I s m m c s o r th e m m a c id and p ep tid e cataly zed BEALDOLZZATXOH OP DIACETONE ALCOHOL II DETEEMINATXON OF THE HmOXYMETHXL (BOW IN SUGARS AND BELATED SUBSTANCES in KINETICS OF THE ACID AND BASE CATALIZED DEGRADATION OF THE TRI0SE3 By A rlin gto n Ardeane F o ria t A THESIS Submitted to th e School o f Graduate S tudiee o f Michigan S ta te C ollege o f A g ricu ltu re and A pplied Science in p a r tia l fu lfillm e n t o f %h® re^uirem ente f o r th e degree o f DOCTOR OF PHILO30PHI Department o f Chemistry 1952 ProQuest Number: 10008303 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008303 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENT The auth or w ishes to express sin c ere ap p reciatio n to Dr* John C# Speck, J r . f o r h is guidance, a s s is ta n c e , and e n th u sia s tic I n te r e s t throughout th e course o f these in v e stig a tio n s* The w rite r I s deeply g ra te fu l to th e Q uarterm aster Food and C ontainer I n s titu te f o r th e Armed Forces whose fin a n c ia l support made th is research possible* ttOtHMHHMMHfr JtiJft it w UiiTiAt *t *> W ww WVW V ****** **«* mt * 343906 TABLE OF CONTENTS FADE X. KBIOTCS OF THE AMINO ACID AND PEPTIDE CATALYZED DEALDQLXZAT2DN OF BXACET( m ALCOHOL INTRODUCTION.. . , .................................................................... HISTORICAL BACKGROUND............ *.......................... 1 2 EXPERIMENTAL M E T H O D S.....*,,............................................ 5 M a te ria ls............................................................ . . . . . . . ..................... 5 P r o c e d u r e , , . , , . . . . . . , . . ......................................... 5 RESULTS AND DISCUSSION................................................... SUMMART,...,,. ............ ................. *.......................... LITERATURE CITED,. 12 .................................... 13 2 1 . DETKHMINATIDN OF THE HHBDXIMETHXL CROUP IN SUGARS AND RELATED SUBSTANCES XHTRDUJCTIGN ......... HISTORICAL BACKGROUND... ................................................. 15 16 20 EXPERIMENTAL METHODS........................................................ ................................... M a te r ia ls .. A p p a r a t u s , . . 20 BsaoHaaondsd Procedure ................................ Sttu^r o f Experim ental C o n d itio n s, .............. 22 Id e n tity o f th e «*ye.............................................................. 22 E ffe c t o f o x id a tio n in acid s o lu tio n , .................... 22 E ffe c t o f s u lfu ric a d d co n cen tratio n used in 4ye d e v e lo p m e n t......................................... E ffe c t o f a e ra tio n , ......... . . . . . . . . . . . . . . . . . . 2h S ta b ility o f fo ra ald e ty d e-au lfit© s o lu tio n , 26 S ta b ility o f th e d y e . ......... . ............ 26 RESULTS AND DISCUSSION SUMMARY 20 20 23 ...................................... ...................................................................... LITERATURE C IT E D ..................................................... 33 X KXHETCC5 OF THE AMXMO ACID AMD PEPTIDE CATAUZED OfiAlOX&lZATXOI o r DOUCETOHE ALCOHOL X IHTfiDIHJCTIDH AXthough c a ta ly s is o f th e aldoX condensation by amino acid s and p ep tid es has beam recognised f o r m m tim e, th e lite r a tu r e o ffe rs no p re c ise d a ta concerning th e r e la tiv e e ffic ie n c ie s o f th e se substances ae c a ta ly s ts f o r a ld o lls a tlo n (o r d o ald o lisa tio n ) o r th e n atu re o f th e c a ta ly tic specie* inv olv ed. Since each inform ation wee d e sire d to sub­ s ta n tia te re c e n t work on th e mechanism o f th e H a llia rd re a c tio n , a s w ell a s to provide a p o ssib le In sig h t In to th a mode o f a c tio n o f th e Smyrna, a ld o la se , th e c a ta ly s is o f th e d e a ld o liz a tlo n o f diaeetone Alcohol by buffered so lu tio n s o f g ly c in e , i& -o c-alan in e, £ -a la n in e , and glycylglyoino and th e ir re sp e c tiv e sodium s a lts has been examined k in e tic a lly . th e e f f e c t o f m e ta llic ions on th e glycine and g ly o y l- glycine ca ta ly se d re a c tio n s has also been determ ined. 2 HISTORICAL BACKGROUND The d aald o H zatio n o f dlaeetooa alcohol (E quation l ) in a lk a lin e so lu tio n s o f both stro n g and weak bases has been w idely In v e stig a te d , and Koelioben (1) and A kerlof (2 ,3 ) have dem onstrated c a ta ly s is o f th is re a c tio n by th e hydroxyl io n . Likew ise, B e ll (h) observed only an OH 0 CHa-C-CHa«cJ-CHA — CH* *“ 9 2 CHa-C-CHft ( l) • iydroxyl Ion c a ta ly s is in k in e tic stu d ie s o f th e ald o l condensation, French (5) found th a t th e phenol-sodiua phenoxide system was ac tiv e o a ta ly tlc a U y only to th e ex ten t o f i t s lydroagrl ic e co n c en tratio n , th us ru lin g o u t th e co n sid eratio n o f a gen eralised aeid«base c a ta ly s is In th e re a c tio n , A kerlof (6) m isin terp reted th e e f f e c t o f th e weaker b a se s, and I t remained f o r H ille r and K ilp a tric k (? ) and W esthsimsr and Cohen (8) to dem onstrate th a t d e a ld o lisa tio n i s s p e c ific a lly cataly sed by ammonia and th e prim ary and secondary amines b u t not by te r tia r y am ines. Thus, th e c a ta ly se s involved a re highly sp e c ific in n a tu re , F isch er and H arseh all (9 ) produced a ld o l condensations in b u ffered so lu tio n s o f amino ac id s (a la n in e ), p ep tid es (le u c y lle u o in e ), and even p ro te in s (egg w hite) b u t found acy lated amino a c id s (h ip p u ric acid ) in a c tiv e . L ikew ise, B udnitskaya (10) observed an a c c e le ra tio n o f th e a ld o l condensation o f acetald ely d e by g ly c in e, a la n in e , and a s p a rtic acid in th a t o rd er w hile am ines, am ides, d ik e to p lp e ra sin e s, pep ton e, and egg 3 albumin v ere e f f w tlv s to a le a s e r extent* F in a lly , Langenbeck and B orth ( U ) in v e stig a te d th a e ffe c ts o f secondary amine ac id s and found sa rc o aln e , I " 6 % 1 g ly c in e , N-mothyl a la n in o , and phenyl-aarcoein© ca taly fc iea lly a c tiv e w hile S to th y l a la n in e , E-benzyl a la n in e , and oc-methyl amino is o b u ty rlc ae ld were n o t c a ta ly sts* The emyme, a ld o la s e , c a ta ly se s th e breakdown o f hexose diphosphate to trio a o phosphates according to Equation 2* In in Y ltro stu d io s o f th is enzyme, Meyerhof, Lohmann, and S chuster (12) found c a ta ly s is o f th o condensation o f dibydroxyacetone phosphate w ith aldehydes o th e r th an glyeeraldehyde. The ernym i s th e re fo re sp e c ific f o r dilydroasyacetone phosphate b u t not f o r th e aldehyde Involved. The m a te ria ls te s te d f o r ald o lase a c tiv ity have been n ea rly as numerous as th e In v e s tig a to rs . T ypical sources have been muscle and y e a st d e tra c ts (1 3 ), tumor e x tra c ts ( l h ) , nervous tis s u e (IS )* b a c te ria (16*17)* blood seruta (1 6 ), molds (1 9 ), h i^ ie r p la n ts (20,21) , and bovine m ilk (2 2 ). T hat th e enzymes is o la te d from v ario u s sources a re id e n tic a l i s a m atter o f c o n je c tu re . Only th e v a rie ty from s k e le ta l muscle has been exystaH lm ed (2 3 ,2 h ), the o th e rs being prepared as e x tr a c ts . The ro le h o f m e ta llic Iona a s a c tiv a to r* o r in h ib ito rs o f th a ald o lase system I s ra th e r confused. The ensyne Iso la te d from muscle toy H erb ert, e t a l . (25) m s s o t a m etallo -^ n o tein and was In h ib ite d by io d ise cod heavy m etals h u t s o t by o x idised o r reduced glutathione* Warburg end C h ristia n (2b) confirm ed th e rep o rted absence o f heavy m etal lo s s from muscle a ld o lase as s e l l a s th e la c k o f in h ib itio n by substances such as pyrophosphate, oC,oc*«4!pyridyl, c y s te in e , o r g lu ta th io n e , capable o f form ing complexes w ith th e heavy m etals* However, in stu d ie s on y e a st e x tra c ts , th e same in v e s tig a to rs observed th a t c y stein e produced an in h ib itio n which vas reversed by th e A ddition o f 2m**, Fa**, Co**, o r Cu** suggesting a p o ssib le m e ta llic io n a c tiv a to r f o r th is p a r tic u la r sp e c ie s. B tu sp f's pea ald o lase (20) displayed no in h ib itio n by Cu**, Hg**, o r Ag* o r by cy ste in e o r oC, <**«dlpyridyi. A ldolase prepared from C lostridium perfrlng en a (17) by Bard and Gunsaloa vas in h ib ite d by oc,oc •-d ip y rid y l and 1 , ffl+pbc~ s a n th ro lia e , an e f f e c t reversed by Fe** and Co**. I t would seem, th e re fo re , th a t a sp e cie s d iffe re n c e e x is ts between th e v ario u s emsymes, and in th is volte th e r o le o f m e ta llic io n s in s is p lif le d system s (g ly cin e and g ly e y lglycine) has bean observed. 5 E3PEK2HEHTAL METHODS M aterials Eastman Kodak Coaqpany dlaeatone alcohol was fre s h ly d is tille d p r io r to each determ ination and b o ile d a t 6 0 62° C/ l k tan, Eastman Kodak Coqpaay glycine was tw ice re o ry a ta llia e d from a m ethanol-w atsr m ix tu re. P fa n stie h l C*P. DL- oc^alanins and Eastman Kodak Company ^ -a la n in e were reezy stallim ed from etbanol-w ater m ix tu res, glycine was n o t f u r r ie r p u r ifie d . P fa n stie h l CJP. g iy o y l- Sodium c h lo rid e , eu p rlc c h lo rid e , and magnesium s u lfa te ware a l l CJP. reagents* Procedure U nless otherw ise In d ic a te d , th e io n ic stre n g th was m aintained a t 1 .0 M w ith sodium c h lo rid e . In some d eterm in atio n s, weighed samples o f th e amino ac id s were added to th e re a c tio n m ixture follow ed by th e amount o f stan d ard sodium hydroxide necessary to give th e d e sire d b u ffe r ra tio * in o th e rs , a liq u o ts o f standard b u ffe r so lu tio n s o f th e amino acid and i t s sodixsa s a l t ware employed. E eactlon v e lo c itie s were measured d U ato m etrieaH y a t a tem perature o f 18.60*0,001° C. In each case th e i n i t i a l diacetone alco ho l concentra­ tio n was 0 .08 m olar. The d ata follow ed a f i r s t o rd er p lo t (F igure 1} , and th e pseudo co n stan ts were evaluated by th e method o f Guggsxtiielm (26) u sin g dseadle logarithm s throughout. curves for o glycine systems o * rate o Typical O O CO N O U N O O rH O O O O n Figure 1. CO ON n n o o > O O _l 6 RESULTS AND DISCUSSION F i r s t o rd er pseudo co n stan ts obtained a t co n stan t b u ffe r r a tio s and varying b u ffe r co n cen tratio n s f o r each system , and, in th e ease o f g ly e ln e , a t te e b u ffe r r a tio s , a re ehoim in Table I« In th e m&im system* stu d ied bar W esthelasr and Cohen (6) , th e k in e tic s expression was found to bet *H k* [A] - Vc* [A] - (fc p g -to rj * l^ tB ]) [A] o r - kOH_[OB“ ] ♦ kg [B] wh.na * co n cen tratio n o f diacetone alcohol a t tim e t [OH*] • co n cen tratio n o f bydroayl io n due to b u ffe r [B] • co n cen tratio n o f n o le eu la r prim ary o r secondary so in s • ex p erh m n tally observed f i r s t o rd er pseudo co n stan t k* 1^** and • re sp e c tiv e c a ta ly tic c o n sta n ts. In th e amino a d d b u ffe rs employed in th ese d eterm in atio n s, th e sp ecies p re se n t a re H-CIIlNH^-COO* (E“ ) and R*CH(8Ha)'*C0C“ (a*) . The conoontra- tio n o f B-CK(NHa)-COOH (R) say be neglected sin ce pK^ « lo g R~/B. as rep o rted by R daall and Blanchard (NT) are $ J i2 , S .Iil, 5.53* and h ,6 l fo r g ly c in e , oC -alanine, /?~ alanlne, and glyeylglycina re sp e c tiv e ly . A ccordingly, th e s d m a c id anion (R~) would be th e p re d ic te d c a ta ly s t and th e k in e tic * expression would become* k* » b^gHOlT] ♦ % -tB r] 7 TABLE I P3QJD0 COHSTABTS 3jrrtm CO^elm P7>~~ ~bTBn1ntt f t -al«ntno O y q r lf lj e ln 1**3 s» lr a /L lte r [**] n o le e A lte r k» x 10* BizT* 0.10 0 JO 16J* o js 0.25 ld»J* 0.30 0 .3 0 53.3 oJo o .bp 73.7 0 .5 0 0 .5 0 9^*9 0 JO 0.067 36.8 OJjO o J33 7U.0 oJ5 0.150 86.0 0J0 0.20 15.5 0J0 0 JO 25.3 0 JO 0 .U0 32.5 0 JO 0 JO 29.2 0.20 0 JO 58.5 0.30 0 .3 0 80.0 0 JO o j5 16.5 ^ X -o*S i to r a ll other* 1*0. 8 Thtm baa been re a lis e d eau> eriae»tally, f o r a p lo t o f psusdo o onatant versus amine a c id anion co n cen tratio n a t co n stan t b u ffe r r a tio (oonatant [OS*]) i s s t r i c t l y lin e a r (F igure 2 ) . In a d d itio n , th e curves fo r glycine a t d if fe re n t b u ffe r r a tio s a re io iis tia g u ls b S b ls . t h i s confirm s th e amine a s id anion a s th e a c tiv e speeds*, and Ira n the slo pee o f th ese cu rv es, th e c a ta ly tic o o n atan t, feg-, f o r each anion has been c a lc u la te d (T able ZZ) . TABLE ZZ CATALZTZC C0BSTA8TS C a ta ly st Vfr. x ID* m r * sin *1 aXaxiin® 26 J t GByeine lfl.5 DSL- °G*alaidLns 8 .1 d y ey lg lyo izie a .3 Due to th e proadjaity o f th e in te rc e p ts to th e o rig in , no attem pt has been mads to ev alu ate pSfs f o r th e system s in v o lv ed . To t e s t th e e f f e c t o f m e ta llie io n s on th e amino a c id cataly sed re ­ a c tio n , determ inatio ns se re made using Cu** and Mg** in th e presence o f glycine b u ffe rs and Cu** in th e presence o f th e gLycylglycine system . The r e s u lts a re aboim in Table ZZZ« A lbert (28) found th a t th e m etal io n ooqplejM w ith glycine in o rd er o f decreasing s ta b ility c o n stan ts were Cu** > Mi** > 2n** > Co ** > Cd** > F« ** ?