ohltooe wltli reasonable aecuraoy, then to moko a study of Ito stability under varying conditions and finally to eetabl conditions under which g].ucooamlnc hydrochloride could be deaminated almost completely to yield eliltoue free ae far 88 poosihle from decomposition or reaction by-prodncte.
(4
Bfstirnstlcm of chit ose, gimmmtmm
mwMjmmw iiinmj imc .
4#
The method of R a w e (Bioohom. J« * 99) as used in. the determination of rediicl^ig augers* was foimd to be Inapplicable in this ease for the extent of ferrloyanlde réduction was not proportional to the volume of ohltooe solution added (Graph 3),
From the result of examination of the rate of oxidation of eh 1 tome by Hanos* solution at 70^0. (Graph 4) It was evident that aftox* the Initial rapid oxidation, probably
representing for the most part the transformation of ohltose to ohltonle aeld, there continued a slower secondary oxidat ion which proceeded at a rate approximately proportional to the emoimt of chitose solution added# This secondary
oxidation might represont a further degradation of oh 1 tonic acid or an oxidation of chitose decomposition products
apparently present In considerable quantity in chltosc aolution.
An attempted modification of Hanes' method* by buffering the solution with sodium acetate In place of sodium cai^bonat# proved imsucccssful. It was hoped that by thus lowering the pH of the reaction medium the secondary oxidations might be eliminated but, as is seen from Graph g* the chit ose
oxidation rate was unduly retarded#
<U9
phenylhydroaoae obtained from obitoee eolutlono in previous experiments gave in owob O0:c)e o rougi* Indiontion of the
obltoee content* and attempts were made to adept this reset- ion to estimate the ehitoee eonteined in e emell volume of Bolution# %%en tbo oondenoetion vme carried out by
dlBeolvlng ehitoee in an aleoholie solution of 8)4 dinitro- phenylhydrasine end evaporating the solution to ameli bulk*
the product \mo obtained eo e red oil from wbiob the hydrexo gradually oryeteliieed* Difficulty le experienced in
separating the oryotalo from snob on uncretellieable oily residue ae remained in this ease without loss of material# However it was found that the hydroaone separated in the form of clean yellow crystals when an aqueous obltoae
solution was ohaken for some time with a saturated solutlozi of the hydras ine in cold nltrobense%%e. Since * at room
température* 2:4dinitrophenylhydrasine is fairly eolublo in nitrobenzene and almost oomple tely Inaolnble in water while ohltoae 2 %4di)iitrophenylhydrmsone is very sparingly soluble in both these solvents, filtration of the product after completion of the condensation should give an almost
quantitative yield of the chitose hydrasone, The value of the obitoss content of a solution thus estimated agreed remarkably well with that obtained by a second method carried out by long shaking of the cold aqueous solution with a weighed excess of finoly powdered 2;4dinltrophenyl- hydrazine and by calculating the ohitose content from the
(5(
Inoreese in weight of the f llto re d end dried prodnot*
e e n e n tie lly a m ixture of o iy n te llin o cliitoeo
8
;2
*. cLinitro- phonyIbydrezone end nnohenged &)W initrophenylhydrezine# Thin sGoond method obviated the neoeeelty fox* w in g re la tiv e larg e volumoe of nitrobenzene to keep the exoeee of unoheng( hydrazine in eo latio n . I t wee cheeked by weohing out tho exoGoe2
iW initrophenylhydrezine w ith nitrobew eno* leaving a reanonebly pure eemplo o f ohitoue2
;W in i troplienylhydrazoi: the dry weig%3
t of which oorreeponded oloaely w ith the value GGtimated from tho weight o f the mixed product,The ra te of eondeneotion in th is method wee dotermined by shaking each of a numboi' of email meaaurod volumes (
0,5
rrof a ohitoae solution w ith an exeeaa of the powdered reagent fo r d iffe re n t lengths of time* and measuring the inoreaee in weight of the dried s o lid product in each ooee (Graph G),
Reasonably eloae duplicate values (w ith in
20
) were obtained from those m ixturee which had been ahaken fo r a e u ffio lo n t length of tim e. The main disadvantage arose from the fa c t th a t, owing to the in s o lu b ility of2
:4
d ln itro -phenylhydraaine in w ater, the mixture required ehaking fo r at le aa t
2*.0
hours before the reaction was completed.In the hope of obtaining a a im ila r but more rapid method of estim ation a eearoli fo r fu rth e r o ry e ta llin e
hydrazoneo of ohitoae waa conducted, The condensation of ohitoae in oold aqueoua solution w ith phonyIhydrasine, p-tolylbydrazine* p"*bromopheny Ihy dr an ino * mothylphenyl-
(51)
hydrazine and phenyIhydrazine-p-aulphonlo acid resulted in the isolation of uncryatalXisahle oily products only.
Chi lose henzylphenylhydrazone separated as a fine white crystalline mass when an aqueous solution of ohitose was shaken at room temperature with henzyIphenyIhydrazine
dissolved in benzene# The compound* purified by recr^ystalll ing from aqueous alcohol* melted sharply at 83^0.* and had a constant rotation in absolut alocohol of + 50,25^#
Zlcroanalysis of this compound has apparently presented some difficulty for in four estimations the following range of results were obtained,
0. H. H
63.180 -64.150 6.340-7.330 7.24-8.1 Calculated for an anhydro-
hexose derivative, 66,670 6,430 8,190
A crystalline chitose hydrazone was also prepared with p-nitrophenylhydrazlne and was purified by recrystallising from absolute alcohol. The compound melted sharply at 185^0, It had a constant rotation in absolute alcohol of + 7.09°.
(52
G H H
Values found by micro- 48# 350# g.OOgL 14.15 onalyslB#
Theoretical for en onhydro- ,
hex000 derivative. 4o*48/</ j, 05/o Î4#14
Theoretical for a normal
hexooe derivative, 45.710 5*400 13*33
Attempts at eatlmatloa of the ehltooe content of a
Golution by ohaklng it ?;lth an exoeee of hmizylphenylhydrazi dlBsolved In benzene* filtering* drying and weighing the eryatalline produet* Indloatod that the reaotion vm8 much more rapid (complete after 5 hours shaking) than when
2 % 4dini trophenylhyd raz ine wee employed# Difficulty wae
exporlenced hew ever in filtering off and cleaning the finely matted cryetelline product and a difforence in value aa grea ae 100 WGo Obtained for duplicate eetimationck.
% uoing pure finely ground p-nitrophenylhydrazlue in place of 2:4dinitrophenylhydrazine the condensation proceedc# comparatively rapidly, the separation of the product being complete after 8-3 hours (Graph 7). Oloec duplicate valueo (vithin 20) were obtained using ae little ac 1 ml# of chitoae
solution (containing 0#1 g# of doomlnated glucosamine hydrochloride * i.e.* a theoretical chitoae oonteat of
0.075 g. ) though the values were 2-30 lower than those o^)taiâ by uaing 2;4dinitrophenylhydrazine duo to the greater solubl!