ,.*+ 7o. 8), )9+, )*9 , .99 7o. <*, )99 7o. :<, ),., .,:, .,+, .)), .;,, .++ 7o. +:, )8<, );*, .,., .)8, .)9, 8,., 8,8 7o. )8, 7o. ));, ):<,.,),.,:, .,+,.,*,.),, 8.+ 7o. )<), .+: 7o. ),.,.,: 7o. :8 7o. 8,) 7o. )99 7o. )+8,.;8 7o. .+,,8,, 7o. *9 7o. )9:,)**,.+: 7o. .+, 7o. ),) 7o. :;, :9, +), )8+, )+;, )+<, .8:, .:< 7o. )9<, )9+, .<; 7o. .*, ):., ..,, 8,9 7o. .<9, .+,

%admium ions %affeine %alcium ions %arbamate esters %ardiac glycosides %arotenoids %arotenoid aldehydes %atechins %atecholamines %ations, inorganic %hloramines %holesterol, #esters %holine %hromium ions %obalt ions %opper ions %orticosteroids %oumarines %reatine %reatinine %yanamide %ysteine, %ystine Dehydroascorbic acid Deo&y sugars Deo&yribonucleosides Dicarbo&ylic acids Digitalis glycosides Dimethylamino acids 8,9#DinitrobenDoic acid esters ),.#Diols Diterpenes Epo&ides Ergot al"aloids Essential oils Esters Estrogens Ethanolamine Ethyl'anillin 0la'onoids 0luorine ions

7o. .98, 8): 7o. 9+ 7o. ).), )9<, )9+ 7o. );: 7o. ))., 8,+, 8.. 7o. .: 7o. )9*, .<8 7o. 8)+, 8.< 7o. )8;, )89 7o. ., ).8, )9,, .9< 7o. <), <. 7o. ) 7o. ).< 7o. 8), 89 7o. .98, .<9 7o. ),<, ).), .98, .<9 7o. ).9, )9;, 8),, 8.8 7o. +., .;8 7o. .8., .*,, .*. 7o. .*,, .*. 7o. *9, .*. 7o. .+: 7o. ..9, ..+ 7o. +9, .+9, .*9 7o. +; 7o. ;; 7o. 9*, .8,, 8,<, 8., 7o. 8)8 7o. *+,.,; 7o. )+,,)+),.+.,.+8 7o. .+ 7o. .8) 7o. ),9 7o. ).;,8.* 7o. )99 7o. )*; 7o. .9: 7o. )9) 7o. 8, .<, 88, +., ):*, 8,*, 8)+ 7o. 88;

Germanium ions Gibberellins Glycals Glycocyanamidine Glycolipids Glycosides Gold ions Guanidine and deri'ati'es Halogen ions Halogen o&yacids Hea'y metal ions Hemloc" al"aloids Heterocyclic compounds Hydrastine HydraDine Hydrocarbons, aromatic Hydrocarbons, chlorinated Hydrocarbons see also sugars Hydro&amic acids Hydro&yamino acids Hydro&ylamine #Hydro&y acids Hypnotics, bromine#containing 5ndole and deri'ati'es 5nsecticides 5odine#containing compounds 5ron ions 5sothiocyanates Keto acids Ketones Ketoses Ketosteroids $actones $ead ions $ipids $ipoids $ithium ions $ysine

7o. ..: 7o. 8,9 7o. )9. 7o. .8. 7o. )): 7o. .:, 9*, ))., .8,, 8,<, 8,+, 8.,, 8.., 889 7o. 8)9 7o. )9:, )**, .*,, .*., .*< 7o. ;9, .<* 7o. .) 7o. ).+, .;; 7o. .+* 7o. );), )+*, .)8, 8,., 8,8, 8,: 7o. <* 7o. .8; 7o. );<, 8,: 7o. );), .:9 7o. )<< 7o. .+; 7o. )<;, .8; 7o. ), 7o. ),+, );, 7o. <,, :), ),;, ),:, );;, )<+, ):8, ..), .+<, 88) 7o. ;,, ),+, ))*, )*,, .:; 7o. 9;, ):, 7o. .)<, .8< 7o. .;, 7o. ),, *8, ..< 7r. +< 7o. .9, **, ))9 7o. )), )::, ..;, .;+, .+) 7o. )99 7o. ).), .98, .<9 7o. ;+, +8, *,, )8:, )*8, .<., 8.; 7o. 9, 7o. 8) 7o. 8.+

(agnesium ions (anganese ions (ercaptans (ercury ions (etal ions (ethyl "etones (ethyl sugars (orphine 7arcotine 7ic"el ions 7icotinic acid 7icotinamide 7itrogen compounds 7itrogen compounds, aliphatic 7itro compounds, aromatic 7itrosamines Bils Brganic compounds Brnithine B&imes @enicillins @ero&ides @ersulfates @henols @henol carbo&ylic esters @henol ethers @henothiaDines @hosphate esters @hosphoric acids @hthalate esters @iperonal @lasticisers @lasto?uinones @olyalcohols @olyethylene glycols and deri'ati'es @olyphenols @olyphenyls @olysaccharides

7o. )9+, .99 7o. 89, ).), .98, .<9 7o. ), 7o. ),<, ).), .<9, .+, 7o. ):+ 7o. .*; 7o. )), 7o. .;. 7o. <* 7o. .98, .<9 7o. <9 7o. <9 7o. )8), )8., )88, )<9, )+*, .;: 7o. .*. 7o. 8); 7o. ))+, 8,; 7o. 8, 7o. <+, )<8 7o. 8.+ 7o. :* 7o. )<) 7o. ):, ),*,):;, .;9 7o. 8. 7o. ;, 8;, 8:, ;:, ++, )8<, );8, )<<, )*), .)8, .);, .)9, .;,, .<<, 8,., 8,:, 8)<, 8.* 7o. .)8 7o. )*) 7o. );9, )<* 7o. :. 7o. );, )< 7o. .9+ 7o. )9) 7o. .).,.9+ 7o. )+. 7o. *, .9,, .:*, .+., .+8 7o. ).*, )<9 7o. 8)< 7o. 99 7o. 98, 8)*

@otassium ions @roaDulenes @roline @urines @yridine compounds @yrimidines @yrones Guinolines 2educing compounds 2esins Sapogenins Serine Sesamine Sil'er ions Sodium ions Sorbic acid Steroids Steroid al"aloids Steroid glucuronides Steroid glycosides Steroid sapogenins Steroid sulfates Sterols Strontium ions Sugars Sugar phosphates Sugars, reducing Sulfides Sulfonamides Sulfonic acids Sulfur#containing compounds Terpenes Terpene aldehydes Tetracyclines ThiaDoles

7o. :8, .** 7o. ),) 7o. 8.+ 7o. )8+, .:., .:+ 7o. ;<, <8, +,, .8; 7o. )8+ 7o. )., 7o. 8* 7o. 8:, )*8, .;,, .;*, .<+, .::, 8),, 8.8, 8.; 7o. 8, 7o. .:, )9; 7o. .+; 7o. <* 7o. ),<, ).), .+, 7o. 888 7o. 8)) 7o. ), .., .:, .+, <:, )*8, ..,, ..*, .88, .9*, .+., .+8, 8,9, 8)<, 8)+, 8.,, 8.), 8.;, 8.:, 8.*, 88. 7o. 9:, );9, )+9, .)* 7o. .9; 7o. .: 7o. 8,, 9:, :,, );9, .)*, 88. 7o. )++ 7o. ), 8+, <:, )*8, .,8, ..*, 8,9, 8)<, 8.; 7o. )9<, )9+, .<; 7o. 9, :, )*, .., 88, 8<, 9., )*<, )*:, )*+, .,,, .):, ..., .9,, .<:, .:), 8)., 8.9 7o. )9 7o. <, )+, .), .8, .;, ))8, ))<, ..8, 8.8 7o. )<) 7o. <,, +:, .*8 7o. .89,.:8 7o. ;*, )<+, .)+, .+: 7o. .., .:, 8, 7o. 88 7o. + 7o. .+<

Thioacids Thiobarbiturates Thiophene deri'ati'es Thiophosphate esters Thiosulfates Thiourea and deri'ati'es Threonine Thyroid hormones Tin ions Tocopheryl?uinones Triterpenes Triterpene glycosides Tryptophan Tungstate ions Abi?uinones Ansaturated compounds Aranyl ions Area Aranic acids 3anillin 3eratrum al"aloids 3inca al"aloids 3itamin 3itamin 6) 3itamin 6< 3itamin % 3itamin D 3itamin E =anthine deri'ati'es Hinc ions

7o. .:, 7o. +), *: 7o. ):9 7o. ):), .)+ 7o. .;, 7o. .*,, .*), .*. .*< 7o. .+; 7o. ):, 7o. ).) 7o. )+. 7o. <:, 8)< 7o. ) 7o. 88) 7o. .99 7o. )+. 7o. )8* 7o. .98, .99 7o. .*,, .*., .*< 7o. )*+ 7o. )9) 7o. 8., 7o. )8 7o. .:, 8, 7o. ).:,.8* 7o. <9 7o. 9), *;, )+: 7o. .:, 8,, 8., 7o. 8,, 8: 7o. )<: 7o. ).), .98

Reagents
-" !" cetic anhydride - sulfuric acid for #-$-sterols %cholesterol and esters&' steroids and triterpene glycosides %Liebermann-(urchard reagent&" Spray solutionC (i& carefully and with cooling freshly before use 9 ml acetic anhydride with 9 ml *:I sulfuric acid and add the mi&ture with cooling to 9, ml ethanol. After-treatmentC Heat ), min at )),J%. %haracteristic fluorescence in long#wa'e A3 light. Literature: %. (ichalec, 6iochim. et biophys. -cta !), )+: 4)*9<>. 2. Tscheche, /. %hromatog. #, .): 4)*<)>. K. Ta"eda, S. Hara, -. Wada, 7. (atsumoto, /. %hromatog. !!, 9<. 4)*<8>. Chemicals: Acetic anhydride GR ACS, ISO, Ord. No. .!!!"# Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *" li+arin for Cations" Spray solutionC Saturated ethanolic aliDarin solution. After-treatmentC @lace the moist chromatogram into a chamber saturated with ammonia 'apours. Literature: G. de 3ries, G.@. SchuetDe, E. 'an Dalen, /. %hromatog. !$, ))* 4)*<;>. Chemicals: Ali,arin indicator -C.I. %+!!!. )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# $" luminium chloride for fla,onoids" Spray solutionC )I ethanolic solution of aluminium chloride. Fellow fluorescence in long#wa'e A3 light Literature: T.G. Gage, %.D. Douglas, S.H. Wender, -nal. %hem. *$, )9+. 4)*9). Chemicals: Aluminium chloride he/ahydrate e/tra pure 0h )ur, 1S0, Ord. No. #"

.! !+" )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -- minoantipyrine - potassium hexacyanoferrate%III& for phenols %.merson reaction&" Spray solution IC .I ethanolic solution of ;#-minoantipyrine. Spray solution IIC +I a?ueous potassium he&acyanoferrate4555> solution. 0rocedureC Spray with 5, then with 55, and subse?uently place the chromatogram into a chamber saturated with ammonia 'apours. Literature: G. Gabel, K.H. (ueller, /. Scho"necht, Dtsch. -poth. Htg. !/*, .*8 4)*<.>. Chemicals: "-Amino-#,(-dimethyl- -phenyl-(-pyra,olin-%-one GR, Ord. No. .!&#$( 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Ammonia solution #%' GR, Ord. No. .!%"(# o- minodiphenyl - phosphoric acid for sugars %modif" reagent acc" to Le0is-Smith&" Spray solutionC Dissol'e ,.8 g o#aminodiphenyl and 9 ml +9I phosphoric acid in *9 ml ethanol. After-treatmentC Heat )9#., min at )),J%. Sugars show brown spots. Literature: T.E. Timell, %.@./. Glandemanns, -nal. %hem. *1, )*)< 4)*9<>. Chemicals: o-Aminodiphenyl ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( )thanol a*s. GR, Ord. No. .!!$&# 2" -- minohippuric acid for reducing sugars" Spray solutionC ,.8I ethanolic ;#aminohippuric acid solution. After-treatmentC Heat + min at );,J%. %haracteristic spots in long#wa'e A3 light. Literature: $. Sattler, 0.W. Herban, -nal. %hem. *-, )+<. 4)*9.>. Chemicals: "-Aminohippuric acid, Ord. No. .!!!+" )thanol a*solute GR ACS, ISO, Ord. No. .!!$+(

3"

o- minophenol - phosphoric acid for sugars" Spray solutionC Dissol'e ,.)9 g o#aminophenol in ., ml ethanol shortly prior to use. -dd ), ml 9,I phosphoric acid to the solution. Literature: $. 3igyKD#3Kmos, (agyar KLm. 0olyMrat #), )+8 4)*98>. S. Hirase, %. -ra"i, S. 7a"anishi, 6ull. %hem. Soc. 4/apan> *2, )+8 4)*98>. Chemicals: #-Aminophenol, Ord. No. +.!!" $ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&(

5ip solutionC Dissol'e ., g ammonium cerium453> nitrate in 9, ml ,.9 7 nitric acid. Dilute freshly before use ) part of this solution with 8 parts water. 0rocedureC -fter drying dip the chromatogram into the dip solution and place it on a clean filter paper. White spots on yellow bac"ground. Literature: (. Trop, (. Sprecher, -. @ins"y, /. %hromatog. $*, ;.< 4)*<+>. Chemicals: Ammonium cerium-l2. nitrate GR ACS, Ord. No. .!##&3 Nitric acid 3%' GR, Ord. No. .!!"%3 !!" mmonium cerium%I4& sulfate for 4inca al7aloids" Spray solutionC )I solution of ammonium cerium453> sulfate in +9I phosphoric acid. Literature: 5.(. /a"o'lEe'ic, $. D. Seay, 2. W. Shaffer, /. @harm. Sci. #$, 998 4)*<;>. Chemicals: Ammonium cerium-l2. sulfate dihydrate GR, Ord. No. .!##&( ortho-0hosphoric acid. +%' GR ISO, Ord. No. .!!%&( !*" mmonium iron%III& sulfate for fla,onoids" Spray solutionC ,..I a?ueous solution of ammonium iron4555> sulfate. Literature: E.-.H. 2oberts, D./. Wood, 6iochem. /. -), ;); 4)*9)>. Chemicals: Ammonium iron-III. sulfate dodecahydrate GR ACS, ISO, Ord. No. .!(&&3 !$" mmonium iron%III& sulfate ,or 4inca al7aloids" Spray solutionC Dissol'e ) g ammonium iron4555> sulfate in ),, ml phosphoric acid 4:9 or +9I>. Spray the reagent on to heated chromatogram 4),,J%>. Literature: 5.(. /a"o'lEe'ic, $.D. Seay, 2.W. Shaffer, /. @harm. Sci. #$, 998 4)*<;>. Chemicals: Ammonium iron-III. sulfate dodecahydrate GR ACS, ISO, Ord. No. .!(&&3 ortho-0hosphoric acid. +%' GR ISO, Ord. No. .!!%&( !-" mmonium molybdate - crystal ,iolet for phosphoric acid" PC"

1"

mmonia for tetracyclines" 0rocedureC @lace the chromatogram into a chamber saturated with ammonia 'apours. Tetracyclines show yellow spots in long#wa'e A3 light. Literature: (. Ar&, /. 3ondrKc"o'K, $. Ko'arN", B. Hors"O, (. Herold, /. %hromatog. !!, <. 4)*<8>. Chemicals: Ammonia solution #%' GR, Ord. No. .!%"(#

)"

mmonium cerium%I4& nitrate 5 6'6-dimethyl-!'--phenylenediammonium dichloride for polyalcohols" Solution aC )I solution of ammonium cerium453> nitrate in ,.. 7 nitric acid. Solution *C Dissol'e ).9 g 7,7#dimethyl#),;#phenylenediammonium dichloride in ).+ ml methanol, .9 ml water and ).9 ml glacial acetic acid. Spray solutionC (i& ) part a with ), parts b freshly before use. After-treatmentC Heat ), min at ),9J%. Fellowish green spots on red bac"ground. Literature: E. Knappe, D. @eteri, /. 2ohdewald, H. anal. %hem. !)), .:, 4)*<;>. Chemicals: Ammonium cerium-l2. nitrate GR ACS, Ord. No. .!##&3 N,N-dimethyl- ,"-phenylenediammonium dichloride GR, Ord. No. .!(!3& 4ethanol GR ACS, ISO, Ord. No. .!3!!$ Acetic acid $3' GR, Ord. No. .!!!3# Nitric acid 3%' GR ISO, Ord. No. .!!"%3

!/"

mmonium cerium%I4& nitrate - nitric acid for -hydroxy acids' -7eto acids and mercaptans" PC"

Spray solutionC (i&ture of 9 ml )I a?ueous ammonium molybdate solution, 9 ml .9I hydrochloric acid and *, ml acetone. Solution aC Dissol'e . g crystal 'iolet 4or brilliant green or iodine green> in 89, ml water. Solution *C Dissol'e with heating ; g ammonium molybdate in water, add 9, ml ), 7 hydrochloric acid and fill up to ),, ml with water. 5ip solutionC (i& a and b, wait at least 8 hours and filter the solution. 0rocedureC Spray the chromatogram with the spray solution, heat 8#< min at +9J%, dip into the dip solution and place immediately on a prepared clean filter paper. NoteC ,.,. Pg of phosphorus are detectable. %rystal 'iolet Q blue spots on yellow bac"ground 6rilliant green Q green spots on orange bac"ground 5odine green Q tur?uois spots on colourless bac"ground Literature: 0. /ungnic"el, /. %hromatog. $!, <): 4)*<:>. Chemicals: Ammonium heptamoly*date tetrahydrate GR ACS, ISO, Ord. No. .! +# Crystal 6iolet -C.I. "#%%%. indicator ACS, Ord. No. .! "!+ 7rilliant 8reen -C:I: "#!"!., Ord. No. .! ( ! 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & Acetone GR ACS, ISO, Ord. No. .!!! " !#" mmonium molybdate - perchloric acid %8anes reagent& for phosphate esters %sugar phosphates&" Spray solutionC Dissol'e ,.9 g ammonium molybdate in 9 ml water, add ).9 ml .9I hydrochloric acid and ..9 ml :,I perchloric acid. -fter cooling to room temperature fill up to 9, ml with acetone. -llow the solution to stand for at least one day prior to use. The solution is stable for about three wee"s. After-treatmentC 5rradiate the chromatogram for . min with an 52 lamp from a distance of 8, cm and subse?uently with long#wa'e A3 light for : min or heat 9# ), min at )),J%. Literature: %.S. Hanes, 0.-. 5sherwood, 7ature !2-, )),: 4)*;*>. T.H. 6e'an, G.5. Gregory, T. (al"in, -.G. @oole, /. %hem. Soc. !)#!, +;). S. 6urrows, 0.S.(. Grylls, /.S. Harrison, 7ature !3/, +,, 4)*9.>. %.W. Stanley, /. %hromatog. !2, ;<: 4)*<;>. Chemicals: Ammonium heptamoly*date tetrahydrate GR ACS, ISO, Ord. No. .! +# 9ydrochloric acid #%' GR, Ord. No. .!!( 3

0erchloric acid &!-&#' GR ACS, Ord. No. .!!% $ Acetone GR ACS, ISO, Ord. No. .!!! " !2" mmonium molybdate - tin%II& chloride for phosphoric acids" Spray solution IC )I a?ueous ammonium molybdate solution. Spray solution IIC )I solution of tin455> chloride in ),I hydrochloric acid. 0rocedureC Spray with 5, dry the chromatogram and spray with 55. Heat, if necessary, at ),9J% for 8 # 9 minutes. Literature: H. Seiler, Hel'. %him. -cta --, ):98 4)*<)>. Chemicals: Ammonium heptamoly*date tetrahydrate GR ACS, ISO, Ord. No. .! +# :in-II. chloride dihydrate GR ACS, Ord. No. .!&+ % 9ydrochloric acid #%' GR, Ord. No. .!!( 3 !3" mmonium thiocyanate - iron%II& sulfate for peroxides" Spray solution IC Dissol'e ,.; g ammonium thiocyanate in 8, ml acetone. Spray solution IIC Dissol'e ).. g iron455> sulfate in 8, ml water. 0rocedureC Spray with 5, dry the chromatogram and spray with 55. Literature: (.H. -braham, -.G. Da'ies, D.2. $lewellyn, E.(. Thain, -nal. %him. -cta !3, ;** 4)*9:>. Chemicals: Ammonium thiocyanate GR ACS, ISO, Ord. No. .! # ( Iron-II. sulfate heptahydrate GR ACS, ISO, Ord. No. .!($3% .! # ( Acetone GR ACS, ISO, Ord. No. .!!! " !1" niline - diphenylamine - phosphoric acid for reducing sugars" Spray solutionC Dissol'e ; g diphenylamine, ; ml aniline and ., ml +9I phosphoric acid in .,, ml acetone. After-treatmentC Heat ), min at +9J%. %haracteristic coloursC ),;#aldohe&ose oligosaccharides turn blue. Literature: 2.W. 6ailey, E./. 6ourne, /. %hromatog. -, .,< 4)*<,>. /.$. 6uchan, 2./. Sa'age, -nalyst 33, ;,) 4)*9.>. S. Schwimmer, -. 6e'enne, Science !*$, 9;8 4)*9<>. Chemicals: Aniline GR, Ord. No. .! #3

5iphenylamine GR and redo/ indicator, Ord. No. .!(!+3 Acetone GR ACS, ISO, Ord. No. .!!! " ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( !)" niline - phosphoric acid for sugars" Spray solutionC (i& ) part . 7 aniline solution in )#butanol saturated with water with . parts . 7 phosphoric acid in )#butanol. After-treatmentC Heat the chromatogram ), min at ),9J%. Literature: 5.$. 6ryson, T.5. (itchell, 7ature !23, +<; 4)*9)>. Chemicals: Aniline GR, Ord. No. .! #3 ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( -7utanol GR ACS, ISO, Ord. No. .! $$! */" niline phthalate" ),, ml ready to use spray solution for chromatography 4c Q ca. 8..I in .# propanolRmethanol>. After-treatmentC Heat the chromatogram ), min. at ),9J%. Ord. No. .! #3$ *!" niline phthalate for reducing sugars and anions of halogen oxy-acids" Spray solutionC Dissol'e ,.*8 g aniline and ).<< g phthalic acid in ),, ml )# butanol saturated with water. After-treatmentC Heat ), min at ),9J%. Literature: S.(. @artridge, 7ature !2-, ;;8 4)*<9>. W. @esch"e, /. %hromatog. */, 9:. 4)*<9>. Chemicals: Aniline GR, Ord. No. .! #3 0hthalic acid GR, Ord. No. .!$3 -7utanol GR ACS, ISO, Ord. No. .! $$! **" nisaldehyde - sulfuric acid for sugars' steroids' terpenes" Spray solutionC @repare freshly before use a solution of ,.9 ml anisaldehyde in 9, ml glacial acetic acid and ) ml *:I sulfuric acid. *#"

After-treatmentC Heat to ),,#),9J% until ma&imal 'isualisation of the spots. The bac"ground may be brightened by water 'apour. $ichen constituents, phenols, terpenes, sugars and steroids turn 'iolet, blue, red, grey or green. 4odified spray solutionC 0or 'isualisation of sugars mi& freshly before use ,.9 ml anisaldehyde, * ml ethanol, ,.9 ml *:I sulfuric acid and ,.) ml acetic acid. After-treatmentC Heat the sprayed chromatogram 9#), min at *,#),,J%. Literature: E. Stahl, A. Kaltenbach, /. %hromatog. #, 89) 4)*<)>. 6.@. $isboa, /. %hromatog. !2, )8< 4)*<;>. Chemicals: "-4etho/y*en,aldehyde -anisaldehyde. Rea8. 0h )ur, Ord. No. .%$3!+ Acetic acid $3' GR, Ord. No. .!!!3# Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *$" p- nisidine for reducing sugars" Spray solutionC Dissol'e ) g p#anisidine hydrochloride in ), ml methanol, fill up the solution to ),, ml with l#butanol and sha"e well after addition of ) g sodium dithionite. After-treatmentC Heat ), min at )8,J%. Literature: 2.%. 6ean, G.G. @ortwe, -nal. %hem. $!, )*.* 4)*9*>. $. Hough, /.K.7. /ones, W.H. Wadman, /. %hem. Soc. !)#/, ):,.. Chemicals: p-Anisidinium chloride, Ord. No. +.#! !( Sodium dithionite LA7, Ord. No. .!3%!& 4ethanol GR ACS, ISO, Ord. No. .!3!!$ -7utanol GR ACS, ISO, Ord. No. .! $$! *-" p- nisidine phthalate for reducing sugars" Spray solutionC ,.) ( solution of p#anisidine and phthalic acid in *<I ethanol. After-treatmentC Heat ), min at ),,J%. Chemicals: p-Anisidine, Ord. No. +.!!"%+ 0hthalic acid GR, Ord. No. .!$3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( nthrone for 7etoses"

Spray solutionC Dissol'e ,.8 g anthrone in ), ml acetic acid and add to the solution ., ml *<I ethanol, 8 ml +9I phosphoric acid and ) ml water. The solution is stable for se'eral wee"s in the refrigerator. After-treatmentC Heat 9#< min at )),J%. Ketoses and oligosaccharides containing "etoses show yellow spots. Literature: 2. /ohanson, 7ature !3*, *9< 4)*98>. Chemicals: Anthrone for synthesis, Ord. No. +.! "3 Acetic acid $3' GR, Ord. No. .!!!3# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( *2" ntimony%III& chloride for fla,onoids" Spray solutionC ),I solution of antimony4555> chloride in chloroform. 0luorescing spots in long#wa'e A3 light. $iteratureC $. Hoerhammer, H. Wagner, K. Hein, /. %hromatog. !*, .89 4)*<;>. 2. 7eu, @. Hagedorn, 7aturwissenschaften -/, ;)) 4)*98>. Chemicals: Antimony-III. chloride GR, Ord. No. .!&+(+ Chloroform GR ISO, Ord. No. .!#""% *3" ntimony%III& chloride for ,itamin and D' carotenoids' steroids' sapogenins' steroid glycosides' terpenes %Carr-Price reagent&" Spray solutionC Dissol'e .9 g antimony4555> chloride in :9 ml chloroformS generally a saturated solution of antimony4555> chloride in chloroform or carbon tetrachloride is used. After-treatmentC Heat ), min at ),,J%. 5nspect the chromatogram in long#wa'e A3 light. Literature: E. Stahl, %hemi"er#Htg. 1*, 8.8 4)*9+>. K. Ta"eda, S. Hara, -. Wada, 7. (atsumoto, /. %hromatog. !!, 9<. 4)*<8>. Chemicals: Antimony-III. chloride GR, Ord. No. .!&+(+ Chloroform GR ISO, Ord. No. .!#""% Car*on tetrachloride GR, Ord. No. .!#### *1" ntimony%III& chloride - acetic acid for steroids and diterpenes" $/"

Spray solutionC Dissol'e ., g antimony trichloride in a mi&ture of ., ml glacial acetic acid and <, ml chloroform. After-treatmentC Heat 9 min at ),,J%. Diterpenes show red#yellow to blue#'iolet spots. 5nspect in long#wa'e A3 light. Literature: H.@. Kaufmann, -.K. sen Gupta, %hem. 6er. )3, .<9. 4)*<;>. Chemicals: Antimony-III. chloride GR, Ord. No. .!&+(+ Acetic acid $3' GR, Ord. No. .!!!3# Chloroform GR ISO, Ord. No. .!#""% *)" ntimony%III& chloride - sulfuric acid for bile acids" Spray solutionC Dissol'e ., g antimony4555> chloride in 9, ml anhydrous )# butanol and mi& this solution with ), ml *:I sulfuric acid and ., ml glacial acetic acid. The solution should be prepared freshly before use. After-treatmentC -fter drying for )9 min in the air heat the chromatogramC conEugated bile acids for .9#8, min, free bile acids for ;9#9, min at )),J%. %olours from yellow to green. Literature: W.$. -nthony, W.T. 6ehr, /. %hromatog. !$, 9<: 4)*<;>. Chemicals: Antimony-III. chloride GR, Ord. No. .!&+(+ -7utanol GR ACS, ISO, Ord. No. .! $$! Acetic acid $3' GR, Ord. No. .!!!3# Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( ntimony%4& chloride for ,itamin ' D and .' terpenes' oils' resins' steroid sapogenins" Spray solutionC (i& freshly before use ) part antimony43> chloride with ; parts carbon tetrachloride or chloroform. After-treatmentC Heat the chromatogram until the spots appear. 5nspect in long# wa'e A3 light. Literature: /.(. (ac(ahon, 2.6. Da'is, G. KalnitD"y, /. -m. %hem. Soc. :;, ;;+8 4)*9.>. E. Stahl, %hemi"er#Htg. 1*, 8.8 4)*9+>. 2. 5"an, /. Kashman, E.D. 6ergmann, /. %hromatog. !-, .:9 4)*<;>. H.G. Hen"el, W. Ebing, /. %hromatog. !-, .+9 4)*<;>. Chemicals: Antimony-2. chloride GR, Ord. No. .!&+(&

Car*on tetrachloride GR, Ord. No. .!#### Chloroform GR ISO, Ord. No. .!#""% $!" urin tricarboxylic acid % luminon& for aluminium' chromium' and lithium ions" Spray solutionC ,.)I solution of aurin tricarbo&ylic acid ammonium salt in )I a?ueous ammonium acetate solution. After-treatmentC @lace the chromatogram into a chamber saturated with ammonia 'apours. Literature: G.@. Heisig, 0.H. @ollard, -nal. %him. -cta !2, .8; 4)*9:>. Chemicals: Aurin tricar*o/ylic acid ammonium salt GR -rea8ent for aluminium. ACS, Ord. No. .!! #+ Ammonium acetate GR ACS, Ord. No. .! 3 Ammonia solution #%' GR, Ord. No. .!%"(# $*" (en+idine for persulfates" Spray solutionC Dissol'e ,.,9 g benDidine in ),, ml ) 7 acetic acid. @ersulfates show blue spots immediately after spraying. Caution: (en+idine is carcinogenic9 Literature: F. Ser'igne, %. Du'al, %ompt. 2end. *-#, )+,8 4)*9:>. Chemicals: 7en,idine Acetic acid $3' GR, Ord. No. .!!!3# $$" (en+idine for terpene aldehydes' fla,onoids' carbohydrates" Spray solutionC Dissol'e ,.9 g benDidine in ., ml glacial acetic acid and +, ml ethanol. Caution: (en+idine is carcinogenic9 After-treatmentC Heat )9 min at ),,J%. Spraying with dilute hydrochloric acid after heating intensifies the colour of the spots of some substances. Literature: /.K7. /ones, /.6. @ridham, 6iochem. /. #1, .++ 4)*9;>. Chemicals: 7en,idine Acetic acid $3' GR, Ord. No. .!!!3# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid #%' GR, Ord. No. .!!( 3

