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Spectrophotometric study of the formaldehyde-leuco-methylene blue reaction
ASALYTICA
ClIIMICA ACTA
SI-‘~CTROI’I~OTOMI5’1’IC1C STUI1Y LI~UCO-Mlfl’~JI-II,l~N~
S. r\l’.J31-IhTTACI-IAIIYA
VOL.
10 (1954)
OF THE FORMALDEI-IYDEl3LU15 REACTION
AND
A.
GHOSE
Mcthylcnc blue is known to form a lcuco-base on reduction, but the stability of the latter against oxidation by air or oxygen appears to vary with the reducing agents used. Possibly the stability is caused by an association of the base with certain groups dcrivcd from the reducing agents themselves. That such is the case with the lcuco-base obtained by reduction of methylcne blue with HCl and Na,S,O, has been suggcstcd by MATIN AND POPESCO~, who are also the authors of the method of reduction itself. The possible stabilising group in this particular instance is - S.SO,H, which maintains the lcuco-base up to 12 hours even in the presence of atmospheric air. We have consiclercd it expedient to accept this suggestion as a working hypothesis. LANC? observed that formaldehyde turns the lcuco-base blue and also suggested the use of the latter as a reagent for detecting formaldehyde, but, as LANG also noted, the test is far from sensitive. This reaction is apparently the reverse of what one cxpccts, but it is to be pointed out that formaldehyde is incapable of turning the leuco-base blue in the absence of air or oxygen. It is thus obvious that formaldehyde only hastens the reaction with oxygen, which is normally held up, in this particular cast for about 12 hours, owing possibly to the presence of the stabilising group - SS0,I-I. The colour characteristics of this oxidiscd compound does not seem to have been systematically studied. The authors have used the spcctrophotomctric measurements for this purpose and have also observed the effect of scnsitising substances on this reaction. In this connection the work of TOUIIS on Schiff’s reagent, although not quite analogous, has been very suggestive. EXl'ERIhIENTAL
The leuco-base was prepared according to MATINS by the reduction of methylene blue (Merck, recrystallised) with Na,S,O, and HCl. For every 15 ml of 3.125211 methylene blue solution were added 3 ml of O.IN HCl and 0.1 ml of o.og7iV References
p.
566.
VOL.
10
(1954)
FOnh¶ALDEHYDE-LEUCO~S~T~I~LE~~
BLUE
REACTIOS
563
Na,S,O,. The mixture on being kept in the dark for z hours becomes colourlcss. The thiosulphate used in the above is just about the minimum necessary to cause decolorisation. All the chemicals used were of reagent quality. The blue colour developed on adding formaldchydc to the Ieuco-base in the presence of air or oxygen was spectrophotometrically analysccl over the entire wavelength (480 - 800 m,u) by means of the Beckman Quartz Spectrophotometcr. Only one absorption maxirinim is shown at GgG.5 rnp which is exactly the same as that of pure mcthylene blue solution*. The following factors determining tltc sensitivity of tbc blue colour developed in the above manner were studied.
The leuco-base, as already pointed out, is stabiliscd by the - S.SO,H group against oxidation. The content of thiosulphate is, therefore, likely to influence the development of the blue colour. In fact the results given in Table I will show that the maximum sensitivity is obtained with the solution of the lcuco-base containing- the minimum amount of Na2S,03 used for reduction. For the purpose of these experiments increasing amounts of Na,S$I, were addecl while preparing the lcuco-base. The maximum absorption and the approximate time required to attain it, under the circumstances, by the addition of formaldehyde are noted in each case. {ii) Effects
of sensitising
szibstanccs
Preliminary experiments have shown that substances like acctonc, ethyl alcohol, diethyl ketone, methyl ethyl ketone increase the sensitivity of the reaction in so far as the colour is intensified, although the wavclength of absorption maximum is not altered at all. Similar sensitisation by alcohols and ketones was observed by ‘~OBIE~ and HOWPAUIR et CXZ.~,in their work on Schiff’s reagent. References
p. 566.
l:ig,
I.
Curve Curve Curve
I 2
-
3 -
scnsitisecl by Mc,CO sensitised l,y EtOH without scnsitiscr
VOL.
10
(19 54)
FORMALDEHYDE-LEUCOhlETH\‘LENE
TABLE EFFECT _
OF
INCREASIl’ZG
______._.___
Acetone Max.
