M E T H O D S F O R T H E Q U A N T I T A T I V E D E T E R M I N A T I O N

O F F O R M A L D E H Y D E A N D T H E I R U S E

I N T H E A N A L Y S I S O F N A T U R A L C O M P O U N D S

S. V. I s a i a n d V. E . V a s ' k o v s k i i UDC 661.727.1+047.1+543.420.62+547.281

In recen t y e a r s , attention to the development and per fec t ion of analyt ical methods for the c h e m i s t r y of na tura l compounds has g rea t ly increased . The methods used in this f ield can be divided into two groups: those used for the analysis of subs tances of s e v e r a l c l a s s e s (to these must be ass igned, in the f i r s t p lace , methods of de termining he te roa toms) and na r rowly spec ia l ized methods pe rmi t t ing the analys is of a single c lass or even a single compound.

The f i r s t group undoubtedly includes methods for the quanti tat ive de te rmina t ion of formaldehyde , which a re widely used in s t ruc tu ra l invest igat ions of subs tances of var ious c l a s s e s , e spec ia l ly ca rbohy- d ra t e s , and in the quantitat ive analys is of ca rbohydra t e s , l ipids, and some other groups of natural c o m - pounds. Voluminous l i t e r a tu re ex is t s on methods of de te rmin ing formaldehyde [1-4], but mos t of the r e - views a re somewhat old, and in the overwhelming number of cu r r en t publicat ions the use of one of the known methods of analys is is desc r ibed for concre te purposes .

Although the p re sen t rev iew is given p r i m a r i l y for the at tention of spec ia l i s t s in the field of the chem- i s t r y of na tura l compounds and b iochemis t ry , it b r ie f ly desc r ibes the whole analyt ical c h e m i s t r y of f o r m a l - dehyde. However, in this emphas i s is p laced on such sect ions as c o l o r i m e t r i c methods of analys is and the de te rmina t ion of formaldehyde in the p r e s e n c e of pe r ioda te , i .e. , those which a re mos t impor tan t in working with na tura l compounds. The rev iew covers the l i t e r a t u r e up to and including 1971.

Exist ing methods of de termining formaldehyde can be roughly divided into three groups: a) g r a v i - m e t r i c , b) vo lumet r i c , and c) phys icochemica l .

The g r a v i m e t r i c methods [1, 2] a re based on the fo rmat ion of insoluble organic de r iva t ives which can be col lected on a f i l t e r and weighed The organic reagents mos t widely used in the g r a v i m e t r y of f o r m a l d e - hyde include 2 ,4-din i t rophenylhydrazine and dimethylcyclohexanedione (dimedone, methone [2]);the second reagent is used more frequently.

The g r a v i m e t r i c method is e x t r e m e l y lengthy: it r equ i r e s not l e s s than 12 h for comple te p r ec ip i t a - tion. Nei ther of the prec ip i tan ts mentioned above give quanti tat ive r e su l t s at low concentra t ions of f o r m a l - dehyde. At the p r e s en t t ime , g r a v i m e t r i c methods a re used compara t i ve ly r a r e ly .

Volumetr ic methods of de termining formaldehyde a re widely used, thanks to the i r s impl ic i ty and ac - curacy , pa r t i cu l a r l y in industr ia l l abo ra to r i e s [6-9]. Of the many methods of vo lumet t i c ana lys i s , we may mention those which (in the opinion of the major i ty of authors) a re s imple , r e l i ab le , and economica l in r e - la t ion to t ime, appara tus , e tc . : 1) the sulfi te method; 2) the method using a luminum chloride; 3) the eyano- hydrin method; 4) the methone (dimedone) method; 5) the a l k a l i - p e r o x i d e method; 6) the iodometr ic method; and 7) the m e r c u r y method.

Methods 1-4 are based on those reac t ions c h a r a c t e r i s t i c for aldehydes and ketones which a re mos t sens i t ive in re la t ion to formaldehyde (thanks to its high reac t iv i ty) and a re l e a s t sens i t ive for ketones and higher aldehydes. Such compounds as methanol , ethanol, and acetone do not in te r fe re with the d e t e r m i n a - tion but in r e t u rn the r e su l t of the ana lys i s is affected by the p r e s e n c e of even smal l amounts of ace ta lde -

Pac i f ic Ocean Insti tute of Bioorganic Chemis t ry , F a r - E a s t e r n Scientific Center , Academy of Sciences of the USSR. Trans l a t ed f r o m Khimiya Pr i rodnykh Soedinenii, No. 4, pp. 465-480, Ju ly-August , 1973. Original a r t i c le submit ted August 20, 1972.

© 19 75 Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part o f this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission of the publisher. A copy o f this article is available from the publisher for $15.00.

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hyde and fo rmic and acet ic acids. Some authors [10, 11] single out the cyanohydrin method as the mos t r e - l iable in the analys is of formaldehyde in the p r e s e n c e of acetaldehyde, benzaldehyde, and other higher a lde- hydes and ketones. Some modif icat ions of this method a re mainly d i rec ted to increas ing its spec i f ic i ty [12, 131.

Methods 5-7 a re based on oxidizing p r o c e s s e s . In the opinion of the ma jo r i t y of w o r k e r s , ne i the r methanol , ethanol, nor acetone in te r fe res with the de terminat ion , while acetaldehyde gives high resu l t s . The iodometr ic method is sui table for the de te rmina t ion of smal l amounts of formaldehyde only in solutions p rac t i ca l l y f r ee f r o m any organic impur i t i es capable of being oxidized.