$-" (en+idine dia+otised for phenols" 7en,idine solutionC Dissol'e 9 g benDidine in ); ml 8:I hydrochloric acid and fill up to ),, ml with water. Caution: (en+idine is carcinogenic9 Nitrite solutionC 0reshly prepared ),I a?ueous sodium nitrite solution. Spray solutionC (i& ., ml of the benDidine solution with ., ml of the nitrite solution at ,J% with constant stirring. 7oteC The reagent is stable for .#8 hours. The colours appear 'ery rapidly or after some hours depending on the phenol present. Literature: /. Sherma, $.3.S. Hood, /. %hromatog. !3, 8,: 4)*<9>. Chemicals: 7en,idine Sodium nitrite GR ACS, Ord. No. .!3%"$ 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & $#" (en+idine - peroxide for chromium and manganese ions" Spray solution IC 9I a?ueous sodium pero&ide solution. Spray solution IIC )I benDidine solution in glacial acetic acid. Caution: (en+idine is carcinogenic9 0rocedureC Spray consecuti'ely with 5 and 55. Literature: 5.(. $adenbauer, $.K. 6radacs, 0. Hecht, (i"rochim. -cta !)#-, 8++. Chemicals: Sodium pero/ide 8ranular GR ACS, Ord. No. .!3%3( 7en,idine Acetic acid $3' GR, Ord. No. .!!!3# $2" (en+idine - trichloroacetic acid for sugars" Spray solutionC Dissol'e ,.9 g bcnDidine in ), ml glacial acetic acid, add ), ml ;,I a?ueous trichloroacetic acid and fill up to ),, ml with ethanol. Caution: (en+idine is carcinogenic9 After-treatmentC 5rradiate the chromatogram ).9 min with A3 light. Sugars show greyish#brown to deep reed#brown spots. Literature: /.S.D. 6acon, /. Edelmann, 6iochem. /. -1, )); 4)*9)>. G. Harris, 5.%. (acwilliam, %hem. T 5nd. 4$ondon> !)#-, .9;. Chemicals: 7en,idine

:richloroacetic acid GR ACS, Ord. No. .!!+!& Acetic acid $3' GR, Ord. No. .!!!3# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $3" *'*:-(ipyridine - iron%III& chloride for phenols' ,itamin . and other reducing compounds" Solution aC ,.9I ethanolic iron4555> chloride solution. Keep in the dar". Solution *C ,.9I ethanolic solution of .,.U#bipyridine. Spray solutionC (i& e?ual parts of a and b before use. Literature: G. (. 6arton, /. %hromatog. */, )+* 4)*<9>. 2. Strohec"er, H.(. Henning, 3itaminbestimmungen, 3erlag %hemie Weinheim )*<8, p. 8)). Chemicals: #,#;-7ipyridine GR, Ord. No. .!(!$+ Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $1" (ismuth chloride for sterols" Spray solutionC 88I ethanolic bismuth4555> chloride solution. After-treatmentC Heat at )),J% until ma&imal fluorescence of the spots in long# wa'e A3 light. Literature: /.W. %opius#@eereboom, Thin $ayer %hromatography, Ed. G.6. (arini#6ettolo, Else'ier -msterdam, )*<;, p. )**. Chemicals: 7ismuth-III. chloride LA7, Ord. No. . #"!( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $)" (oric acid - citric acid for ;uinolines" Spray solutionC Dissol'e ,.9 g boric acid and ,.9 g citric acid in ., ml methanol. After-treatmentC Heat at ),,J%. 5nspect in A3 light. Literature: 2. 7eher, -. Wettstein, Hel'. %him. -cta $#, .:< 4)*9.>. Chemicals: 7oric acid cryst. GR ACS, ISO, Ord. No. .!! 3% Citric acid monohydrate GR ACS, ISO, Ord. No. .!!#"" 4ethanol GR ACS, ISO, Ord. No. .!3!!$

-/" (romine - fluorescein - sil,er nitrate for insecticides" Spray solutionC 0ill up ) ml of a ,..9I solution of fluorescein in 7,7#dimethyl# formamide to 9, ml with ethanol. Spray solution IIC Dissol'e ).: g sil'er nitrate in 9 ml water, add ), ml ethylene glycol monophenyl ether and fill up the solution to .,, ml with acetone. 0rocedureC @lace the chromatogram 8, s into a chamber with a 9I solution of bromine in carbon tetrachloride. Spray the chromatogram with 5, then with 55 and irradiate : min with long#wa'e A3 light. Literature: K.%. Wal"er, (. 6eroDa, /. -ssoc. Bff. -gr. %hemists -2, .9, 4)*<8>. Chemicals: 7romine GR ISO, Ord. No. .! $"+ <luorescein -C.I. "%(%!. N,N-5imethylformamide GR ISO, Ord. No. .!(!%( )thylene 8lycol monophenyl ether for synthesis, Ord. No. +.!&#$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Acetone GR ACS, ISO, Ord. No. .!!! " -!" (romocresol green" ),, ml ready to use spray solution for chromatography 4c Q ,.)I in .#propanol>. Ord. No. .! $$" -*" (romocresol green - indicator reagent" Spray solutionC Dissol'e ,.,; g bromocresol green in ),, ml ethanol. -dd sodium hydro&ide solution 4c Q ,.) molR$> until blue colour appears. Literature: 0. 6ryant, 6.T. ,'erell, 6iochim. et biophys. -cta !/, ;:) 4)*98>. Chemicals: 7romocresol 8reen indicator p9 (.+-%." ACS, Ord. No. .!+ # )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sodium hydro/ide solution !. mol=l :itrisol> Ord. No. .!$$%$ -$" (romocresol green - bromophenol blue - potassium permanganate for organic acids" Solution aC Dissol'e ,.,:9 g bromocresol green and ,.,.9 g bromophenol blue in ),, ml ethanol. Solution *C Dissol'e ,..9 g potassium permanganate and ,.9 g sodium carbonate in ),, ml water.

Spray solutionC (i& * parts a and ) part b prior to use and spray immediately. The mi&ture is stable for 9#), minutes only. Literature: /. @Ks"o'K, 3./. (un", /. %hromatog. -, .;) 4)*<,>. Chemicals: 7romocresol 8reen indicator p9 (.+-%." ACS, Ord. No. .!+ # 7romophenol *lue indicator p9 (.!-".3 ACS, Ord. No. .!+ ## 0otassium perman8anate GR ACS, Ord. No. .!%!+# Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( --" (romocresol purple for dicarboxylic acids on polyethyleneglycol impregnated layers" Spray solutionC Dissol'e ,.,; g bromocresol purple in ),, ml 9,I ethanol and adEust the solution to pH ),., with sodium hydro&ide solution 4c Q ,.) molR$, glass electrode>. 0rocedureC De'elop the chromatogram with the eluent di#iso#propyl ether # formic acid #water 4*,V:V8> and heat subse?uently ), min at ),,J%. Spray after cooling to room temperature. Fellow spots on blue bac"ground. Literature: E. Knappe, D. @eteri, H. anal. %hem. !11, )+; 4)*<.>. Chemicals: 7romocresol purple indicator, Ord. No. .!(!#% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sodium hydro/ide solution !. mol=l :itrisol>, Ord. No. .!$$%$ -#" (romocresol purple for halogen ions" 5ndicator reagent for use of acetone # )#butanol # ammonia 4.9I> # water 4<9V.,V),V9> as eluent. Spray solutionC ,.)I ethanolic bromocresol purple solution. -dEust the solution with some drops of ),I ammonia solution until the colour change Eust appears. Literature: H. Seiler, T. Kaffenberger, Hel'. %him. -cta --, ).+. 4)*<)>. Chemicals: 7romocresol purple indicator, Ord. No. .!(!#% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(#

-2" (romocyan - --aminoben+oic acid %reagent acc" to <oenig& for tertiary pyridine compounds 0ith at least one free -position" 0rimary treatmentC 6efore spraying place the chromatogram for ) hour into a chamber with a solution of bromocyan 4Caution' ,ery poisonous9>. 0or preparation of the bromocyan solution add ),I a?ueous solution of sodium cyanide to saturated bromine water, cooled in ice, until the colour of bromine has disappeared. Spray solutionC Dissol'e . g ;#aminobenDoic acid in :9 ml ,.:9 7 hydrochloric acid and fill up the solution to ),, ml with ethanol. Literature: E. Kodice", K.K. 2eddi, 7ature !21, ;:9 4)*9)>. Chemicals: 7romine GR ISO, Ord. No. .! $"+. Sodium cyanide pure, Ord. No. .!3"(& "-Amino*en,oic acid e/tra pure 1S0, Ord. No. .!! !# 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 2ariationC (i&ture of e?ual parts of a .I ethanolic ;#aminobenDoic acid solution and phosphate buffer 4c Q ,.) molR$, pH :.,>. 0rocedureC -fter spraying dry the chromatogram )9 min at room temperature and place subse?uently into a chamber with some crystals of bromocyan. Literature: E. Hodgson, E. Smith, 0.E. Guthrie, /. %hromatog. */, ):< 4)*<9>. Chemicals: 7romocyan "-Amino*en,oic acid e/tra pure 1S0, Ord. No. .!! !# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 7uffer Solution concentrated :itrisol p9 &.! -phosphate., Ord. No. .!$++& -3" (romophenol blue - methyl red - Pauly reagent for phenols" Spray solution IC (i& ),, ml ,.).I a?ueous bromophenol blue solution, ),, ml ,.,<I ethanolic methyl red solution and ),, ml phosphate buffer acc. to Sorensen 4pH :..>. Spray solution IIC See reagent 7o 8,8C Sulfanilic acid diaDotised. 0rocedureC Spray the chromatogram consecuti'ely with 5 and 55. Literature: /.W. %opius#@eereboom, H.W. 6ee"es, /. %hromatog. !-, ;): 4)*<;>.

Chemicals: 7romophenol *lue indicator p9 (.!-".3 ACS, Ord. No. .!+ ## 4ethyl red -C.I. (!#!. indicator ACS, Ord. No. .!3!&3 0otassium dihydro8en phosphate solution = %, mol=l, Ord. No. .!"+&% di-Sodium hydro8en phosphate solution = % mol=l, Ord. No. .!3%+& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -1" (romosuccinimide - fluorescein for lipids" Spray solution IC Dissol'e ,.,) g 7#bromosuccinimide in ),, ml glacial acetic acid. Spray solution IIC Dissol'e ,.,) g fluorescein in ),, ml ethanol. 0rocedureC Spray consecuti'ely with 5 and 55. 5nspect in day light and in long# wa'e A3 light. Literature: -. @opo', 3. Gade'a, /. %hromatog. !2, .9< 4)*<;>. /. (ice', -. @opo', $. 7edelce'a, /. %hromatog. *-, ;8. 4)*<<>. Chemicals: N-7romosuccinimide, Ord. No. +.! $"$ <luorescein -C.I. "%(%!. Acetic acid $3' GR, Ord. No. .!!!3# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -)" (romosuccinimide - fluorescein for sulfur containing compounds" Spray solution IC ,.,89I solution of 7#bromosuccinimide in ),),)#trichloro# ethane. Spray solution IIC 0ill up 8 ml ,.88I solution of fluorescein in sodium hydro&ide solution 4c Q ,.) molR$> to ),, ml with ethanol. 0rocedureC Spray with 5, dry at room temperature and spray with 55. Literature: /.W. %oo", /. -ssoc. Bff. -gr. %hemists $3, *+8 4)*9;>. Chemicals: N-7romosuccinimide for synthesis, Ord. No. +.! $"$ <luorescein -C.I. "%(%!. Sodium hydro/ide solution !. mol=l :itrisol>, Ord. No. .!$$%$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( , , -:richloroethane for synthesis, Ord. No. +. +&%( #/" (romothymol blue for lipoids"

Spray solutionC Dissol'e ,.,; g bromothymol blue in ),, ml sodium hydro&ide solution 4c Q ,.,) molR$>. Literature: H. /atD"ewitD, E. (ehl, Hoppe#Seylers H. physiol. %hem. $*/, .9) 4)*<,>. Chemicals: 7romothymol *lue indicator ACS, Ord. No. .!(!#3 Sodium hydro/ide solution !.! mol=l :itrisol>, Ord. No. .!$$3 #!" Cacotheline for ,itamin C" Spray solutionC .I a?ueous cacotheline solution. After-treatmentC Heat at )),J%. 3iolet spots. Literature: 6. Tegethoff, H. 7aturforsch. 1b, 8:; 4)*98>. Chemicals: Cacotheline #*" Carba+ole - sulfuric acid for sugars" Spray solutionC Dissol'e ,.9 g carbaDole in *9 ml ethanol and add 9 ml *:I sulfuric acid. @repare freshly before use. After-treatmentC Heat ), min at ).,J%. 3iolet spots on blue bac"ground. Chemicals: Car*a,ole for synthesis, Ord. No. +.#!#%% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( #$" Carmine for polysaccharides" PC" Stoc? solutionC Heat ) g carmine, ,.9 g anhydrous aluminium chloride and . ml water .#8 min, add the solution to ),, ml 9,I ethanol and filter after .; hours. The filtrate must be stored at 9J%. Spray solutionC Dilute 9 ml of stoc" solution with ): ml ethanol and 8 ml water. 0rocedureC 6efore drying it is ad'antageous to fi& the polysaccharides. Dip the chromatogram )9 min into a mi&ture of ., ml formaldehyde and +, ml ethanol and dry at room temperature. Literature: /.0. Heremans, /.@. 3aerman, %lin. %him. -cta $, ;8, 4)*9+>. D. Hamerman, Science !**, *.; 4)*99>.

Chemicals: Carmine -C.I. &%"&!. Aluminium chloride -anhydrous, su*limed., Ord. No. +.! !+# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( <ormaldehyde solution min. (&' GR, Ord. No. .!"!!( #-" Cerium%I4& sulfate - arsenite for organic and inorganic iodine containing compounds" PC" Solution aC -dd ), g cerium453> sulfate to ),, ml ) 7 sulfuric acid, which has been cooled to ,#9J%. The mi&ture is cooled for another hour and then filtered or centrifuged. Store the clear solution until use in the refrigerator. Solution *C Dissol'e 9 g sodium arsenite in 8, ml sodium hydro&ide solution 4c Q ) molR$>. -dd the solution dropwise with stirring to <9 ml . 7 sulfuric acid cooled to ,#9J% and fill up to ),, ml with water. Spray solutionC (i& e?ual parts of a and b prior to use. 0rocedureC Spray the chromatogram with the spray solution by placing it on a glass plate. This permits uniform spraying. @lace a second glass plate of e?ual siDe o'er the moistened chromatogram and press. Within 8, minutes white spots on yellow bac"ground will appear at the sites of iodine compounds. @otassium iodide turns chocolate#brown. After-treatmentC 0or greater contrast the chromatogram may be sprayed before drying with )I solution of o#phenylenediamine in acetone. Thus the entire chromatogram turns brown and the white spots are more pronounced. Dry the chromatogram in iodine#free air. Literature: %.H. 6owden, 7.0. (ac$agan, /.H. Wil"inson, 6iochem. /. #$, *8 4)*99>. Chemicals: Cerium-I2. sulfate tetrahydrate GR, Ord. No. .!##&" Sodium metaarsenite ),.-0henylenediamine for synthesis, Ord. No. +.!$&# Sulfuric acid !.% mol=l :itrisol>, Ord. No. .!$$+" Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Sodium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$%3 Acetone GR ACS, ISO, Ord. No. .!!! " ##" Cerium%I4& sulfate - nitric acid for polyphenyls" Spray solutionC Dissol'e ,.8 g cerium453> sulfate in ),, ml <9I nitric acid. After-treatmentC Heat )9#., min at ).,J%. 5nspect in long#wa'e A3 light. Literature: 0. Geiss, H. Schlitt, Euratom#6ericht EA2#5#)* d 47o'. )*<)>.

Chemicals: Cerium-I2. sulfate tetrahydrate GR, Ord. No. .!##&" Nitric acid 3%' GR ISO, Ord. No. .!!"%3 #2" Cerium%I4& sulfate - sulfuric acid for al7aloids and iodo-organic compounds %modified reagent acc" to Sonnenschein&" Spray solutionC Slurry ,.) g cerium453> sulfate in ; ml water. -fter addition of ) g trichloroacetic acid boil and add dropwise *:I sulfuric acid until the solution becomes clear. After-treatmentC Heat some minutes at )),J% until the spots appear. NoteC The reagent dyes the al"aloids apomorphine, brucine, colchicine, papa'erine and physostigmine. Brganic iodine compounds also can be detected. Literature: B.#E. SchultD, D. Strauss, -rDneimittel#0orsch. #, 8;. 4)*99>. Chemicals: Cerium-I2. sulfate tetrahydrate GR, Ord. No. .!##&" :richloroacetic acid GR ACS, Ord. No. .!!+!& Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( #3" Cerium%I4& sulfate - sulfuric acid for solanum steroid al7aloids and steroid sapogenins" Spray solutionC Saturated solution of cerium453> sulfate in <9I sulfuric acid. After-treatmentC Heat )9 min at ).,J%. NoteC 7ot applicable with aluminium o&ide layers. Literature: K. Schreiber, B. -urich, G. Bss"e, /. %hromatog. !*, <8 4)*<8>. Chemicals: Cerium-I2. sulfate tetrahydrate GR, Ord. No. .!##&" Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( #1" Chloramine T for caffeine" Spray solution IC ),I a?ueous chloramine T solution. Spray solution IIC ) 7 hydrochloric acid. 0rocedureC Spray with 5 and after short drying with 55. Heat at *<#*+J% until the smell of chlorine has disappeared. @lace the chromatogram into a chamber saturated with ammonia 'apour and heat subse?uently for a short time until the ma&imal 'isualisation of the spots.

Literature: H. Gaenshirt, -. (alDacher, -rch. @harm. *)$, *.9 4)*<,>. Chemicals: 9ydrochloric acid mol=L :itrisol>, Ord. No. .!$$&! Chloramine : trihydrate GR, Ord. No. .!#"#3 Ammonia solution #%' GR, Ord. No. .!%"(# #)" Chloramine T - trichloroacetic acid for digitalis glycosides" Spray solutionC (i& ), ml of a freshly prepared 8I a?ueous chloramine T solution with ;, ml .9I solution of trichloroacetic acid in ethanol. Trichloroacetic acid solution is stable for se'eral days. 0rocedureC Heat : min at )),J%. 6luish and yellow fluorescence in long#wa'e A3 light. Literature: D. Waldi, -rch. @harm. *)*, .,< 4)*9*>. Chemicals: Chloramine : trihydrate GR, Ord. No. .!#"#3 :richloroacetic acid GR ACS, Ord. No. .!!+!& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 2/" Chlorine - pyra+olone - cyanide for indoles' amides' sulfonamides" ChlorinationC @lace the chromatogram for about .#8 min into a chlorine atmosphere 4prepared from potassium permanganate and .9I hydrochloric acid>.To remo'e e&cess chlorine heat the plate at ),,J%. Spray solutionC E?ual 'olumes of ,.. ( solution of 8#methyl#)#phenyl#8# pyraDo5on#9#one in pyridine and potassium cyanide solution 4c Q ) molR$>. 0rocedureC -fter remo'al of the e&cess chlorine spray the chromatogram until beginning transparency. Caution' poisonous9 The respecti'e compounds show bright red spots which turn blue after . min. Literature: @ri'ate communication G. 6ohnstedt, 5nst. f. Brgan. %hemie, Ani'ersitaet des Saarlandes. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# 9ydrochloric acid #%' GR, Ord. No. .!!( 3 0yridine GR ACS, Ord. No. .!$&#+ 0otassium cyanide GR ACS, ISO, Ord. No. .!"$3& 2!" Chlorine - tolidine for compounds con,ertible into chloramines"

ChlorinationC @lace the chromatogram into a chlorine atmosphereS 9#), min with chlorine from a bomb, )9#., min with chlorine prepared from a ).9I, solution of potassium permanganate and ),I hydrochloric acid 4)V)>. 0or remo'ing e&cess chlorine allow the plate to stand for 9 min in the air. Spray solutionC Dissol'e ,.)< g o#tolidine in 8, ml glacial acetic acid, fill up the solution to 9,, ml with water and add ) g potassium iodide. NoteC Spray a corner of the chromatogram to establish that chlorine has been remo'ed completely. 5f no blue colour appears spray the whole plate. Literature: 0. 2eindl, W. Hoppe, %hem. 6er. 13, )),8 4)*9;>. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Acetic acid $3' GR, Ord. No. .!!!3# o-:olidine 0otassium iodide GR ISO, Ord. No. .!%!"( 2*" Chlorine - tolidine %modif" act" to =reig and Leabac7&" Spray solution IC .I a?ueous solution of potassium hypochlorite. Spray solution IIC (i& before use e?ual 'olumes of a saturated solution of o#tolidine in .I acetic acid and ,.+9I a?ueous potassium iodide solution. 0rocedureC Spray lightly with 5, dry at room temperature for )#. hours, and spray with 55. Literature: %.%. Greig, D.H. $eabac", 7ature !11, 8), 4)*<,>. Chemicals: Acetic acid $3' GR, Ord. No. .!!!3# 0otassium iodide GR ISO, Ord. No. .!%!"( 0otassium hypochlorite o-:olidine 2$" Chlorocyan - --aminoben+oic acid for tertiary pyridine compounds 0ith at least one free -position" Spray solutionC 9I methanolic solution of ;#aminobenDoic acid. 0rocedureC @lace the sprayed chromatogram into a chamber with a freshly prepared mi&ture of ., ml .+ I a?ueous slurry of chloramine T, ., ml ) 7 hydrochloric acid and ), ml ),I a?ueous potassium cyanide solution. Caution' poisonous9 The spots will appear after a short time.

Literature: E. 7uernberg, Dtsch. -pothe"er#Htg. !/!, );. 4)*<)>. Chemicals: "-Amino*en,oic acid e/tra pure 1S0, Ord. No. .!! !# Chloramine : trihydrate GR, Ord. No. .!#"#3 0otassium cyanide GR ACS, ISO, Ord. No. .!"$3& 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 2-" !-Chloro-*'--dinitroben+ene - indicator reagent" Spray solutionC ,.9I ethanolic solution of ) #chloro#.,;#dinitrobenDene. Chemicals: -Chloro-#,"-dinitro*en,ene GR, Ord. No. .!#"#& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 2#" !-Chloro-*'--dinitroben+ene for nicotinic acid' nicotinamide' pyridoxol" Spray solution IC )I methanolic solution of )#chloro#.,;#dinitrobenDene. Spray solution IIC Sodium hydro&ide solution 4c Q 8 molR$>. 0rocedureC Spray subse?uently with 5 and 55. Literature: $. (aiwald, H. (as"e, Hoppe#Seylers H. physiol. %hem. $/2, );8 4)*9<>. Chemicals: -Chloro-#,"-dinitro*en,ene GR, Ord. No. .!#"#& Sodium hydro/ide solution min. #&' - .(. GR, Ord. No. .!%%$ 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 22" Chlorophenol red - indicator reagent" Spray solutionC ,.,;I ethanolic solution of chlorophenol red. -dEust the solution with sodium hydro&ide solution 4c Q ,.) molR$> to pH :.,. Literature: -.2. /ones, E./. Dowling, W./. S"roba, -nal. %hem. *#, 8*; 4)*98>. Chemicals: Chlorophenol red indicator, Ord. No. .!(!#" Sodium hydro/ide solution !. mol=L :itrisol>, Ord. No. .!$$%$ 23" Chlorosulfonic acid - glacial acetic acid for triterpenes' sterols' steroids" Spray solutionC Dissol'e 9 ml chlorosulfonic acid in ), ml glacial acetic acid with cooling.

:reatmentC -fter spraying heat 9#), min at )8,J%. 5nspect in long#wa'e A3 light. Literature: 2. Tscheche, G. Wulf, %hem. 6er. )-, .,)* 4)*<)>. 2. Tschesche, /. %hromatog. #, .): 4)*<)>. K. Ta"eda, S. Hara, -. Wada, 7. (atsumoto, /. %hromatog. !!, 9<. 4)*<8>. Chemicals: Chlorosulfonic acid, Ord. No. +.!!##! Acetic acid $3' GR, Ord. No. .!!!3# 21" Chromosulfuric acid as uni,ersal detectant for organic compounds" Spray solutionC Dissol'e 9 g potassium dichromate in ),, ml ;,I sulfuric acid. NoteC The reagent is suitable for charring organic compounds, in particular, lipids, by heating the chromatogram at )9,J%. Literature: /. 6ertetti, -nn. %him. 42ome> --, ;*9 4)*9;>. Chemicals: 0otassium dichromate GR ACS, ISO, Ord. No. .!"+3" Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( 2)" Chromotropic acid for methylenedioxyphenyl-type compounds %e"g" narcotine' hydrastine' sesamine and other compounds splitting off formaldehyde&" Solution aC ),,I a?ueous solution of chromotropic acid sodium salt. Solution *C -dd 9 parts *:I sulfuric acid to 8 parts water and cool to room temperature. Spray solutionC @repare freshly before use a mi&ture of ) part a and 9 parts b. After-treatmentC Heat 8, min at ),9J%. Literature: (. 6eroDa, -gricult. and 0ood %hemistry !!, 9) 4)*<8>. Chemicals: Chromotropic acid disodium salt dihydrate GR, Ord. No. .!#"$+ Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( 3/" Cinnamaldehyde - acetic anhydride - sulfuric acid for steroid sapogenins" Spray solution IC )I ethanolic cinnamaldehyde solution. Spray solution IIC @repare freshly before use a mi&ture of ). parts acetic anhydride and ) part *:I sulfuric acid.

0rocedureC Spray with 5, dry 9 min at *,J% and spray with 55. -fter )#. min at room temperature, the chromatogram is heated at *,J% until the spots appear. Chemicals: Cinnamaldehyde for synthesis, Ord. No. +.!#%!% Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Acetic anhydride GR ACS, ISO, Ord. No. .!!!"# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 3!" Cinnamaldehyde - hydrochloric acid for indole deri,ati,es" Spray solutionC Dissol'e 9 ml cinnamaldehyde in ),, ml ethanol and add 9 ml 8:I hydrochloric acid freshly before use. After-treatmentC @lace the plate into a hydrogen chloride atmosphere. 2ed spots. Literature: D. /erschel, 2. (ueller, 7aturwissenschaften $1, 9<) 4)*9)>. Chemicals: Cinnamaldehyde for synthesis, Ord. No. +.!#%!% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & 3*" Cobalt%II& chloride for organic phosphate esters" Spray solutionC )I anhydrous cobalt455> chloride solution in acetone. After-treatmentC Heat at ;,#9,J%. 6lue spots. The reaction is not sensiti'e. Literature: 2. Donner, K. $ohs, /. %hromatog. !3, 8;* 4)*<9>. Chemicals: Co*alt-II. chloride he/ahydrate GR ACS, Ord. No. .!#%($ Acetone GR ACS, ISO, Ord. No. .!!! " 3$" Cobalt%II& - lead nitrite for ammonium and potassium ions" PC" Spray solution IC Dissol'e 9 g cobalt455> nitrate and 9 g lead nitrate in ),, ml water and add )#. drops nitric acid. Spray solution IIC Saturated sodium nitrite solution in acetic acid 4c Q . molR$>. 0rocedureC Spray with 5, and after drying with 55. Then rinse with water and dry again. Literature: E. 6eerstecher, -nal. %hem. **, ).,, 4)*9,>. 2.A. (agee, /.6. Headridge, -nalyst 1*, *9 4)*9:>.

Chemicals: Lead-II. nitrate GR ACS, Ord. No. .!&($+ Co*alt-II. nitrate he/ahydrate GR, Ord. No. .!#%(3 Sodium nitrite GR ACS, Ord. No. .!3%"$ Acetic acid $3' GR, Ord. No. .!!!3# Nitric acid 3%' GR ISO, Ord. No. .!!"%3 3-" Cobalt%II& nitrate - ammonia for barbiturates %>0i77er reagent&" Spray solutionC )I ethanolic cobalt455> nitrate solution. After-treatmentC Dry and place into a chamber saturated with ammonia 'apours. Literature: E./. Shellard, /.3. Bsisiogu, $ab. @ractice !$, 9)< 4)*<;>. Chemicals: Co*alt-II. nitrate he/ahydrate GR, Ord. No. .!#%(3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# 3#" Cobalt%II& nitrate - lithium hydroxide for barbiturates" Spray solution IC .I cobalt455> nitrate solution in absolute methanol. Spray solution IIC ,.9I methanolic lithium hydro&ide solution. 0rocedureC Spray with 5 and after drying at room temperature with 55. Literature: H. Weidmann, Dissertation, 6erlin )*<). Chemicals: Co*alt-II. nitrate he/ahydrate GR, Ord. No. .!#%(3 Lithium hydro/ide -a*out $+' LiO9. LA7, Ord. No. .!%3$ 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 4ethanol dried SeccoSol6, Ord. No. .!3! # 32" Cobalt%II& thiocyanate for al7aloids and amines" Spray solutionC Dissol'e 8 g ammonium thiocyanate and ) g cobalt455> chloride in ., ml water. NoteC -l"aloids and amines show blue spots on white to pin" bac"ground. The colours grow pale after . hours and can be restored by spraying with water or by placing the chromatogram into water 'apours. Literature: E.S. $ane, /. %hromatog. !1, ;.< 4)*<9>.