-_.____-_--
__
AMOUNTS OI’ ACI:TOSI: COFZTAIxIxG 0.45 _____ ._____.L__ _.-_-~--__-.-
0
1r1 ml opt. -
density
0.780
- _ -... __ ..- ._-_ ..-.. - -- --- --
BLUE
565
RISACTIObi
II I’ISII
mg
-)
OF
Xl11 01: J.ISUCO-MISTIIYLENE JICSIO -_----_. -.. .- .-.--- --_
0.025
0.050
0.075
0.1
0.SO.j
0.H2g
O.H_52
0.554
-.--_
.._
BLUE _- --. -0.125
0.s5.1
. -__---
DISCUSSION
The following points emerge from the above results. Formaldehyde, which is normally capable of reducing mcthylcnc blue to the leuco-basea, helps oxidation of the latter by air or oxygen. In the abscncc of oxygen formaldehyde is without any effect. Again, in abscncc of formnldchydc the lcucobase as prcparcd by reduction with Na,S,O, and HCl is stabiliscd by an - S. SO,H group for about 12 hdurs against oxidation by air or oxygen. Obviously, formaldehyde reacts with the stabilising group thus undoing the cffcct of the latter. That the lcuco-base dots not form any complex compound with formaldehyde, as it has been shown to be the cast with Schiff’s rcagcnt’, is clcmonstratcd by the fact that the coloured compouncl has always the absorption maximum at wavelength 656.5 mp, exactly corresponding to that of mcthylcnc blue itself. even in prcscncc of formnldchyde, is Moreover, stabilisation against oxidation, increased in presence of a large concentration of thiosulpllatc, which supplies the ncccssary stabilising group. The exact function of the sensitizers, like acetone and ethyl alcoh~, is not clear, but it is shown that they serve a two-fold purpose: they dccpcn the colour, Lo. absorption maximum and at the same time stabilisc the maximum; further, the colour development is rendered slowest with the most cffcctivc scnsitiser, as shown in Fig. 2. The kinetics appears to be dccidcd by the slower of at least two consecutive reactions, e.g. (i) that of the removal of the stabilising group from the leuco-base by formaldchydc and (ii) that of oxidation of the true lcuco-base dcprivcd of of tllc work is now being the stabilising group, by means of osygcn. This aq)cct studiccl. ACKNOWLEDGEMENT
The wnsitlvity of the reaction bctwecn fwmalclchyde ant1 lcuco-mcthylcnc b111c has been found to be increasccl in the prcscncc of ethyl alcol~ol, acctonc, tllcthyl Acetone has been fount1 tu Ix tnmt cffcctlvc. I.&one ant1 rncthyl ethyl kctonc. ir’c~~~~cmes p.
566.
566
S.
N.
BHATTACHARYA,
A.
GHOSE
VOL.
10
(1954)
Sensitiscrs increase the ccbsorption maximum and stabilise it, while the rate of development of mcthylene blue colour is slowed down. Sensitivity also depends upon the content of thiosulphstc used to prepare the a smaller amount of thiosulphatc increases greater sensitivity. lcuco-base;
La scnsibilit6 dc la rtiaction entre la formald&hydc ct lc d&rivd leuco du bleu de mdthylc?nc augmentc cn prdscncc d’alcool Bthyliquc, d’acbtone, de dMth l&tone *y: et de mdtflylc’:thyIcdtc)ne. L’acdtone semble la plus active. Ccs sensibl,xsateurs augmcntent l’absorption maximum et stabilise la reaction, tandis que la vltcsse de formation du l)lcu de mJthyl0ne diminue. La sensibilitc! ddpend cgalement de Ia utiliscSc pour pr&parer la lcuco-base. quantitd dc thiosulfatc, ZUSAMMENFASSUNG Es wurdc gcfundcn, dass die Empfindlichlccit.der Rcalction zwischcn Formaldehyd und Lcukomcthylenblau in Gegenwart von Athylalkohol, Accton, J>i%thyi$eton und ~ctl~yl~tJ~y~l~eto~~ zunimmt. Am wirkungsvollsten crwies sich Accton. Scnslbilisatoren crhijhen das hbsor tionsmaximum und stabilislcrcn es und setzen die hcrab. Die EmpfindGcschwindigkcit dcr Entwic E lung dcr Mcthylcnblauf:irbung lichkeit hiingt ebenfalls von der Mengc des bci tier lierstellung dcr Leukobase bcniitztcn Thiosulfats ab. Eine geringcrc Mengc ‘Thiosulfat crhiiht die Empfindlichkeit.
