Capitulo VII Desarrollo
7.2 Análisis de la estructura del control de la organización
DBF hydrasones have been classified on t A basis of tAir sAorption bonds in t A 9#9-10#4^ and 6#05«^#1
jm
r a n g e s T A presenoe of a band in t A 9*9*40#4^
range immediately oharaoterises trans isomers of unsaturated DIP hydrasones while an additional bandin t A 6#05-6#1
jm
range distinguisAs trens allc-2-enal DSP hydrasones# Slight diffbrences have also been observed in t A spectra of alkanals, alkanones, ois-alAnals and aUoenones#T A mass speotra of DIP hydrasones show very intense parent ions, as well as oharaeteristio fragment ions typical of t A AAffbrty
rearrangement t ^ #+ B E IE IE Ar
\ / .
OH ^I
CE I ^ I - SI# Ar — > R.CH • CHj, ♦ // OBg V * 224 At - 2,4 (»2>a ®6®3“— 26 —
ao that m P hydrazonea of alkanala C4) Aagnant to give strong ions at */e 224 while t A BMP hydrazones of methyl ketones (R GO Me) and ethyl ketones (H CO St) show ions at ® A 238 and 252 respectively* SHP hydrasones of alk-2-enals undergo & 6 fission to produce
characteristic ions at "/e 235 and ®/e 231
In addition, the relative abundances of the ions differ for cis and trans alk-2-enal W P hydrazones which in turn are different from those of alkenal DflP hydrazones %dth the double band not in the 1- position*
Chromatographic systems have been descriAd which, in tAory, should allow the isolation of individual DNP hydrazones* lAse usually involve (i) adsorption chromatography on zinc carAnaA or magnesium oxide to separaA according to class (i.e* saturaAd A Anes, saturaAd aldehydes, unsaturaAd ketones and unsaturaAd aldehydes), (ii) partition chromaAgrai^ which sépara A s the SNP hydrazones according A tAir chain length, and (iii) silver ion chromaAgraiday A separaA t A compounds according to A e numAr of double bonds and tAir configuration* In A e first separation t A several classes of Arivatives give different colours under alkaline conditions, viz*
Rf value
Methyl Atones grey ;
Alkanals brown ^
Acreases
Alkenals brick red j
Alkadienals violet
UnfortunaAly, this system cannot A guaranteed A compleAly separaA t A complex mixtures of unsaturaAd and saturaAd CMP hydrazones tAt can A isolaAd from natural sources. For example, alAnals %riA t A double A n d not A conjunction w i A t A carbonyl grovgp run %riA t A saturaAd CMP hydrazones during class separation^ while
long chain molecule# often run InA shorAr CMP hydraxones of another da#a. This latAr limiAtion was important A this specific flavour problem Aeause the large amounts of formaldehyde present in cold sAred ood swamped the unsaturaAd short chain alkanals. Moreover, it appeared that the CMP hydrazones were not as stable as antieipaAd since the
frequent refractionation necessary during the separation of the complex CMP hydrasone mixture of A n left a deposit at the origin of the TLC piste. It was also extremely difficult to coaqa.eAly separaA the mixtures, entrainaent Aing a major problem. In addition A tAse fractionation difficulties, it waus found Aat regeneration of t A original carbonyl is not quanti Ative.
TAse drawbacks are not so critical if «me has sufficiently large quantities of t A carbonyls in t A foodstuff unAr analysis^ but ^ as this investigation proceeAd, It A came obvious tAt this was not the situation in this case.
4.2 m hr*nam»a of th* mnnocTtanyl» at eold ■tor.d cod
T A CMP hydrasones prepared during t A oharacArlsation of t A flavour volatiles from 3 year frozen ood were separaAd by ILC on zinc carAnaA plaAs and t A p a t A m compared w i A tAse obtained w i A
hydrazones from fresh ood and from cod sAred for one year (Fig 1). ] i Visual examination of tAse plaAs showed tAt t A composition of t A i
CMP hydrazones was not t A same and in particular t A concentration of 1 t A asArisked fraction increased w i A increasing time of oold sArage.
T A position of this band on t A chroaunAgrem compared w i A standard CMP hydrazones indicaAd tAt t A Arivative was tAt of a low molecular weight carAnyl such as formaldehyde or aoetaldehyde. It was already known Aat tAse aldehydes were present in t A fish, tAt tAy increased w l A sArage time but tAt tAy did not confer t A typical eold sArage
m 26 —
flavour teonietaristiom* The CMP hydraaooes %#er# ragenermted waiag oC -4-a toglutArio add aod th# zvodtiag Aad o^boo vapours prssantsd to tA panel for aaeesmaaat* Disappointingly this gave 00 conelusiw
lAritiJiads odd storage odoui *^otoa# Thia negative result could arise from a relatively ioefiidaat method of isolation of As volatilu^. : xwii uatic-ii ai tlie pro tain residue from t A orlglsal distillation aWwed that aose odour was still present and reinforced tA opinion tAt tm was relatively iaefficient as previous
4: i.
work el& jyiiore -ndic&tnd*
Fresh
■ ' lyr
3yr ^
f Ivi. 1
Zinc carbonate aepfu atio;. vf LL* carbonyl ilh hydrasmm# prepared from the vacuum distilla tea uT cooiced fresh, 1 year and 3 year oold stored cod. ^Ivcz t : i yridiiva
— 29 —
A modified Bligh and Byer extraction technique was therefore applied and the BNP hydrasones were again prepared* It was Aped tAt this procedure would reduce A t h the loss of caurbonyls and their production by oxidation in t A wozk up procedures* This product was very much more complex than tAt obtained previously and contained a large amount of lipid, some of which appeared to have reacted with the BNPH reagent* These unwanted components were removed by column
chromatography on Dowex-1 (1 x 4#100) in the basic form and the mono- oarAnyl BNP hydrasones finally taken up in hexane*
%ese BNP hydrasones were chromatographed on preparative plates of sino carbonate and a number of bands were obtained, one of which on regeneration produced an odour of cold stored ood* Chain length fractionation of the components of this band was carried out by preparative TLG on polyethylene glycol 400, the BNP hydrasones from each section were extracted, the carbonyls regenerated and their odours evaluated (Fig 2)* Oily Oily. Petrol
D
Total Hydrazones. Blank.Rancid / Painty / Coldstore. Sharp I Fruity.
Oily /Sharp. Oily.
Sharp.
Paint / Rancid /Coldstore. Nothing.
FIG 2* Separation of the monocarAnyl BNP hydrazones prepared from cold stored cod (1-)- years at -lyc) on PEG 400 using the modified Bligh
— 30 “
One or more mteriels in bend D seened to
tm reepooeible fbr t A
oold storage odour# T A band waa IbrtAr fAotionated on ellver nitrate plates and an odour reaedbling oold storage odour waa regeneratedfrom
a eeotiw of low toere no band was viaibA#Coqpariaon with t A separation of standards on these thin layer ayatens suggested that t A oonponent(s) reaponaible fbr t A oold storage odour might A an unsaturated 0^-0^ oazbonyl ooqpound but mass spectral analysA of t A D W hydrasones
tnm
this seotion showed only t A presenoe of acetone and pentanal DIP hydrasones#A i t A r acetone nor pentanal (Table 3) possess t A oharaoteristA