Chemicals: Co*alt-II. chloride he/ahydrate GR ACS, Ord. No. .!#%($ Ammonium thiocyanate GR ACS, ISO, Ord. No. .! # ( 33" Copper acetate - potassium hexacyanoferrate%II& for the identification of higher fatty acids acc" to <aufmann" PC" 5ip solution IC (i& ), ml saturated a?ueous copper acetate solution with .;, ml water. 5ip solution IIC 0reshly prepared ).9I a?ueous potassium he&acyanoferrate455> solution. 0rocedureC -fter separation of the fatty acids on petroleum# or undecane# impregnated paper heat the chromatogram . hours at ).,J% to remo'e the impregnation. Then place the chromatogram ;9 min into dip solution 5. Subse?uently remo'e the e&cess copper acetate with running water by rinsing for )9 min. Then place the chromatogram into dip solution 55 where the acids show red#brown spots. Literature: H.@. Kaufmann, W.H. 7ietsch, 0ette u. Seifen, -nstrichmittel #2, )9; 4)*9;>. Chemicals: Copper-II. acetate monohydrate GR, Ord. No. .!#& 0otassium he/acyanoferrate-II. trihydrate GR ACS, ISO, Ord. No. .!"$+" 31" Copper acetate - rubeanic acid for the identification of higher fatty acids acc" to <aufmann" PC" 5ip solution C Dilute ), ml saturated copper455> acetate solution to ) ) with water. 5ip solution IIC ,.)I ethanolic rubeanic acid solution with ,.9 I ammonia. 0rocedureC @lace the chromatogram ;9 min into dip solution 5 and remo'e e&cess copper salt by rinsing with water for ).9 hours. Dip the moist chromatogram 8, min into 55, then rinse again 8, min with running water and dry. Literature: @.E. 6allance, W.(. %rombie, 6iochem. /. 2), <8. 4)*9+>. Chemicals: Copper-II. acetate monohydrate GR, Ord. No. .!#& Ru*eanic acid -dithioo/amide. GR, Ord. No. .!!3#$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(#

3)" Copper chloride for oximes" Spray solutionC ,.9I a?ueous copper455> chloride solution. NoteC #B&ime comple& compounds show green spots immediately after spraying, #B&ime comple& compounds show wea" green spots after heating ), min at )),J%. Literature: (. Hranisa'lEe'ic#/aco'lEe'ic, 5. @e&E"o'ic#Tadic, -. StoEilE"o'ic, /. %hromatog. !*, :, 4)*<8>. Chemicals: Copper-II. chloride dihydrate GR ACS, Ord. No. .!#&(( 1/" Copper sulfate - ben+idine for pyridine monocarboxylic acids" Spray solution IC Dissol'e ,.8 g copper455> sulfate in ),, ml ;9I ethanol. Spray solution IIC ,.)I solution of benDidine in 9,I ethanol. Caution: (en+idine is cancerogenic9 0rocedureC Spray with 5, dry the chromatogram at <,J% and spray with 55. 6lue spots. Chemicals: Copper-II. sulfate pentahydrate GR ACS, ISO, Ord. No. .!#&$! 7en,idine )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 1!" Copper sulfate - ;uinine - pyridine for barbiturates and thiobarbiturates" Spray solution IC Dissol'e ,.. g copper455> sulfate and ,.,. g ?uinine hydrochloride in 9, ml water, add . ml pyridine and fill up to ),, ml with water. Spray solution IIC ,.9I a?ueous potassium permanganate solution. 0rocedure aC Spray with 5 and dry at room temperature. White, yellow or 'iolet spots in daylight, dar" spots on fluorescent bac"ground in long#wa'e A3 light. 0rocedure *C Spray subse?uently with 55. Fellow or white spots. Literature: (. 0rahm, -. Gottesleben, K. Soehring, @harm. -cta Hel'. $1, :+9 4)*<8>. Chemicals: Copper-II. sulfate pentahydrate GR ACS, ISO, Ord. No. .!#&$! 0otassium perman8anate GR ACS, Ord. No. .!%!+# @uinine hydrochloride 0h )ur, Ord. No. +. &!(& 0yridine GR ACS, Ord. No. .!$&#+

1*" Copper%II& sulfate - sodium citrate for fla,onoids and coumarins 0ith o-dihydroxy groups %(enedict:s reagent&" Spray solutionC Dissol'e ).8 g copper455> sulfate, ):.8 g sodium citrate and ), g anhydrous sodium carbonate in water and fill up to ),, ml. NoteC The fluorescence in long#wa'e A3 light of coumarins with o#dihydro&y groups is ?uenched by 6enedictUs reagent. %ompounds without o#dihydro&y groups "eep or show stronger fluorescence, often connected with a change of colour. Literature: H. 2eDni", K. Egger, H. anal. %hem. !1$, )*< 4)*<)>. Chemicals: Copper-II. sulfate pentahydrate GR ACS, ISO, Ord. No. .!#&$! Sodium car*onate anhydrous GR ISO, Ord. No. .!3($# tri-Sodium citrate dihydrate GR ACS, ISO, Ord. No. .!3""+ 1$" -Cyclodextrin for straight-chain lipids" Spray solutionC 8,I ethanolic solution of #cyclode&trin. 0reparationC K. 0reudenberg et al., $iebigs -nn. %hem. ##1, ) 4)*;:>. D. 0rench et al., /. -m. %hem. Soc. 3!, 898 4)*;*>. After-treatmentC Dry the chromatogram at room temperature and place it into a chamber containing iodine 'apour. Literature: D.%. (alins, H.K. (angold, /. -m. Bil %hemists Soc. $3, 9:< 4)*<,>. H.K. (angold, /.$. Gellermann, H. Schlen", 0ederation @roc. !3, .<* 4)*9+>. H.K. (angold, 6.G. $amp, H. Schlen", /. -m. %hem. Soc. 33, <,:, 4)*99>. Chemicals: Iodine resu*limed GR ACS, IAO, Ord. No. .!"&3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -Cyclode/trine, Ord. No. .!# #3 1-" Cysteine - sulfuric acid for desoxyribonucleosides %modif" reagent acc" to Dische&" Spray solutionC (i& freshly before use ) part of a ,.9I cysteine hydrochloride solution in 8 7 sulfuric acid with * parts acetone. 0rocedureC Spray the chromatogram with the solution or dip into it, then heat 9#), min at +9J%. Deso&yribonucleosides and their phosphates turn green or grey, purines are dyed more rapidly than pyrimidines.

Literature: G. 6uchanan, 7ature !21, ),*) 4)*9)>. Chemicals: L-Cysteine hydrochloride monohydrate, Ord. No. .!#+($ Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Acetone GR ACS, ISO, Ord. No. .!!! " 1#" $'#-Diaminoben+oic acid - phosphoric acid for *-deoxy-sugars" Spray solutionC Dissol'e ) g 8,9#diaminobenDoic acid in .9 ml +,I phosphoric acid and dilute with <, ml water. After-treatmentC Heat )9 min at ),,J%. The spots fluoresce green#yellow in long# wa'e A3 light. -mounts more than . g are 'isible as brown spots in daylight. Literature: (. @eseD, 6ull. soc. chim. biol. $*, :,) 4)*9,>. Chemicals: ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( (,%-5iamino*en,oic acid for synthesis, Ord. No. +.#!"!% 12" o-Dianisidine for aldehydes and 7etones" Spray solutionC Saturated solution of o#dianisidine in glacial acetic acid. NoteC 5n some cases .,:#diaminofluorene may be used instead of o#dianisidine. Good differentiation of colours. Literature: 2. Wasic"y, B. 0rehden, (i"rochim. -cta !, 99 4)*8:>. Chemicals: o-5ianisidine -(,(;-dimetho/y*en,idine. Acetic acid $3' GR, Ord. No. .!!!3# #,&-5iaminofluorene 13" Dia+otisation and coupling 0ith !-naphthol for aromatic primary amines and sulfonamides %(ratton-?arshall reagent&" Spray solution IC 0reshly prepared )I sodium nitrite solution in hydrochloric acid 4c Q ) molR$>. Spray solution IIC 0reshly prepared ,..I )#naphthol solution in potassium hydro&ide 4c Q ) molR$>. 0rocedureC Spray with 5 and after ) min with 55. Dry the chromatogram at <,J%. NoteC 5nstead of l#naphthol a ,.;I methanolic solution of 7#4l#naphthyl>ethylene diammonium dichloride may be used as coupling agent.

Literature: -.%. 6ratton, E.K. (arshall, /. 6iol. %hem. !*1, 98: 4)*8*>. -. Wan"mueller, 7aturwissenschaften $), 8,. 4)*9.>. G. Wagner, -rch. @harm. *1#, ;,* 4)*9.>. T. 6ican#0ister, 3. KaEgano'ic, /. %hromatog. !!, ;*. 4)*<8>. Chemicals: -Naphthol GR, Ord. No. .!3##( Sodium nitrite GR ACS, Ord. No. .!3%"$ N-- -Naphthyl.ethylenediamine dihydrochloride GR, Ord. No. .!3#(& 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! 0otassium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$ + 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 11" *'2-Dibromo;uinone chlorimide for phenols %=ibbs@ reagent&" Spray solutionC 0reshly prepared ,.;I methanolic solution of .,<#dibromo# ?uinone chlorimide. :reatmentC Spray the chromatogram first with the spray solution and then respray with a ),I a?ueous sodium carbonate solution or place it in a chamber saturated with ammonia. Literature: E. 7uernberg, Dtsch. -pothe"er#Htg. !/!, .<+ 4)*<)>. Chemicals: #,3-5i*romoAuinone chlorimide Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ Ammonia solution #%' GR, Ord. No. .!%"(# 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 1)" *:'3:-Dichlorofluorescein" ),, ml ready to use spray solution for chromatography 4c Q ,..I in .#propanol>. Ord. No. .!$# $ )/" *:'3:-Dichlorofluorescein fluorescence indicator for saturated and unsaturated lipids" A. Spray solutionC ,.. ethanolic solution of .U,:U#dichlorofluorescein. 7. Spray solution -for 6itamin ).C ,.,)I ethanolic solution of .U,:U#dichloro# fluorescein. NoteC -fter drying with warm air it is sometimes ad'isable to place the chromatogram in a current of steam, or to spray it with water. 5nspect in long#wa'e A3 light.

Literature: D.%. (alins, H.K. (angold, /. -m. Bil %hemists Soc. $3, 9:< 4)*<,>. @./. Dunphy, K./. Whittle, /.0. @ennoc", %hem. T 5nd. 4$ondon> !)2#, ).):. Chemicals: #;,&;-5ichlorofluorescein, Ord. No. .!$3&3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )!" *:'3:-Dichlorofluorescein - aluminium chloride - iron%III& chloride for free fatty acids %specific detection&" Spray solution IC ,.,9I ethanolic solution of .U,:U#dichlorofluorescein. Spray solution IIC ) I ethanolic solution of aluminium chloride. Spray solution IIIC )I a?ueous solution of iron4555> chloride. 0rocedureC Spray with 5, dry some minutes at ),,J%, spray with 55, dry again some minutes at ),,J% and spray with 555. @in"#'iolet spots on fallow bac"ground. Literature: -.E. DudDins"y, /. %hromatog. $!, 9<, 4)*<:>. Chemicals: #;,&;-5ichlorofluorescein, Ord. No. .!$3&3 Aluminium chloride anhydrous, Ord. No. .! !+# Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )*" *'2-Dichlorophenolindophenol- sil,er nitrate for al7ali chlorides" Spray solutionC ,..I ethanolic solution of .,<#dichlorophenolindophenol sodium salt. 0ilter after addition of 8 g sil'er nitrate and sha"ing. @repare freshly before useW Literature: T. 6arnabas, (.G. 6ad'e, /. 6arnabas, 7aturwissenschaften -!, ;:+ 4)*9;>. Chemicals: #,3-5ichlorophenol-indophenol sodium salt dihydrate GR, Ord. No. .!(!#+ Sil6er nitrate GR ACS, ISO, Ord. No. .! % # )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )$" *'2-Dichlorophenolindophenol sodium salt for organic acids and 7eto acids" Spray solutionC ,.)I ethanolic solution of .,<#dichlorophenolindophenol sodium salt.

After-treatmentC -fter brief warming the acids appear as red spots on light blue bac"ground. Literature: %. @assera, -. @edrotti, G. 0errari, /. %hromatog. !-, .+* 4)*<;>. Chemicals: #,3-5ichlorophenol-indophenol sodium salt dihydrate GR, Ord. No. .!(!#+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )-" *'2-Dichlorophenolindophenol sodium salt for ,itamin C %Tillman reagent&" Spray solutionC ,.,9I solution of .,<#dichlorophenolindophenol sodium salt in 9,I ethanol. NoteC %olourless spots on blue bac"ground. Literature: F.#T. %hen, 0.-. 5sherwood, $.W. (apson, 6iochem. /. ##, +.) 4)*98>. Chemicals: #,3-5ichlorophenol-indophenol sodium salt dihydrate GR, Ord. No. .!(!#+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )#" *'2-Dichloro;uinone chlorimide for antioxidants' adrenaline and deri,ati,es' cyanamide and deri,ati,es" Spray solutionC @repare freshly before use a ,.) to l I solution of .,<#dichloro# ?uinone chlorimide in ),, ml absolute ethanol. The spots appear after about )9 minutes. 7ot to be used for urea. Some antio&idants show characteristic change of colours after being sprayed with a .I solution of sodium tetraborate in ;,I ethanol. Literature: -. Seher, 0ette u. Seifen, -nstrichmittel 2!, 8;9 4)*9*> 2.0. '. d. Heide, B. Wouters, H. $ebensm.#Anters. u. 0orsch. !!#S ).* 4)*<.>. 2. Segura#%ardona, K. Soehring, (ed. E&p. !/, .9) 4)*<;>. Chemicals: #,3-5ichloroAuinone chlorimide GR, Ord. No. .!(!(& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( di-Sodium tetra*orate !-hydrate GR ACS, ISO, Ord. No. .!3(!+ )2" Dicobalt octacarbonyl for acetylene compounds" Spray solution IC Dissol'e ,.9 g dicobalt octacarbonyl in ),, ml petroleum benDine.

Spray solution IIC Hydrochloric acid 4c Q ) molR$>. 0rocedureC Spray with 5, wait ), min, spray with 55 and remo'e the layer with 7eatan after drying. Wash out e&cess reagent with water and place the chromato gram into a bromine atmosphere. The spots show yellow colours. Literature: K.E. Schulte, 0. -hrens, E. Sprenger, @harm. Htg. !/1, ))<9 4)*<8>. Chemicals: 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! 7romine GR ISO, Ord. No. .! $"+ Neatan> 5i-Co*alt octacar*onyl for synthesis, Ord. No. +.#!&"+ 0etroleum *en,ine - !!- "!BC., Ord. No. .! &&! )3" Diethylamine - copper%II& sulfate for thiobarbiturates" Spray solutionC Dissol'e ,.9 g copper455> sulfate in ),, ml methanol. -dd 8 ml diethylamine to the solution. NoteC Sha"e prior to useS stable for only a few days. Thiobarbituric acids show green spots. Literature: W. DietD, K. Soehring, -rch. @harm. *)/, +, 4)*9:>. Chemicals: Copper-II. sulfate pentahydrate GR ACS, ISO, Ord. No. .!#&$! 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 5iethylamine for synthesis, Ord. No. +.!(! ! )1" Diethyl malonate for $'#-dinitroben+oic acid esters" Spray solution IC ),I ethanolic solution of diethyl malonate. Spray solution IIC ),I a?ueous sodium hydro&ide. 0rocedureC Spray with 5 and then with 55. Heat 9 min at *9J%. 2ed#'iolet spots. Literature: /. %erny, %hem. listy -), )+** 4)*99>. Chemicals: 5iethyl malonate for synthesis, Ord. No. +.!!+$+ Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( ))" Dimedone - phosphoric acid for 7eto sugars"

Spray solutionC Dissol'e ,.8 g 9,9#dimethylcyclohe&ane#),8#dione 4dimedone> in *, ml ethanol and add ), ml +9I phosphoric acid. After-treatmentC Heat )9#., min at )),J%. 5n daylight yellow spots on a white bac"ground, in long#wa'e A3 light blue fluorescent spots. Literature: S. -dachi, -nal. 6iochem. ), ..; 4)*<;>. Chemicals: 5imedone GR, Ord. No. .!3! ( ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !//" --Dimethylaminoben+aldehyde" ),, ml ready to use spray solution for chromatography 4c Q ca. )..I in .# propanol>. Ord. No. .!(&## !/!" --Dimethylaminoben+aldehyde - acetic acid - phosphoric acid for proa+ulenes and a+ulenes %.P reagent&" Spray solutionC Dissol'e ,..9 g ;#dimethylaminobenDaldehyde in a mi&ture of 9, g glacial acetic acid and 9 g +9I phosphoric acid. -fter dissolution is complete, add ., ml water. Stable for months in a brown bottle. NoteC -Dulenes turn deep blue at room temperature. @roaDulenes show blue spots only after heating for ), min at +,J%. The colours grow pale and become green to yellow. 6y e&posure to steam o'er a water bath the spots show again their intense blue colour. Literature: E. Stahl, Dtsch. -pothe"er#Htg. )$, )*: 4)*98>. H. Kaiser, G. Hasenmayer, -rch. @harm. *13, 9,8 4)*9;>. Chemicals: "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ Acetic acid $3' GR, Ord. No. .!!!3# ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( !/*" --Dimethylaminoben+aldehyde - acetylacetone for amino sugars %?organ.lson reagent&" Spray solution IC -dd 9 ml of a mi&ture of 9 ml 9,I a?ueous potassium hydro&ide and ., ml ethanol immediately prior to use to ), ml of a solution of ,.9 ml acetylacetone and 9, ml )#butanol.

Spray solution IIC Dissol'e ) g ;#dimethylaminobenDaldehyde in 8, ml ethanol. -dd 8, ml 8:I hydrochloric acid. 5f re?uired dilute with )+, ml )#butanol. 0rocedureC -fter spraying with 5 heat 9 min at ),9J%, spray with 55 and dry 9 min at *,J%. 2ed spots. Literature: $.-. Elson, W.T./. (organ, 6iochem. /. *3, )+.; 4)*88>. 2. 6elcher, -./. (utten, %.(. Sabroo", -nalyst 3), .,) 4)*9;>. Chemicals: "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ Acetylacetone GR, Ord. No. .!$3!! 0otassium hydro/ide pellets GR, Ord. No. .!%!(( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -7utanol GR ACS, ISO, Ord. No. .! $$! 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & !/$" --Dimethylaminoben+aldehyde - hydrochloric acid for amines %.hrlich@s reagent&" Spray Solution AC Dissol'e ) g ;#dimethylaminobenDaldehyde in a mi&ture of .9 ml 8:I hydrochloric acid and :9 ml methanol. After-treatmentC 5n some cases it is necessary to warm the plate. Spray solution 7C )I ethanolic solution of ;#dimethylaminobenDaldehyde. :reatmentC @lace the sprayed chromatogram 8#9 min in a chamber saturated with hydrochloric acid 'apours or respray with .9I hydrochloric acid. Sometimes it is necessary to warm the plate. Literature: 2.-. Heacoc", (.E. (ahon, /. %hromatog. !3, 88+ 4)*<9>. Chemicals: "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ 9ydrochloric acid #%' GR, Ord. No. .!!( 3 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & 4ethanol GR ACS, ISO, Ord. No. .!3!!$ )thanol a*solute GR ACS, ISO Ord. No. .!!$+( !/-" --Dimethylaminoben+aldehyde - hydrochloric acid according to Stahl for indole deri,ati,es %,an Ar7 reagent&" Spray solutionC Dissol'e ) g ;#DimethylaminobenDaldehyde in 9, ml 8:I hydrochloric acid and add 9, ml ethanol. NoteC 5n case of eluents with 'olatile al"aline reacting components it is necessary to heat the plate to about 9,J%, until these compounds ha'e disappeared.

0rocedureC Spray intensi'ely until transparency. Subse?uently blow 'apours of a?ua regia o'er the layer. Literature: E. Stahl, H. Kaldewey, Hoppe#Seylers H. physiol. %hem. $*$, )+. 4)*<)>. Chemicals: "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !/#" --Dimethylaminoben+aldehyde - sulfuric acid for ergot al7aloids" Spray solutionC Dissol'e ,.).9 g ;#dimethylaminobenDaldehyde in a cooled mi&ture of <9 ml *:I sulfuric acid and 89 ml water and add ,.,9 ml 9I a?ueous iron4555> chloride solution. Stable for about a wee". Literature: (. Hinser, %. 6aumgaertel, -rch. @harm. *)3, )9+ 4)*<;>. Chemicals: "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( !/2" Dimethylaminoben+ylidenerhodanine for sil,er' copper and mercury ions" Spray solutionC )I ethanolic solution of 9#4;#dimethylaminobenDylidene># rhodanine. :reatmentC 2espray with .9I ammonia solution or place into a chamber saturated with ammonia 'apours. @in" to 'iolet spots. Literature: 0.W.H.(. (er"us, @harm. Wee"blad )1, *99 4)*<8>. Chemicals: %--"-5imethylamino*en,ylidene.rhodanine GR, Ord. No. .!(!%$ )thanol a*solute GR ACS, ISO Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# !/3" Dimethylaminocinnamaldehyde for indoles" Stoc? solutionC Dissol'e . g ;#dimethylaminocinnamaldehyde in a mi&ture of ),, ml hydrochloric acid 4c Q < molR$> and ),, ml ethanol. Store the solution in the refrigerator. Spray solutionC ) part stoc" solution and ; parts ethanol.

After-treatmentC Heat 9 min at ),9J%. The colours of the spots are intensified by blowing 'apours of a?ua regia o'er the layer. NoteC Ansuitable with ammonia#containing eluents because the bac"ground becomes coloured. 6y brief heating 4), min at ),9J%> this can be e'aporated before spraying. Literature: /. Harley#(ason, -.-.@.G. -rcher, 6iochem. /. 2), <, 4)*9+>. Chemicals: "--5imethylamino.cinnamaldehyde for synthesis, Ord. No. +.##!(" 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !/1" 6'6-Dimethyl-!'--phenylenediammonium dichloride for bromine-containing hypnotics and chlorinated insecticides" Spray solutionC Dissol'e ,.9 g 7,7#dimethyl#).;#phenylenediammonium dichloride in ),, ml sodium etho&ide 4) g sodium in ),, ml ethanol>. 0rocedureC -fter spraying moisten the chromatogram with a water spray and irradiate ) min with unfiltered A3 light. This liberates free halogen which o&idises the reagent to WursterUs red. Literature: /. 6aeumler, S. 2ippstein, Hel'. %him. -cta --, ))<. 4)*<)>. Chemicals: N,N-5imethyl- ,"-phenylenediammonium dichloride GR, Ord. No. .!(!3& Sodium rods, Ord. No. .!3#3! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !/)" 6'6-Dimethyl-!'--phenylenediammonium dichloride for peroxides" Spray solutionC Dissol'e ).9 g 7,7#dimethyl#).;#diphenylenediammonium dichloride in a mi&ture of ).+ ml methanol, .9 ml water and ) ml glacial acetic acid. @ero&ides show purple spots. Literature: E. Knappe, D. @eteri, H. anal. %hem. !)/, 8+< 4)*<.>. Chemicals: N,N-5imethyl- ,"-phenylenediammonium dichloride GR, Ord. No. .!(!3& Acetic acid $3' GR, Ord. No. .!!!3# 4ethanol GR ACS, ISO, Ord. No. .!3!!$

!!/" 6'6-Dimethyl-!'--phenylenediammonium dichloride - trichloroacetic acid for methyl-sugars" Spray solutionC Dissol'e ,.; g 7,7#dimethyl#),;#phenylenediammonium dichloride in ),, ml .I a?ueous trichloroacetic acid solution. After-treatmentC Heat )#. min at ).,J%. NoteC The colour spots may be eluated for colorimetric determination. Literature: W.%. Schaefer, /.W. 'an %le'e, -nal. %hem. *1, ).*, 4)*9<>. $. 6oggs, $.S. %uendet, 5. Ehrenthal, 2. Koch, 0. Smith, 7ature !22, 9., 4)*9,>. Chemicals: N,N-5imethyl- ,"-phenylenediammonium dichloride GR, Ord. No. .!(!3& :richloroacetic acid GR ACS, Ord. No. .!!+!& !!!" !'$-Dinitroben+ene for !3-7etosteroids" Solution aC .I ethanolic solution of ),8#dinitrobenDene. Solution *C (ethanolic potassium hydro&ide solution 4c Q ..9 molR$>. Spray solutionC (i& e?ual parts of a and b. After-treatmentC Heat )#. min at +,J%. 3iolet spots. Literature: T. 0eher, (i"rochim. -cta !)2#, ),9. 6.@. $isboa, /. %hromatog. !2, )8< 4)*<;>. 2. 7eher, Steroid %hromatography, Else'ier )*<;, -msterdam, $ondon, 7ew For". Chemicals: ,(-5initro*en,ene for synthesis, Ord. No. +.###&# 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 2ariation for 0C: 5ip solution IC (i& ) part 8,I a?ueous potassium hydro&ide with ) part ethanol. 5ip solution IIC .I ethanolic ),8#dinitrobenDene solution. 0rocedureC -fter dipping into 5 press off e&cess between filter paper. Then dip into 55, press off and heat slowly at <9J%. ):#Ketosteroids turn 'iolet, .#"eto# steroids blue#'iolet and .,#"etosteroids brown.

Literature: /. 6arrolier, /. Heilmann, H. physiol. %hem. $/), ..) 4)*9:>. B. Schindler, T. 2eichstein, Hel'. %him. -cta $-, ),+ 4)*9)>. Chemicals: ,(-5initro*en,ene for synthesis, Ord. No. +.###&# 0otassium hydro/ide pellets GR, Ord. No. .!%!(( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !!*" $'#-Dinitroben+oic acid for cardiac glycosides" A. Spray solutionC Dissol'e ) g 8,9#dinitrobenDoic acid in a mi&ture of 9, ml methanol and 9, ml potassium hydro&ide solution 4c Q . molR$>. 7. Spray solution IC .I methanolic solution of 8,9#dinitrobenDoic acid. Spray solution IIC 9.:I methanolic potassium hydro&ide solution. 0rocedureC Spray lightly with 5 and then with e&cess 55. The spots show blue 'iolet colours. Literature: 2. Tschesche, G. Grimmer, 0. Seehofer, %hem. 6er. 12 ).89 4)*98>. (.$. $ewbart, W. Wehrli, T. 2eichstein, Hel'. %him. -cta -2, 9<9 4)*<8>. Chemicals: (,%-5initro*en,oic acid, Ord. No. .!! (+ 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !!$" $'#-Dinitroben+oic acid for reducing sugars" Spray solutionC )I solution of 8,9#dinitrobenDoic acid in sodium carbonate solution 4c Q . molR$>. After-treatmentC Dry 9#), min at ),,J%. Literature: 0. Weygand, H. Hofmann, %hem. 6er. 1$, ;,9 4)*9,>. Chemicals: (,%-5initro*en,oic acid, Ord. No. .!! (+ Sodium car*onate anhydrous GR ISO, Ord. No. .!3($# !!-" *'--Dinitrofluoroben+ene for amino acids" Spray solution IC Dissol'e +.; g sodium hydrogen carbonate in +, ml water, add ..9 ml ) 7 sodium hydro&ide solution and ma"e up to ),, ml with water. Spray solution IIC ),I methanolic solution of .,;#dinitrofluorobenDene. :reatmentC Spray with 5 and subse?uently with 55.

0rocedureC Scrape off 9 mm from both sides of the plate. @lace two polyethylene strips of same breadth on the margins so that a second glass plate can be laid on the layer. Heat ) hour at ;,J% in the dar", cool the plate and place ), min in an ether bath. -fter drying the spots are outlined. Literature: G. @ata"i, /. %hromatog. !2, 9;) 4)*<;>. Chemicals: -<luoro-#,"-dinitro*en,ene GR, Ord. No. .!#$33 Sodium hydro8en car*onate GR ISO, Ord. No. .!3(#$ Sodium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$%3 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 5iethyl ether GR ACS, Ord. No. .!!$# !!#" *'--Dinitrophenylhydra+ine for free aldehyde and 7eto groups and 7etoses" A. Spray solutionC ,,;I solution of .,;#dinitrophenylhydraDine in hydrochloric acid 4c Q . molR$>. 7. Spray solutionC -dd ), ml 8:I hydrochloric acid to ) g .,;#dinitrophenyl# hydraDine in ),,, ml ethanol. After-treatmentC 0or distinction of the formed .,;#dinitrophenylhydraDones 4D7@H> spray consecuti'ely with ,..I solution of potassium he&acyanoferrate4555> in hydrochloric acid 4c Q . molR$>. Saturated "eto#D7@H show blue colour immediately, saturated aldehyde#D7@H show oli'e#green colour more slowly. Ansaturated carbonyl deri'ati'es change only slowly or not at all. Literature: -. (ehlitD, K. Gierschner, T. (inas, %hemi"er#Htg. 13, 9:8 4)*<8>. Chemicals: #,"-5initrophenylhydra,ine GR, Ord. No. .!(!+ 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( !!2" $'#-Dinitrosalicylic acid for reducing sugars" Spray solutionC ,,9I, solution of 8,9#dinitrosalicylic acid 4.#hydro&y#8,9# dinitrobenDoic acid> in ;I sodium hydro&ide solution. After-treatmentC -fter brief pre#drying at room temperature heat ;#9 min at ),,J%. Literature: -. /eanes, %.S. Wise, 2./. Dimler, -nal. %hem. *$, ;)9 4)*9)>.

