DEVELOPMENT OF RESIN-BOUND REACENTS FOR ANALYSIS OF CARBO XYLIC ACIDS AND AMINES BY ADEWUYI, GREGORY OLUWAFEMI YJV B .S c . (H o n s ) , M .Sc. (A n a ly t ica l C h em is try ) Ibadan A DISSERTATION IN THE DEPARTMENT OF CHEMISTRY, TO THE COLLEGE OF SCIENCE AND TECHNOLOGY I I ^ P A R T IA L FULFILMENT OF THE REQUIREMENTS FOR AWARD OF MASTER OF PHILOSOPHY (M .P h il.) OF THE UNIVERSITY OF iB A D A N , NIGERIA DECEMBER, 1993 ii C E R TIF IC A T IO N T h is is to c e r t i f y th a t M r. G re g o ry Oluwafemi A d ew u y i, a p o s tg ra d u a te S tudent o f the Departm ent o f C h em is try ( A n a ly t ic a l ) , College o f Science and T ech no logy , U n iv e rs i ty o f Ibadan , c a r r ie d O .O .P . Faboya B .S c . Ph .D . O .R . Idow u, B .S c .(H o n s ) ( Iba dan ) M .Sc. Food Sc. ( ieeds) ( Ibadan) P h .D . (S t ra th c y ld e ) I ACKNOWLEDGEMENT Special thanks to the fo llow ing people who rende red th e ir t ime, love and u n d e rs ta n d in g to make th is research w o rk a success. D r . O .R . Idowu, fo r his hea lthy and in te l le c tu a l ly e leva ting s u p e rv is io n . . . . ....................................... / > $ D r . Iweibo and Professor Faboya fo r th e ir o b je c t iv i ty and classical m aste r-p iece o f in te g r i t y . D r . A t i r i , " b r i l l i a n t b u t co n tro v e rs ia l N iy i " , in s p ir in g Oye, fo r in s e r t in g the word courage in to my d ic t io n a ry . D r . O bi, "c le v e r Pau l" , seasoned Segun, fo r many happy hours o f acacemic b ra in s to rm in g . D r . Sarum i, O m agbem i'^ / 'Sho lay fo r b r i l l ia n t d iscuss ion d u r in g social g a th e r tn g s . H a rd w o rk in g and a r t ic u la te Lawal fo r immense f inanc ia l c o n i r ib u t io n d u r in g many times o f need. T im eyin my w ife fo r he r love, immense assistance and s in c e r i t y . Tope, T os in , Yewande my c h i ld re n who have g iven me so m u c h \ ^ t h e i r time by m ainta irnng minimum sta te o f d is o rd e r at home. My m o th e r- in - la w (D r.(N '**s .) V .A . O fuya ) fo r se t t ing the pace in the search fo r know led^e . IV My p a re n ts , A .O . Adewuyi and Late M rs . V . A . Adew uyi fo r t h e i r re s i l ie n t pa tience, moral, f inanc ia l and s p i r i tu a l S u ppo rt. M r. Daramola fo r encourag ing me to t r iu m p h th ro u g h reason and de te rm ina t io n . Faleye's and Adeosun 's fo r re n d e r in g adv ice wheneve» re q u i re d . P ro fessor G .B . Ogunmola (Head o f D epartm ent o f C hem is try ) fo r a llow ing the d isse rta t io n to be ob jec t ive ly exarnined w ith an unb iased dec is ion . F ina lly to Cod th a t g ives me the gu idance to know when to hold on and when to let go and grace to make the r ig h t decision w itn d ig n i ty . w & DEDICATION To the memory o f My M other, M rs . V . A . Adewuyi and My G randm other, Mama Rachel Adeoye. v\ EPIGRAM T c jg h times do not las t, b u t Tough people do. & & VII ABSTR AC T R e s in -b o u n d -re a g e n ts made up o f io n ic a l ly -b o u n d 2- naphtha lene-m ethano l and cova len tly bound and sod ium -benzoxazo le- 2-s u lfo n a te have been synthes ized and u t i l ize d as ana ly t ica l reagents fo r the p re -c h ro m a to g ra p h ic d e r iva t iza t ion o f f a t t y ac ids l i . e . acetic , la u r ic , c a p r ic , hexano ic , octanoic, nonanoic, p a lm it i j^ t ip c o s a n o ic l in o le ic ) , 1° and 2° a l ip h a t ic amines and amino acids r e s p e c t iv e ly . The reagents have been designed to con ta in f luo rescen t moieties a tta ch °d to the anionic and ca t ion ic res in backbones th ro u g h su lfona ted es te r l inkages . These moieties im parted UV and FL de te c to r p ro p e r t ie s to the f ina l d e r iv a t iv e s . The d e r iv a t iz a t io n reactions were pe rfo rm ed be fo re the th in - la ye r Chrom atographie separa tions. S tandards we,-e p re p a re d and were used in m on ito r ing the ex ten t o f reactions on the res in s u p p o r t . The de r iva t iyeV ^w ere chrom atographed and f lu o re sce n t spots were obse rved u n d e r UV l ig h t . These so lid phase de r iva t iza t ions have led to p re l im in a ry in v e s t ig a t ip n o f these two func t iona li t ies be fo re em bark ing on in s t^ m e r f ta t io n ana lys is such as h igh perfo rm ance l iq u id chrom ato - e iraphy w itn UV o r fluorescence detection and HPLC-MS Identification. TA B LE OF CONTENTS Page T IT L E PACE C E R TIF IC A T IO N ACKNOWLEDGEMENT D~D IC ATIO N EPIGRAM A c jTR ACT TABLE OF CONTENTS L :ST OF FIGURES LIST OF TABLES CHAPTER ONE: INTRODUCTIO N 1 1.1 D e r iva t iza t ion in Homoqer.eous Media 2 1 . 2 D e r iva t iza t io n in /T lP le rogeneous Media w ith Resin Eound R^agent 3 1.3 R es in -B ound Reagents 4 mers . . . 5 3 \ < ^ mer P rope rt ie s o f Polymers 8 - P o lym er-S uppo r ied Reagents 12 lo n ic a l ly bound reagents 13 1 .3 .3 .2 C a tion -exchange res ins 13 1 .3 . . 3 An ion exchange res ins 17 TT3.3.1 ix Page 1 .3 .3 .4 C ova len t-bound reagents 18 1 .3 .3 .5 Func tiona liza t ion o f p o ly s ty re n e 18 1.4 A na lys is o f C a rb o xy l ic Acids . . . ^ 2 4 1.5 A na lys is o f Amines . . . . . . ^ 26 1.5.1 Methods o f Analys is ... * „ 1 .5 .2 Dansy la tion Reaction 28 1 .5 .3 O the r Reagents V 29 1.6 Aim and O bjec t ive . . . 35 CHAPTER TWO: EXPERIMENTAL « A / 36 2.1 Prepara tion o f Sod ium -Benzoxazo le-2- Sulphonate 36 2 . 1.1 P repa ra tion o f 2-mercaptobenzoxazole 36 2 .1 .2 P repara tion o f 2 -ch lo robenzoxazo le and sod ium -benzoxazo le-2-s u lp h o n a te . . . 37 2 . 2 Prepara tion o f R es in -B ound Benzoxazo le-2- Su lfonate 38 2 . 2 . 1 te rm ina tion o f exchange capac ity o f n ion -exchange res in (anion exchange res in ( (CI) form . . 38 T i t ra t io n o f e f f lu e n t (NaCI) aga inst Standard 0.1M s i lv e r n i t ra te . . 39 2 .2 .3 C oup ling sod ium -benzoxazo le-2- su lfonate w ith res in Ic h lo r id e fo rm ) . . 40 2.3 Reaction o f Amines w ith Sodium -Benzoxazo le-2- Su lfonate . . . . 43 X Page 2.4 O ptim iza t ion o f So lvent, Tem pera tu re and Time 44 2.5 D e r iva t iza t ion o f Amines w ith R es in -Bound Benzoxazo ie-2-Su lfonate . . . . . . 44 2.6 Reaction o f Amino Acids w ith Benzoxaxo le -2 - Su lfonate . . . • • • / 2.7 D e r iva t iza t ion o f Amino Acids Using Resin-Bound^^^^* Benzoxazo le-2-Su lfonate . . . 45 2.8 D e r iva t iza t ion Using Resin, Sodium Benzoxazo le-2- Su lfonate , Amine Substra tes v v . . . 46 2.9 D e r iva t iza t ion Using Resin, Sodium Benzoxazo le- 2 Su lfona te , Amino Acids S ubs tra tes . . . 46 2 10 T h in -L a y e r Chrom atograph ie Ana lys is o f Amine D e r iva t ives Obta ined T h ro u g h Homogeneous Reaction . . / V \ . . . 47 2.11 T h in -L a y e r Chrom atography yo f Amino Ac id D e r iva t ives Obta ined from Homogeneous Reaction 47 2.12 T h in - - .a y e r Chrom atography o f Amine D e r iva t ives Obta ined T h ro u g h Heterogeneous Reaction . . . . . . 48 2.13 T h in -L a y e r C hrom atography o f D e r iva t ives from Resin (C hhöride fo rm ) , Sodium Benzoxazole-2 Su lfonate and Amine Substra tes i .e . D ie th y ja m ire , D i-n -b u ty la m in e , D i-n -p ro p y la m in e , 4 n i t iy ^ n u in e and B lank . . . 48 2.14 T h i n - L a y e r Chrom atography o f R es in -Bound D e r iva t ives o f Lys ine , C lys ine , C ys te in and B lank . . . . . . 40 2.15 H P LC -U V -F L Ana lys is . . . . . . 49 2.16 Mass Spectrom etr ic Ide n t i f ic a t io n . . . 49 XI Page 2.17 P repara tion o f 2-C h lo rom e thy l Naphthalene . . . 50 2.18 P repara tion o f 2 -Naphtha lene Methanol . . . 50 2.19 P repara tion o f R e s in -S uppo rted S u lph ony lch lo r id e J ; ' 2.20 P repara tion o f Resin Bound Naphthalene Methanol 51 2.21 Reaction o f C a rb o x y l ic Acids w ith 2 -C h lo rom ethy l Naphthalene . . . 52 2.22 D eterm ina tion o f C a rb o x y l ic Ac ids w ith Resin- Bound Reagents . . . v V 55 2.23 T h in -L a y e r C hrom atograph ie A na lys is o f Esters 56 2.24 M elt ing Point De term ination o f S tandard Ester D e r iva t ives . . . . . . 57 2.25 T h in -L a y e r C hrom atograph ie Ana lys is o f D e r iva t ives Prepared Using the Resin-Bound Reagent / . . . . . 37 2.26 T h in -L a y e r C hrom atograph ie Analys is o f S tandard Ester D e r iv a t iv e s and Each C orrespond ing D e r iva t ives O bta ined from R es in-Bound Reaction . . . . . . 58 CHAPTER THREE: RESULT AND DtSCUSSION 3.1 Syn theV jsvo f S odium Benzoxazo le-2-Su lfonate . . . 59 3 * O bse rva tion o f sodium-benzoxazole so lu tion und e r UV . . . 65 E ffec t: o f reaction o f amines w ith the re s in -o o u n d benzoxazo ie-2-su lfona te 67 3 .1 .3 T h in - la y e r ch rom atography o f homoge- neous reaction p ro d u c t and comparing w ith d e r iv a t iv e s from res in -bo und reagen t . . . . . . 81 XII Page 3.1 .4 C onduc t ing reactions th ro u g h one- way process . . . . . . 83 3 .1 .5 E ffec t o f reaction o f amino acids w ith sodium benzoxazo le-2-s u lfo n a te . . . 83 3.2 Prepara tion o f 2 -C h lo rom e thy lnaph tha lene . . . 3.2.1 T h in - la y e r Chromatographie ana lys is (T L C ) o f es te r d e r iv a t iv e s / T , * 87 3.2 2 PreDaration o f 2 -naph tha lene methanol 89 3 .2 .3 P repara tion o f re s m -s u p p o r te d su lphon y l Chloride and coup lm g w ith 2 -naph tha lene methanol . . . 89 3 .2 .4 T h in - la y e r ch rom atography o f re s in - bound d e r iv a t iv e s • • • 91 3 .2 .5 RF values o f S tandard es te r d e r iv a t iv e s and re s in -b o u n d d e r iv a t iv e s . . . 92 CHAPTER FOUR: CONCLUSION 95 96 XIII LIST OF FIGURES Figure Page 1.0 R es in -bound reagent , 1.1 T rans fo rm a t io n o f s ty ren e to p o ly s ty re n e 1 . 2 Condensation reaction between a dialcoho and an o rg a n ic d iac id 1.3 L inear po lym er e .g . po lyethene 1.4 L inear a l te rn a t in g copolymer e .g ny lon 6 . . . 1.5 M inor c ro s s - l in k e d polymer e .g vu lcan ised ru b b>eerr . . . S W • 1.6 M assive ly cross - l in k e d polymer e .g .u re a form a ldehyde 8 1.7 Iso tac tic , syn d io ta c t ic , a: tac t ic c o n f ig u ra t io n s o f a po lym er chain . . . 10 1.8 Equations fo r exchange capac ity . . . 14 1.9 P repara tion o f cation exchanger es in . . . 15 1.10 Exchange process o f ion exchange res in 16 1. 10a splacement o f potassium ions by gnessium ions . . . . . . 16 1.11 ^ S t r u c t u r e o f an an ion-excnange res in . . . 17 1.12 T rea tm en t o f c h lo ro m e thy lpo lys ty rene w ith nu: leophiles . . . . . . 21 1.13 Phase t^a n s fe r process in v o lv in g ch lo rom e thy l p o lys ty ren e 21 xiv Fiqure Page 1 . 1» Prepa ra tion o f po lym eric reagents by s ing le Step F r ie d e l-C ra f ts a lky la t io n o f p o ly s ty re n e . . . 22 1.15 Prepa ra tion o f po lym eric su lfona te es ters and tozy laz ide from ch lo rosu lfona ted p o ly s ty re n e . . . . . . 22 1.16 Prepa ra tion o f po lym eric reagents from metalated p o ly s ty re n e in te rm ed ia te r 23 1.17 S t ru c tu re o f po lym eric a n h y d r id e co ing O -ace ty l as the labe ll ing moiet^v . 31 1.18 D e r iva t iza t io n reaction o f p r im a ry and secondary amines w ith a po lym e r-bou nd a n h y d r id e reagent . . . < Y 31 1.19 Polymer- 3 - m t ro -4 ( ( 9 - f Iu o re n y lm e th o x y ) - c a r b o x y l ) - o x y benzophenone . . . 33 1.20 D e r iva t iza t io n o f typ ic a l amines w ith the po lym er bound n itrobenzophenone ac t iva ted es te r v J • • • . . . 33 1 . 21 S t ru c tu re o f po lym er bound 4 -h y d r o x y I -3 - n itrobenzophenone con ta in ing FM OC-L- P ro l ine . . . . . . 34 1 . 2 2 Equation showing the exchange of Chloride lon by n itra te ion in an anion exchange Yesion 39 S^Ve ( exchange capac ity 40 ” 5 ............ ........... * 1.24 B '^n k c o r re c t io n . . . 42 1.25 C oup ling o f benzoxazoie w ith a.iion exchange res in . . . 