Of the phys icochemica l methods used fo r the de te rmina t ion of formaldehyde we may mention in the f i r s t p lace the po la rograph ic and co lo r ime t r i c methods. The po la rographic method was p roposed as ea r l y as 1935 [14]. It p e r m i t s the de terminat ion of formaldehyde in low concentra t ions with a high accuracy . Ace- taldehyde and higher aldehydes of the al iphatic s e r i e s do not in te r fe re with the determinat ion, s ince they a re reduced at a higher potential than formaldehyde; methanol and ethanol, and acetone and benzaldehyde, do not in te r fe re , e i ther . Several modif icat ions of this method a re known [15-17]. Very impor tan t is the maintenance of an accura te t e m p e r a t u r e in the record ing of the p o l a r o g r a m : a r i se in the t e m p e r a t u r e by I°C changes the height of the half -wave by 6.5%. A defect , pa r t i cu l a r l y for routine ana lyses , is the n e c e s - s i ty for p r e l i m i n a r y dist i l lat ion in o r d e r to f ree the formaldehyde f r o m subs tances in te r fer ing with the anal - ys i s . At the p r e s e n t t ime , po la rographic methods a re used fa i r ly r a r e l y [18-21].

Co lo r ime t r i c methods a re the mos t widely used for the de te rmina t ion of formaldehyde. We shall de- vote specia l at tention to them.

Schiff 's tes t o r Denig~s ' method - the f i r s t method for the c o l o r i m e t r i c de te rmina t ion of smal l amounts of formaldehyde [2, 22] - is based on the fact that in an acid med ium a fuchs ine -b i su l f i t e r eagen t gives a c h a r a c t e r i s t i c colora t ion with formaldehyde. This tes t is specif ic in the p r e s e n c e of both ace ta lde- hyde and o ther al iphatic a ldehydes , but it does not p e r m i t formaldehyde to be dist inguished f r o m acro le in and glycolic acid. The se lec t iv i ty of the reagent can be r a i s ed by changing the pH of the solution. If the so - lution is s t rongly acid, the reagen t is more sens i t ive to formaldehyde , and if it is alkaline it is more s e n s i - tive to higher aldehydes. The sens i t iv i ty of the method is subs tant ia l ly affected by the quality of the fuch- sine used, and a lso by the method of p r epa r ing the reagent . Some authors [23] r ecommended the use of a s m a l l e r amount of the Schtff r eagen t than is r equ i red by a mo lecu la r ra t io to the aldehyde. P ropor t iona l i ty between the density of the colora t ion and the concentra t ion of the aldehyde is obse rved only in a re la t ive ly n a r r o w range. The method p e r m i t s the de te rmina t ion of 0.05 mg of formaldehyde.

The va r i an t s of the method re l a t e mainly to the modif icat ion of the Schiff r eagen t [24, 25]. Veks le r [26] has p roposed a spec t ropho tome t r i e va r i an t of this method. He has shown that the t e m p e r a t u r e of the reac t ion is a fundamental fac tor .

Schiff 's method, in spite of its long h i s to ry and wide use, is one of the mos t complex methods. I ts p e r f o r m a n c e r equ i r e s the daily p r e p a r a t i o n of s tandard solutions and analys is in s t rong sul fur ic acid; the p e r f o r m a n c e of one analys is r equ i r e s a min imum of 6 h.

The Chromot rop ic Acid Method. In 1937, Eegr iwe [27] p roposed the use of 1 ,8-dihydroxynaphthalene- 3 ,6-disulfonic acid (chromotropic acid) for the quanti tat ive de te rmina t ion of formaldehyde. Heating a so lu- tion of formaldehyde with chromot rop ic acid in 72~ sulfur ic acid at 60°C for 10 min gives a purple co lo ra - tion. The b e a r e r of the colora t ion is the product of the condensat ion of formaldehyde with two molecules of ch romot rop ic acid.

;OH OH OH OH OH OH

According to Eegr iwe , this reagent does not f o r m a colorat ion with many al iphat ic and a roma t i c a lde- hydes (acetaldehyde, propionaldehyde, [sobutyraldehyde, butyra ldehyde, benzaldehyde, etc.) or with acetone, g lucose, and g lycero l and fo rm i c , glycolic, gall ic, and levulinic acids. However, when a cons iderable amount of g lycera ldehyde is p r e s e n t in the reac t ion solution a yellow color with a g reen f luorescence ap- p e a r s , and fu r fu ra l g ives a d a r k - b r o w n colorat ion.

Thanks to its spec i f ic i ty , this reagent rapidly came into use also for quanti tat ive de terminat ions of formaldehyde [28].

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In 1945, MacFadyen [29] and Br icker and Johnson [30] s imultaneously and independently of one another proposed the use of chromotropic acid for the spect rophotometr ie analysis of formaldehyde. Their p ro - cedures differ in some experimental details. MacFadyen confirmed Eegr iwe ' s s ta tements concerning the specif ici ty of the chromotropic acid react ion for formaldehyde. He also found that such compounds as methanol, acetaldehyde, and formic acid interfere with the determination of formaldehyde if they are p r e s - ent in amounts g rea te r than 10 : 1. It was shown that important factors for the development of the colorat ion are the concentrat ion of the sulfuric acid, the tempera ture , and the time of the reaction. According to Mac- Fadyen the react ion is pe r fo rmed in 9-10 M sulfuric acid with heating on the boiling-water bath for 30 mill. Br icker and Johnson per formed the react ion in concentrated sulfuric acid with heating at 100°C for at leas t 30 min. They recommended for the per formance of the react ion that a rat io of reagent to formaldehyde of 500 : 1 should be used. These authors showed that any compounds forming formaldehyde on hydrolysis by sulfuric acid give a purple coloration. Higher aliphatic alcohols inhibit the formation of the coloration, and acetaldehyde, acrolein, and f l-hydroxypropionaldehyde give a yel low-brown colorat ion with chromotropic acid and therefore appreciably interfere with the determinat ion of formaldehyde. The development of the coloration is prevented by diacetone alcohol and methyl ethyl ketone.

The methods descr ibed by these authors are fast and accurate and permi t the determinat ion both of f ree formaldehyde and of that formed on acid hydrolysis . The minimum detectable amount is approximately 1 pg of aldehyde in 1 ml of solution. The accuracy of the method is ± 5%.

