INTRODUCTION
Formaldehyde (FA) is a gaseous substance (BP
= -19 °C) with a pungent odor. The California Air Resources
Board identified FA as a toxic air contaminant and that it is
a carcinogen without an identified threshold. FA is highly irritating
to the eyes, skin, and respiratory tract. Exposure to low to
moderate levels of FA in air for even short periods of time can
cause temporary burning or itching of the eyes or nose, stuffy
nose, sore or burning throat, or headaches. Breathing high levels
of FA can cause chest tightness and coughing or wheezing. High
levels may also worsen asthma symptoms. The California Environmental
Protection Agency Office of Environmental Health Hazard Assessment
estimate of the most plausible cancer risk from FA is approximately
7 excess cancers per 1 million people continuously exposed to
1 part per billion. At these ambient levels, this corresponds
to an excess of 930 potential cancer cases for a California population
of 30 million people exposed over a 70 yr lifetime.
In 1981, production of FA in California from
direct and indirect sources was estimated to be 1.9 and 1.2 x
105
tons, respectively. FA is emitted from building materials, furniture,
some cosmetics, paper products, and permanent-press fabrics.
In homes, the major sources of FA are pressed-wood products made
with urea-FA resins. In the ambient air, photochemical oxidation
is the largest source of FA. The largest sources of directly
emitted FA are combustion of fuels, mobile sources (i.e., automobile
exhausts), and process emissions from oil refineries.
Because of the increasing concern about the levels of FA in the environments, there are efforts to develop simple and reliable quantitative methods. Colorimetry is the most common method for FA determination. FA is derivatized to colored products or chromophores and quantitation of these products is based on the absorption at specific wavelengths. A major drawback of colorimetry is that other compounds that absorb at the wavelength of interest can interfere with quantitation.
Chromatographic methods have been extensively used for formaldehyde analysis. These methods commonly involve derivatization of FA with reagents such as cysteamine, 2,4-dinitrophenylhydrazine (2,4-DNPH), semicarbazide, hydroxylamine, or dansyl hydrazine. The resulting derivatives of these reagents and FA can be analyzed by gas chromatography (GC) or liquid chromatography (LC).
FA (and most carbonyl compounds) reacts readily with cysteamine under moderately basic conditions at pH 8 and 25 °C to form thiazolidine as shown in Figure 1. The thiazolidine derivative is more stable and less volatile (B. P. = 164 °C) than FA but is sufficiently volatile for GC. A highly sensitive and specific nitrogen-phosphorus detector (NPD) or flame photometric detection in sulfur mode (FPD(S)) can be used due to the presence of nitrogen or sulfur in the compound. An advantage of the GC-NPD or GC-FPD(S) method for thiazolidine analysis is the absence of interference by common solvent contaminants which can occur with other detectors.
Figure 1. Derivatization of carbonyl
(FA, R1
= R2
= H, MW = 30) with cysteamine to form alkylthiazolidine (thiazolidine,
R1
= R2
= H, MW = 89).
The derivatization reaction of aldehydes and
ketones with 2,4-DNPH is routinely used for analysis. FA (and
most carbonyls) reacts readily with 2,4-DNPH under acidic conditions
to form the corresponding hydrazone as shown in Figure 2. The
DNPH derivatives (hydrazones) are not suitable for GC because
of their high boiling points and thermal instability. The hydrazone
is amenable to reverse-phase HPLC and absorbs very strongly at
wavelengths 360 - 380 nm so that UV detection can be used. Either
an isocratic or gradient elution can be used for the separation
of the hydrazone from other components in complex mixtures.
Figure 2. Derivatization of a carbonyl (FA, R1
= R2
= H, MW = 30) with 2,4-DNPH to form 2,4-dinitrophenylalkylhydrazone
(2,4-DNP-hydrazone, R1
= R2
= H, MW = 210).
The objective of this lab are to apply derivatization,
extraction methods, internal standards, and recovery analysis,
using the thiazolodine dervitization method in concernt with
GC/NPD/FPD analysis for the quantitation of FA in cigarette smoke
in the laboratory. You will use the DNPH LC method in the field
sampling experiment in Smoke II.
