ABSTRACT:
Occurrence of potentially toxic formaldehyde and other carbonyl compounds in indoor air is threat to human life. Woodwork and cigarette smoking are known as extensive sources of emission of carbonyl compounds. Hence a simple and rapid method for determination of carbonyls in indoor air has been developed which includes sampling and analysis. The method involves the use of isocratic reverse phase HPLC to determine carbonyl compounds after derivatization with 2,4-dinitrophenylhydrazine (DNPH). This method is sensitive, cost effective, speedy and relatively specific, since the derivative has an absorbance maximum at 360 nm. A known volume of ambient air is drawn through a pre packed silica gel cartridge coated with alcoholic DNPH at a sampling rate of 0.03 to 1.5 L/min for an appropriate period of time.
INTRODUCTION:
Carbonyl compounds, in particular aldehydes, are reactive volatile substances. Carbonyl compounds have been studied because of their adverse effect viz. Irritation of eyes, lung and mucous membranes of the upper respiratory tract. Formaldehyde has been found to be a major promoter in the formation of photochemical ozone.
REACTION:
The method is based on the specific reaction of organic carbonyl compounds (aldehydes and ketones) with DNPH-coated silica gel cartridges in the presence of a strong acid, as a catalyst, to form a stable coloured hydrazone derivative according to the following reaction:
The derivatization reaction leads to an orange-coloured mixture of DNPH-carbonyls as shown in figure 1.
Figure 1. DNPH derivatization reaction
where R and R1 are organic alkyl or aromatic group (ketones) or either substituent is a hydrogen (aldehydes). The reaction proceeds by nucleophilic addition to the carbonyl followed by 1,2-elimination of water to form the 2,4- diphenylhydrazone derivative.
EXPERIMENTAL:
Apparatus & Materials:
Instrumentation
1. Isocratic High performance liquid chromatograph (HPLC) of Perkin Elmer 200 series
1.1 Pumping system – Gradient, with constant flow control capable of 1.50 mL/min.
1.2 High pressure injection valve with 20-μL loop.
1.3 Column C18.
1.4 UV detector
2. Pre packed silica gel cartridge (SKC Inc, USA)
3. HPLC-grade methanol , acetonitrile, formaldehyde and acetone
4. 2,4- dinitrophenylhydrazine (DNPH)- GR grade , recrystallized twice with
UV grade acetonitrile before use.
5. A portable low flow and constant volume air sampler (SKC Inc., USA)
6. ortrho Phosphoric Acid – GR grade
7. HCl – GR grade
Method
Based on the method EPA TO 11A and NIOSH 2016
Preparation of DNPH coated silica gel Cartridge :
DNPH Coating Solution
2.0 g. of purified 2:4 dinitrophenylhydrazine is suspended in 100 ml methanol and pH is adjusted to 3 using phosphoric Acid.
i) The pre-packed silica gel cartridge package is opened and 10 mL
of acetonitrile is added.
ii) Liquid is drained to waste by gravity.
iii) 7 mL DNPH coating solution is dispensed into the cartridge.
iv) Once the solution flow at the outlet of the cartridge has stopped, 7
mL of the DNPH coating reagent is dispensed again into the
cartridge.
v) Let the coating reagent drain by gravity through the cartridge
until flow at the other end of the cartridge stops.
vi) The excess liquid at the outlet of the cartridge is wiped with clean
tissue paper.
vii) The cartridge is air dried.
viii) Both ends of the coated cartridge is plugged with polypropylene
caps.
Figure 2: DNPH coated absorbent tube
Sampling :
The method used for the sampling is based on the NIOSH 2016. 1 to 15 L of air sample is drawn through the DNPH coated silica gel cartridge using Personal sampling pump (SKC) at a flow rate ranged 0.03 to 1.5 L/min.
Transportation & storage :
As the pump turned off, the tube is immediately removed from the Sampler and two open sides are tightly closed with plastic caps. The tube is securely kept with ice gel in a ice box and immediately send to the laboratory and refrigerate the tubes at <40 C. Samples and blanks should be stored under the same conditions.
Sample Preparation / Extraction:
The time between sampling and extraction should not exceed 2 weeks. Since background levels in the cartridges may change due to adsorption during storage.
1. Acetonitrile solvent is checked for carbonyl content by elution and analysis of a blank cartridge
2. The carbonyl derivative is eluted from the cartridge samplers with 5-mL
quantities of acetonitrile.