All subsequent methods for determining formaldehyde with chromotropic acid are modifications of these methods. Br icker and Vail [31] showed that in the presence of a large excess of chromotropic acid it is possible to eliminate the influence of many compounds interfering with the determination of formalde- hyde. Their modification reduces to the evaporation of the mixture under investigation with chromotropic acid at 170-200°C for 20 min (in an oil bath o r on a hot-plate) with subsequent addition of sulfuric acid and heating the resul t ing solution under the conditions descr ibed previously. It was found that the recommended modification permi t s the determination of one part of formaldehyde in the presence of 20,000 par ts of such solvents as chloroform, carbon te t rachlor ide , methanol and other lower alcohols, methyl ethyl ketone, py r i - dine, and benzene.

Recently, Still et al. [32] have shown that ethanol in terferes with the determination of formaldehyde with chromotropic acid. Their resul ts contradict the old s tatements of Br i cke r and his colleagues. Other authors [33] have shown an adverse influence of phenolic compounds.

Chr is tofferson [34] has somewhat modified the method of Br icker and Vail [31] by considerably lower- ing the tempera ture of evaporat ion of the solution under investigation with the chromotropic reagent (to ll0°C) and using 18 M sulfuric acid; however, under these conditions it was found that the measurement of the optical density of the solution can be pe r fo rmed only 5-44 h af ter the beginning of the reaction. Yet another modification of the chromotropic acid method was published in 1970 [35]. The maximum sensit ivity (0.055 pg/ml of CH20, which is 36 t imes bet ter than the sensi t ivi ty of Br icker and Vail 's method) is achieved by planning the exper iment with the aid of simplex optimization. The analysis of one sample r e - quires less than 10 min, and the mean coefficient of deviation is 1.3%. Unfortunately, other aldehydes in ter - fere with the determination.

The chromotropic acid method is one of the s implest , but it is fa i r ly severe in the conditions of its per formance . It requi res high tempera tures and prolonged heating with concentrated su l fu r i cac id . Con- sequently, any compounds forming formaldehyde under such conditions will give a positive react ion with chromotropic acid. Fu r the rmore , many other substances give a coloration. It is true that some modif ica- tions [36, 37] have made this method highly specific for formaldehyde since they permi t interfer ing impur i - ties to be removed during the react ion or their influence to be eliminated in some other way.

We may note that in the use of chromotropic acid an important factor is its puri ty. A technical prep- arat ion is unsuitable because of the impuri t ies that it contains. However, its purification is difficult and does not always give a good sample. Fu r the rmore , the solutions of the reagent are unstable on s torage. All these fo rm ser ious defects of the method. Never theless , it is the most widely used in the analysis of formaldehyde.

The Phenylhydrazine Method. The oxidation of a phenylhydrazine complex was f i r s t used for the de- terminat ion of formaldehyde by Schryver [38]. He used potass ium ferr icyanlde as the oxidizing agent, and the determination was pe r fo rmed in an acid medium.

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Later , this react ion was used by Desnuelle and Naudet [39]. Roberts [40] who careful ly investigated it, showed that the coloration of the solutions formed in an acid medium was too unstable. It is unsuitable for accurate investigations.

This method began to be widely used after the work of Tanenbaum and Br icker [40], who used it to determine the free aldehyde in the presence of the bound aldehyde and of many organic compounds inter- fering with the chromotropic acid procedure. A s imi la r problem had been. solved previously by the use of the Schiff reagent (the defects of this method are descr ibed above). Tanenbaum and Bricker , in p re l iminary investigations, used the phenylhydrazine complex of diazobenzenesulfonic acid as the oxidizing agent, and the reaction was per formed in an acid medium. They found that under such conditions some aliphatic aldehydes gave a c r imson coloration, while with ketones no charac te r i s t i c coloration whatever was produced, and a ro - matic aldehydes reacted slowly. Fur the rmore , it was observed that the colorat ion with formaldehyde was more stable than that with the higher aldehydes. However, in this fo rm the procedure had a number of de- fects, of which the main one was the poor reproducibi l i ty of the resul ts . The authors connected this with the instability of the diazobenzenesnlfonic acid. To improve the method, they tested a whole ser ies of ox- idizing agents and for a number of reasons they chose potass ium ferr icyanide. The time neces sa ry for the per formance of one analysis is 30 min, and the accuracy of the method is of the o rde r of 2%.

The method descr ibed was la ter improved by Stankovi~ [41]. The essence of the modification con- sists in the salting out of isopropanol (which is used to improve the solubility of the reagents) f rom the so- lution. In this p rocess , the colored complex passes into the organic layer . The sensit ivity of the method is increased - it is possible to determine 0,02-1.0 mg of formaldehyde in one l i ter . The time for one de- termination is of the o rder of 25 min. The analysis can be per formed in the presence of many substances (methanol, ethanol, phenol, etc.); the presence of hexamethylenetetramine in the mixture is impermissible . Acetaldehyde, sulfite ion, and salts of divalent iron interfere with the determination, beginning at low con- centrations.

Mari et al. [42] showed that it is more convenient to use the oxygen d issolved in the reagents as the oxidizing agent. In these c i rcumstances , the procedure is considerably less laborious. The react ion is per formed in an alkaline medium. These authors investigated the influence of various factors on the p ro - cess. They showed that the maximum coloration develops in approximately 80 min.

According to Marl et al. [42], the react ion is specific and sensit ive for formaldehyde; thus, 10 pg of it give an absorption magnitude of 0.450 under the conditions of the experiment, while 50 pg of acetaldehyde give only 0.030. Moreover, the absorption for 10 pg of formaldehyde in the chromotropic acid procedure of Br icker and Johnson is only 0.200. The accuracy of this modification is 5-10% and the minimum amount of formaldehyde detectable is 1 pg.

