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Journal of Saudi Chemical Society (2016) 20, S352S356

King Saud University

Journal of Saudi Chemical Society


www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

A simple spot test quantication method to


determine formaldehyde in aqueous samples
Amara Dar a,*, Umer Shaque a, Jamil Anwar b, Waheed-uz-Zaman b,
Arooj Naseer a
a
b

Center for Undergraduate Studies, University of the Punjab, New Campus, Lahore-54590, Pakistan
Institute of Chemistry, University of the Punjab, New Campus, Lahore-54590, Pakistan

Received 26 April 2012; accepted 5 December 2012


Available online 20 December 2012

KEYWORDS
Chromotropic acid;
Densitometry;
Formaldehyde;
Micro-analysis;
Quantication of spot test;
Spot test analysis

Abstract A simple, accurate, and economical method has been proposed to measure formaldehyde in
aqueous samples. The method is based on quantication of classical chromotropic acid formaldehyde violet spots developed on TLC. Different parameters such as concentration of chromotropic
and sulfuric acid, time of heating and order of application of reagents have been studied to nd out
the optimum working procedure. Spots have been quantied by scanning the spotted TLC and analyzing the image in computer with Visual Basic 6.0 based graphic application. The study consisted of
developing an appropriate calibration line, analyzing articial and real samples, and comparing the
new method with a standard spectrophotometric method (NIOSH Method-3500, 1994). It has been
concluded that the present method had the capability for measuring formaldehyde in aqueous samples
at trace level with high precision and accuracy, particularly when dealing with turbid and small volume
samples where the standard method failed.
2012 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Formaldehyde or methanal (HCHO) is the simplest aldehyde
that is an important precursor to many chemical compounds.
Yearly production of formaldehyde around the globe has
exceeded 23 million tons during last few years (Lide, 2004).
* Corresponding author. Tel.: +92 4299232068; fax: +92 4299232057.
E-mail address: research.group.pu@gmail.com (A. Dar).
Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

Formaldehyde is a gas at room temperature, but readily converts into a variety of other gaseous derivatives like trioxane.
Formaldehyde occurs in the environment up to 0.03 ppm parts
of air. Materials having formaldehyde, such as ureaformaldehyde foam insulation (UFFI) can release it in the form of gas or
vapor. Pressed-wood products are the major source of formaldehyde indoor pollution. Cigarette smoke, gas stoves, woodburning stoves, and kerosene heaters can also release formaldehyde. It is highly toxic to both plants and animals, and is particularly dangerous for the human eyes (Swenberg et al., 1980).
When present in the air at levels exceeding 0.1 ppm, it causes
severe irritation in the eyes, nose, and throat and usually results
in watery eyes with burning sensations, coughing, wheezing,
nausea, and skin irritation (Sakai et al., 2002). There is some

http://dx.doi.org/10.1016/j.jscs.2012.12.002
1319-6103 2012 Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Determination of formaldehyde in water


