Lightcolouranddifferentdyestuffs

Dyes and bonding

Thecomponentsinvolvedinhistologicalstainingaredyesandproteins.
Thefundamentalprocessinvolvedisthechemicalbondingbetweenthecarboxylgroupsofone
andtheaminogroupsoftheother.
Thecommonestbondsinvolvedareionicbonds,althoughthereareexceptions

What dyes are

Dyesarecoloured,ionising,aromaticorganiccompounds.

Itmustbeappreciatedthattheyareindividualchemicals,andlikeallchemicals,theyare
similarintheirreactionstosomeotherchemicals,anddistinctlydifferentfromothers.

Itmayseemthatthisisastatementoftheobvious,butwesometimesappeartoviewdyes
assomethingotherthanordinarychemicals,andIwanttostressthatthesamerulesthat
applytosodiumchloride,aceticacid,benzidine andahostofothersalsoapplytodyes.

What is a colour

Dyesarearomaticorganiccompounds,andassucharebasedfundamentallyonthestructure
ofbenzene. Tous,benzeneappearstobeacolourlessfluid.Infactitabsorbs
electromagneticradiationjustasdyesdo,butitdoessoatabout200nmsothatwedonot
seeit.

Theperceptionofcolourisanabilityofsomeanimals,includinghumans,todetectsome
wavelengthsofelectromagneticradiation(light)differentlyfromotherwavelengths.Normal
daylight,orwhitelight,isamixtureofallthewavelengthstowhichwecanrespondand
sometowhichwecannot,inparticulartheinfraredandultravioletrays.Werespondto
wavelengthsbetweenabout400700nm.Whenanobjectabsorbssomeoftheradiation
fromwithinthatrangeweseethewavesthatareleftover,andtheobjectappearscoloured.
Inrealitythisrangeweseemakesuponlyaverysmallfractionoftheelectromagnetic
spectrum.

How is color descibed?

Inscientifictermsthereisnothingspecialaboutthewavelengthsinthevisiblerange,otherthanbeing
themajorcomponentsofsunlightwhicharenotremovedbytheearth'satmosphere.Theirspecial
importanceisbasedexclusivelyontheabilityofhumanretinastorespondtothem,andtodiscriminate
betweenthemtoasignificantdegree.Thesediscriminationsarewhatwecallcolour.

Wavelengthsjustoutsidethevisiblerangeareconsideredcolourless,eventhoughthereisno
substantivedifferencebetweenthemandthelimitingwavelengthsinsidetherange.Someanimals
(bees,forexample)canseetheseotherwavelengthsbut,becausehumansdonot,weconsiderthem
colourless.Thepointisthatcolourisasubjectivephenomenon,andthinkingofcolourassomething
objectiveismisleading.Forthatreason,weshouldrefertothewavelengthsinvolvedratherthan
describethehumanresponsetothem.

Why dyes are coloured

ColourindyesisinvariablyexplainedasaconsequenceofthepresenceofaChromophore.Since,by
definition,dyesarearomaticcompoundstheirstructureincludesarylringswhichhavedelocalised
electronsystems.

Theseareresponsiblefortheabsorptionofelectromagneticradiationofvaryingwavelengths,
dependingontheenergyoftheelectronclouds.

Forthisreason,chromophores donotmakedyescolouredinthesensethattheyconferonthemthe
abilitytoabsorbradiation.Rather,chromophores functionbyalteringtheenergyinthedelocalised
electroncloudofthedye,andthisalterationresultsinthecompoundabsorbingradiationfromwithin
thevisiblerangeinsteadofoutsideit.

Oureyesdetectthatabsorption,andrespondtothelackofacompleterangeofwavelengthsbyseeing
colour.

Visible range

Wavelengthsjustoutsidethevisiblerangeareconsideredcolourless,eventhoughthereisno
substantivedifferencebetweenthemandthelimitingwavelengthsinsidetherange.Someanimals
(bees,forexample)canseetheseotherwavelengthsbut,becausehumansdonot,weconsiderthem
colourless.Thepointisthatcolourisasubjectivephenomenon,andthinkingofcolourassomething
objectiveismisleading.Forthatreason,weshouldrefertothewavelengthsinvolvedratherthan
describethehumanresponsetothem.

