Enzymatic Methods for Determining Formaldehyde Release from

10 Enzymatic Methods for Determining Formaldehyde Release from Wood Products Mat H. Ho and Jui-Lin Weng

Downloaded by CORNELL UNIV on October 21, 2016 | http://pubs.acs.org Publication Date: August 8, 1986 | doi: 10.1021/bk-1986-0316.ch010

Department of Chemistry, University of Alabama, Birmingham, AL 35294 Two sensitive fluorometric enzymatic methods for the determination of formaldehyde release from wood products were described. These methods were developed using the enzyme formaldehyde dehydrogenase to catalyze the oxidation of formaldehyde to form formic acid and NADH in the presence of oxidized nicotinamide adenine dinucleotide (NAD ). The increase in NADH, which is directly proportional to the concentration of formaldehyde, is measured fluorometrically at λ = 348 nm and λ = 467 nm. The NADH produced can also be reacted with resazurin in the presence of diaphorase to form resorufin, a highly fluorogenic compound. The fluorescence production is measured at λ = 575 nm and λ = 590 nm. The optimal conditions as well as the sensitivity and linear range of these methods will also be described. +

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During the past decade, urea formaldehyde and phenol formaldehyde resin binders have contributed greatly to the progress of wood industries. Formaldehyde is widely used as a major component in the production of building materials, such as particleboard and plywood, and in urea formaldehyde foam insulation. However, the emissions of formaldehyde from these products create considerable concerns not only in the working environments but also in residences, mobile homes, and office buildings. These concerns have also been stimulated by reports on the health effects and carcinogenicity associated with formaldehyde exposure. Recently, numerous particleboard manufacturers have initiated programs to reduce formaldehyde release from their products, thus "low emission" urea formaldehyde resins were introduced (1,.2)« The emissions of formaldehyde from wood products have been addressed by several authors in this volume. This paper will focus on the development and application of two sensitive and specific analytical procedures for the determination of formaldehyde. The measurements of formaldehyde release from wood products usually involves two steps: sampling and analysis. For sampling, 0097-6156/86/0316-0116$06.00/0 © 1986 American Chemical Society

Meyer et al.; Formaldehyde Release from Wood Products ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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Methods for Determining

Formaldehyde

Release 1 1 7

formaldehyde e m i s s i o n s were c o l l e c t e d i n water o r sodium b i s u l f i t e absorbing solution using a s u i t a b l e t e s t such as l a r g e s c a l e t e s t chamber, mobile home s i m u l a t o r test chamber, q u i c k t e s t , o r desiccator t e s t ( 2 ) . C h r o m o t r o p i c a c i d i s t h e most w i d e l y used and recommended method f o r t h e a n a l y z i n g o f t h e c o l l e c t e d f o r m a l d e h y d e . However, t h e c h r o m o t r o p i c a c i d i s p o t e n t i a l l y s u b j e c t e d t o numerous interferences such as phenols, alcohols, olefins, aromatic h y d r o c a r b o n s , n i t r i t e s , and n i t r a t e s ( 3 , 4 J . Because of inherent interferences i n t h e nonenzymatic reactions, such as c h r o m o t r o p i c a c i d , t h e r e i s a need f o r a more specific test which will yield a better estimation of actual formaldehyde levels release from wood p r o d u c t s . The purpose o f this paper i s t o i n t r o d u c e the use o f an enzyme as an a n a l y t i c a l reagent f o r formaldehyde d e t e r m i n a t i o n and e x p l o r e i t s p o t e n t i a l utility f o r measuring formaldehyde e m i s s i o n l e v e l s . The use o f an enzyme i n t h e d e t e r m i n a t i o n o f formaldehyde i s an a t t r a c t i v e approach for a number o f reasons including s p e c i f i c i t y and sensitivity. The tremendous p r o g r e s s i n enzyme t e c h n o l o g y t o g e t h e r with the advent o f a n a l y t i c a l i n s t r u m e n t a t i o n , encourages t h e use of enzymes f o r quantitation of various substrates, i n h i b i t o r s , activators and enzymes themselves. The growing analytical applications of enzymes has been reflected i n extensive publications i n r e c e n t y e a r s (5_f6_), w i t h most o f t h e s e a p p l i c a t i o n s in clinical chemistry. Enzymes have found l i t t l e o r no p r a c t i c a l application i n e n v i r o n m e n t a l c h e m i s t r y . T h i s work r e p r e s e n t s t h e first attempt t o use enzyme f o r the s p e c i f i c and s e n s i t i v e determination o f formaldehyde. P r i n c i p l e o f E n z y m a t i c Method f o r Formaldehyde D e t e r m i n a t i o n Enzymes are proteins which have t h e c a p a b i l i t y t o c a t a l y z e many complex chemical reactions. Outstanding properties of these biological catalysts a r e t h e i r s p e c i f i c i t y and t h e i r c a p a b i l i t y o f catalyzing t h e r e a c t i o n o f a s u b s t r a t e a t v e r y low c o n c e n t r a t i o n . Many enzymes a r e s p e c i f i c f o r a p a r t i c u l a r r e a c t i o n o f a p a r t i c u l a r substrate even i n t h e p r e s e n c e o f o t h e r isomers o f t h a t s u b s t r a t e or similar compounds. Some o t h e r enzymes are s p e c i f i c for a p a r t i c u l a r c l a s s o f compounds. In 1974, U o t i l a and K o i v u s a l o (J_) r e p o r t e d t h a t t h e o x i d a t i o n of formaldehyde to formate c a n o c c u r i n a l l t i s s u e s , and formaldehyde derived from methanol appears t o be o x i d i z e d by glutathione-dependent formaldehyde dehydrogenase i n the c y t o s o l . Cinti e t a l . ( 8 ) showed that formaldehyde derived from the microsomal N-demethylation reactions is oxidized by a non-glutathione-requiring formaldehyde dehydrogenase i n the mitochondria. In this s t u d y , a n o n - g l u t a t h i o n e - d e p e n d e n t enzyme was u s e d . Two novel fluorometric methods f o r the d e t e r m i n a t i o n of formaldehyde were developed using the enzyme formaldehyde dehydrogenase. The p r i n c i p l e o f t h e s e methods i s based on t h e quantitative oxidation o f formaldehyde w i t h n i c o t i n a m i d e adenine dinucleotide (NAD ) , c a t a l y z e d by formaldehyde dehydrogenase,to form f o r m i c a c i d and NADH as shown i n t h e f o l l o w i n g r e a c t i o n :