evidence that formaldehyde become carcinogen when inhaled
(Leonard, 1999). Epidemiological investigations of the fatality
of factory workers following prolonged occupational exposure
to formaldehyde showed a slight excess of lung cancer (Acheson et al., 1984a, b). Safe exposure limit to formaldehyde for
30 min is 100 lg m 3 (0.08 ppm) (Sawada, 2006).
Formaldehyde in water comes mainly from oxidation of organic matter during ozonation (Glaze et al., 1989) and chlorination (Becher et al., 1992). In drinking water, it can arise from
leaching from plastic ttings and water treatment processes. In
the dissolved form, formaldehyde converts into a diol
CH2(OH)2. An aqueous solution of formaldehyde is called formalin. Pure formalin is a saturated solution of formaldehyde in
water (nearly 37% by weight) (Turoski, 1985; Keilson and
Newell, 1990). It does not persist in water for long time because it is broken down within few hours by sunlight or by
bacteria. However, when ingested to a level above 2.6 mg l 1
(NOEL) via contaminated food or water, formaldehyde has
been shown to cause vomiting, abdominal pain, dizziness,
and in extreme cases can cause even death.
Li et al., 2007 developed a uorophotometric method for
formaldehyde determination in environmental waters, which
was founded on the reaction of formaldehyde with acetoacetanilide and ammonia. The method was simple, rapid, economical, and highly sensitive. Roz_ yo et al., 2002 determined
the levels of formaldehyde in human saliva. Formaldehyde
was determined as the dimedone adduct (formaldemethone)
using OPLC. Tomkins et al., 1989 determined total formaldehyde in drinking water samples. Formaldehyde present in 1 liter water was derivatized with 2,4-dinitrophenylhydrazine in
an acidic media and subsequently extracted with chloroform.
After separation by solvent extraction, the product was quantied using reverse-phase liquid chromatography. Keyvanfard,
2010 proposed a simple and rapid catalytic kinetic method
(based on the catalytic effect of formaldehyde on the oxidation
of cresyl violet by bromate in the presence of sulfuric acid) for
the determination of trace amount of formaldehyde. The method was exercised for the measurement of formaldehyde in
water samples.
It is obligatory to develop simple and precise analytical
methods to determine formaldehyde because of its widespread
use, toxicity, and volatility. There are several methods available
for the detection of formaldehyde in air and water. The most
widely used methods for air samples are based on spectrophotometry, while for aqueous samples high-performance liquid
chromatography (HPLC) is a preferred choice. Other methods
include colorimetry, uorimetry, polarography, gas chromatography (GC), infrared detection, ow injection analysis and
gas detector tubes (Cogliano et al., 2004). HPLC or GC coupled
with the mass spectrometer (MS) are most sensitive techniques.
However, these techniques are very expensive. Besides, difculties arise with the monitoring of turbid samples that can block
columns or membranes or precipitations can demand considerable maintenance effort (Papaefstathiou et al., 1996).
This paper describes a simple, rapid, sensitive, selective, and
reproducible analytical method of formaldehyde analysis based
on the quantitative spot test technique. The violetred spots of
formaldehyde with chromotropic acid were developed on TLC.
Chromotropic acid has been widely used as an analytical
reagent in organic as well as in biological chemistry after the discovery of violet spots with formaldehyde in 1937 (Eegriwe,
1937). In view of the fact that the color density of spot is directly

S353
proportional to the concentration of formaldehyde, a simple
analytical method has been developed for precise quantication
of formaldehyde in aqueous samples at trace levels. The color
densities of the spots were measured with simple software by
taking the image of spotty TLC in computer subsequent to
scanning. There are other techniques to quantify chromotropic
acid formaldehyde spots (Boyd and Logan, 1942; Boos, 1948);
the present method is only one of its kind in terms of measuring,
quantity of sample, simplicity and accuracy. The most appropriate application of the method is on-eld analysis of trace
amounts of formaldehyde in aqueous samples.
2. Materials and methods
2.1. Stock solution and standards
Stock solution of formaldehyde (1000 mg l 1) was prepared by
diluting analytical grade 10% formalin solution (SigmaAldrich, Inc.) with proper quantity of double distilled water.
Standard solutions of desired concentrations (210 mg l 1)
were prepared by appropriate dilution of the stock solution.
2.2. Chemicals
Technical grade chromotropic acid disodium salt dihydrate,
(HO)2C10H4(SO3Na)22H2O and reagent grade concentrated
sulfuric acid (SigmaAldrich, Inc.), 9598%, 1.840 g ml 1 at
25 C (lit.) were the chemicals that were used as it is or as doubled distilled water diluted solutions in order to evaluate the
effect of these on color intensity of spots. Desired solutions
of chromotropic acid were prepared in 50% sulfuric acid.
2.3. Procedure
TLC plate (Merck, Aluminum sheet, Silica gel 60F254,
3 5 cm) was used to develop spots since paper could not be
employed (burnt because of concentrated sulfuric acid). One
micro-liter drop of each, chromotropic acid, sulfuric acid
and aldehyde solution was employed with micropipette (Pipettman) one over the other. The order of application of reagents and aldehyde solution was altered to check the effect
on color density of the spot. Later, the TLC was placed in
an oven for 220 min at 60 C to notice the effect of time. Violet color spots intensifying on cooling were obtained.
2.4. Quantication of spots and analysis of samples
The spotty plate was scanned on a atbed scanner (HP 3670
CCD Reective Flatbed Scanner) and the image was imported
in Visual Basic 6.0 based graphic application to measure the
color density of spots. Detail of graphic application to read
color densities of the spots is given in (Anwar et al., 2010).
A calibration line was plotted taking concentration of formaldehydes standards as abscissa against the corresponding color
density of spots (ordinate). Slope, intercept, standard error of
estimates and correlation coefcient were measured. Finally,
synthetic as well as true aqueous samples of formaldehyde
were analyzed using the same set of conditions by measuring
their color densities and interpolating the corresponding concentrations from standards calibration line.