42 XV Figure Page 1.26 Equation o f reaction between c a rb o x y l ic acids and ch lo rom e thy lnaph tha lene . . . 54 1 . 2 7 Equation to show the reaction o f 2- ch lo rom e thy l naphthalene w ith la u r ic acid 54 1 . 2 8 S tru c tu re o f benzoxazole 2 1 . 2 9 Reaction between aldehydes and compou, n d 5 w ith ac t ive methylene g roups in the presence o f an o rgan ic base 62 1 30 Reaction o f methyl g roups in ketones w ith p y r id in e s . . . _ \ . . . 63 1 . 3 1 Equation fo r p repa ra t ion o f sodium- benzoxazo le-2-su lfona te 64 1 . 3 2 Equation showing the add it ion o f n - bu ty lam ine to so lu tion c f benzoxazole to g ive f luo resce n t p ro d u c t . . . 65 1 . 3 3 Equation fo r po lymer suppo rted benzoxazole moiety ( a n a ly t ^ ^ / r e a g e n t ) . . . 66 1.34 Eauation fo r d e r iva t iza t io n o f amine on res in s u p p o r t . . . . . . 68 1.35 I l lu s t ra t io n o f reaction o f sodium b^hzoxazo le -2-su lfo n a te w ith amines (homögeneous approach) 70 1.35a- ass spectra o f 2 - (N , N -d ia lky lam ino ) benzoxazole formed from the reaction o f d ia lky lam ines w ith benzoxazo le-2-su fonate 7 5 - 7 7 Mass Spectrum o f benzoxazole-2-su lfo n a te 78 1.35e A chrom atogram o f a reaction m ix tu re o f amines w ith sodium benzoxazole-2- su lfona te . . . 79 XV Figure Page 1 .35f Z w it te r io n i l lu s t ra t io n in amino acid d e r iv a t iv e s . . . . 79 1.36 Equation fo r the p repa ra t ion o f es te r d e r iv a t iv e o f c a rb o x y l ic acid . 86 1.37 Equation i l lu s t ra t in g the reaction between ■ x la u r ic acid and 2-ch lo rom e thy l naphtha lene 86 1.38 Equation i l lu s t ra t in g convers ion o f 2- c i lo rom e thy lnaph tha lene to 2-n a p h th a l methanol . . . d 89 1.39 Equation i l lu s t ra t in g convers ion o f su lfonated ion-exchange res in (sodium form) to the s u l fo n y l Chloride form . . . 90 Equation i l lu s t ra t in g the condensation of ion exchange resin in sulfor/l Chloride form with 2-naph the lene methanol . . . 90 1.41 Equation i l lu s t ra t in g the app lica tion o f re s in -b o u n d 2-naph tha lene methanol in d e r iv a t iz in g c a rb o x y l ic ac id , e .g . la u r ic acid t . . . . . . 91 xvi i LIST OF TABLES Table Page Functions and Classification of covalent bound reagents . . . . 19 Determination of Optimum time of exchange between Chloride ion and benzoxazole moiety in anicn exchange resin (chloride form) . . . Ul Sto ichom etr ic p repa ra t io n o f es te r d e r iv a t iv e s o f fa t ty acids . . . . 53 O bserva tion o f f luo rescen t d e r iv a t iv e s of amines u n d e r UV l ig h t V . 68 T h in - Ia v e r Chrom atographie re s u lt o f amine d e r iv a t iv e s ob ta ined by heterogeneous reaction 69 UV Observation of the (a) amine derivatives obtained by hcmogerveous reaction of amines with sodium benzoxazole-2-sulfonate, and (b ) underivatized äirilne . . 80 Comparison of th in -laye r Chromatographie analvsis of derivatives obtained by homoge- necus approach and those obtained by heterogeneous approach . . 82 n of u ltra violet light Observation -bound benzoxazole derivatives . . 84 parison o f UV light Observation of amino * id s and amino acid bezoxazole derivatives 85 ^ . 10 Thin layer Chromatographie analysis of ester derivatives of carboxylic acids and 2- chloromethyl naphthalene showing retentive factors values . . . . . 88 11 T h in -la y e r Chromatographie analysis of derivatives of carboxylic acids obtained by homogeneous and heterogeneous approaches 93 CHAPTER ONE 1. INTRODUCTIO N F a tty acids and amines are o f immense im portance . Some fa t t y acids are known p recu rso rs o f the b io log ica l ly im po rtan t p ro s ta g la d in s . 1 Some o the r a l iphatic amines and polyamin« wh ich are odorous substances are known precursors of the c genic and to x ic N -n itrosam in es . '2 The a b i l i ty to m onitor these two g ro u p s o f compounds is v e ry im portan t and severa l techn iques to detect o r con’fuir-m th e i r p re - sence are needed. A number of ana ly t ica ! methods have been used fo r th e i r ana lys is rang ing frcm classicaf p i in s trum en ta l m e thods .J However, each o f these methods own l im ita tions and d is ­ advan tages. Classical methods o f ana lysis have the d isadvan tages o f iow s e n s i t iv i ty o f de tec t ion , n o n -s e le c t iv i ty and n o n -s p e c i f ic i t y . The l im ita tions o f the classical approach have been overcome by modern chromatograpl u^w eiin iques, in particu lar gas liquid chromatography and high perfOE/nance liquid chromatography. Problei is that are encountered in Chromatographie analysis of these tw!o funciionalities can, for example, be iüustrated with the determination of aliphatic amines. Cas Chromatographie dete mina- tion of these *n ines at low concentration is hamoered by adsorp­ tion and decomposition, column ghosting phenomena, tailed elution 2 peaks and low detector sensltiv ity . To d iffe ren t degrees, one or more of these problems are encountered in the analysis of carboxylic acids using any of the available Chromatographie techniques. A general approach to these problems has been the prepara - tion of suitable derivatives of the analytes, because derivatization can be used to improve the Chromatographie behavit as well as the sensitiv ity and specificity of detection. 1.1 D e r iva t iza t io n in Homogeneous The importance of Chemical derivatization techniques in Chromatographie analysis cannot be exaggerated. However, most of i.he derivatizations performed have involved the use of f miogeneous reactions in which the reagent is present in solution and is mixed with a solution of Substrate of in terest. Many books have been published on homogeneous derivatization reaction that can be performed prior to Chromatographie analysis of trace organic compounds. When an analyte is derivatized in an homoge­ neous medii um, a large excess of derivatizing reagent would be preseift*r in reaction solution. The earliest approach to the problem of removal of excess reagents during pre-chrom atographic derivatization was the development of volatile reagents which could be easily evaporated a fte r the derivatization process. 3 Use o f such reagents seems to have been lim ited to gas Chromatographie ap p lica t io n s . The re may be many stages invo lved in such a reaction in c lu d in g p u r i f ic a t io n and recove ry o f des ired de r iva t ize d p ro d u c t . A major d raw back o f homogeneous d e r iv a t iz a - t ion techn iques is associated w ith the need to remove excess o f reagent because the reagent often has sim ilar behav iou r to the d e r iv a t iv e in terms o f i ts response to the chrom atoqraph ic de tec t ion . In the presence o f the excess o f reagent, thereforfe»» the response o f the System to th e i r d e r iv a t iv e may be swamped by tha t o f the excess reagen t. T h is problem o f excess reagenl tt Iis Oomne th a t could be con- ve n ie n t ly overcome i f d e r iv a t izta ttiio n was dorne in a heterogeneous medium. 1.2 D e r iv a t iz a t io n in Heterogeneous Media w ith Resin Bound Reaqen t To a v e rb p rob iem s p resen t in homogeneous d e r iv a t iz a t io n , heterogeneous o e r iv a t iz a t io n have been employed. In he te roge­ neous d e r iv a t iz a t io n , the reagent is inso lub le in the reaction medi T h is is possib le because i t is a ttached to a p re form ed polymer o f h igh molecular mass. Such attachments may be at reno te po in ts along a po lym er chain o r anchored in close p ro x im i ty . The re are a num ber o f s ig n if ic a n t advantages in us ing heterogeneous n reactions to d e r iv a t is e ; fo r instance, i t allows excess o f reagent and Substrates to be separated from the react ion p ro d u c t by simple f i l t r a t io n th e re b y avo id ing complex Chromatographie p ro c e d u re , 5 wh ich are ted ious and t im e-consum ing . In add it ion , reactions th a t are not possib le in so lu tion because o f the low s o lu b i l i ty o f one o r more o f the reagents may be ca r r ie d ou t in h igh e f fe c t ive reagent concen tra t ion on a po lym eric s u p p o r t . A lso, heterogeneous reactions o ften are more se lective and g ive fewer side p ro d u c ts . In some ins tances, the b y -p ro d u c ts can remain a ttached to an inso lub le po lym er, these eart then be reconve rted in to the o r ig in a l va luab le reaqents th a t can la te r be reused. As a re s u lt o f all the possib le advantages enumerated above, the re is tne^e fo re a need to develop more read i ly accessible reagents fo r heterogeneous d e r iv a t iz a t io n s o f c a rb o x y l ic acids, am ints and o th e r commonly encountered fu n c t io n a l i t ie s . 1.3 Resin-I Reagents esLr**cound reaqents are formed when low molecular we igh t 9 9 reage i. is are a ttached to p re fo rm ed re s in . The bound reagents v . p ' ^ S S e s s a com binatio r o f the phys ica l p ro p e r t ie s o f a h igh polymer and Chemical p ro p e r ies o f the a ttached reagent.** t 5 f 7 W polymer o r res in low molescular :wWe igh t reagent F ig . 1 .0 : Res in -bound reagent 1 . 3.1 Polymers These are la rge molecules cons o f repea ting small u n i ts called mers, the small u n i ts are cova len t ly bonded to g e th e r to form the la rge molecuies. The simple molecule from wh ich a po lymer is made is called a monomert For example, s ty re n e , a monomer, can repea ted ly react w i t f > ^ ^ e l f and be trans fo rm ed in to p o ly s ty re n e , a la rge po lym eric Chain as shown in F igu re 1.1 below. « H H H H I I O s 1 I 1 1 / 1 I I | c = c — > w c — c — c — c — — c — : — \ — c — c ■ ü h A J M U J A S ly rcn e Pol y styrene F ig . 1 .1 : T rans fo rm a t io n o f s ty re n e to p o ly s ty re n e . s Chemica lly , th e re are fundam en ta l ly two modes o f po lym eriza- t ions , namely a dd it ion and condensation po lym eriza t ions . In add i­ t ion to po lym eriza t ion , the p ro d u c t is th e o re t ica l ly an in teg ra l m u lt ip le of the monomeric molecule, e .g . po ly thene and p o ly s ty re n e . Condensation process a lways re s u lt in the form ation o f copolymer such as d im ers, t r im e rs and te tram ers , examples o f such copolymer are organosil.con po lym ers (s i l ic o n e s ) ; pheno l- fo rm a ldehyde res in , Nylon 66. The in i t ia l p a r t o f condensation po lym eriza t ion , reaction , invo lve the conve rs ion o f all the monomers to low molecular we igh t po lym ers , and no h igh molecular wei ers are p resen t. T h e re a f te r , the lower molecular w e ig h t^species condense in a s tep - wise manner fo rm ing po lym er o f la rge degree o f po lym e r iza t io n . 7 Th is process can be f u r t h e r exp la ined in the F igu re 1.2 below. H H O i 0 H H 0 Q ! l »l »I I I H \\ HO - C - t C H O - C - O H +• HO - C - ( C H O - C - O H - ^ H O - C - C C H ) - C- O- C- f CH^. C- .I * * , V N ' I ' \ ’ H H < & H + h 2 o F ig . 1.2^- C- •ondensation reaction between a dialcohol ■ r and an o rgan ic d iac id massively c ro s s - l in k e d s t ru c tu re is also formed i f one o f the reactan ts is more than b i fu n c t io n a l , in th is fo rm , po lym eriza t ion occurs in the th re e d imensions. 7 In po lym ers, monomeric u n i ts can be jo ined to g e th e r in d i f fe re n t form s. In l inea r po lymers, monomeric u n i ts are jo ined to g e th e r in a s t ra ig h t open-cha in fashion as seen in the f ig u re b e low : C H2 Cv' "H2- F ig . 1 .3 : L inear po lym er e .g . po lye thene . ln l inea r a l te rn a t in g copo lym er, two d i f fe re n t types o f monomers are a rra nged in a s t ra ig h t open-cha in a l te r a t ing fashion as in the f ig u re to fo llow : _ N H ^ _ C — ( C H , )_ - C - ( CH — |l 1 5 II 1 b 0 0 F ig . 