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Formaldehyde hydrogenase • > Formic a c i d + NADH ( 1 )

Formaldehyde + NAD

In the f l u o r o m e t r i c method I , t h e NADH produced i s monitored spectrofluorometrically a t an e x c i t a t i o n w a v e l e n g t h ( \ ) °f 348 nm and an e m i s s i o n w a v e l e n g t h (\ ) o f 467 nm. The f l u o r e s c e n c e intensity i s proportional t o t h e c o n c e n t r a t i o n o f formaldehyde. Alternatively, t h e f o l l o w i n g c o u p l e d r e a c t i o n c a n be used f o r more s e n s i t i v e a n a l y s i s o f formaldehyde i n t h e ppb c o n c e n t r a t i o n s : ex

e

Diaphorase


NADH + R e s a z u r i n

>

, NAD + R e s o r u f i n

(2)

The NADH produced i n r e a c t i o n 1 i s i n t u r n o x i d i z e d by r e s a z u r i n . This reaction i s c a t a l y z e d by d i a p h o r a s e which a c t s as an e l e c t r o n carrier. The r e d u c e d form of resazurin i s a highly fluorogenic compound called r e s o r u f i n . The f l u o r e s e n c e p r o d u c t i o n i s measured at \ of 575 nm, and Aem ->90 nm, and i s l i n e a r l y p r o p o r t i o n a l to t h e c o n c e n t r a t i o n o f t h e f o r m a l d e h y d e . The concentrations o f formaldehyde p a r t i c i p a t i n g i n these enzymatic r e a c t i o n s c a n be d e t e r m i n e d by two d i f f e r e n t methods: t h e equilibrium method and the k i n e t i c method (5_,6„)I n the e q u i l i b r i u m method, t h e r e a c t i o n i s a l l o w e d t o go t o c o m p l e t i o n and the product formed i s measured, p r o v i d e d t h e p r o d u c t i s c h e m i c a l l y and/or p h y s i c a l l y d i s t i n g u i s h a b l e from t h e s u b s t r a t e . NADH i n t h e enzymatic method I and r e s o r u f i n i n t h e enzymatic method I I a r e measured fluorometrically and they are proportional to the concentration o f f o r m a l d e h y d e . The e q u i l i b r i u m method i s g e n e r a l l y more precise and reliable, particularly i n t h e manual and non-automated procedures. However, t h i s method r e q u i r e s a l a r g e amount o f enzyme t o ensure r e l a t i v e l y r a p i d r e a c t i o n ; o t h e r w i s e t h e time r e q u i r e d t o r e a c h e q u i l i b r i u m becomes r e l a t i v e l y l o n g . In the kinetic method, t h e i n i t i a l rate o f t h e enzymatic r e a c t i o n i s measured w i t h o u t w a i t i n g f o r t h e r e a c t i o n t o go t o c o m p l e t i o n . The initial rate method i s fast, however, t e m p e r a t u r e , pH and i o n i c strength of buffer, stirring rate and t i m i n g must be c a r e f u l l y controlled f o r good results. I f t h e time required to reach equilibrium i s l o n g , l a r g e q u a n t i t y o f enzyme i s needed and i n t h i s case t h e k i n e t i c method i s p r e f e r r e d o v e r t h e e q u i l i b r i u m method. o

