Chromotropic acid�/formaldehyde reaction in strongly acidicmedia. The role of dissolved oxygen and replacement of

concentrated sulphuric acid

E. Fagnani, C.B. Melios, L. Pezza, H.R. Pezza *

Instituto de Quımica-UNESP, P.O. Box 355, CEP 14801-970 Araraquara, SP, Brazil

Received 4 November 2002; received in revised form 3 February 2003; accepted 3 February 2003

Abstract

The procedure for formaldehyde analysis recommended by the National Institute for Occupational Safety and Health

(NIOSH) is the Chromotropic acid spectrophotometric method, which is the one that uses concentrated sulphuric acid.

In the present study the oxidation step associated with the aforementioned method for formaldehyde determination was

investigated. Experimental evidence has been obtained indicating that when concentrated H2SO4 (18 mol l�1) is used

(as in the NIOSH procedure) that acid is the oxidizing agent. On the other hand, oxidation through dissolved oxygen

takes place when concentrated H2SO4 is replaced by concentrated hydrochloric (12 mol l�1) and phosphoric (14.7 mol

l�1) acids as well as by diluted H2SO4 (9.4 mol l�1). Based on investigations concerning the oxidation step, a modified

procedure was devised, in which the use of the potentially hazardous and corrosive concentrated H2SO4 was eliminated

and advantageously replaced by a less harmful mixture of HCl and H2O2.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Formaldehyde; Chromotropic acid; Spectrophotometry; Dissolved oxygen

1. Introduction

Formaldehyde (CH2O, methanal, formic alde-

hyde, oxomethane) is uniquely important because

of its widespread use and toxicity and it is

recognized as one of the most important air

pollutants. Due to large volume production and

usage of formaldehyde and its possible exposure-

related health effects, much concern has arisen

over the sensitivity and accuracy of analytical

methodology for this compound.

Small amounts of formaldehyde and formalde-

hyde-releasing compounds are commonly ana-

lyzed by spectrophotometric methods [1�/3] and,

one of these, the chromotropic acid (CA) method,

especially through its P&CAM 125, P&CAM 235

[4] and 3500(2) [5] versions, was established as an

international reference method and, despite the

advent of more sophisticated techniques, it is still

widely used because it is simple, sensitive, inex-

pensive and very selective. The major drawback

presented by the CA method has been the use of* Corresponding author. Fax: �/55-16-222-7932.

E-mail address: hrpezza@iq.unesp.br (H.R. Pezza).

Talanta 60 (2003) 171�/176

www.elsevier.com/locate/talanta

0039-9140/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0039-9140(03)00121-8

hot concentrated sulphuric acid, which is poten-

tially hazardous and corrosive. Attempts to re-

place the aforementioned acid by less hazardous

ones, e.g. glacial acetic, concentrated hydrochloric

and phosphoric acids, were unsuccessful. It was

reported, without details, that the reaction’s sensi-

tivity was greatly reduced as compared with that

observed by employing concentrated H2SO4 [6,7].

In order to reduce the hazard brought about by

concentrated H2SO4, the use of a 50% (v/v)

solution of that acid has been recommended [8],

but, again, significant loss of sensitivity was

encountered by adopting this procedure, along

with other drawbacks [8].

The chemistry of the color reaction between CA

and formaldehyde, in strongly acidic media, is not

yet known with certainty. The most often-quoted

reaction path [7,9,10] involves a two step process,

as shown in Scheme 1, where (I) would be

responsible for light absorption at lmax�/570�/

580 nm [7,9,10].

According to Feigl [9], H2SO4 participates in

both steps as dehydrant and, in the last one, also

as oxidant, being reduced to sulfurous acid. It is

perhaps worth noting that H2SO4 rarely acts as an

oxidizing agent [11]. An alternative structure

proposed for the chromogen arising from the

reaction of CA and formaldehyde is (II) [7,8]:

The nature of this chromogen has never been

unambiguously proven but evidence has been

obtained [7] using NMR techniques and calibra-

tion line studies to support the hypothesis that the

chromogen formed in the analytical procedure has

a mono-cationic dibenzoxanthylium structure (II)

and not the p-quinoidal one (I) that is commonly

cited. A likely mechanism for the reaction is

presented [7], involving a final oxidation step.

Scheme 1.

E. Fagnani et al. / Talanta 60 (2003) 171�/176172

However, the oxidizing agent is not mentioned [7].Finally, these authors state that concentrated

H2SO4 appears to be the optimal acid to use and

that no simple modification would seem feasible in

order to avoid the use of concentrated H2SO4 in

the analytical method [7].