>Mn**> Kg** , The two extrem es have th e re fo re been esantned* Mg**, incapable o f stro n g ly eomplaadng g ly c in e , 90 80 70 60 50 40 30 20 S y s te m • o © ® I0 0 .1 0.2 0.3 G ly c in e G ly c in e DL-OC- a l a n i n e ^ -a la n in e 0.4 1 3 1 1 0.5 ( R" / F ig u r e 2 . R e la t io n o f pseudo c o n s ta n t amino a c i d a n i o n c o n c e n t r a t i o n ( R“ ) . (k 1 9 bad U t l l i e ffe c t on th e re a c tio n w hile Ca++ produced in h ib itio n In bo th ay atoms* Assuming th a t Cu( g ly e in ate) l a formed (29) and la c a ta ly tic * a lly in a c tiv e , th e c a lc u la te d pseudo co n stan t l a b8«p x ID*15 mln** which l a i s e x c e lle n t agreem ent w ith th e observed v alu e o f 1*8*1 x 10*"* niiT** The a f f e c t o f Cu** o s glycylglyoin© i s < iu a llta tiv 3ly th e san e, b ut th e magnitude I s somsshat g re a te r th an eapactad* TABLE I I I METALLIC IOK SPFXTS System <% cinc (B ycylglyeine Si x 30* ■tri"1 IB’ ]* [S*J* Son 0.30 0.30 —— ------ 53.3 0,30 0 JO Me*" 0.01 51.5 0 .3 0 OJO Cxf* 0 .0 1 1*8.3 0.20 0.0$ 0.20 0.05 k* 16.5 Cu*" OjDl 9 .8 A ll concentrations eyre In H o le s/lite r, These o b serv atio n s o f th e ro le o f n a ta l io n s I n th e sim p lifie d "aldolas© ~like* system s engxLoyed are s te l l a r to those nade by S e ib e rt, e t a l . ( 25) with Ag*, in * * , and Cu** and the ald o lase ©nayae from s k e le ta l muscle* The f a c t th a t muscle a ld o la se e x h ib its naotibmza a c tiv ity a t pH 7 *t ( 3° ) , above i t s is o e le c tr ic p o in t o f 6.05 ( 23) , eo ^L sd w ith th e knowledge th a t th e b a sic amino a c id s comprise approxim ately 20$ o f th e to t a l amino a d d co n ten t o f th e p ro te in ( 31) , stro n g ly sug gests th a t th e a c tio n o f th e ©raym© may be one o f ap eo lflo amine c a ta ly s is . The m etals m entioned by B exbert, a t s i . (2j>) are a l l re a d ily coxaplexed by m alm s and t he ir In h ib ito ry a c tio n may be due to removal o f th e a c tiv e c e n te r a s a r e s u lt o f complex form ation such a s was re a lis e d cjg>e?Jjtental« 3y in tlie case o f glycin e and ^lycylglycino w ith Cu**, The mechanism o f th e mains cataly sed d e a ld o lisa tlo n has never been e lu c id a te d , W esthstmar and Jones (32) shoved th a t th e r a te o f th e methyl* amine ca taly sed d e a ld o lla a tio n e f diaeetone alco h o l i n aqueous methanol and eth an o l was independent o f th e d ie le c tr ic o f th e medium th u s ru lin g o u t th e deccraposition o f a d ip o la r io n a s th e r a te determ ining step* The form ation o f a S ah iff *e base type in teim ed iate between th e amine and d la s s to s s a lcohol has bean suggested* On th e b a s is o f th e d a ta o f W esthelnar anti Jones (32) and tb s o b serv atio n th a t th e v e lo c ity co n stan ts f o r th e frfeutylantm s ca taly sed re a c tio n are th e same in w ater and in SDf vater-diojeane m ix tu res, Koob, H ille r and Bay (33) have concluded th a t th e form ation o f such a 3 ch lff* s base i s th e r a te dotorsdxdag ste p and have w ritte n th e follow ing equations* (CE8)y5^CH*-^-CH# *aHH3 £ s& Cfl*«^ ^ a l a n i n e , ^ a l a n in e , end giyuylglycina end th e ir re sp e ctiv e sodium s a lts has been stu d ie d k in e tie a lly . %. f he andno a c id anion has been shown to be th e ca laly tlc aX ly a c tiv e sp e c ie s, th e o rd er o f a c tiv ity being ^ -a la n in e > glycine ^DL-oc« alaidne-glycylglyoin© • 3* M e tallic lo ne have been shown to have no a c c e le ra tin g e f f e c t on th e re a c tio n ; lon e capable o f forming oosapleaees w ith es&ir&s in h ib it th e reactio n * h . A p o ssib le mode o f a c tio n o f th e emyme, a ld o la se , i s suggested , 5. The m echanlm o f an ise cataly sed dealdol& sation i s d iscu ssed . 13 LZS£SmsS CITED (1 ) S . Koelichon, Z . phyoik. Ctxsa., JJJ, 129 (19°°) . (2 ) 0 . Akarlof , 2 . An. Chaa. S o o ., Ij0, J0U6 (1 926 ). (3 ) 0 . A karlof, J . Aa. Cham. S o e ., i £ , 295S (1 92 7). (It) R. P . M U , J . Chaa. S o o., 1937. 1637, (5) c. (6 ) Q. A karlof, 2 . Am. Chaa. R oe., «» , 733 (19 28 ). (7 ) J . 0 . M iller and H. KilpatrlOk, J r ., J . t a , Chaa. S o o ., S3. 3217 (1931) . (8 ) 7 . B. W M ttttlnr mod H. Cohen, J . Am. Chea. S oo ., 60. 90 (19 38 ). (9 ) 7 . 0 . Piacbor and A, M araehall, B o r., 6liB. 2825 (1931). C. Fronoh, J . Aa. Cha«. S o o ., £L, 3215 (1 92 9). (10) B. f . Budnitakaya, BlokMaijra, 6 , 1U6 (1 9 itl). (U ) w. Langaidaeek and 0 . Borth, B a r., 75B. 951 (19ti2). (12) 0 . H m it o f , X, Lofcaana, and P . Schuster, Bloohem. Z „ 266. ----301) 319 (1 9 3 6 ). (13) 0 . 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Guggonhei» , P h il. M ag., [ 7 ) f £ , 533 (1926). (27) J . T . EdaaH and M. H. B lanchard, (1933)* (26) A. Alfcarfe, Bloofana. J . , j £ , 531 O S P ) . (29) B. M. Kooffcr, 4 , Aa. Cham. B oo., 6EJ, 2329 (19b6). Asa. Cham. B oo., 5 5 , 2337 (3D) A. L . Dounce, 6 , 11. B a rn e tt, and G. T . B oyar, J . B io l. C han., 165. vk il950) . ( 31) s. (32) F . H. V aath elser andW . A. Jon es. J . As. cban. S oo ., 63 , 3283 F» T tU sk and E . K onioni, J« B io l, C b n ,, 173. 627 (191*8). (a m ). (33) £ . P . Keob, J . 0 . M illa r, aad A. B , Day, 3 . A s. Chea. S o e., 73. 5775 (19 51 ). (3ii) 3 , B . Conant and P . D. B a r tle tt, J . A s. Cham. S o e., 5U, 2681 (1932). (^ ) 6 . B . t t o m & ^ J r ., and 0 . S . S c h a ffe l, J . An. Chen. S a c ., 6§, 15 IHTRODflCTlOH I n th a course o f an in v e stig a tio n o f th e degradation o f g ly o eraldehyde, i t became n&cem&xy to deviae an a n a ly tic a l schema f o r d e te r­ mining to t a l sugars (glycoraldoljytS® p lu s dihydroxyacetone) in the p m m o f pyruvaldehyde. A method combining accuracy w ith speed and ease o f m anipulation man needed f o r uao in k in e tio a s tu d ie s involving la rg e nottbera o f sam ples. A procedure has been developed efcieh c o n s ists o f an o x id a tio n o f th e sugars in a p erio d ic aeid-sodlxmi b icarbonate b u ffe r, d e stru c tio n o f th e a ssess p erio d a te and io d a te by a d d itio n o f sodium s u l f i t e , and stfteeqpsnfc spectrophotom etries determ ination o f th e formaldehyde p rese n t in th i s so lu tio n by means o f th e chronotropic acid re a c tio n . The method has sheen g en eral a p p lic a b ility in th e determ ination o f angers and re ­ la te d sdbstanoes. 16 EX&OfOm BACKGROUND H alaprade (1 ,2 ) f i r s t esta b lish e d th a t tha l^dro syn etb yl grotap o f su g ars and polyol© v a i o x id ised to formaldehyde by periodate* t h i s re*> a c tio n was l a t e r extended to th e <*-«a±no alcoho ls by B lco let and Shinn (5 ) * The formaldehyde go fbzwsd has been found u se fu l in th e determ ination o f many su b stan ces, and, a s a r e s u lt, nary schemes have been devised f o r I t s m sasuransnt* In e a rly stu d ie s o f the a c tio n o f p erio d ic acid on p o ly iyd roaylated ocnpounds, F leury and iange (It) determ ined formaldehyde by neans o f N esalerfa reag en t according to th e method o f Bougault and Gpqs ( 5 ) , and also g ra v lse tric ik lly w ith V orlaader1© dlnsdon reagent ( 6 ) , The p re c ip ita tio n o f fbrnaldebyd© w ith dlaedon, 5 ,£-dii»etbyl~ l ,>* cyelohaaanedlono, as m odified by loneecu and Bodea (7) and by lo e and Bead (8) has been w idely and e ffe c tiv e ly used* K arrer and P fa eh le r (9) removed th e formaldehyde fom ed d arin g p erio d ate o x id atio n by steam d is t illa tio n a t reduced p ressu re and p re c ip ita te d th e d&nssdon d e riv a tiv e from th e d is tilla te * As many in v e stig a to rs have observed, low r e s u lts were obtained when tb s ©addatlnna were c a rrie d o ut in acid so lu tio n * Beeves (10) , in determ ining a s e rie s o f su g ars, p re c ip ita te d th e dimedon d e riv a tiv e d ir e c tly from th e re a c tio n m ixture a f te r d e s tru c tio n o f th e a sse ss p erio d ate and io d ate w ith sodium a rsa n lte* T h is in v e stig a tio n Showed th a t th e th e o re tic a l amount o f formaldehyde was produced and recovered i f th e p erio d ate o x id atio n was c a rrie d o u t in so lu tio n s 17 b u ffered a t pH 7*5 by sodium b icarb o n ate. T hia was confirm ed by Jearilcs (11) wfcie, however, was unable to o b ta in the th e o re tic a l amount o f fo raald ely d e from d&hydroxyacetona by th ia procedure* In a m o d ificatio n o f Baevas* ache®©, B a ll (12) and B e ll, Palm er, and Jones (13) conducted th a p e rio d a te o x id atio n s in phoaphate b u ff e ra a t pH 7*k*7.5. th ia vas s a tis fa c to ry f o r sim ple sugars b u t w ith h ighly m ethylated d e riv a tiv e s gave somewhat low and e r r a tic re s u lts * Dimedon p re c ip ita tio n o f form* aldehyde h as lik ew ise been employed follow ing p erio d ate o x id atio n o f s e rin s In bicarb on ate a o lu tio n by B lco le t and Shine (H i) and in phosphate b u ffe r by Bees (15) , w hile Head aad B ertron (16) u tilis e d a s im ila r procedure in d etem in ln g formaldehyde produced on o x id atio n o f a e a rie a o f hydroayaB&no alk an es. Polarograpliic detexm ination o f formaldehyde d is t ille d from ao lu tio n follow ing p erio d ate o x id atio n wax used by Boyd and Baa&aoh (17) in th e dstesw dnatton o f merino aad by Warahowaky and E lving (16) in th e an aly aia o f m ixtures o f ethylene and 1,2-propyleno g ly c o ls. I n determ ining m ixtures o f g ly o o la, D esnuello and Baudot (19) p re o ip lta te d tl»a excess p erio d a te by the a d d itio n o f potaaalu n n itr a te and measured th e formaldehyde in th a sup ern atant by th e re a c tio n w ith phenylhydraaine hydrochloride and potassium fe rric y a n id e . Tanaaxabaum and B riok er (20) re c e n tly made a e r i t i e a l study o f th ia method fo r determ ini» g formaldehyde and found th a t th e operation* muat be very clo aely tim ed , a n e c e ssity which g re a tly c u rta il* I t s usefulness* fh s re a c tio n o f foiM ldehyde w ith chroiaotropic acid (1 ,6 -d H y d ro ^r naphthalene-3 ,6 - d i s u l f o ^ sold ) in s u lfu ric ac id s o lu tio n , f i r s t 18 d escrib ed Segriwe (21) , ha* been developed Into e very s e n s itiv e and e x c e lle n t c o lo rim e tric method f o r foraaldebyd© by MaoFadyaa, W atkins, and Anderson (2 2 ), and by B rlek er and Johnson (23) , Boyd and Logan (Sit) measured se rin e by d is t illi n g formaldehyde from an o x id atio n m ixture and condensing i t w ith cljroinotro p ic a d d . B risk er and Roberta (25) u tilis e d a permanganate o x id a tio n , follow ed by a p erio d ate o x id a tio n , and a fa n saldehyde d e te m ln a tio n on th e d i s t i l l a t e in measuring term inal unsatura* tio n in organic congpounda. In d eto m in in g m anaitol in plasma and u rin e , Corcoran and Page (26) o x id ised th e sample w ith p e rio d ic acid fo r e ig h t to te n m inutes, destroyed excess o xidant w ith standard stannous c h lo rid e , and developed th e ciirODaotropic aeid^fom aldalyde dye d ire c tly on th e re a c tio n m ix tu re» Under th ese c o n d itio n s, glucose sag converted to about fiv e p e r can t o f th e th e o re tic a l fom aLdehyds, Lambert and ftsish (2?) detais& ned g ly cero l in fo m e n ta tio n so lu tio n s by a flveHsdx&t® o x id atio n w ith p erio d ic a d d , red u ctio n o f excess p erio d a te and lo d a ts to io d id e by a la rg e excess o f sodium a r s e d t e , and a d ir e c t measure o f formaldehyde In th e o x id atio n m ixture by th e co lo r re a c tio n w ith c h rts^ tro p ic a c id , Glucose gave about fiv e p e r cen t o f the th e o re tic a l fom aldehyde. Of th e many methods d escrib ed , only th e l a s t two are a ttra c tiv e a s being a p p lica b le to la rg o numbers o f s a s p ls s , In both c a se s , an acid o x id a tio n i s used whieh mm found slow and u n sa tisfa c to ry in th is in v e s tig a tio n . The use o f stannous ch lo rid e i s un desirable sin ce i t i s u n s ta b le . I t s co n cen tratio n must be known accu rately because an excess 19 o f stannous ch lo rid e produces a lo s s o f fbraaldaijyde due to red u ctio n . In th is in v e s tig a tio n , use o f a rs e n lte to d estro y excess p erio d ate and lo d a te baa in v a ria b ly le d to a copious co elu tio n o f io d in e xxpon a d d itio n o f s u lfu ric ac id to th e s o lu tio n . Since io d in e in te rfe re s w ith th e dy© form ation (2 8 ), th is i s highly u n d esira b le. 20 EXPERIMENTAL METHODS M aterials Tbe D-glucos© used in these ea^periments was National Bureau of Standards Dextrose (Lot $*560), B-ayloee, D-faannltoX, and maltose wore a ll Ffanstlehl G*P* reagents* Diljydroxyaeotone was a N utritional Bio-* chemical s product while IH*~glyQeraldsiyde was synthesised by the method o f Fischer and Baer (29) • Pfaaatiehl C*P. DL-serine was recxystallised from $Q% ethanol* Victor Chemical Works glycolic sold was reoiy sta lU sed from methyl ethyl ketone, Matheaon Company practical ohromotroplo sold was xwevystallised from $0% ethanol* The periodic acid was a 0 . Frederick Smith Chemical Company product while the arsanious oxide was a Baker9* C*P, analysed material* The sulfuric add was Merck re~ agent grade and the sodium bicarbonate f sodium s u lfite , potassium iodide, and iodine were a ll C*P, reagents* Apparatus Beckman Model B and Model DU spectrophotometers equipped