1 .4 : L inear a l te rn a t in g copolymer e .g . ny lon 6 . In m inor c ro s s - l in k e d po lym er, th e re ex is ts a l im ited num ber o f c ross - l in kages between each po lym eric chain as seen in the f ig u re be low : ^3 CH “ C H = ^ V C H - C H — CH C H — CH L CH — c ^4/ ~ C H 2— C H — C = C H — C H - F ig . 1 .5 : Mi nor c ro s s - l in k e d po lym er e .g . J \vt ut lcan ised ru b b e r T h e re i ilso the massively c ro s s - l in k e d po lym er as obse rved in u rea - fo rm a ldehyde res in in the f ig u re below: 8 CO NH- / N n H2 S SH2 H NOCN NCONH, 2 " 'C l I F ig . 1 .6 : Massively c ro s s - l in k e d polymerye>gy urea fo rm aldehyde. F in a l ly , th e re is the b lock polymers made uJpP o ,f monomers in b lock o f in d iv id u a l monomers. 1 .3 .2 P rope rt ie s o f Polymers Polymerie substances w he the r they are o f na tu ra l o r ig in such as co t to n , wool, na tu ra l r u b b e r , p ro te in s , po lysaccharides , nucle ic acids o r p roduced s y n th e t ic a l ly in the la bo ra to ry e .g . po lye thy le ne , po ly/m etrhy lm e thacry la te are easily recogn ised by th e ir phys ica l appearance and some spec if ic p ro p e r t ie s ; these p ro p e r t ie s in c lude lo w ^ J ^ Q e g l ig ib le so lub i l i t ie s in common so lven ts , mechanical s t re n g th , >^>esticity, f ib re - fo rm in g p ro p e r t ie s and dimensional s ta b i l i t y ; how ever, they may s t i l l be d i f fe re n t when th e ir phys ica l p ro p e r t ie s are cons ide red . Polymers may be o f va r ious forms ra n g in g from read i ly soluble l iq u id s , o r low -m e lt ing , w axy , o r v e ry hard and b r i t t l e so lids . 9 The anomalous p ro p e r t ie s o f po lymers in comparison to the low-molecu lar we igh t compound can be exp la ined in terms o f molecular size and s ta b i l iz in g fo rces. There are two major bonding forces in polymers, they are the prim ary Chemical bonds which occur along polymer chains and secondary bond forces such as vander waals, dipole interactions, < ? y and h yd rog en b o n d in g . Un like the small molecules, the secondary bond forces p lay an ex trem e ly im po rtan t role in po lym ers , th is is because the h igh molecu lar we igh t o f the polymer perm its these forces to b u i ld up s u f f ic ie n t s t re n g th to im part mechanical s t re n g th ano r ig id i t y in the po lym ers . These in te rm ctecu la r forces also in f luence o th e r p ro p e r t ie s o f po ly mer-s such as sw e ll ing , ge la t ion , m isc ib i l i ty and s o lu b i l i t y in ce r ta in so lven ts . C ry s ta l i in e reg ions a re 'fö rm e d by po lym er molecules possess- ing symr i r y . T ho uigh pto lymers may be am orphous, ye t have reg ions o f c r y s t a l l i n i t y . The c ry s ta l i in e and amorphous reg ions o f polymer are cons ide rab ly d i f f e r e n t in p ro p e r t ie s , fo r instance the fo rm er has increased mechanical s t re n g th . C r y s ta l l in i t y and o th e r phys ica l p ro p e r t ie s o f a po lym er are dependent upon the s u b s t i tu e n t 's c o n f ig u ra t io n . C ons ide r ing the mode o f po lym eriza t ion , polymers can be e i th e r iso tac t ic in wh ich 10 s u b s t i tu e n ts a round the polymer backbone are in an o rde red c o n f ig u ra t io n , o r a tac t ic , th a t is when s u b s t i tu e n ts are fashioned in a random m anner. S ynd io tac t ic po lym ers have th e i r S u b s t i tu e n t g ro u p s like a lte rn a te ly above and below the p lane. The iso tac t ic , synd io tac t ic and a tac t ic c o n f ig u ra t io n s o f a po lym er chain are shown in the f ig u re below: X X I H 'c F ii ; . 1 .7 : Iso tac tic , syn d io ta c t ic , a tac t ic c o n f ig u ra t io n s o f a po lymer cha in . n O f equal im portance are the f i r s t - and secon d -o rd e r t ra n s i ­ t ion tem pera tu res . These tem pera tures are not as " s h a rp " as those o f low -m o lecu la r-w e ig h t so l ids . In c ry s ta l l in e po lym ers , these tem pera tu res re fe r to the melting tem pera tu res o f c ry s ta l reg ions , and to the so ften ing tem pera tu re in amorphous reg ions . Polymers are so f te n ing when the re is increase k in e t ic ene rgy o f the molecules as i t becomes large enough to overcome secondary bond fo rces. v w > O the r p ro p e r t ie s tha t are de term ined by in term olecula. forces are m isc ib i l i ty and s o lu b i l i t y respec tive l y . Poly mers d isso lu t ion u n l ik e the low-m o lecu lar we igh t substances is a slow process o c c u r r in g in two s tages. In the f i r s t stage, the so lven t molecules s low ly d i f fu s e in to the po lymer leading to swe ll ing and ge la t ion . T h is may be the on ly stage i f s t ro n g po lym er-po lym er in te rm o lecu la r forces are p re s e n t. T h is may be due to c ro s s - l in k in g , c r y s ta l l in i t y , and s tron g h y d ro g e n bon d in g . However, in l inea r po lym er, the f i r s t stage is a ^ o m p a n ie d by a second stage in wh ich a t r u l y homogeneous so lu t ion re s u lts from d i f fu s io n o f so lvated po lymer moiecules in to the so lven t . H f l ymers th a t are used as inso lub le reagents possess the re q u ire c p ro p e r ty o f swe ll ing ra th e r than s o lu b i l i ty . 12 The usual c o l l ig a t ive p ro p e r t ie s o f Solutions are exh ib ite d by d i lu te Solutions o f complete ly so luble po lym ers . The p rope rt ie s have been employed in the de te rm ina t ion o f po lymer molecular w e igh ts . The Chemical react ions o f po lym ers can be c lass if ied as fo llows: The f i r s t d a s s are those a f fe c t in g the degree o f po lym eriza t ion (D .P ) wh ich inc lude f u r t h e r po lym eriza t ion o f a lready formed po lymers and the syn thes is o f a g ra f t o r ^ b l o c k co-po lym er and deg rada tion reaction c lass if ied as macromolecule. The second dass are those not a f fe c t in g tne degree o f po lym eriza t ion (D P ) , b u t in vo lve the reac t ion o f func t iona l g ro u p a lready conta ined in the po lym er-m ed ia ted o rgan ic sy n th e s is . 1 .3 .3 Polym er-Supported Reagents P o lys ty rene has been the most w ide ly used po lymer fo r va r ious reac t ions . & s ty re n e can be func t iona lized by e i th e r Convert ing the po lymer d i re c t ly in to the des ired reagent o r it serves as a handle to wh ich a low-m o lecu lar w e igh t reagent is then at;ach d. Reagent a ttachm ent to the po lym er backbone can be by ionic bond re fe r re d to as io n ica lly bound reagent, while those tha t are attached by cova len t bond ing are known as cova lent bound reagents respec t ’ v e l y . 13 1. 3 . 3 .1 lon ica tlv bound reagents These are based on ion-exchange res ins and are easy to p re p a re . ln ion -exchange ions o f l ike charges are exchanged between solu tion and an inso lub le solid in con tact w ith the so lu t ion . For exchange to occu r, the io n -e xch a n g e r (so lid ) must c a r ry its own ion and must be s t ru c tu re d to fa c i l i ta te movement o f the ions. In a d d it io n , no apprec iab le phys ica l e^ange must occu r in the materia ls d u r in g exchange p r o c e s s ^ J ^ h e ion- exchanger is complex and po lym eric and ca rr ie s an e le c tr ic Charge th a t is exac t ly neu tra l ized by charges on the cou n te r ions. Some exchangers are used as so lu tion in a so lven t immiscible w ith w a te r ; these are l iq u id io n -e xchang e rs . In cation exchangers , the ac t ive o r cou n te r ions are cations and ihe res ins o r po lymers are an ion ic while in anion exchangers , the coun te r o r a c t iv e ^ Q S are anions while the res ins o r po lymers are c a t io n ic . 10 1 .3 .3 .2 Cat»on-exchange res ins These are h igh m o lecu la r-w e igh t c ro s s - l in k e d po lymers con- ta in in q su lphon ic , c a rb o x y l ic , pheno lic g 3ups as an in te g ra l p a r t o f the res in and equ iva len t amount o f cations l ike hyd ro g e n or sodium. T he re are s t ro n g ly acid ic cation exchangers and weakly acid ic exchangers . 14 S tro n g ly ac id ic ca t ion -exchange rs are usua lly supp lied in the h yd rog en o r sodium forms as in p o lys ty re n e su lphon ic acid res in w r i t te n as (Res S O .)H T fo r the hyd rogen form and as (Res SO^JNa4 fo r the sodium form . Weakly ac id ic ca t ion -exchange rs such as po ly m e th y la c ry l ic acid res in also come in hyd rog en form . In a d d it io n , some weak acid cation exchangers conta in the ca rboxy la te g ro u p , i .e . - + W (Res COO 1H . The exchange capac ity fo r s t ro n g ly ac id ic cation res ins is pH in dependen t. 10 While fo r weak acid cation exchangers . exchange on ly occu rs in a lka line sol ution. The equation fo r the exchange capac ity are shown in F iqu re 1.3 below. ph independent (Res S O ")H + + Na~CI SOlrf (Res S 0 3)Na + H CI SOIn a lka line (Res COO ) Ht + Na*Ö1 SOIn N medium (Res COO ) Na h 2o F ig . Equations fo r exchange capa c ity . A\ w ide l'y used cc t ion -exchange resin is p repa red by co- p o ly r f t ^ ^ ä t io n s ty re n e and a small p ro p o r t io n o f d iv in y lb e n ze n e as shown in F ig . 1.9 below. 15 Fig . 1.9: P repara tion o f catiop, exchange res in . “ 9 The po lym eric ske le to n *in the above s t ru c tu re is held toge- th e r by l in k in g s Crossing fron ’ one po lym er chain to the n e x t ; the ion exchange g roup s i ^ ^ c a r r i e d on th is ske le ton . The degree o f l in k in g determ ines th e phys ica l p ro p e r t ie s . T hu s , h ig h ly c ross - l inked res ins are h a rd e r , more b r i t t l e , and im perv ious than l ig h t ly c ro s s - I in k e d c o u n te -p a r t . Swelling and p re fe re nce o f res in fo r one ion ove r ano ther are in f luenced by degree o f c ro s s - l in k in g s . In the above f ig u re , d iv iny lbenzene u n i ts weld the p o ly s ty re n e chain to g e th e r , th is p reven ts i t from sw e ll ing in d e f in i te ly . The d isp e rs in g and 16 sp o n g e -iike n e tw o rk a rrangem ent p ro v id e s a fram ew ork in w h ich the n e g a tive ly charged su lphonate ions are a tta ch e d . The fixe d nega tive charges are balanced by an e q u iva le n t num ber o f cations such as hyd rog en ions o r sodium ions re s p e c t iv e ly . These ions are mobile and move fre e ly w ith in the w a te r f il le d po res . > lh e ions can exchange w ith o th e r ions, as seen in a S itua tion w here a cation exchange r co n ta in in g mobile ions X + is b ro u g h t in to close con tact w ith a so lu tion co n ta in in g cations Y + w h ich d iffu s e in to the res in + . O v s tru c tu re and ca tions X d iffu s e ou t u n t i l e q u ilib r iu m is a tta in e d . A t the end o f exchange process, the so lid and the so lu tion conta in bo th ca tions X and Y4 in num bers depend ing upon the pos ition o f e q u ilib r iu m . The same process holds fo r exchange o f anions in an anion e xcna nge r. The exchange process is exp la ined in F igu res 1. 10a anc 1. 10b below . (Res .A )X ♦ + ?xS^SSoln) ( R es.A )Y + + X (So ln) Figq . 1. Iflfffr ^Exchange process o f ion exchange res in t ty p ic a l exaßmple is the d isp lacem ent o f potassium ions in a su lpho ­ nate res in by magnesium ions 2 (R e s .S 0 3)K + Mg" (S o ln ) (Res.SC>3) 2 Mg"2 + + 2K Sein F ig . 1 .10b: D isplacem ent o f potassium ions by magnesium ion 17 1 .3 .3 .3 A n ion exchange res ins T h is is p re p a re d by co -p o lym e riz in g s ty re n e and a l i t t le d iv in y lb e n z e n e fo llow ed by ch lo ro m e lh y la tio n l i . e . in tro d u c in g 18 1 .3 .3 .« C o v a le n t-b o u n d -re a a e n ts These are reagents a ttached to the po lym eric S upport by sim ple cova len t b o n d in g . They are c la ss ifie d acco rd ing to ty jes and fu n c tio n s o f reagents th a t a re a ttached to the po lym er s u p p o rt. Tab le 1 below g ives an exp lana tion on th e ir Q . C lass ifica tion . 