LABORATORY SECTION
SAMPLE PREPARATION.
All samples must be prepared in the fume hoods.
1. Cysteamine Derivatization of FA in Cigarette
Smoke. To a 1 L separatory funnel, add 200 mL of deionized
water containing 0.7 g cysteamine hydrochloride (0.03 M, adjusted
to pH 8 with 6 N NaOH) for preparation of the GC sample. Attach
the stopcock end of the capped separatory funnel containing the
derivatizing solution to the vacuum line, leaving the stopcock
opened to the vacuum, and adjust the vacuum to ca. 25 in
Hg. Allow 10 - 15 minutes for air evacuation.
2. Controls. There will be two control blanks for the GC (water and smoke) and one for the HPLC (smoke) experiments.
Water Blanks.
Since FA is extremely water soluble and endogenous in water,
water blanks must be prepared to determine the amount of FA in
the water matrix. For the GC water blank, prepare 200 mL of deionized
water, dissolve 0.7 g cysteamine hydrochloride, and adjust to
pH 8 with 6 N NaOH. This sample will not be smoke-exposed.
Smoke Blanks.
To determine if the thiazolidine (cysteamine derivative of FA)
is naturally found in the samples (which seems unlikely but you
want to be sure) or is a contaminant (due to poor cleaning of
the glassware or residual effects, which seems likely), prepare
a separatory funnel for the GC blank, and a C-18 SPE cartridge
for the HPLC analysis. For the GC, the funnel containins 200 mL
of deionized water adjusted to pH 8 with 6 N NaOH (cysteamine
smoke blank) . These samples will be smoke-exposed, therefore
proceed to the vacuum evacuation procedure.
3. Smoke Exposure Procedure. GC: After
air evacuation, close the stopcock of the separatory funnel, immediately
turn off the vacuum, and detach the separatory funnel from the
rubber tubing. Insert the filter end of the cigarette to the
stopcock and place it securely. Light the cigarette and open
the stopcock quickly. Allow the cigarette to burn completely.
Not all of the smoke will be pulled into the separatory funnel.
Close the stopcock and remove the cigarette butt. Allow the
cigarette smoke and the derivatizing or smoke blank solution to
mix. Gently hold the separatory funnel until the smoke and the
solution are thoroughly homogeneous; then carefully open the stopcock
(point the stopcock into the fume hood and away from you and your
lab partners). Close the stopcock, and allow your samples to
settle for 5 minutes. Transfer your solutions to separate 250
or 500 mL Erlenmeyer flasks. Proceed to the respective GC for
analysis.
SPIKE ANALYSIS
(GC) You will be given a FA spike solution. Prepare 200 mL cysteamine solution (pH 8) into a 250-500 mL Erlenmeyer flask. Add the appropriate volume of the spike solution (the spike concentration and volume will be determined during the lab session). Add magnetic stir bars and allow the solutions to react for 30-45 minutes.
GC PROCEDURE
Refer to Figure 3 for a schematic diagram
for the determination of FA using cysteamine derivatization and
gas chromatography with either nitrogen-phosphorus or flame photometric
(in sulfur mode) detection.
Liquid-liquid Extraction.
Place fluted filter paper in a filter funnel. Add ca.
5 g magnesium sulfate or sodium sulfate to the filter paper as
a drying agent. Insert the filter funnel into a 500 mL round
bottom flask. To the cysteamine smoke, water or smoke blanks,
and spiked sample, extract each three times with 50 mL each of
chloroform. After each 50 mL extraction, filter the chloroform
extract through the magnesium sulfate funnel into the round bottom
flask. Pour ca. 25-50 mL of chloroform to rinse the magnesium
sulfate funnel and combine with the other extracts. After the
three extractions and rinse, reduce the volume extract to 100
mL using a rotary evaporator; transfer this solution quantitatively
to a clean 100 mL volumetric flask, and bring the final volume
to 100 mL with chloroform. Add the internal standard (1,3,5-trimethylthiazole,
the amount and concentration will be told to you on experiment
day). The purpose of the internal standard is for quantitation
purposes and to eliminate injection volume differences. Analyze
for the amount of FA as the thiazolidine derivative by GC with
NP or FP (S mode) detection.