with Corex c e lls were used for op tical density measurements* Becomaended Procedure To a mixture o f 2,0 ml* o f 0*3 H periodic acid and 2,0 ml* of 1*0 M sodium bicarbonate in a 100 ml* volumetric flask is added 1.0 ml, of solution containing from 0*01 to 0,10 m illim ole of the -CBOH-CH^CSI group 21 and capable o f reducing no more than 1 .0 m illle ^ u iv a le n t o f p e rio d a te . The o x id atio n i s allowed to proceed fo r a period o f one hour. At the end o f th a t tim e, 5*0 s i , o f 0 ,5 H sodium s u lf ite so lu tio n i s added to the m ixture and th e re s u ltin g form aldehyde-sulfite so lu tio n d ilu te d to 130 m l. A blank i s prepared con tainin g th e same amounts o f sodium b icarb o n ate, p e rio d ic a c id , and.sodium s u lf ite . One m i l l i l i t e r a liq u o ts o f th e form aldehyde-sulfite so lu tio n and of tb s blank are then p ip e tte d in to sep arate 50 m l. volum etric fla s k s equipped w ith g lass sto p p e rs. To each i s added 0.5 ml. o f 10# chromo- . tro p ic so ld and 5 m l. o f 1^ M s u lfu ric acid (7 p a rts s u lfu ric acid to 2 p a rts w ater by volume). The fla s k s are Immersed in a b o ilin g w ater b a th , th e sto p p ers in s e rte d , and th e h eatin g continued fo r 30 m inutes. At th e end o f th is tim e th e fla s k s are cooled and approxim ately hO m l. o f die* t i l l e d w ater added to each f la s k . The fla sk s are again cooled and the so lu tio n s d ilu te d to 50 m l. w ith d is tille d w ater. Excess s u lfu r dioxide i s then removed by bubbling a ir sa tu ra te d w ith w ater vapor through the so lu tio n s a t a ra te o f 79^-1^00 m l. o f a ir p er m inute. The o p tic a l d en sity o f th e unknown i s measured ag ain st th e blank a t 570 using the Beckman Model 3 spectrophotom eter. A c a lib ra tio n curve i s prepared by determ ining the o p tic a l d e n s itie s obtained by th e above procedure w ith so lu tio n s o f known glucose c o n ten t. Such a curve i s s t r i c t l y lin e a r over th e recommended co n cen tratio n range (F igure 1 ) . 0.3 0 0 LOG l o / l 5 7 0 MU 0.200 00 10 5 15 GLUCOSE (MG) ( F ig u r e 1 . C a lib r a tio n c u r v e . 20 22 Study of Experim ental C onditions Id e n tity o f th e dye. The spectrum o f th e dye produced from 0 .1 mi l l i mo le o f d i hydroxy acetone by th e recommended procedure was measured using th e Beckman Model DU spectrophotom eter and compared w ith th a t found by Speck ( 30) f o r th e foraaldehyde-chroiaotropic acid dye (F igure 2 ). The two sp e c tra were id e n tic a l. E ffe c t o f o x id atio n in acid so lu tio n . To te s t th e e ffe c t o f an acid o x id atio n on formaldehyde prod uctio n, samples o f D -glucose, D -xylose, and DL-glyceraldehyde were oxidised by 2.0 ml. o f G.3 M p erio d ic acid in th e absence o f sodium b icarb o n ate, and fcrmaldeigrtie was determ ined by the recomaended procedure. In a d d itio n , p erio d ate consumption was measured by a m o dification o f th e method of F lsu ry and Lange (31) as follow si One m i l l i l i t e r a liq u o ts o f th e sugar so lu tio n s were mixed w ith 2.00 m l. o f 0 .3 H p erio d ic a c id , th e o x id atio n allowed .to proceed fo r th e in d icated tim e s, th e so lu tio n s n e u tra liz e d w ith so lid sodium b ic arb o n ate, 2.0 m l. o f $% potassium iodide follow ed by $.00 m l. o f 0.1311*2 N sodium a rse n ite so lu tio n added, and the excess a rse n ite titr a te d w ith 0.01 M io d in e to a sta rc h endpoint. The r e s u lts are shown in Table I . Since about fo u r hours i s required fo r complete o x id atio n to the th e o re tic a l m ount o f formaldehyde in acid so lu tio n w hile th is i s aocomp* lis h e d in one hour o r le s s In buffered so lu tio n a t pH 7 .5 , th e p erio d ic acid-sodium bicarbonate b u ffe r described in the recommended procedure has been employed throughout th is in v e stig a tio n . Figure 2. iO in in o io I 1— I — m «H I O o CM Comparison of spectra m •H 23 TABLE I EFFECT OF ACID OXIDATION ON FOKMALDE2GTDE PKODUCTION AND PERIODATE CONSUMPTION O xidation Tima (hours) D-glucose DL-glyceraldehyde D-xylose * «->i * _ *# HCHO* 101** HCHO 101 HCHO 63 63 99.0 99.2 $9 91 • 98.lt 98 .It 98.X 2 88 66 99.2 99.3 .... **mmm mm —• 3 97 97 99 J* 100 .... 9 9 .k 100 100 99.1i 100.0 1 h «... .... 10* 100 * P er cen t o f th e o re tic a l formaldehyde formed* ** P er cen t o f th e o re tic a l p erio d ate consumed. A ffect of g u lf u ric acid co ncentratio n used In dye development. In prelim in ary work, the chrom otropic acid-form aldehyde dye was developed in concentrated s u lfu ric acid (16 M) aa p rescribed by B risk er and Johnson (2 3 ). Since g ly c o lic acid la an end product of th e o x id atio n o f dibydrojyacetone, i t vaa te s te d aa a p o ssib le In te rfe re n c e . l .o Table I I shows the e ffe c t o f ml* o f 0*001 M g ly c o lic acid (th e amount p rese n t a f te r oxida­ tio n o f 0 .1 m illim ole o f dihydroxyacetone by the recommended procedure) w ith O S m l. o f 10% chrom otropic acid and 5 m l. o f 18 M su lfu ric acid fo r the in d ic a te d tim es. Subsequent to th is o b serv atio n , an a n a ly tic a l method fo r g ly c o lic acid based on th is co lo r form ation was described by F leu ry , C o u rto is, and P e rle s (3 2 ). 2k TABLE I I PBO0OCTIOB OF DIE BI GLYCOLIC ACID Time o f H eating (m inutes) Log 10/1 a t 570 m/y 30 o.ob3 60 0.077 150 0.128 Speck ( 30) had previously suggested th a t concentrated s u lfu ric acid o x idised d ia c e ty l to produce formaldehyde, and i t was thought th a t a sim ila r o x id atio n occurred w ith g ly co lic a c id . T herefore, the e ffe c t o f b eatin g 0.001 m illim ole o f g ly c o lic acid w ith 0 .5 m l. o f 10$ chronotropic acid and $ m l. o f s u lfu ric acid o f varying concentration s fo r th ir ty m inutes was measured. T his I s shown in Table I I I . Since tile g ly c o lic acid in te rfe re n c e could be elim inated by using 12* M s u lfu ric a c id , sasg&es o f DL-glyc©raldalyde were c a rrie d through tb s recommended procedure and th e o p tic a l d e n s itie s compared w ith those obtained using 18 H s u lfu ric a c id , Table IV . I t i s obvious th a t the s e n s itiv ity i s unim paired. E ffe c t o f a e ra tio n . I t was observed e a rly in th is in v e stig a tio n th a t the presence o f excess s u lfu r dioxide in so lu tio n re su lte d in considerable e r r a tic bleaching o f th e c h a ra c te ris tic dye. U tilis a tio n o f the evapora­ tio n technique o f B risk er and V ail (28) fa ile d to give reproducible r e s u lts . I t was th e re fo re decided to attem pt a e ra tio n o f th e sam ples. 25 TABLE H I EFFECT OF SOLFUKIC ACID CONCEHTBATIOH OB QLICQLIC ACID DIE FQfMATXOB S u lfu ric acid C oncentration Log 10/1 a t S70 VkjA 18 M 0 .0L3 16 0.005 Jit 0.000 12 0.000 TABLE XV EFFECT OF mFtmXC ACID CONCENTRATION ON INTENSITY OF CHEC»TEQPIG ACID-FOKMALDEHIDE DIE D L -glycerald«l3jyde (m g. o x id iz e d ) Log V I a t 570 mu * 18 M BaSD4 lit M 838)4 9 .0 0.300 0.301 7 .2 0 .2 3 9 0 .2 3 8 5.1* 0 .1 7 9 0 .1 7 9 3 .6 0.120 0 .1 2 1 * Average o f th ree samples . 26 T able V above th e e ffe c t o f tim e o f a e ra tio n on th e dye produced from 0 .1 m illim o le o f D~glucoee by th e recom ended procedure. A th ir ty minute a e ra tio n produces maximum o p tic a l d e n sity . TABLE V SFFBCT OF AEEATICH TBSE ON ZNTi^SSIXX OF BIS A eration tim e (m inutes) &>g V 1 a t 570 0 0.175 10 0.270 20 0.289 30 0.301 h$ 0.305 60 0.302 ISO 0.301 S t a b i l i t y o f fom aldeh ard e-sulfite a o lu tlo n . To te a t i t s s ta b ili ty , 1.0 m l. a liq u o ts o f th e fonraaldehyde-sulfit© so lu tio n from th e o x id atio n o f 0 .1 m illim ole o f D-glucose were removed a t th e tim es in d icated in Table VI and c a rrie d through th e reeoanended procedure. Such a so lu tio n i s p e rfe c tly sta b le f o r a t le a s t fo rty -e ig h t ho urs. S ta b ility o f th e dye^ The dye produced from 0 .1 m illim ole o f D-glucose by th e recommended procedure was sto red in th e dark and the o p tic a l d en sity measured a t th e tim es in d ic ated in Table V II. a ls o , i s sta b le f o r a t le a s t fo rty -e ig h t hours. The dye, 27 ta b u n STABILITX OP FOKM AUmDE~mPIT£ SOLUTION Age o f S olu tion (hour#) Log X0/ t a t 570 k /l{ 0 0.323} 0.32L 2li 0.325} 0.325 U8 0.323} 0.32li TABLE TXX STABILITY OF DTE Age o f Bye (h o u rs; Log Ic/1 a t $70 0 0.323 2h 0.325 m 0.323 28 KKSULTS AMD 8ISCUSSI0M The procedure h e re in describ ed has boon su c cessfu lly applied to th e determ ination o f D -glueose, D -aylose, DL-glyceraldeiyd© , dihydroxyacetcne, D-iwuanltol, end m alto se. The r e s u lts ere in d icated In T able V U I. In th e a n a ly sis o f th ir ty - e ig h t samples o f D -glucoae, th e p re c isio n was l l . l t . In th e ease o f DL-aerin®, low re s u lts were always obtained (Table IX ). Extended o x id atio n s produced no higher formaldehyde v alues thu s e lim in a t­ in g th e p o s s ib ility o f incom plete re a c tio n . Van S lyke, H ille r , and MacFadyen ( 33) in studying th e p erio d ate o x id atio n o f s e rin e , obtained low v alu es fo r ammonia a t n e u tra l to s lig h tly a lk a lin e pHfs and b eliev ed th is due to a re a c tio n between ammonia and form aldehyde, T his may account f o r th e low and reproducible values obtained in th is in v e s tig a tio n . To t e s t th e recommended procedure on a d lsa ec h arid e, m altose was se le c te d . T his sugar was oxidised w ith in th ir ty m inutes to produce one mole o f formaldehyde p er mole o f disaccharide w ith no fu rth e r o x id atio n a f te r fo u r hours (T able X)« T his in d ic a te s th a t no h y d ro ly sis occurred during o x id a tio n in a n e u tra l b u ffe r and suggests the p o s s ib ility o f apply­ in g th is procedure to end group determ inations on high m olecular weight polysaccharides . The method presented here i s su p erio r to those o f Corcoran and Page (26) and Lambert and N eish (2?) sin ce by u tiliz in g o x id atio n a t pH 7 .5 , th e more slow ly o x idised sugars may be determ ined as w ell as rap id ly oxidised m a te ria ls such as gly cero l and m anzdtol. Use o f sodium s u lf ite 29 TABU m i REMITS OBTAINED SX RSCOHMSfDED PROCEDURE Compound D-glucoee B-*yloee Taken (g g .) Pound ta g .) 18.0 lli.ii 10.6 7.2 1 8 .0 | 17.9} 18.0} 16.0 Ui.51 lii.1 } 11.1} H .1 1 0 .9 i 10.9} U.O} 10.9 7 .5 i 7.1} 7.1} 7.1 18.0 11.1 10.6 7.2 18.1} 18.1} 18.0 U».5i 11.1} 11.9 10.6} 10.8} 11.0 7.0} 7.1} 7.3 18.0 lli.li 10.8 7.2 3 .6 1 7 .7 | 17.8 Hi .5} H .5 10.9} 10.8 7 M 7.1 3.6} 3.5 15.0 12.0 9.0 6.0 11.9} 15.0} 15.0 11.9} 12.0} 12.0 8.9} 9.0} 9.0 5.9} 5.9} 6.1 DL-glyceraldeRyde 9.0 7.2 5.1* 3 .6 9.0} 7.2} 5.1} 3.6} 9.0} 7.2} 5.3} 3.6} 9.0 7.2 5.1 3 .6 DiljydraxyACGtcne 9.0 9.0} 9.0} 9.0 DHnannltoX 9.1 1 .6 9.0} 1.5} 9.0} 1.5} 9.0 1 .6 M altose 36.0 32.5 35.0} 35.1 33.3} 33.3} 33.2 30 TABLE IX D&rEftHXBATIQH Of DU3&OH& f g Found ( o s .) Per cent o f Theory 10.5 9*5 9*5 9M 91 1.83 93 5.25 ii.fio TABLE X FOKMALDEHTDB PRODUCTION FROK MALTOSE Tlae of Oxidation (houre) Holee BSHO/aole Maltoee 0 .5 1*02 0.97 1.0 1.02 0.98 1.5 1.03 - 2 .0 1.02 mm h.0 - 0.97 to d e stro y e x o iia p erio d ate and io d ate elim in a tes th e n ec e ssity o f a Oteadard reag en t such aa i t i m w ch lo rid e and avoids in te rfe re n c e by Iodine produced when a re e n lte l a used. The a d d itio n a l a e ra tio n atep n ee ea eita te d by th e nee o f sodium s u lf ite la not a se rio u s dravbaok sin ce la rg e numbers o f weepies may be aerated sim ultaneously. th e s ta b ility o f the fo rm ald eh y d e-su lfite so lu tio n coupled w ith th e s ta b ility o f th e dye s ilo e s extrem e f le x ib ility o f o p eratio n . A complete a n a ly sis re q u ire s about 2 1/2 hours, and many sa sp lsa may be run co n cu rren tly . Obviously any m a te ria l which in te rfe re s w ith th e formaldehyde d e te r­ m ination by chrom otropic a d d c o n s titu te s an in te rfe re n c e in th is method. In a d d itio n , axy substances producing formaldehyde on p erio d a te o x id atio n w ill in te r f e r e . The g ly co lic ac id In te rfe re n c e has been elim inated by th e choice o f s u lfu ric acid co n cen tratio n used during th e dye development. D iacety l ( 30) and pyruvaldebyde (3U) which are known to re a c t w ith chrono­ tro p ic a c id cause no in te rfe re n c e sin ce they are cleaved by p e rio d a te . The r e s u lts obtained by an »acid oxidation* are shown in Table 1 . A t le a s t fo u r hours i s required f o r complete re a c tio n as measured by formaldehyde p ro d u ctio n . However, p erio d ate conjunction as measured by th e method o f Floury and Lange (31) i s complete in one h our. Hughes and H evall (35) observed sim ila r r e s u lts in a study o f th e o x id atio n o f glucose by p e rio d a te . They found p erio d ate consumption as measured by th e method o f F leury and Lange to be very rap id w hile form ic acid pro­ d u ctio n was much slo w er. I f , however, p erio d ate consumption was measured by th e a d d itio n o f a c id ifie d potassium iodide and a ti t r a t i o n o f th e lib e ra te d io d in e w ith sodium th io s u lfa te , i t then p a ra lle le d form ic acid 32 p ro d u ctio n , These in v e stig a to rs considered th a t th e o x id atio n proceeded by *®y o f in term ed iates which were store sta b le in a lk a lin e than In a d d s o lu tio n s • Our d ata would agree w ith th is h yp oth esis. A pparently p e rio d a te re a c ts ra p id ly w ith th e reducing sugars both in acid and in n e u tra l m edia, Th© interm ediate® so formed must re a c t only slowly w ith a rse n ite in n e u tra l so lu tio n o r , in o th er w ords, behave as io d a te , th u s accounting f o r th e apparent complete consumption o f p e rio d a te , Zn acid so lu tio n , however, th is interm ediate breaks down in th e presence o f io d id e and allow s measurement of unreduced p e rio d a te . T h erefo re, th e statem ent o f Hughes and N ovell regarding the r e la tiv e s ta b ility o f th e in term ed iates In so ld o r a lk a li i s tru e only in th e presence o f io d id e . Zn th e absence o f io d id e , th e in te m e d la te e would appear to be le s s sta b le in a lk a lin e so lu tio n since th e products o f th e re a c tio n are re* leased more ra p id ly . Z t i s p o ssib le th a t d iap ro p o rtio n at io n o f the p erio d ate-red u cin g sugar complex involves p a rtic ip a tio n o f th e hydroxyl io n . 