Whensomeofthewavelengthsfoundinwhitelightareabsorbed,thenweseewhatisleftoveras
colouredlight.Thecolourthatweseeisreferredtoasthe complementarycolourof thecolourthatwas
removed.Forinstance,iftheredraysareremovedfromwhitelight,thecolourwedetectisbluegreen.
Bluegreeniscomplementarytored,andrediscomplementarytobluegreen.

Chromophore
Chromophores areatomicconfigurationswhichcanaltertheenergyindelocalisedsystems.Theyare
composedofatomsjoinedinasequencecomposedofalternatingsingleanddoublebonds.Double
bondsinorganiccompoundscanbeoftwotypes.Iftheatomswithdoublebondsarenotadjacent,they
aretermed isolated doublebonds,andexistindependentlyofotherdoublebondsinthesame
molecule.Ifadjacentatomshavedoublebondstheyaretermed conjugated doublebondsandthe
bondsinteractwitheachother.Chromophore configurationsoftenexistasmultipleunits,having
conjugateddoublebonds,andaremoreeffectivewhentheydoso.Thisisduetotheinteraction
betweenthedoublebonds,whichcausespartialdelocalisationoftheelectronsinvolvedinthebonds.In
thiscase,althoughspecificatomsareinvolvedinthebonds,theelectronsaredistributedoveralarger
areathanthespecificatomsandalsoinvolveadjacentatomsthathavedoublebonds.

Conjugated systems

Thepointofthisisthatconjugatedsystemshavepartiallydelocalisedelectrons,andtheenergyinthese
delocalisedelectronscanimpactontheenergyofthedelocalisedelectronsoftheparentaromatic
compoundbyextendingthenumberofelectronsinvolvedinthesystemandtheenergyneededtokeep
thewholesysteminplace.

Conjugateddoublebonds

Anothercommonchromophore isthenitrogroup.Thischromophore isanitrogenwithtwooxygen

atomsattached.Oneoxygenisshownattachedwithasinglebond,theotherwithadoublebond.Infact,
likethecarbonatomsinbenzene,thesetwooxygenatomsareattachedtothenitrogenwithbondsof
equalstrength.Theextraelectronsaredelocalisedbetweenthethreeatoms.

Complementary Colours
Removed

Observed

Removed

Observed

Violet

Yellowgreen

Yellowgreen

Violet

Blue

Yellow

Yellow

Blue

Cyan

Orange

Orange

Cyan

Bluegreen

Red

Red

Bluegreen

Green

Purple

Purple

Green

Auxochromes

Auxochromes aregroupswhichattachtononionisingcompoundsyetretaintheirabilitytoionise.While
thisdefinitionislargelycorrect,itisalsoinadequate.Thisisbecauseitrestrictsthedefinitionofthe
auxochrome toionisation,anddoesnotcommentontheeffectofauxochromes ontheabsorbanceof
theresultingcompound.

Theword auxochrome isderivedfromtworoots.Theprefix auxo isfrom auxein,and

means increased. Thesecondpart, chrome means colour, sothebasicmeaningoftheword
auxochrome is colourincreaser. Thiswordwascoinedbecauseitwasnotedoriginallythattheaddition
ofionisinggroupsresultedinadeepeningandintensifyingofthecolourofcompounds.

Colour enhancing by an auxochrome

Totheleftis naphthalene,acolourlesscompound.

Theadditionofasinglehydroxylgrouptonaphthaleneproduces 1
naphthol whichisalsoacolourlesscompound,butonewhichcanionise.

Ifinsteadofahydroxylgroupweaddthenitrogroup,whichisa
chromophore,wegetthecompound 2,4dinitronaphthalene.Theaddition
ofthischromophore hascausedittobecomepaleyellow.

Ifinsteadofahydroxyl or nitrogroups, both ahydroxyland nitrogroupsare

added,wegetthedeepyellowdye, martius yellow.

