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��ࡱ�>��	�����������{�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	��B)bjbj����	4Pΐΐ(��������		CCCCC����WWWW�S�W�8������pB��P�7�7�7�7�7�7�7$�:�U=N�7�C.��..�7CC���m8�#�#�#.FC�C��.��#.�7�#�#V~+@�+�����`�Ck�9�Wt"��+�.�80�8�+0�=�"��=�+�+\�=CV,�..�#.....�7�7�#...�8....���������������������������������������������������������������������=.........			:	@COLORIMETRIC ANALYTICAL PROBE FOR DETERMINATION OF FORMALDEHYDE AND ITS VALIDATION USING A SINGLE REAGENT

ANANTHARAMAN SHIVAKUMAR*
PG Department of Chemistry and Research Centre, St. Philomena�s College (Autonomous), Bangalore-Mysore Road, Bannimantap, Mysore 570015, India
*Corresponding Author: 
E-mail: - HYPERLINK "mailto:shivakem77@yahoo.co.in" shivakem77@yahoo.co.in and shivakem77@gmail.com   (http://orcid.org/0000-0003-1132-7087)
Phone: -             +919986137477
Postal Address: - Associate Professor 
PG department of Chemistry and Research Centre 
St. Philomena�s College, 
Bangalore-Mysore road, 
Bannimantap, 
Mysore 570015, 
India 

ABSTRACT:
A simple and sensitive method for the determination of formaldehyde is proposed involving Terbutaline Sulphate with the formation of yellow coloured product with absorption maximum at 460 nm in the presence of concentrated sulphuric acid is proposed. The method lies in a calibration range 0.038 - 0.76 �g ml-1 and molar absorptivity 2.6 � 104 M-1 cm-1. The method has been validated with a phloroglucinol method in its determination in fruits, vegetable and water bodies. 