33 mmm 1* A new oathed Tor determ ining th e i^d ro a^seth y l group in sugars and re la te d substances i s described which c o n sists o f a p erio d ate oxide** tio n in n e u tra l so lu tio n b uffered by b icarb o n ate, d e stru c tio n o f excess p erio d a te and lodat© w ith sodium s u lf ite f and subsequent determ ination o f formaldehyde d ir e c tly on th e re a c tio n m ixture by the re a c tio n w ith chromotro p ic acid* 2. The e ff e c ts o f (a) a c id ity o f th e o x id atio n medium, (b) s u lfu ric acid co n cen tratio n during dye development, and (c ) tim e o f a e ra tio n have been in v e stig a te d . 3« The s ta b ili ty o f th e fofinaldshyde*sulfite so lu tio n and o f th e 3ye has been determ ined. k 0 The method has been su c cessfu lly applied to th e determ ination o f D~glu0o s e , D**sylose, QL^glyceraldehyde f diiydroxy acetone, D~®annitol, and m alto se. 5, DL-eerine has been found to give low y ie ld s o f m easurable form aldehyde, 6 , M altose has been shown to y ie ld one mole o f formaldehyde p e r mole o f d issco h arld e suggesting use o f th is method in end groxjp a n a ly sis o f poly sacch arid es * 7, The method i s compared w ith sim ila r procedures. 8, The anomalous “acid oxidation* i s discussed 3k LXTSBATDKE CITED (1 ) L . Malaprad®, Conpi. re n d ., 186 . 382 (1928). (2 ) L* Malaprad®, B u ll. eoc, ohim ., 1 ^, 683 (1928). (3 ) B. H. H ico let and L . A. Shinn, J . A®. Chaa. S o c., (k ) P . Floury and J* Lange, J . Pham* Chim ., 17 , 196 (1933)* (5 ) 4 . Bougault and B. Groa, «?. Pham* Chlm ., 26, 5 (1922)* 1615 (1939). ( 6) D. V orlander, C* X hle, and H. Volkhol®, Z. a n a l. Ohara., 77 , 321 (1929)* (7 ) &• V. Xowsecu and G* Bod®a, B a ll. ao c. chlra., h 7 , 11*08 (1931)« (8 ) J* H* To® and L . C . Bead, I n i. Eng. Chraa*, A nal. E d ., 1 3 , 238 a m ). (9) P . K arror and K. F fa a h le r, H«lv. Chin* A cta, 1 J , 766 (193k)• (10) H. S . Beevea, d . As. Chao* See*, J £ , 11*76 (1910.). (11) &. Jo an lo a, H elv. C h is. A cta, 2 J, 1509 (19kb). (12) D. J . B e ll, J . Chew. So©*, 191*8 . 992* (13) D. *?. B e ll, A* F a ls e r, and A, T . donee, d . Chesa. doc*, 191*9* 1536* (Ih ) B* K* H leo let and L . A* Shinn, 4 . B io l. C has., 139. 68? (1910.). (15) M. v , E eee, Biochera. d . , ] £ , 632 (19k6). (16) J . P . Mead and S. A. B a rtro n , d . As. Cham. S e c ., JO, 1286 (191*8). (17) M. J . Boyd and K. Basbach, In d . Eng. Chora., Anal, Ed*, 1 £ , 31k (19k3). (18) B. WarslKwsfcy and P . d . Klving, Xnd. Eng. Chera,, Anal. E d ., 1 8, 253 (19L6). (19) P* DeanueUe and M. Kautiet, B a ll. aoc. ©him., 12, 671 (19k5). (20) H. Tstnmnbaxm and C. E* Q rick er, Anal. C h es., 2£, 35k (1951). yJ (21) E. Eegriw e, 2 . a n a l. C h e* ., H O . 22 (1937). (22) D. A, MacPadyen, H, D. '.'atk in a, and F . R. Anderson, J . B io l. O ban.. 156. 10? (191*5). (S3) C . E . B ricker and H. B. Johnson. In d . En*;. O lios.. A nal. E d ., 17. !*£» (191*5). (21*)J . S . Boyd and M. A. Logan, J . B io l. Cham., 11*6. 279 (191*2). (25) C . E, B riokar and K. B. R oberta, A nal. C ha*., 21 , 1331 (191*9). (26) A. C. Corcoran and J . R. Fags, J . B io l. Cham., 170. 165 (191*7). (27) M. L a m (26) C . E. B riokar andW. A. T a ll, A nal. Ch«n., 22, 720 (1950). b e rt and A.C. H alah, Can. J . R esearch, 28B. 83 (1950). (29) H. 0 . L . F isch e r an i 3 . B aar, B d v . C hin. A cta, IB , 5U* (1935). (30) J . C . Speck, J r . , A nal. Clues., 20, 61*7 (191*8). (» ) P . Floury and J . Langs, J , Phans. C h i* ., 1£, 107 (1933). (32) P . F lo u ry , J . C o u rto ls, and ft. P er le a , HlkrooboBla v e r. Mikrochim. A ota, 36/3 7. 663 (1951). (33) 0 . 0 . Tan Slyko, A. H ille r , and D. A. MacFadyen, J . B io l. C hon., 11*1. 661 (191*1). (3ti) B. J . Thornton and J . C . Speck, J r . , A nal. C he*., 22, 899 (1950). (35) 0 . Hughes and T . P . Have1 1 , T rans. Faraday S o e., W*, 91*1 (191*8). zn KINETICS OF THE ACID AND BASE CATALYZED DEGRADATION OP THE T&IGSKS # » 36 IBT&DDUCTIOH A lthough c o n s id e ra b le e f f o r t has been devoted to an e lu c id a tio n o f th e mechanism o f th e Lobry de Brqyn-van E k en ste in tra n s fo rm a tio n i n a lk a lin e s o l u t io n , l i t t l e In fo rm a tio n I s a v a ila b le co n cern in g th e o c c u rre n c e o f t h i s r e a c tio n i n d i l u t e a c id s o lu tio n o r th e e x a c t n a tu re o f th e c a ta ly s e s In v o lv ed . L ikew ise, pyruvaldehyde p ro d u c tio n from th e t r i o s e s ( a s w e ll a s from th e liig h er su g a rs) i n b o th a c id and a lk a lin e s o lu tio n s h a s lo n g been re c o g n ise d , y e t l i t t l e i s known o f th e c a t a l y s t s f o r t h i s r e a c tio n o r i t s mechanism. Knowledge o f th e c a ta ly s e s o p e ra tiv e In th e s e p ro c e ss e s was con­ s id e re d n e c e ssa ry to th e study o f th e mechanism o f red u c in g su g ar d e g ra d a tio n i n amino a c id system s (M a illa rd r e a c t i o n ) . T h e re fo re , th e I n te r c o n v e rs io n o f 0L-glyc@ raidehyde and dihydroxyaceton© , a s w e ll a s th e sim u lta n eo u s d e h y d ra tio n to py ruvaldofcyde , has been examined k i n e t i c a l l y i n b u ffe re d s o lu tio n s o f fo rm ic , a c e t i c , and tr lm e th y la c e tic a c id s and t h e i r r e s p e c tiv e sodium s a l t s . th e s e r e a c tio n s has a ls o been o b se rv ed . The e f f e c t o f calcium io n on 37 HISTORICAL BACKGROUND The Lobry de Br\jyn-van L k sn sta in T ran sfo rm atio n The r e v e r s i b l e t r a n s f o r a a tio n o f a ld o s e s in to k e to s e s o c c u rrin g i n d i l u t e a lk a li n e s o lu tio n was f i r s t d e sc rib e d by Lobry de Bruyn and van E k e n s te in ( 1 , 2) and b e a rs th e names o f th e s e i n v e s ti g a t o r s . G lucose, f r u c t o s e , and mannoao were each c o n v erted to th e o th e r s i n d i l u t e so lu ­ t i o n s o f sodium , p o ta ssiu m , o r ammonium h y d ro x id e, calcium o r magnesium o x id e , o r sodium o r potassium c a rb o n a te . I n a d d itio n , non-fem aentable s u b s ta n c e s (3*ketohexoses?) were form ed. These s tu d ie s a ls o in clu d ed th e s i m il a r tra n s fo rm a tio n s o f g a la c to s e , m a lto s e , la c to s e and m e lib io s e . O th er r e s e a r c h e r s have a p p lie d t h i s is o m e riz a tio n to glyceraldeliytie and dihydroxy acetone ( 3 ) , la c to s e and la c tu lo s e ( b ) , glucoheptose and g lu c o h s p tu lo s e ( 5) » x y lo se and a ra b in o se ( 6 ) , and c e llo b io s e ( 7 ) . Calcium h ydroxide h a s b een th e u su a l c a t a l y s t . I n most c a s e s , th e sim ple in te rc o n v e rs io n o f su g a rs has been com pli­ c a te d by fra g m e n ta tio n r e a c tio n s and a c id fo rm a tio n . G o ttf r ie d and Benjamin (6 ) r e c e n tly conducted an e x h a u stiv e study o f th e fo rm atio n o f k e to s e s , o rg a n ic a c i d s , unferm entable s u b s ta n c e s , and c o lo r d u rin g tre a tm e n t o f glucose w ith b a se s and a rriv e d a t a s e r i e s o f e m p iric a l e x p re s s io n s which p e rm itte d c a l c u l a t i o n o f each p ro d u c t. U t i l i z i n g improved te c h n iq u e s in v o lv in g ra d io is o to p e d i l u t i o n a n a ly s e s f o r g lu co se and f r u c to s e and a c o rre c te d phenylhydrazone p r e c i p i t a t i o n p ro ced u re f o r mannose, Sowden and S c h a ffe r (9) have 38 r e i n v e s t ig a t e d th e r e a c tio n s o f th e s e su g a rs i n Q .035 H sodium hydroxide a t 35° C . A l t e r extended r e a c tio n tim e s , summation o f a n a ly se s f o r th e s e th r e e su g a rs accounted f o r about e ig h ty p e rc e n t o f th e s t a r t i n g m a te r ia l | th e rem ainder had been co n v erted to a m ix tu re o f n o n -ferm en ta b le su g a r p ro d u c ts o f unknown n a tu r e . G a rb u tt and Hubbard (10) dem onstrated in te rc o n v e rs io n o f g lu c o s e , f r u c t o s e , and mannose i n b o ilin g aqueous s o lu tio n and i n b o ilin g s o lu ­ t i o n s b u ffe re d a t n e u t r a l i t y w h ile M ursohhauser (11) re p o rte d th e is o m e r iz a tio n o f glu co se on b o ilin g w ith a lk a lin e e a r th c a rb o n a te s . Spoehr and co-w orkers observed s im ila r c o n v ersio n s i n th e p re se n c e o f n e u tr a l ( 12) and s l i g h t l y a c id ( 13) phosphate b u f f e r s . T h at o rg a n ic b a s e s could produce th e lo b ry de Bruyn-van E k en steln r e a c tio n was shown by F is c h e r , T aube, and B aer ( lb ) who co n v erted glyceraldebyd© I n to dihydroaqy a ceto n e by b o ilin g w ith anhydrous p y r id in e . D a n ilo v , e t ajL. ( 1 $ ) , found t h a t glucose was iaom erieed to f r u c to s e w ith o u t mannose p ro d u c tio n by anhydrous p y r id in e . Mannose was form ed, how ever, when aqueous p y rid in e and aqueous a lc o h o lic q u in o lin e were em ployed. M ldorlkawa and Takeshima (16) confirm ed th e s e o b s e rv a tio n s and extended th e r e a c tio n to In clu d e ^ u in a ld in e ( 1 7 ) . Anhydrous p y rid in e h as been a v a lu a b le t o o l i n th e s y n th e s is o f mary k e to s e s (1 6 -2 $ ). The mechanism o f th e Lobry de Bruyn-van E k en ste ln tra n s fo rm a tio n h as been th e s u b je c t o f much ex p erim e n tal work. I n 1900, Vohl and Neuberg ( 3) e x p la in e d th e c o n v e rsio n o f g lyceraldehyde to dihydroxy ace ton© i n a lk a li n e s o lu tio n on th e b a s is o f an e n e d io l fo rm a tio n , and t h i s 3? soehA B istlc soaowpt i» s t i l l th e accepted one. In general form , th is may be p ic tu re d schem atically as follows* # § [K«8, CBapH, e tc . ; H»-«, CH^OH, e tc .] On th e b a s is o f th e fragm entation products obtained on o xid ation o f v ario u s sugars in a lk a lin e s o lu tio n , Kef (26) p o stu lated th e form ation o f a s e rie s o f en sd io ls capable o f being s p li t by th e oxidants* The spontaneous rearrangem ent of an "aldosate* anion suggested as th e mechanism by M iehaelis and Bona (27) and by CJroot (28) also Im plied previous en sd lo l form ation* Zn an exhaustive s e rie s o f in v e stig a tio n s o f carbohydrate o x id a tio n s, Evans and co-workers (29-ti3) were le d to th e conclusion th a t an equilibrium ex isted between th e sugars and a s e rie s o f e n sd io ls in a lk a lin e so lu tio n . Gustos and Lewis (44) examined th e ox id atio n o f tetram etbyl glucose in a lk a lin e so lu tio n and obtained r e s u lts in d ic a tin g th e presence o f 1 ,2 -e n e d io l. a Extension o f th ese stu d ie s to th e a lk a lin e rearrangem ent o f tetram eth y l glucose (4 5 ), tetram ethyl mannose (4 6 ), trim sth y l xylose (4 7 ), and trim e th y l arabinose (48) showed, In each c a se , form ation o f th e corresponding epim eric m ethylated aldose w ith no ketosie p ro d u ctio n . In a d d itio n , high iodine absorbing su bstances, b elieved to be th e enediol hO lf ltf ir a td ia tti, were p rsso o t in a lk a lin e so lu tio n b u t rap id ly disappeared on a c id if ic a tio n . Under abdlai* co n d itio n s, 3« se tl9 l glucose (i»9) vaa converted to JHsstfayl fru c to se w ith no d e te c ta b le enedlol co n cen tratio n (a ls o tru e fo r g lu co se). E lectro rad u ctio n o f sugars In a lk a lin e so lu tio n by Wolfrom and co-w orkers (50-£ii) gave isciaerlc poly ols in d ic a tin g th e presence o f 1 ,2 - and 2 ,3 -e n e d lo ls. On th e o th e r hand, ea rly stu d ie s o f the Lobry de Bruyn-van E kenstein transform atio n in a lk a lin e deuterium oxide so lu tio n s produced r e s u lts co n trary to th e p re d ic tio n s o f th e enedlol mechanism. Fredenhagen and Bonhoeffer (55) rep orted no in co rp o ratio n o f carbon-bound deuterium w ith glucose a t 25° C w hile a t high er tem peratures non-reproducible d ata were o b tain ed . On th e b a s is o f these r e s u lts , a "dim er Interm ediate" mechanism was proposed fo r th e low tem perature re a c tio n and a k sto -en o l mechanism a t higher tem peratures. Goto (56) reported comparable fin d in g s. In search o f an explanation fo r th is repo