Chemicals: #-9ydro/y-(,%-dinitro*en,oic acid, Ord. No. +.!! " Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ !!3" Diphenylamine for glycolipids " Spray solutionS (i&ture of ., ml ),I ethanolic diphenylamine solution, ),, ml 8:I hydrochloric acid and +, ml glacial acetic acid. After-treatmentC Heat 9#), min at ),,J%. 6lue#grey spots. Literature: H. /atD"ewitD, Hoppe#Seylers H. physiol. %hem. $*/, .9) 4)*<,>. Chemicals: 5iphenylamine GR and redo/ indicator, Ord. No. .!(!+3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & Acetic acid $3' GR, Ord. No. .!!!3# !!1" Diphenylamine - palladium%II& chloride for nitrosamines" Spray solutionC (i& 9 parts ).9I ethanolic diphenylamine solution and ) part ,..I sodium chloride solution containing ,.) g palladium455> chloride. After-treatmentC -fter e&posure to short#wa'e A3 light the substances show 'iolet spots. Literature: 2. @reussmann, D. Daiber, H. Hengy, 7ature */!, 9,. 4)*<;>. 2. @reussmann, G. 7eurath, G. Wulf#$orentDen, D. Daiber, H. Hengy, H. anal. %hem. */*, )+: 4)*<;>. Chemicals: 5iphenylamine GR and redo/ indicator, Ord. No. .!(!+3 0alladium-II. chloride anhydrous for synthesis, Ord. No. +.!& ! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sodium chloride GR ACS, ISO, Ord. No. .!3"!" !!)" Diphenylamine - +inc chloride for chlorinated insecticides" Spray solutionC Dissol'e ,.9 g diphenylamine and ,.9 g Dinc chloride in ),, ml acetone. After-treatmentC Heat 9 min at .,,J%. %olour reaction. Literature: D. Kath, /. %hromatog. !#, .<* 4)*<;>.

Chemicals: 5iphenylamine GR and redo/ indicator, Ord. No. .!(!+3 Cinc chloride GR ACS, ISO, Ord. No. .!++ 3 Acetone GR ACS, ISO, Ord. No. .!!! " !*/" - minoethyl diphenylborate for - and -pyrones %6eu@s reagent&" Spray solutionC )I methanolic #aminoethyl diphenylborate XQ .# 4Diphenylborylo&y>ethylamineY solution. 0rocedureC Spray about ), ml of the solution and inspect the fluorescence in long wa'e A3 light. Literature: 2. 7eu, 7aturwissenschaften --, )+) 4)*9:>. E. Stahl, @./. Schorn, Hoppe#Seylers H. physiol. %hem. $*#, .<8 4)*<)>. Chemicals: #--5iphenyl*orylo/y.ethylamine Rea8. 0h )ur, Ord. No. 1.59626 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !*!" Diphenylcarba+ide for sil,er' lead' mercury' copper' tin' +inc' and calcium ions" Spray solution IC )#.I ethanolic diphenylcarbaDide solution. Spray solution IIC .9I ammonia solution or a chamber saturated with ammonia. NoteC 0or mercury acetate adducts heat some minutes at +,J%, causing the spots to turn blue#'iolet. Literature: 0.W.(.H. (er"us, @harm. Wee"blad )1, *;: 4)*<8>. Chemicals: 5iphenylcar*a,ide GR and redo/ indicator, Ord. No. .!(!$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# !**" Diphenylcarba+one for addition compounds of unsaturated fatty acids" Spray solutionC ,..I ethanolic solution of diphenylcarbaDone. NoteC -ddition compounds of unsaturated acids 4e. g. with Hg> are dyed purple. %olour intensification may be obtained by respraying with ethanolic nitric acid 4c Q ,.,9 molR$>. Literature: F. 5nou'e, (. 7oda, B. Hirayama, /. -m. Bil %hemists Soc. $*, )8. 4)*99>.

Chemicals: 5iphenylcar*a,one, Ord. No. .!(!+& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !*$" Diphenylcarba+one for cations" Spray solutionC Saturated solution of diphenylcarbaDone in methanol. Literature: G.6. Heisig, 0.H. @ollard, -nal. %him. -cta !2, .8; 4)*9:>. Chemicals: 5iphenylcar*a,one, Ord. No. .!(!+& 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !*-" Diphenylpicrylhydra+yl for essential oils" Spray solutionC Dissol'e ,.,< g diphenylpicrylhydraDyl in ),, ml chloroform. After-treatmentC Heat 9#), min at )),J%. Fellow spots on 'iolet bac"ground. Literature: %. 6ergstrom, %. $agercrantD, -cta %hem. Scand. !1, 9<, 4)*<;>. Chemicals: #,#;-5iphenyl- -picrylhydra,yl Chloroform GR ISO, Ord. No. .!#""% !*#" *'#-Diphenyl-$-%--styrylphenyl&tetra+olium chloride %TPT>& for reducing steroids %corticosteroids&" Solution aC 0reshly prepared )I methanolic solution of T@TH. Solution *C 8I a?ueous sodium hydro&ide solution. Spray solutionC (i& e?ual parts of a and b freshly before use. Literature: @./. Ste'ens, /. %hromatog. !-, .<* 4)*<;>. Chemicals: #,%-5iphenyl-(--"-styrylphenyl.tetra,olium chloride 4ethanol GR ACS, ISO, Ord. No. .!3!!$ Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++ !*2" Dipicrylamine for choline %non-specific&" Spray solutionC Dissol'e ,.. g dipicrylamine in a mi&ture of 9, ml acetone and 9, ml water. NoteC %holine and its deri'ati'es appear as red spots on yellow bac"ground.

Literature: K.6. -ugustinsson, (. Grahn, -cta %hem. Scand. 3, *,< 4)*98>. Chemicals: 5ipicrylamine Acetone GR ACS, ISO, Ord. No. .!!! " !*3" Dipicrylamine for ,itamin ( ! " Stoc? solutionC -dd ) g dipicrylamine to ,.). g magnesium carbonate and )9 ml water, heat the mi&ture )9 min on a boiling water bath and filter. Spray solutionC -dd to ,.. ml of the dipicrylamine solution 9, ml methanol, ;* ml water and ) ml .9I ammonia solution. Literature: K.6. -ugustinsson, (. Grahn, -cta %hem. Scand. 3, *,< 4)*98>. Chemicals: 5ipicrylamine 4a8nesium car*onate 4ethanol GR ACS, ISO, Ord. No. .!3!!$ Ammonia solution #%' GR, Ord. No. .!%"(# !*1" Dithi+one for ions of hea,y metals" Spray solution IC ,.,9I solution of dithiDone in carbon tetrachloride. Spray solution IIC Spray with .9I ammonia solution or place the chromatogram in a chamber saturated with ammonia 'apours. Literature: T. 6arnabas, /. 6arnabas, 7aturwissenschaften --, <) 4)*9:>. 0.W.H.(. (er"us, @harm. Wee"blad )1, *99 4)*<8>. Chemicals: 5ithi,one GR - ,%-diphenylthiocar*a,one., Ord. No. .!(!$# Car*on tetrachloride GR, Ord. No. .!#### Ammonia solution #%' GR, Ord. No. .!%"(# !*)" Dragendorff reagent for polyethylene glycols' polyethylene glycol ethers and polyethylene glycol esters" Solution aC Dissol'e ).: g bismuth4555> nitrate in a mi&ture of ., ml glacial acetic acid and +, ml water, add a solution of ;, g potassium iodide in ),, ml water and .,, ml glacial acetic acid and ma"e up to ),,, ml with water. Solution *C .,I a?ueous barium chloride solution. Spray solutionC (i& . parts a with ) part b before use.

Literature: K. Thoma, 2. 2ombach, E. Allmann, Sci. @harm. $*, .)< 4)*<;>. Chemicals: 7ismuth-III. nitrate *asic GR, Ord. No. .! +&+ 0otassium iodide GR ISO, Ord. No. .!%!"( 7arium chloride dihydrate GR ACS, ISO, Ord. No. .! & $ Acetic acid $3' GR, Ord. No. .!!!3# !$/" Dragendorff reagent acc" to (regoff-Del0ische for ;uaternary bases" Stoc? solutionC Dissol'e +., g bismuth4555> nitrate in .,#.9 ml .9I nitric acid. -dd this solution slowly with stirring to a slurry of ., g potassium iodide and ) ml < 7 hydrochloric acid and 9 ml water. -dd water to the dar" precipitate until an orangered colour de'elops. The 'olume of the solution should be *9 ml. -ny solid residue present is filtered off and the solution made up to ),, ml with water. The solution is stable for se'eral wee"s in the refrigerator when stored in an amber flas". Spray solutionC (i& in this orderC ., ml water, 9 ml hydrochloric acid 4c Q < molR$>, . ml stoc" solution and < ml sodium hydro&ide solution 4c Q < molR$>. 5n case bismuth hydro&ide is not completely dissol'ed by sha"ing, add se'eral drops of hydrochloric acid 4c Q < molR$>. NoteC The spray solution is stable for about ), days in the refrigerator. Literature: H.(. 6regoff, E. 2oberts, %.%. Delwiche, /. 6iol. %hem. */#, 9<9 4)*98>. Chemicals: 7ismuth-III. nitrate *asic GR, Ord. No. .! +&+ Nitric acid 3%' GR ISO, Ord. No. .!!"%3 9ydrochloric acid #%' GR, Ord. No. .!!( 3 0otassium iodide GR ISO, Ord. No. .!%!"( Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++ !$!" Dragendorff reagent acc" to ?unier for al7aloids and other nitrogencontaining compounds" Solution aC Dissol'e ).: g bismuth4555> nitrate and ., g tartaric acid in +, ml water. Solution *C Dissol'e )< g potassium iodide in ;, ml water. Stoc? solutionC (i& e?ual parts of a and b. The stoc" solution is stable for se'eral months, if refrigerated. Spray solutionC Dissol'e ), g tartaric acid in 9, ml water and add ), ml of the stoc" solution. NoteC 0or detecting 'itamin 6) spray with the stoc" solution.

Literature: 2. (unier, 6ull. soc. chim. biol. $#, )..9 4)*98>. Chemicals: 7ismuth-III. nitrate *asic GR, Ord. No. .! +&+ 0otassium iodide GR ISO, Ord. No. .!%!"( L-D.:artaric acid GR ACS, ISO, Ord. No. .!!+!" !$*" Dragendorff reagent acc" to ?unier and ?acheboeuf for al7aloids and other nitrogen-containing compounds" Solution aC Dissol'e ,.+9 g bismuth4555> nitrate in ), ml glacial acetic acid and ;, ml water. Solution *C Dissol'e + g potassium iodide in ., ml water. Stoc? solutionC (i& e?ual parts of a and b. The mi&ture can be stored in a dar" bottle for a long time. Spray solutionC (i& ) ml stoc" solution with . ml glacial acetic acid and ), ml water before use. Literature: 2. (unier, (. (acheboeuf, 6ull. soc. chim. biol. $$, +;< 4)*9)>. H. /atD"ewitD, Hoppe#Seylers H. physiol. %hem. *)*, ** 4)*98>. Chemicals: 7ismuth-III. nitrate GR, Ord. No. .! +&+ 0otassium iodide GR ISO, Ord. No. .!%!"( Acetic acid $3' GR, Ord. No. .!!!3# !$$" Dragendorff reagent acc" to Thies and Reuther' modif" by 4aguBfal,i' for al7aloids and other nitrogen-containing compounds" Stoc? solutionC 6oil ..< g bismuth carbonate and : g sodium iodide with .9 ml glacial acetic acid for a few minutes. -fter ). hours filter off the precipitated sodium acetate. Then mi& ., ml of the red#brown filtrate with +, ml ethyl acetate and add ,.9 ml water. Store in a dar" bottle. Spray solutionC (i& ), ml stoc" solution with ),, ml glacial acetic acid and .;, ml ethyl acetate. -fter spraying of 9#), ml al"aloids and some other compounds containing no nitrogen show orange spots. After-treatmentC - more sensiti'e detection is a'ailably by subse?uent spraying with sulfuric acid 4c Q ,.,.9#,.,9 molR$>. The spots are bright orange to red on a grey bac"ground. Literature: H. Thies, 0.W. 2euther, 7aturwissenschaften -!, .8, 4)*9;>. D. 3aguEfal'i, @lanta (ed. 1, 8; 4)*<,>. E. Tyiha", /. %hromatog. !-, ).9 4)*<;>.

Chemicals: 7ismuth-III. car*onate Sodium iodide e/tra pure 70, 0h )ur, 1S0, Ord. No. .!3%#! Acetic acid $3' GR, Ord. No. .!!!3# )thyl acetate GR, Ord. No. .!$3#( Sulfuric acid !.!% mol=l :itrisol>, Ord. No. .!$$+" !$$a Dragendorff reagent"

),, ml ready to use spray solution for chromatography 4Sol'entC ethyl acetate, acetic acid, water>. Ord. No. .!#!(% !$-" .thylenediamine for catecholamines" Spray solutionC (i&ture of e?ual parts of ethylenediamine with water or diluted sodium hydro&ide solution. 0rocedureC Heat the chromatogram ., min at 9,#<,J%. 5nspection in short# or long#wa'e A3 light. Literature: 2. Segura#%ardona, K. Soehring, (ed. E&p. !/, .9) 4)*<;>. Chemicals: )thylenediamine for synthesis, Ord. No. +.!!$"& Sodium hydro/ide solution # mol=l, Ord. No. .!$ (3 !$#" .thylenediamine - potassium hexacyanoferrate%III& for catecholamines %adrenaline' noradrenaline and acetyl deri,ati,es&" Spray solutionC Solution of ,.) g potassium he&acyanoferrate4555> in 9 ml ethylenediamine, ;9 ml ethanol and 9, ml water. After-treatmentC Heat the chromatogram ), min at ),9J%. 5nspection under A3 light. Literature: /.S. Stern, (./. 0ran"lin, /. (ayer, /. %hromatog. $/, <8: 4)*<:>. Chemicals: )thylenediamine for synthesis, Ord. No. +.!!$"& 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !$2" Cast blue salt ( for phenols and coupling amines %dia+onium reagent&" Spray solution IC - freshly prepared ,.9I a?ueous fast blue salt 6 solution.

Spray solution IIC Sodium hydro&ide solution 4c Q ,.) molR$>. :reatmentC Spray with 5 and then with 55. Literature: H. /atD"ewitD, A. $enD, Hoppe#Seylers H. physiol. %hem. $/#,98 4)*9<>. Chemicals: <ast *lue salt 7, Ord. No. .!( $ Sodium hydro/ide solution !. mol=l :itrisol>, Ord. No. .!$$%$ !$3" Cluorescein for lipids" Spray solutionC ,.,)I ethanolic solution of fluorescein. After-treatmentC Dry with warm air and handle subse?uently with water 'apour or spray lightly with water. Chemicals: <luorescein -C.I. "%(%!. )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !$1" Cluorescein - ammonia for purines' pyrimidines and barbiturates" Spray solutionC ,.,,9I solution of fluorescein in ,.9 7 ammonia solution. 5nspect the chromatogram in long# and short#wa'e A3 light. Literature: T. Wieland, $. 6auer, -ngew. %hem. 2$, 9)) 4)*9)>. Chemicals: <luorescein -C.I. "%(%!. Ammonia solution #%' GR, Ord. No. .!%"(# !$)" Cluorescein - bromine for unsaturated compounds" Spray solutionC ,.)I ethanolic fluorescein solution. 7romine solutionC 9I bromine in carbon tetrachloride. 0rocedureC -fter spraying with the fluorescein solution place the chromatogram into a chamber containing the bromine solution. 0luorescein is con'erted to eosin which shows no fluorescence in long#wa'e A3 light. %ompounds adding on pre'ent the formation of eosin and the fluorescence remains. $arger amounts of substance show yellow spots on reddish bac"ground. Literature: 0. 2unge, -. /umar, 0. Koehler, /. pra"t. %hem. *!, 8* 4)*<8 Chemicals: <luorescein -C.I. "%(%!.

7romine GR ISO, Ord. No. .! $"+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Car*on tetrachloride GR, Ord. No. .!#### 2ariationC With self#coated plates ta"e ,.,;I a?ueous fluorescein sodium solution instead of water. After-treatmentC -fter de'elopment of the chromatogram blow bromine 'apours o'er the dried plate. Literature: E. Stahl, %hemi"er#Htg. 1*, 8.8 4)*9+>. Chemicals: <luorescein sodium -C.I. "%(%!. e/tra pure, Ord. No. .!($$# 7romine GR ISO, Ord. No. .! $"+ !-/" Cluorescein - hydrogen peroxide for hypnotics containing bromine. Spray solution IC ,.)I fluorescein solution in 9,I ethanol. Spray solution IIC (i& e?ual parts of 8,I hydrogen pero&ide and glacial acetic acid. 0rocedureC Spray with 5 and then with 55, heat finally ., min at *,J%. NoteC 6romine formed by o&idation reacts with fluorescein under formation of eosin. Literature: H. Weichsel, (i"rochim. -cta !)2#, 8.9. Chemicals: <luorescein -C.I. "%(%!. Acetic acid $3' GR, Ord. No. .!!!3# 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !-!" Cluorescein - rhodamine ( - sodium carbonate for chlorinated hydrocarbons and heterocyclic compounds" Spray solution IC ,.9I ethanolic rhodamine 6 solution. Spray solution IIC ),I a?ueous sodium carbonate solution. 0rocedureC Asing plates impregnated with fluorescein sodium spray the chromatograms after de'elopment first with 5, dry and spray liberally with 55. 5nspect in daylight and in long#wa'e A3 light. Chemicals: <luorescein sodium -C.I. "%(%!. e/tra pure, Ord. No. .!($$# Sodium car*onate anhydrous GR ISO, Ord. No. .!3($#

Rhodamine 7 -C.I. "% &!. GR, Ord. No. .!&%$$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !-*" Cluorescence indicators and luminescent substances as general ,isualisation reagents" A. Spray rea8ents: ). .U,:U#Dichlorofluorescein, spray reagent 7o. +*. .. 0luorescein, spray reagent 7o. )8:. 8. (ethylumbelliferone, spray reagent 7o. )+*. ;. (orin, spray reagent 7o. )*9. 9. 2hodamine 6, spray reagents 7o. .<,, .<). 7. Additi6es to adsor*ents: <. 0luorescein sodium ,.,;I in water added to the adsorbent suspension, Brd. 7o. ).,8*... :. 0luorescence indicator 0.9; for thin layer chromatography 4)#.I added to the adsorbent> for detection in short#wa'e A3 light 4ma& Q .9; nm>, Brd. 7o. ).,*)+.. The following adsorbents contain an additional fluorescence indicator for detection in long# and short#wa'e A3 light 4ma& .9; nm and ma& 8<< nm>C Silica gel <, H0.9;V8<<, Brd. 7o. ).,::;) Silica gel <, @0.9;V8<<, Brd. 7o. ).,::;+ -luminium o&ide <, @0.9;V8<< 4Type E>, Brd. 7o. ).,)),; !-$" Colin Ciocalteau reagent for phenols" Stoc? solutionC Dissol'e ), g sodium tungstate and ..9 g sodium molybdate in :, ml water, add 9 ml +9I phosphoric acid and ), ml 8:I hydrochloric acid and reflu& the mi&ture for ), hours. -dd subse?uently )9 g lithium sulfate, 9 ml water and ) drop bromine, heat again )9 min and ma"e up to ),, ml with water after cooling. The solution shall not show green colouring. Spray solution IC .,I a?ueous sodium carbonate solution. Spray solution IIC Dilute freshly before use ) part of the stoc" solution with 8 parts water. 0rocedureC Spray with 5, dry for a short while and spray with 55. Literature: 2.W. Keith, D. le Turneau, D. (ahlum, /. %hromatog. !, 98; 4)*9+>. Chemicals: Sodium moly*date dihydrate GR, Ord. No. .!3%# Sodium tun8state dihydrate GR, Ord. No. .!33&(

Lithium sulfate monohydrate GR ACS, Ord. No. .!%3$" Sodium car*onate anhydrous GR ISO, Ord. No. .!3($# 7romine GR ISO, Ord. No. .! $"+ ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & !--" Cormaldehyde - hydrochloric acid for indoles and indole deri,ati,es %Procha+7a reagent&" Spray solutionC 0reshly prepared mi&ture of ), ml formaldehyde solution 489I>, ), ml hydrochloric acid 4).).9> and ., ml ethanol. After-treatmentC Heat 9 min at ),,J%. The yellow#orange#greenish fluorescence colours become stronger by blowing 'apours of a?ua regia o'er the layer. Literature: H. @rochaD"a, %hem. $isty -3, )<;8 4)*98>. E. Stahl, H. Kaldewey, Hoppe#Seylers H. physiol. %hem. $*$, )+. 4)*<)>. Chemicals: <ormaldehyde solution min. (&' GR, Ord. No. .!"!!( 9ydrochloric acid #%' GR, Ord. No. .!!( 3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !-#" Cormaldehyde - phosphoric acid for steroid al7aloids' steroid sapogenins and phenothia+ine deri,ati,es" Spray solutionC Dissol'e ,.,8 g paraformaldehyde in ),, ml +9I phosphoric acid with stirring at room temperature. The reagent is stable for se'eral wee"s. Literature: K. Schreiber, B. -urich, G. Bss"e, /. %hromatog. !*, <8 4)*<8>. E.G.%. %lar"e, 7ature !1!, ))9. 4)*9+>. Chemicals: 0araformaldehyde e/tra pure 5AC, 70C, 1S0, Ord. No. .!"!!% ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( !-2" Cormaldehyde - sulfuric acid for aromatic compounds" Spray solutionC (i&ture of ,.. ml 8:I formaldehyde solution and ), ml *:I sulfuric acid. 0rocedureC Spray the chromatogram directly after ta"ing out of the de'eloping chamber. 3ariously coloured spots. Literature: 7. KucharcDy", /. 0ohl, /. 3ymetal, /. %hromatog. !!, 99 4)*<8>.

Chemicals: <ormaldehyde solution min. (&', Ord. No. .!"!!( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( !-3" Curfural - sulfuric acid for carbamate esters" Spray solution IC )I solution of furfural in acetone. Spray solution IIC ),I solution of sulfuric acid in acetone. 0rocedureC Spray with 5 and subse?uently with 55. Literature: -. Heyndric"&, (. Schau'liege, -. 6lommel, /. pharm. 6elg. */, )): 4)*<9>. 5. Sunshine, -m. /. %lin. @athol. -/, 9:< 4)*<8>. Chemicals: <urfural GR, Ord. No. .!"! ( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Acetone GR ACS, ISO, Ord. No. .!!! " !-1" =lucose - aniline for acids %Sch0eppe reagent&" Spray solutionC Dissol'e . g glucose in ., ml water and also . ml aniline in ., ml ethanol. (i& both solutions and ma"e up to ),, ml with )#butanol. 0rocedureC -fter spraying heat the chromatogram 9#), min at ).9J%. Dar" brown spots on white bac"ground. Literature: H. Schweppe, Dissert. (uenster )*9;. Chemicals: 5--D.-Glucose monohydrate, Ord. No. .!+("# Aniline GR, Ord. No. .! #3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -7utanol GR ACS, ISO, Ord. No. .! $$! !-)" =lucose - phosphoric acid for aromatic amines" Spray solutionC Dissol'e . g glucose in ), ml +9I phosphoric acid and ;, ml water. -dd 8, ml ethanol and 8, ml )#butanol. After-treatmentC Heat for about ), min at ;9J%. Literature: 0. (icheel, H. Schweppe, (icrochim. -cta !)#-, 98. Chemicals: 5--D.-Glucose monohydrate, Ord. No. .!+("# ortho-0hosphoric acid min. +%' - .&. GR, Ord. No. .!!%&(

)thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -7utanol GR ACS, ISO, Ord. No. .! $$! !#/" =lyoxalbis-%*-hydroxyanil& for cations" Spray solutionC Dissol'e ) g glyo&albis#4.#hydro&yanil> and 8 g potassium hydro&ide in ),, ml methanol. 0rocedureC Spray the dried chromatogram and dry again with a stream of air at 9,J%. Literature: H.G. (oeller, 7. Heller, /. %hromatog. !-, 9<, 4)*<;>. Chemicals: Glyo/al*is-#-hydro/yanil. GR, Ord. No. .!" $ 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !#!" 8ydra+ine sulfate for piperonal' ,anillin and ethyl ,anillin" Spray solutionC (i& *, ml of a saturated a?ueous solution of hydraDine sulfate with ), ml hydrochloric acid 4c Q; molR$>. NoteC 5nspect the moist chromatogram in long#wa'e A3 light before and after e&posure to ammonia 'apour. Literature: K.G. 6ergner, H. Sperlich, Dtsch. $ebensm.#2undschau -3, )8; 4)*9)>. Chemicals: 9ydra,inium sulfate GR ACS, Ord. No. .!"3!( 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Ammonia solution #%' GR, Ord. No. .!%"(# !#*" 8ydrochloric acid for glycals" Spray solutionC (i& ) part 8<I hydrochloric acid with ; parts ethanol. 0rocedureC Glycals appear as pin" spots on heating to *,J%. NoteC To be used also as general spray reagent for T$%. Literature: /.T. Edward, D.(. Waldron, /. %hem. Soc. !)#*, 8<8). Chemicals: 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !#$" 8ydrogen peroxide for aromatic acids"

Spray solutionC ,.8I a?ueous hydrogen pero&ide solution. After-treatmentC 5rradiate the chromatogram with long#wa'e A3 light until ma&imal blue fluorescence. Literature: D.W. Grant, /. %hromatog. !/, 9)) 4)*<8>. Chemicals: 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ !#-" --8ydroxyben+aldehyde - sulfuric acid for sapogenins and corticosteroids %<omaro0s7y reagent&" Solution aC 9,I sulfuric acid. Solution *C .I methanolic solution of ;#hydro&ybenDaldehyde. Spray solutionC (i& freshly before use 9 ml a with 9, ml b. After-treatmentC Heat 8#; min at ),9J% or ), min at <,J%. Fellow to pin" spots. Literature: @./. Ste'ens, /. %hromatog. !-, .<* 4)*<;>. Chemicals: "-9ydro/y*en,aldehyde for synthesis, Ord. No. +.!"%(3 Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !##" 8ydroxylamine - iron%III& chloride for lactones' esters' amides and anhydrides of carboxylic acids" Solution aC Dissol'e ., g hydro&ylammonium chloride in 9, ml water, ma"e up to .,, ml with ethanol. Store the solution in the refrigerator. Solution *C Dissol'e 9, g potassium hydro&ide in as little water as possible and ma"e up to 9,, ml with ethanol. Spray solution IC (i& e?ual parts of a and b and filter off the precipitated potassium chloride. @lace the solution in the refrigerator 4stable for about . wee"s>. Spray solution IIC Dissol'e ), g finely powdered iron4555> chloride in ., ml 8<I hydrochloric acid. Sha"e with .,, ml diethyl ether until a homogenous mi&ture is obtained. The solution 55 is stable for some time only well sealed. 0rocedureC Spray with 5, dry at room temperature and spray with 55. Literature: 3.@. Whitta"er, S. WiEesundera, 6iochem. /. #!, 8;+ 4)*9.>. Chemicals: 9ydro/ylammonium chloride GR ACS, ISO, Ord. No. .!"3 3

0otassium hydro/ide pellets GR, Ord. No. .!%!(( Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 5iethyl ether GR ACS, Ord. No. .!!$# !#2" 1-8ydroxy;uinoline for barium' strontium and calcium ions" Spray solutionC Dissol'e ,.9 g +#hydro&y?uinoline in ),, ml <,I ethanol. :reatmentC 2espray with .9 I ammonia solution or place the chromatogram into a chamber with ammonia 'apours. 5nspect in long#wa'e A3 light. Literature: W.-. 2ee'es, T.6. %rumler, -nal. %hem. *$, )9:< 4)*9.>. T.3. -rden et al., 7ature !2*, <*) 4)*;+>. Chemicals: +-9ydro/yAuinoline GR ACS, Ord. No. .!&!$+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# !#3" 1-8ydroxy;uinoline - hypobromite for arginine and other guanidine deri,ates %Sa7aguchi reagent&" Spray solution IC ,.)I solution of +#hydro&y?uinoline in acetone. Spray solution IIC (i&ture of ,.. ml bromine and ),, ml sodium hydro&ide solution 4c Q ,.9 molR$>. 0rocedureC Spray with 5 and after drying with 55. The spots show orange to red colour. Literature: /.6. /epson, /. Smith, 7ature !3*, )),, 4)*98>. /. KalouZe", (. KutKce", /. 6Nle", %es"oslo'. farm. -, )++ 4)*99>. Chemicals: +-9ydro/yAuinoline GR ACS, Ord. No. .!&!$+ 7romine GR ISO, Ord. No. .! $"+ Sodium hydro/ide solution !.% mol=l :itrisol>, Ord. No. .!$$%& Acetone GR ACS, ISO, Ord. No. .!!! " !#1" 1-8ydroxy;uinoline - 7oBic acid for aluminium' magnesium' calcium' strontium and barium ions" Spray solution IC Solution of ..9 g +#hydro&y?uinoline and ,.9 g "oEic acid in 9,, ml *,I ethanol.