In this paper, investigations concerning the

oxidation step associated with the CA spectro-

photometric method for formaldehyde determina-tion were carried out. Based on these

investigations, a modified procedure was devised,

in which the use of concentrated sulphuric acid

was eliminated and advantageously replaced by a

mixture of HCl and H2O2, without significant loss

of sensitivity.

2. Experimental

2.1. Apparatus

A Hewlett Packard Model HP8453 spectro-

photometer with 1 cm matched silica cells wasused for all absorbance measurements. Micropip-

ettes Brand and Eppendorf were used to measure

the smaller volumes in the experiment. A fast

oxygen detection system from Hanna Instruments

mod, HI 9142 was used to study the oxygen

consumption in the reactional medium. A gas

meter GF 2200 HR, from Gilmont Instruments

was used to monitor the flux of gases in thesaturation of solutions. All experiments were

performed in a thermostated room (25.09/0.1 8C).

2.2. Reagents

. All reagents utilized were of analytical grade

(Carlo Erba, Merck or Mallinckrodt Co.). For

the preparation of the solutions and samples,

deionized water (Milli-Q plus) and grade ‘A’

glassware were used throughout. Nitrogen wasused to deaerate solutions where required. The

solutions were flushed with pure N2 until the O2

concentration was 4.1�/10�5 mol l�1 or less,

as determined by an official method [12]. In

many experiments, air- and oxygen-saturated

solutions were employed for which the deter-

mined oxygen concentrations [12] were 2.3�/

10�4 and 8.6�/10�4 mol l�1, respectively.

. Formaldehyde stock standard solution, 1000

mg l�1, prepared by appropriate dilution of

commercially available analytical-reagent grade

formaldehyde solution (37%, Mallinckrodt

Co.), standardized by a AOAC method [13].

The standard formaldehyde solutions used for

constructing the calibration graph were freshlyprepared by appropriate dilution of the stock

solution with water.

. Chromotropic acid (disodium salt dihydrate,

C10H6O8S2Na2 �/ 2H2O, Merck): a 5% (m/v)

aqueous solution was freshly prepared.

. Hydrogen peroxide (2.5�/10�2 mol l�1): pre-

pared from perhidrol 30% by convenient dilu-

tion and standardized as described in theliterature [14].

. Sulphuric acid solution: (50% v/v; 9.42 mol

l�1), prepared in the usual way, from the

concentrated acid (96%).

2.3. Recommended procedure

2.3.1. Calibration curves

2.3.1.1. NIOSH procedure. The basic NationalInstitute for Occupational Safety and Health

(NIOSH) procedure was followed with modifica-

tions as described by Georghiou et al. [7]. Curves

with concentrated sulphuric, phosphoric and hy-

drochloric acids and diluted sulphuric acid were

made under oxygen-, air- and nitrogen saturated

solutions. Calibration graphs are prepared by

plotting absorbance against formaldehyde concen-tration for each acid.

2.3.1.2. Proposed method using hydrochloric acid

and hydrogen peroxide. A calibration curve is

prepared as follows: transfer 630 ml of formalde-hyde working standard solution (comprising 0.80�/

4.80 mg of formaldehyde) into 25 ml glass tubes,

add 300 ml of 5% CA solution, 70 ml of 2.5�/10�2

mol l�1 H2O2 and 4.00 ml of 12 mol l�1 HCl

(under stirring). The tubes are sealed with PTFE

tape and heated for 1 h in a steam bath (100 8C).

Afterwards, they are cooled at 25 8C. The absor-

E. Fagnani et al. / Talanta 60 (2003) 171�/176 173

bances are recorded at 575 nm (b�/1 cm) againstthe reagent blank.

3. Results and discussion

The absorption spectra of solutions obtained by

using concentrated sulphuric or hydrochloric acids

are superimposable within the 400�/700 nm range,

suggesting that the same chromogen is produced in

both procedures.

The results obtained for the calibration curves

carried out with concentrated sulphuric, hydro-

chloric and phosphoric acids, varying the oxygen

concentrations, are shown in Fig. 1.