rted isom erization w ithout exchange o f carbon-bound hydrogen by deuterium , Bothnsr-By and Gibbs (57) employed l-C ^-D -g lu co se to te s t th e p o s s ib ility o f carbon chain re ­ arrangem ent during th e re a c tio n . Bo such rearrangem ent was observed. Topper and S te tte n (56) , in a re in v e st!g a tio n o f the re a c tio n s of glucose in heavy w ater, observed deuterium exchange a t both 25° and 35° C in agreement w ith th e enedlol neehanian, These r e s u lts also in d icated th a t th e mannose produced when glucose was tre a te d w ith sa tu ra te d lim e w ater was derived ex clu siv ely from fru c to se , and th e follow ing scheme was presented! n glucose tra n s-en ad lo l fpuotoae ^—- c is-e n e d lo l biqodm Sovden and S chaffer (59) have re c e n tly presented fu rth e r evidence o f deuterium exchange a t 25° C h u t concluded th a t fruotoee vas not a necessary in term ed iate In th e conversion o f glucose to maxmose. Although th e enediol mechanism f o r th e Lobry de Bruyn-van E kensteln tran sfo rm atio n has apparently been w ell e sta b lish e d , th e nature and mode o f a c tio n o f th e c a ta ly s ts involved have received considerably le s s a tte n tio n . Most stu d ie s have been conducted in a lk a lin e so lu tio n s and H leb aalls and Sons (2?) repo rted glucose decom position p ro p o rtio n al to th e hydroxyl io n co n cen tratio n , V arious bases including th e a lk a li and a lk a lin e e a rth hydroxides and carbonates as v e il as phosphate uystems have been employed, b u t th e a c tiv ity has been a ttrib u te d to th e hydroxyl io n p re se n t in these so lu tio n s. L ittle Inform ation I s a v a ila b le concerning sugar ln tereo n v ersio n s in a c id so lu tio n s under th e in fluence o f bases o th e r th an the hydroxyl Io n , A conversion o f fru c to se in to glucose in s lig h tly acid phosphate system s vas rep o rted by Spoehr and S tra in (1 3 ), Ashmarin and co-w orkers (60-62) examined th e re a c tio n s o f glucose and fru c to se In form ate, a c e ta te , and su ccin ate b u ffe rs and found an apparent c a ta ly s is o f the in terco n v ersio n (and a lso o f hydroxymethyl fu rfu ra l form ation) by the anions o f th e ac id s involved, A sim ila r a c e ta te ion cataly sed Iso* m arisatio n o f arabinose vas repo rted by Braun and Konnova (6 3 ). In each c a se , a b is u lf ite fra c tio n a tio n scheme (6k) vas eaployed to separate ald o ses from k eto see, and th e isomer produced vas id e n tifie d as th e osasone o f th e s ta rtin g m a te ria l. U tilis in g aqueous pyrid in e syetam e, hz Mldorikawa (65) observed th a t w ith In creasin g p y rid in e co n cen tratio n s, th e conversion o f glucose to fru c to se Increased to a subsequently decreased. value and Due to th e s im ila rity o f th ese re s u lts to those obtain ed by Lowry and Faulkner (66) in th e inu taro tatio n o f glucose, an acld*»base c a ta ly s is was proposed. S everal in v e stig a to rs have reported apparent v a ria tio n s in the n atu re o f th e Lobry de Bruyn-van Skenateln transform ation dependent on th e p a r tic u la r c a tio n ic species involved. Lobry do B nyn and van B ksnstein (6?) found th a t lead hydro adds converted glucose to mannose w ith no d e te c ta b le fru c to s e , and th a t, under those c o n d itio n s, fru c to se was not iaom erized to th e corresponding ald o ses. Kef (26) reported th a t calcium and lead a c e ta te s and lead ch lo rid e caused no enoU zatlon o f th e hsxoses. A com parative study o f the ac tio n o f calcium and sodium hydroxides on glucose a t 25° C mas conducted by Kuzin (6 8 ). With calcium hydroxide, a substance (believed to be th e e n o l), capable o f reducing io d in e and dichlorophenolindophenol, vas p resen t which disappeared on a c id ific a tio n . produced no such r e s u lt. Under sim ila r co n d itio n s, sodium hydroxide Mannose form ation vas favored by th e calcium hydroxide; fru c to se form ation by sodium hydroxide • From th ese observa­ tio n s , Kusin concluded th a t a t low tem peratures, calcium hydroxide produced a c y c lic enol w ithout ru p tu re of the pyranose rin g whereas a s tr a ig h t ch ain enol was formed in the presence o f sodium hydroxide. At high er tem p eratu res, no d iffe re n ce in the a c tio n o f th e two bases was d e te c ta b le . Sovden and S chaffer (9) have re c e n tly confirmed th a t th e i n i t i a l course o f iso m erizatio n i s d iffe re n t in mono* and d iv a le n t bases (calci.u a and barium hydroxides) o f O.SK co n cen tratio n . At O.G35H c o n c e n tra tio n s, however, these in v e stig a to rs found no apparent d iffe r* on es. Wind (69) observed th a t o x id atio n o f glycer&ldehyde and dihydroxy— acetone in phosphate so lu tio n s was cataly sed by heavy m e tals, e sp e c ia lly copper, Ahletrom and von E uler (70) likew ise found th a t oxygen con* sumption by b uffered so lu tio n s o f th e trio s e s was cataly sed by c u p rio , f e r r i c , and fe rro u s s u lf a te s . Assuming th e enediol as th e o x id iseab le S p ecies, th e se d a ta may in d ic a te increased a b ility fo r enediol form ation in th e presence o f th ese m etal io n s. Pyruvaldehyde Formation Pyruvaldehyde has been in d icated as a degradation product o f carbo* hy d rates when sub jected to a wide v a rie ty o f co n d itio n s, hsuberg and O ertel (71) obtained pyruvaldehyde from glucose, fru c to s e , and mannose by warming w ith d ilu te sodium carbonate as did Fernbaoh and Sohoen (7 2 ). F isc h le r (73) and F isc h le r and Lindner (7 l) found pyruvaldei^ydo in th e d i s t illa te s from d ilu te a lk a lin e so lu tio n s o f glucose, fru c to s e , g a la c to se , m altose and la c to s e . T riose form ation and subsequent dehydration to pyruvaldeljyde was p o stu la te d . Neuberg and Eswald (75) rep o rted pyruvaldehyde form ation on heating arab in o se, xylose riiamnose, glucose, fru c to s e , g a la c to se , and glucosamine w ith te n p er cen t ammonia. Bsrnhauer and Wolf (76) heated glucose w ith calcium and magnesium carbonates and obtained pyruvaldehyde, supposedly through a trios© in te rm ed ia te. Evans and co-workers found th a t a lk a lin e degrada­ tio n re su lte d in pyruvaldehyde production from glucose and galactose (31) hh fru c to se (3 5 ), mannose ( 3? ) , arabinose and xylose ( 39) , m altose ( 36) , o e lld b io se , la c to s e , m ellblose, and gentlobiose (1*0) , and rhamnoao (Ii2) aa v a i l aa glyeeraldehyde ( 33) and dthydrosgraestone (3W . A gain, trio s e form ation vas considered th e f i r s t ste p In tb s degradation o f th e higher sugars* Indore and M arquardt ( 77) observed pyruvaldehyde In th e d is t illa te s from aqueous so lu tio n s o f glucose and xylose. Cameron (76) obtained pyruvaldehyde from glucose In th e presence o f a c e tic acid and bensylsmlne w hile T aufel and B urm elster (79) found th a t a f te r extended acid hydrolyses sucrose so lu tio n s contained pyruvaldelyde. Enders (80) In v estig ated the production o f pyruvaldehyde from m altose over a vide range o f pH v alu es. A minimum vas observed a t pH 1 , Increased a c id ity Increased the conversion u n til a maximum vas reached in te e n ty p er c e n t su lfu ric s o ld . As th e pH was ra ise d , th e conversion Increased to a second maximum a t pH 11*12. Above th a t p o in t conversion o f pyruvaldehyde to la c tic acid became more rap id th an pyruvaldehyde form ation. Fyruvaldeiyde was u su a lly id e n tifie d as i t s osasone. The observe* tio n s th a t a e e to l was produced along w ith pyruvaldehyde (same osasones) on d is t i l l a t i o n o f hexcses in phosphate (8l*8h) and In d ilu te sodium carbonate so lu tio n s (65 >66) as v e il as on the d is tilla tio n o f aqueous glucose and m altose so lu tio n s (67) c a s t doubt on some o f th e e a r lie r r e s u lts . However, u tilis in g paper p a r titio n chromatography, Speck (6 8 ), has re c e n tly unequivocally Id e n tifie d pyruvaldehyde as a product In d i s t i l l a t e s from amino aeld*reduclng sugar re a c tio n m ixtures as v e il as In d i s t i l l a t e s from potassium hydroxide so lu tio n s o f glucose. 1»S The re a d / conversion o f the tr lo s e s to pyruvaldehyde has long been known, end, e» noted p rev io u sly , maigr workers have conaldered th e trio e e a as p recu rso rs o f pyruvaldehyde in l i e production from ttie higher su g ars. Bentgee (89) rep o rted th e form ation o f pyruveldetyde by the a c tio n o f s u lfu ric ac id on dlhydroayaeetone, and Rsuberg, jet s i , (90) u tilis e d th is re a c tio n in determ ining th e tr lo s e s , Evans and co-w orkers observed pyruvaldehyde production from gjXyceraldehyde (33) and dlhydroayace tone (3b) in potassium hydroxide ao lu tlo n a. F isch er and Taube (91) wore able to dehydrate tiihydrojgracetone to pyruvaldehyde by a d is tilla tio n w ith phosphorous p en to xlde. L i t t l e inform ation i s a v a ila b le concerning the mechanism o f th e dehydration o f th e tr lo s e s o r th e c a ta ly se s involved. Diache and Bobbins (92) rep o rted an a c c e le ra tio n o f pyruvaldehyde fo m atio n in n e u tra l glyeeraldehyde o r dihydroayacetone so lu tio n s on ad d itio n o f phosphate o r a rse n a te , Ca**, Cu**, Fe** and Fe*** tied no apparent e f f e c t, Evans and Corntftw aite (3b) have suggested th a t pyruvaldehyde a ris e s from the enediol cozsson to th e tr lo s e s , Xn a study o f th e e ffe c t o f amines on th e conversion o f tr lo s e s in to pyruvaldehyde, S tra in and Spoehr (93) found th a t se v eral weakly basic amines cataly sed ti& s re a c tio n in d ilu te a c e tic ac id s o lu tio n s . These in v e stig a to rs also thought th a t a common en ediol m od ificatio n o f th e tr lo s e s might be e s s e n tia l to th e deljydratlon. A nalysis o f th e ir d ata in d ic a te s th a t the amines which were ac tiv e were weak bases such th a t, a t the a c id itie s employed, they e x iste d la rg e ly in th e form o f the fre e am ine. Tide suggests a sp e c ific amine e f f e c t, a base c a ta ly s is and perhaps Smith and Anderson (9U) have re c e n tly U6 in v e stig a te d th e dehydration o f th e 3-C -p h en y ltrio see in d ilu te a c e tic a o id # The a ld o trlo se required th e presence o f a prim ary amine fo r dehydration, w hile th is was unnecessary f o r th e k e to trlo s e s . in term ed iate was proposed. An en ediol Speck (68) has dem onstrated th e presence o f pyruvaldehyde in d i s t i l l a t e s from glyeeraldehyde-amino acid m ixtures. The p re se n t in v e stig a tio n was designed (a) to confirm the occurrence o f th e lo b ry de Bruyn-van Ekenateln transform ation in acid m edia, (b) to t e s t th e p o s s ib ility o f a gen eralised aeid-base c a ta ly s is in th is re ­ a c tio n and in th e sim ultaneous form ation o f pyruvaldehyde, (c ) to examine th e e ffe c ts o f d iv a le n t ca tio n s on these p ro cesses, and (d) to gain as much inform ation as p o ssib le concerning th e re a c tio n mechanisms involved. QL*g3yoeraldehyde and dlhydrojQraoetone were chosen fo r th is study sin c e , i n each c a se , th e re i s only one p o ssib le iso m erlsatlo n o r dehydration p ro d u ct, an d , in a d d itio n , th e number o f d istu rb in g side re a c tio n s i s few . hi EXPERIMENTAL METHODS M a te ria l* The DL~glyceraldehyde employed in th ese measurements was synthesized by th© method o f F isch e r and Baer (95) j th e dlhydroagracetone was a N u tritio n a l Biochemical® p ro du ct. The a c e tic acid was Bakerfa C .P . an aly sed . Eastman Kodak Company p ra c tic a l formic acid was fra c tio n a lly d is t ille d and th e fra c tio n b o ilin g a t 99.5° C used in th is in v e stig a tio n . The trim a tly la c e tic acid was an Eastman Kodak Company p ro d u ct. Standard sodium p erc h lo ra te was prepared by n e u tra liz in g standard 2 M p erch lo ric acid so lu tio n to pH 7.0 w ith standard sodium hydroxide so lu tio n . Calcium p e rc h lo ra te and calcium a c e ta te were prepared in so lu tio n by n e u tra liz a ­ tio n o f weighed sample® o f Baker1s C .P . calcium carbonate w ith th e re sp e ctiv e a c id s . The n -b u tan o l, n -h eptanol, and hydro^ylamine hydro- ch lo rid e were Eastman Kodak Company p ro d u cts. P eriod ic a c id , p erch lorato e e rie a c id , and n ltro fe rro in were obtained from th© 0 . F rederick Smith Chemical Company. Sodium o x a la te , arsenious ox id e, and p e rch lo ric acid were Baker1® C .P . analyzed m a te ria ls. Matheson Company p ra c tic a l chromo- tro p ic acid was re c ry s ta llis e d from $0% eth an o l. Merck reagent grade. S u lfu ric acid was S ilv e r n itr a te , nlckelous s u lf a te , sodium a c e ta te , sodium s u l f ite , sodirat b icarb o n ate, potassium Io d id e, io d in e , aaraonluB hydroxide, and sodium i^ydroxide used in th is in v e stig a tio n were a l l C. P . re a g e n ts. M Apparatus A Beckman Hod e l DU spectrophotom eter equipped v lth q u arts c e lls was used f o r u ltr a v io le t s p e c tra l measurements w hile a Beckman Model B spectrophotom eter w ith corex c e lls was employed fo r measurements in th e v is ib le range. A Beckman pH m ater w ith an o u tsid e g la ss electro d e was used f o r pH