The phenylhydrazine method is very sensit ive but is specific for formaldehyde only in Mari ' s modi- fication, which, in spite of its positive features , has not found wide use. Indeed, Tanenbaum and Br i cke r ' s method, in spite of its ser ious defects (in addition to lower aliphatic aldehydes, lower aliphatic alcohols also interfere with the reaction), is used far more frequently [43-46].

The acetylacetone method, which was proposed by Nash [47] in 1953, is based on the Hantzsch r e a c - tion - the formation of lutidine derivatives f rom a fl-diketone, an aldehyde, and an amine. On condensation with acetylacetone and ammonia, formaldehyde gives 3,5-diacetyl-2,6-dimethyldihydrolut idine, which has

C..R Ill~ NII R, R.j

R 3 R~

a yellow color [47, 48].

Nash made a detailed investigation of the react ion conditions - the influence of the pH of the medium, the composit ion and stabili ty of the reagent and of the colored complex formed with formaldehyde, the s ta- bility of the coloration, and the influence of various substances. It was found that for low concentrat ions of formaldehyde the react ion takes place quantitatively at pH 5.5-6.5. Ammonium acetate and phosphate were

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used as the amine components of the react ion, the la t ter proving to be more suitable. It was shown that the diacetyldimethyldihydrolutidine is formed only if the formaldehyde and acetylacetone are presen t in small amounts while the ammonium acetate is p resen t in enormous excess . In prac t ice , the optimum concent ra- tions of these components were as follows: ammonium acetate 0.1-1.0 lVl, and acetylacetone 0.1-0.001 M. The reagent (acetylacetone in ammonium acetate buffer) is stable for at least two weeks. The colored com- plex does not change its color for two days. The influence of the major i ty of substances is reduced to a minimum because of the mild conditions of the analysis .

Under the conditions of the react ion, acetaldehyde also fo rms diacetyldihydrolutidine derivat ives, but the absorption maximum of their solution is in the 388-nm region, and not at 412 nm as in the case of fo r - maldehyde. Fu r the rmore , the rate of formation of the colored complex f rom acetaldehyde is ve ry smal l in compar ison with formaldehyde (at the same concentration). Some amines may compete with the ammonia in the Hantzsch react ion, although the ra tes of these react ions are also low. Acetone, chloral , furfural , and glucose do not interfere with the reaction. Per ioda tes and sulfites des t roy the color to an appreciable ex- tent, especial ly sulfites which decrease the resul ts by 90~c in ve ry low concentrat ions (0.001 M).

Hasegawa has modified this method for the microdeterminat ion of formaldehyde, introducing the ex- traction of the colored complex with an organic solvent [49]. The influence of various fac tors on the r e - action was checked. The most suitable of the solvents tested (benzene, cyclohexane, chloroform, xylene, bu tan - l -o l , and n-hexane) was butanol. At 60°C, the maximum colorat ion develops in 10 min. This author showed that there are no fundamental changes in the absorption of the solutions in the pH range f rom 4 to 8. The colored complex is stable for severa l days. This method permi t s the determinat ion of formaldehyde in the presence of colored compounds. Hasegawa also investigated the influence of some substances on the determinat ion of formaldehyde and showed that a 500-fold excess of acetone or of phenylacetaldehyde, a 50- fold excess of p-chlorobenzaldehyde or propionaldehyde, and a fivefold excess of acetaldehyde do not in ter- fere with the determination. In Hasegawa's opinion, ketones and aromat ic aldehydes should not interfere with the development of the colorat ion, and the influence of aliphatic aldehydes decreases with an increase in the chain length.

Belman has proposed a f luor imet r ic modification of Nash 's procedure which permi t s the determina- tion of 0.01 pg of formaldehyde [50].

The acetylacetone method of determining formaldehyde is undoubtedly the best method for the major - ity of cases . It is ex t remely select ive and sensit ive. The formation of the complex takes place in a neu- tral medium, which makes it par t icu la r ly valuable for the analysis of free aldehydes in the presence of bound aldehydes and also for the investigation of biological mater ia ls . In addition to the methods for the co lor imet r ic determinat ion of formaldehyde that have been considered, many others have been proposed with different degrees of sensi t ivi ty and specif ici ty [1, 2, 4]. We shall l imit ourse lves s imply to mention- ing them, since they have not found wide use and, unfortunately, to a large extent are more suitable for the qualitative detection of formaldehyde than for its quantitative analysis. Various react ions of formaldehyde are used: with phloroglucinol, with resorc inol , with a lkal i -metal and copper sal ts , with hydroxamic, n i t ro- hydroxamic, and benzenesulfohydroxamic acids, with alkaloids, with an ammoniacal solution of s i lver ni- t rate, and with fe r r ic chloride in concentrated hydrochlor ic acid. The main disadvantage of these methods is their low specifici ty.

Below we shall consider methods that have appeared in the l i t e ra tu re in the las t decade and have not appeared in ea r l i e r reviews. We shall not dwell on them in detail, since none of them, in our opinion, pos- s e s ses the advantages of Nash 's method and they are not so widely used as the chromotropic acid and phen- ylhydrazine methods.

In the f i r s t place we may mention the work of Sawicki and his colleagues [51-53] Fo r the co lor lmet r ic determinat ion of formaldehyde they have proposed 2-hydrazinobenzothiazole , p-ni t robenzothiazonium flu- orobora te [52], and 3-methylbenzothtazol in-2-one hydrazone (MBTH) [53]. Each of these reagents is non- specific for formaldehyde, since they all give a positive react ion with other aliphatic, a romat ic , and hetero- cyclic aldehydes. Another disadvantage is the high values of the absorption for blank experiments .