Keywords: Formaldehyde, terbutaline sulphate, Single reagent, validation









1. 	INTRODUCTION:  Formaldehyde, a colourless foul smelling gas is intensively used in the preparation building materials and household products. It is photo chemically broken down in the air, generally within hours. It also quickly dissolves in water easily. When dissolved in water in 40% ratio, called formalin, used as a industrial disinfectant in several process such as in funeral homes and medical labs. It is also used as a preservative in foods, and in pharma products such as antiseptics, medicines, cosmetics and such others. It also acts as a preservative in food. Naturally minute quantities of formaldehyde are always produced during the metabolic processes in human and other living organisms. Formaldehyde can be generated autoxidation degradation by polyvinylpyrrolidone and polythyleneglycol  ADDIN EN.CITE <EndNote><Cite><Author>Gullapalli</Author><Year>2015</Year><RecNum>2061</RecNum><DisplayText>[1]</DisplayText><record><rec-number>2061</rec-number><foreign-keys><key app="EN" db-id="d5ezsxra85zezrep5vepzs9ut0vx9rw95s99">2061</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gullapalli, Rampurna P.</author><author>Mazzitelli, Carolyn L.</author></authors></contributors><titles><title>Polyethylene glycols in oral and parenteral formulations�A critical review</title><secondary-title>International Journal of Pharmaceutics</secondary-title></titles><periodical><full-title>International Journal of Pharmaceutics</full-title></periodical><pages>219-239</pages><volume>496</volume><number>2</number><keywords><keyword>Polyethylene glycols</keyword><keyword>Solubilization</keyword><keyword>Bioavailability</keyword><keyword>Precipitation</keyword><keyword>Autooxidation</keyword><keyword>Degradation</keyword></keywords><dates><year>2015</year><pub-dates><date>2015/12/30/</date></pub-dates></dates><isbn>0378-5173</isbn><urls><related-urls><url>http://www.sciencedirect.com/science/article/pii/S0378517315303628</url></related-urls></urls><electronic-resource-num>https://doi.org/10.1016/j.ijpharm.2015.11.015</electronic-resource-num></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_1" \o "Gullapalli, 2015 #2061" 1].  
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Bearing all these the chromatography have serious disadvantages such as, the instruments should be operated by a trained persons only, less economical, error due to overloading of the samples, parts are expensive and sensitive, more solvent for separation, high pressure may be required in times. Furthermore, the fluorescence instrumentation, assay reagents, light sources are not economical. Scattering process is one of the serious concerns, as could contribute to positive interference. All the samples should be deaerated as it can result in erratic or fluctuating readings, reaction pH is key in carrying out the reaction as it leads to inaccurate measurements. 
The proposed analytical methodology adopted is very simple, sensitive compared to some of the available methods already reported. The method uses only two reagents namely concentrated sulphuric acid and terbutaline sulphate, which are not only economical but also easy available. The reaction between the two reagents involves the formation of yellow coloured product with an wavelength of absorption at 460 nm due to the coupling and dehydration between two molecules of terbutaline sulphate in the presence of formaldehyde and concentrated sulphuric acid. 
2.	Chemicals and Reagent preparation: All the chemicals that were used in the assay are of analytical grade. Formaldehyde and terbutaline sulphate was purchased for Himedia, Mumbai. All the interfering ion solution was tested by using 1000 ppm of the stock. Double distilled water was used throughout the experiment for the preparation of solution and requisite dilution was prepared as per the need. Stock solution (19 ppm) was prepared by 1:1 dilution with water and concentration was determined by carrying out a titration with standard potassium permanganate.  0.2 % Terbutaline sulphate was prepared dissolving 0.2 g in 100 ml of double distilled water. 
	Fruit, vegetable, plant and water samples were collected locally. The fruit, vegetable and plant were peeled, dried for 2 h, weighed and homogenised using a blender and diluted with sufficient quantity of double distilled water and squeezed using a muslin cloth. This solution was used as a stock. The suitable dilution was prepared as per the calibration graph. Aqueous extract was prepared just before the start of the experiments so as to prevent undesired degradation reaction, then assayed at least in triplicate and results are averaged out. All the infusion were analysed as fresh as possible for reliability of the results. Water sample were collected nearby ponds of city of Mysore, Karnataka, India
3.	Instrumentation:  A SYSTRONICS model 2202 PC based double beam ultraviolet�visible (UV�Vis) spectrophotometer with 1.0 cm matched cells was used for all absorbance measurements. All the glass apparatus were neatly washed with a mixture potassium dichromate and sulphuric acid during each step of the optimisation to avoid unwanted side reaction. 
4.	Optimisation of the reaction condition and reagents for the colour development: Optimisation of the reaction condition was carried out one-factor-at-a-time method. An attempt was made to carry out the reaction in various concentrations of sulphuric acid (2N to concentrated). Finally it was seen that the use concentrated sulphuric acid showed a maximum colour development (Graphical plot not shown). 0.5 mL of Terbutaline Sulphate solution (0.2%) was needed for the development of maximum colour. Higher concentration gave lesser absorption values.
5.	Preparation of the calibration curve: To a series of 10 ml standard flask, aliquots containing 0.38 to 7.6 �g formaldehyde solutions, resulting in the concentration range between 0.038 to 0.76 �g ml-1, was added. To each of the standard flask, 0.5 ml of terbutaline sulphate and 4.0 ml concentrated sulphuric acid was added. The mixture was shaken well and diluted to volume by double distilled water. The so developed yellow colour, after cooling, instantaneously was measured at 460 nm against the reagent blank containing all the reagent except formaldehyde. The repeatability of the calibration graph was checked by carrying out a triplicate analysis. The unknown concentration of formaldehyde was determined by the calibration graph. The Table 1 indicates the spectral characteristics of the coloured product
6.	Proposed reaction mechanism: The proposed mechanism is suggested as described below. The first step is the formation protonated formaldehyde. The protonated formaldehyde undergoes stepwise formation of the yellow coloured product which has extended conjugation as shown in the Scheme 1. The minimum interference by acetone, there by overruling the suggested mechanism to acetone
 