1 .3 .3 .5 -F-u-n c-t-io-n-a-li-za-t io —n o f p^o ly s ty r—e n e vVv > A p a rt from a tta c h in g reagents to re s in s , res ins can d ire c t ly be conve rted to the des ire d re a g e n t; such $a conv e rs ion invo lves p rim a ry tra n s fo rm a tio n o f p o lys ty ’ene th ro u qg h F riede i C ra fts re a c tio n 11 w ith ch lo ro m e th y la tio n be ing the s ta r t in g Step. C h lo rom e thy la tion is a p'-ocess w hereby a h yd ro g e n atom o f an arom atic compound is rep laced b y a ch lo ro m e thy l g ro u p , CH^CI. The in tro d u c tio n o f a ch lo ro m e thy l g ro u p is v e ry u se fu l s ince th is g ro u p is re a d ily c o inwve rt ed in to o th e r g ro u p s such as a ldehydes a lcohol, e th e r ^ a n d am ines. 19 Tab le 1- F un c tions and C la ss ifica tio n o f C o va le n t-B o u n d Rcagonts F un c tio n Reagents i ca tion O x id iz in g reagen ts P e rac ids , chrom ium co n ta in in g In epox ida tion o f a lkene o x id n tio n o f po lym er su p p o rte d chrom ic acid p r im a ry and secondary c a rb o n y l chem isorb c h ro m y lc h lo r id e com pound. P re p a ra tio n o f a ldehydu and (C r 0 2 C l2) on s ilica alum ina) ke tone from a lly l ic and b e n z y lic ha lides s ilv e r c a rb o n a te -c e lite . o x id a tio n o f a lcohol and lac tones . Selec- Po lym erie th io -n is o ly l re s in . t iv e o x id a tio n o l a lcoho l. O x id a tio n - Po lym erie q u inon es . D e hyd ronen a tion and o x id a tio n o f o rg a n ic rec iuc tion reagents om pounos. Po lym erie- A iu m in a -s u p p o rte d m a te ria l. R eduction o f d is u lp h id e s o f th io ls . re d u c in g re igen ts R eduetion o f c a rb o n y l com pounds. P o lym eric - Po lym erie l in h y d r id e S e lec tive re d u c tio n o f ha lides in the re d u c in g reagen ts presence o f o lh e r fu n c tio n a l g ro u p s e .g . brom oacelo phenone lo acetopheuone. S ilica -g e l a b s o rb in g t r i - S e lec tive o x id a tio n o f a ld e h yd e . b u ty lt in h y d i ido . Polym erie g ro u p H a logenating age n ts . In tro r iu c t io n o f ha logen in to molaeule t ra n s fe r reagen ts u ith e r by n u e le o p h ilic d i3p lacen it.n l o r e le c tro p h ilic a c k lit io n . Polym erie g ro u p A n io n -e xch a n g e res in c a r ry in y Use in exchange o f one Im lide frern t ra n s fe r agen ts f l / o r t f c , c h lo r in e b ro in ine ano the r in a lk a lh a lid e . fo d itu ^ a n io n s . A c y la tin g agen ts Po!lyn ie rs in c o rp o ra tin g m ixed Uscd in p e p tid e s y n th e s is . a nh ycdlrriid e o f c .n b o x y lic and benzuic ac id s . 20 Tab le 1 (C o n td .) F un c tion Reagents A lk y la t in g agen ts N -a lk y l-n a ry lt r ia z in e su p p o rte d on po lym er m a te ria l. Polym er s u p p o rte d CN e tc su p p o rte d on a n io n - N u c leo ph ilic S u b s titu tio n as in p re p a ra - nuc leoph iles e xch a n g e r. t io n o f b e n zy lcya n id e by the reac tion o f b e u z y l-b ro m id e w ith an an ion exchange n . S epara tion o f main p ro d u c t from ro d u c t is eas ily a th ie v e d . W ittin g reagen ts and o th e r n v e is io n o f low -m oler u la r w e ig h t- y lid s po lym e ric w it t in g reage c a rb o n y l g ro u p to alu ;e . po lym er in c o rp o ra tin g a tr ip h e n y l phosph ine g ro u p . Po lym eric s u lp h u r y lid S yn th e s is o f epoxidos from ca rb o n y l reagen ts co n ta in in g d im e th y l com pound p ro d u c t wei e e a s i l y iso la ted . su lfon iu in m e th y lid e syn thes ized from a co -p o lym e r o f v in y lm e th y l th io e th e r, s ty re n e and DVB ( D iv in y lb e r iz e n e ). Po lym eric co u p lin g C arbodim ii des p o ly mene Use in pep tides syn th e s is and in the agents ca rbod im i p re p a ra tio n o f ca io x y lic a n h y r ir id e s ÖS used in o x id a tio n o f a lcohol to a ldehyd es , s ta r t in g reagen ts can he regen e ra ted th ro u g h less a c tiv e than the o r ig in a l re a g e n ts . Po lym eric co u p lin g \ I Chlorides polymeric- P eptide syn th e s is v ia the po lvm e ric agents ne s u lfo i iy l Chloride. m ixed c a rb o x y lic s u lfo n y l a itn y u iid e s . ily meiric aryl sulfonyl S yn th e s is o f o lig o n u c le o tid e s . Chlorides 21 Such conve rs ions can be c a rr ie d ou t in o rg a n ic so lven ts most o fte n sw e lling so lven ts and the reaction can also proceed u n d e r phase t ra n s fe r co n d itio n s . © ~ f - ' v v CH2 CI 0 R ' - > © ---- CH: OR I C N ‘ ( c ^ C ^ C N r n _ i— Dm SO "> ( p )*̂ v v CHO F ig . 1 . 12: T rea tm e n t o f ch lo rom e thy l p o ly s ty re n e w ith nuc leoph iles Cl & CH ~ rr3~CrOCH2 COCH3 , 7 ~ --------------— > ( pV ^ ch?ch ( coch. CH2C52 /B U a U + Br* Nq OH W 2 V « f f : h 3c o c h ;C 6 CH-CH - COCH. Ü4 Hf“ Br “ NqOH ( 3 C5H5 ^ ( p \ —/v»/ C H . N H — CjH^Cl / C 7H7 We N+ 0H,N50H s ' NO ; F g. 1 .13 : Phase tra n s fe r process in v o lv in g ch io ro m e thy l p o ly s ty re n e . »O 22 P o lys ty re n e can also be a lk y la te d u n d e r F r ie d e l-C ra fts cond ition s The reaction is v e rs a tile in C o nve rting p o ly s ty re n e to va rio u s reagen ts . T h is rou te is coup led to the po lym er th ro u g h a s tab le C -C bond as shown in F ig u re 1.14. CHOH AI CI . V N° 2 cich?—(Q>-oh — z F ig . 1 .14: P repa ra tion o f po lym eric reagents by- s ing le Step F r ie d e l-C ra fts a lk y la tio n o f p o ly s ty re n e Polym eric su lfona te es te rs and to sy laz ide were p repa red from ch lo ro su lfona ted p o ly s ty re n e as shown in F ig u re 1.15. 0 -----+ CI SO2H O )— S02 CI S 0 2 N — ( o ) ~ so2or F ig . 1.1 V e pa ra tion o f po lym eric su lfon a te es te rs and to xy la z id e from ch lo ro su lfo n a te d p o ly s ty re n e . In fo rm ing the lith ia te d po lym er, p o ly s ty re n e is halogenated to ob ta in an in te rm ed ia te th a t p roduces the lith ia te d po lym er. B u ty lith iu m N, N , N , 1 N , 1 -te tra m e th y le n e diam ine is a good m eta lating agent fo r c ro s s - lin k e d p o ly s t' ’-ene. The metalated 23 p c ly s ty re n e in te rm ed ia te is a good rou te in the p re p a ra tio n o f po lym e ric reage n ts . T h is is shown in F igu re 1 .16 . BULt © --------- <□> T MEDA © — <0>_Li C 0 3 X E > — < 0 > - c o j 4 MeSSMe F ig . 1 .16: P repa ra tion o f po lym eric reagents from metalated p o ly s ty re n e in te rm ed ia te V a rious research w o rke rs have endeavoured to use some o f the above mc n tioned reagents fo r the i r '»S0 rk s w h ile some have m odified the reagents to su it th ei iir pu rposes. The a p p lica tio n o f these reagents in heterogeneous d e r iv a tiz a tio n has been exp lo ited by K ru ll and c o -w o rk e rs .*1 In one o f th e ir w o rks they used p o lym e r-b o u n d a n h y d rid e s as acy la tin g agents fo r p rechrom ato- g ra p h ic d e r iv a tiz a tio K ^o f amines. An in te re s t in g aspects o f th e ir w o rk is th a t th ^ ^ ä re p a re d po lym er-bound reagen ts w h ich cou ld be used fo r \p f th e r o f f- I in e or o n - lin e d e r iv a tiz a tio n fo r h igh ne rfo rm anc:^ liq u id ch ro m a to g ra p h y . T h is has p rom pted us to io o k \n t£ the p o s s ib ility o f deve lop ing re a d ily ava ilab le reagents not on ly fo r a m iiv s b u t fo r o th e r commonly encoun te red fu n c tio n a - lit ie s such as a lcohols, amino acids and c a rb o x y lic ac ids . 24 M oreover, lite ra tu re s u rv e y revea led th a t in the ana lys is o f c a ro o x y lic acid such heterogeneous d e r iv a tiz a tio n techn iques have not been fu l ly e x p lo ite d . Most w o rke rs tend to c a r ry ou t th e ir d e r iv a tiz a tio n th ro u g h homoaeneous processes. 1.4 A n a lys is o f C a rb o x y lic A c ids A number o f liquid Chromatographie m ethods have been employed fo r the ue te rm ina tion o f fa t ty ac ids . For instance , , 2 Pei et a! used m e th y le s te r d e r iv a tiv e s fo r h ig h perfo rm ance liq u id ch ro m a to g rp h ic ana lys is o f c a rb o x y lic ac ids . In a d d itio n , m ethylester d e r iv a tiv e s o f 11- and 12-h y d ro x y la u r ic acid p ro - duced by microsomal metabolism o f sodium la u ra te have been analysed by re ve rse phase h igh perfo rm ance liq u id c h ro m a to g ra p h y . A n o th e r w o rk e r 13 , alsoJ in v e s tig a te d the Separation o f fa t ty ac ic m e th y les te rs us ing co ra il 11 w ith a s ta tio n a ry phase o f s i lv e r -n it ra te e thy len& g ly c o l. In p re p a n n g m ethyl e s te r d e r iv a tiv e s o f the ac ids , c a rb o x y lic acids a re e s te r if ie d in methanol w ith h yd ro g e n C hloride o r boron trich te rnde as the c a ta ly s t. In all cases, de tec tion leve ls were poor a ltho ugh sucn d e r iv a tiv e s are c e r ta in iy use fu l fo r p re p a ra tiv e w o rk . Hence in an a ttem pt to p roduce su itab le d e r iv a tiv e s fo r h in h perfo rm ance liq u id C hrom atographie ana lys is , acids were 25 e s te r if ie d w ith arom atic d e r iv a tiz in g age n ts , th is p roduced derivatives with improved Chromatographie properties and increase s e n s it iv .i ty o f d e te c t.i o n . Some w o rke rs 14 were abie to p re p a re the phenacyl e s te r d e r iv a tiv e s by re a c tin g the acid w ith 2 brom oacetophenone us ing tr ie th y la m in e as base. B orch in h is work reported the Separation of long chain fatty acids as i phenacyl es te rs by HPLC. Cooper and A n d e r used 2-n a p h th a c y l-b ro m id e to d e r iv a tiz e c a rb o x y lic acids to ob ta in th e ir 2-n a p h th a c y l e s te r th a t was ana lysed by h ig h perform ance liq u id ch rom a tog raphy and mass s p e c tro m e try . In h is d e r iv a tiz a - tio n scheme, m ix tu re s o f the acid (lO^Anoles), 2-n a p h th a c y l b rom ide (20i/moles) and N ,N -d iis o p ro p y le th y la m in e (40^moles) all in 1ml d im e thy l-fo rm am ide were heated a t 60°C fo r 10 m inutes. The a liq u o t was then ana lysed by liq u id ch ro m a to g ra p h y . He how ever obse rved good Chromatographie properties o f the n ap h th acy l es te rs us fng a reve rsed phase column b u to ^ - and 'jf- lin o le n ic ac ids cou ld not be reso lved . O th e r Ww o -k e rs , 15 re p o rte d the d e te rm ina tio n o f c a rb o x y lic acids b y tfq u id ch rom a tog raphy a fte r p h a s e - tra n s fe r cata lysed f lu o ro ^S ^ ic la b c llin g . In th e ir w o rk , te trabu ty lam m on ium mediated tra n s fe r o f ana ly te as an anion in to e th y le n e d ich lo rid e (c rg a n ic phase) co n ta in in g the flu o ro g e n ic re a g e n t. Even though 26 d e r iv a tiz a tio n p roved successfu l, th e re was th e need to e lim ina te excess o f d e r iv a tiz a tio n reagent w h ich m igh t in te r fe re w ith the d e te rm in a tio n . F a tty acids have also been separated b y a d so rp tio n and 16 - 1 p a r t it io n m ethods. While the Chrom atographie Separation by a d so rp tio n p ro ve d d i f f ic u l t , th a t o f p a r t it io n y ie ld e d e xce lle n t re s u lts . T ho ugh each w o rke r tended to m od ify an)d vimnpfro ve on the w o rk o f the p re v io u s w o rk e r, no a ttem pt had been made to ob ta in d e r iv a tiv e s fo r fa tty acid de te rm ina tion th ro u g h h e te ro - geneous te ch n iq u e s , so a lso, some o f the reagen ts em ployed fo r homogeneous d e r iv a tiz a tio n by some w o rke rs cou ld not be eas ily o b tsm e d . Thus th e re is the need to deve lop reagen ts w h ich a re accessib le and can be used to p repa re f lu o re s c e n t d e r iv a tiv e s th ro u g h heterogeneous techn iques fo r the ana lys is o f c a rb o x y iic ac id . C r fs is o f Amines r 'n e s are ammonia d e riv a tiv e s in w h ich one o r more hydrcJ'gen atoms have been replaced by a lk y l and o r a ry i g ro u p s g attachv^d to the r.itro g e n atom, A lip h a tic amines can be p resen t o r can be p roduced th ro u g h na tu ra l decom position processes in a v a r ie ty c f Systems such as in many d a ily fo o d s .1' 27 A lip h a tic amines can by them selves be to x ic o r become toxic via Chemical reactions as in reaction with n itrites , fo rm in g n itrosam in es . Some n itrosam ines are h ig h ly to x ic c a rc i- nogenic com pounds. Some m icroorganism s p roduce n itrosam ines 'in v iv o ' d u r in g in fe c tio n . 