Kamata modified the MBTH method by the photometry of the colored complex after extract ion with a solvent [54]. As extractant he proposed a mixture of ch lo roform and acetone (1 : 1). The colored com- plex, af ter it has passed into the organic solvent, is stable for 2 h. In extract ion, Kamata made use of sa l t - ing out with sodium chloride, since without this more than 10% of the colored product remains in the aque- ous medium. The s t r i c t maintenance of cer ta in t ime intervals is important. Thus, the ext rac t must be

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dried rapidly, otherwise the absorption decreases . One of the most important advantages of Kamata ' s method is the fact that the presence of 1 mg of sugar in the sample gives a hardly distinguishable absorp- tion while, for example, formaldehyde cannot be determined by the chromotropic acid method in the p r e s - ence of appreciable amounts of carbohydrates .

Sawicki et al. have proposed another two spectrophotometr ic methods of determining the free fo rmal - dehyde f rom various organic compounds during chemical react ions. They a re based on the react ion of for- maldehyde with 6-amino- l -naphthol -3-su l fonic acid (J acid} [55] and with 6-ani l ino- l -naphthol -3-sul fonic acid (phenyl J acid) [56]. Both methods are 2-2.5 times more sensit ive than the chromotropic acid method. As with chromotropic acid, the react ion with J acid is pe r fo rmed in the presence of sulfuric acid, and therefore any compounds giving a coloration under these conditions will interfere with the determination.

The authors mentioned immediately suggested four modifications of the determination of formalde- hyde with J acid, distinguished f rom one another by the concentrat ions of sulfuric acid used and the condi- tions of heating the react ion mixture.

In the per formance of the determination with phenyl J acid, the choice of solvent in which the react ion is per formed proves to be very important. Thus, the use of water as solvent led to a substantial sca t te r in the resul ts of the determination. The best resul ts were obtained in methylcellosolve.

In working with phenyl J acid, sulfuric acid is neces sa ry only to dissolve the main reagent. However, this amount is sufficient for the determination of the free formaldehyde l iberated on acid hydrolysis .

These authors consider both methods to be highly specific for formaldehyde (like the chromotropic acid method), re fe r r ing to the work of Kamel and Wizinger [57], according to whom the specifici ty of the react ion between formaldehyde and the reagents mentioned is the resul t of the s te r ic effect of the two sulfo groups.

In addition to the methods descr ibed by Sawicki et al, severa l others have been proposed. Thus, Custa [58] has used the react ion with / - tyros ine hydrochloride in 50% sulfuric acid. Heating to 150-160°C is nec- e s s a r y to develop a green coloration. In these c i rcumstances , for the determination of formaldehyde at least 40 mg of it must be present in the sample. In this form, the method can hardly be considered suitable for the chemis t ry of natural compounds.

Dokladova and Stankova [24] have proposed a method based on the formation of the addition product of sulfur dioxide to formaldehyde. As the SO 2 donor they use the complex [HgC12SO3] 2- (dichloridosulfito- mereuriate) the react ion of which with parafuchsine hydroehloride forms the colored quinoid fo rm of pa ra - fuchsine methyl sulfonie acid (p-rosaniline methyl sulfonic acid}. The sensi t ivi ty of the method can be changed by varying the concentration of the complex formed. This method is in fact a modification of Schiff 's test. Later , the same authors proposed another variant of this method [25].

Ekberg and Silver [59] recommend the determination of low concentrat ions of formaldehyde in the presence of ethanol with the aid of the peptone proteose. They consider that their rapid method has a sub- stantial advantage for their purposes over the chromotropic acid method [60] and Sawicki and Hauser ' s method [51]. The colored complex formed in this method is stable for 1 h. Small amounts of acetaldehyde, butyraldehyde, propionaldehyde, and diacetone alcohol interfere with this method, while they do not inter- fere in the chromotropic acid method.

Korenman et al. have proposed four color react ions for formaldehyde [61]. They are based on the r e - actions of formaldehyde with derivatives of chromotropic acid and azo dyes containing free hydroxy groups. The concentrat ion of the reagents and the time of heating the react ion mixture have marked effects on the intensity of the coloration.

Bailey [62] has developed a method for determining formaldehyde which is based on its catalytic ac - tion in the oxidation of fl-phenylenediamine with hydrogen peroxide.

In the development of new procedures , some authors make use of various modifications of known methods. Of such procedures we may mention the following: the spect rophotometr ic determination of for - maldehyde with dimedone [63]; the determination of formaldehyde in cellulose formals and methylcellulose formals by a modification of Schiff 's method [64]; and the automatic spec t rof luor imet r ic determination of formaldehyde by a modification of Nash 's method [65].

It can be seen f rom the l i te ra ture information considered in our review that at the presen t time col- o r imet r ic methods (especially spect rophotometr ic and spec t rof luor imet r ic methods) are most widely used

444

for the determinat ion of formaldehyde as being the most accurate and rapid and, which is par t i cu la r ly im- portant , requir ing only small amounts of the s tar t ing mater ia l for their pe r fo rmance , which is essential in the solution of many chemical and biochemical p roblems.

It can be seen f r o m a shor t account of the cha rac te r i s t i c s of the co lor imet r ic methods that the most select ive for formaldehyde are the chromotropic acid, the phenyl J acid, and the acetylacetone methods. Of these the most widely used is the chromotropic acid method, although it is considerably infer ior to the other two in many respec t s . The resul ts of a compar ison of the chromotropic acid and the phenyl J acid methods shows that while their select ivi t ies for formaldehyde are s imi la r , the l a t t e r is considerably super ior to the chromotropic acid method both in sensi t ivi ty and in the mildness of the conditions of per forming the analy- sis. Never theless , it has s ca rce ly come into use so far.

The acetylacetone method, with a select ivi ty as high as the two just mentioned, is super ior to all known methods in the mildness of the conditions of analysis. However, it is little used. Consequently, at the present t ime the problem of the development of new and the modernizat ion of old methods of determining formaldehyde is an urgent one. This is par t icu lar ly important for the solution of the problem of the analy- sis of products of periodate oxidation.