Standard curve.
Prepare your calibration curve by injecting 1-3 µL of each
of the thiazolidine standards provided (25, 50, 75, and 100 pmol/µL)
spiked with the internal standard, 1,3,5-trimethylthiazole. Obtain
duplicate injections of each standard. Plot the GC peak area
ratio of thiazolidine/1,3,5-trimethylthiazole versus the concentration
of thiazolidine (pmole/µL) injected.
Sample. Once
a satisfactory standard curve is obtained, inject 1-3 µL
each of the chloroform extract of the cigarette, blanks, and spiked
samples. Obtain duplicate injections of the cigarette and spiked
samples only. Compare the GC peak area ratio of the sample with
the standard curve, and obtain the concentration of thiazolidine
injected. You may need to do some range-finding for quantitation
of your sample. Calculate the mass of thiazolidine in the sample,
determine the concentration of FA in the unknown sample, and the
% recovery of the spiked sample, knowing that 1 mole of FA corresponds
to 1 mole thiazolidine.
REFERENCES
1. T. Hayashi; C. Reece; T. Shibamoto (1986).
Gas chromatographic determination of Formaldehyde in coffee
via thiazolidine derivative. J. Assoc. Off. Anal. Chem.,
69(1), 101.
2. A. Yasuhara; T. Shibamoto (1989).
Formaldehyde quantitation in air samples by thiazolidine derivatization:
Factors affecting analysis. J. Assoc. Off. Anal. Chem.,
72(6), 899.
3. M. B. Rogozen (1984). Formaldehyde:
A survey of airborne concentrations and sources. Final report;
Science Applications; Hermose Beach, CA.
4. M. Colli; A. Gironi; V. Molina; R. Marchetti; G. Melzi D'Eril; C. Lucarelli (1991). Improved HPLC methodology in occupational exposure studies on fomaldehyde. Chromatographia, 32 (3/4), 113.
5. C. H. Riser; P. Martin (1994). Quantification
of formaldehyde, acetaldehyde, and acetone in sidestream cigarette
smoke by high-performance liquid chromatography. J. Chrom.
Sci., 32(3), 76.
6. A. Larsen; N. A. Jentoft; T. Greibrokk (1992).
Determination of ppb levels of formaldehyde in air. Science
of the Total Environment, 120(3), 1428.
7. California Environmental Protection Agency,
Air Resources Board Program Update (Sept. 1992). Air
Toxics Update #8, Sacramento, CA.
8. California Air Resources Board (Sept. 1991).
Indoor Air Quality Guideline, No. 1, Sacramento, CA.
Figure 3. Schematic procedure for FA determination in cigarette smoke using cysteamine derivatization and GC-NPD or GC-FPD(S).
APPENDIX
Lab Report
I. Abstract.
Key words: formaldehyde; cysteamine; thiazolidine;; gas chromatography
with nitrogen-phosphorus detection; gas chromatography with flame
photometric detection in sulfur mode; spike recovery. You must
mention numerical results, overall conclusion(s), comments about
analysis, and statements comparing instrumental and extraction
methods. Keep this section as brief, concise, and thorough as
possible.
II. Introduction. Complete,
thorough, and informative according to the guidelines listed in
the syllabus.
III. Materials and Methods.
A numerated format of the procedures will not be accepted
for lab reports. All procedures must be summarized in text.
You may refer to the handout but briefly summarize the procedures
and any changes from the handout. List materials in text form
only. Experimental conditions must be clear and complete.
IV. Results and Discussion.
Summarize your results in a paragraph. Use appropriate table
and figure captions and use complete sentences for these captions.