1 8 In the fie ld o f m edicine, and the in d u s t ry , the s tu d y o f a lip h a tic p o ly fu n c tio n a l amines are o f g re a t im portance , fo r in s tance , the presence o f sperm ine, spe rm id ine , the p u tre s c in e detected a t trace leve is in u r in e a llowed cancer d iagnos is a t an unu sua lly e a r ly s tage . 1 .5 .1 M ethods o f A n a lys is Many a n a iy tica l methods haavve beenr employed fo r the analysis of amines by many w orkers. However, most of the methods have been Chemical derivatization techniques in which the amine Substrate o f interest is reacted with reagents to form derivatives that can be monitored by spectrophotometry fiuot imetry or colorim etry. Some w orkers17 used dansylchloride as the derivatising agent and detecticn was by fluorim etry . Such a reaction provides an o tu n ity for Identification and Separation of aliphatic monamines, diamines and polyamines with fluorescence detectors. 28 1 . 5 . 2 D a n s y 'a tio n R eaction D a n sy lch lo rid e w h ich is 5 -d im e thy lam ino -nap h th a le ne -1 - s u lfo c h lo r id e has been used to form amides th a t a re cha rac te rized by h ig h a b s o rb iv ity as well as fluo rescence em ission. in the re a c tio n , m ix tu re o f 5.0ml o f 0.25M sodium hyd rogen carbona te s o lu tio n , 1.0ml o f d a n s y lc h lo r id e 0.02m and 0.5m l o f each amine so lu tion o f des ired co n ce n tra tio n were reacted in about 10.5ml acetone. The reaction was c a rr ie d ou t in iO le a le d reaction vessel at 60°C fo r 20 m inutes a f te r w h ich the d e r iv a tiv e s were ob ta ined and ana lysed . The major lim ita tio n o f th is method is the need to avo id the C _ .. use o f excess d a n s y lc h lo r id e w h ich can fluo rescence as the d e r iv a ­ tive s thus in te r fe r r in g w ith the ana lys is by swam ping th e response o f the System to the d e r iv a!tiv e s to be de te rm in e d . In a d d itio n , Schwedt and Bossemas 19 co n v e rte d dopamine and n o rad e rn a line to f lu o re sce n t d e r iv a tiv e s b y the reaction w ith d a n s y lc h lo r id e a lso, n itrogeneous Compounds re la te d to amines such as carbam ates have been examined as the d a n sy l d e r iv a tiv e s o f N -roethyicarbam ates and m onitored by h ig h perfo rm ance liq u id ch rom a to g raphy (H P L C ). 20 F u rth e rm o re , b a rb itu ra te s have been d e riv a tiz e d by d a n sy lch lo rid e s and de tec ted b y fluo resce nce . 29 1 .5 .3 O ther Reaqents O th e r u se fu l reagents th a t have been used in C onve rting amines and re la ted compounds in to f lu o re sce n t d e r iv a tiv e s inc lude fluorescam ine and O -p h th a ld e h yd e (O P A ). 21 Fluorescam ine w h ich was s u b s titu te d fo r n in h y d r in has been used fo r bo th post and precoium n d e r iv a tiz a tio n o f catecholam ines. dopam ines and n o ra d re n a lin e .2' 2,23 A lso o f equal im portance is O -p h th a ld e h y dJe3 and a un ique aspect o f th is reagen t is th a t its e lf and the p ro d u c t have e n t ire ly d if fe re n t e le c tro n ic sp e c tra , th u s Separation o f flu o re sce n t p ro d u c ts from equ a lly fluo resce n t excess reagen t is avo ided in the re a c tio n . OPA undergoes condensation reac tion w ith the amine S ubstra te in the presence o f a s tro n g nuc leop h ile ( i .e . a th io l) to fo rm an iso indo le thJa t fluorescences in te n s e ly . 21 H ow ever, the lim ita tio n o f th is reac tion is th a t the p ro d u c t i .e . iso indo le , is h ig h ly uns tab le w ith respec t to l ig h t , acid a tta c k o r a ir o x id a tio n . -N itra b e n zoyl Chloride, chloroacetaldehyde have been em pleyVed as d e r iv a tiz in g agents 24 ' 25 fo r amines and they form ed d e r iv a tiv e s th a t were eas ily de tected by f'uo rescen ce o r spec tropho tom e te r. 30 D iastereo isom eric d e r iv a tiv e s were p roduced by reac ting o p tic a lly a c tive amines w ith (s ) -o -m e th y l m andeloyi C h loride . The re so lu tio n o f the R - and S -m e th y l benzylam ine d e r iv a tiv e s revea led the o p tica l p u r i t y o f the am ines. 2 6 Amines have also been id e n tif ie d by spectropho tom e tr ic m ethod. In th is aspec t, amines and re la ted Compounds were reacted w ith reagents to p roduce co loured species th a t could be de te rm ined s p e c tro p h o to m e tr ic a lly . 2 -K A n th o n y Benson and William S p illance de te rm ined amines and sulfam ates ( i .e . a r t i f ic ia l sw eetner) w ith 1, 4 -benzoqu inone us ing s p e c tro p h o to m e try . In th e ir re ac tion , the amine S ubstra te in C hloroform was reacted w ith 1% ethano lic , 1 ,4 -benzoqu inone at 60°C fo r d if fe re n t reaction tim es to ob ta in reac tion p ro d u c ts th a t were de te rm ined sp e c tro p h o to m e trica lly by measurement o f absorbance a t / \ m a x (w ave leng th o f a b so rp tio n ) 478-510nm. Most o f the re p o rte d w o rks have been c a rr ie d ou t th ro u g h homogeneous d e r iv a tiz a tio n tech n iq u e s , how ever, the app lica tion o f heterogeneous d e r iv a tiz a tio n in the ana lys is o f amines has been lim ite d to the re p o rte d w o rk o f K ru ll and c o -w o rk e rs . The w o rke rs p re p a re d d if fe re n t bound a n h y d r id e s . The po lym eric a n h y d r id e con ta ins 0-a c e ty l sa lic y ' as the labe lled m oiety to d e r iv a tiz e secondary and p r im a ry am ines. The s tru c tu re o f the 31 g ro u p to g e th e r w ith its reaction w ith an amine com pound is p resen ted in F ig u re 1.17. © -----CHjO - C - 0 - © = Polystyrene back bone 0 - C - C H - If 0 r ig . 1 .17 : S tru c tu re o f po lym eric a n h y d r id g e co n ta in in g 0-a c e ty l as the [abe lled m oie ty. 0 0 ^ o II CH O - C - O — C J. n l CH-^CN , II+ R NH —------ - R fv|h - C-r' 10 Amine 5 0 ° 20mins \0 V - C - C H , < / . II 3 O -C -CH-, &' „ Ä / \ . CH , CN , ,, IIII / “ \ * R ' R U NH -- ------------ > f i f r ' n - C - ( o ) 5 0° 2 ° Amine 2 0 mins O - C - C H , II 0 /F ig . 1 .18 : D e riv a tiz a tio n reaction o f p r im a ry and secondary amines w ith a p o lym e r- bound a n h y d rid e re age n t. 32 The same g ro u p o f w o rke rs 2 8 also in v e s tig a te d the use o f (a) a po lym eric benzo triazo le ac tiva ted e s te r re a g e n t, and (b ) a po lym er f lu o re n y le s te r ac tiva te d by O -n itro -benzophenone fo r the d e r iv a tiz a tio n o f amines fo r HPLC ana lys is w ith U V /F L d e te c tio n . The lim ita tions o f the po lym eric ben zo tria zo le reagents are its s e n s it iv ity to m oisture and e levated te m p e ra tu re due to the uns tab le tr ia zo le r in g . For these reasons i t is not easily used in o n - lin e d e r iv a tiz a t io n , though it can be used su cce ss fu lly fo r the o f f - l in e d e r iv a tiz a tio n . The co n tin u a l use o f these reag en ts in a p reco lum n o n -lin e m anner leads to g ra d u a l degrada tion o f the m ate ria ls and loss o f a ll ta g g in g p ro p e rt ie s because o f the in s ta b il ity o f the reagen ts . On the o th e r hand , the O -n itrobenzophenone a c tiv a te d es te r reagen t is more s tab le to m oisture at bo th room and e levated te m p e ra tu re . I t is h ig h ly reac tive tow ards n u c le o p h ilic a tta ck and X can th e re fo re be used fo r o n - lin e d e r iv a tiz a tio n in HPLC. The s tru c tu re o f the f lu o re n y l a ttached po lym e ric o- n itrobenzophenor.e reagents in F ig . 1 .19, to g e th e r w ith its re ac tion w ith an amino compound is re p resen ted in F ig u re 1.19 and . 1.20. 33 NO- o 0 — C - O - CH- F ig . 1 .19: P o ly m e r-3 -n itro -4 [ 19 - f lu ° r en y Im e th o xy t;- -c--a--r-b---o--x-1y--l-]---o---x--yu ,b enzoph. enone. +- r n h 2 OH +■ R - NH - C - 0 - CH2 F ig . 1 .20: D e riv a tiz a tio n o f ty p ic a l amines w ith the po lym er bound n itrogenzophenone ^ C l iv a t e d es te r A lso <<^ u r , r Cao, C rin b e rg and K ru il have u t il iz e d a p o ly m e t j^ lc h is c o v a le n tly a ttached to a C h ira l molecule such as amino acid in re s o lv in g and q u a n tita t in g b io lo g ic a lly ac tive enan tiom ers. In th e ir app roach , 9 - flu o re n y lm e th y l m oiety (FM O G ), a d e te c to r se n s tiv ie m olecule, was co va le n tly bonded an amino acid (a c h ira l rro lecu le) w h ich has a lrea dy been c o v a le n tly a ttached to a po lym er. T h is approach had been m ain ly a p p licab le in the d e te rm ina tio n o f enantiom eric p u r ity and com position as well as Chemical p u r ity o f v ir tu a lly all s tro n a nuc leoph iles such as p r im a ry and secondary o p tic a lly ac tive amines and amino like Compounds. B u t the lim ita tions o f the approach is th a t the p a r t ic u la r reagents employed have not been success fu l fo r so ft nuc leoph iles such as a lcohols, th io ls , c a rb o x y la te an ions . The s tru c tu re o f po lym er bound 4 -h y d ro x y l-3 - n itrobenzophenone co n ta in in g FMOC L -p ro lin e is shown in F ig u re 1.21 below . , N °j 0 ® ^ > - c ^ o - c 0 < C IIc - o - c h 2 S t J o ) F ig . 1.2 1 - 51I ru c tu re o f po lym er bound *» -h y d ro x y l-3 - n itrobenz ophenone co n ta in in g FM O C -L- {< / p ro lin e . Though a lo t o f s y n th e tic w o rk has been c a rr ie d ou t by va rio u s w o rke rs w ith th is app roach , the reagen ts deve loped by these w o rke rs cannot be easily p^epared in a r o u t ir e la b o ra to ry . T h e re is there fo re a need to deve lop more re a d ily accessib le 35 reage n ts , no t o n iy fo r amines, bu t also fo r o th e r commonly encoun te red fu n c tio n a lit ie s such as c a rb o x y lic ac ids , a lcoho ls , ca rb o n y l Compounds and so on. Such reagents cou ld be based on a v a r ie ty o f o th e r ideas apa rt from the a c tiv a te d a n h y d r id e approach repea ted ly adopted by K ru ll and c o w o rk e rs . 2 ' 4 ' 28 ' 29 A lso , because o f the ready a v a ila b ility o f io n -e xchang e re s in s , bound reagents based on these res ins as s u p p o rt w ould be expected to be more re a d ily accessible than the p re v io u s ly re p o rte d reagents based on the use o f spec ia lly p re p a re d po lym ers as s u p p o rt. 1 • 6 Aijm and Objective The aim o f the prese; n t w o rk is th e re fo re the deve lopm ent o f nove l io m ca lly o r covaJentty bound reagents fo r the p re - chrom atogra ; tic d e r iv a tiz a tio n o f c a rb o x y lic acids and am ines. These reagents w ill be based m ostly on the use o f re a d ily ava iiab le ion--aeexxcchhange res ins as S upports . 36 CHAPTER TWO EXPERIMENTAL o f the p e lle ts in 10ml o f d is t i l le d w a te r, was added. 25ml o f carbon d is u lp h id e was c a re fu lly added to the m ix tu re and re flu x e d fo r tw o and h a lf h o u rs , a fte r w h ich a fu r th e r 10ml o f carbon d is u lp h id e was fu r th e r 30 m in. A f te r 3 h rs , the m ix tu re was allowed to cool fo r 5 m inutes, d e co lo riz in g charcoa l was added and fu r th e r re flu x e d fo r 10 mins be fo re f i l te r in g hot th ro u g h flu te d f i l t e r p a p e r. The f i l t r a te was evapora ted to d ry n e s s in b o ilin g w a ter b a th . The res idue was re d iso lve d in 75-100ml o f d is t i l le d w a te r and the so lu tion tre a te d c a re fu lt ) , w ith v ig o ro u s sh a k in g , w ith 30ml o f g lac ia l ace tic acid m ixed w ith an equal volume o f w a te r. 2-m ercaptobenzoxazo le was im m ecia te ly p re r 'n ita te d as a w h itish so lid . T h is was f ilte re d by suc tion and o’ r ie d in a»r. The m elting po in t was de te rm ined . 