 EMBED ChemDraw.Document.6.0  

Scheme 1: Proposed reaction mechanism for the quantification of formaldehyde


7.	Effect of foreign chemicals
Interference from foreign substances was tested by analyzing their effect in a fixed concentration of 0.57 �g ml-1 of formaldehyde. The experimental results are tabulated in the Table 2. It has been seen that the method has a minimum interference from alcohol, acetone, acetaldehyde, dextrose which are part of the analysis in fruit, vegetables. It also recorded that except nitrate and magnesium, other chemicals used in the reaction did not interfere significantly with the proposed method. Nitrate interference was suitably eliminated by pre-treatment of the solutions with copperised zinc and treating the solution with 0.5% sulphamic acid. The minimum interference from carbonyl containing species such as acetone, acetaldehyde, and dextrose clearly indicates the reaction is highly specific for formaldehyde.  
8.	Application for the determination of formaldehyde in fruit, vegetables and water: 
The proposed reaction in the determination was applied for formaldehyde quantification in the spiked fruit, vegetables and water samples. The samples were collected from the local bodies in and around Mysore city, Karnataka, India. The percentage recovery were reported as presented in the Table 3
9.	Conclusion: 
A colorimetric method has been proposed for the determination of formaldehyde using terbutaline sulphate has been suggested. The method involves a single reagent, which involves the intramolecular coupling of terbutaline sulphate in the presence of concentrated sulphuric acid. The linearity of the proposed assay lies in the range of 0.038 to 0.76 �g ml-1. The method has been successfully applied for formaldehyde determination in fruits, vegetables and local water bodies samples with recovery ranging between 95.3 to 101.8%. As formaldehyde, a serious pollutant in the water bodies and household edible products its colorimetric quantification makes a great impact on the scientific community. The proposed assay would thus be a method which can be adopted in economically weaker analytical laboratories.  
ACKNOWLEDGEMENTS:  Author Dr. Anantharaman Shivakumar (A.S.K) thanks St. Philomena�s College for providing facilities to carry research.

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Figure 1. Calibration graph for the quantification of formaldehyde in the linearity range

Table 1. Optical parameters for the determination of formaldehyde 
ParametersCharacteristicsColourYellow�max460 nmStability5 hrsBeer s law range �g ml-10.038 - 0.76 Molar absorptivity (104 M-1cm-1)2.6Detection limit0.009Limit of quantification0.030Regression equationaCorrelation co-efficient0.998Slope (a) (�M)-1 cm-10.026Intercept (b)-0.005Relative standard deviationb (%)2.0Reaction timeInstantaneousa Y=ax+b where x is concentration in �M  and Y is absorbance  
b Ten replicate measurements

Table.2. Effect of interfering species on the determination of formaldehyde
Foreign speciesTolerance limit* (�g/mL)Alcohol25000Acetone20000Acetaldehyde5000Dextrose6000Sucrose, lactose5000Oxalate, acetate, Ascorbic acid4000Na+ , phosphate 2500Zn2+ , sulphate, sulphite,1000Co2+, tartarate900Fe2+, Ca2+, carbonate850Ni2+, Cl-, F- EDTA, I-725Mn2+, Cu2+ , Hg2+450Fe3+, dichromate, Ce4+, Al3+300Nitrate**100Magnesium4
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Table. 3. Determination of formaldehyde in fruit, vegetables and water 
	
Sl NoSamples tested%Recovery �R.S.D% Recovery �R.S.D  ADDIN EN.CITE <EndNote><Cite><Author>Gayathri</Author><Year>2000</Year><RecNum>2074</RecNum><DisplayText>[3]</DisplayText><record><rec-number>2074</rec-number><foreign-keys><key app="EN" db-id="d5ezsxra85zezrep5vepzs9ut0vx9rw95s99">2074</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gayathri, N.</author><author>Balasubramanian, N.</author></authors></contributors><titles><title>Spectrophotometric Determination of Formaldehyde</title><secondary-title>Analytical Letters</secondary-title></titles><periodical><full-title>Analytical Letters</full-title></periodical><pages>3037-3050</pages><volume>33</volume><number>14</number><dates><year>2000</year><pub-dates><date>2000/01/01</date></pub-dates></dates><publisher>Taylor &amp; Francis</publisher><isbn>0003-2719</isbn><urls><related-urls><url>https://doi.org/10.1080/00032710008543240</url></related-urls></urls><electronic-resource-num>10.1080/00032710008543240</electronic-resource-num></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_3" \o "Gayathri, 2000 #2053" 3]01Malus domestica95.3 � 0.999.6 � 0.4502Citrus limon97.3 � 1.4101.1 � 0.5603Musa paradisiaca103.3  � 2.5100.5 � 0.3604Averrhoa carambola96.8  �0.90102.3 � 0.2905Manilkara zapota101.8  � 0.9699.6  � 0.4606Citrullus lanatus99.9 �  0.8999.1 � 0.5607Daucus carota98.7  � 1.998.9 � 0.2908Cucurbita pepo97.6  � 1.56  101.3 � 0.8909Moringa oleifera96.8 � 1.89102.3 � 0.9210Water sample from the local water bodies95.6  � 0.78101.8 � 0.86
Determination has been made after adding three different amounts of formaldehyde to the fixed concentration of pre-analysed analyte 



 
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