If you plan to use tables and figures, they must be referred
to in your text. Your results must include:
a) Standard curves for each detector on the
GC. Figures are complete and appropriately labeled.
b) Representative chromatograms of a standard,
sample, and spike for each GC method. You may show the blanks
if necessary. Absolutely no raw data with chromatograms. These
chromatograms must be labeled properly, figures numerated, and
figure captions must be as detailed but brief as possible.
c) Calculated amount of FA in cigarette smoke
using appropriate units. Complete calculations of FA in the unknown
sample and the blanks from each method, which must be provided
in the Appendix, properly labeled.
d) Spike recovery must be reported. Calculations
must be presented in the Appendix, properly labeled.
Your discussion must be written in paragraph
form (no numerations). Below are
some suggestions for a thorough paper. (Remember, these are
just the minimum requirements for the Discussion section.)
a) Discuss the advantages and disadvantages
of the derivatization method.
b) Discuss the advantages and disadvantages
of the extraction methods.
c) Compare your GC-NPD and GC-FPD(S) results.
Are there any differences?
d) Discuss your spike recovery. What do these
values reveal? If your recoveries were high or low, explain why.
e) Interpret what the results mean. Do both
methods give the same value of FA in the sample? If not, discuss
the possible reasons. Do not only say "human error"
as your reason without sufficient explanation.
f) Propose the general reaction mechanisms
for the formation of alkylthiazolidines from a carbonyl and cysteamine.
For the alkylthiazolidine formation, you may assume an initial
attack of the sulfhydryl group (rather than the amine) to the
electrophilic carbonyl carbon since sulfur is more electronegative
than nitrogen.
g) What are the advantages and disadvantages
of an internal standard.
h) Significance of results. What does the
amount of FA mean? Is this value significant or a concern considering
that there are other toxic compounds in cigarette smoke.
V. Conclusion.
Summarize the results and provide an overall "big picture"
of the experiment.
VI. References.
Follow the syllabus or any accepted format.
VII. Appendix.
Sample calculations of FA from cigarette smoke, blanks, field
sampling, and spike recoveries from the GC method. Present your
calculations clearly and neatly with the appropriate units. This
is a mandatory requirement.
Worksheet for GC/NPD Analysis
Total volume of cigarette extract:_____ mL
Total volume of smoke blank: _____ mL
Total volume of water blank: _____ mL Total
volume of spike: _____ mL
Internal standard: _________________________
GC Conditions:
Instrument: ____________________________________
Detector: ______________ Injector Temp:
______________ Detector Temp: ___________
Column (Manufacturer, Type, Dimensions):
Temperature Program:
Integrator: _______________________________________
Conditions:
Gas Flowrates:
Carrier (Type: ________ ) ___________ Make-up
(Type: _________ ) __________
Fuel Gases:
Calculation Worksheet
I. GC Analysis- NPD
1. Determine your standard curve. Plot the
Average Peak Area Ratio (Peak Area of Analyte/Peak Area Internal
Standard) versus Concentration. The equation of the standard
curve:
where m and b are the slope and y-intercept.
The x-axis is in units of moles (pmole/µL) and the y-axis
is in units of the Peak Area Ratio (no units)
2. Substitute your average (or single) peak
area ratio value of your blank, sample, or spike into the equation
of your standard curve and solve for your concentration:
The result is the concentration of your extract.
3. Adjust for the amount of the total volume of the extract to obtain the pmol carbonyl in the cigarette:
4. Multiply pmol by the molecular weight to
calculate the mass carbonyl/cigarette.
5. Subtract blank values (follow same calculation
procedures) to calculate the adjusted mass/cigarette (removal
of residual carbonyl).
II. GC Analysis-FPD
1. Determine your standard curve. Plot the
Average Peak Area Ratio (Peak Area of Analyte/Peak Area Internal
Standard) versus Concentration. The expression of the standard
curve is a quadratic equation:
2. Substitute your average (or single) peak
area ratio of your blank, sample, or spike into the equation of
your standard curve and solve for your concentration using the
quadratic expression:
The result is the concentration of your extract.
3. Adjust for the amount of the total volume of the extract to obtain the pmol carbonyl in the cigarette:
5. Multiply pmol by molecular weight to calculate
the mass carbonyl/cigarette.
6. Subtract blank values (follow same calculation
procedures) to calculate the adjusted mass/cigarette (removal
of residual carbonyl).