37 2 .1 .2 P repa ra tion o f 2 -ch lo robenzoxazo le and sod ium - benzoxazo le -2-su lp h o n a te 2-m ercaptobenzoxazo le (5 .0 g ) was m ixed w ith about 15g o f phospho rus p e n ta c h lo r id e . M ix tu re was heated on a w a te r bath fo r 1 ho u r and then on a hot p la te fo r 1- 1? h o u rs , to re f lu x . The m ix tu re was allowed to cool to room tem p e ra tu re aand trnea ted w ith 50ml o f 251 sodium s u lp h ite so lu tio n . A f te r e ffe rv e s c e n re , the m ix tu re was tre a te d w ith a fu r th e r 50ml o f sa tu ra te d sodium s u lp h ite so lu tio n . I t was then re flu x e d fo r abou t 2 hou rs on hot p la te , a f te r w h ich it was f llte re d hot th ro u g h f lu te d f i l t e r p a p e r. C ry s ta ls appeared as w h ite needles. T h is was f it te re d by suc tion and d r le d irn a ir . T h io n y lc h lo r id e may be used in place o f phosph orus p e n ta c h lo r id e . When 2- m ercaptobenzoxazole (4 .5 g ) was tre a te d w ith 20ml o f th io n y l C h loride (S O C I^), a v ig o ro u s reaction w ith e v o lu tio n o f gas and a d a rk g reen s o lu tio n W a s ob ta in e d . The so lu tion was re flu x e d in w a te r ba th fo r 2 hou rs d u r in g w h ich th re e 10ml p o rt io n o f th io n y lc h lo r id e were added at in te rv a ls . The excess th io n y l C h loride was d is t i l le d o f f and a b row n o il was le f t . The brow n e il was pou red c a re fu lly in to 50ml o f 25% W /V sodium su lph ide s o lu tio n . T h :s was then re flu x e d fo r 2 -2 .5 h o u rs . A t the end o f re f lu x , so lu tion was f ilte re d hot and w h ite c ry s ta ls p re c ip ita te d . The b row n so lid in the ro u n d b o tto n fla sk was 38 re pea ted ly d iges ted 4 tim es w ith 20-30mI o f d is t i l le d w a te r by hea ting o ve r a ho t p la te fo r 15-20 m inutes on each occasion, the ho t so lu tion was f ilte re d in to the o r ig in a l liq u id , more o f the w h ilte p ro d u c t p re c ip ita te d . T h is was then f i lte re d by suc tion to ob ta in w h ite so lid . 2.2 Preparation of Resin-Bound- 2.2.1 Determination of exchange capacity of an ion­ exchange resin (anion exchange resin( (C I) form 1. 0g o f a ir d r ie d anion exchange re s in (c h lo r id e fo rm ) was w e igh ted in to a w atch g lass , and th is was t ra n s fe r re d c a re fu lly th ro u g h a d r y clean funne l in to a 50ml clean b u r re t te . S u ff ic ie n t ly d is t i l le d w a te r was added to cove r the re s in and all traces o f a ir bubb les th a t s t ic k to the res in and the b u re tte were d is lo d g e d . 0.24M o f sodium n itra te (NaNO^) so lu tion was p re p a re d and t ra n s fe r re d 250cm3 sepa ra ting fu n n e l w h ich was m ounted on the top o f the b u re tte such th a t the t ip o f the fu n n e l made close con tac t w ith the opening o f the b u re tte as th e so lu tion d ropp ed in to the column a t the rate o f about 2cm3 p e r m inu te , the e ff lu e n t was co llected in a 500cm3 conica l f la s k and t it ra te d w ith Standard 0.1M s ilv e r n itra te so lu tion (A gN O ^) us ing potassiurt chromate as in d ic a to r . 39 The fo llo w in g reaction o ccu rre d as exp la ined in F ig . 1.22 below . + N 0 3 R + N 0 3 + CI R = res in C I" = co u n te r ion N 0 3 = exchang ing ion F ig . 1 .22 : Equation show ing the exchange o f Chloride ion by n itra te ion in an an ion -pxchange res in 2.2.2 Titration of effluent (NaC I) agai nst Standard 0.1M silver nitrate T itra t io n : E ff lu e n t (NaCI) vs ^ y iM AgNC>3 P ipe tte (25cm3) con ta ins 0.25M NaCI B u rre tte (50cm3) con ta ins 0 . 1M AgNO^ In d ic a to r 0 .5cm 3 fo r each 25cm3 o f the f in a l volume o f the te s t so lu tio n A verage nesu lts o f th re e d if fe re n t t it ra t io n s _ 29.70 cm3 In d ic a to r b la n k = Is t 1.65cm3, 2nd 1.25cm 3. Exchange capa c ity o f res in = M illi-e q u iv a ie n t p e r gram = bV/W where VCM3 o f BMAgNO^ are re q u ire d by Wg o f the re s in . The ca lcu la tion is shown in F ig . 1.23 below . bV/M 0.1 x 29.70 1 2.970 Meq/g 2.97 x 10"3Moles 2 1g 2.97 Moles = 100g 0.00297 = lg M eq/g o f anion exchange res in (C h lo ride f< = 0.00297 F ig . 1 .23 : Equation e xp re ss in g the d e te rm in a tio n o f exchange capac ity 2 .2 .3 Co u p lin g sod ium -benzoxazo>l le-2-su Ilfonate with re s in (c h lo r id e fo rm )30 Resin (2 .9 7m e q /g ) used fio r p re p a ra tio n o f re s in bound agent as fo llow s. The e q u iva le n t am ount o f sod ium -benzoxazo le -2 -su lphona te th a t w ill coup le w ith lg o f res in (c h lo r id e fo rm ) is ob ta ined as fo llow s . 0.00297 x 221g (m olar mass scdium benzoxazo le -2 - su lpha te = 0.69615g. 0 .5g o f re s in was s t ir re d w ith sod ium -b enzoxazo le -2 -su lfon a te (0 .35gm s) in so lu tion o f warm d is t i l le d w a te r. To e s tab lish the Optimum time fo r exchange, m ix tu re was s t ir re d fo r 3 h o u rs , so lu tion from m ix tu re was co llected a t tim e in te rv a ls o f 15 m inutes, 30 m ir.s; 1 h o u r; 1 ho u r 30 m ins; 2 h o u rs ; 2 hou rs 30 m ins; and 3 h o u rs , and t it ra te d aga ins t a S tandard so lu tio n o f s ilv e r n itra te 41 Tab le 2: D e te rm ina tion o f Optimum time o f exchange between C h loride ion and benzoxazole m oiety in an ion e x c h a n g e ^ e t jn (c h lo r id e fo rm ) Time In te rv a l 15 mins 30 mins 1 h r I h r 30m 2 h r 30m 2 h r 45m 3.0 h r F inal re a d - ings cm3 11.20cm3 11.60cm3 12.60 2 2 .G rN O 1 .6 0 25.00cm 3 36.60 12 .7 ü ^n 3 In it ia l re a d - in g cm3 0.0 0.0 1.00 11*40 0.00 13.35 25.00 1.50 Volume o f A gN 0 3 used in cmJ 11.20 11.60 .60 11.25 11.60 11.65 11.60 11.20 « 5 < r us ing potassium d ich rom a te /ch rom a te m ix tu re as in d ic a to r . Same p ro ce d u re was c a rr ie d o u t fo r b la n k . The p ro ce d u re used in d e te rm in in g Optimum tim e is summarized in Tab le 2 below . C o rre c tio n was also done fo r b la n k , i .e . d is t i l le d wate T h is is shown in F ig . 1.2 below . B lank d is t i l le d w ater F ina l read ings cm3 1 3 . 5 5 ^ In it ia l read ings cm3 Volume o f AgNO^ used cm3 S - 5 5 je F ig . 1 .24 : B lank c o rre c tio n . The O p t i m u m t i m e fo r e x c h a n g e w a s 3 0 m i n u t e s . E q u a t i o n fo r coup ling is shown in F ig . 1.25 below . ( J H ^ ch^ - n - chJ- er + No+ so3 —^ ] Q ) CH- disti l led F ig . 1 .25: C oup ling o f benzoxazole W a le r w ith anion 6 3 - 7 0 °C exchange res in C H i+ 0 - ^ ^ - c h 2 - n - c h J S 0 3 ------ 43 2.3 Reaction of Amines with Sodium-Benzoxazole- 2 -S u lfo n a te 10|jl o f the fo llow ing amines, D i-n -b u ty lam ine, d ie th y la m in e , D i-n -p ro p y la m in e , 4 n itro a n ilin e and b lank were added to f iv e d if fe re n t te s t tu b e s . S o d ium -benzoxazo le -2 -su lfona te ( 0 . 1g) was added to each o f the te s t tu b e s . The m outh o f the te s t tubes were sealed w ith m asking tape, and m ix tu re s were warmed at 60°C fo r 5 m inutes in a w a te r b a th . A t the end o f 30 m inu tes , the reac tion p ro d u c t were each obse rved u n d e r u lt r a - v io le n t l ig h t (254nm and 360nm ). A f te r was e x tra c te d w ith 5ml o f C hloroform by shak ing on a v o r te x m ixe r fo r 1-2m in. The aqueous la ye r was removed w ith a p a s te u r p ip e t and the C hloroform d r ie d w ith a n h yd rous sodium s u lfa te . The C hloroform e x tra c t was then t ra n s fe r re d to a n o th e r te s t tu b e s and the C hloroform removed u n d e r a stream o f n itro g e n . Some ot the res idue ob ta ined w a a /red isso lved in methanol fo r T L C ; HPLC and mass sp e c tro m e tric ana lys is . The methanol so lu tion was also exam ined u n d e r UV lig h t . The same p ro ce d u re was also c a rr ie d ou t u s ing a c e to n itr ile as medium fo r reaction ins tead o f d is t i l le d w a te r. 2.4 Optimization of Solvent, Temperature and Time Among the reaction perfo rm ed was to in v e s tig a te w h ich o th e r so lve n t a p a rt from water cou ld be used as a good medium fo r d e r iv a tiz a tio n reactions o f the an a ly te . In th is re g a rd op iim a l reac tion co n d itio n s fo r the fo rm ation o f the d e r iv a tiv e s were de te rm ined by com paring the fluo rescence o f the p ro d u c ts u n d e r UV lig h t as a fu n c tio n o f so lve n t. A ll o th e r reactions co n d itio n s were he ld co n s ta n t; so lven ts tes ted in c lud ed m ethanol. e th y la ce ta te , hexane and a c e to n itr ile . T em pe ra tu re was optim ized by v^«a ry ing 1from 30°C ( la b o ra to ry te m p .) to 100°C h o ld in g time cons tan t a t 30 m inu tes . N ext to be op tim ized was time in the p rocess , the Optimum te m p e ra tu re a t 60°C was held constan t and tim e v a r ie d from 0 m inutes to 1 h o u r. 2.5 Derivat» of Amines with Resin-Bound benzo 2-S u ifon a te To a on o f 100pl o f d ie th y l- o r d i-n -p ro p y l o r d i - n - bu ty la m ine o r 4 -n itro a n ilin e in 1. 0ml o f methanol was added 1. 0g o f r e N ^ ta o g e d w ith ben zoxazo le -2 -su lfona te . A f te r w arm ing in a w a te r ba th (60°C ) fo r 5 min the so lu tion was exam ined u n d e r UV lig h t and ana lysed by T LC , HPLC and HPLC-M S. The p ro ce d u re was repeated using acetonitrile in place of methanol. The procedure was repeated, with the m ixture being kept at room tem perature before Chromatographie analysis. The same p ro ce d u re was done fo r b la n k . 2.6 Reaction of Amino-Acids with Benzoxazole-2I-Su lfo n a te To 100mg o f g ly c in e (o r 1 - ly s in e o r 1 -cys te in e ) d isso lved in 1. 0ml o f w a te r was added a so lu tion o f 100mg o f sodium ben zoxazo le -2 -su lfona te in 2.0m l o f w a te r. T he m ix tu re was exam ined u n d e r UV lig h t and then warmed in a w a te r ba th (60°C ) fo r 5 mins be fo re be ing re -exam ined u n d e r UV l ig h t . A p o rt io n o f the reaction m ix tu re was b a s ifie d by d is s o lv in g about 200mg o f sodium b ica rb ona te in i t and so lu tion re -exam ined u nd e r UV lig h t . The same p ro ce d u re was done fo r b la n k . 2.7 D eri ion of Amino-Acids Using Resin-Bound te n z o x a z o Ie -2- iS u lfon a te mg o f g ly c in e (o r 1 lys in e o r 1 cys te in e ) d isso lved in 1. 0ml o r methanol was added a 1 . 0g o f re s in -ta g g e d w ith b e n zo xazo le -2 -su lfona te . A f te r warm ing in a w a te r ba th (60°C ) fo r 5 min the so lu tion was exam ined u n d e r UV lig h t . An a liq u o t o f the reaction m ix tu re was tre a te d w ith 200mg o f sodium carbonate and obse rved u n d e r UV lig h t and ana lysed by T L C . The same p roced u re was done fo r b la n k . 2.8 Derivatization Using Resin, Sodium Benzoxazole-2- Sulfonate, Amine Substrates To lOOpI o f d ie th y l o r d i-n -p ro p y l o r d i - n - b u ty l o r 4 - nitroaniline in 1.0ml of methanol was added solution of 0.1g sodium benzoxazole in methanol and 0.1g of anion exchange resin Chloride fo rm . The m ix tu re was warmed in a w a te r ba th a t 60°C fo r 5 m inutes be fo re be ing observed u n d e r UV l ig h t . The same p ro - / > v cedu re was c a rr ie d ou t fo r b la n k . 2.9 Derivatization Using Resin, Sodium Benzoxa.. Süifonate, Amino Acids Substrate To 100mg o f g ly s in e o r 1 lys in e o r 1 cys te in e in methanol was added so lu iio n o f 0 . 1g sodium benzoxazole in m ethanol and 0 .1g o f an ion exchange res in C h loride fo rm . The m ix tu re was warmed w ater ba th at 60°C fo r 5 m inutes be fo re be ing observed u n d e r UV l ig h t . An a liq u o t o f reac tion m ix tu re was tre a te d w ith 200mg o f sodium carbonate and exam ined u n d e r UV l ig h t . The same p roced u re was pe rfo rm ed fo r b la n k . 