Spray solution IIC .9I ammonia solution. The spots fluoresce in long#wa'e A3 light. Literature: 0.H. @ollard, /.0.W. (cBmie, 5.5.(. Elbeih, /. %hem. Soc. !)#!, ;<<. Chemicals: +-9ydro/yAuinoline GR ACS, Ord. No. .!&!$+ EoFic acid G%-hydro/y-#-hydro/ymethylpyrone--".H )thanol a*s. GR, Ord. No. .!!$&# Ammonia solution #%' GR, Ord. No. .!%"(# !#)" Indanedione for carotenoid aldehydes" Spray solutionC Dissol'e ,.9 g .#diphenylacetyl#),8#indanedione#)#hydraDone in ., ml water, filter after short warming and add ,.8 ml 8<I hydrochloric acid. After-treatmentC Dry with cold air. Literature: H. Thommen, B. Wiss, H. Ernahrungswiss. !)2$, Suppl. 8, )+. Chemicals: #-5iphenylacetyl- ,(-indanedione- -hydra,one 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & !2/" Iodine as general detection reagent" @lace the chromatogram into a chamber in which some crystals of iodine ha'e been placed. 5odine 'apour is more ?uic"ly generated by gently warming the chamber. (any organic compounds show brown spots. 4odificationC @lace the chromatogram 9 min into a strong iodine atmosphere or spray with a 9I solution of iodine in chloroform. E&cess iodine e'aporates on standing in the air. -fter spraying with )I a?ueous starch solution the spots turn blue. The bac"ground also turns blue if there is too much iodine still on the layer 4test on a corner or part of the co'ered layer>. Literature: G.%. 6arret, 7ature !)-, )):) 4)*<.>. -. 6ettschart, H. 0luec", @harm. -cta Hel'. $!, .<, 4)*9<>. G. 6rante, 7ature !2$, <9) 4)*;*>. 2. (unier, (. (acheboeuf, 6ull. Soc. chim. biol. $!, ));; 4)*;*>. 2. (unier, 6ull. Soc. chim. 0rance !), +9. 4)*9.>. Chemicals: Iodine resu*limed GR ACS, ISO, Ord. No. .!"&3 Chloroform GR ISO, Ord. No. .!#""% Starch solu*le GR ISO, Ord. No. .! #%#

!2!" Iodine a+ide for sulfur-containing amino acids' sulfides and penicillins" Iodine a,ide solutionC 0reshly prepared solution of 8 g sodium aDide in ),, ml iodine solution 4c Q ,.,9 mol 5.R$>. Dry iodine aDide is e&plosi'eW Iodine a,ide-starch rea8entC Spray solution IC 0reshly prepared solution of ) g sodium aDide in ),, ml iodine solution 4c Q ,.,,.9 molR$>. Spray solution IIC )I a?ueous starch solution. 0rocedureC Spray with 5 and subse?uently with 55. Literature: E. %hargaff, %. $e'ine, %. Green, /. 6iol. %hem. !3#, <: 4)*;+>. W. -we, 5. 2einec"e, /. Thum, 7aturwissenschaften -!, 9.+ 4)*9;>. Chemicals: Sodium a,ide e/tra pure, Ord. No. .!33++ Iodine solution !.!% mol I#=l :itrisol>, Ord. No. .!$$ ! Starch solu*le GR ISO, Ord. No. .! #%# !2*" Iodine - potassium iodide acidic for al7aloids" Spray solutionC Dissol'e ) g iodine and ), g potassium iodide in 9, ml water and add . ml glacial acetic acid. (a"e up this solution to ),, ml with water. Literature: 0. Santa'y, not published. Chemicals: Iodine resu*limed GR ACS, ISO, Ord. No. .!"&3 0otassium iodide GR ISO, Ord. No. .!%!"( Acetic acid $3' GR, Ord. No. .!!!3# !2$" Iodine - potassium iodide for organic compounds" Spray solutionC Dissol'e ,.. g iodine and ,.; g potassium iodide in ),, ml water. Literature: -. Haffaroni, 2.6. 6urton, H. Kentmann, Science !!!, < 4)*9,>. -. 6ettschart, H. 0luec", @harm. -cta Hel'. $!, .<, 4)*9<>. /. 6uechi, H. Schumacher, @harm. -cta Hel'. $*, )*; 4)*9:>. Chemicals: Iodine resu*limed GR ACS, ISO, Ord. No. .!"&3 0otassium iodide GR ISO, Ord. No. .!%!"(

!2-" Iodine - sulfanilic acid - 6-%!-naphthyl&ethylenediamine for hydroxylamines %Csa7y reagent&" Solution aC ).8I solution of iodine in acetic acid. Solution *C )I sulfanilic acid solution in 8,I acetic acid. Spray solution IC @repare freshly before use a mi&ture of e?ual parts of a and b. Spray solution IIC ,.)I a?ueous solution of 7#4)#naphthyl>ethylenediammonium dichloride. 0rocedureC Spray with 5 and subse?uently with 55. Literature: /.(. 6remmer, -nalyst 3), )8+ 4)*9;>. Chemicals: Iodine resu*limed GR ACS, ISO, Ord. No. .!"&3 N-- -Naphthyl.ethylenediamine dihydrochloride GR, Ord. No. .!3#(& Sulfanilic acid GR ACS, Ord. No. .!!3+3 Acetic acid $3' GR, Ord. No. .!!!3# !2#" Iodine - sulfuric acid for organic compounds containing nitrogen' polyethylene glycols and polyethylene glycol deri,ati,es" Spray solutionC (i& e?ual parts of iodine solution 4c Q ,.9 mol 5.Rl> and ),I sulfuric acid. Literature: H. 0elt"amp, 0. Koch, /. %hromatog. !#, 8); 4)*<;>. Chemicals: Iodine solution !.!% mol I#=l :itrisol>, Ord. No. .!$$ ! Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( !22" Iron%III& chloride for phenols and hydroxamic acids" Spray solutionC )#9I solution of iron4555> chloride in hydrochloric acid 4c Q ,.9 molR$.> NoteC Hydro&amic acids turn red, phenols blue or greenish. Literature: K. 0in", 2.(. 0in", @roc. Soc. E&pl. 6io. (ed. 3/, <9; 4)*;*>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 9ydrochloric acid !.% mol=l :itrisol> Ord. No. .!$$& !23" Iron%III& chloride - iodine for xanthine deri,ati,es"

Spray solutionC Dissol'e 9 g iron4555> chloride and . g iodine in a mi&ture of 9, ml acetone and 9, ml .,I a?ueous tartaric acid solution. Literature: /. Harna", S. @feiffer, @harmaDie !), .)< 4)*<;>. Chemicals: lron-III. chloride GR, Ord. No. .!($"( L-D.:artaric acid GR ACS, ISO, Ord. No. .!!+!" Iodine resu*limed GR ACS, ISO, Ord. No. .!"&3 Acetone GR ACS, ISO, Ord. No. .!!! " !21" Iron%III& chloride - perchloric acid for indoles %Sal7o0s7y reaction&" Spray solutionC (i& ) ml a?ueous iron4555> chloride solution 4c Q ,.9 molR$> with 9, ml 89I perchloric acid. After-treatmentC Heat 9 min at <,J%. 6low 'apours of a?ua regia o'er the layer for intensi'ation of the colours. Literature: S.-. Gordon, 2.@. Weber, @lant. @hysiol. *2, )*. 4)*9)>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 0erchloric acid 3!' GR ACS, ISO, Ord. No. .!!% + 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !2)" Iron%III& chloride - perchloric acid for phenothia+ines" Spray solutionC (i& 9 ml 9I a?ueous iron4555> chloride solution with ;9 ml .,I perchloric acid and 9, ml 9,I nitric acid. %olour reaction. Literature: -. 7oirfalise, (.H. GrosEean, /. %hromatog. !2, .8< 4)*<;>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 0erchloric acid 3!' GR ACS, ISO, Ord. No. .!!% + Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !3/" Iron%III& chloride - potassium hexacyanoferrate%III& - arsenite for thyroid hormones and other iodine containing compounds" Solution aC Dissol'e ..: g iron4555> chloride in ),, ml hydrochloric acid 4c Q ,.. molR$>. Solution *C 8.9I a?ueous potassium he&acyanoferrate4555> solution.

Solution cC Dissol'e 9 g sodium metaarsenite in 8, ml sodium hydro&ide solution 4c Q ) molR$> at ,J% and mi& with <9 ml hydrochloric acid 4c Q . molR$> with stirring. Spray solutionC (i& 9 parts a, 9 parts b and ) part c. :reatmentC Dry the chromatogram with precaution at 9,J% and spray, co'er with a second glass plate and store in dar"ness for )9 min. 5odine containing compounds show light blue spots on yellowish bac"ground. Literature: E. Happi, /. %hromatog. $!, .;) 4)*<:>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Sodium metaarsenite Sodium hydro/ide solution mol=l, Ord. No. .!$$%3 9ydrochloric acid #%' GR, Ord. No. .!!( 3 !3!" Iron%III& chloride - sulfosalicylic acid for thiophosphate esters" Spray solution IC ,.)I solution of iron4555> chloride in +,I ethanol. Spray solution IIC )I solution of sulfosalicylic acid in +,I ethanol. 0rocedureC @lace the chromatogram ), min into a bromine atmosphere and spray subse?uently with 5. Dry )9 min at room temperature and spray with 55. White spots on 'iolet bac"ground. Literature: (. SalamL, /. %hromatog. !2, ;:< 4)*<;>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( %-Sulfosalicylic acid dihydrate GR ACS, Ord. No. .!!3$ 7romine GR ISO, Ord. No. .! $"+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !3*" Iron%III& chloride - sulfuric acid for bile acids" Spray solutionC Dissol'e . g iron4555> chloride in +8 ml anhydrous )#butanol and mi& with )9 ml *:I sulfuric acid. After-treatmentC -fter drying for )9 min at room temperature heat .9#8, min with conEugated bile acids, ;9#9, min with free bile acids. Literature: W.$. -nthony, W.T. 6eher, /. %hromatog. !$, 9<: 4)*<;>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"(

-7utanol GR ACS, ISO, Ord. No. .! $$! Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( !3$" Iron%III& chloride - sulfuric acid for indole deri,ati,es %Sal7o0s7y reaction&" Spray solutionC (i& 8 ml a?ueous iron4555> chloride solution 4c Q ).9 molR$> with ),, ml water and add <, ml *:I sulfuric acid. After-treatmentC Heat 9 min at <,J%. 6low 'apours of a?ua regia o'er the layer for intensi'ation of the colours. Literature: @.E. @ilet, 2e'. gLn. bot. 2-, ) 4)*9:>. Chemicals: Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Nitric acid 3%' GR ISO, Ord. No. .!!"%3 !3-" Iron%II& thiocyanate for peroxides" Solution aC ;I a?ueous iron455> sulfate solution. Solution *C ).8I solution of ammonium thiocyanate in acetone. Spray solutionC (i& freshly before use ), ml a and )9 ml b. NoteC 0ast appearence of brown#red spots 4iron4555> thiocyanate> shows the presence of pero&ide compounds. Literature: E. Stahl, %hemi"er#Htg. 1*, 8.8 4)*9:>. E. Knappe, D. @eteri, H. anal. %hem. !)/, 8+< 4)*<.>. Chemicals: Iron-II.sulfate heptahydrate GR ACS, ISO, Ord. No. .!($3% Ammonium thiocyanate GR ACS, ISO, Ord. No. .! # ( Acetone GR ACS, ISO, Ord. No. .!!! " !3#" Isatin - sulfuric acid for thiophene deri,ati,es" Spray solutionC Dissol'e ,.; g isatin in ),, ml *:I sulfuric acid. After-treatmentC Heating to ).,J% is occasionally needed. 3ariously coloured spots. Literature: 2.0. %urtis, G.T. @hillips, /. %hromatog. ), 8<< 4)*<.>.

Chemicals: Isatin for synthesis, Ord. No. +.#!&!$ Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( !32" Isatin - +inc acetate for amino acids" Spray solutionC Dissol'e ) g isatin and ).9 g Dinc acetate in ),, ml *9I isopropanol by warming to +,J% and add ) ml glacial acetic acid after cooling. The reagent is stable stored in a refrigerator. After-treatmentC Heat 8, min at +,#+9J% or better inspect the chromatogram after standing ., hours at room temperature. Literature: /. 6arrolier, /. Heilman, E. WatD"e, Hoppe#Seylers H. physiol. %hem. $/-, .) 4)*9<>. Chemicals: Isatin for synthesis, Ord. No. +.#!&!$ Cinc acetate dihydrate GR, Ord. No. .!++!# Acetic acid $3' GR, Ord. No. .!!!3# #-0ropanol GR ACS, ISO, Ord. No. .!$3(" !33" Isonicotinic acid hydra+ide for --$-<etosteroids" Spray solutionC Dissol'e ) g isonicotinic acid hydraDide 457H> and ) ml glacial acetic acid in ),, ml ethanol. 0rocedureC Dry after spraying at room temperature. Spots show yellow fluorescence in long#wa'e A3 light. Literature: 6.@. $isboa, -cta Endocrinol. -$, ;: 4)*<8>. 6.@. $isboa, /. %hromatog. !2, )8< 4)*<;>. Chemicals: Isonicotinic acid hydra,ide Acetic acid $3' GR, Ord. No. .!!!3# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !31" <oBic acid for metal ions" Spray solutionC Dissol'e ,.) "oEic acid in ),, ml <,I ethanol. NoteC 5nspect fluorescence under A3 light. Literature: 0 H. @ollard, /.0.W. (cBmie, 5.5.(. Elbeih, 7ature !2$, .*. 4)*;*>.

Chemicals: EoFic acid G%-hydro/y-#-hydro/ymethylpyrone--".H )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !3)" Lead acetate basic for fla,onoids" Spray solutionC .9I a?ueous solution of basic lead acetate. 0luoresceing spots in long#wa'e A3 light. Literature: $. Hoerhammer, H. Wagner, K. Hein, /. %hromatog. !$, .89 4)*<;>. 2. 7eu, @. Hagedorn, 7aturwissenschaften -/, ;)) 4)*98>. Chemicals: Lead-II. hydro/ide acetate anhydrous, Ord. No. .!&" " !1/" Lead%I4& acetate for !'*-diol groups" Spray solutionC )I solution of lead453> acetate in benDene. 4@repare freshlyW> After-treatmentC Heat 9 min at )),J%. White spots on brown bac"ground. Literature: /. Wright, %hem. T 5nd. 4$ondon> !)2$, )).9. Chemicals: Lead-I2. acetate for synthesis, Ord. No. +.!&" + 7en,ene GR ACS, ISO, Ord. No. .! &+( !1!" Lead%I4& acetate - rosaniline for !'*-diol groups" Spray solution IC Dissol'e 8 g lead 455, 53> o&ide in ),, ml acetic acid with occasional stirring until completely dissol'ed. Spray solution IIC Dissol'e ,.,9 g rosaniline base in a mi&ture of ), parts glacial acetic acid and *, parts acetone. ,.)I methanolic fuchsine solution may be used e?ually. 0rocedureC Spray with 5 and after ;#9 min with 55. Literature: K. Sampson, 0. Schild, 2./. Wic"er, %hem. T 5nd. 4$ondon> !)2!, +.. K.G. 6ergner, H. Sperlich, H. $ebensm.#Antersuch. u. 0orsch. )3, .98 4)*98>. Chemicals: Lead-II,I2. o/ide e/tra pure, Ord. No. .!3!+! NeI <uchsin -C.I. "#%#!., Ord. No. .!"!" Acetic acid $3' GR, Ord. No. .!!!3# Acetone GR ACS, ISO, Ord. No. .!!! " 4ethanol GR ACS, ISO, Ord. No. .!3!!$

!1*" Leu7omethylene blue for ubi-' plasto- and tocopheryl;uinones" Spray solutionC -dd a suspension of ,..9 g Dinc powder in ) ml glacial acetic acid to 9 ml ,.,.I solution of methylene blue in acetone. Literature: T.W. Goodwin, $ab. @ractice !)2-, .*9. Chemicals: Cinc poIder GR, Ord. No. .!+&+$ 4ethylene *lue 7 -C.I. %#! %. Acetic acid $3' GR, Ord. No. .!!!3# Acetone GR ACS, ISO, Ord. No. .!!! " !1$" ?agnesium acetate for anthra;uinone glycosides and their aglucones" Spray solutionC ,.9I methanolic magnesium acetate solution. 0rocedureC -fter spraying dry 9 min at *,J%. Brange to 'iolet colour. Literature: S. Shibita, (. Ta"ido, B. Tana"a, /. -m. %hem. Soc. 3*, .:+* 4)*9,>. Chemicals: 4a8nesium acetate tetrahydrate GR ACS, Ord. No. .!%+ $ 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !1-" ?ercury%II& chloride - diphenylcarba+one for barbiturates" A. Solution aC .I ethanolic mercury455> chloride solution. Solution *C ,..I ethanolic diphenylcarbaDone solution. Spray solutionC (i& freshly before use e?ual parts of a and b. @in" spots on 'iolet bac"ground. Literature: E.K./. %hristensen, T. 3os, T. HuiDanga, @harm. Wee"blad !//, 9): 4)*<9>. 7. Spray solution IC ,.)I ethanolic diphenylcarbaDone solution. Spray solution IIC ,.88I mercury455> nitrate solution in nitric acid 4c Q ,.,9 molR$>. 0rocedureC Spray with 5 until the plate is faintly pin", then spray with 55. @in" spots on 'iolet bac"ground, the latter is bleached by sunlight or A3 light and the spots turn 'iolet. Literature: /. $ehmann, 3. Karamustafauglu, Scand. /. %lin. T $ab. 5n'est. !-, 99; 4)*<.>.

C. Spray solution I -4ercuryGIIHsulfate solution.C Suspend 9 g mercury455> o&ide in ),, ml water and add ., ml *:I sulfuric acid with stirring. -fter cooling fill up to .9, ml with water. Spray solution IIC ,.,)I diphenylcarbaDone solution in chloroform. 0rocedureC Spray with 5, dry and spray with 55. Literature: 5. Sunshine, E. 2ose, /. $e 6eau, %lin. %hem. ), 8). 4)*<8>. Chemicals: 4ercury-II. chloride su*limed GR ACS, Ord. No. .!"" $ 5iphenylcar*a,one Rea8. 0h )ur, Ord. No. .%$3$$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 4ercury-II. nitrate monohydrate GR ACS, Ord. No. .!""($ Nitric acid 3%' GR ISO, Ord. No. .!!"%3 4ercury-II. o/ide yelloI GR ACS, Ord. No. .!""3 Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Chloroform GR ISO, Ord. No. .!#""% !1#" ?ercury%II& chloride - potassium iodide for steroid al7aloids %?eyer reagent&" PC" Spray solution IC Dissol'e )8.99 g mercury455> chloride and ;*.+ g potassium iodide separately each in ., ml water. (i& both solutions and fill up with water to ) ). 6efore spraying add ) part ):I hydrochloric acid to ), parts of this solution. Spray solution IIC Dissol'e 9 g Dinc chloride in +, ml water and add )9 ml 8<I hydrochloric acid. Spray solution IIIC )9I ammonia solution. 0rocedureC -fter spraying with 5, the steroid al"aloids appear as faint yellow spots. 2inse the chromatogram ), min with water and, after remo'al of the water, spray with 55 and subse?uently with 555. NoteC The resulting dar" brown spots are not stable for a prolonged period. Literature: 2. Tschesche, 2. @etersen, %hem. 6er. 13, .<* 4)*98>. Chemicals: 4ercury-II. chloride su*limed GR ACS, Ord. No. .!"" $ 0otassium iodide GR ISO, Ord. No. .!%!"( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & Cinc chloride GR ACS, ISO, Ord. No. .!++ 3 Ammonia solution #%' GR, Ord. No. .!%"(# !12" ?ercury%I& nitrate for barbiturates"

Spray solutionC )I a?ueous mercury45> nitrate solution. Literature: /. 6aeumler, (itt. Gebiete $ebensm. u. Hygiene -1, )89 4)*9:>. 2. Deininger, -rDneimittel#0orsch. #, ;:. 4)*99>. Chemicals: 4ercury-I. nitrate dihydrate GR ACS, Ord. No. .!""(& !13" --?ethoxy-*-nitroaniline dia+otised for the identification of ,itamin C" Solution aC Dissol'e ,.9 g ;#metho&y#.#nitroaniline in ).9 ml glacial acetic acid. Dilute the solution to .9, ml with ),I sulfuric acid. Solution *C ,..I a?ueous sodium nitrite solution. Spray solution IC (i& e?ual parts of a and b. Spray solution IIC Sodium hydro&ide 4c Q . molR$>. 0rocedureC Spray with 5, and then with 55. 6lue spots on orange bac"ground. Literature: 7. Schmall, %.W. @ifer, E.G. Wollish, -nal. %hem. *#, );+< 4)*98>. Chemicals: "-4etho/y-#-nitroaniline for synthesis, Ord. No. +.!3##% Acetic acid $3' GR, Ord. No. .!!!3# Sodium hydro/ide solution # mol=l, Ord. No. .!$ (3 Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Sodium nitrite GR ACS, Ord. No. .!3%"$ !11" ?ethylene blue for sulfate esters of steroids" Spray solutionC Dissol'e ,.,.9 g methylene blue in ),, ml sulfuric acid 4c Q ,.,.9 molR$>. 6efore use dilute ) part of the spray solution with ) part acetone. NoteC The sulfate esters show differently coloured spots on blue bac"ground. Bn de'elopment with chloroform the formed colour comple&es migrate and lea'e white spots on blue bac"ground. Literature: B. %rLpy, B. /udas, 6. $achese, /. %hromatog. !2, 8;, 4)*<;>. Chemicals: 4ethylene *lue 7 -C.I. %#! %. Acetone GR ACS, ISO, Ord. No. .!!! " Chloroform GR ISO, Ord. No. .!#""% !1)" ?ethylumbelliferone for heterocyclic compounds containing nitrogen %fluorescence indicator&"

Spray solutionC Dissol'e ,.,. g ;#methylumbelliferone in 89 ml ethanol and fill up to ),, ml with water. After-treatmentC @lace the chromatogram into a chamber saturated with ammonia 'apours and inspect in long#wa'e A3 light. Literature: 5.(. Hais, K. (ace", Handbuch der @apierchromatographie 5, p. :9*, G. 0ischer, /ena )*9+. Chemicals: "-4ethylum*elliferone )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# !)/" ?ethyl yello0 for chlorinated insecticides" Spray solutionC Dissol'e ,.) g methyl yellow in ),, ml :9I ethanol. 0rocedureC -fter spraying dry the chromatogram at room temperature and irradiate with A3 light without filter for 9 min. 2ed spots on yellow bac"ground. Literature: $.0. KrDemins"y, W.-. $andmann, /. %hromatog. !/, 9.9 4)*<8>. Chemicals: "-5imethylaminoa,o*en,ene -C.I. !#!. indicator, Ord. No. .!(!%% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !)!" ?illon:s reagent for phenols' phenol ethers and phenol ether glycosides " Spray solutionC Dissol'e 9 g mercury in ), g fuming nitric acid and add ), ml water. After-treatmentC Heating at ),,#)),J% often produces colour changes. Literature: E. Stahl, @./. Schorn, Hoppe#Seylers H. physiol. %hem. $*#,.<8 4)*<)>. Chemicals: 4ercury GR, Ord. No. .!""!( Nitric acid fumin8 !!' - .%#. GR, Ord. No. .!!"%% !)*" ?olybdatophosphoric acid %Phosphomolybdic acid&" ),, ml ready to use spray solution for chromatography 4c Q +I in .#propanol>. Ord. No. .!!"+! !)$" ?olybdatophosphoric acid for reducing compounds' lipids' sterols and steroids"

A. Spray solutionC 9#),I ethanolic molybdatophosphoric acid. After-treatmentC Heat at ).,J% until ma&imal 'isualisation of the spots. NoteC Treatment with ammonia 'apour produces a colourless bac"ground. 7. Spray solutionC .,I solution of molybdatophosphoric acid in ethanol or ethylene glycol monomethylether 4.#metho&yethanol>. -ntio&idants show blue spots after )#. min. Literature: D. Kritsche's"y, (.%. Kir", -rch. 6iochem. 6iophys. $#, 8;< 4)*9.>. -. Seher, 0ette u. Seifen, -nstrichmittel 2!, 8;9 4)*9*>. Chemicals: 4oly*datophosphoric acid hydrate GR ACS, Ord. No. .!!%(# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( )thylene 8lycol monomethyl ether for synthesis, Ord. No. +.!!+%+ Ammonia solution #%' GR, Ord. No. .!%"(# !)-" ?olybdatophosphoric acid al7aline for estrogens" Spray solution IC +I methanolic solution of molybdatophosphoric acid. Spray solution IIC ..9I a?ueous potassium hydro&ide or 8I a?ueous sodium hydro&ide solution. 0rocedureC Spray with 5 and subse?uently with 55. NoteC 5nstead of spraying with 55 place the chromatogram into a chamber saturated with ammonia. Literature: 6. Hoffmann, /. %hromatog. $-, .<* 4)*<+>. Chemicals: 4oly*datophosphoric acid hydrate GR ACS, Ord. No. .!!%(# 0otassium hydro/yde pellets GR, Ord. No. .!%!(( Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ Ammonia solution #%' GR, Ord. No. .!%"(# 4ethanol GR ACS, ISO, Ord. No. .!3!!$ !)#" ?orin for aluminium ions' Spray solutionC )I solution of morin in glacial acetic acid. @ronounced light green fluorescence in long#wa'e A3 light. Literature: T.3. Toribara, 2.E. Sherman, -nal. %hem. *#, )*9; 4)*98>.

Chemicals: 4orin dihydrate -C.I. &%33!. GR, Ord. No. .!3!$+ Acetic acid $3' GR, Ord. No. .!!!3# !)2" !'$-6aphthalenediol - phosphoric acid for sugars" Spray solutionC (i&ture of ),, ml ,..I ethanolic ),8#naphthalenediol solution with ), ml +9I phosphoric acid. After-treatmentC Heat 9#), min at ),,#),9J%. Literature: 3. @rey, H. 6erbae", (. KausD, (i"rochim. -cta !)2!, *<+. Chemicals: ,(-Naphthalenediol ortho-0hosphoric acid. +%' GR ISO, Ord. No. .!!%&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !)3" !'$-6aphthalenediol - sulfuric acid for sugars" Solution aC ,.. I ethanolic solution of ),8#naphthalenediol. Solution *C .,I sulfuric acid. Spray solutionC @repare freshly before use a mi&ture of e?ual parts a and b. After-treatmentC Heat 9#), min at ),,#),9J%. Literature: (. $ato, E. 6runelli, G. %iuffini, /. %hromatog. $-, .< 4)*<+>. Chemicals: ,(-Naphthalenediol Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( !)1" !'$-6aphthalenediol- trichloroacetic acid for sugars and uronic acids" Solution aC ,..I ethanolic ),8#naphthalenediol solution. Solution *C .,I a?ueous trichloroacetic acid solution. Spray solutionC (i& freshly before use e?ual parts of a and b. After-treatmentC 0or "etoses heat 9#), min at ),,#),9J%, for uronic acids ),#)9 min in a moist atmoshphere 4water bath> at :,#+,J%. NoteC The presence of collidine and pyridine interferes with the colour reaction. 5nstead of ),8#naphthalenediol resorcinol, orcinol 48,9#dihydro&ytoluene>, phloroglucinol or )#naphthol may be used. Bne part trichloroacetic may be replaced by )R), part phosphoric acid.

Literature: S.(. @artridge, 6iochem. /. -*, .8+ 4)*;+>. Chemicals: ,(-Naphthalenediol :richloroacetic acid GR ACS, Ord. No. .!!+!& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -Naphthol GR, Ord. No. .!3##( (,%-5ihydro/ytoluene monohydrate for synthesis, Ord. No. +.#!$(( 0hloro8lucinol - ,(,%-trihydro/y*en,ene. GR, Ord. No. .!&!3$ Resorcinol GR, Ord. No. .!&%$( ortho-0hosphoric acid. +%' GR ISO, Ord. No. .!!%&( !))" !-6aphthol - hypobromite for arginine and other guanidine deri,ati,es %Sa7aguchi reagent&" Spray solution IC Solution of ,.)I )#naphthol in sodium hydro&ide solution 4c Q ) molR$>. Spray solution IIC (i&ture of ),, ml 9I a?ueous sodium hydro&ide and . ml. bromine. 0rocedureC Spray with 5 and then with 55. NoteC 0or the detection of streptomycine it is recommended to spray with a mi&ture of 9, ml a?ueous sodium hypochlorite solution 4)8 I acti'ated chlorine> and 9, ml ethanol instead of spraying with 55. Literature: 2. -cher, %. %rac"er, 6iochem. biophys. -cta ), :,; 4)*9.>. Chemicals: -Naphthol GR, Ord. No. .!3##( Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ 7romine GR ISO, Ord. No. .! $"+ Sodium hypochlorite solution, Ord. No. .!%3 " )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *//" !-6aphthol - sulfuric acid for sugars" Spray solutionC (i& ),.9 ml )9I ethanolic solution of )#naphthol, <.9 ml *:I sulfuric acid, ;,.9 ml ethanol and ; ml water. After-treatmentC Heat 8#< min at ),,J%. Literature: H. /acin, -.2. (ish"in, /. %hromatog. !1, ):, 4)*<9>. Chemicals: -Naphthol GR, Ord. No. .!3##(

Sulfuric acid. min. $%-$&' - .+". GR, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( */!" !'*-6aphtho;uinone-sulfonic acid sodium salt for amino acids %Colin reagent&" Spray solutionC @repare freshly a solution of ,.. g ),.#naphtho?uinone#;#sulfonic acid sodium salt in ),, ml 9I a?ueous sodium carbonate solution. 0rocedureC Spray and dry the chromatogram at room temperature. 7o further Treatment.-mino acids show 'arious colours. Literature: D. (ueting, 7aturwissenschaften $), 8,8 4)*9.>. K.3. Giri et al., 7aturwissenschaften $), 9;+ 4)*9.>. Chemicals: ,#-NaphthoAuinone-"-sulfonic acid sodium salt GR, Ord. No. .!3%( Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ */*" !'*-6aphtho;uinone-sulfonic acid sodium salt for aromatic amines" Spray solutionC Dissol'e ,.9 g ),.#naphtho?uinone#;#sulfonic acid sodium salt in *9 ml water and add 9 ml glacial acetic acid. 0ilter off from insoluble parts. NoteC 5nspect the colour of the spots after 8, min. Literature: 2.6. Smyth, G.G. (cKeown, /. %hromatog. !2, ;9; 4)*<;>. Chemicals: ,#-NaphthoAuinone-"-sulfonic acid sodium salt GR, Ord. No. .!3%( Acetic acid $3' GR, Ord. No. .!!!3# */$" !'*-6aphtho;uinone-sulfonic acid - perchloric acid for sterols" Spray solutionC Dissol'e ,.) g ),.#naphtho?uinone#;#sulfonic acid in a mi&ture of 9, ml ethanol, .9 ml <,I perchloric acid, .9 ml 8:I formaldehyde solution, and ...9 ml water. 0rocedureC Heat at :,#+,J% and inspect the de'elopment of the spots. 0irst pin", after prolonged heating blue spots. Literature: E. 2ichter, /. %hromatog. !1, )<; 4)*<9>. %.W.(. -dams, 7ature !)*, 88) 4)*<)>. Chemicals: ,#-NaphthoAuinone-"-sulfonic acid sodium salt GR, Ord. No. .!3%( 0erchloric acid 3!' GR ACS, ISO, Ord. No. .!!% +

<ormaldehyde solution min. (&' GR, Ord. No. .!"!!( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( */-" l-6aphthylamine for $'#-dinitroben+oic acid esters and dinitroben+amides" Spray solution IC ,.9I ethanolic )#naphthylamine solution. Spray solution IIC ),I methanolic potassium hydro&ide solution. 0rocedureC Spray with 5 and then with 55. Spots show red#brown colour. Literature: 2.G. 2ice, G./. Keller, /.G. Kirchner, -nal. %hem. *$, )*; 4)*9)>. Chemicals: -Naphthylamine GR, Ord. No. .!3#"% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 0otassium hydro/ide pellets GR, Ord. No. .!%!(( */#" 6essler:s reagent for al7aloids" Spray solutionC 7esslerUs reagent 4s. spray reagent 7o. .+;>. NoteC -pomorphine, hydrastinine and physostigmine show colour reaction. Literature: B.E. SchultD, D. Strauss, -rDneimittel#0orsch. #, 8;. 4)*99>. Chemicals: Nessler;s rea8ent */2" 6inhydrin ),, ml ready to use spray solution for chromatography 4c Q ca. ,..I in .# propanol>. Ord. No. .!3&!% */3" 6inhydrin for amino acids' amines and amino-sugars" A. Spray solutionC Dissol'e ,.8 g ninhydrin in ),, ml )#butanol and add 8 ml glacial acetic acid. 7. Spray solutionC ,..I ethanolic ninhydrin solution. After-treatmentC Heat at )),J% until ma&imal 'isualiDation of the spots. 0or pantothenic acid heat at )<,J%. Literature: 2.-. 0amy, -. 7iederwieser, G. @ata"i, (. 6renner, Hel'. %him. -cta --, .,..