d e te m in a tlo n s. Id e n tific a tio n of Products (Eyoeraldshyds y ie ld s pyruvaldehyde In acid so lu tio n (8 9 , $ 0 ). In p relim in ar y stu d ie s u tilis in g th e c e ra te o x id atio n technique de­ scrib ed below, degradation o f gSyeeroldehyda in 2 .5 M p erch lo ric acid a t £0° C in d ic a te d pyruvaldehyde as th e only p ro d u ct. T h erefo re, pyruvaldehyde was a n tic ip a te d as th e predominant product o f glyceraldehyde degradation in m ildly so ld b u ffe r system s such as a c e ta te a t 50° C. The p o s s ib ility o f th e tran sfo naation o f glyceraldehyde to dliydroj^acetone was n o t overlooked In view o f the ln terco n v srsio n o f hexoses (60-62) and pentoses (63) reported to occur under sim ila r co n d itio n s. E ith e r o x id atio n w ith p e rio d ic acid and subsequent determ ination o f the p e rio d a te consumed by th e method o f Floury and Lange (96) o r oxida­ tio n w ith p erch lo ra te c e ra te in I M p erch lo ric acid follow ed by determ ina­ tio n o f th e te tra v a le n t cerium consumed by th e procedure o f Speck, F o r is t, and Heely (9?) would perm it determ ination o f glyceraldehyde and pyruvaldehyde in m ixtures o f th ese substances provided th e i n i t i a l glyceraldehyde co n cen tratio n I s known. In a p erio d ate o x id a tio n , h9 glyceraldehyde and pyruvaldehyde consume fo u r and two eq u iv alen ts o f oxidant p e r mole re sp e c tiv e ly whereas w ith c e ra te the consumption I s s ix snd two eq u iv alen ts p er m ole. At th e beginning o f th is in v e stig a ­ tio n , th e c e ra te method was chosen due to th e g re a te r d if f e r e n tia l in eq u iv alen ts o f oxidant consumed. C erate oadLdatlon s tu d ie s . I f pyruvaldehyde were the only pro d u ct, c e ra te consumption should decrease w ith tim e. T herefore, prelim inary stu d ie s o f th e degradation o f glyceraldehyde in a c e ta te b u ffe rs a t $0° C were made by measuring c e ra te consumed by a liq u o ts o f th e re a c tio n m ixture a t vario u s tim es as fo llo w si 2.00 m l. a liq u o ts o f th e re a c tio n m ixture ( i n i t i a l l y 0,2M in glyceraldehyde) were withdrawn a t d e fin ite tim es, added to 20.0 m l. o f 0.268? H p erch lo rato c e ra te in U H p erch lo ric a c id , and th e oxid ation allowed to proceed fo r a t le a s t fo u r hours. At th e end o f th a t tim e, 25.0 m l. o f 0.1825 H sodium o x alate in 0 .1 If p erch lo ric acid was added and th e excess o x alate tit r a te d w ith 0.02703 8 c e ra te in 2 H p erch lo ric acid to a n itro -fo rro in end p o in t. In prelim inary experim ents, q u a n tita tiv e o x id atio n o f pure glycer­ aldehyde had been observed in a period o f fo u r hours. However, th e re a c tio n m ixtures containing a c e ta te b u ffe rs were incom pletely ox idised a f te r fo u r hours (low values a t sero tim e ). On th e o th e r hand, over o x id a tio n occurred i f th e o x id atio n s were allowed to proceed fo r longer p e rio d s. The presence o f a c e ta te may have caused a redu ction in th e s p e c if ic ity o f th e o x id a n t, o r a c e ta te and form ate may have been slow ly 50 o x id is e d * t h l a l e d to th e d is c a r d in g o f t h i s method f o r q u a n ti t a t iv e H o te ith e ta n d in g t h i s , th e c e r a te o x id a tio n s tu d ie s con** t r i b u t e d v a lu a b le q u a l i t a t i v e info rm atio n * In s te a d o f th e expected d rop i n c e r a t e consum ption a s d e g ra d a tio n o f th e t r i o s e i n a c e ta te b u f f e r p ro ceed ed , th e r e was a ra p id r i s e follow ed by o f f a n d , a f t e r p rolonged r e a c t i o n tim e s, a decrease* a g rad u a l le v e lin g A lthough q u a n ti t a - t i v e c a l c u l a t i o n s c o u ld n o t be made from th e s e d a t a , i t v a s a p p a re n t t h a t some p ro d u c t, cap ab le o f a g r e a te r c e r a t e consum ption th an g ly c e rald eh y d e v a s b e in g form ed. v a s dihydrojQ racetone* I t seemed p ro b ab le t h a t t h i s su b sta n c e The e v e n tu a l drop i n c e r a te consum ption su g g ested a slow c o n v e rsio n o f one o r b o th o f th e t r l o s e s to pyruvaldehyde. U ltra v io le t s p e c tra l s tu d ie s . C e ra te o x id a tio n s tu d ie s in d ic a te d dibydroay acetone a s w e ll a s pyruvaldehyde a s r e a c tio n p ro d u c ts . I n an e f f o r t to f u r t h e r i d e n t i f y th e s e o r o t h e r p ro d u c ts th e fo llo w in g experim ent was c a r r i e d o u ti A t y p i c a l r e a c tio n m ixture was p rep ared which was 0 . 2 0 M i n DiL~ g ly ce rald eh y d e and 0*1*0 M each i n a c e tic a c id and sodium a c e ta te * T h is s o lu tio n was p la c e d i n a th e rm o sta t a t 50° C , an d , a t d e f i n i t e tim e s , 2 ,0 ml* a li q u o t s were removed and d ilu te d to 10 ml* The a b s o rp tio n s p e c tr a o f th e s e sam ples i n th e re g io n 21*0-32) mu were th e n measured u s in g th e Beekman Model DU spectrophotom eter* F ig u re 1 shows th e a b s o rp tio n s p e c tr a o f p u re Q L -g ly cerald ely d e, dihydroayaceton© , and pyruvaldehyde w hereas F ig u re 2 g iv e s th e r e s u l t s ©f th e above experim ent o v e r a p e rio d o f tw e n ty -fiv e h o u rs. The i n i t i a l g ly ce rald eh y d e spectrum changes f a i r l y ra p id ly w ith a stro n g peak 0.400 0.300 0.250 0.200 0.150 0 .1 0 0 0.050 0 .1 0 M D L -g ly c e r a ld e h y d e 0 .0 2 M D ih y d ro x y a ceto n e 0 .0 2 M ly r u v a ld e h y d e 250 F ig u r e 1 . 275 300 325 MJU U l t r a v i o l e t a b so r p tio n s p e c t r a . <5 0.800 0.700 0.600 0.500 LOG U / l 0.400 0.5 0 0 0 250 HOURS 275 300 325 MJL) F ig u re 2. S p e c t r a l ch a n g es d u rin g th e d e g r a d a tio n o f D L -g iy c e r a ld e h y d e i n a c e t a t e b u f f e r a t 5 0 ° C. SI developing near 210 mi a f te r tw elve hours. On fu rth e r re a c tio n , th e re I® * alow e h if t toward th e lo n g er wave le n g th s, th e naxlsun occurring a t 275 a f te r tw en ty-fiv e houre. Comparison o f these curves w ith those o f F igure 1 suggests a rap id form ation o f dihydroayacetone (maximum a t 270 mu) w ith a slow conversion to pyruvaldehyde (naadjsum a t 260 mu). Id e n tific a tio n o f dihydrogyacatone by paper chromatography. The r e a c tio n m ixture employed f o r th e s p e c t r a l s tu d ie s was used f o r chrom atography. f o u r h o u rs . The sample was removed f o r t h i s experim ent a f t e r tw en ty - Two drops o f t i d e s o lu tio n were p la c e d on f i l t e r p aper (G'hatman Ho. 1) and an ascending chromatogram developed u sin g n -b u ta n o l s a tu r a te d w ith w a ter a s th e so lv e n t (9 6 ) . S im u ltan eo u sly , p u re samples o f D L -glyceraldehyde, dihydroxy ace tone , pyruvaldehyde, and a s y n th e tic m ix tu re o f th e s e th re e ware chrom atographed under i d e n t i c a l c o n d itio n s . A f te r th ir ty - tw o h o u rs , th e chromatograms were removed, th e so lv e n t f r o n t m arked, and th e s h e e ts d rie d b e fo re an e l e c t r i c f a n . They were th e n sp rayed w ith a s o lu tio n c o n ta in in g @*ual volumes o f 5 M ammonium hydroxide and 0 .1 M s i l v e r n i t r a t e , d rie d a t U C ° G f o r ab o u t f iv e m in u te s, washed w ith d i s t i l l e d w a te r, and d rie d once more ( 9 9 ) . s p o ts were c i r c l e d and h f v a lu e s c a lc u la te d . T ab le I . Hie The r e s u l t s a re shown i n I n every c a s e , g ly ceraldehyde produced a poor chromatogram and t a i l e d c o n s id e ra b ly . G ibydroxyacetone gave a s h a rp , w e ll d e fin e d sp o t and was e a s i l y seen even when p a r t i a l l y o v e rla p p in g th e g ly cerald eh y d e ta il. I t was obvious t h a t pyruvaldehyde could n o t be d e te c te d by t h i s p ro c e d u re , probably due to i t s v o l a t i l i t y . The t h i r d component o f low h f 52 found a f t e r tw enty— fo u r h o u rs i n th e r e a c tio n m ixture v as n o t i d e n t i f i e d b u t l a b e lie v e d to be a polym er formed from pyruvaldehyde. Anomalous p e r io d a te consum ption by pyruvaldehyde i n a c e ta te b u f f e r s a f t e r ex­ ten d ed tim e s ( c i t e d below) su p p o rts t h i s p o in t o f view . D ihydroxyacetone v a s th e r e f o re e s ta b lis h e d a s a p ro d u c t o f th e d e g ra d a tio n o f gly cerald eh y d e under th e c o n d itio n s o f t h i s I n v e s tig a tio n . I d e n t i f i c a t i o n o f pyruvaldehyde by paper chrom atography. Pyruvaldsi^ydo was i d e n t i f i e d by chrom atography o f I t s d io x in e a c c o rd in g t o th e method o f Speck (66) a s o u tlin e d below . TABLE X IDENTIFICATION OF DIHXD&OXXAChTQNii M a te r ia l Chromatographed DL-glyc e r aldehyde Diljydroxy aceto n e ** —0.35 0.3X q u a lity o f Spot Very d if f u s e ; d i f f i c u l t to e s tim a te h f W ell d e fin e d No sp o t Fyruvaldehyde Known M ixtures DL-glyceraldeliyd@ D iljydroxyacetone Pyruvaldehyde — 0 .3 6 0.33 ** Poorly d e fin e d W@H d e fin e d No sp o t h e a c tio n M ixtures DL-glyceraldetiyde D ihydroxyacet >ne Unknown — 0 .3 6 0 .3 3 0.05 D iffu se W ell d e fin e d w e ll d e fin e d 53 F o r ty - e ig h t m l. o f a r e a c tio n m ix tu re , I d e n t i c a l w ith th e on® d e s c rib e d ab o v e, was removed a f t e r f i f t y - f o u r h o u rs , c o o le d , and mixed w ith 50 m l. o f s o lu tio n c o n ta in in g 1 gn. o f hydroxy lam ine h y d ro ch lo rid e and 2 gm* o f sodium a c e ta te t r i h y d r a t e . A fte r s ta n d in g o v e rn ig h t, th e s o lu tio n was s a tu r a te d w ith sodium c h lo r id e and e x tra c te d s i x tim es w ith 2*> m l, p o r tio n s o f d ie th y l e t h e r . The combined e th e r e x tr a c t s were d r ie d o v e r anhydrous sodium s u l f a t e , f i l t e r e d , and th e e th e r removed on th e w a te r pump. The sm all amount o f re s id u a rem aining was e q u ilib r a te d w ith 2 m l. o f d i s t i l l e d w a te r, th e s o lu tio n f i l t e r e d , and s i x d ro p s p la c e d on f i l t e r p aper (Vhatman Ho. 1 ) . An ascending chromatogram was developed u s in g n -h e p ta n o l s a tu r a te d w ith w ater aa th e s o lv e n t, S im u lta n e o u sly , a known sample o f m ethylglyoxim e was chrom atographed under i d e n t i c a l c o n d itio n s . A f te r t h i r t y —s i x h o u rs, th e ohromatograms were removed and d r ie d b e fo re an e l e c t r i c fa n i n th e absence o f h e a t . They w ere th e n sprayed w ith a 3% s o lu tio n o f n lc k e lo u s s u l f a t e hexah y d ra te c o n ta in in g 0 .1 m l. o f 26% ammonia p e r 100 m l. and once more d rie d . The r e s u l t s a re shown I n T able X I. T hus, both dibydroxyacetone and pyruvaldehyde were e s ta b lis h e d a s r e a c tio n p ro d u c ts . TABLE XI IBEHTIFICATIOH OF PIHUVALDEHIDE M a te ria l Chromatographed % d u a lity o f Spot M©th y lglyoxim e 0 .6 1 K e ll d e fin e d ; orange w ith ro se c e n te r . R e a c tio n m ix tu re oxime 0.60 K e ll d e fin e d ; orange w ith ro s e c e n te r . 51* Q u an titativ e A nalysis o f R eaction Mixture* Since two product* were being formed, two a n a ly tic a l method* were necessary in ord er to estim ate the th ree components, glyceraldehyde, dlhydro^yacetone, and pyruvaldehyde, a t any time* I t was hoped th a t c e ra te o x id atio n s could to© employed, tout, due to the anomalous o xidation s discussed above, th is method was d iscard ed . A second a ttra c tiv e procedure co n sisted o f o x id atio n w ith p erio d ic acid and subsequent determ ination o f p erio d ate consumed by th e method o f Floury and Lange (9 6 ). To te s t the a p p lic a b ility o f th is method to th e problem a t hand, samples o f DLgLyesraldehyde and dihydrosyaeetone o f co ncentrations expected to be en­ countered were o xid ised fo r varying tim es toy approxim ately 0 .1 N p erio d ic acid and th e p erio d ate consumption determ ined. The r e s u lts are given in Table I I I . TABLE I I I PERIODATE OXIDATION OF TRIQSE3 O xidation Time (Hours) 1 2 3 k 6 Theory E quivalents 3304~ consumed p er mole trio se * ir-Elyceraldetsrde Djhydroaoracetone . . 3 91*8 3 91*1 3.956 3.956 3*962 1.987 1.992 1.996 2.008 2.008 ii.0 0 0 2 .0 0 0 *» Average of d u p licate analyses 55 From th e s e d a ta , I t l a se e n t h a t p e rio d a te consum ption by b o th s u g a rs l a com plete a f t e r th r e e hours under th e c o n d itio n s employed. T h o rn to n and Speck (100) , showed t h a t pyruvaldehyde was com pletely o x id is e d by p e r io d ic a c id i n one h o u r. T h e re fo re , i n t h l a I n v e s tig a tio n , p e r io d a te o x id a tio n s have been allow ed to proceed f o r a t l e a s t fo u r h o u rs , tim e in s u r in g com plete r e a c t i o n . On p erio d ate o x id a tio n , glyceraldehyde consumes fo ur equ ivalents o f oxidant p e r mole whereas dihydro ayacetone and pyruvaldehyde each consume two eq u iv alen ts p er m ole. I f 0 , 0 , and F rep rese n t m illim oles o f glyceraldehyde, dihydroxyaceto n e, and pyruvaldehyde resp e ctiv e ly p er m l. o f so lu tio n analysed, Go re p re se n ts I n i t i a l glyceraldehyde co n cen tratio n , and I rep resen ts m illieq u lv alen te o f p erio d ate consumed by one m l. o f re a c tio n m ix ture, the follow ing equations may he w ritten* 0 + D + ? • Go UG ♦ 2D + 2P • (1) I (2) From th ese ex p ressio n s, equation (3) may be obtained. 