All the methods given permi t the determination ei ther of free formaldehyde or of formaldehyde for - mation in the hydrolysis and oxidation of organic compounds. A special case is the analysis of formalde- hyde produced by the oxidation of substances with periodate [66-70], since per iodates and iodates fundamen- tally influence the course of color react ions.

We shall consider in more detail methods of determining formaldehyde formed as the resul t of pe r i - odate oxidation - one of the most important methods in the chemis t ry of natural compounds.

In this section we shall not dwell in detail on the methods of determining formaldehyde themselves , since this would be to repeat what has a l ready been said. It is more desirable to consider their features connected with periodate oxidation and relat ing to the elimination of the periodate and the iodate, which in- t e r fe re with the color react ions.

Some authors determine formaldehyde after its p re l iminary distil lation f rom the react ion mixture [28, 71]. The quantitative distil lation of micro amounts of a substance is a difficult problem. Fu r the rmore , the products of the oxidation of a number of substances are unstable and may decompose on distillation, forming ei ther an additional amount of formaldehyde or substances prevent ing its determination.

In the major i ty of cases , however, various precipi tants and reducing agents are used to eliminate periodate and iodate ions.

Various methods are used to determine formaldehyde freed f rom interfer ing substances [47, 72, 73], but the chromotropic acid method [67] o r a modification of it is the one used more frequently. Success has been achieved in the use of the l a t t e r method after the appropriate select ion of reagents quantitatively p re - cipitating periodate and iodate.

F leury et al. [74] have shown that it is possible to eliminate iodine-containing ions in the f o r m of in- soluble salts . However, the use for this purpose of bar ium hydroxide and s i lver acetate did not give good resul ts because of the impossibil i ty of select ing a pH of the medium which would simultaneously be the op- t imum for the determination of the formaldehyde and for the precipi tat ion of the interfering ions. P r e c i p - itants that have been used are salts of mercury , zinc, magnesium, aluminum, beryl l ium, titanium, bismuth, calcium, etc. , but the desi red effect was not achieved with these ions either.

The most promis ing resul ts have been given by the use of lead salts as precipitants [72]. It has been shown that it is not any lead sal t that can be used for this purpose: for example, the ni trate in terferes with the react ion and the acetate is more suitable but the la t ter gives a yellow colorat ion with a solution of chro- motropic acid. The most successful reagent proved to be lead dithionate, thanks to its high solubility and the capacity of the dithionate ion for decomposing in an acid medium into sulfate and sulfur dioxide. The lead sulfate can be eliminated by centrifuging, and the sulfur dioxide does not interfere with the de termina- tion of the formaldehyde - on the cont rary , it protec ts the chromotropic acid f rom the oxidizing action of light and air . Strontium hydroxide may also be used as precipi tant [75].

The use of ion-exchange res ins for the elimination of periodate and iodate ions is known [67]. Here the res in must be careful ly prepared for the elimination of var ious impurit ies that may interfere with the determination. Trea tment of the react ion mixture with the ion-exchange res in leads to a sharp change in its pH. The formaldehyde may be par t ia l ly lost by adsorption on the resin.

445

In addit ion to p rec ip i t an t s , reducing agents may be used for e l imina t ing in te r fe r ing ions. Among these, a r s en i t e , su l f i tes , iodides , and ch lor ides have found the wides t use.

Unfortunately, the a r sen i t e ion is the most widely used [67-69, 77]. When a r sen i t e is used, the p r e s - ence of l a r g e amounts of minera l acids or a lka l i s is i m p e r m i s s i b l e . It has been e s t ab l i shed that the r e - duction of per ioda te with a r sen i t e takes p lace quant i ta t ively at pH 8 and room t e m p e r a t u r e . At this pH value, the p r e s e n c e of iodate (lO~) does not i n t e r f e re with the de te rmina t ion . However, under such condi- tions the r eac t ion r equ i r e s s e v e r a l hours for its complet ion. The addit ion of iodide to the r eac t ion mixture d e c r e a s e s the t ime of the r eac t ion to a few seconds (at pH 8).

Since this reducing agent is unsuitable for working under acid condi t ions, it cannot be used for the chromot rop ic acid method. F u r t h e r m o r e , ch romot rop ic acid gives a co lora t ion with a r sen i t e [66]. The a r - senic oxide p r e sen t in the r eac t ion mixture fo rms a col lo ida l suspension if solut ions containing ch romo- t ropic acid a re cooled below 60~C. It is not s u r p r i s i n g that the l i t e r a t u r e contains s t a t emen t s accord ing to which the use of a r s e n i t e as reducing agent does not a lways give r ep roduc ib le r e s u l t s [69]. There a r e a lso r epo r t s that the use of a r s en i t e does not always give good r e s u l t s even in a s soc ia t ion with o ther methods of de te rmin ing formaldehyde [65]. Very recen t ly , it has been proposed to r ep l ace a r sen i t e as reducing agent by a sco rb ic acid, as a more acce s s ib l e and nonpoisonous reagent [78].

Since a r sen i t e could not in fact be used in the ch romot rop ic acid method, some authors have p roposed to use sulf i te to reduce pe r ioda te [79], s ince it does not affect reac t ions with ch romot rop ic acid. However, in its or ig ina l va r ian t this p rocedure p o s s e s s e d no s m a l l e r defects than the one using a r s e n i t e [72]. With the use of sulf i te as reducing agent, the iodide fo rmed in the r eac t ion was e l imina ted in the fo rm of the s i l - ve r sa l t , l a rge amounts of r e l a t i ve ly expensive s i l v e r sa l t being r equ i r ed and, m o r e o v e r , col lo idal s i l v e r being formed photochemica l ly , which i n t e r f e r ed with the photometry .