4)*<)>. -.2. @atton, @. %hism, -nal. %hem. *$, )<+8 4)*9)>. Chemicals: Ninhydrin GR, Ord. No. .!3&3# Acetic acid $3' GR, Ord. No. .!!!3# -7utanol GR ACS, ISO, Ord. No. .! $$! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Stabilisation of ninhydrin spots" Spray solutionC (i& ) ml saturated a?ueous copper455> nitrate solution with ,.. ml ),I nitric acid and ),, ml ethanol. 0rocedureC Spray the ninhydrin spots with the spray solution and place the chromatogram into a chamber with ammonia. The red copper comple& is stable as long as no free hydrogen ions or strong comple& forming compounds are present. Literature: E. Kawerau, T. Wieland, 7ature !21, :: 4)*9)>. Chemicals: Copper-II. nitrate trihydrate e/tra pure, Ord. No. .!#&%# Nitric acid 3%' GR ISO, Ord. No. .!!"%3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# */1" 6inhydrin - cadmium acetate for amino acids and amines" Spray solutionC 0ill up to 9,, ml with ethanol a solution of ) g ninhydrin, ..9Jg cadmium acetate and ), ml glacial acetic acid. After-treatmentC Heat ., min at ).,J%. This method is more suitable for detecting heterocyclic amines than the procedure using reagent 7o. .,:. lternati,e: 5ip solutionC Dissol'e ,.) g cadmium acetate in ), ml water, add 9 ml glacial acetic acid and ),, ml acetone and dissol'e ) g ninhydrin. This order of the reagents for the preparation of the dip solution must be obser'ed. The solution is stable in the refrigerator. 0rocedureC -fter dipping place the chromatogram for colour de'elopment 8, min into a chamber containing concentrated sulfuric acid. Literature: /. 6arrolier, /. Heilmann, E. WatD"e, Hoppe#Seylers H. physiol. %hem. $/), .)* 4)*9:>.

Chemicals: Cadmium acetate dihydrate GR, Ord. No. .!#!!( Ninhydrin GR, Ord. No. .!3&3# Acetic acid $3' GR, Ord. No. .!!!3# Acetone GR ACS, ISO, Ord. No. .!!! " )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( */)" 6inhydrin - copper%II& nitrate for amino acids %polychromatic detection&" Solution aC Dissol'e ,.) g ninhydrin in 9, ml ethanol and add ), ml glacial acetic acid and . ml .,;,<#trimethylpyridine. Solution *C )I ethanolic copper455> nitrate solution. Spray solutionC 6efore use mi& solution a and b in the proportion 9,C8. After-treatmentC Heat the chromatogram until the colour de'elopment is Eust beginning. 5n transmitted light the gradual intensification of colours on the warm plate can be obser'ed. Some amino acids show first small points of colours only, they should be mar"ed with a sharp pencil. 5n this way one can often detect indi'idual spots which later merge into each other. Some amino acids show characteristic colours. They differ amongst themsel'es also in the speed with which coloured products are formed. Literature: (. 6renner, -. 7iederwieser, E&perientia !2, 8:+ 4)*<,>. Chemicals: Ninhydrin GR, Ord. No. .!3&3# Copper-II. nitrate trihydrate e/tra pure, Ord. No. .!#&%# Acetic acid $3' GR, Ord. No. .!!!3# #,",3-:rimethylpyridine GR, Ord. No. .!#3(% )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *!/" 6inhydrin - tin%II& chloride for amines" Stoc? solutionC Dissol'e by heating . g ninhydrin in ;, ml water. -dd a solution of ,.,+ g tin455> chloride in 9, ml water and allow to stand. -fter filtration of the precipitate store in the refrigerator. Spray solutionC -dd 9, ml water and ;9, ml .#propanol to .9 ml of the stoc" solution. Literature: 2./. 6loc", -nal. %hem. **, )8.: 4)*9,>. Chemicals: Ninhydrin GR, Ord. No. .!3&3#

:in-II. chloride dihydrate GR ACS, Ord. No. .!&+ % #-0ropanol GR ACS, ISO, Ord. No. .!$3(" *!!" 6itric acid for al7aloids and amines" Spray solutionC -dd 9, drops <9I nitric acid to ),, ml ethanol. NoteC 5nspect in A3 light. The spray solution may be used in this or higher concentration also in T$% for the identification of other organic compounds. 0re?uently fluorescent spots appear only after prolonged heating at ).,J%. Literature: H. Schmid, /. Kebrle, @. Karrer, Hel'. %him. -cta $#, )+<; 4)*9.>. Chemicals: )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Nitric acid 3%' GR ISO, Ord. No. .!!%"3 *!*" --6itroaniline dia+otised %acidic& for plasticisers" Spray solution IC @otassium hydro&ide solution in ethanol 4c Q ,.9 molR$>. Spray solution IIC Dissol'e ,.+ g ;#nitroaniline in .9, ml water, add ., ml .9I hydrochloric acid and dropwise 9I a?ueous sodium nitrite solution until the solution is colourless. 0rocedureC Spray with 5, dry )9 min at <,J% and spray with 55. Fellow to orange spots. Literature: /.W. %opius#@eereboom, /. %hromatog. -, 8.8 4)*<,>. D. 6raun, %himia !), :: 4)*<9>. Chemicals: "-Nitroaniline, Ord. No. .!3&3! Sodium nitrite GR ACS, Ord. No. .!3%"$ 9ydrochloric acid #%' GR, Ord. No. .!!( 3 0otassium hydro/ide pellets GR, Ord. No. .!%!(( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *!$" --6itroaniline dia+otised for phenols' phenol carboxylic acids' coupling amines and heterocyclic compounds" Spray solutionC (i& ), ml ,.)I a?ueous ;#nitroaniline solution with ), ml ,..I a?ueous sodium nitrite solution and ., ml ),I a?ueous potassium carbonate solution. %oloured products are formed. Literature: -. Sturm, H.W. ScheEa, /. %hromatog. !2, )*; 4)*<;>.

Chemicals: "-Nitroaniline, Ord. No. .!3&3! Sodium nitrite GR ACS, Ord. No. .!3%"$ 0otassium car*onate GR ACS, ISO, Ord. No. .!"$#+ *!-" --6itroaniline dia+otised %buffered& for phenols" Spray solutionC (i& under cooling 9 ml ,.9I ;#nitroaniline solution in hydrochloric acid 4c Q . molR$> with ,.9 ml 9I a?ueous sodium nitrite solution and add )9 ml .,I a?ueous sodium acetate solution. Literature: H.G. 6ray, W.3. Thorpe, K. White, 6iochem. /. -2, .:) 4)*9,>. T. Swain, 6iochem. /. #$, .,, 4)*98>. %.0. 'an Sumere, G. Wolf, H. Teuchy, /. Kint, /. %hromatog. */, ;+ 4)*<9>. Chemicals: "-Nitroaniline, Ord. No. .!3&3! Sodium nitrite GR ACS, Ord. No. .!3%"$ Sodium acetate GR, Ord. No. .!3#3& 9ydrochloric acid #%' GR, Ord. No. .!!( 3 *!#" --6itrophenyldia+onium fluoborate for phenols and coupling amines" "-Nitrophenyldia,onium fluo*orateC Dissol'e ); g ;#nitroaniline in 8, ml 8<I hydrochloric acid and 8, ml water by warming. -fter cooling at 9J% add a solution of + g sodium nitrite in ., ml water and then <, ml ;,I hydrofluoboric acid 4tetrafluoroboric acid>. 0ilter off the yellow precipitate, wash with hydrofluoboric acid, ethanol and ether and dry in a 'acuum desiccator. Spray solution IC @repare freshly a )I ;#nitrophenyldiaDonium fluoborate solution in acetone. Spray solution IIC ,.)I methanolic potassium hydro&ide solution. 0rocedureC Spray with 5, then with 55. Literature: /.H. 0reeman, -nal. %hem. *-, *99 4)*9.>. H. Seeboth, H. Goersch, %hem. Techn. !#, .*; 4)*<8>. Chemicals: "-Nitroaniline, Ord. No. .!3&3! Sodium nitrite GR ACS, Ord. No. .!3%"$ :etrafluoro*oric acid, Ord. No. .!! & 0otassium hydro/ide !.% mol=l in methanol, Ord. No. .!$(% Acetone GR ACS, ISO, Ord. No. .!!! " )thanol a*solute GR ACS, ISO, Ord. No. .!!$+(

5iethyl ether GR ACS, Ord. No. .!!$# 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & *!2" *-6itroso-!-naphthol---sulfonic acid for iron ions" Spray solutionC ,.,9I solution of .#nitroso#)#naphthol#;#sulfonic acid in :,I ethanol. After-treatmentC 2espray with .9I ammonia solution or place the chromatogram into a chamber with ammonia 'apours. Green spots. Literature: G.6. Heisig, 0.H. @ollard, -nal. %him. -cta !2, .8; 4)*9:>. Chemicals: #-Nitroso- -naphthol-"-sulfonic acid Ammonia solution #%' GR, Ord. No. .!%"(# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *!3" Drcinol - iron%III& chloride - sulfuric acid for sugars" Solution aC Dissol'e ) g iron4555> chloride in ),, ml ),I sulfuric acid. Solution *C <I ethanolic orcinol 48,9#dihydro&ytoluene> solution. Spray solutionC (i& freshly before use ), ml a and ) ml b. After-treatmentC Heat ),#)9 min at ),,J%. Chemicals: (,%-5ihydro/ytoluene monohydrate for synthesis, Ord. No. +.#!$(( Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *!1" Palladium%II& chloride for thiophosphate esters and other sulfur compounds" Spray solutionC Dissol'e ,.9 g palladium455> chloride in ),, ml water containing a few drops .9I hydrochloric acid. Literature: /. 6aeumler, S. 2ippstein, Hel'. %him. -cta --, ))<. 4)*<)>. Chemicals: 0alladium -II. chloride anhydrous, Ord. No. +.!& ! 9ydrochloric acid #%' GR, Ord. No. .!!( 3 *!)" Paraformaldehyde - phosphoric acid for Solanum steroid al7aloids and steroid sapogenins"

Spray solutionC Dissol'e ,.,8 g paraformaldehyde in ),, ml +9I phosphoric acid under sha"ing. The reagent is stable for se'eral wee"s. Literature: K. Schreiber, B. -urich, G. Bss"e, /. %hromatog. !*, <8 4)*<8>. Chemicals: 0araformaldehyde e/tra pure 5AC, 70C, 1S0, Ord. No. .!"!!% ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( **/" Perchloric acid for steroids and bile acids" A. Spray solution 4for steroids>C .,I a?ueous perchloric acid solution. 7. Spray solution 4for bile acids>C <,I a?ueous perchloric acid solution. After-treatmentC Heat the chromatogram for about ), min at )9,J% until ma&imal 'isualisation of the spots. 5nspect also in long#wa'e A3 light. Literature: H. (etD, 7aturwissenschaften -1, 9<* 4)*<)>. S. Hara, (. Ta"euchi, /. %hromatog. !!, 9<9 4)*<8>. Chemicals: 0erchloric acid 3!' GR ACS, ISO, Ord. No. .!!% + **!" Perchloric acid - iron%III& chloride for indole deri,ati,es" Spray solutionC (i& ),, ml 9I a?ueous perchloric acid solution with . ml ,.,9 ( iron4555> chloride solution. NoteC 7o reaction with isatin and other o&indole deri'ati'es. Literature: T.-. 6ennet#%lar", (.S. Tambiah, 7.@. Kefford, 7ature !2), ;9. 4)*9)>. Chemicals: 0erchloric acid 3!' GR ACS, ISO, Ord. No. .!!% + Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( ***" Phenol - sulfuric acid for sugars" Spray solutionC Dissol'e 8 g phenol and 9 ml *:I sulfuric acid in *9 ml ethanol. After-treatmentC Heat ),#)9 min at )),J%. 6rown spots. Literature: S. -dachi, /. %hromatog. !3, .*9 4)*<9>. Chemicals: 0henol GR ACS, Ord. No. .!!#!3

Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( **$" m-Phenylenediamine for reducing sugars" Spray solutionC Dissol'e 8.< g m#phenylenediamine dihydrochloride in ),, ml :,I ethanol. After-treatmentC Heat briefly at ),9J%. NoteC 5ntensely fluorescent colours in A3 light. Literature: S.S. %hernic", 5.$. %hai"off, S. -braham, /. 6iol. %hem. !)$, :*8 4)*9)>. Chemicals: m-0henylenediamine dihydrochloride )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( **-" p-Phenylenediamine - phthalic acid for conBugated $-7etosteroids" Spray solutionC Dissol'e ,.* g p#phenylenediamine and ).< g phthalic acid in ),, ml )#butanol, saturated with water. After-treatmentC Heat at ),,#)),J%. Fellow to orange spots. Literature: 6.@. $isboa, -cta Endocrinol. -$, ;: 4)*<8>. 6.@. $isboa, /. %hromatog. !2, )8< 4)*<;>. Chemicals: p-0henylenediamimonium dichloride for synthesis, Ord. No. +.###$& 0hthalic acid GR, Ord. No. .!$3 -7utanol GR ACS, ISO, Ord. No. .! $$! **#" !'*-Phenylenediamine - sulfuric acid for dehydroascorbic acid" Spray solutionC Dissol'e ,.) g ),.#phenylenediamine in a mi&ture of 9, ml sulfuric acid 4c Q ,.,9 molR$> and 9, ml ethanol. Literature: S. Bgawa, /. @harm. Soc. /apan 3$, 9* 4)*98>. Chemicals: ,#-0henylenediamine, Ord. No. +.!$&# Sulfuric acid !.!% mol=l, Ord. No. .!$$+" )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( **2" !'*-Phenylenediamine - trichloroacetic acid for -7eto acids"

Spray solutionC Dissol'e ,.,9 g ),.#phenylenediamine in ),, ml ),I a?ueous trichloroacetic acid solution. 0rocedureC Heat the chromatogram at ),,J% for not more than . min. Green fluorescent spots in long#wa'e A3 light. Literature: T. Wieland, 0. 0ischer, 7aturwissenschaften $2, .)* 4)*;*>. B. Wiss, Hoppe#Seylers H. physiol. %hem. *)$, ),< 4)*98>. Chemicals: ,#-0henylenediamine, Ord. No. +.!$&# :richloroacetic acid GR ACS, Ord. No. .!!+!& **3" Phenylfluorone for germanium" Spray solutionC ,.,9I solution of phenylfluorone in a mi&ture of 8 parts ethanol and ) part 8:I hydrochloric acid. Literature: 5.(. $adenbauer, K. 6radacs, 0. Hecht, (i"rochim. -cta !)#-, 8++. Chemicals: 0henylfluorone )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & **1" Phenylhydra+ine for dehydroascorbic acid" Spray solutionC Dissol'e ,.8 g phenylhydraDine and ,.;9 g sodium acetate in ), ml water. Chemicals: 0henylhydra,ine GR, Ord. No. .!&#% Sodium acetate anhydrous GR ACS, Ord. No. .!3#3+ **)" Phosphoric acid for sterols and steroids" A. Spray solutionC (i& +9I phosphoric acid with water )C) 4'olume>. 7. Spray solutionC )9I methanolic phosphoric acid solution. 0rocedureC Spray the layer thoroughly until transparent and heat )9#8, min at ).,J%. The indi'idual sterols or steroids re?uire 'arying heating times for attainment of ma&imal colour intensity or fluorescence. NoteC -ll compounds of this class show fluorescence in long#wa'e A3 light. $arger amounts of substance yield spots which are 'isible in daylight. Literature: 2. 7eher, -. Wettstein, Hel'. %him. -cta $-, ..:+ 4)*9)>.

Chemicals: ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ *$/" Phosphoric acid - bromine for digitalis glycosides" Spray solution IC ),I a?ueous phosphoric acid solution. Spray solution IIC (i& . ml saturated a?ueous potassium bromide solution, . ml saturated a?ueous potassium bromate solution and . ml .9I hydrochloric acid. 0rocedureC Spray with 5 and heat the plate ). min at ).,J%. Digitalis glycosides of the series 6, D and E show blue fluorescence in long#wa'e A3 light. Heat the plate again at ).,J% and spray lightly with 55. Glycosides of the series - show now orange, of the series % grey#green to grey#blue fluorescence in A3 light. Literature: $. 0auconnet, (. Waldesbuehl, @harm. -cta Hel'. $1, ;.8 4)*<8>. Chemicals: ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( 0otassium *romide GR ACS, Ord. No. .!"$!% 0otassium *romate GR ACS, ISO, Ord. No. .!"$ # 9ydrochloric acid #%' GR, Ord. No. .!!( 3 *$!" Picric acid for epoxides" Spray solutionC ,.,9 ( ethanolic picric acid solution. After-treatmentC @lace the sprayed chromatogram 8, min into a chamber with etherRethanolRglacial acetic acid 4+,V.,V)> and subse?uently )#. min into a chamber with ammonia 'apours. Brange spots on yellow bac"ground. Literature: /.-. 0ioriti, 2./. Sims, /. %hromatog. $*, :<) 4)*<+>. Chemicals: 0icric acid desensiti,ed -cont. a*out (!' Iater. GR, Ord. No. .!!3#( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 5iethyl ether GR ACS, Ord. No. .!!$# Acetic acid $3' GR, Ord. No. .!!!3# Ammonia solution #%' GR, Ord. No. .!%"(# *$*" Picric acid - al7ali for creatinine' glycocyamidine %Eaffe reagent&" Spray solution IC )I ethanolic picric acid solution. Spray solution IIC 9I ethanolic potassium hydro&ide solution. 0rocedureC Spray with 5, dry and spray with 55. Brange colour.

Literature: 2. Williams, 6iochem. 5nst. Stud. 53, Ani'ersity of Te&as, @ubl., -ustinRTe&as 7o. 9),*, .,9 4)*9)>. Chemicals: 0icric acid desensiti,ed -cont. a*out (!' Iater. GR, Ord. No. .!!3#( 0otassium hydro/ide pellets GR, Ord. No. .!%!(( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *$$" Picric acid - perchloric acid for #$-hydroxysteroids" Spray solutionC Dissol'e ,.) g picric acid in a mi&ture of 8< ml glacial acetic acid and < ml :,I perchloric acid. 0rocedureC Heat 8#9 min at :,#+,J%. Fellow#red spots. Literature: W.2. Eberlein, /. %lin. Endocrinol. *#, .++ 4)*<9>. Chemicals: 0icric acid desensiti,ed -cont. a*out (!' Iater. GR, Ord. No. .!!3#( Acetic acid $3' GR, Ord. No. .!!!3# 0erchloric acid &!-&#' GR ACS, ISO, Ord. No. .!!% $ *$-" Picryl chloride for hydroxylamines' hydra+ines and pyridine deri,ati,es" Spray solutionC ,.9#).9I ethanolic picryl chloride solution. After-treatmentC @lace the chromatogram into a chamber with ammonia. Literature: W.0./. %uthbertson, D.(. 5reland, W. Wolff, 6iochem. /. ##, <<* 4)*98>. /.(. 6remner, -nalyst 3), )*+ 4)*9;>. Chemicals: 0icryl chloride -#-chloro- ,(,%-trinitro*en,ene. )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# *$#" Pinacryptol yello0 for al7yl- and arylsulfonic acids" Spray solutionC ,.,9#,.)I a?ueous pinacryptol yellow solution. Fellow to orange fluorescence in long#wa'e A3 light. Literature: /. 6orec"y, /. %hromatog. *, <). 4)*9*>. Chemicals: 0inacryptol yelloI LA7, Ord. No. .!$&#(

*$2" Potassium hexacyanoferrate%II& for iron%III&ions" Spray solutionC 0reshly prepared .I a?ueous solution of potassium he&acyanoferrate 455>. Literature: 0.H. 6urstall, G.2. Da'ies, 2.@. $instead, 2.-. Wells, /. %hem. Soc. !)#/, 9)<. Chemicals: 0otassium he/acyanoferrate-II. trihydrate GR ACS, ISO, Ord. No. .!"$+" *$3" Potassium hexacyanoferrate%II& - hydrogen peroxide for barbiturates" Spray solution IC Dissol'e ,.) g potassium he&acyanoferrate455> in ),, ml water containing ,.9 ml 8:I hydrochloric acid. -dd to ), ml of this solution 9 g ammonium chloride and ma"e up to ),, ml with water. Spray solution IIC 8,I hydrogen pero&ide solution. Spray solution IIIC ),I a?ueous potassium carbonate solution. :reatmentC Spray with 5 and dry at ),,J%. -fter cooling spray with 55 and heat 8, min at )9,J%. Spray with 555 for intensi'ation of the yellow and red spots. This reaction may be applied after detection with mercury45> nitrate. Literature: H. Weichsel, (i"rochim. -cta !)2#, 8.9. Chemicals: 0otassium he/acyanoferrate-II. trihydrate GR ACS, ISO, Ord. No. .!"$+" Ammonium chloride GR, Ord. No. .! "% 0otassium car*onate GR ACS, ISO, Ord. No. .!"$#+ 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ *$1" Potassium hexacyanoferrate%III& for adrenaline and deri,ati,es" Spray solutionC Dissol'e ,.) g potassium he&acyanoferrate4555> in ),, ml ,.9I sodium hydro&ide solution. Spots show red colour. Literature: -.H. 6ec"ett, (.-. 6ea'en, -.E. 2obinson, /. @harm. @harmacol. !*, .,8 T 4)*<,>. Chemicals: 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++

*$)" Potassium hexacyanoferrate%III& for ,itamin (! %thiochrome reaction&" Solution aC )I a?ueous potassium he&acyanoferrate4555> solution. Solution *C )9I a?ueous sodium hydro&ide solution. Spray solutionC (i& ).9 ml a with ., ml water and add ), ml b. -fter drying inspect in long#wa'e A3 light. Literature: D. Siliprandi, 7. Siliprandi, 6iochim. et biophys. -cta !-, 9. 4)*9;>. Chemicals: 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Sodium hydro/ide solution min. #&' - .(!. GR, Ord. No. .!%%$ *-/" Potassium hexacyanoferrate%III& - iron%III& chloride for reducing compounds' phenols' amines' thiosulfates' isothiocyanates" Solution aC )I a?ueous potassium he&acyanoferrate4555> solution. Solution *C .I a?ueous iron4555> chloride solution. Spray solutionC (i& freshly before use e?ual parts of a and b. After-treatmentC Spray with hydrochloric acid 4c Q . molR$> for intensi'ation of colours. Literature: G.(. 6arton, 2.S. E'ans, /.-.0. Gardner, 7ature !3/, .;* 4)*9.>. (. Gillio#Tos, S.-. @re'itera, -. 3imercati, /. %hromatog. !$, 9:) 4)*<;>. H. Wagner, $. Hoerhammer, H. 7ufer, -rDneimittel#0orsch. !#, ;98 4)*<9>. Chemicals: 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Iron-III. chloride he/ahydrate GR, Ord. No. .!($"( 9ydrochloric acid #%' GR, Ord. No. .!!( 3 *-!" Potassium hexacyanoferrate%III& - phosphate buffer for adrenaline" Spray solutionC ,.;;I solution of potassium he&acyanoferrate4555> in phosphate buffer solution, pH :,+. NoteC 7oradrenaline appears as brown red spots, adrenaline as light red and methyladrenaline as white spots on yellow#brown bac"ground. Literature: S. Senoh, 6. Wit"op, /. -m. %hem. Soc. 1!, <... 4)*9*>. Chemicals: 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( di-Sodium hydro8en phosphate solution = % mol=l, Ord. No. .!3%+&

*-*" Potassium hexacyanoferrate%III& - potassium hexacyanoferrate%II& for morphine" Spray solutionC Dissol'e 9: mg potassium he&acyanoferrate4555> and :.+ mg potassium he&acyanoferrate455> in ),, ml water. Literature: H./. Kupferberg, -. 6urghalter, E.$. Way, /. %hromatog. !2, 99+ 4)*<;>. Chemicals: 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( 0otassium he/acyanoferrate-II. trihydrate GR ACS, ISO, Ord. No. .!"$+" *-$" Potassium hydroxide methanolic for coumarins' anthra;uinone glycosides and their aglucones" Spray solutionC 9I methanolic potassium hydro&ide solution. 5nspect the chromatogram after drying in daylight and in long#wa'e A3 light. Literature: H. $edino'a, 5.(. Hais, %es"olo'. farm. ), ;,) 4)*<,>. $. Hoerhammer, H. Wagner, G. 6ittner, -rDneimittel#0orsch. !$, 98: 4)*<8>. Chemicals: 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ *--" Potassium iodide - hydrogen sulfide for hea,y metal ions" Spray solutionC .I a?ueous potassium iodide solution. 0rocedureC Dry the plate after spraying and place it into a chamber saturated with ammonia 'apours. -fter a few minutes place the plate into a second chamber with hydrogen sulfide gas. Caution9 8ydrogen sulfide is poisonous and explosi,e9 Literature: H. Seiler, (. Seiler, Hel'. %him. -cta -$, )*8* 4)*<,>. Chemicals: 0otassium iodide GR ISO, Ord. No. .!%!"( Ammonia solution #%' GR, Ord. No. .!%"(# Iron-II. sulfide fused stic?s, Ord. No. .!($%3 9ydrochloric acid #%' GR, Ord. No. .!!( 3 *-#" Potassium iodide - starch for peroxides"

Spray solution IC -dd to a mi&ture of ;, ml glacial acetic acid and ), ml ;I a?ueous potassium iodide solution a spatula#tipful of Dinc powder. Spray solution IIC 0reshly prepared )I a?ueous starch solution. 0rocedureC -fter filtering off Dinc powder, spray with 5, dry 9 min at room temperature and spray with 55 until the layer is transparent. @ero&ides show blue spots by formation of free iodine. Literature: E. Stahl, %hemi"er#Htg. 1*, 8.8 4)*9+>. Chemicals: 0otassium iodide GR ISO, Ord. No. .!%!"( Cinc poIder GR, Ord. No. .!+&+$ Starch solu*le e/tra pure, Ord. No. .! #%( Acetic acid $3' GR, Ord. No. .!!!3# *-2" Potassium iodine platinate for al7aloids" Spray solutionC -dd to 9 ml 9I he&achloroplatinic453> acid solution ;9 ml ),I a?ueous potassium iodide solution and ),, ml water. @repare freshly before use. Literature: /. Smith, %hromatographic and Electrophoretic Techni?ues, W. Heinemann, $ondon )*<*, 3ol. 5, p. 9)*. Chemicals: 0otassium iodide GR ISO, Ord. No. .!%!"( 9e/achloroplatinic-I2. acid solution a*out !' GR, Ord. No. .!&(" *-3" Potassium iodine platinate for al7aloids and other organic compounds containing nitrogen" Spray solutionC -dd to 8 ml ),I he&achloroplatinic453> acid solution *: ml water and ),, ml <I a?ueous potassium iodide solution. @repare freshly before use. Literature: 2. (unier, 6ull soc. chim. 0rance !), +9. 4)*9.>. 2. HilD, 0.0. %astano, G.-. $ightbourne, /. $ab. %lin. (ed. #-, <8. 4)*9*>. Chemicals: 9e/achloroplatinic-I2. acid solution a*out !' GR, Ord. No. .!&(" 0otassium iodide GR ISO, Ord. No. .!%!"( *-1" Potassium iodine platinate for 7etosteroids" PC"

Spray solutionC -dd to 9 ml 9I he&achloroplatinic453>acid solution in hydrochloric acid 4c Q ) molR$> ;9 ml ),I a?ueous potassium iodide solution and ),, ml water. The reagent is stable for some time when stored in the dar". After-treatmentC -fter spraying rinse out the e&cess reagent with water. Literature: 2.T. 6urton, -. Haffaroni, E.H. Keutmann, /. %lin. Endocrinol. 1, <)+ 4)*9+>. Chemicals: 0otassium iodide GR ISO, Ord. No. .!%!"( 9e/achloroplatinic-I2. acid he/ahydrate, Ord. No. +.!&("! 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! *-)" Potassium permanganate al7aline for reducing compounds and aromatic polycarboxylic acids" Spray solutionC -dd to )I a?ueous potassium permanganate solution an e?ual 'olume of 9I a?ueous sodium carbonate solution. Literature: B.6. (a&imo', $.S. @anthin"hina, /. %hromatog, */, )9, 4)*<9>. 5.(. Hais, K. (ace", @apierchromatographie 5, G. 0ischer, /ena )*9+, p. :89. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# Sodium car*onate anhydrous GR ISO, Ord. No. .!3($# *#/" Potassium permanganate al7aline for sugars and polyalcohols" Spray solutionC Dissol'e ,.9 g potassium permanganate in ),, ml sodium hydro&ide solution 4c Q ) molR$>. After-treatmentC -fter spraying heat the plate at ),,J%. Literature: G.W. Hay, 6.-. $ewis, 0. Smith, /. %hromatog. !!, ;:* 4)*<8>. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# Sodium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$%3 *#!" Potassium permanganate neutral for easily oxidisable compounds" Spray solutionC ,.,9I a?ueous potassium permanganate solution. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# *#*" Potassium permanganate - sulfuric acid %uni,ersal reagent&"

Spray solutionC Dissol'e ,.9 g potassium permanganate in )9 ml *:I sulfuric acid. Caution9 ?anganese heptoxide is explosi,eW Literature: H. Ertel, $. Horner, /. %hromatog. 3, .<+ 4)*<.>. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( *#$" !-%*-Pyridyla+o&-*-naphthol %P 6& for lead' cadmium' cobalt' copper' manganese' nic7el' +inc and uranyl ions" Spray solutionC ,..9I ethanolic solution of @-7. After-treatmentC @lace the plate into a chamber with ammonia 'apours. Literature: H. Seiler, (. Seiler, Hel'. %him. -cta --, *8* 4)*<)>. 0.W.H.(. (er"us, @harm. Wee"blad )1, *;: 4)*<8>. Chemicals: --#-0yridyla,o.-#-naphthol -0AN. metal indicator, Ord. No. .!&%( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# *#-" !-%*-Pyridyla+o&-*-naphthol %P 6& - cobalt%II& nitrate for glucuronides of steroids" Spray solution IC (i& freshly before use ) part ,.;I ethanolic @-7 solution and ; parts methylene chloride 4by 'olume>. Spray solution IIC (i& + ml ,.+I a?ueous cobalt455> nitrate solution with ; ml acetate buffer solution 4c Q ,.. molR$S pH ;.<> and fill up to ),, ml with water. 0rocedureC Spray with 5 until the layer is e'enly yellow, dry and spray with 55. Glucuronides show rapidly fading 'iolet spots, the colours of which turn greenish on drying. Literature: B. %rLpy, B. /udas, 6. $achese, /. %hromatog. !2, 8;, 4)*<;>. Chemicals: --#-0yridyla,o.-#-naphthol -0AN. metal indicator, Ord. No. .!&%( Co*alt-II. nitrate he/ahydrate GR, Ord. No. .!#%(3 Acetate *uffer solution p9 ".33, Ord. No. .!&+#& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 5ichloromethane GR ACS, ISO, Ord. No. .!3!%!