ClIIMICA ACTA
SI-‘~CTROI’I~OTOMI5’1’IC1C STUI1Y LI~UCO-Mlfl’~JI-II,l~N~
S. r\l’.J31-IhTTACI-IAIIYA
VOL.
10 (1954)
OF THE FORMALDEI-IYDEl3LU15 REACTION
AND
A.
GHOSE
Mcthylcnc blue is known to form a lcuco-base on reduction, but the stability of the latter against oxidation by air or oxygen appears to vary with the reducing agents used. Possibly the stability is caused by an association of the base with certain groups dcrivcd from the reducing agents themselves. That such is the case with the lcuco-base obtained by reduction of methylcne blue with HCl and Na,S,O, has been suggcstcd by MATIN AND POPESCO~, who are also the authors of the method of reduction itself. The possible stabilising group in this particular instance is - S.SO,H, which maintains the lcuco-base up to 12 hours even in the presence of atmospheric air. We have consiclercd it expedient to accept this suggestion as a working hypothesis. LANC? observed that formaldehyde turns the lcuco-base blue and also suggested the use of the latter as a reagent for detecting formaldehyde, but, as LANG also noted, the test is far from sensitive. This reaction is apparently the reverse of what one cxpccts, but it is to be pointed out that formaldehyde is incapable of turning the leuco-base blue in the absence of air or oxygen. It is thus obvious that formaldehyde only hastens the reaction with oxygen, which is normally held up, in this particular cast for about 12 hours, owing possibly to the presence of the stabilising group - SS0,I-I. The colour characteristics of this oxidiscd compound does not seem to have been systematically studied. The authors have used the spcctrophotomctric measurements for this purpose and have also observed the effect of scnsitising substances on this reaction. In this connection the work of TOUIIS on Schiff’s reagent, although not quite analogous, has been very suggestive. EXl'ERIhIENTAL
The leuco-base was prepared according to MATINS by the reduction of methylene blue (Merck, recrystallised) with Na,S,O, and HCl. For every 15 ml of 3.125211 methylene blue solution were added 3 ml of O.IN HCl and 0.1 ml of o.og7iV References
p.
566.
VOL.
10
(1954)
FOnh¶ALDEHYDE-LEUCO~S~T~I~LE~~
BLUE
REACTIOS
563
Na,S,O,. The mixture on being kept in the dark for z hours becomes colourlcss. The thiosulphate used in the above is just about the minimum necessary to cause decolorisation. All the chemicals used were of reagent quality. The blue colour developed on adding formaldchydc to the Ieuco-base in the presence of air or oxygen was spectrophotometrically analysccl over the entire wavelength (480 - 800 m,u) by means of the Beckman Quartz Spectrophotometcr. Only one absorption maxirinim is shown at GgG.5 rnp which is exactly the same as that of pure mcthylene blue solution*. The following factors determining tltc sensitivity of tbc blue colour developed in the above manner were studied.
The leuco-base, as already pointed out, is stabiliscd by the - S.SO,H group against oxidation. The content of thiosulphate is, therefore, likely to influence the development of the blue colour. In fact the results given in Table I will show that the maximum sensitivity is obtained with the solution of the lcuco-base containing- the minimum amount of Na2S,03 used for reduction. For the purpose of these experiments increasing amounts of Na,S$I, were addecl while preparing the lcuco-base. The maximum absorption and the approximate time required to attain it, under the circumstances, by the addition of formaldehyde are noted in each case. {ii) Effects
of sensitising
szibstanccs
Preliminary experiments have shown that substances like acctonc, ethyl alcohol, diethyl ketone, methyl ethyl ketone increase the sensitivity of the reaction in so far as the colour is intensified, although the wavclength of absorption maximum is not altered at all. Similar sensitisation by alcohols and ketones was observed by ‘~OBIE~ and HOWPAUIR et CXZ.~,in their work on Schiff’s reagent. References
p. 566.
l:ig,
I.
Curve Curve Curve
I 2
-
3 -
scnsitisecl by Mc,CO sensitised l,y EtOH without scnsitiscr
VOL.
10
(19 54)
FORMALDEHYDE-LEUCOhlETH\‘LENE
TABLE EFFECT _
OF
INCREASIl’ZG
______._.___
Acetone Max.