47 2.10 Th in-Layer Chromatographie Analysis of Amine Derivatives Obtained Through Homogeneous Reaction Working Procedure 1 - 2|jI o f d ie th y la m in e , d i-n -b u ty ia m in e , d i-n -p ro p y la m in e , 4 n itro a n ilin e d e r iv a tiz e d in aqueous medium (S tandard amine d e r iv a tiv e s ) were spo tted along w ith b lank on a th in la ye r p la te coated w ith s ilica g e l. Spots were allowed to d ry in an oven a f te r w h ich the p la te was developed in a ta n k co n ta in in g a c e to n itr i le :w a te r (8 5 :1 5 ). A t the end o f developm ent the p la te was removed from the bank and a llowed to d ry in a ir ; a f te r w h ich the spots were obse rved u n d e r a UV lig h t . The, same p ro ce d u re was also c a rr ie d ou t fo r S tandard amine d e r iv ^ ^ e s p re p a re d us ing a c e to n itr ile as the medium fo r re a c tio n . 2.11 T h in Laye r C h rom atography of Am ino A c id D e riv a tiv e s O b ta ined from Homogeneous Reaction Ä ? ------------------ W orkin g Procedure /^Hquots o f L - ly s in e , c y s tin e , g ly c in e d e r iv a tiz e d in aqueous medium were spo tted along w ith the a p p ro p ria te b la n k . The p ro ce d u re above as describ ed fo r amines was fo llow ed . 48 2.12 Th in-Layer Chromatography of Amine Derivatives Obtained Throuqh Heterogeneous Reaction A liq u o t o f the d e r iv a tiz e d p ro d u c ts (d ie th y la m in e , d i- n - b u ty la m ine , d i-n -p ro p y la m in e , 4 n itro a n ilin e ) and b lank were spo tted on a h ig h la ye r coated w ith s ilic a . Spots were allowed to d ry a fte r w h ich p la te was developed in a ta n k con ta in in g a c e to n itro le :w a te r (8 5 :1 5 ). A t the end o f deve lopm ent the p la te was removed and so lve n t was allowed to d ry a f te r w h ich i t was obse rved u n d e r a UV lig h t . 2.13 T hin Layer Chromatography of Derivatives from Resin (C hlorideform), S ric a»ciicd (30m!) were m ixed . The m ix tu re was heated by immensing in a w a te r ba th at 90°C - 100°C fo r 3 h o u rs ; w ith shak ing a t in te rv a ls . A yeüow ish oil was form ed on the top o f the m ix tu re a fte r hea ting fo r 2* h o u rs , the o il la ye r was c a re fu lly decanted and the yeüow ish m ix tu re heated fo r fu r th e r 30 m inu tes, more o ily la ye r energed and was c a re fu lly decanted. The o il remoxed soon so tid ifie d and th is was d igcS ted w ith acetone by hea ting on a w a te r ba th to g ive a pale yeüow ish so lid . V .ix tu re o f the pale ye llow so lid separa tes as the main b u ik o f the reaction m ix tu re s .ooled. 'he rr.elting int w, the end o f deve lop ­ ment the p la te was allowed to d ry in a ir , a f te r w h ich i t was obse rved u n d e r u lt ra v io le t l ig h t . ( ü ) To vafidate the resul t further T h in la y e r ch rom a to giraSphy was c a rr ie d o u t in an o th e r so lven t medium i.e . 1ml ammonia so lu tion and 100ml o f 95% e thano l. The p la te was also obse rve d u n d e r UV lig h t . The Chromatographie p roced u re was pe rfo rm ed 3 times and each reaction m ix tu re W2S spo tted in t r ip lic a te fo r a to ta l (b = 9 ). HPLG q u a n tita tio n to be c a rr ie d ou t in an ou ts id e la b o ra to ry . 57 2.2*1 M e ltin g P o in t D e te rm in a tio n o f S ta n d a rd E s te r D e r iv a t iv e s The m elting p o in ts o f the va riou s es te r d e r iv a tiv e s were de term ined by two m ethods. The f i r s t in vo lve d the use o f p a ra ff in and c a p illa ry tubes a ttached to a ce lsu is therm om eter ( 100° C ) . The second p ro ce d u re in vo lve d the use o f in s tru m e n t (e le c tro th e rm a l) . In both cases w ith lim its o f e x p e r i­ mental e r ro r the re s u lt ob ta ined were re p ro d u c ib le when com pared. The va rio u s m e lting po in ts are lis te d u n d e r re s u lts and d iscuss ions sec tion . 2 .2 5 T h i n -L a y e r C h rom at o g rs p h ic A n a ly s is o f D e r iv a t iv es Pre p a re d U s in g th e R esin B ound R eag en t Follow ing the same p roced u re app lied fo r C hrom atographie ana lys is o f s tanda rs i*« « te r d e r iv a tiv e s . The th in la ye r Chromato­ g ra p h ie ana lys i i Q r re s in -b o u n d d e r iv a tiv e s and the a p p ro p ria te b lank w e r^ c a rr ie d o u t . The re su lts ob ta ined were compared w ith tnose ob ta ined fo r S tandard es te r d e r iv a tiv e s . T h is is d iscussed u n d e r the section fo r re s u lts and d iscuss io n . 58 2 .2 6 T h in -L a y e r C h ro m a to g ra p h ie A n a ly s is o f S ta n d a rd Es te r D e r iv a t iv e s an d Each C o rre s p o n d in q D e r iv a t iv e s O b ta in e d from R e s in -B o u n d R eaction On same p la te each Standard e s te r d e r iv a tiv e s was spo tted a longside its c o rre sp o n d in g re s in -b o u n d d e r iv a tiv e s . T h is was also done fo r the b la n k . Plates were deve loped as exp la ined p re v io u s ly and obse rved u n d e r UV l ig h t . The re s u lts are d iscussed u n d e r the section fo r re s u lt and d iscCuUSsSsKio n . & < r 59 C H A P T E R T H R E E R E S U L T A N D D IS C U S S IO N 3 .1 S y n th e s is o f Sodium B e n z o x a z o le -2 -S u lfo n a te For d e r iv a tiz a tio n reagents to be ideal fo r HPLC on TLC w ith UV o r FL de tec tion i t shou ld possess UV o r FL c haraic te r is t ic s th a t a re com ple te ly d if fe re n t from th a t o f the d e r iv a tiv e s , th is w ill take care o f excess reagent th a t may in te r fe re w ith the de tec tion o f tne d e r iv a t iv e . H ow ever, i f reagen t and the de r iv a tiv re have s im ila r spectra c h a ra c te r is t ic s , th e ir C hrom atographie b e h a v io u r shou ld be w ide ly d if fe re n t ; th is w ill allow easy Separation o f excess reagen t from the d e r iv a tiv e i f th is cannot & s ily ach ieved b y sim ple so lven t e x tra c t ic n . The reagent shou ld react re a d ily w ith the ana ly te w ith o u t any com pücating s ide reac tions . ln a d d itio n2 d e r iv a tiv e s obta ined from reac tion shou ld be s tab le , and d e r iv a tiz a tio n reac tion shou ld be possib le in a v a r ie ty o f so lven ts .ä^cT ^o Iven t v.ombinations th a t are lik e ly to be encoun te red d u r in g the in ten ded C hrom atographie a p p lica tio n s . These are im p o rta n t requ ire m en ts i f the reagent is to be app licab le to o n - lin e p re - o r post-co !um n d e r iv a tiz a tio n in a possib le autom ation o f the a n a ly tica l m ethod. The reagent should be cheap. so Most a n a ly tic a l reagents p re v io u s ly used by va rio u s w o rke rs e ith e r have close s im ila r ity in th e ir spectra o r flu o re sce n t c h a ra c te r is t ic s when com pared to the d e r iv a tiv e s they form ed, w ith amine, amino acids and c a rb o x y lic acid e .g . D a nsy lch lo ride (5 -d im e th y la m in o -n a p h th a le n e -1 -s u lfo c h lo r id e ). As a re s u lt , the in e v ita b le presence o f excess reagent in the reac tion m ix tu re , th e re fo re , causes h igh b a c k g ro q ^ ^ e s p o n s e and hence an inadequate de tec tion lim it fo r the an a lly te . In tro d u c tio n o f Steps to remove excess reagent o fte n makes th e method more ted ious and more p rone to e r ro r . D Some reagents such as fiuorescam ine, NBD Chloride (7 ,c h lo ro -4 -n itro b e n z o -2 -o x a -1 , 3 -d ia zo le ), OPA (o -p h th a la ld e h yd e are n o n -flu o re s c e n t o r p o o rly f lu o re s c e n t; though th e y react w ith amines to g ive flu o re s c e n t d e r iv a tiv e s , are in th is respect b e tte r than a flu o re sce n t reagen t such as dansy l C h lo ride . These reage n ts , how ever, share the d isadvan tage th a t they can on ly be used in homoaeneous media, m oreover, they sometimes unde rgo undes irab je h y d ro ly t ic decom position d u r in g d e r iv a tiz a tio n reactions w ith öffcve; besid-_s, th e ir flu o re sce n t d e r iv a tiv e s are h ig h ly unsta& te to l ig h t , tem p e ra tu re and m o is tu re . 61 Amines are known as fa ir ly s tro n g nuce loph iles , chrom oaenic d e r iv a tiv e s o f amines have been p re p a re d . Such p re p a ra tio n was based on th e ir reaction w ith reagents bea ring su lfona te g roup s ac tiva te d tow ards nu c le o p h ilic S u b s titu tio n . The reagent tha t ia t were used fo r such p re p a ra tio n in c lud ed sodium 2 , 4 -d in itro b e n ze n e su lfona te 35““38 and sodium 2 ,6 -d in itro -4 - tr if lu o ro m e th y lb e n z e n e su lfona te . 39 These reagents have the d es ira b le p ro p e r ty o f be ing usable in aqueous media. The reage n ts , how ever, g iv e n o n -flu o re sce n t d e r iv a tiv e s since the e le c tro n -w ith d ra w in g n it r o g roup s re q u ire d fo r the a c tiva tio n o f the su lfona te m olety tow ards n u ce lop h ilic S u b s titu tio n are also s tro n g in h ib ito rs o f fluo resce nce . For the p re p a ra tio n o f f lu o re sce n t d e r iv a tiv e s o f amines, i t was th c u g h t th a t a lia b ile su lfona te g ro u p a ttached to a flu o re sce n t arom atic nucleus would g ive a w a te r-s o lu b le reagent possessing the des ira b le c h a ra c te r is t ic o u t lin e d . A z o le s ^ re m onocylic arom atic com pound w ith more than one hete roa tom s. \ VTney a re 5 membered r in g s w ith 2-heteroatom s w ith n itro g one o f the heteroatom s. In oxazo le, the 3 rd carbon atom o f a fu ra n is rep laced by n itro g e n and when th is oxazole is jo ined to a benzene r in g a üenzcxazole re s u lts as shown in the F ig u re 1.28 below . 62 : C U F ig . 1 .28 : S tru c tu re o f Benzoxazole. The n itro g e n atom is h ig h ly u n s a tu ra te d , and as Longuet and co -w o rk e rs 31 have show n, the 2 orC’C-position is re la t iv e ly low in e le c tro d e n s ity . T hus the re a c t iv ity o f a m ethylene g ro u p in th is p o s ition is s im ila r to th a t o f ac tiva te d m ethylene g ro u p o f 2 ,4 ,6 , t r in it ro to lu e n e o r e th y l acetoacetate. T hus 2 -m e thy l benzoxazole w ill p a rt ic ip a te in knoevenagel 32 f j reac tions i .e . a genera l method o f p re p a rin g ^ -u n s a tu ra te d acids as shown in F iq u re 1.2: 9 below . RCHO + CH. C C ^ C 2H5 )2 -M H .^ R C H =C (C 02C2H5) 2 + H20 RCH = C IC C T C -H .) , H) KOH__ v r c h = C (C O ,H ) , 150-200°C v 2 5 2 ( i i ) HCl * 2 2 y PXH = C H C 02H + C 0 2 F ig . 1 .29 : Reaction between a ldehydes and compounds w ith ac tive m ethylene g ro u p s in the presence o f an o rg a n ic base. 63 ln a d d it io n , 2 m ethylbenzoxazo le w ill take p a r t , in O rto leva K ing reac tion 33 ju s t lik e m ethyl o r m ethylene g ro u p s in ketones as shown in the exam ple below . I 2 / P y rid in e HO' C 0 C H 3 60 V . ^ 0 NJ~ \ _ K> - C 0 C H 2 Py 1“_j> Py-f H f OH F ig . 1 .30 : Reaction o f m ethyl g roup s in w ith p y r id in e A iso i t has been shown th a t the ac tiva teadc ^J^uullfon ic g ro u p in 2 ,4 ,6 , tr in itro b e n z e n e su lphon ic acid can be sub jec ted to nu c le o p h iiic Substitution. Besides, the reagent was used to determ ine amino com pounds spe c tro pDhhoottoomrre-' tr ica lly . Such a reagen t canno t how ever be used fo r ana lys is o f amines by s p e c tro flu o r im e try due to the presence o f n it ro g ro u p s th a t is fluorescence in h ib it in g . T nus from a ll the enum erated ana log ies, i t was im agined th a t a su lphon ic ac id g ro u p in the a c tiva ted 2-p o s it io n o f benzo- xazole would1 . be lia; b le to nucleophiiic Substitution. Besides, benzoxxaa fte^v is p o te n tia l ly a flu o re sce n t m oiety w h ich w ill be non - ftuo resT prit o r w eakly flu o re sce n t when a su lfona te g ro u p is a ttach • J due to e le c tro n -w ith d ra w in g /f lu o re s c e n t in h ib it in g e ffe c t o f th is group, h cw eve r, Substitution by an amine will give a f lu -^e s c e n t p ro d u c t. 30 In conc lus ion , due to a n tic ip a te d h ig h re a c t iv ity o f the a c tiva te d s u lfo n ic g ro u p tow ards amines and the po ten tia l fluorescence o f the benzoxazole m oie ty , sodium benzoxazo le -2- su lfona te was p re p a re d and in v e s tig a te d as a poss ib le w ater so lub le reagent fo r f lu o r im e tr ic ana lys is o f amines and aminoA ac i.ds. Sodium benzoxazo le -2-s u lfo n a te has on ly been m entioned in an old pa ten t l i te ra tu re . 