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Method and P r o c e d u r e Reagents. Formaldehyde dehydrogenase s o l u t i o n , 10 u n i t s / m l , was prepared in phosphate buffer (pH 7.5, 0.1M). Formaldehyde dehydrogenase (EC 1.2.1.1) from Pseudomonas p u t i d a was o b t a i n e d from Sigma Chemical Co., S t . l^ouis, Missouri. Oxidized nicotinamide a d e n i n e d i n u c l e o t i d e (NAD ) s o l u t i o n , 5 mg/ml, was prepared using doubly distilled deionized water. Diaphorase Solution, 7 J units/ml, was p r e p a r e d i n phosphate b u f f e r (pH 7.5, 0.1M). NAD and diaphorase (EC 1.6.4.3, from C l o s t r i d i u m kluyveri) were also obtained from Sigma. Formaldehyde dehydrogenase, NAD , and diaphorase solutions should be prepared fresh daily and s t o r e d a t 4 C when they a r e n o t i n use. Resazurin was d i s s o l v e d i n d o u b l y d i s t i l l e d d e i o n i z e d water to g i v e a f i n a l c o n c e n t r a t i o n o f 30 mg/1 s o l u t i o n i n a dark b o t t l e . +


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Resazurin was purchased from A l d r i c h Chemical Co., Milwaukee, Wisconsin. Formaldehyde stock solution was p r e p a r e d by d i l u t i n g 2.7 ml o f 37% formaldehyde s o l u t i o n to 1 l i t e r w i t h d e i o n i z e d water and standardized using the sulfite method ( 3 , 9 ) . T h i s s o l u t i o n remained s t a b l e f o r s e v e r a l months. Formaldehyde" s o l u t i o n was ACS reagent grade and obtained from F i s h e r S c i e n t i f i c , P i t t s b u r g h , Pennsylvania. Formaldehyde s t a n d a r d s o l u t i o n s f o r the c a l i b r a t i o n were prepared daily from the s t o c k s o l u t i o n . Other c h e m i c a l s f o r formaldehyde s t a n d a r d i z a t i o n and buffer preparations were a l l a n a l y t i c a l r e a g e n t s and were used w i t h o u t f u r t h e r p u r i f i c a t i o n . Apparatus. Fluorescent measurements were made w i t h an AMINCO SPF-125 s p e c t r o f l u o r o m e t e r (American Instrument Co., S i l v e r S p r i n g , M a r y l a n d ) equipped with a thermostated cuvette. A s t r i p chart recorder (Omnigraphic-2000, Houston I n s t r u m e n t , A u s t i n , T e x a s ) was used to record the f l u o r e s c e n t i n t e n s i t y as a f u n c t i o n o f t i m e . Temperature was controlled with a LAUDA t h e r m o s t a t e d water b a t h circulator (Model K-2/R, F i s h e r S c i e n t i f i c Company, P i t t s b u r g h , Pennsylvania). Analytical Procedure. For the enzymatic method I, 0.98 ml o f phosphate b u f f e r (pH 8) and 50 y l o f formaldehyde dehydrogenase were p i p e t t e d i n t o a cuvette. To this 400 y l o f formaldehyde sample, or standard, were added, and mixed by shaking for 5 seconds. The cuvette was p l a c e d i n the s p e c t r o f l u o r o m e t e r (^ ^ 348 nm and A = 467 nm) and a s t a b l e b a s e l i n e was o b t a i n e d b e f o r e proceeding. The r e a c t i o n was initiated by injecting a 50 y l solution of NAD into the cuvette with the increase in fluorescence recorded as a f u n c t i o n of time. The f l u o r e s c e n t intensity was measured one minute a f t e r i n j e c t i o n , or at the s t e a d y state. e