*##" Fuercetin for cations of the hydrogen sulfide group' aluminium' magnesium' uranyl and tungstate ions" Spray solutionC ,..I ethanolic ?uercetin solution. After-treatmentC Spray with .9I ammonia solution or place into a chamber with ammonia. 5n long#wa'e A3 light fluorescing spots. Literature: -. Weiss, S. 0allab, Hel'. %him. -cta $3, ).98 4)*9;>. E. @feil, -. 0riedrich, T. Wachsmann, H. anal. %hem. !#1, ;.* 4)*9:>. Chemicals: @uercetin cryst. LA7, Ord. No. .!&%"3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# *#2" Fuinali+arin for cations" Spray solutionC ,.,9I solution of ?uinaliDarin in :,I ethanol. After-treatmentC @lace the chromatogram into a chamber saturated with ammonia 'apours. Literature: B.H. /ohnson, H.H. Krause, -nal. %him. -cta !!, ).+ 4)*9;>. Chemicals: ,#,%,+-:etrahydro/yanthraAuinone -Auinali,arin. )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# *#3" p-Fuinone for ethanolamine" Spray solutionC Dissol'e ,.9 g p#benDo?uinone 4p#?uinone> in a mi&ture of ), ml pyridine and ;, ml )#butanol. NoteC -fter spraying red spots of ethanolamine will appear immediately. %holine shows no reaction. Chemicals: p-7en,oAuinone for synthesis, Ord. No. +.!#" ! -7utanol GR ACS, ISO, Ord. No. .! $$! 0yridine GR ACS, Ord. No. .!$&#+ *#1" Resorcinol - +inc chloride - sulfuric acid for plasticisers %especially phthalate esters&" Spray solution IC -dd to a .,I ethanolic resorcinol solution some Dinc chloride. Spray solution IIC Sulfuric acid 4c Q . molR$>.

Spray solution IIIC ;,I a?ueous potassium hydro&ide solution. 0rocedureC Spray with 5, heat ), min at )9,J%, spray with 55, heat ), min at ).,J% and spray with 555. Brange spots on yellow bac"ground. Literature: /.W. %opius#@eereboom, /. %hromatog. -, 8.8 4)*<,>. D. 6raun, %himia 4SwitD.> !), :: 4)*<9>. Chemicals: Resorcinol GR, Ord. No. .!&%$( Cinc chloride GR ACS, ISO, Ord. No. .!++ 3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( 0otassium hydro/ide pellets GR, Ord. No. .!%!(( *#)" Resorcyl aldehyde - sulfuric acid for !2-dehydrosteroids" Solution aC ,.9I solution of resorcyl aldehyde in acetic acid. Solution *C 9I sulfuric acid solution in glacial acetic acid. Spray solutionC (i& freshly before use e?ual parts of a and b. After-treatmentC Heat at ),,#)),J% until ma&imal 'isualisation of the spots. Literature: D.6. Gower, /. %hromatog. !-, ;.; 4)*<;>. Chemicals: Resorcyl aldehyde Acetic acid $3' GR, Ord. No. .!!!3# Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( *2/" Rhodamine ( ),, ml ready to use spray solution for chromatography 4c Q ca. ,..I in ethanol>. Ord. No. .!&3!# *2!" Rhodamine (' general spray reagent" Spray solutionC ,.,.9#,..9I ethanolic solution of rhodamine 6. 5nspect in long# wa'e A3 light. Literature: H.@. Kaufmann, /. 6udwig, 0ette u. Seifen, -nstrichmittel #$, 8*, 4)*9)>. Chemicals: Rhodamine 7 -C.I. "% &!. GR, Ord. No. .!&%$$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+(

*2*" Rhodamine 2 = for lipids" Spray solutionC Dissol'e ,.,,) g rhodamine < G in ),, ml acetone. 5nspect in long#wa'e A3 light. Literature: 2.0. Witter, G.3. (arinetti, -. (orrison, -rch. 6iochem. 6iophys. 21, )9 4)*9:>. Chemicals: Rhodamine 3 G Acetone GR ACS, ISO, Ord. No. .!!! " *2$" Rhodanine for carotenoid aldehydes" Spray solution IC )#9I ethanolic solution of rhodanine. Spray solution IIC .9I ammonia solution or .:I a?ueous sodium hydro&ide solution. 0rocedureC Spray with 5, then with 55 and dry the chromatogram. Literature: -. Winterstein, 6. Hegedues, %himia 4SwitD.> !-, )+ 4)*<,>. Chemicals: Rhodanine Ammonia solution #%' GR, Ord. No. .!%"(# Sodium hydro/ide solution min. #&' - ,(. GR, Ord. No. .!%%$ *2-" Rhodi+onic acid sodium salt for barium and strontium ions" Spray solution IC )I a?ueous solution of sodium rhodiDonate. Spray solution IIC .9I ammonia solution. Literature: T.3. -rden, 0.H. 6urstall, G.2. Da'ies, /.-. $ewis, 2.@. $instead, 7ature !2*, <*) 4)*;+>. Chemicals: Rhodi,onic acid disodium salt indicator, Ord. No. .!3%$% Ammonia solution #%' GR, Ord. No. .!%"(# *2#" Rubeanic acid for lead' cobalt' copper' manganese' nic7el' mercury and bismuth ions" Spray solution IC ,.9I ethanolic solution of rubeanic acid. Spray solution IIC .9I ammonia solution.

0rocedureC Spray with 5, dry briefly, then spray with 55 or place the chromatogram into a chamber with ammonia 'apours. Literature: 0.W.H.(. (er"us, @harm. Wee"blad )1, *99 4)*<8>. /.-. $ewis, /.(. Griffiths, -nalyst 32, 8++ 4)*9)>. Chemicals: Ru*eanic acid GR, Ord. No. .!!3#$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# *22" Sil,er nitrate for phenols" Spray solutionC -dd with stirring ) ml saturated a?ueous sil'er nitrate solution to ., ml acetone, then add water dropwise until the precipitated sil'er nitrate has Eust dissol'ed. $ight pin" to deep green spots are yielded. Literature: W./. 6ur"e, -.D. @otter, 2.(. @ar"hurst, -nal. %hem. $*, :.: 4)*<,>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Acetone GR ACS, ISO, Ord. No. .!!! " *23" Sil,er nitrate - ammonia for sugars and sugar alcohols %Dedonder reagent&" PC" Spray solutionC -dd with stirring ) ml saturated a?ueous sil'er nitrate solution to ., ml acetone, then add water dropwise until the sil'er nitrate Eust dissol'es. 0rocedureC Spray the chromatogram liberally from both sides. :reatmentC @lace the moist chromatogram ) hour into a chamber saturated with ammonia 'apours 4protected against light>. Then heat the chromatogram at +,J% until the paper bac"ground has turned light brown, and remo'e the e&cess sil'er nitrate with ),I sodium thiosulfate solution. -fter rinsing for . hours under running water dry the chromatogram. Literature: %. @etronici, G. Safina, %hem. -bstr. -3, )).*: 4)*98>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Sodium thiosulfate pentahydrate GR ACS, ISO, Ord. No. .!3% 3 Acetone GR ACS, ISO, Ord. No. .!!! " Ammonia solution #%' GR, Ord. No. .!%"(#

*21" Sil,er nitrate - ammonia for reducing substances %Tollens or >affaroni reagent&" Solution aC Sil'er nitrate solution 4c Q ,.) molR$>. Solution *C -mmonia solution 4c Q 9 molR$>. Spray solutionC (i& a and b in the ratio )C9 freshly before use. Caution9 Cormation of explosi,e sil,er a+ide by prolonged standing. After-treatmentC Heat 9#), min at ),9J% until the dar" spots ha'e become most intense. Literature: -.%. 6ath#Smith, 2.G. Westall, 6iochim. et biophys. -cta -, ;.: 4)*9,>. Chemicals: Sil6er nitrate solution !. mol=l, Ord. No. .!$!+ Ammonia solution #%' GR, Ord. No. .!%"(# *2)" Sil,er nitrate - ammonia - fluorescein for halogen ions" Spray solution IC Dissol'e ) g sil'er nitrate in ),, ml ammonia solution 4c Q ,.9 molR$>. Spray solution IIC ,.)I ethanolic fluorescein solution. 0rocedureC Spray with 5, dry briefly and spray with 55. Literature: H. Seiler, T. Kaffenberger, Hel'. %him. -cta --, ).+. 4)*<)>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # <luorescein -C.I. "%(%!. Ammonia solution #%' GR, Ord. No. .!%"(# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *3/" Sil,er nitrate - ammonia - sodium chloride for thioacids" Spray solution IC (i& freshly before use 9, ml sil'er nitrate solution 4c Q ,.) molR$> with 9, ml ),I ammonia solution. $onger standing may lead to formation of explosi,e sil,er a+ideW Spray solution IIC ),I a?ueous sodium chloride solution. 0rocedureC Spray with 5, then dry and spray with 55. E&pose the chromatogram to daylight until the yellow#brown spots ha'e attained ma&imum colour intensity. Chemicals: Sil6er nitrate solution !. mol=l, Ord. No. .!$!+ Ammonia solution #%' GR, Ord. No. .!%"(# Sodium chloride GR ACS, ISO, Ord. No. .!3"!"

*3!" Sil,er nitrate - ammonia - sodium methoxide for sugars" Solution aC ,.8I methanolic sil'er nitrate solution. Solution *C -mmonia gas saturated methanol. Solution cC Dissol'e : g sodium in ),, ml methanol. Spray solutionC (i& freshly before use ., ml a, ; ml b and + ml c. After-treatmentC Heat ), min at )),J%. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Sodium rods, Ord. No. .!3#3! 4ethanol GR ACS, ISO, Ord. No. .!3!!$ Ammonia solution #%' GR, Ord. No. .!%"(# *3*" Sil,er nitrate - bromophenol blue for purines %Good reagent&" Spray solutionC Dissol'e ,.. g bromophenol blue in 9, ml acetone and add 9, ml .I a?ueous sil'er nitrate solution. The reagent is stable for about one wee". 0rocedureC -fter de'elopment in acidic sol'ents dry the chromatogram and place into a chamber with ammonia. Then remo'e the e&cess ammonia by hot air and spray. Literature: H. (ichl, 0. Harberler, (h. %hem. 1#, ::* 4)*9;>. Chemicals: 7romophenol *lue indicator p9 (.!-".3 ACS, Ord. No. .!+ ## Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Acetone GR ACS, ISO, Ord. No. .!!! " *3$" Sil,er nitrate - fluorescein for al7yl- and arylsulfonic acids" Solution aC ),I a?ueous sil'er nitrate solution. Solution *C ,..I ethanolic fluorescein sodium solution. Spray solutionC (i& freshly before use ), ml a and 9, ml b. Fellow spots on salmon#pin" bac"ground. Literature: 0.H. @ollard, G. 7ic"las, K.W.%. 6urton, /. %hromatog. 1, 9,: 4)*<.>. %.(. %oyne, G.-. (aw, /. %hromatog. !-, 99. 4)*<;>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # <luorescein sodium -C.I. "%(%!. e/tra pure, Ord. No. .!($$# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+(

*3-" Sil,er nitrate - formaldehyde for chlorinated insecticides %e"g" dieldrin' aldrin and lindane&" Spray solution IC Sil'er nitrate solution 4c Q ,.,9 molR$>. Spray solution IIC 89I formaldehyde solution. Spray solution IIIC (ethanolic potassium hydro&ide solution 4c Q . molR$>. Spray solution I2C 0reshly prepared mi&ture of e?ual 'olumes of 8,I hydrogen pero&ide and <9I nitric acid. 0rocedureC Spray with 5, dry 8, min, spray with 55 and dry again 8, min. Spray with 555 and heat 8, min at )8,J%. Spray with 53, allow the chromatogram to stand in the dar" for ). hours, and e&pose to daylight. Dar" green spots on light grey bac"ground. Literature: $.%. (itchell, /. -ssoc. Bff. -gr. %hemists $#, *., 4)*9.>. Chemicals: Sil6er nitrate solution !. mol=l, Ord. No. .!$!+ <ormaldehyde solution min. (&' GR, Ord. No. .!"!!( 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 4ethanol GR ACS, ISO, Ord. No. .!3!!$ 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ Nitric acid 3%' GR ISO, Ord. No. .!!"%3 *3#" Sil,er nitrate - hydrogen peroxide for chlorinated hydrocarbons" Spray solutionC Dissol'e ,.) g sil'er nitrate in ) ml water, add ), ml ethylene glycol monophenyl ether, fill up to .,, ml with acetone and add ) drop 8,I hydrogen pero&ide. After-treatmentC 5rradiate with unfiltered A3 light. 5f long#wa'e A3 light is used e&pose alumina layers about 9, min and silica gel layers up to )9 min. Dar" spots are formed. Literature: (.0. Ko'acs, /. -ssoc. Bff. -gr. %hemists -2, ++; 4)*<8>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Acetone GR ACS, ISO, Ord. No. .!!! " 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ )thylene 8lycol monophenyl ether for synthesis, Ord. No. +.!&#$ *32" Sil,er nitrate - potassium dichromate for barbiturates" Spray solution IC -dd .9 ml saturated a?ueous sil'er nitrate solution to a mi&ture of 9, ml acetone and . ml water.

Spray solution IIC ,.8I a?ueous potassium dichromate solution. Spray solution IIIC .I methanolic sodium hydro&ide solution. 0rocedureC Spray liberally with 5 and dry in the air. Then spray with 55, dry, re# spray with 55 and re#dry again in the air. Then spray with 555. Literature: H. Weidmann, Dissertation 6erlin )*<). Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # 0otassium dichromate GR ACS, ISO, Ord. No. .!"+3" Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ Acetone GR ACS, ISO, Ord. No. .!!! " 4ethanol GR ACS, ISO, Ord. No. .!3!!$ *33" Sil,er nitrate - potassium permanganate for reducing compounds" Solution aC (i& freshly before use ) part sil'er nitrate solution 4c Q ,.) molR$>, ) part ammonia solution 4c Q . molR$> and . parts sodium hydro&ide solution 4c Q . molR$>. Solution *C Dissol'e ,.9 g potassium permanganate and ) g sodium carbonate in ),, ml water. Spray solutionC (i& freshly before use e?ual parts of a and b. NoteC 2educing compounds show light yellow spots on green#blue bac"ground immediately after spraying. Literature: /. Kellen, %hem. listy #!, *:8 4)*9:>. Chemicals: 0otassium perman8anate GR ACS, Ord. No. .!%!+# Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ Sil6er nitrate solution !. mol=l, Ord. No. .!$!+ Sodium hydro/ide solution # mol=l, Ord. No. .!$ (3 Ammonia solution #%' GR, Ord. No. .!%"(# *31" Sil,er nitrate - sodium dichromate for purines" PC" 5ip solution IC .I a?ueous sil'er nitrate solution. 5ip solution IIC ,.9I a?ueous sodium dichromate solution. 5ip solution IIIC 7itric acid 4c Q ,.9 molR$>. 0rocedureC Dip into 5, dry the chromatogram in the air ), min and dip into 55. Dip the red#dyed chromatogram into 555, thus discolouring the bac"ground, lea'ing the purines as red spots.

Literature: 2.(. 2eguera, 5. -simo', /. -m. %hem. Soc. 3$, 9:+) 4)*9,>. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Sodium dichromate dihydrate GR, Ord. No. .!3((3 Nitric acid 3%' GR ISO, Ord. No. .!!"%3 *3)" Sil,er nitrate - sodium hydroxide for sugars and polyalcohols" Spray solution IC 0ill up ) ml saturated a?ueous sil'er nitrate solution to .,, ml with acetone and add 9#), ml water to dissol'e the resulting precepitate. Spray solution IIC Sodium hydro&ide solution 4c Q ,.9 molR$> in a?ueous methanol 4dissol'e ., g sodium hydro&ide in a minimum of water and fill up to ) l with methanol>. 0rocedureC Spray with 5 and 55 and heat )#. min at ),,J%. Chemicals: Sil6er nitrate GR ACS, ISO, Ord. No. .! % # Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ Acetone GR ACS, ISO, Ord. No. .!!! " 4ethanol GR. Ord. No. .!3!!$ *1/" Sodium dithionite for antimony' arsenic' mercury' sil,er and bismuth ions" Spray solutionC ,.)I a?ueous sodium dithionite solution. Literature: 0.H. @ollard, /.0.H. (cBmie, %hromatographic (ethods of 5norganic -nalysis, 6utterworths Scientific @ublications, $ondon, )*98, p. ;:. Chemicals: Sodium dithionite LA7, Ord. No. .!3%!& *1!" Sodium hydroxide for --$-7etosteroids" Spray solutionC ),I sodium hydro&ide solution in <,I methanol. After-treatmentC Heat ), min at +,J%. ;#8#"etosteroids show yellow fluorescence in long#wa'e A3 light. Literature: 5.E. 6ush, 6iochem. /. #/, 8:, 4)*9)>. Chemicals: Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ 4ethanol GR ACS, ISO, Ord. No. .!3!!$

*1*" Sodium meta-periodate - ben+idine for compounds 0ith !'*-diol groups %sugars' polyalcohols&" Spray solution IC ,.9I a?ueous sodium meta#periodate solution. Spray solution IIC -dd 9, ml water, ., ml acetone and ), ml ,.. 7 hydrochloric acid to a solution of ).+ g benDidine in 9, ml ethanol. 0rocedureC Spray with 5 and after 9 min with 55. White spots on blue bac"ground. Caution: (en+idine is carcinogenic9 Literature: /.-. %ifonelli, 0. Smith, -nal. %hem. *2, ))8. 4)*9;>. Chemicals: Sodium metaperiodate GR ACS, Ord. No. .!3%$& 7en,idine 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Acetone GR ACS, ISO, Ord. No. .!!! " *1$" Sodium meta-periodate - ben+idine - sil,er nitrate for substances 0ith !'*diol groups %sugars' polyalcohols&" Spray solution IC ,.)I a?ueous sodium meta#periodate solution. Spray solution IIC -dd :, ml water, 8, ml acetone and ).9 ml hydrochloric acid 4c Q ) molR$> to a solution of ..+ g benDidine in +, ml ethanol. Spray solution IIIC (i& ) ml a?ueous saturated sil'er nitrate solution with stirring with ., ml acetone and add water dropwise until the precipitated sil'er nitrate dissol'es. 0rocedureC Spray with 5 and dry the chromatogram at room temperature. Spray with 55 and place it into a chamber saturated with ammonia 'apours. -dditionally you can spray with 555, the white spots turn dar". Caution: (en+idine is carcinogenic9 Literature: D. Waldi, /. %hromatog. !1, ;): 4)*<9>. Chemicals: Sodium metaperiodate GR ACS, Ord. No. .!3%$& 7en,idine Sil6er nitrate GR ACS, ISO, Ord. No. .! % # 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Acetone GR ACS, ISO, Ord. No. .!!! " Ammonia solution #%' GR, Ord. No. .!%"(#

*1-" Sodium meta-periodate - 6essler:s reagent for hydroxyamino acids %serine' threonine&" Spray solution IC )I a?ueous sodium meta#periodate solution. Spray solution IIC 7esslerUs reagent. (a"e a paste with ), g mercury455> iodide and a little water and add 9 g potassium iodide. -dd a solution of ., g sodium hydro&ide in +, ml water. -fter complete dissolution fill up to ),, ml with water. -llow to stand for some days and decant after deposition of the precipitate. 0rocedureC Spray with 5, dry the chromatogram at room temperature and spray with 55. Literature: 2. %onsden, -.H. Gordon, -./.@. (artin, 6iochim. /. -/, 88 4)*;<>. Chemicals: Sodium metaperiodate GR ACS, Ord. No. .!3%$& 4ercury-II. iodide red e/tra pure, Ord. No. .!""#! 0otassium iodide GR ISO, Ord. No. .!%!"( Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ *1#" Sodium meta-periodate - --6itroaniline for deoxy-sugars" Spray solution IC (i& ) part saturated a?ueous sodium meta#periodate solution with . parts water. Spray solution IIC (i& ; parts )I ethanolic#;#nitroaniline solution with ) part 8:I hydrochloric acid. 0rocedureC Spray with 5, wait ), min, then spray with 55. NoteC Deo&y#sugars and glycals show yellow spots which fluoresce strongly in long#wa'e A3 light. The colour changes to green by spraying with 9I methanolic sodium hydro&ide solution. Literature: /.T. Edward, D.(. Waldron, /. %hem. Soc. !)#*, 8<8). Chemicals: Sodium metaperiodate GR ACS, Ord. No. .!3%$& "-Nitroaniline, Ord. No. .!3&3! )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO,Ord. No. .!!( & Sodium hydro/ide pellets, Ord. No. .!3"$+ 4ethanol GR ACS, ISO, Ord. No. .!3!!$ *12" Sodium nitrite - hydrochloric acid for indoles and thia+oles"

Spray solutionC 0reshly prepared solution of ) g sodium nitrite in ),, ml hydrochloric acid 4c Q ) molR$>. Heat at ),,J%. NoteC 5ndoles turn red and thiaDole deri'ati'es light green. lternati,e: Spray solutionC ,.9I a?ueous sodium nitrite solution. After-treatmentC @lace the chromatogram into a chamber with hydrogen chloride 'apours. Literature: D. '. Denffer, (. 6ehrens, -. 0ischer, 7aturwissenschaften $), .9+ 4)*9.>. Chemicals: Sodium nitrite GR ACS, Ord. No. .!3%"$ 9ydrochloric acid mol=l :itrisol>, Ord. No. .!$$&! 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & *13" Sodium nitroprusside for compounds 0ith S8-group %cysteine&' 0ith S-Sgroup %cystine& and arginine" Spray solution IC Dissol'e ).9 g sodium nitroprusside in 9 ml hydrochloric acid 4c Q . molR$>. 0ilter after addition of *9 ml methanol and ), ml .9I ammonia solution. NoteC SH#%ompounds show red spots, arginine turns orange and later grey#blue. Spray solution IIC Dissol'e . g sodium cyanide in 9 ml water and fill up to ),, ml with methanol. NoteC 2espraying with 55 'isualises compounds with #S#S#lin"age as red spots on yellow bac"ground. Caution 0hen using this highly toxic reagentW 4ariation for -S-S-compounds: Spray solution IC Dissol'e 9 g sodium cyanide and 9 g sodium carbonate in ),, ml .9 I ethanol. Spray solution IIC Dissol'e . g sodium nitroprusside in ),, ml :9I ethanol. 0rocedureC Spray with 5, dry briefly in the air and spray with 55. Caution 0hen using this highly toxic reagent9 Literature: G. Tonnies, /./. Kolb, -nal. %hem. *$, +.8 4)*9)>. 4ariation for thiolactones: Spray solution IC Sodium hydro&ide solution 4c Q ) molR$>. Spray solution IIC Dissol'e . g sodium nitroprusside in ),, ml :9I ethanol.

0rocedureC Spray with 5, dry briefly in the air and spray with 55. Literature: 0. Korte, /. 3ogel, /. %hromatog. ), 8+) 4)*<.>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ 9ydrochloric acid #%' GR, Ord. No. .!!( 3 Ammonia solution #%' GR, Ord. No. .!%"(# 4ethanol GR ACS, ISO, Ord. No. .!3!!$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sodium cyanide pure, Ord. No. .!3"(& Sodium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$%3 *11" Sodium nitroprusside - acetaldehyde for secondary aliphatic and alicyclic amines" Spray solutionC Dissol'e 9 g sodium nitroprusside in ),, ml ),I a?ueous acetaldehyde solution. 6efore use mi& ) part of this solution with ) part .I a?ueous sodium carbonate solution. Literature: 0. 0eigl, Spot Test in Brganic -nalysis, Else'ier @ub. %o., :th Ed., )*<<, p. .9). K. (ace", /. Hacaper"o'K, 6. Ka"ai#, @harmaDie !!, 988 4)*9<>. E. Stein, 3. Kamiens"i, @lanta (ed. #/, .*) 4)*9:>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ Acetaldehyde for synthesis, Ord. No. +.!!!!" *1)" Sodium nitroprusside - ammonia for hemloc7 al7aloids" Spray solution IC )I a?ueous sodium nitroprusside solution. Spray solution IIC ),I ammonia solution. 0rocedureC Spray with 5 and then with 55. NoteC #%oniceine turns red. Literature: 0. (all, -rch. @harm. *)2, .,9 4)*<8>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Ammonia solution #%' GR, Ord. No. .!%"(#

*)/" Sodium nitroprusside - hydrogen peroxide for guanidine' urea' thiourea and deri,ati,es' creatine and creatinine" Spray rea8entC (i& . ml 9I a?ueous sodium nitroprusside, ) ml ),I a?ueous sodium hydro&ide and 9 ml 8I a?ueous hydrogen pero&ide and dilute with )9 ml water. The solution can be stored se'eral days in the refrigerator. Literature: E. Hofmann, -. Wuensch, 7aturwissenschaften -#, 88+ 4)*9+>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++ 9ydro8en pero/ide (!' 9#O# -0erhydrol>., Ord. No. .!&#!$ *)!" Sodium nitroprusside - hydroxylamine for thiourea deri,ati,es %=rote reagent&" Spray solutionC Dissol'e ,.9 g sodium nitroprusside in ), ml water, add ,.9 g hydro&ylamine hydrochloride and ) g sodium hydrogen carbonate. -fter gas generation is complete, add . drops bromine and fill up to .9 ml with water. The reagent is stable for about . wee"s. Literature: 5.W. Grote, /. 6iol. %hem. )$, .9 4)*8)>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium hydro8en car*onate GR ISO, Ord. No. .!3(#$ 7romine GR ISO, Ord. No. .! $"+ 9ydro/ylammonium chloride GR ACS, ISO, Ord. No. .!"3 3 *)*" Sodium nitroprusside - potassium hexacyanoferrate%III& for aliphatic nitrogen compounds' cyanamide' guanidine' urea' thiourea and deri,ati,es' creatine and creatinine" Spray solutionC (i& in the ratio )C)C)C8 ),I a?ueous sodium hydro&ide solution, ),I a?ueous sodium nitroprusside solution, ),I a?ueous potassium he&acyanoferrate4555> solution and water. The mi&ture is allowed to stand at least ., min at room temperature before use. Stable for se'eral wee"s when stored in the refrigerator. 6efore use mi& with an e?ual part of acetone. Literature: /. 2oche et al., 6iochim. et biophys. -cta !-, :) 4)*9;>. $. 0ishbein, (.-. %a'anaugh, /. %hromatog. */, .+8 4)*<9>. $. 0ishbein, 2ec. tra'. chim. 1-, ;<9 4)*<9>.

Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" 0otassium he/acyanoferrate-III. GR ACS, ISO, Ord. No. .!"$&( Sodium hydro/ide solution min. !' - . . GR, Ord. No. .!%%++ Acetone GR ACS, ISO, Ord. No. .!!! " *)$" Sodium nitroprusside - potassium permanganate for sulfonamides %Roux reagent&" Spray solutionC Dissol'e ), g sodium nitroprusside in ),, ml water, add . ml 88I a?ueous sodium hydro&ide and 9 ml potassium permanganate solution 4c Q ,.,. molR$> and filter after mi&ing. 0rocedureC Spray and inspect in A3 light. Literature: E. 3itolo, 6ull. %him. 0arm. 1), 89) 4)*9,>. G. Wagner, @harmaDie ), *:* 4)*9;>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ 0otassium perman8anate solution !.!# mol=l :itrisol>, Ord. No. .!$$(% *)-" Sodium nitroprusside - sodium hydroxide for methyl 7etones and acti,ated methylene groups" Spray solutionC Dissol'e ) g sodium nitroprusside in ),, ml of a mi&ture of sodium hydro&ide 4c Q . molR$> and ethanol 4)V )>. 2ed to 'iolet spots. Literature: 0. 0eigl, Spot Tests in Brganic -nalysis, Else'ier @ubl. %o., )*<<, :th Ed., p. .,+. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sodium hydro/ide solution # mol=l, Ord. No. .!$ (3 *)#" Sodium nitroprusside - sodium meta-periodate for deoxy-sugars" Spray solution IC ..9I a?ueous sodium meta#periodate solution. Spray solution IIC (i&ture of ) part :I a?ueous sodium nitroprusside solution, 8 parts water and ., parts of a saturated solution of piperaDine in ethanol. 0rocedureC Spray with 5, dry ), min at room temperature, then spray with 55. Literature: /.T. Edward, D.(. Waldron, /. %hem. Soc. !)#*, 8<8).

Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium metaperiodate GR ACS, Ord. No. .!3%$& 0ipera,ine he/ahydrate 0h )ur, 70, Ord. No. .!&(#& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( *)2" Sodium pentacyanoamineferrate%II& for urea' thiourea and guanidines %Cearon reagent&" Sodium pentacyanoamineferrate-II.C Dissol'e ), g sodium nitroprusside in ;, ml .9I ammonia solution. -llow the solution to stand at ,J% until all nitroso iron4555> cyanide is decomposed. This is the case if se'eral drops of the mi&ture added to a solution of creatinine in sodium carbonate solution 4c Q ,.9 molR$> produce no longer any red colour. Then filter and add ethanol to the clear filtrate until no further precipitate is formed. 0ilter off the resulting precipitate, wash with absolute ethanol and dry o'er sulfuric acid in a 'acuum desiccator. The salt is stable when stored protected from light and moisture. Spray solutionC -dd to 9 ml ),I sodium hydro&ide )9 ml )I a?ueous sodium pentacyanoaminoferrate455> solution and ) drop @erhydrol[. Stable for about .; hours. Literature: @.H. $ist, Hoppe#Seylers H. physiol. %hem. $/#, .: 4)*9<>. Chemicals: Sodium nitroprusside dihydrate GR ACS, Ord. No. .!3%" Sodium car*onate anhydrous GR ISO, Ord. No. .!3($# )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Ammonia solution #%' GR, Ord. No. .!%"(# 9ydro8en pero/ide (!' 9#O# -0erhydrol>., Ord. No. .!&#!$ Creatinine, Ord. No. .!%#!+ Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( *)3" Sodium sulfide solution for ions of the hydrogen sulfide group" Spray solutionC 0reshly prepared ,.9 I a?ueous sodium sulfide solution. Literature: 0.W.H.(. (er"us, @harm. Wee"blad )1, *9: 4)*<8>. Chemicals: Sodium sulfide hydrate GR, Ord. No. .!33(+ *)1" Sodium tetraphenylboron %<alignostH& for al7aloids" Spray solution IC )I sodium tetraphenylboron 4sodium tetraphenyl borate> solution in ethyl methyl "etone, saturated with water.

Spray solution IIC ,.,)9I methanolic solution of fisetin or ?uercetin. 0rocedureC Spray with 5, dry at room temperature, then spray with 55 and dry again at room temperature. Brange to red spots which fluoresce in long#wa'e A3 light. Literature: 2. 7eu, /. %hromatog. !!, 8<; 4)*<8>. Chemicals: Sodium tetraphenyl *orate -Eali8nost>. GR ACS, Ord. No. .!333$ @uercetin cryst. LA7, Ord. No. .!&%"3 <isetin 4ethanol GR ACS, ISO, Ord. No. .!3!!$ )thyl methyl ?etone GR ACS, Ord. No. .!$&!+ *))" Sodium tetraphenylboron%<alignostH& - rhodamine ( for potassium ions" Spray solution IC Sodium hydro&ide solution 4c Q ,.) molR$>. Spray solution IIC ) I ethanolic Kalignost[ solution. Spray solution IIIC ,.9I ethanolic rhodamine 6 solution. 0rocedure Spray with 5, dry, spray with 55, and then with 555. 5ntense dar" blue fluorescence in long#wa'e A3 light. $arger amounts of potassium appear in daylight as light red spots on dar" red bac"ground. Chemicals: Sodium tetraphenyl *orate -Eali8nost>. GR ACS, Ord. No. .!333$ Sodium hydro/ide solution !. mol=l :itrisol>, Ord. No. .!$$%$ Rhodamine 7 -C.I. "% &!. GR, Ord. No. .!&%$$ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $//" Sodium thiosulfate - copper%II& acetate for antimony ions" PC" Spray solution IC Saturated a?ueous sodium thiosulfate solution. Spray solution IIC Dissol'e ,.; g copper455> acetate in a mi&ture of . ml glacial acetic acid and ;+ ml water. 0rocedureC Spray with 5, heat briefly, rinse out e&cess sodium thiosulfate with water and spray with 55. Literature: G.@. Heisig, 0.H. @ollard, -nal. %him. -cta !2, .8; 4)*9:>. Chemicals: Copper-II. acetate GR, Ord. No. .!#& Sodium thiosulfate pentahydrate GR ACS, ISO, Ord. No. .!3% 3 Acetic acid $3' GR, Ord. No. .!!!3#

$/!" Starch for amylases" Spray solution IC .I a?ueous starch solution. Spray solution IIC 5odine solution 4c Q ,.,,9 mol 5.R$>. 0rocedureC Spray with 5, then place the chromatogram into a moist chamber at ;,#9,J% for ) hour. -fter drying at room temperature spray with 55. NoteC -mylases will appear as white spots on 'iolet or brown bac"ground. Literature: K. Wallenfels, E. '. @echmann, -ngew. %hem. 2$, ;; 4)*9)>. Chemicals: Starch solu*le GR ISO, Ord. No. .! #%# Iodine solution !.!% mol I#=l, Ord. No. .!$$ ! $/*" Sulfanilamide dia+otised for phenols' coupling amines and heterocycles %Pauly reagent acc" to <utace7&" Spray solution IC Dissol'e 8 g sulfanilamide in .,, ml water, < ml 8<I hydrochloric acid and ); ml )#butanol. -dd freshly before use to ., ml ,.8 g sodium nitrite. Spray solution IIC ),I a?ueous sodium carbonate solution. 0rocedureC Spray with 5, and after 9#), min with 55. Literature: 5.(. Hais, K. (ace", Handbuch der @apierchromatographie 5, G. 0ischer, /ena, )*9+, p. :;8. Chemicals: Sulfanilamide e/tra pure, Ord. No. .!+!(% Sodium nitrite GR ACS, Ord. No. .!3%"$ Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & -7utanol GR ACS, ISO, Ord. No. .! $$! $/$" Sulfanilic acid dia+otised for phenols' coupling amines and heterocycles %Pauly reagent&" Spray solutionC Dissol'e ;.9 g sulfanilic acid in ;9 ml hydrochloric acid 4c Q ). molR$> with warming and fill up the solution to 9,, ml with water. %ool ), ml of the diluted solution with ice and add ), ml of cold ;.9I a?ueous sodium nitrite solution. -llow to stand for )9 min at ,J% 4it is stable for )#8 days at this temperature> and add freshly before use e?ual parts of ),I, a?ueous sodium carbonate solution.

Literature: H. /atD"ewitD, Hoppe#Seylers H. physiol. %hem. *)*, ** 4)*98>. 7.2. Grimmett, E.$. 2ichards, /. %hromatog. */, ):) 4)*<9>. Chemicals: Sulfanilic acid GR ACS, Ord. No. .!!3+3 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & Sodium nitrite GR ACS, Ord. No. .!3%"$ Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ $/-" Sulfanilic acid - !-naphthylamine for nitrosamines" Solution aC )I sulfanilic acid solution in 8,I a?ueous acetic acid. Solution *C ,.)I )#naphthylamine solution in 8,I a?ueous acetic acid. Spray solutionC (i& freshly before use e?ual parts of a and b. 0rocedureC 5rradiate the chromatogram for about 8 min with short#wa'e A3 light, then spray with the spray solution. NoteC -liphatic nitrosamines show red#'iolet spots, aromatic nitrosamines turn green to blue. Literature: 2. @reussmann, D. Daiber, H. Hengy, 7ature */!, 9,. 4)*<;>. 2. @reussmann, G. 7eurath, G. Wulf#$orentDen, D. Daiber, H. Hengy, H. anal. %hem. */*, )+: 4)*<;>. Chemicals: Sulfanilic acid GR ACS, Ord. No. .!!3+3 -Naphthylamine GR, Ord. No. .!3#"% Acetic acid $3' GR, Ord. No. .!!!3# $/#" Sulfuric acid as general ,isualisation reagent %in particular for sterols' steroids' bile acids and gibberellins&" Spray solutionsC -C (i& e?ual parts of *9I sulfuric acid and methanol with cooling. 6C 9I ethanolic solution of *9I sulfuric acid. %C )9I solution of *9I sulfuric acid in )#butanol. DC 9I solution of *9I sulfuric acid in acetic anhydride. EC (i& e?ual parts of *9I sulfuric acid and glacial acetic acid. 0rocedureC Spray the chromatogram with one of these reagents, allow to dry for )9 min in the air and heat to )),J% until ma&imal 'isualisation of the spots. NoteC %holesterol and 'itamin -, their esters and many isoprenoid lipids show characteristic colours after spraying with spray solution - during subse?uent heatingC cholesterol and esters first turn red, then red#'iolet and brown while

'itamin - and esters first turn blue. (ost compounds may be subse?uently charred, yielding blac" spots. Heating with sulfuric acid on layers impregnated with sil'er nitrate may be followed by complete o&idation to %B.. Literature: D.0. /ones, /. (c(illan, (. 2adley, @hytochemistry *, 8,: 4)*<;> 4gibberellins>. W.$. -nthony, W.T. 6eher, /. %hromatog. !$, 9:, 4)*<;>. H. /atD"ewitD, E. (ehl, Hoppe#Seylers H. physiol. %hem. $*/, .9) 4)*<,> H. (etD, 7aturwissenschaften -1, 9<* 4)*<)>. Chemicals: Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -7utanol GR ACS, ISO, Ord. No. .! $$! Acetic anhydride GR ACS, ISO, Ord. No. .!!!"# Acetic acid $3' GR, Ord. No. .!!!3# $/2" Sulfuric acid - hypochlorite for digitalis glycosides" Spray solutionC (i& ), ml sulfuric acid 4c Q ) molR$> and 8 ml sodium hypochlorite solution. After-treatmentC Heat ),#)9 min at ).9J%. NoteC Digitalis glycosides of series - # E show fluorescence of 'arious colours in long#wa'e A3 light. Literature: $. 0auconnet, 2. 0aDan, 6ull. Soc. 'aud. sci. nat. 22, 8,: 4)*9<>. $. 0auconnet, (. Waldesbuehl, @harm. -cta Hel'. $1, ;.8 4)*<8>. Chemicals: Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( Sodium hypochlorite solution -a*out (' acti6ated chlorine., Ord. No. .!%3 " $/3" Tetracyanoethylene for aromatic hydrocarbons' phenols and herocyclic compounds" Spray solutionC ),I solution of tetracyanoethylene in benDene. 0rocedureC Spray directly after de'elopment of the chromatogram. NoteC -romatic hydrocarbons show 'arious colours, some of them for a short time only. /anK" recommends warming at ),,J%. Literature: @.3. @eurifoy, S.%. Slayma"er, (. 7ager, -nal. %hem. $!,):;, 4)*9*>. /. /anK", /. %hromatog. !#, )9 4)*<;>. 7. KucharcDy", 0. 0ohl, /. 3ymLtal, /. %hromatog. !!, 99 4)*<8>.

Chemicals: 7en,ene GR ACS, ISO, Ord. No. .! &+( :etracyanoethylene for synthesis, Ord. No. +.!+#"! $/1" Tetranitrodiphenyl for cardiac glycosides" Spray solution IC Saturated solution of .,.U,;,;U#tetranitrodiphenyl in benDene. Spray solution IIC ),I potassium hydro&ide solution in 9,I a?ueous methanol. 0rocedureC Spray with 5, dry at room temperature and spray with 55. 6lue spots. Literature: /. 6in"ert, E. -ngli"er, -. '. Wartburg, Hel'. %him. -cta -#, .).. 4)*<.>. Chemicals: 0otassium hydro/ide pellets GR, Ord. No. .!%!(( 7en,ene GR ACS, ISO, Ord. No. .! &+( #,#;,",";-:etranitrodiphenyl 4ethanol GR ACS, ISO, Ord. No. .!3!!$ $/)" Tetraphenyldiboroxide for fla,ones" PC" @repare tetraphenyldiboro&ide according to the directions by 2. 7eu from 8 g sodium tetraphenylboron 4Kalignost[>, +.9 ml . 7 hydrochloric acid and +.9 ml water. 0or details see 2. 7eu, %hem. 6er. 13, +,. 4)*9;>. 5ip solution IC Saturated solution of tetraphenyldiboro&ide in petroleum benDine. 5ip solution IIC ) # .I a?ueous solution of a ?uaternary ammonium base 4e.g. $audacit[>. 0rocedureC Dip into 5, dry briefly at room temperature and then dip into 55. Subse?uently dry at room temperature. Literature: 2. 7eu, H. anal. %hem. !-$, 8, 4)*9;>. 2. 7eu, H. anal. %hem. !#!, 8.) 4)*9<>. Chemicals: Sodium tetraphenyl *orate -Eali8nost>. GR ACS, Ord. No. .!333$ 9ydrochloric acid #%' GR, Ord. No. .!!( 3 0etroleum *en,ine GR *oilin8 ran8e "!-3!BC, Ord. No. .! &&% $!/" Tetra+olium blue for corticosterids and other reducing compounds" Spray solutionC (i& freshly before use e?ual parts of ,.9I methanolic tetraDolium blue solution and sodium hydro&ide solution 4c Q < molR$> in water or water#methanol mi&ture. 3iolet spots at room temperature or after short warming.

Literature: B. -damec, Steroids !, ;*9 4)*<8>. T. 0eher, (i"rochim. -cta !)2#, ),9. A. 0reimuth, 6. Hawta, (. 6uechner, -cta 6iol. et (ed. Ger. !$, <.; 4)*<;>. B. 7ishi"aDe, 2. -braham, H. Staudinger, /. 6iochem. 4To"yo> #-, ;.: 4)*<8>. 5.E. 6ush, (. Willoughby, 6iochem. /. 23, <+* 4)*9:>. Chemicals: :etra,olium *lue, Ord. No. .!+ !( Sodium hydro/ide pellets GR ISO, Ord. No. .!3"$+ 4ethanol GR ACS, ISO, Ord. No. .!3!!$ $!!" Thiobarbituric acid for sorbic acid" Spray solutionC Saturated a?ueous solution of thiobarbituric acid. Sorbic acid shows red spots. Literature: /.W. %opius#@eereboom, H.W. 6ee"es, /. %hromatog. !-, ;): 4)*<;>. Chemicals: #-:hio*ar*ituric acid, Ord. No. .!+ +! $!*" Thymol - sulfuric acid for sugars" Spray solutionC Dissol'e ,.9 g thymol in *9 ml ethanol and add 9 ml *:I sulfuric acid with caution. After-treatmentC Heat )9#., min at ).,J%. Sugars show pin" spots. Literature: S. -dachi, /. %hromatog. !3, .*9 4)*<9>. Chemicals: :hymol cryst. e/tra pure 0h )ur, Ord. No. .!+ 3& )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( $!$"' Thymol blue for dimethylamino acids" Spray solutionC Dissol'e ,.,; g thymol blue in a mi&ture of .9 ml )#butanol, .9 ml ethanol and 9, ml sulfuric acid 4c Q ,.,,9 molR$>. Fellow spots on red bac"ground. Literature: 3.(. 5ngram, /. 6iol. %hem. */*, )*8 4)*98>. Chemicals: :hymol *lue indicator ACS, Ord. No. .!+ &3

)thanol a*solute GR ACS, ISO, Ord. No. .!!$+( -7utanol GR, Ord. No. .!$$$! Sulfuric acid !.!!% mol=l :itrisol>, Ord. No. .!$$+# $!-" Tin%II& chloride - hydrochloric acid - --dimethylaminoben+aldehyde for aromatic compounds containing nitro groups" Spray solution IC @repare freshly before use a mi&ture of 8 ml )9I a?ueous tin455> chloride and )9 ml 8:I hydrochloric acid and dilute with )+, ml water. Spray solution IIC Dissol'e ) g ;#dimethylaminobenDaldehyde in a mi&ture of 8, ml ethanol, 8 ml 8:I hydrochloric acid and )+, ml )#butanol. :reatmentC Spray with 5, dry at room temperature and spray with 55. Fellow spots after re#drying at room temperature. Literature: (. /urece", /. %hurKce", 3. %er'in"a, (i"rochim. -cta !)2/, ),.. Chemicals: :in-II. chloride dihydrate GR ACS, Ord. No. .!&+ % "-5imethylamino*en,aldehyde GR ACS, Ord. No. .!(!%+ )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & -7utanol GR ACS, ISO, Ord. No. .! $$! $!#" Tin%II& chloride - potassium iodide for gold ions" Spray solutionC Dissol'e 9.< g tin455> chloride in ), ml 8:I hydrochloric acid. -fter dilution with water to ),, ml, add ,.. g potassium iodide to the solution. 6lac" spots. Literature: 0.H. 6urstall, G.2. Da'ies, 2.@. $instead, 2.-. Wells, /. %hem. Soc. !)#/, 9)<. Chemicals: :in-II. chloride dihydrate GR ACS, Ord. No. .!&+ % 0otassium iodide GR ISO, Ord. No. .!%!"( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & $!2" Tin%I4& chloride for triterpenes' sterols' steroids' phenols and polyphenols. Spray solutionC -dd ), ml tin453> chloride to )<, ml of a mi&ture of e?ual 'olumes of chloroform and glacial acetic acid. After-treatmentC -fter spraying heat the chromatogram 9#), min at ),,J% and inspect subse?uently in daylight and in long#wa'e A3 light. Literature: /./. Scheidegger, E. %herbulieD, Hel'. %him. -cta $1, 9;: 4)*99>.

Chemicals: :in-I2. chloride e/tra pure, Ord. No. .!&+ ! Chloroform GR ISO, Ord. No. .!#""% Acetic acid $3' GR, Ord. No. .!!!3# $!3" Titan yello0 for cadmium ions" Spray solutionC ,.)I a?ueous titan yellow solution. After-treatmentC Spray either with .9I ammonia solution or place the chromatogram sprayed with titan yellow solution into a chamber saturated with ammonia 'apours. Literature: 5.5.(. Elbeih, (.-. -bou#Elnaga, -nal. %him. -cta !3, 8*: 4)*9:>. Chemicals: :itan yelloI -C.I. $%"!. GR, Ord. No. .! (!& Ammonia solution #%' GR, Ord. No. .!%"(# $!1" p-Toluenesulfonic acid for steroids' fla,onoids and catechins" Spray solutionC .,I solution of p#toluenesulfonic acid in chloroform. After-treatmentC -fter spraying heat a few minutes at ),,J%. 5nspect the spots in long#wa'e A3 light. Literature: D.G. 2ou&, 7ature !1/, *:8 4)*9:>. H./. Heitler, /. %hromatog. !1, )+, 4)*<8>. H. Silbermann, 2.H. Thorp, /. @harm. @harmacol. 2, 9;< 4)*9;>. Chemicals: "-:oluenesulfonic acid monohydrate GR, Ord. No. .!$3 ( Chloroform GR ISO, Ord. No. .!#""% $!)" Toluidine blue for acidic polysaccharides" PC" <i/in8 solutionC ., ml 89I formaldehyde solution in +, ml ethanol. Spray solutionC Dissol'e ,.,; g toluidine blue in +, ml acetone and ., ml water. 5ip solutionC 9I acetic acid solution. 0rocedureC @lace the chromatogram )9 min into the fi&ing solution. -fter drying, spray with the spray solution and rinse the e&cess dye first with dip solution, then with water. Literature: D. Hamerman, Science !**, *.; 4)*99>.

Chemicals: <ormaldehyde solution min. (&' GR, Ord. No. .!"!!( :oluidine *lue ! Acetone GR ACS, ISO, Ord. No. .!!! " )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( Acetic acid $3' GR, Ord. No. .!!!3# $*/" Trichloroacetic acid for steroids' Digitalis glycosides' 4eratrum al7aloids and ,itamin D" A. Spray solutionC .9I solution of trichloroacetic acid in chloroform. 7. Spray solution -for 6itamin 5.C )I trichloroacetic acid solution in chloroform. C. Spray solution -for 5i8italis 8lycosides.C Dissol'e 8.8 g trichloroacetic acid in ), ml chloroform and add )#. drops hydrogen pero&ide. After-treatmentC Heat 9#), min at ).,J%. 5nspect the spots in daylight and in long#wa'e A3 light. $iteratureC 6./. -ldrich, (.$. 0rith, S.E. Wright, /. @harm. @harmacol. 1, ),;. 4)*9<>. H./. Heitler, /. %hromatog. !1, )+, 4)*<8>. Chemicals: :richloroacetic acid GR ACS, Ord. No. .!!+!& Chloroform GR ISO, Ord. No. .!#""% 9ydro8en pero/ide (!' 9#O# -0erhydrol>. GR ISO, Ord. No. .!&#!$ $*!" Trifluoroacetic acid for steroids" Spray solutionC )I trifluoroacetic acid in chloroform. After-treatmentC Heat 9 min at ).,J%. Chemicals: :rifluoroacetic acid for synthesis , Ord. No. +.!+#3! Chloroform GR ISO, Ord. No. .!#""% $**" *'-'2-Trinitroben+oic acid for cardiac glycosides" Spray solution IC ,.)I solution of .,;,<#trinitrobenDoic acid in a mi&ture of water and dimethylformamide. Spray solution IIC 9I a?ueous sodium carbonate solution. Spray solution IIIC 9I a?ueous sodium dihydrogen phosphate solution. 0rocedureC Spray with 5, then with 55, heat ;#9 min at *,#)),J%, cool and spray finally with 555. %ardiac glycosides show orange#red spots.

Literature: T. (omose, T. (atsu"uma, F. Bh"ura, /. @harm. Soc. /apan 1-, :+8 4)*<;>. Chemicals: N,N-5imethylformamide GR ISO, Ord. No. .!(!%( Sodium car*onate !-hydrate GR ISO, Ord. No. .!3($ Sodium dihydro8en phosphate GR, Ord. No. .!3("3 #,",3-:rinitro*en,oic acid $*$" *'$'#-Triphenyltetra+olium chloride %TTC& for reducing sugars' corticosteroids and other reducing compounds" Spray solutionC (i& freshly before use one part ;I methanolic TT% solution with one part sodium hydro&ide solution 4c Q ) molR$>. After-treatmentC Heat 9#), min at ),,J%. 2educing compounds show red spots. NoteC TetraDolium blue is more sensiti'e. Literature: 0.G. 0ischer, H. Doerfel, Hoppe#Seylers H. physiol %hem. *)3, )<; 4)*9;>. Chemicals: #,(,%-:riphenyltetra,olium chloride, Ord. No. .!+(+! 4ethanol GR ACS, ISO, Ord. No. .!3!!$ Sodium hydro/ide solution mol=l :itrisol>, Ord. No. .!$$%3 $*-" Tungstophosphoric acid for reducing compounds' lipids' sterols and steroids" Spray solutionC .,I ethanolic solution of tungstophosphoric acid. After-treatmentC Heat at ).,J% until ma&imal 'isualisation of the spots. Literature: H.@. (artin, 6iochim. et biophys. -cta *#, ;,+ 4)*9:>. Chemicals: :un8stophosphoric acid hydrate GR, Ord. No. .!!%+( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $*#" Area - hydrochloric acid for sugars" Spray solutionC Dissol'e 9 g urea in ., ml hydrochloric acid 4c Q . molR$> and add ),, ml ethanol. After-treatmentC Heating at ),,J% promotes reaction. Ketoses and oligosaccharides containing "etoses turn blue. Literature: 2. Dedonder, 6ull. soc. chim. biol. $-, ;; 4)*9.>.

Chemicals: 1rea GR ACS, Ord. No. .!+"+& 9ydrochloric acid #%' GR, Ord. No. .!!( 3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $*2" 4anillin - hydrochloric acid for catechins" Spray solutionC Dissol'e ,.9 g 'anillin in 9, ml 8:I hydrochloric acid. After-treatmentC Dry the chromatogram at room temperature. %atechols show red spots. Literature: E.-.H. 2oberts, 2.-. %artwright, D./. Wood, /. Sci. 0ood -gr. 3, <8: 4)*9:>. Chemicals: 2anillin, Ord. No. .!+% ! 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & $*3" 4anillin - phosphoric acid for steroids" Spray solutionC Dissol'e ) g 'anillin in ),, ml 9,I a?ueous phosphoric acid. After-treatmentC Heat ),#., min at ).,J%. Literature: H. (etD, 7aturwissenschaften -1, 9<* 4)*<)>. Chemicals: 2anillin, Ord. No. .!+% ! ortho-0hosphoric acid +%' GR ISO, Ord. No. .!!%&( $*1" 4anillin - potassium hydroxide for amino acids %ornithine' lysine' proline& and amines" Spray solution IC .I 'anillin solution in .#propanol. Spray solution IIC )I ethanolic potassium hydro&ide solution. 0rocedureC Spray with 5 and heat the chromatogram ), min at )),J%. Brnithine then fluoresces intensi'ely green#yellow in long#wa'e A3 light, lysine only wea"ly green yellow. -fter spraying with 55, heat again in the same manner. Brnithine first shows a salmon colour and then fades, while proline, hydro&yproline, pipecolic acid and sarcosine turn red after se'eral hours. Glycine turns brown#green, the other amino acids faintly brown, Literature: G. %urDon, /. Giltrow, 7ature !3*, 89< 4)*98>.

Chemicals: 2anillin, Ord. No. .!+% ! 0otassium hydro/ide pellets GR, Ord. No. .!%!(( )thanol a*s. GR, Ord. No. .!$&## #-0ropanol GR ACS, ISO, Ord. No. .!$3(" $*)" 4anillin - sulfuric acid for higher alcohols' phenols' steroids and essential oils" A. Spray rea8entC Dissol'e ) g 'anillin in ),, ml *:I sulfuric acid. After-treatmentC Heat the chromatogram at ).,J% until the spots attain ma&imum colour intensity. Literature: E. TyihK", D. 3KguEfal'i, @.$. HKgony, /. %hromatog. !!, ;9 4)*<8>. -.$. le 2osen, 2.T. (ora'e", /.K. %arlton, -nal. %hem. *-, )889 4)*9.>. 7. Spray rea8entC Dissol'e ,.9 g 'anillin in ),, ml of a mi&ture of *:I sulfuric acid and ethanol 4;,V),>. After-treatmentC Heat the chromatogram at ).,J% until the spots attain ma&imum colour intensity. Literature: /.S. (atthews, 6iochim. et biophys. -cta 2), )<8 4)*<8>. Chemicals: 2anillin, Ord. No. .!+% ! Sulfuric acid $%-$&' GR ISO, Ord. No. .!!&( )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( $$/" 4ioluric acid for al7ali and al7aline earth metal ions" Spray solutionC ).9I a?ueous 'ioluric acid solution. 3ioluric acid must not be heated abo'e <,J%. After-treatmentC Heat ., min at ).,J%. Literature: H. Erlenmeyer, H. '. Hahn, E. Sor"in, Hel'. %him. -cta $-, );)* 4)*9)> Chemicals: 2ioluric acid $$!" Ianthydrol for tryptophan and other indole deri,ati,es" Spray solutionC Dissol'e ,.) g &anthydrol in *, ml ethanol and add ), ml 8:I hydrochloric acid freshly before use. After-treatmentC Heat at )),J% until ma&imal 'isualisation of the spots.

Literature: S.2. Dic"mann, -.$. %roc"ett, /. 6iol. %hem. **/, *9: 4)*9<>. Chemicals: Janthydrol Rea8. 0h )ur, Ord. No. .!+3$3 )thanol a*solute GR ACS, ISO, Ord. No. .!!$+( 9ydrochloric acid fumin8 (&' GR ISO, Ord. No. .!!( & $$*" >inc chloride for steroid sapogenins and steroids" Spray solutionC Dissol'e 8, g Dinc chloride in ),, ml methanol and filter off from the insoluble matter. After-treatmentC Heat ) hour at ),9J% and co'er the layer immediately with a glass plate for protection against the influence of moisture. The spots fluoresce in long#wa'e A3 light. Literature: @./. Ste'ens, /. %hromatog. !-, .<* 4)*<;>. Chemicals: Cinc chloride GR ACS, ISO, Ord. No. .!++ 3 4ethanol GR ACS, ISO, Ord. No. .!3!!$ $$$" >inc uranyl acetate for sodium ions" Spray solutionC Dissol'e ), g uranyl acetate in < ml 8,I acetic acid and fill up to 9, ml with water. (i& 8, g Dinc acetate with 8 ml 8,I acetic acid and fill up to 9, ml with water. (i& e?ual 'olumes of both solutions, allow to stand for one day and filter off. NoteC 5nspect in A3 light. Literature: H.H. 6arber, 5.(. Kolthoff, /. -m. %hem. Soc. #/, )<.9 4)*.+>. Chemicals: 1ranyl acetate dihydrate GR, Ord. No. .!+"&( Cinc acetate dihydrate GR, Ord. No. .!++!# Acetic acid $3' GR, Ord. No. .!!!3# $$-" >irconyl chloride - ali+arin - hydrochloric acid for fluorine ions" Spray solutionC Dissol'e ,.,9 g Dirconyl chloride and ,.,9 g aliDarinsulfonic acid sodium salt 4aliDarin red S> in ),, ml hydrochloric acid 4c Q . molR$>. Literature: H. Seiler, T. Kaffenberger, Hel'. %him. -cta --, ).+. 4)*<)>.

Chemicals: Circonium-I2. o/ide chloride octahydrate GR, Ord. No. .!+$ & Ali,arin red S -C.I. %+!!%. GR and indicator, Ord. No. .!3#&$ 9ydrochloric acid #%' GR, Ord. No. .!!( 3 $$#" >irconyl chloride - citric acid for glycosides" PC" Spray solution IC .I methanolic Dirconium453> o&ide chloride solution. Spray solution IIC 9I a?ueous citric acid solution. 0rocedureC Glycosides are first hydrolysed on the chromatogram which has been placed into a co'ered bea"er with boiling .9I hydrochloric acid. -fter drying, spray with 5, dry again and spray 'igorously with 55. Literature: $. Hoerhammer, K.H. (ueller, -rch. @harm. *13, 8), 4)*9;>. Chemicals: Circonium-I2. o/ide chloride octahydrate GR, Ord. No. .!+$ & Citric acid monohydrate GR ACS, ISO, Ord. No. .!!#"" 9ydrochloric acid #%' GR, Ord. No. .!!( 3 4ethanol GR ACS, ISO, Ord. No. .!3!!$