0 - 1/2 - Go (3) T h erefo re, beginning w ith pure glyceraldehyde, G a t any tim e may be ca lc u la te d from Go and the p erio d ate consumption, 1 . However, th is method o ffe rs no d is tin c tio n between G and P , giving only th e ir sum. Since 0 could be determ ined a t any tim e, a second method allow ing determ ination o f to ta l tr lo s e s would perm it an estim atio n of a l l th ree components. The method se lec ted was devised fo r tid e purpose and i s describ ed in P a rt I I o f th is th e s is . 56 The o p e ra tin g p ro ced u re employed i s b e s t d e sc rib e d by o u tlin in g a t y p i c a l eaq p eriasn t, The c a lc u la te d Q u an tity o f sta n d a rd s a id ( o r a weighed sample i n th e c a s e o f t r i s e t h y l a e e t i c a c id ) was mixed i n a 50 ml • v o lu m e tric f l a s k w ith s u f f i c i e n t sta n d a rd sodium hydroxide to p ro v id e th e d e s ire d b u f f e r r a t i o and c o n c e n tra tio n * To t h i s was added enough sta n d a rd sodium p e r c h lo r a te s o lu tio n to b rin g th e io n ic s tr e n g th to O .h . T h is s o lu tio n was p la c e d i n th e c o n s ta n t tem p eratu re b a th o p e ra tin g a t 56° 1 0 .0 1 ° C and b ro u g h t to th e b a th te m p e ra tu re , A sam ple o f p u re D L-glyeeraldeljyde ( u s u a lly 0.9008 gm,) was weighed on an a n a l y t i c a l b a la n c e , d is s o lv e d i n r e d i s t i l l e d w a ter a t th e b a th te m p e ra tu re , and q u a n ti t a t iv e l y tr a n s f e r r e d to th e r e a c tio n f l a s k . The s o lu tio n was m ixed, bro u g h t n e a rly to th e mark w ith r e d i s t i l l e d w a te r a t th e b a th te m p e ra tu re , and r e tu rn e d to th e b a th f o r about f i f t e e n m in u te s. I t was th e n d i lu t e d t o th e m ark, th o roughly m ixed, re tu rn e d to th e b a th , and th e f i r s t sam ples removed. I n m ost e x p e rim e n ts, s o lu tio n s 0 .2 0 M i n g ly ceraldehyde were used and 1 .0 0 m l, a li q u o t s were removed a t th e d e s ire d tim es f o r a n a l y s i s . To d e te m in e p e rio d a te consum ption, a 1 ,0 0 m l. a liq u o t o f th e re * a c tio n m ix tu re was t r a n s f e r r e d to a 300 m l, erlenm eyer f l a s k c o n ta in in g 1 5 .0 0 m l. o f 0 .1 0 8 N p e r io d ic a c id and th e o x id a tio n allow ed to proceed f o r a t l e a s t f o u r h o u rs. The s o lu tio n was th e n n e u tr a lis e d and s a tu r a te d w ith s o l i d sodium b ic a rb o n a te and 2 m l. o f 5% potassium io d id e follow ed by 10.00 m l. o f 0.13112 N a rsen it© added. A f te r about f i f t e e n m in u te s, th e e x ce ss a r s e n i t e was t i t r a t e d w ith 0 .0 1 N io d in e s o lu tio n to a s ta r c h St en d p o in t. M illle ^ u iv a le n ts o f period ate consimsd may be ca lcu late d from th e expression* I « IS x - IP x 0.1311*2 - m l. I 8 x KI# (U) To determ ine to ta l trio e e a , a 1.00 m l. a liq u o t o f the re a c tio n m ixture was tra n sfe rre d to a 200 nil. volum etric fla a k containing 1* m l. o f 1 M sodium bicarbonate and 1* m l. o f 0 ,3 M p erio d ic a c id , and th e oxid ation allow ed to proceed f o r one hour. At th e end o f th a t tim e, 10 m l. of 0 .5 M sodium s u lf ite waa added and th e so lu tio n d ilu te d to th e mark. D uplicate 1.00 m l. a liq u o ts o f th is so lu tio n were p ip e tte d in to 5° m l. volum etric f la s k s , follow ed by 0 .5 m l. o f 10# chronotropic acid and 5 m l. o f lh M s u lfu ric a c id . The fla sk s were heated in a b o ilin g w ater bath f o r th ir ty m inutes* They were then removed, coo led , and about i*0 m l. o f d is t ille d w ater added. The fla sk s were cooled once more, and th e solu­ tio n s d ilu te d to th e mark. Excess s u lfu r dioxide was removed by bubbling a i r sa tu ra te d w ith w ater through th ese so lu tio n s a t a ra te o f 750-1000 m l. p er m inute. The o p tic a l d e n s itie s o f these so lu tio n s were then measured a g a in st a reagent blank a t 570 mu using th e Beckman Model B spectrophotom eter. The to ta l tr io s e values were ca lcu lated from a standard curve prepared by sim ila r treatm ent o f e ith e r D-gluooee o r QLglyceraldehyde» SB RESULTS AND DISCUSSION Order o f th e Reaction P re li mi nary stu d ie s in d icated th a t pyruvaldehyde was no t com pletely s ta b le in b u ffe rs of th e type employed in th is in v e stig a tio n . A fre sh so lu tio n o f pyruvaldehyde (obtained by d is tilla tio n o f a s u lfu ric acid so lu tio n o f BL-glyceraldehyde) was prepared in a b u ffe r 0.1& H in sodium a c e ta te and 0.133 M in a c e tic a c id . T his so lu tio n was placed in th e $0° C therm ostat and one m l. a liq u o ts removed p e rio d ic a lly fo r perio d ate oadd atio n. A t th e same tim es # sim ila r a liq u o ts were te ste d fo r form* aldelyde producing m a terial by th e to ta l trio s e a n a ly sis. are shown in Table I ? . The r e s u lts The so lu tio n was colored a f te r eighteen hours, and some m a te ria l capable o f a g re a te r perio d ate reduction than pyruv­ aldehyde i t s e l f was fo m ed . TABU IV INSTABILITY OF PYRUVALDEHYDE IN ACETATE BUFFER tim e o f Standing (Hours) io * Consumed Apparent [P i (HoI sb A ) HCHO Producing 0 18 2h SJ 72 336 0.2679 0.2852 0.2930 0.2958 0.301*7 0.39U* 0.131(0 0.11*26 0.11*65 0.11*79 0.1521* OJ.972 Done Rons Rone * Average o f d u p licate analyses Kate r i a l — — *** 59 In most o f th e reactio n * stu d ie d , th e s ta rtin g m aterial was pure BL-glycoraldehyde, and i t s co n cen tratio n a t any tin e could be ca lcu late d from a knowledge o f th e p erio d ate consumed by an a liq u o t o f th e re a c tio n m ixture aa o u tlin ed above* Sue to the p erio d ate consumption by pyruvaldehyde when p rese n t in appreciable concentrations and in o rd er to d isreg a rd th e reverse re a c tio n in th e interoo aversion o f the tr io se e , only 0i n i t i a l velocity* d ata has been used. R eactions were u su ally follow ed to th e disappearance o f 204*5# o f the i n i t i a l glyceraldehyde. A p lo t o f th e logarithm o f glyceraldehyde concentration versus was lin e a r (F igure 3) in d ic a tin g a f i r s t o rd er disappearance o f glyceraldehyde. In o v er fo rty v e lo c ity determ in ation s, no d ev iation s from th is o rd er were observed. I f a re a c tio n I s o f the f i r s t o rd e r, th e pseudo constant ca lcu late d from th e slope o f th e lo g 0 versus tim e curve i s independent o f i n i t i a l re a c ta n t co n c en tratio n . T herefore, v e lo c ity determ inations were made a t d iffe re n t i n i t i a l glyeeraldelyde co ncentrations in b u ffe rs 0.1*0 M each In a c e tic acid and sodium a c e ta te . The r e s u lts are shown in column two o f Table V. TABLE ? EFFH3T OF IHITIAL GLXCKKALDiiHlDL COKCcRTRATION "• ............ tCtoJ....... ............ (KoleaA) 0.10 0.20 0.1|0 k' * 10* mln”1 (DAH 233 205* 167 a Average of four determ inations Average o f two determ inations 5.0 5.3 - 0.700 - 0.800 LOG G 250 F i g u r e 3* T a b le V I ) . 500 750 TIM E (MINUTES) 1000 T y p i c a l r a t e c u r v e ( E x p e r im e n t 1 1 , 60 A re g u la r v a ria tio n was observed which vas not considered in d ic a tiv e o f a a o rd er o th e r th an th e f i r s t . The decrease in pseudo co n stan t w ith in c re a sin g i n i t i a l glyceraldebyde co n cen tratio n v as believed th e r e s u lt o f decreased a c tiv ity o f th e glyceraldehyde due to th e many p o la r hydroxyl groups p re se n t in so lu tio n . C alcu latio n o f C onstants Since th e o v e r-a ll disappearance o f glyceraldehyde vas o f the f i r s t o rd e r, th is may be expressed as - dO /dt * k 10] (5 ) which on in te g ra tio n y ie ld s or - In 0 • k i ♦ constant (6) lo g 0 * - k 't ♦ con stan t (7) The pseudo co n stan ts ( k 1), employing decadic lo garithm s, were evaluated a t varying b u ffe r con cen tration s and b u ffe r r a tio s fo r fo rm ats, a c e ta te , and trim e th y la c e ta te system s. The r e s u lts are in d icated in column seven o f T ables V I, V II, and V III. The observed v a ria tio n s in pseudo co n stan ts w ith various b u ffer co n cen tratio n s a t co n stan t b u ffe r r a tio , as v e il as a t d iffe re n t b u ffe r r a tio s , suggested an acid and base oatalyzed re a c tio n . I f [HA] and [A*] re p re se n t co n cen tratio n s o f the acid and conjugate base re sp e c tiv e ly o f th e b u ffe r system in vo lved, such a c a ta ly s is may be represented by the expression -dG /dt - (kjj+lH+] ♦ kjyJHA] ♦ 1^- [A -]] [G] (8) 61 TABLE VI PSEUDO CONSTANTS — ACETATE STSTiiMS & p%* EOo] [CHjPOOH] [CH.COONa] [NaC104] A* H3T k fx l0* m ±rrx ( d/ p H 18 0,20 mm mm o.ho U 0.20 0J® 0.20 0.20 1 98 — 5 0.20 0 .20 0.20 0.20 1 99 — 10 0.20 0.20 0.20 0.20 1 101 L .6 Ik 0.20 0.20 0.20 0.20 1 100 lu5 IS 0.20 0.30 0.30 0.10 1 152 fc.8 6 0.20 0.1& O .U j — 1 208 mm 7 0.20 0 .1 ® 0.1i0 — 1 198 — 8 0.20 0.1<0 0.1i0 mm 1 208 $ .k 16 0.20 0.1i0 0 .1)0 — 1 207 L .6 13 0.20 0.067 0.20 0.20 3 96 L .6 12 0.20 0.10 0.30 0.10 3 139 k.B 11 0.20 • 0.133 o.llo 3 193 5.0 0 Average (D ^?)i • iu 8 * b .2 AH co ncentration s In s o le s /1 . 62 TABLE VII PSEUDO CONSTANTS — FOfiHATE SISTEKS E *pt. lOo) [ hcoob] [HCOONa] [HaClQ4 ] 23 2i* 26 29 28 27 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.30 0J*0 0.067 0.10 0.133 0.20 0.30 o.iiO 0.20 0.30 0.20 0.10 0.20 oao — O.JaQ ^ 1 1 1 3 3 3 mln*1 ( d/ p)± 35 60 71 33 51 62 3.5 3.5 2.L 2.5 2.6 h Ji Average (D/P)i - 3.240.7 A ll concentrations In moles/l . TABLE VIH PSEUDO CONSTANTS — TBIMSTHXUCETATE SISTEHS Sap*. [Go] 31a 32 35 33 0 .2 0 0 .2 0 0 .2 0 0 .2 0 [(GHg)gGCOOH] 0 .0 5 0 .1 0 0 .0 2 5 ■ 0 .0 5 [(CH*)BCCQ0Ha] tfi«C104 ] 0.1 0 0.20 oao 0.20 0 .3 0 0 .2 0 0.30 0 .2 0 A- k'xlO * Iff « ln - i 2 2 1* 1* 61* 177 81 166 (D /M i ii .3 6 .1 li.l* 6 .6 Average (D /P )l • 5 . 0 - .1 A ll c o n c e n tra tio n s I n m o le s /I, 63 where th e JkJs are th e resp e ctiv e c a ta ly tic co n sta n ts• No term fo r spontaneous w ater c a ta ly s is has been Included sin c e , In th e absence o f b u ffe r system s, no re a c tio n occurred (Aaqperimant 18, Table V I). The pseudo co n stan t becomes *' *[% tl« * l * (9) or where k* -[kj| ♦ ][« * ] ♦oc [HA] (10) oC » (11) ♦ i^~ [A-/hA] T hereforef a t co n stan t b u ffe r r a tio (co n stan t [H+] ) , a p lo t o f k* v ersus [HA] should be lin e a r . Tide was observed experim entally f o r th e systems stu d ied (F ig u res 2*, 5 , and 6 ), In every case th e in te rc e p t was aero in d ic a tin g n e g lig ib le c a ta ly s is by the hydronium ion as w ell as w ater, A p lo t o f th e slopes (oC) o f these curves ag a in st the b u ffe r ra tio [A*yilAj should be lin e a r . Moreover, th e slopes and in te rc e p ts should equal th e c a ta ly tic co n stan ts fo r the bases and acid s involved re sp e c tiv e ly , Such a p lo t i s shown in F igure ? , and th e c a ta ly tic con stan ts so ca lcu late d a re given in Table IX. From th ese d a ta , i t i s seen th a t th e o v e r-a ll disappearance o f glyceraldekyde i s su b je c t to an acid and base c a ta ly s is . The b asic c a ta ly s is by trim e th y la c e ta te , a c e ta te , and formate ion s l a in q u a lita tiv e agreement w ith th e ir r e la tiv e stre n g th s as Bronsted b a se s. The c a ta ly tic c o n sta n ts f o r th e re sp e c tiv e a c id s do not follow th e expected p a tte rn . However, th e e rro r in th e ir estim atio n may b© considerable due to th e proxim ity o f th e in te rc e p ts to th e o rig in . No explanation i s evident fo r the apparent f a ilu r e o f th e hydronium ion to fu n ctio n as a c a ta ly s t. 200 150 125 MIN" 100 75 0.10 0.20 (CH3 - C O O H } 0.30 F igu re 4. R e l a t i o n o f pseudo c o n s ta n t a c e t i c a c i d c o n c e n t r a t i o n (CH^-COOH). 0.4 0 ( k f ) to 100 75 50 0.10 0.20 0.30 0.40 (HCOGH) F igu re 5. R e l a t i o n o f pseudo c o n s t a n t f o r m i c a c i d c o n c e n t r a t i o n (HGOOH)• ^k*) t o 75 K' X 10 M IN' * 100 75 50 A"/HA 0.025 0.0 50 0.075 0 .1 0 0 ((CH3 ) 3 C C00H) F ig u re b. R e l a t i o n o f p s e u d o c o n s t a n t ^kf ) t o t r i m e t h y l a c e t i c a c i d c o n c e n t r a t i o n ( (CH^)^C-COOH). System 30 0 o © 3 F orm ate A cetate T rim eth ylacetate 250 200 a x MOL1 MIN 1 50 00 50 5 2 3 4 A 7 HA F igu re 7 . R e l a t i o n o f ©c t o b u f f e r r a t i o (A /H A ). 