Theadditionof both anauxochrome and achromophore resultsinamuchstrongeralterationoftheabsorption

maximumofthecompound.Thehydroxylgroupmusthavedeepenedthecolour,showingthatauxochromes ar
alsochromophores.

Trinitrobenzene
Trinitrobenzene,althoughcoloured,isstillnotadye,asitwillnotbindtotissues.Treatingthesection
withtrinitrobenzene willtemporarilycolourityellowinthesamewaythataplasticspongeappears
colouredwhenitissoakedinacolouredliquidbutthecolourwillwashoutassoonasthetissueis
rinsedinasolvent.
Toturnacolouredcompoundintoadyerequirestheadditionofanionizable groupthatwillallow
bindingtothetissues.Suchbindinggroupsarecalledauxochromes.
Theadditionofanionizable OHgroupturnstrinitrobenzene intothedyetrinitrophenol,whichismore
commonlycalledpicricacidinhistology.Picricacidisanaciddye(theOHgroupisphenolic andionizes
bylosingahydrogenion)andisveryuseful

Colour

Thecolourofthedyeiscausedbytheabsorbanceofelectromagneticradiation.Wehaveconstantly
referredtothe wavelengththatisabsorbed inthesingular,butasimplescanofadyesolutionwitha
spectrophotometershowsthatdyesdonotremoveasinglewavelength.Rathertheyabsorbradiation
oneithersideofthewavelengthmostcompletelyremoved(theabsorptionmaximum).Plottingthe
wavelengthabsorbedagainstthedegreeofabsorbanceusuallyresultsinadisplayresemblingabell
curve.Ifanypartofthiscurveisinthevisiblerange,thedyewillappearcoloured.

Whitelightisamixtureofwavelengths.Someofthesehavearelationshiptotheenergyinthe
delocalisedelectroncloudofthedyemolecule.Bytheprocessofresonance,previouslydescribed,the
electroncloudwillrespondtotheenergycontainedinthatradiationbyabsorbingit,andremovingit
fromthespectrum.Asaconsequencethewhitelightwillceasetobewhiteandwilldisplaythecolours
ofthewavelengthsleftover.Thetransmittedlightwillhavethecomplementarycolourtothe
wavelengthsremoved.

Structure and colour depend

Theexplanationoftherelationshipbetweenstructureandcolourdependsonthebasicatomicstructure
ofthearylring,andthesharedordelocalisedelectronsthatthisatomicarrangementhas.Theabilityto
absorbradiationisinherentinthisstructure.Theeffectofotheratomicconfigurationsistomodifythe
energycontainedinthedelocalisedelectroncloudsothatthecompoundabsorbselectromagnetic
radiationatawavelengthinthevisiblerange.Somealsoionise,enablingthecompoundtochemically
reactwithionisingtissuegroups.

Colour,fading,fluorescenceandphosphorescenceareallseentobedifferenteffectsofthesame
fundamentalprocess.

Effect of light

Whenlightilluminatesadye,someofitisabsorbedasenergy.Sinceenergyisnotdestroyed,something
mustthenhappen.Wecoulduseananalogyofheatingwater thewater'stemperaturerises,molecular
vibrationincreases.However,wedonotseeanythingelse,asthewaterjustsitstherebeingwater.

Thesamecanhappenwithdyes,wemaynotobserveanythingparticularastheeffectmaybeatthe
atomiclevel.Thereareseveralpossibilities,however.

Theenergylevelintheelectronsinanunaffecteddyeiscalledthegroundstate.Whenelectromagnetic
radiation,aslightenergy,isabsorbedtheelectronsbecomemoreenergised.

Withmostdyes,thereisthenagradualdecayandtheelectronsreturntothegroundstate.Wedonot
seeanything.Nevertheless,somethingmayhappenthatwedonotsee.Possiblythereisanincreasein
temperature,orsomechemicalchangesoccurwhichdisruptthedye'sstructureandcauseittolose
colour fading.