-_.____-_--
__
AMOUNTS OI’ ACI:TOSI: COFZTAIxIxG 0.45 _____ ._____.L__ _.-_-~--__-.-
0
1r1 ml opt. -
density
0.780
- _ -... __ ..- ._-_ ..-.. - -- --- --
BLUE
565
RISACTIObi
II I’ISII
mg
-)
OF
Xl11 01: J.ISUCO-MISTIIYLENE JICSIO -_----_. -.. .- .-.--- --_
0.025
0.050
0.075
0.1
0.SO.j
0.H2g
O.H_52
0.554
-.--_
.._
BLUE _- --. -0.125
0.s5.1
. -__---
DISCUSSION
The following points emerge from the above results. Formaldehyde, which is normally capable of reducing mcthylcnc blue to the leuco-basea, helps oxidation of the latter by air or oxygen. In the abscncc of oxygen formaldehyde is without any effect. Again, in abscncc of formnldchydc the lcucobase as prcparcd by reduction with Na,S,O, and HCl is stabiliscd by an - S. SO,H group for about 12 hdurs against oxidation by air or oxygen. Obviously, formaldehyde reacts with the stabilising group thus undoing the cffcct of the latter. That the lcuco-base dots not form any complex compound with formaldehyde, as it has been shown to be the cast with Schiff’s rcagcnt’, is clcmonstratcd by the fact that the coloured compouncl has always the absorption maximum at wavelength 656.5 mp, exactly corresponding to that of mcthylcnc blue itself. even in prcscncc of formnldchyde, is Moreover, stabilisation against oxidation, increased in presence of a large concentration of thiosulpllatc, which supplies the ncccssary stabilising group. The exact function of the sensitizers, like acetone and ethyl alcoh~, is not clear, but it is shown that they serve a two-fold purpose: they dccpcn the colour, Lo. absorption maximum and at the same time stabilisc the maximum; further, the colour development is rendered slowest with the most cffcctivc scnsitiser, as shown in Fig. 2. The kinetics appears to be dccidcd by the slower of at least two consecutive reactions, e.g. (i) that of the removal of the stabilising group from the leuco-base by formaldchydc and (ii) that of oxidation of the true lcuco-base dcprivcd of of tllc work is now being the stabilising group, by means of osygcn. This aq)cct studiccl. ACKNOWLEDGEMENT
The wnsitlvity of the reaction bctwecn fwmalclchyde ant1 lcuco-mcthylcnc b111c has been found to be increasccl in the prcscncc of ethyl alcol~ol, acctonc, tllcthyl Acetone has been fount1 tu Ix tnmt cffcctlvc. I.&one ant1 rncthyl ethyl kctonc. ir’c~~~~cmes p.
566.
566
S.
N.
BHATTACHARYA,
A.
GHOSE
VOL.
10
(1954)
Sensitiscrs increase the ccbsorption maximum and stabilise it, while the rate of development of mcthylene blue colour is slowed down. Sensitivity also depends upon the content of thiosulphstc used to prepare the a smaller amount of thiosulphatc increases greater sensitivity. lcuco-base;
La scnsibilit6 dc la rtiaction entre la formald&hydc ct lc d&rivd leuco du bleu de mdthylc?nc augmentc cn prdscncc d’alcool Bthyliquc, d’acbtone, de dMth l&tone *y: et de mdtflylc’:thyIcdtc)ne. L’acdtone semble la plus active. Ccs sensibl,xsateurs augmcntent l’absorption maximum et stabilise la reaction, tandis que la vltcsse de formation du l)lcu de mJthyl0ne diminue. La sensibilitc! ddpend cgalement de Ia utiliscSc pour pr&parer la lcuco-base. quantitd dc thiosulfatc, ZUSAMMENFASSUNG Es wurdc gcfundcn, dass die Empfindlichlccit.der Rcalction zwischcn Formaldehyd und Lcukomcthylenblau in Gegenwart von Athylalkohol, Accton, J>i%thyi$eton und ~ctl~yl~tJ~y~l~eto~~ zunimmt. Am wirkungsvollsten crwies sich Accton. Scnslbilisatoren crhijhen das hbsor tionsmaximum und stabilislcrcn es und setzen die hcrab. Die EmpfindGcschwindigkcit dcr Entwic E lung dcr Mcthylcnblauf:irbung lichkeit hiingt ebenfalls von der Mengc des bci tier lierstellung dcr Leukobase bcniitztcn Thiosulfats ab. Eine geringcrc Mengc ‘Thiosulfat crhiiht die Empfindlichkeit.