61* TABLE XX CATALXTIC COHSTAHTS %A x 10* im Sri airT * kA- x 10® s»i~A adrT1 Formate 6 li* A cetate 6 SfymUm T rim eth ylacetate 19 18 Dibydroayacetone — Pyruvaldebyde R atios Since an aold and baas c a ta ly s is was estab lish ed fo r the gross disappearance o f g^ceraldebyd® , th e form ation o f dihydro ay acetone and pyrusaldefcgrde must be su b je c t to such c a ta ly s is . n o t perm it a choice between two p o ssib le pathways) However, th ese d ata do (a ) form ation o f dilydroasy acetone and pyruvald@ljyd® by two sep arate f i r s t o rd er processes ( 12) o r (b) by th e breakdown o f a cowmen interm ediate 0 ■& “> U 3) Rev ersa p ro cesses a re ignored sin ce only i n i t i a l v e lo c ity d a ta have been employed. In an e f f o r t to decide between th ese p o s s ib ilitie s , th e r e la tiv e r a te s o f form ation o f dltaydroayacetone and pyruvaldetyde were determ ined. 65 C oncentration* c f these products were c a lcu late d a* follow* • Value* I b r [0 ] from p erio d ate consumption d ata and fo r [G ♦ DJ from to ta l trio e e analy ses were p lo tte d ag a in st tin e and th e b ea t smooth curve* drawn through th ese p o in ts (Figure ©). C orrected values fo r [G] and [G ♦ D] were th en c a lc u la te d from th e se curv es. From these co rrected v a lu e s, [B] m d [F] were ca lcu late d a t v arious tim es. Assuming th a t during i n i t i a l Stage* o f th e re a c tio n a l l th e pyruvaldehyds comes frost glycer aldehyde, a p lo t o f [£>] v ersu s th e corresponding [P] should be lin e a r a t low con* c e n tra tio n s o f products* T his was observed experim entally (Figure 9) and th e slo p e s o f th e se cu rv es, designated a s (D ^ H , a re shown in column th re e o f Table V and column e ig h t o f T ables V I, VXX, and V III. Although th e v a ria tio n s in (D /P )i are la rg e In some c a se s , these r a tio s a re e s s e n tia lly co n stan t f o r a given acid-base systeei and are independent o f b u ffe r r a tio and co n cen tratio n . I f equation (12) re p rese n ts th e re a c tio n p ro cess, (D /P )i *> k&Aa ***& i t i s im plied th a t th e c a ta ly tic co n stan ts f o r the a c id s and bases involved in th e two re a c tio n s are always p ro p o rtio n al to each o th e r. T his seems highly u n lik e ly . The re a c tio n in d ic a te d in equation (13) , in which gXyoeraldehyde i s slow ly converted to an intexm ediate E which ra p id ly produces dihydroxy acetone and pyruvaltietyde seems more p la u s ib le . The value (D /P )l would then rep resen t the r a tio o f two f a s t p ro cesses e s s e n tia lly in s e n s itiv e to amounts and r a tio s o f c a ta ly s ts p re se n t. 0.20 0 c o n g 'n 0.150 0 .1 0 0 a 50 50 C 7 50 1000 1250 TIME ( MINUTES) F ig u r e 8T y p ic a l c o n c e n tr a t io n v e r s u s tim e c u r v e s ( E x p e r i m e n t 1 1 , T a b l e VI ) . 0 .0 6 0 0 .0 5 0 0 ,0 4 0 0.030 0.020 0 .0 1 0 0 .0 0 7 5 0.0025 0 .0 1 0 0 ( P) F ig u re 9T y p i c a l (D) v e r s u s (E x p erim en t 1 1 , Table VI ) . (P) c u r v e 66 Glyc&raldehyde —* ftiiydroajyacetona Equilibrium E ffo r ts to ev alu ate an s ^ i i l l b d m co n stan t f o r the re a c tio n between glyoeraldehydo and dihydroa^acetone were not e n tire ly euoceeeful. The eq u ilib riu m was w all in fav o r o f th e k eto se. Experiments in which glyceraldahyds wae th e s ta r tin g m a terial gave r e s u lts th a t were in v a lid due to th e anomalous p erio d a te uptake by the concentrated so lu tio n s o f pyruvaldehyde p re se n t a t o r near eq u ilib riu m . A lte rn a tiv e ly , pure dihydroaQraeetone (0*10 M) was allowed to re a c t f o r tw enty-four hours in a b u ffe r 0 ,30 M each in a c e tic acid and sodium a c e ta te a t *30° C, The u su a l an aly ses and c a lc u la tio n s were made, A p lo t o f [D]/[G] versus tim e was made and i s shown in Figure 10. T his curve approaches a value o f 1? which does a c t compare unfavorably w ith values o f about 20 reported f o r th e corresponding trios© phosphates (101,102). E ffe c t o f Calcium Ion Since d iv a le n t c a tio n s rep o rted ly produce anomalous reac tio n s in the in tereo n v crsio n o f th e hesosas (se e above) , Ca*+ was te s te d fo r i t s e ffe c t on th e re a c tio n s o f glyosraldeiiyds, Varying concentrations o f Ca** were added to re a c tio n system s 0,20 If each in a c e tle acid and ac e ta te io n . The r e s u lts a re in d ic a te d In Table X, In n e u tra l s o lu tio n , calcium p erch lo rate produced no re a c tio n , whereas in an a c e ta te b u ffe r th e presence o f Ca** produced an augmentation o f r a te p ro p o rtio n al to Ca** co ncen tration over the range in v e stig a te d . The (D /P )i r a tio was n o t a ffe c te d . The ro le o f Ca*+ as an ac c e le ra to r o f 40 35 30 25 20 300 6 00 900 1200 1500 1800 TIME (MINUTES) F igu re 10. E v a l u a t i o n o f DihiydroxyacetoneO lyeeraldehyde eq u ilib riu m c o n sta n t. 67 TABLE X EFFECT OF CALCIUM ICR Eapt. 16 39 10 Ik hi to [Go] 0 ,20 0.20 0.20 0.20 0.20 0.20 [CHjCOOH] [CH.OOD*] mm mm mm mm 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 [Cm**] 0.10* — — 0 .05®* 0.10** kf x 10® (D/]p)i Min"1 0 0 101 100 111 120 mm mm k.6 It. 5 h.6 U.5 AH eo n een tratlo n s In moles/ ! ; u m aintained a t 0 .6 by ad d itio n o f KaCID*. Calcium p erch lo ra te ®* Calcium a c e ta te th e re a c tio n probably involves a complex between glyceraldetyde and Ca** which forma ra p id ly and which la e n a b le o f a more rap id reac tio n w ith acid o r base than l a fre e glyeeraldefayde. The increased re a c tiv ity o f m etal io n complexes has been dem onstrated by S teinberger and Westhelmer (103) In th e decorboxylation o f dim ethyloxaloaoetic ac id and by K roll (lO idT in th e h y d ro ly sis o f amino acid e a te rs . Mechanism o f R eaction I t baa been found th a t th e o v e rfa ll disappearance of glyceraldehyde i s su b je c t to an acid and base c a ta ly s is . C onstant (D /P )i values fo r a given acid -base system stro n g ly suggest form ation o f a common in te r ­ m ediate capable o f y ie ld in g both d t^d ro sy aeeto n a and pyruvaldeliyde, The n atu re o f th e c a ta ly se s support the form ation o f tli© enediol common to 66 th e tr lo s s s a s th e ra ta determ ining step according to the follow ing schemest Acid c a ta ly s is — SI £i -OH H\ q /OH HC-QH I CHgPH HA CH^OH ♦A (1U> ♦ HA H 5) <• HA (16) ^ 0S XC I bc- ob ♦ A i -OH ■* I CHaOH CH^OH Base c a ta ly s is H.V v H v° / O I HC-GH C-OH I GHaOH I c a jm H. v° I C-OH I CB^DB II HA .... ♦ A" C-OH (17) I UHgOH I t i s a lso p o ssib le th a t these processes are not separate b u t occur by a concerted mechanism as suggested by. Strain (1 0 $ ). K - *■* C c - oh ................ CH^OH .OH (M ' a i'-OH * HA .ACHaOH (18) The v ilo e ( 0 /P ) i I s b elieved to bo a measure o f th e re la tiv e ra te s o r too rap id pro cesses by which th e enedlol i s converted to dlhydrosyacetone and pyruvaldahgrde re sp e c tiv e ly , th e d ata do not perm it an e v a lu atio n o f th e ro le o f a c id s and bases in these processes although I t i s suspected th a t th ese substances do p a r tic ip a te . The fa c t th a t a d if fe re n t (D /P )i value i s obtained in d iffe re n t acid-base systems im plies th e ir p a rtic ip a tio n in th e rap id s te p s . The constancy o f the (D /P )i value f o r a given system , independent o f b u ffe r r a tio and co n cen tratio n f may also insply p a rtic ip a tio n o f acid and/or base in th e rap id steps* The degradatio n o f glyoeraldehyde mgr be considered as occurring as follows* glyceraldahyda -tia s L * slow en sdiol Cr« irt dlhy&noxyacetone T SR fa st pyruvaldehyd© The tran sfo rm atio n o f e n sd ic l to y ie ld pyruvaldehyde may involve a p ro cess such asi c!-OH I Hf-OH H (19) * nA *■ i ~ * H ,0 (2 0 ) T his s o r t o f mechanism la supported by th e f a c t th a t pyruvildebyde la ap p aren tly rele ase d aa i t a e n o l, F reshly prepared pyruvaldehyde (by d i s t i l l a t i o n o f glyceraldebyd© in a u lfu rlc acid) d isp lay s a strong ab so rp tio n maximum a t approxim ately 250 mu In d ic a tiv e o f an en o llc group* in g . T his peak disap p ears on standing and th e curve given In Figure 1 la th a t o f an e q u ilib ra te d s o lu tio n . The apparent increased ra te o f enedlol form ation in the presence o f Ca** i s probably th e r e s u lt o f a glyceraldehyde-Ca*+ eoaplex such as I t l a conceivable th a t th e e le c tro s ta tic e ffe c t produced by an e le c tro p h ilio Ca** would in crease th e a c id ity o f th e hydrogen attached to the c e n tra l carbon atom and th u s render I t more e a sily attacked by a basic c a ta ly s t. 71 ssm m Xm The is tifo o n re rs lo n oT glyooraldehyde and dihydroxyacetonat as w ell a s th e sim ultaneous conversion to pyruvaldehyde hae been examined k in e tio a lly In b u ffered so lu tio n s o f fo rm ic, a c e tic , end trirao tb y la c e tic ecld e end th e ir re sp e c tiv e sodium s a lts , 2* The degradation o f glyceraldehyde hae been found f i r s t order in the tr lo s e , 3* C&yceraldshyde degradation has been shown su b ject to an acid and base c a ta ly sis* b , The r e la tiv e r a te s o f dihydroayaeetona and pyruvaldehyde form ation have been found independent o f b u ffe r r a tio and con centration fo r a given system ; th e r e la tiv e ra te s are d iffe re n t In d iffe re n t a c id base system s, 5 # The equ ilib riu m co n stan t f o r the re a c tio n between glyeeraldehyde and dibydrosyacetone has been ev alu ated , 6 , An in c re ase in th e ra te o f glyeeraldehyde degradation in the presence o f Ca+* in a c e ta te b u ffe rs has been observed; th e in crease was p ro p o rtio n a l to Ga** co n cen tratio n , 7 , The p o ssib le mechanisms o f the processes involved are discussed. 72 LITERATURE CITED (1 ) C. 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S o c., ] 2 , b57$ (195c). z KXHmcs or th e akxho a c id and p ep tid e catalzze d DEALDOLXZATXW Of DIACETONE ALCOHOL XX DKTEE&INATIDN OF THE HZD&OXXHETHZL GBOUF IN SUGARS AND BELATED StJBSTAHCES m KINETICS OF THE AGIO AMD BABE GATALXZED DEGRADATION OF THE TKXOSES By / A rlington Ardpane Forlart ,?• AH ABSTRACT Submitted to th e School o f Graduate S tu d ies o f M ichigan S ta te C ollege o f A g ricu ltu re and A pplied Science in p a r tia l fu lfillm e n t o f th e requirem ents fo r th e degree o f DOGTOB OF PHILOSOm Department o f Chemistzy Tear Approved 1952 A rlin g to n A. F o r i s t THESIS ABSTRACT The c a ta ly s is o f th e d e a ld o lisa tio n o f diacetone alcohol in b uffered so lu tio n s o f g ly c in e, D L-oc-alanine, (3-a la n in e , and glycylglycine and th e ir re sp e c tiv e sodium s a lts vas examined k in e tic a lly by a d ila to m e tric teciuilque a t 18,60° C, The amino ac id o r p ep tid e anion vas th e c a ta ly tic * a lly a c tiv e sp e c ie s, th e o rd er o f a c tiv ity being ft -ala n in e > glycine > DLc^-alan in e“g ly cy lg ly cin e, M etallic io n s did not ac c e le ra te th e re a c tio n . Ions capable o f forming amine complexes in h ib ite d th e re a c tio n . The d is - p ro p o rtlo n atio n o f a c y c lic in term ed iate involving th e amine c a ta ly s t and diacetone alco h o l vas p o stu lated a s th e ra te determ ining ste p In th e p ro c e ss, A nev method fo r determ ining the hydroxymetliyl group in sugars and re ­ la te d substances vas developed which c o n s ists o f a p erio d ate o x id atio n in n e u tra l so lu tio n buffered by b icarb o n ate, d e stru c tio n o f excess p erio d ate and io d a te by a d d itio n o f sodium s u lf ite , and subsequent spectrophotoro etric determ ination o f formaldehyde in th e re a c tio n m ixture a f te r re ­ a c tio n w ith elirom otropic a c id . G lycolic acid in te rfe re n c e vas elim inated w ithout reduction in s e n s itiv ity by using Ih M s u lfu ric acid fo r develop­ ment o f th e dye, A th ir ty m inute a e ra tio n was s u f fic ie n t to remove excess s u lfu r dioxide from th e dye so lu tio n . Both the form aldehyde-sulfit® solu­ tio n and th e form aldeiyde-chrom otropic ac id dye were sta b le fo r a t le a s t fo rty -e ig h t h o u rs. D -glucose, D -xylose, D L-glyceraldeliyde, dil^ydroxyacetone, D -m annitol, and m altose were su c cessfu lly determ ined by th is procedure. B L-serine gave c o n s iste n tly low y ie ld s o f measurable form aldelyde. M altose produced one mole o f formaldeiiyde per mole o f d isacch arid e suggesting use o f th is method in end group a n a ly sis o f p o ly sacch arid es. A rlin g to n A. F o r i s t DL-glyceraldehyde was found to pro dues dihydroxyaeatone and pyruvaldehyde in a c id ic b u ffe rs . These re a c tio n s o f DL-glyceraldehyde were in v e stig a te d k in e tic a lly in b u ffered so lu tio n s of form ic, a c e tic , and trira e th y la c e tic acid s and th e ir resp e c tiv e sodium s a lts a t $0° C. The re a c tio n components were determ ined by sim ultaneous measurement o f p erio d ate consumption and estim atio n o f to ta l trio s e s (th e l a t t e r by means o f th e method described above) . The degradation was o f th e f i r s t o rd er In th e tr io s e and was sub* je c t to an acid and base c a ta ly s is . The re la tiv e r a te s o f dihydroxyaoetone and pyruvaldehyde form ation were independent o f b u ffe r r a tio and concentra­ tio n fo r a given system but were d iffe re n t in d iffe re n t acid-base system s. The equilibrium co n stan t fo r th e conversion o f glyceraldehyde to dihydroxyacetone was approxim ately 17. Calcium io n in creased th e ra te o f g ly c erald e - hyde d eg rad atio n , in p ro p o rtio n to th e calcium io n co n c en tratio n . The form ation o f an enediol was suggested as th e r a te determ ining step in th e degradation o f DL-glyceraldehyde. Tills enediol was thought to re a c t by two se p a ra te , rap id processes to y ie ld dihydroxyacetone and pyruvaldehyde. Form ation o f a trio e e -c aleium io n complex more re a c tiv e than the fre e sugar was p o stu la te d . ~2