Speck and F o r i s t [79] have proposed to use su l fur dioxide in an acidic medium for the e l imina t ion of per ioda te . In this p r o c e s s , however, the dye that ch romot rop ic acid fo rms with fo rmaldehyde p a s s e s into the leuco form. They found that this leuco fo rm is unstable and may be decomposed by pass ing a i r into the r eac t ion mixture to e l imina te SO 2. This s tage compl i ca t e s the p rocedu re for de te rmina t ion .

Mitchell and P e r c i v a l [80] have used sodium bisul fa te for the des t ruc t ion of per ioda te . They s t a r t e d f rom unpublished r e p o r t s by Rees. In the i r paper , the authors show that with w e l l - d e v i s e d blanks this method enables r e su l t s to be obtained which agree well with the r e su l t s of g r a v i m e t r i c de te rmina t ion .

While sul f i tes do not i n t e r f e r e apprec iab ly in the ch romot rop ic acid method, they cannot be used for the ace ty lace tone method. In very low concent ra t ions (0.001 M) su l f i tes cause the lo s s of 90~ of the f o r m a l - dehyde de t e rmined [47].

The iodide ion has a lso been used as reducing agent [68, 69, 75]. In this case , both s t rong ly acid and s t rong ly a lkal ine reac t ion solut ions can be analyzed. In acid solution, both pe r ioda te and iodate a r e reduced to f ree iodine.

The use of stannous chlor ide as reducing agent has been de sc r ibed [81-87]. Inves t iga t ions in which this reducing agent is used a re s l ight modif ica t ions of the ch romot rop ic acid p rocedure . Modifications of the phenylhydraz ine [88] and ace ty lace tone [89] methods for the de te rmina t ion of formaldehyde a f te r p e r i - odate oxidation have been developed in our l a b o r a t o r y .

A de ta i led inves t igat ion of the phenylhydraz ine method has shown that when pe r ioda te is used as oxi- dizing agent an excess of it within a definite range of concent ra t ions does not i n t e r f e r e with the d e t e r m i n a - tion of formaldehyde [88]. However, this p rocedure is not comple te ly s a t i s f a c t o r y , p r i m a r i l y because of the subs tant ia l influence of va r ious impur i t i e s (lower a ldehydes , e s p e c i a l l y aceta ldehyde) . This impel led us to change to the ace ty lace tone method, the advantages of which have been desc r ibed . We have shown that to e l imina te an excess of pe r ioda te it is poss ib le to use polyols - b u t a n e - 2 , 3 - d l o l , rhamnose , and inosi tol [89]. Butanediol and rhamnose a r e more su i tab le for a rap id reac t ion , but if for any r e a s o n the in t roduc- tion of aceta ldehyde into the r eac t ion mix ture is undes i rab le it is poss ib le to use inosi tol .

Methods for de te rmin ing formaldehyde can be used for two p u r p o s e s : to deduce the s t r u c t u r e of com- pounds containing a - g l y c o l groupings in the molecule and to de te rmine subs tances of var ious c l a s s e s quan- t i ta t ive ly . The f i r s t d i rec t ion is the most impor tant for the c h e m i s t r y of c a rbohydra t e s and has been ex- amined in detai l in ava i lab le r ev iews [66-70].

446

Among the quanti tat ive de te rmina t ions based on the analys is of formaldehyde , the s imp le s t case is the analys is of polyols . In the per ioda te oxidation of va r ious ca rbohydra t e s , formaldehyde is fo rmed f r o m f ree pentoses and hexoses , and a lso f r o m pentoses and hexoses p r e sen t a t the reducing end of a po ly- o r o l igo- sacchar ide chain (if this chain is at tached to the hexose in the 2, 3, o r 4 posi t ion and to the pentose in the 2 or 3 position). However , the oxidation of f ree o r bound sugars takes place compa ra t i ve ly s lowly and can be compl ica ted by side reac t ions . Consequently, as a rule , it is bes t to reduce the f r ee o r bound po lysaccha - r ide to the cor responding polyol before oxidation. Analys is by the method of r e d u c t i o n - p e r i o d a t e oxida- t i o n - d e t e r m i n a t i o n of formaldehyde is of g rea t impor tance for de termining the mo lecu l a r weights of ol igo- and po lysacchar ides [90]. In the invest igat ion and de termina t ion of ca rbohydra te s by this p rocedure , v a r i - ous methods a r e used. The dimedone [91-93] and phenylhydrazine [94] methods are used compara t i ve ly r a r e l y . The ch romot rop ic acid method is the mos t widely used [95-100]. Although Nash ' s method has been desc r ibed in a handbook of ca rbohydra te c h e m i s t r y [67], only recen t ly have pape r s appeared in the l i t e r a - ture descr ib ing the r e su l t s of the use of this method o r modif icat ions of it [101-102]. Thus, the use of a modificat ion of Nash ' s method for the automat ic spec t ro f luor i ine t r i c analys is of ca rbohydra tes a f t e r p e r i - odate oxidation is known [65].

The methods of de termining formaldehyde may find and a re finding wide use in the c h e m i s t r y of l ipids [103]. In actual fact , g lycerol ip ids , sphingolipids, and glycolipids, i .e. , p r ac t i c a l l y all c l a s s e s of l ipids, con-

tain f r agmen t s f r o m which, a f t e r appropr ia te t r ans fo rma t ions , the >CH--CH.~ grouping that is n e c e s s a r y /

h OH OH

fo r analys is can be obtained. Such f ragments may be g lycerol and o ther polyols , sphingosine bases