3. The eluent is collected from the cartridge in a screw capped vial .
4. The vial is then sonicated for 15 min.
5. Sample elutes are stable at 40C for up to one month.
HPLC Analysis:
The operating parameters are as follows:
Column: 4.6 x 250-mm, stainless steel, packed with 5-μm C-18
Mobile Phase: 70% acetonitrile/30 % water, isocratic.
Detector: ultraviolet, operating at 360 nm.
Flow Rate: 1.0 mL/min.
Retention Time: 5.45 minutes for formaldehyde , 6.75 minutes for acetone
Sample Injection Volume: 20 μL.
Calibration of Instruments:
1) Preparation of DNPH-Carbonyl Derivative
2N HCl is sufficiently added to a portion of the recrystallized DNPH, to obtain an approximately saturated solution. Formaldehyde and acetone are added to this solution in molar excess of the DNPH . It is allowed to dry in air.The coloured precipitate is filtered, washed with 2N HCl and water and let the precipitate air dry.
2) Preparation of DNPH-Carbonyl Standards
A standard stock solution of the DNPH-carbonyl (formaldehyde and acetone) derivative is prepared by dissolving accurately weighed amounts in acetonitrile. A working calibration standard mix i.e. 0.5 , 2.5 and 5 ppm are prepared from the standard stock solution. The concentration of the DNPH-carbonyl compounds in the standard mix solutions is adjusted. All standard solutions can be stored in a refrigerator for at least one month.
3) HPLC Calibration
The external calibration is performed using above three working standards. A recovery study has been done using the calibration curve prepared from the above mentioned standards. In order to standardize the method of sampling and to avoid matrix effect another calibration curve is prepared using DNPH coated silica gel cartridge. A mix standard of formaldehyde and acetone is prepared using acetonitrile. Using the stock solution, working calibration standards are prepared and are spiked into the DNPH coated silica gel cartridge. The calibration curve is then prepared using the eluents of these three cartridges.For analysis of the samples we have used the second calibration curve. A blank cartridge is also analyzed.
A representative chromatogram of the DNPH carbonyl derivative is shown in figure 3.
Fig. 3 Chromatogram of DNPH carbonyl derivative
The limits of detection (LOD) lie in the range of 0.1 ppm for formaldehyde and 0.5 ppm for acetone. Calibration is realized for two carbonyl compounds and the linearity (r²) for formaldehyde and acetone are 0.999 and 0.987 respectively. As an example, the calibration curve for formaldehyde is shown in figure 4.
Fig. 4 Calibration curve for DNPH- formaldehyde
RESULTS & DISCUSSIONS:
Formaldehyde is classified as a Group one carcinogen by the International Agency for Research on Cancer.
The Occupational Exposure Limits (“OELs”) and the threshold limit value (TLV) recommended by the American Conference of Governmental Industrial Hygienists (ACGIH) has been furnished in Table 1
Table 1
| Chemical Name | Exposure Limit In Indoor Air/Work place | |||
| Current OEL (ppm) | ACGIH TLV (ppm) | |||
| TWA | STEL | TWA | STEL | |
| Formaldehyde | 0.3 | C 1 | —– | C 0.3 |
TWA – the 8-hour time weighted average (TWA) limit.
STEL- the short-term exposure limit or STEL
“C” indicates a ceiling limit, which refers to the concentration of a chemical or material that no person should be exposed to for any period of time to prevent ill effects or death.
A no. of samples has been collected from the major contributor sources of Carbonyl compounds in indoor air , however two of them are furnished in Table 2. The sampling had been carried out during the month of September.
Table 2
| Sample | Unit | Formaldehyde | Acetone |
| No.1 (Inside a Shopping Mall) | ppm | 2.4 | 1.5 |
| No. 2 (Inside a public vehicle) | ppm | 6.23 | 8.05 |
The recovery of each DNPH derivative ranged from 88% to 104%.
CONCLUSION:
It is a rapid, cost effective and routine approach for the analysis of carbonyls in assessing the environmental situation in industry. The method consists of derivatization with DNPH followed by separation and detection of DNPH-carbonyl derivatives using HPLC and UV absorption using C18 column. Formaldehyde and acetone have been used as representative aldehyde and ketone. Other carbonyl compounds, can also be determined by this method. It is suggested to check up the HPLC system including all used chemicals and solvents and especially the derivatization agent. Otherwise the the generated data may loose its reliability.
The work done under the guidance of Prof. Barun Gupta.
Contributed by : Ms. Sutapa Bhowmik & Mr. Ranajit Maji