It was found that the reaction of CA with

formaldehyde, developed as recommended by

Georghiou et al. [7], is not dependent on the

concentration of dissolved oxygen, as shown in

curve 1, Fig. 1, supporting Feigl’s proposal [9]

concerning the oxidation step promoted by con-

centrated (18 mol l�1) H2SO4. However, if that

acid is replaced by concentrated hydrochloric (12

mol l�1) or phosphoric (14.7 mol l�1) acids, the

results are significantly dependent on the concen-

tration of dissolved oxygen, in addition to the

already reported drop in sensitivity [6,7], as shown

Fig. 1. Dependence of oxygen concentration and nature of the

concentrated inorganic acid on the CA�/formaldehyde reaction.

Before heating, the reaction solutions were saturated with O2

([O2]�/8.6�/10�4 mol l�1), air ([O2]�/2.3�/10�4 mol l�1) or

N2 ([O2]�/4.1�/10�5 mol l�1). For all solutions, the color

reaction was developed as described in [7]. Curve 1: H2SO4 (18

mol l�1)�/O2 (j), H2SO4 (18 mol l�1)�/air (�/), H2SO4 (18

mol l�1)�/N2 (k) (r�/0.999; pooled points); Curve 2: HCl (12

mol l�1)�/O2; Curve 3: HCl (12 mol l�1)�/N2; Curve 4: H3PO4

(14.7 mol l�1)�/O2; Curve 5: H3PO4 (14.7 mol l�1)�/N2 (r�/

0.998).

Fig. 2. Dependence of oxygen concentration and nature of the

concentrated inorganic acid on the CA�/formaldehyde reaction.

Before heating, the solutions comprising H2SO4 were saturated

with O2, air or N2 as described in Fig. 1, and the color reaction

was developed as recommended in [7]. For solutions comprising

HCl, the color reaction was developed as described in the text

and freshly distilled/deionized water was used throughout.

Curve 1: H2SO4 (18 mol l�1)�/O2 (r�/0.999); Curve 2: HCl

(12 mol l�1)�/H2O2 (r�/0.999); Curve 3: H2SO4 (9.4 mol

l�1)�/O2; Curve 4: H2SO4 (9.4 mol l�1)�/N2.

E. Fagnani et al. / Talanta 60 (2003) 171�/176174

in Fig. 1, curves 2�/5. The same is true if lowerH2SO4 concentrations are used (e.g. 9.4 mol l�1)

cf. curves 3 and 4 of Fig. 2. These facts could

explain the lack of reproducibility of the measure-

ments and consequently the non linearity of the

calibration curves reported in the literature [8]

when 9.4 mol l�1 H2SO4 is utilized, which would

probably be associated with the lack of control

over oxygenation conditions. No color at allappears in the absence of oxygen (e.g. in sodium

dithionite rich solutions).

Curves 2 and 3 of Fig. 1 show a departure from

linearity for a formaldehyde concentration higher

than approximately 2.4 mg l�1 and a drop of

analytical sensitivity with decreasing of dissolved

oxygen content in the reaction medium comprising

HCl. Furthermore, departure from linearity ismore pronounced for the solutions comprising

lower oxygen concentrations (curve 3). Taken

together, these observations provide, for the first

time, compelling indirect evidence regarding the

O2 participation in forming the purple chromogen.

The curves obtained by using concentrated and

diluted sulphuric acid (in this last case, in oxygen-

and nitrogen-saturated solutions), along with thatobtained by using HCl and H2O2, are presented in

Fig. 2. The adopted hydrogen peroxide concentra-

tion was found to be sufficient to yield the oxygen

needed to promote the oxidation step of the

reaction and to avoid the oxidation of chromo-

tropic acid to the corresponding quinone and/or

formaldehyde to formic acid.

Very good analytical results were achieved whenconcentrated HCl was used in combination with

hydrogen peroxide (see curve 2, Fig. 2). Based

mainly on this observation, a modified CA method

for the determination of formaldehyde was pro-

posed in which the hazardous H2SO4 is replaced

by a mixture of hydrochloric acid and hydrogen

peroxide. This method proved to be equivalent in

color developing time and temperature, simplicity,inexpensiveness and reproducibility*/with only

about 5% loss in sensitivity*/as compared with

widely accepted CA procedures [4,5,7]. The slope

of that line leads to an apparent molar absorptivity

of (1.719/0.02)�/104 mol l�1 cm�1 for the

chromogen, which is close to that reported for

the reaction developed with concentrated H2SO4,

i.e. 1.8�/104 mol l�1 cm�1 [7] and (1.839/0.03)�/

104 mol l�1 cm�1 (this laboratory).