S354

A. Dar et al.

2.5. Comparison with standard method


The present method was compared with the standard method
of formaldehyde analysis recommended by the National Institute for Occupational Safety and Health (NIOSH). It is NIOSH
Method-3500, published in NIOSH Manual of Analytical
Methods in 1994. The detailed procedure of the standard spectrophotometric method using chromotropic acid and concentrated sulfuric acid is given in Georghiou and Ho (1989) and
Fagnani et al. (2003). The method employs chromotropic acid
(1%), sulfuric acid (H2SO4, 96%), formalin solution (37%),
and deionized water. To the solution of formaldehyde, small
amount of chromotropic acid is added followed by addition
of sulfuric acid in excess. Solution is heated and cooled and subjected to spectrometric analysis. The absorbances were recorded on a double beam spectrophotometer (Labomed,
UVD-3500) at 580 nm against the reagent blank. Real and articial formaldehyde-contaminated water samples were tested by
both methods, and the results were compared.
3. Results and discussion
Spot tests are simple chemical procedures in which an essential
feature is to employ a drop of the analyte on the reagent/s onto
paper or TLC that will produce a spot of specic color. These
detection methods are responsive and highly selective. The
detections are micro-analytical and are used to identify a wide
range of organic and inorganic compounds. These procedures
are elementary and reasonably fast (Feigl and Anger, 1998).
Spot test was originally developed for qualitative analysis
but in a near past, efforts have been made to quantify the process (Anwar et al., 2010). Working on the same principle, the
classical chromotropic acid spot test method of formaldehyde
detection has been quantied to nd out the formaldehyde
content of water at trace level with no trouble.

SO 3

SO 3H

HO

- H2 O

HO3S

HO

OH

HO

OH

HCHO

HO
SO 3

Chromotropic acid is the most widely used reagent for


formaldehyde analysis because it is highly selective. Despite
the advent of sophisticated methods such as HPLC and GC,
spectrophotometric method based on chromotropic acid is still
an international reference method of formaldehyde analysis
because of simplicity and sensitivity. The classical method uses
concentrated sulfuric acid but modications have been made
to replace the aforementioned hazardous and corrosive acid
with some less dangerous acids like glacial acetic acid, hydrochloric acid, and phosphoric acid. However, it has been found
that the sensitivity reduces to a signicant extent in the absence
of sulfuric acid (Fagnani et al., 2003). Therefore, the present
method was developed with sulfuric acid. The drawback of
using concentrated sulfuric acid was that it burnt the paper
and corroded the TLC. For that reason, a test was performed
to nd out an optimum concentration of sulfuric acid that can
give maximum color to spot and would not corrode the TLC.
Effect of sulfuric acids strength on color density of spot was
studied working with 10 mg l 1 solution of formaldehyde
and 1% solution of chromotropic acid in 50% sulfuric acid.
It was found that the color density of spot increased with an
increase in the strength of acid. However, after 50%, the
TLC started to corrode. Therefore, the optimum strength of
acid chosen was 50%.
The chemistry of the reaction of chromotropic acid with
formaldehyde in strongly acidic media is very complex and is
not known completely. The often-quoted reaction consists of
two steps that are shown in the Scheme 1. Sulfuric acid acts
as a dehydrant in the rst step and as an oxidant in the next.
The p-quinoidal product is assumed to absorb light in the
range of 570580 nm. However, this product has never been
conrmed experimentally by any valid mean. Conversely,
NMR analysis suggested that the nal product responsible
for violet color is not p-quinoidal but mono-cationic dibenzoxanthylium (Scheme 2) (Georghiou and Ho, 1989). However,

SO 3H

HO3S

-H2O

+O

SO 3H

HO3S

HO

HO

OH
SO 3H

HO3S

Violet-red
Scheme 1

Seses.

Determination of formaldehyde in water


-

OH

O3 S

HO
O

SO 3

SO 3

SO 3

Scheme 2

S355

Asasa.

the nature of this chromogen and its formation has never been
conclusively proven.
The effect of concentration of chromotropic acid on color
intensity of the spot was studied. It was found that the color
intensity had increased with an increase in the concentration
of chromotropic acid up to 2%. However, afterward there
was no prominent increase in color density with further increase in concentration. In the same way, time to heat TLC
at 60 C was changed from 2 to 20 min and it was found that
14 min were optimum to obtain maximum intensity of the spot.
To check the selectivity of the method, different compounds
were tested separately and with formaldehyde to study the
interference if any. It was noted that among acetaldehyde,