Effect of light

Anotherpossibilityisthatthereturntothegroundstateisnotgradual,butsudden.Ifthisis
accompaniedbyemissionofanyresidualenergyintheformoflight,weobservethedyeglowing
fluorescence.Sincetheemittedlightmustalwayscontainlessenergythantheabsorbedlight,assome
wasusedtoenergisetheelectrons,theemittedradiationisalwaysatlongerwavelengthsthanthe
absorbedradiation.Bymanipulatingthelightavailable,wecancauseultravioletlighttobeabsorbed
andvisiblelighttobeemitted.

Athirdpossibilityisthattheelectronsstabiliseintheirnewlyenergisedstate.Afterapassageoftime
theythenreturntothegroundstate.Ifthishappensgradually,wemayobservenothing,withthesame
possibilitiesregardingfadingandtemperatureincreaseasbefore.

Ifreturntothegroundstatehappenssuddenly,andtheresidualenergyisemittedaslight,weonce
againseethedyeglowing phosphorescence.Aswithfluorescence,thelightemittedisalwaysalonger
wavelengththanthelightabsorbed,butthedisparityisgreaterwithphosphorescenceduetothe
greaterenergyconsumedinkeepingtheelectronsintheexcitedstate.

Notethatthedifferencebetweenfluorescenceandphosphorescenceisinwhethertheelectrons
stabiliseintheexcitedstatebeforereturningtothegroundstate.Withanystability,nomatterhowlong
(orshort),itisconsideredphosphorescence.

Color Modifiers

Colourmodifierssuchasmethylorethylgroupsalterthecolourofdyesbyalteringthe
energyinthedelocalisedelectrons.Bythemselvestheycannotdothisenoughtocause
absorptioninthevisiblerange,buttheycanaffecttheshadesignificantlywhenabsorptionis
alreadyinthatrange.Addingmoreofaparticularmodifierresultsinaprogressivealteration
ofcolour.Compoundsthatdifferfromeachotherinthiskindofregularfashionare
called homologues.AverygoodexampleisseenwiththeMethylvioletseries.

Alteration of colour by modifiers

Withoutanymethylgroupstheparentdyeis
calledpararosanilin andisred.

Whenfourmethylgroupsareaddedwegetthe
reddishpurpledye methylviolet.

Asmoremethylgroupsareaddedwegetthepurple
bluedye crystalviolet whichhassixsuchgroups.

Ifaseventhmethylgroupisadded,theresultingdye
ismethyl green.

Resonance

Theprocessbywhichelectronsarestimulatedbyradiationis resonance.Itshouldbemadeclearatthe
outsetthatresonanceisnotthesameasvibration.

Resonanceistheinductionofaresponseinoneenergysystemfromanotherenergysysteminclose
proximity,whichisoperatingatthesameenergylevel(frequency).Inaromaticorganiccompounds,
includingdyes,thetwoenergysystemsare electromagneticradiation,andthe delocalisedelectron
cloud.Donotconfuseresonancewiththeimaginary resonancehybrids usedinanolderexplanationfor
thestructureofbenzene.

Dyesareproducedmainlyforindustrialusessuchastextiledyeing,soawidevarietyofdifferentdyeshave
beensynthesizedtogivealargerangeofcolours.Dyemanufacturersusuallygivethedyestheyproduce
commonnamessuchaseosinorCongoredratherthantheirfullchemicalnameandsomeofthesenames
arecopyrighted.The dyestructureshownbelowillustratesthecomplexityofmanyofthesedyes.Thefull
chemicalnameofthisstructureis3,3((biphenyl)4,4diylbis(azo))bis(4amino1naphthalenesulphonic
acid)disodiumsalt,whilst itscommonnameisCongored.Thecommonnameiseasiertorememberandto
say,somosthistologistssticktousingthese.