40 It is re a d ily ob ta ined p u fe V ro m 2 -ch lo ro benzoxazole as describ ed in F ig u re 1.31 be low . A lth ough we had to p re p a re 2-ch lo rob enzoxazo le , th is com pound is a p p a re n tly ava ilab le com m erc ia lly . The reagen t was p re p a re d as seen in> the equa tion 30 as shown in F igu re 1.31 below . NH- . & K O H /M e c Hf r C M SH PC15 CI 65 Each stage o f the reaction was m onitored by th in - Ia y e r c h ro m a to g ra p h y . When th in Iayer ch rom a to g raphy was c a rr ie d ou t in C h loro form . S ing le b u t d if fe re n t spots were ob ta ined fo r 2 m ercaptobenzoxazole, 2 ch lorobenzoxazo le and sodium benzoxazole 2-su lfona te 3 .1 .1 O bse rva tion 100mg o f the sod ium -benzoxazo le -2-s u lfo n a te was d isso lved by warm ing in d is t i l le d w a te r. T h is s o lu tio r i'w a s obse rved u n d e r UV lig h t th e re was no fluo rescence . Then about 100pl o f N - bu ty la m ine was added to the sol “ nd obse rved u n d e r UV lig h t . The so lu tion gave an in tense b lue fluo resce nce . T h is in d ica te d th a t the amine w ill react v e ry fas t w ith benzoxazole so lu tion to g ive a flu re s c e n t p ro d u c t. The equa tion fo r such reac tion is shown in F ig u re 1.32 below . H / N \ r Equation show ing the a d d itio n o f CH3 ( c h 2 n -b u ty la m in e to so lu tion o f benzoxazole to g ive flu o re sce n t p ro d u c t 66 One o f the in it ia l e xpe rim en t was to de te rm ine the exchange capac ity o f the anion exchange re s in s . Based on the de te rm ina ­ t io n , i t was es tab lished th a t 0.69615g o f benzoxazole reagent w ill exchange fo r C h loride ions in lg o f an ion ic re s in s . M oreover, com parison o f t i t r e va lues showed th a t the am ount o f sodium C hloride released in to so lu tion as a re s u lt o f the d isp lacem ent o f C hloride ions by the ben zoxazo le -2 -su lfona te ion remained cons tan t a f te r 30 min o f s t i r r in g the res in w ith sodiilm benzoxazo le -2 - su lfo n a te . T h is was taken to mean th a t the exchange was complete w ith in 30 m in, and subsequen t p re p a ra tio n s o f the re s in -b o u n d benzoxazo le -2-s u lfo n a te s im p ly in v o lv e d s t ir r in g a warm so lu tion o f the reagent w ith th e an ion -excha nge res in fo r SO-^Smin. The equation fo r co u p lin g is exp la ined in F ig u re 1.33 below . F in . 1.33: E q u rtio n fo r po lym er su p p o rte d benzoxazole m oiety (a n a ly tic a l reagen t) 67 The op tim iza tion o f so lv e n t, te m p e ra tu re and time e x p e r i­ ments showed th a t methanol and a c e to n itr ile so lven ts gave the best f lu o re sce n t p ro d u c t. A lso the d e r iv a tiv e s ob ta ined at 60°C and above fluo resced in te n se ly w ith no s ig n if ic a n t d iffe re n c e in t ne fluorescence at h ig h e r te m p e ra tu re . The time indicated that the fluorescent derivatives nave been formed in 5 minutes of reacting the benzoxazole with the Substrate. As a re s u lt o f the above o b se rva tio n s , d e r iv a tiz a tio n reac tion can be pe rfo rm ed in the fo llo w in g m edia: (a) d is t i l le d w a te r (b ) mwthanol (c ) ace ton itr i le at tem p e ra tu re between 60°-65° ho ld in g time cons tan t at 5 m inutes o r 10 m inu tes. 3 .1 .2 reaction of amines with the resin-bound jzcie-2-sulfonate A ll fu r th e r reactions o f the amines were pe rfo rm ed u nd e r op tim ized tim e, tem p era tu re and so lven t as de te rm in e d . The volume o f sample d e r iv a tiz e d was 100p! as re p o rte d in the expe rim enta l sec tion . The fo llo w in g amine were d e r iv a tiz e d : 63 1. D ie thy lam ine 2. D i-n -b u ty la m in e 3. D i-n -p ro p y la m in e 4 . 4 n itro a n ilin e . The equa tion fo r d e r iv a tiz a tio n on the po lym eric S upport is shown in F ig u re 1.34 below . CH- INH. © ----( o ) “ C H ; - N '- C H j S O ; RNH acetonitrile o CH 3 Sr 60 mins F ig . 1 .34: E quation fo r d e r iv a tiz a tio n o f amine on re s in S upport The same reac tion p roced u re was c a rr ie d o u t fo r the b lank and all the s u p e rn a ta n t were co ltected each in d if fe re n t te s t- tu b e s and obse rved u n d e r th e U V . The re s u lt ob ta ined are p resen ted in Tab le 4 below . Tab le 4: O bse rva tion o f f lu o re sce n t d e r iv a tiv e s o f amines u nd e r UV lig h t A n a lv t i cal S u p e rn a ta n t O bserva ­ Reagent Amine Substrate tio n u n d e r UV R esin -bound D ie thy lam ine B lue fluo rescence weak benzoxazole D i-n -b u ty la m in e In tense b lue fluo rescence s tro n g D i-n -p ro p y la m in e B lue fluo resence 4 n itro -a n ilin e D ark red fluorescence B lank No fluo rescence 69 The re s u it ob ta ined are p resen ted in Tab le 5 below . Tab le 5: T h in - la y e r C hrom atograph ie re s u it o f amine d e r iv a tiv e s ob ta ined by heterogeneous reaction D e riva tive s o f C o lou r o f spot Spot ce n tre u n d e r UV FF va lue=Solvent f ro n t 2- ( N, N -d ie th y lam in o ) B lue FL weak <(.3/7.2 = 0.5 97cm benzoxazole 2 - f N ,N -d i-n - In tense b lue FL 3 4cm b. ylam ino) s tro n g benzoxazole 2 -(N , N -d i-n - B lue 3 .9 /7 .2 = 0.542cm p ophylam ino) benzoxazole 2 -( N, N-4 5 .7 /7 .2 = 0.792cm n itro a n iiin o ) benzoxazole B lank ls fi\ fluo rescence No RF va lue (sodium benzoxazole 2-s u lfo n a te ) The reactioni o f eaml:nes wiith sodium ben zoxazo le -2 -su lfona te is i l lu s tra te d i rt F ig u re 1.35 below . Form ation o f the d e r iv a tiv e s was con firm ed from th e ir mass s p e c tra . The mass spec tra o f the d e r iv a tiv e s o f d ie th y l- d i- n -p ro p y l and d i-n -b u ty la m in e are shown in F igu res 1.35a, 1.35b and 1.35c re s p e c t iv e ly . 70 k X o ^ s°5 No+ +| « 3 (ch2)3' nh ------ =* Q C oV n x ‘ { ! ] Di - n - b u ty la m in e ^ X o ^ S 0 ' N a » + | c H j C H > NH - I ' ‘ ' I [ 2 ) D ie thy l amine c h 2c h 3 N / CH2CH2 CH3 I X 1] y — SO “ Na+ + ( C H 3 CH2 CH^ )2 NH ( 3 ) Di - n - propylamine c h 2c h 2 c h 3 Q Q / - s ° S N ^ ^ o ,-c 6 h , . NH- I M t v * t roa niline sc: N o * ( Blank ) W V ~ S 0 3 N a * in so lu tion F ig . 1 .35: I llu s tra t io n o f reac tion o f sodium benzoxazo le -2 - su lfona te w ith amines (homogeneous approach) A ll the 2 - (N , N -d ia lky la m in o ) benzoxazole, gave spectra w h ich e x h ib it in tense [M +N H ^]+ ions due to the presence o f acetate in the mobile phase used fo r the HPLC-MS System. The o th e r peaks in the mass spec tra o f these com pounds may be re a d ily ra tio n a lise d as shown in F ig u re 1 .35d . The ion w ith m/z 135 is common to the mass spectra o f 2 -(N , N -d ia lky la m in o ) benzoxazoles and a p p a re n tly re s u lts from loss o f th e two a lk y l 4. g ro u p s from the [M+H] ion as the co rre s p o n d in g o le fin e s . T ha t is , loss o f the a lk y l g ro u p s accompanied by H -re a rra n g e m e n t. F ission o f the bond between the O-atom and the 2 -ca rbon atom is known to be the p r im a ry fra g m e r't a t io n Ss tep in the mass spectra o f cxazo les. 41 T h is seems to be tru e fo r these 2 -d ia lky la m in o benzoxazoles too as shown in F ig u re 1 .35d. The app a ren t s p e c if ic ity o f the frag m en ta tio n mode«^^eading to the ions be a rin g the a lk y l g roup s th a t th e benzoxazole d e r iv a tiv e s may also be u se fu l in the q u a lita ­ t iv e id e n tif ic a tio n o f unknow n am ines. The mass spectrum o f the rc ^g e n t jV ^WP lhrw n itn F ig u re 1 .35d. A p p a re n tly the base peak ion (m / 136) resu llts from e lim ina tion o f S0 2 from the s u lfo n ic ac id . Sodium benzoxazo le -2 -su lfona te was found to be non - f lu o re s c e n t, p ro b a b ly because o f the fluo rescence in h ib it in g e ffe c t 72 o f the su lfona te g ro u p . The 2 - (N , N -d ia lky lam ino ) benzoxazole how ever, e x h ib its an in tense b lue fluo rescence when th e ir Solu­ tion were obse rved u n d e r UV lig h t . Reaction o f the amines w ith sodium benzoxazo le -2-s u lfo n a te occu rs ra p id ly , w ith the blue fluorescence o f the 2 - (N , N -d ia lky la m in o ) benzoxazoles appearing w ith in a m inute o r two o f add ing an aqueous o r methanol so lu tion o f the amine to an aqueous o r m ethanol so lu tion o f the reagen t. The d e r iv a tiv e s were rie tec tab le a t low m icrogram levels by TLC fo llowed by exam ination o f the p la te u n d e r UV lig h t . The r f o f the d e r iv a tiv e s o f d ie th y l- , d i - n - p r o p ^ 4 ^ ^ id d i-n -b u ty la m in e were 0 .60 , 0 .54 , 0.51 re s p e c t iv e ly . D ire c t HPLC ana lys is o f the reaction m ix tu re was found poss ib le w ith o u t in te r fe re n c e from excess reagent w h ich , be ing a § a lt, is p o o rly re ta ined on the reve rsed phase co lum n. A chrom atogram o f a reaction m ix tu re o f amines w ith sodium benzoxazo le -2 -su lfona te is shown in F igu re ’ - 35e- , f o f excess rea9ent in ,he so lu tion to iV s m * am atographed may be avo ided a lto g e th e r by add ing a l i t t le o f a s tro n g an ion -exchange res in in the Chloride form reac tion m ix tu re . A n y excess reagent is sequestered o r "möpped u p " by the re s in . The d e r iv a tiv e s may also be e x tra c te d w ith C h loroform w ith o u t in te r fe re n c e from the reagen t. 73 To examine the p o s s ib ility o f us ing the reagen t fo r o n - lin e p re - o r post-co lum n d e r iv a tis a tio n o f am ines, the re s in -b o u n d form o f the reagent was p repa red as d e s c r ib e d . In th is form the reagent s t il l reacted as re a d ily w ith amines as when the resaacttiion was c a rr ie d ou t in a homogeneous medium. The reac tion o f amines w ith re s in -b o u n d benzoxazo le -2-s u lfo n a te has been il lu s tra te d in F ig u re 1.34 above. When the tagged res in was packed in a p a s te u r p ip p e t and a so lu tion o f the amines passed th ro u g h the column o f the re s in , the e luate e x h ib ite d the b lue fluo rescence o f the 2 - (N ,N - d ia lky lam ino jben zoxazo les . A chrom atogram o f an e lua te con ta in ing a m ix tu re o f the d e r iv a tiv e s is id en t ica l as shown in F ig u re 1.35e, excep t th a t th e re is no peak due to excess re a g e n t. The re s in - bound benzoxazo le-2-s u lfo n a te would th e re fo re be u se fu l fo r on­ line HPLC deriva tizage 342. 33. PROF. DR. KROHNKE, F . , 1963. "New M Is o f P re p a ra tive O rga n ic C h e m is try " . Angew Chemie, 225-275. 3«, SA TAK E, K . , OLENYAM A, T . , O HASHI, M . , SHIHOELA, T . AND P H ILL IP S , A .P . , 1954. "S p ec troph o tom e tric D e te rm ina tion o f A m in o g ly c o s id e ^ h t ib io t ic s " . J A .C .S . 76, 3986. 35. SM ITH, A .D . AND JEPSON, A n a l, Biochem, 18:36 (1967). 35. EDWARDS, D .J . AND B LA U , K . A n a l. B iochem .. 45; 387 (1972). 37. IS H IT O Y A , Y . , B A B A , S. AND HASHIMOTOR, I . C lin . Chim . A c ta , 4£;55 (1973). 38. B A B A , S .,C *tyS H IM O TO , I . AND ISHO TO YA, J . C h ro m a toq r. 88^:373 (1974). 39. DOSHI, P .S . AND EDWARDS, D .J . J . C h rom a toq r. 176, 359*41979). 40. ISH, PATENT 418291 (1934); Chem. A b s t r . , 29:819 ^ • 9 3 5 ) . “ 41. AU D IER , H -E ., FETIZO N, Y .H . AND PRANGE, T . O rg . Mass S p e c tr . , 1J_:1047 (1976).