e m

+

For formaldehyde analysis using method I I , 0.83 ml of phosphate b u f f e r was p i p e t t e d i n t o a sample c u v e t t e . To t h i s 50 y l of formaldehyde dehydrogenase, 50 y l o f d i a p h o r a s e , and 100 y l o f r e s a z u r i n were added. Next 400 y 1 of formaldehyde sample, or standard, were added, then mixed by s h a k i n g f o r 5 seconds. The cuvette was p l a c e d i n the s p e c t r o f l u o r o m e t e r ( X x 575 nm a n d X » 590 nm) and a s t a b l e b a s e l i n e was o b t a i n e d b e f o r e p r o c e e d i n g . The r e a c t i o n was initiated by the a d d i t i o n o f 50 y l o f NAD solution to the c u v e t t e , w i t h the f l u o r e s c e n c e i n t e n s i t y measured one minute after i n j e c t i o n , or a t the s t e a d y s t a t e . The i n c r e a s e i n f l u o r e s c e n c e was a l s o r e c o r d e d as a f u n c t i o n o f t i m e . a

e

R e s u l t s and

e m

Discussion

Enzymatic F l u o r o m e t r i c Method I . There a r e s e v e r a l f a c t o r s , such as enzyme c o n c e n t r a t i o n , s u b s t r a t e c o n c e n t r a t i o n , pH o f b u f f e r , and temperature, which can a f f e c t the k i n e t i c s o f the enzyme c a t a l y z e d reaction. These factors should be optimized and carefully controlled i n order to o b t a i n the most s e n s i t i v e and r e p r o d u c i b l e results. The r e s u l t s o f the o p t i m i z a t i o n s t u d i e s a r e summarized i n Table I. Figure 1 shows the p l o t s o f the f l u o r e s c e n c e i n t e n s i t y v e r s u s time f o r s e v e r a l d i f f e r e n t c o n c e n t r a t i o n s o f f o r m a l d e h y d e . About 75% o f the f l u o r e s c e n c e can be o b t a i n e d w i t h i n the f i r s t minute and


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Table

I.

O p t i m a l C o n c e n t r a t i o n s o f the Reagents Used i n the E n z y m a t i c F l u o r o m e t r i c Methods


Reagent Formaldehyde Dehydrogenase NAD Diaphorase Resazurin

Amount/Determination Method I I Method I 0.50 u n i t s 0.50 u n i t s 0.25 mg 0.25 mg 3.6 u n i t s 3.0 yg —

the s t e a d y s t a t e i s a c h i e v e d i n about 5 m i n u t e s . After the o p t i m a l c o n d i t i o n s of the a s s a y were i n v e s t i g a t e d , a series of calibration plots were prepared using different formaldehyde concentrations. Figure 2 shows t y p i c a l c a l i b r a t i o n plots f o r 30 seconds, 1 m i n u t e , and a t the s t e a d y s t a t e . I f the f l u o r e s c e n c e was measured at the steady s t a t e , the c a l i b r a t i o n curve fits the g e n e r a l e q u a t i o n Y =» 69.67(X) + 9.82 where Y i s the fluorescent intensity and X i s the corresponding formaldehyde c o n c e n t r a t i o n . A l i n e a r dynamic range was observed up to 0.54 ug/ml. The lowest concentration of formaldehyde i n the a s s a y solution which can be determined w i t h t h i s method i s l i m i t e d by experimental r e p r o d u c i b i l i t y and i n s t r u m e n t a l r e s o l u t i o n , which was found to be 0.02 yg/ml. F i g u r e 2 a l s o shows the c a l i b r a t i o n p l o t s in which f l u o r e s c e n c e was measured a t 30 seconds and 1 minute a f t e r the r e a c t i o n had s t a r t e d , and the d a t a f i t the f o l l o w i n g e q u a t i o n s : Y - 60.87 (X) + 7.58 and Y - 43.18 (X) + 5.38, r e s p e c t i v e l y . Enzymatic Fluorometric Method II. In this method, the concentration of formaldehyde dehydrogenase, d i a p h o r a s e , NAD , resazurin and the pH o f b u f f e r were o p t i m i z e d . The r e s u l t s o f the optimized parameters a r e a l s o shown i n T a b l e I . The times r e q u i r e d to obtain the steady state ( o f about 3 minutes) at d i f f e r e n t formaldehyde c o n c e n t r a t i o n s a r e shown i n F i g u r e 3. The calibration curve was obtained using optimized concentrations o f formaldehyde dehydrogenase, NAD , d i a p h o r a s e , resazurin and b u f f e r pH. The calibration curve measured a t 1 minute after injection fits the equation Y - 120 (X) + 4 . 6 8 as shown i n F i g u r e 4. T h i s f i g u r e a l s o shows the extended c a l i b r a t i o n plot at low c o n c e n t r a t i o n s and the d a t a f i t the e q u a t i o n Y • 0.437 (X) + 11.3. The l o w e s t c o n c e n t r a t i o n o f formaldehyde i n an a s s a y s o l u t i o n which can be determined w i t h t h i s method i s 0.27 ng/ml. The slopes of the c a l i b r a t i o n p l o t s , 60.87 f l u o r e s c e n c e u n i t per ug/ml f o r enzymatic f l u o r o m e t r i c method I and 120 f l u o r e s c e n c e unit per ug/ml f o r enzymatic f l u o r o m e t r i c method I I , show t h a t method I I i s a p p r o x i m a t e l y t w i c e as s e n s i t i v e as method I . T h i s i s due to formation o f the i n t e n s e l y f l u o r o g e n i c r e s o r u f i n i n method II. The higher sensitivity and lower detection limit o f the enzymatic f l u o r o m e t r i c method I I w i l l have p o t e n t i a l a p p l i c a t i o n s in a i r sampling o f formaldehyde e m i s s i o n s s i n c e sampling time can be r e d u c e d . Several i n o r g a n i c and organic compounds such as nitrite, nitrate, phenols, alcohols, organic s o l v e n t s , and aromatic hydrocarbons are known to be i n t e r f e r e n t s i n the c h r o m o t r o p i c a c i d method were investigated. No i n t e r f e r e n c e s were observed from these compounds even a t high c o n c e n t r a t i o n (1,000 y g / m l ) . Some +