The Colour Index

Toovercomealloftheconfusionthereisastandardlistofalldyes,theirsynonymsandtheirstructures.This
iscalledtheColourIndex(CI).ThisisamonumentalworkofreferenceproducedbyTheSocietyofDyersand
Colouristsandeachdyeisgivenanindividualnumberandlistedalongwithitsname(s)andproperties.Since
eachdyeonthelisthasauniquenumbertoidentifyit,thislististhemostreliablewayofidentifyingadye.
Whennamingadyeinthedescriptionofatechniques,theCInumbershouldbegiventoavoidambiguity,
e.g.eosinY(CI45380).CInumbersarearrangedaccordingtotheirstructure,withthemostimportant
featurebeingtheirchromophoric group.Forexample,allnitroso dyeshavenumbersbetween10000and
10299,nitrodyeshavenumbersbetween10300and10999,monoazo dyeshavenumbersbetween11000
and19999,andsoon.There are31groupsinall,withCInumbersupto78000.Notallofthesegroups
includeimportanthistologicaldyes;afewofthemoreimportant
groupsarelistedbelowwithexamplesofhistologicaldyesfromthegroup.

Nitrodyes.ThesehavethenitrogroupNO2asthechromophore,e.g.picricacid,martius yellow.

Azo dyes.ThesehavetheN=N group(azo)asthechromophore,e.g.orange G

Triaryl methane dyes

Triaryl methanedyes.Theseincludethequininoid arrangementastheactualchromophore.The

quininoid ringbutsinceallthreebenzeneringsareequivalenttherecanberearrangementofthebonds
andanyofthebenzeneringscouldtakeupthisarrangement.

Therearealargenumberofdyesusedinhistologythatfallintothiscategory;afewexamplesare
fuchsins,methylviolet,methylblueandanilineblue

Anthraquinone.Herethequininoid ringisseenasthemiddleofthethreefusedrings.Examplesare
alizarinandcarmine.

Xanthene.Herethequininoid ringistherighthandoneofthethreefusedringsandtheringistilted
comparedwiththepreviousexample.Examplesincludeeosinandxanthene.

Thiazine.Thisisverysimilartothepreviousexampleinoverallstructure,butthemiddleringnowhasS
andNasconstituentatoms.Thisgroupcontainsmanyimportantmetachromatic dyes,suchastoluidine
blue,methylene blueandazureA.

Basic Dyes

Basicdyesarecationicandwillstainanionicoracidicmaterialssuchascarboxylates,sulphates(many
complexcarbohydratesaresulphated)andphosphates(particularlythephosphatesinnucleicacids).
Mostareusedasnuclearstainsandstainingofcytoplasmic carboxylgroupsisdeliberatelysuppressed
byusingaslightlyacidpH. Acidicsubstancesthatstainwithbasicdyesaretermedbasophilic.

Acidic and Neutral dyes

Acidicdyesareanionicandwillcolor cationicorbasicgroupssuchasaminogroups.Mostareusedto
stainproteinsinthecytoplasmandconnectivetissues.Substancesthatstainwithaciddyesarecalled
acidophilic.

Neutraldyesaresimplycompoundsofbasicandaciddyes.Inthiscase,bothionsarecoloured.Suchdye
complexeswillstainbothnucleusandcytoplasmfromasingledyebath.Romanowsky stainsareneutral
dyesmadefrommorecomplexmixtures.Thesearethecommonestdyesusedinhaematology.Theyare
lesscommoninhistologybutstillveryusefulandincludeGiemsa,Leishman andWrightsstains.

Amphoteric dyes

Amphoteric dyesalsohavebothanionicandcationicgroups,buttheseareonthesameion.

Amphoteric dyeshavebothpositivelychargeablegroupsandnegativelychargeablegroupspresenton
themolecule.Dependingonthechargeactuallypresent,thesedyesmayinteractaseitherpositively
chargedionsbasicdyes ornegativelychargedionsaciddyes

Natural dyes
Naturaldyesaresimplydyesubstancesextractedfromnaturalsources.Althoughthemainsourceof
dyesforearlytimes,theyhavelargelybeenreplacedbysyntheticdyes,whichareusuallymorereliable,
cheaperandcanbesuppliedmorereadily.Naturaldyesstillinuseincludehaematoxylin,carmine,orcein
andlitmus,althoughsyntheticvarietiesarealsoavailableforsomeofthese.