In principle, the O2 consumption could be

directly monitored in the solutions where the

analytical reaction is carried out. These solutions,

however, contain large excess (100�/600 fold) of

CA relative to microgram amounts of formalde-

hyde (see curve 2, Fig. 1), thereby precluding the

use of even the most sensitive O2 sensors. Anexperiment was, therefore, carried out with a much

larger formaldehyde amount (0.032 mmol), where

the same CA and HCl concentrations were main-

tained, permitting direct measurement of O2 con-

centration during the reaction course (before and

after heating) by employing a waterproof oxygen

sensor, mod HI 9142, from Hanna Instruments,

Germany. The results obtained can be seen in theTable 1.

The consumption of O2 in the reaction tube was

greater than that observed in the blank tube

confirming that, even though there is an oxygen

loss due to heating, oxygen is consumed in the

chemical reaction. This is in keeping with what

was observed in the spectrophotometric measure-

ments, i.e. the oxygen participates in the reactionwhen it occurs in a concentrated hydrochloric acid

medium. In these experimental conditions, con-

sumption of O2 was, therefore, undoubtedly con-

firmed.

4. Conclusion

The results of the present work provide a

significant contribution regarding the oxidation

step of the reaction between formaldehyde andchromotropic acid in a strongly acidic media.

Feigl’s hypothesis [9] was confirmed for the first

Table 1

Determination of oxygen consumption

Tube ([O2]before heating�/[O2]after heating) (mg l�1)a

Blank 12.29/0.3

Reaction 16.19/0.2

a Triplicate determinations.

E. Fagnani et al. / Talanta 60 (2003) 171�/176 175

time, namely that for the reaction in concentratedsulphuric acid this acid acts as oxidizing agent

(step 2, Scheme 1); furthermore, the reaction does

not depend on the concentration of dissolved

oxygen. However, when concentrated hydrochlo-

ric- and phosphoric acids or diluted sulphuric acid

(9.4 mol l�1) are used, the participation of

dissolved oxygen is crucial, as the results are

significantly dependent on its concentration. Re-cognition of the important role of oxygen in the

reaction promoted the development of the pro-

posed analytical procedure, in which the use of

concentrated H2SO4 was eliminated and replaced

by a mixture of HCl and H2O2.

Preliminary experiments have shown that the

utilization of concentrated phosphoric acid and

hydrogen peroxide can also be successfully carriedout, with changes in the procedure and appro-

priate heating. Investigations along these lines are

currently in progress.

Acknowledgements

We would like to thank FAPESP, CNPq and

CAPES Foundations (Brazil), for financial sup-

port.

References

[1] E. Sawicki, T.R. Hauser, S. McPherson, Anal. Chem. 34

(1962) 1460.

[2] A.A. Cardoso, E.A. Pereira, Anais Assoc. Bras. Quım. 48

(1999) 63.

[3] K.A. Sakiara, L. Pezza, C.B. Melios, H.R. Pezza, M. de

Moraes, Il Farmaco 54 (1999) 629 (References therein).

[4] NIOSH Manual of Analytical Methods, second ed., vol. 1,

US Department Health, Education, and Welfare, Publ.

(NIOSH) 77-157-A, 1977.

[5] NIOSH Manual of Analytical Methods, fourth ed., US

Department Health, Education, and Welfare, method

3500(2), 1994.

[6] C.E. Bricker, W.A. Vail, Anal. Chem. 22 (1950) 720.

[7] P.E. Georghiou, C.K. Ho, Can. J. Chem. 67 (1989) 871.

[8] A.D. Pickard, E.R. Clark, Talanta 31 (1984) 763.

[9] F. Feigl, Spot Tests in Organic Analysis, seventh ed.,

Elsevier, Amsterdam, 1966, pp. 434, 435.

[10] E. Jungreis, Spot Test Analysis, second ed., Wiley, New

York, 1997, pp.125, 126.

[11] M. Hudlicky, Oxidations in Organic Chemistry, ACS

Monograph 186, Am. Chem. Soc., Washington, DC,

1990, p. 20.

[12] Association of Official Analytical Chemists, Official meth-

ods of Analysis of AOAC international, vol. 2, 16th ed,

AOAC International, Arlington, 1995, p. 5.

[13] Association of Official Analytical Chemists, Official meth-

ods of Analysis of AOAC international, vol. 2, 16th ed,

AOAC International, Arlington, 1995, p. 99.

[14] O.A. Ohlweiler, Quımica Analıtica Quantitativa, vol. 2,

third ed, LTC-Livros Tecnicos e Cientıficos Editora SA,

Rio de Janeiro, 1982, p. 190.

E. Fagnani et al. / Talanta 60 (2003) 171�/176176