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(A):0.270 ug/ml (B):0.189 ug/ml (C):0.135 ug/ml (D):0.027 ug/ml (B) (C)


(D)

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1

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Time, Minute F i g u r e 1. P l o t s o f f l u o r e s c e n c e i n t e n s i t y v e r s u s time i n enzymatic method I .

°l 0

i 0.1

i 0.2

i 0.3

i 0.4

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Concentration of Formaldehyde, F i g u r e 2. C a l i b r a t i o n c u r v e f o r the enzymatic I.

f l u o r o m e t r i c method


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Time, Minute F i g u r e 3. P l o t s o f f l u o r e s c e n c e enzymatic method I I .

i n t e n s i t y versus

time i n

Concentration of Formaldehyde, ng/ml, (#)

£

•

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o 2

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0.1

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Concentration of Formaldehyde, /ig/ml,(0) F i g u r e 4. C a l i b r a t i o n p l o t s f o r the enzymatic f l u o r o m e t r i c method I I i n ppm and ppb l e v e l s .


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higher aldehydes, such crotonaldehyde, benzaldehyde high concentrations.

as and

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acetaldehyde, propionaldehyde, acrolein, s l i g h t l y i n t e r f e r e at


Use o f E n z y m a t i c Methods f o r D e t e r m i n a t i o n o f Formaldehyde E m i s s i o n from Wood P r o d u c t s . The measurement o f formaldehyde r e l e a s e from wood p r o d u c t s i n v o l v e s the collection of formaldehyde vapor i n the t e s t chamber u s i n g a suitable absorbing solution and t h e n a n a l y z i n g the formaldehyde collected. F o r many y e a r s , formaldehyde e m i s s i o n measurements were carried out u s i n g the d e s i c c a t o r t e s t s a m p l i n g method (1,10) due t o its simplicity. In this method, specimens of p a r t i c l e b o a r d or paneling, after c o n d i t i o n e d o v e r n i g h t a t 25 C and 50% r e l a t i v e humidity, are placed i n a clean, dry d e s i c c a t o r containing distilled w a t e r . F o r 24 hours t e s t , 300 ml o f d i s t i l l e d water were used i n p l a c e o f 25 ml used i n the 2 hours t e s t . A t the end o f the testing period, the water solution i s a n a l y z e d f o r formaldehyde content. R e c e n t l y , Lehmann (2) i n v e s t i g a t e d many t e s t p r o c e d u r e s such as l a r g e s c a l e t e s t chamber, m o b i l e home s i m u l a t o r t e s t , q u i c k test, q u i c k a i r t e s t and d e s i c c a t o r t e s t , and found t h a t the l a r g e scale test chamber i s the most a c c u r a t e and r e l i a b l e means o f e s t i m a t i n g formaldehyde e m i s s i o n from wood p r o d u c t s . These test chambers can be incorporated to the e n z y m a t i c methods f o r formaldehyde d e t e r m i n a t i o n . Formaldehyde e m i s s i o n s o f a p r o d u c t , o r mix o f p r o d u c t s , to the ambient a i r can be c o l l e c t e d in distilled water or 1% sodium bisulfite as the a b s o r b i n g solution. After collection, formaldehyde samples a r e a n a l y z e d as described above. In the m o b i l e home s i m u l a t o r t e s t method ( 2 J , double or triple i m p i n g e r s , which a r e p l a c e d i n s e r i e s , s h o u l d be used i n o r d e r to c o l l e c t a l l o f the formaldehyde v a p o r . The t e s t conditions s h o u l d s i m u l a t e the a c t u a l e n v i r o n m e n t . Several factors such as temperature and r e l a t i v e h u m i d i t y o f the system i n c l u d i n g the specimens and background o f formaldehyde i n the t e s t chamber, affect the p r e c i s i o n and accuracy of the r e s u l t s . I t has been shown t h a t a 7 C change i n temperature d o u b l e s the e m i s s i o n level (1_). The temperature of the test chamber s h o u l d be maintained a t + 0 . 