(R--CH--CH--CH=OH), and monosacchar ide r e s idues . I I

OH NH~

Methods of' de te rmin ing sphingolipids can be divided into two groups. The f i r s t includes methods based e i ther on the de terminat ion of sphingosine n i t rogen [104] or on the color reac t ion between the f r ee amino group fo rmed in the acid hydro lys i s of sphingosine ba se s and a reagen t giving a colored complex. Such reagen ts a re Methyl Orange (sodium p-d imethylaminoazobenzenesul fona te) [105], f luorodini t robenzene [106], t r in i t robenzenesul fonic acid [107, 108], and 1-aminonaphtha lene-4-su l fon lc acid [109]. A second group of methods is based on the de te rmina t ion of the degradat ion products fo rmed a f t e r the oxidation of the sphingosine bases by per iodate o r by lead t e t r aace ta t e . While lead ace ta te is used compara t i ve ly r a r e l y , s ince it leads to overoxidat ion and to o ther side reac t ions [110-111], per iodate is widely used. Its advantage cons is t s in the poss ib i l i ty of p e r f o r m i n g oxidation in var ious media: aqueous [112], acet ic acid [113], aque- ous methanol [112], and o ther mixed solvents [114]. Recent ly , Baumann and Schmid [111] have shown that the oxidation of l ipids takes place spec i f ica l ly in absolute pyr id ine at r oom t empera tu r e . The use of p y r i - dine enables side reac t ions to be excluded, and many l ipids a r e read i ly soluble in it; f u r t h e r m o r e , amino alcohols a re more eas i ly c leaved in a bas ic medium.

The products of degradat ion by per ioda te oxidation a r e de te rmined e i the r by gas ch roma tog raphy (long-chain aldehydes, N - a c e t y l - O - t r i m e t h y l s i l y l de r iva t ives of f ree bases [111, 112, 115, 116]), o r col- o r ime t r i c a l l y . In the l a t t e r case , the formaldehyde fo rmed is de te rmined mos t f requent ly by ch romot rop ic acid method [117, 118], and more r a r e l y by o ther methods [119].

Although in the de te rmina t ion of t r i g lyce r ides gas ch roma tog raphy [120-123] or o ther physical meth- ods [124] have been used e v e r more frequent ly in r ecen t y e a r s , until now the main method for the i r quanti- tat ive analys is has been the de te rmina t ion of formaldehyde a f t e r saponif icat ion and per ioda te oxidation, and this by the mos t var ious methods. The phenylhydrazine method is used compara t ive ly r a r e l y [125-126], and the ch romot rop ic acid method is used mos t f requent ly [130-141].

The ace ty lace tone method has become widely used for the analys is of t r i g lyce r ides in all its poss ib le modif icat ions: c o l o r i m e t r i c [142-144], spec t ropho tomet r i c [145, 146], f l uo r ime t r i c [143, 147-152]. The fact that it is just on the bas i s of the acety lacetone method that a semiau tomat ic p rocedu re for de termining t r i g lyce r ide s is used [145, 147-154] shows the high re l iab i l i ty and s tabi l i ty of the method. Recently, Weigel [153] has published a pape r on the de te rmina t ion of g lycerophosphat ides as g lycerol . The bas i s of the method is the t he rm a l c leavage of the g lycerophosphates to g lycerol , the oxidation of the g lycerol with p e r i - odate, and the subsequent de te rmina t ion of the resu l t ing formaldehyde by automat ic acety lacetone method.

447

For the co lo r ime t r i c determinat ion of t r i g lyce r ides , some authors use 2 -hydraz ino -3 -me thy lbenzo- thiazoline [154] or , ,sulfophosphovanillin" [155]. Hoef lmayr et al. de te rmined t r ig lycer ides f r o m the dif- ference between the total amount of e s t e r s remain ing a f te r the separa t ion of f ree choles te ro l and phospha- tides and the choles te ry l e s t e r s [156, 157]. In recen t y e a r s , enzymat ic methods, which pe rmi t the de t e r - mination of individual t r ig lyce r ides , have come into use for the s t ruc tu ra l ana lys is of the t r ig lycer ides [158-163].

A specia l case in the c h e m i s t r y of l ipids is the analysis of g lyceryl e the rs . Genera l ly , g lycery l e thers a re de te rmined in the unsaponifiable f rac t ion of lipid ex t r ac t s [164, 165]. It is known that they are a com- ponent pa r t both of neutra l lipids and of phospholipids. In the major i ty of previous invest igations the i so la - tion of the glyceryl e thers f rom the phospholipid f rac t ion included its dephosphorylat ion, saponification, and ext ract ion of the unsaponifiable products , the analysis of which gave information on the p r e sence of e thers in them [165-169]. However, in recen t yea r s reduction with l i thium te t rahydroa lumina te is being used eve r more frequently for the isolation of g lyceryl e thers [167, 170-172]. Other questions re la t ing to the analysis of g lyceryl e thers have been cons idered in reviews [173, 174].

In the major i ty of cases , the chromot rop ic acid p rocedure (the defects of which we have discussed) is used for the analysis of g lyceryl e thers . The per iodate oxidation is mos t f requent ly p e r f o r m e d in ethanol [175] or aqueous ethanol with the addition of other solvents [176]. Marinett i has used 90% acet ic acid for this purpose [177].

We have shown the appl icabi l i ty for the analysis of g lycery l e thers of a modification of the ace ty lace - tone method that we have developed [89]. In this, the per iodate oxidation of the p r e p a r a t i v e l y isolated glyc- e ry l e ther f ract ion was p e r f o r m e d in aqueous isopropanol .

As can be seen f rom the las t sect ions of this review, which have been devoted to the analys is of na t - ural compounds using methods of determining formaldehyde , up to the p re sen t t ime the chromot rop ic acid p rocedure has been the mos t widely used. However, in the nea r future it will probably be rep laced by o ther methods, espec ia l ly Nash ' s method. This has a l ready p rac t i ca l ly taken place in individual f ields, such as the determinat ion of t r ig lyce r ides .

We have br ie f ly d iscussed a sma l l f rac t ion of the questions re la t ing to the p rospec t s for the develop- ment of the analytical c h e m i s t r y of formaldehyde. There is no doubt that this analysis is an impor tant p rob l em in the c h e m i s t r y of natural compounds.

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*As in Russian original. The journal of this name did not begin publication until 1970 - Publ isher .

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