Figure 1

Table 1

propanal, butanal, isovaleric aldehyde, chloral hydrate, glyoxal, aromatic aldehydes, formic acid, phenoxyacetic acid
and isopropyl ester, only the last two produced violet spots
at optimum conditions (2% chromotropic acid, 50% sulfuric
acid, and heating at 60 C for 14 min), while furfural, fructose
and sucrose produced yellow spots. The order of application of
reagents and aldehyde solution was also changed and it was
noted that the correct order was to apply formaldehyde solution foremost, then sulfuric acid followed by chromotropic
acid. When chromotropic acid was applied foremost, the nal
color of spot was brownish rather than violet. It may be because of silica or some impurity on TLC that interfered with
chromotropic acid and did not allow it to produce violet chromogen (Scheme 2) on contact with formaldehyde.
TLC containing violet spots was imported to specially designed Visual Basic 6.0 based graphical application using atbed scanner at 300dpi, 24-bit RGB mode. Each spot was
selected inside a selection marquee and the color density was
measured. The software measured the red, green, and blue components of each pixel inside the margin and sums up the values
to give a nal color density. Calibration lines (Fig. 1) were plotted for formaldehyde standards in the range of 210 mg l 1 by
the present method (color density against concentration) and
the standard NIOSH method (absorbance against concentra-

Calibration lines of the present and standard method using Microsoft Excel 2007.

Comparison of calibration lines of the present and standard method using Microsoft Excel 2007.

Parameter

Standard method*

Present method

Slope
Y-intercept when X = 0.0
X-intercept when Y = 0.0
1/slope

0.049 0.002
0.086 0.015
1.778
20.580

18.700 0.627
38.000 4.160
2.032
0.053

95% Condence intervals


Slope
Y-intercept when X = 0.0
X-intercept when Y = 0.0

0.041 to 0.056
0.038 to 0.135
3.219 to 0.692

16.700 to 20.700
24.760 to 51.240
3.043 to 1.206

Goodness of t
r2 **
Sy.x ***
Result for 5 mg l
Percentage error

0.993
0.014
4.745
5.100%

0.997
3.967
5.044
0.880%

solution

Spectrophotometric method (NIOSH Method-3500 (1994), NIOSH Manual of Analytical Methods (NMAM), ALS Columbia).
Coefcient of determination.
***
Standard error of estimates.
**

S356
tion, Method-3500 published in 1994). The calibration data are
compared in Table 1. Coefcient of determination for the present method (0.997) was better than the standard method
(0.993). The coefcient of determination is useful as it gives
the amount of the uctuation of one variable that is predictable
from the other variable. In addition to the standard statistical
analysis of calibration, an articial sample (5 mg l 1) was tested
by both methods. The output of the present method was
5.044 mg l 1 (0.88% ) whereas the standard method found it
to be 4.745 mg l 1 (5.10% ). The present method used only
1 ll of the sample while the standard method used 5 ml. Moreover, present method was applicable to turbid samples but the
standard method could only analyze clear solutions.
Actual samples (tap and canal water from university campus,
and textile efuent from local industry) were also tested by both
methods. Tap water was used as such and no formaldehyde was
detected by both methods. Canal water and efuent were allowed to settle for 30 min and then ltered ahead of analysis with
the standard method, whereas the sample was used without ltration in case of the present method. The formaldehyde content
of canal water was 2.546 and 2.429 mg l 1 while the concentrations of formaldehyde in textile efuent were 8.926 and
8.654 mg l 1 by the present and standard method, in that order.
The present method is not only economical but also accurate,
and it can be used for on-eld analysis with the help of portable
scanners and laptops/smart phones (having color density measuring software) since it does not involve painstaking sample
preparations and sophisticated instruments.
4. Conclusion
A trouble-free, precise, and inexpensive method of formaldehyde analysis for aqueous samples has been proposed. The
method was based on quantication of classical chromotropic
acid formaldehyde violet spots developed on TLC. Optimum
working conditions were 2% chromotropic acid, 50% sulfuric
acid, and 14 min heating at 60 C to get utmost color intensity
spots. The correct order of reagents application was sample
(formaldehyde), followed by sulfuric acid and then chromotropic acid (1 ll drop of each). Spots were quantied by scanning
the spotted TLC and analyzing the image in computer with
homemade Visual Basic 6.0 based graphical application. Calibration line of the present method was compared with that of
the standard spectrophotometric method. The present method
was found to have better capability for measuring formaldehyde in aqueous samples at trace level with high precision
and accuracy, especially when dealing with turbid and small
volume samples where the standard method was inapplicable.
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