1 C. S i n c e formaldehyde i n aqueous s o l u t i o n s is unstable, a l l samples s h o u l d be a n a l y z e d w i t h i n one hour a f t e r collection. The enzymatic methods d e s c r i b e d i n t h i s paper a r e not o n l y more s p e c i f i c but also more s e n s i t i v e than the c h r o m o t r o p i c a c i d method. These methods can be used f o r the measurement of formaldehyde e m i s s i o n from wood p r o d u c t s as w e l l as formaldehyde exposure i n the w o r k p l a c e and i n i n d o o r e n v i r o n m e n t s . Conclusion We have d e v e l o p e d two n o v e l new e n z y m a t i c f l u o r o m e t r i c methods f o r the trace analysis of formaldehyde. Due to t h e i r s i m p l i c i t y , sensitivity and s p e c i f i c i t y , t h e s e methods should f i n d wide applications i n the m o n i t o r i n g o f formaldehyde r e l e a s e d from wood products. As we stated above, e n z y m a t i c f l u o r o m e t r i c method I I does offer h i g h e r s e n s i t i v i t y and better detection l i m i t over enzymatic fluorometric method I . However, method I I r e q u i r e s two


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enzymes and is more expensive than method I, which only uses one enzyme. So the choice between use of method I or method II depends upon your need. If you are not concerned about the sensitivity and the low detection limit, you may simply use method I. Furthermore, the enzymes can be immobilized and can then be reused many times, up to several hundred assays, thus substantially reducing the cost of analysis. An obvious application of the immobilized formaldehyde dehydrogenase is in the automated flow injection system for analysis of large numbers of environmental samples. Such extension of the work described here is already in progress in our laboratory.


Acknowledgments This work was supported by the University of Alabama at Birmingham, Faculty Research Grant 2-12726. Literature Cited 1.

McVey, D. T. Proc. 16th Wash. State Univ. Int. Sym. Particleboard, 1982, p. 21. 2. Lehmann, W. F. Proc. 16th Wash. State Univ. Int. Sym. Particleboard, 1982, p. 35. 3. "Methods of Air Sampling and Analysis," American Public Health Association: Washington,D.C.,1977, 2nd ed.,pp.303-307. 4. Krug, E. L. R.; Hirt, W. E. Anal. Chem. 1977, 98, 1865. 5. Guilbault, G. G. "Handbook of Enzymatic Methods of Analysis"; Marcel Dekker: New York, 1976 6. Carr, P. W.; Bowers, L. D. "Immobilized Enzymes In Analytical and Clinical Chemistry"; John Wiley & Sons: New York, 1980. 7. Uotila, L. Koivusalo, M. J. Biol. Chem. 1974,249, 7653. 8. Cinti, D. L.; Keyes, S. R.; Lemelin, M. A.; Denk, H.; Schenkman, J. B. J. Biol. Chem. 1976, 251, 1571. 9. Walker, J. F. "Formaldehyde"; Reinhold Pub. Co.: New York, 1964; p. 486-7. 10. Newton, L. R. Proc. 16th Wash. State. Univ. Int. Sym. Particleboard, 1982, p. 45. RECEIVED January 14, 1986


Enzymatic Methods for Determining Formaldehyde Release from

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