Atomic Absorption Method for Determining Micromolar Quantities of Aldehydes Philip J. Oles and Sidney Siggia Department of Chemistry, University of Massachusetts. Amherst. Mass. 0 1002
The quantitative analysis of aldehydes using atomic absorption spectrophotometry is described. Silver-ammonia complex (Tollen's reagent) is used to oxidize aldehydes to their corresponding carboxylic acids. The reduced silver is separated from excess reagent, dissolved in nitric acid, and the resultant solution is analyzed for silver content using conventional atomic absorption spectrophotometry. A Hammett relationship for substituted benzaldehydes is shown to exist and its use enables an estimation of the reactivities of substituted benzaldehydes not reported in this study. The effects of multiple substituents are additive and substituted benzaldehydes with a net Hammett constant value of - 0 . 2 do not react quantitatively by this procedure. One calibration curve was found to be suitable for all of the aldehydes encountered. The relative standard deviations are 1.2-5.8% in the 1-4 wmol/ ml range.
The oxidation of aldehydes by silver(1) has formed the basis of several procedures for the quantitative determination of aldehydes. This reaction has been recognized as being specific for aldehydes although some ketones and hydroxy ketones have been reported to react to various degrees (1, 2). T h e use of solid silver oxide (3, 4 ) , silverammonia complexes (Tollen's reagent) ( 5 ) and silver-tertbutylamine complexes (6) have resulted in successful titrimetric procedures for the determination of millimolar quantities of aldehydes. Radiochemical methods (7, 8) have included the use of l1OrnAgN03 to prepare the reagent and measurement of the reduced llomAg produced in the reaction, or reaction of the silver metal with K1311 and K I 0 3 and radioassay of the resultant Ag1311. These procedures have been applied to the determination of 0.1 pmol quantities of glucose and fructose. The present work evaluates the silver-ammonia complex and the silver-tert-butylamine complex as oxidizing agents for aldehydes at the micromolar level. Rate data are presented which indicate the practical limits of detection for aliphatic aldehydes and substituted benzaldehydes. A Hammett relationship is shown to exist for the rate of oxidation of substituted benzaldehydes and its use enables an estimation of the reactivities of compounds not investigated in this study. Data are presented which show that the net effect of more than one substituent is additive in some cases. The determination of chloride-containing samples and samples of aldehyde in ethanol are described. The reactivity of aldehydes is shown to decrease significantly in ethanol. The titration procedures described previously involving the determination of unreacted silver complex precludes the use of reagents more con(1)
(21 (3)
(41 (51 (6) (7) (81
"Instrumental Methods of Organic Functional Group Analysis," s. Siggta. Wiley-Interscience, New York, N . Y . , 1972. pp 98-100. "Quantitative Organic Anaiysis via Functional Groups," S Siggla, John Wiley 8, Sons lnc., New York, N Y . , 1963, p 94 H . C. Bailey and J. H . Knox. J . Chem. SOC..London. 1951, 2741 J Mitchell.Jr.. and D M Srntth. Anal Chem. 2 2 , 746 (1950) S Siggia and E. Segal, A n a l . Chem 25, 640 (1953) J. Mayes E. Kuchar, and S. Siggta,A n a / . Chem . 36, 934 (1964) M . Jaarena. Acta Chem. S c a q d . 8, 860 (1954) J . Z Beer. Talanta. 8, 809 (1961)
centrated than approximately 0.1M Ag+. In this study, however, 0.5-1.OM AgN03 is used to prepare the Tollen's reagent. Since this reagent has been reported (2) to be dangerously explosive a t lower concentrations of silver and ammonia after 24 hours, all rate data and most determinations were carried out using reagent prepared from 0.5M AgN03. I t was observed t h a t this reagent is stable for at least 4 hours. Reagent prepared from 1M AgN03 is stable for a t least 2 hours. The reaction is pseudo-first order with respect to aldehyde concentration; therefore, addition of smaller amounts of more concentrated reagent minimizes the dilution of the aldehyde in the sample, thereby decreasing the time required for quantitative recovery. This procedure will be particularly useful for determining total aldehyde content in many systems in which a number of aldehydes are known to be present-e.g., distilled liquors, flavorings, or perfumes. All values of Hammett constants were obtained from reference (9).
EXPERIMENTAL Apparatus. Absorbances were measured a t 328.1 nm with a Perkin-Elmer 403 Atomic Absorption Spectrophotometer. A fine frit (4-5.5 micrometer) Pyrex glass funnel was used for all filtering. The use of a medium frit (10-15 micrometer) funnel results in significant losses of the silver precipitate and, therefore, was not used. Reagents. The modified Tollen's reagent previously described by Mayes et al. (6) was used in all cases except where otherwise stated and is prepared as follows: 5.00 ml of 0.50M AgNO, are pipetted into a 50-ml beaker; exactly 1.00 ml of 3M NaOH is added and the contents are agitated. Sufficient 1:l NH3 (specific gravity 0.90):HzO is then added dropwise to dissolve all of the AgZO present. It was determined that 2.00 ml of 1:l NH3:HzO would just dissolve the AgzO present, and subsequently all rate data were obtained using exactly this amount. This reagent must be prepared fresh and should be used immediately after its preparation. The formation of a highly explosive black precipitate has been reported ( 2 ) when a less concentrated reagent was allowed to stand for 24 hours and, therefore, all unused reagent was discarded within 4 hours of its preparation. Butyraldehyde, propionaldehyde, p-nitrobenzaldehyde, rn-nitrobenzaldehyde, and p-chlorobenzaldehyde were purchased from Eastman Organic Chemicals. Formaldehyde (36.8% in HzO) and benzaldehyde were purchased from Fisher: rn-cyanobenzaldehyde, rn-methoxybenzaldehyde, vanillin (4-hydroxy-3-methoxybenzaldehyde), p-methoxybenzaldehyde, 3,5-dimethoxybenzaldehyde, p-acetamidobenzaldehyde, and p-tolualdehyde were purchased from Aldrich. All liquid aldehydes with boiling points greater than 100 "C were vacuum distilled and sealed under nitrogen. Lower boiling aldehydes were distilled a t atmospheric pressure under nitrogen and immediately sealed in an appropriate container. All solid aldehydes were purified by recrystallization twice from ethanol with the exception of rn-cyanobenzaldehyde which, because of the small amount of material available was used as received (Aldrich analyzed, 97%). Distilled. deionized water was used throughout and was found to result in a reproducible blank value equivalent to 0.06-0.08 Fmol RCHO. It is most probable that this blank value arises from carryover of silver from the reagent and not oxidation of trace amounts of organic material in the water. Aqueous solutions of substituted benzaldehydes (91
"Physical Organic Chemstry , J Hine. McGraw-Hlll Book Company, Inc New York, N Y , 1962, p 8 7
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Table I. Analysis of Aldehydes b y O x i d a t i o n w i t h Tollen’s Reagent
Compound
Formaldehyde Propionaldehyde Butyraldehyde p-N02-benzaldehyde m-NOy-benzaldehyde m-CN-benzaldehyde p-Cl-benzaldehyde m-OCH3-benzaldehyde Benzaldehyde 3,5-Dimethoxybenzaldehyde p-N02-benzaldehyde p-N02-benzaldehyded p-NO?-benzaldehydec
Concentration, pmol/ml
Taken, @mol
Found, rmol
3.28 3.47 1.13 0,990 1.029 1.004 1.007 0.985 3.93 1.001
1.64 1.74 1.13 0.990 1.029 1.004 1.007 0.985 1.96 1,001
1.65 1.72 1.13 1.00 1.03 0.97 1.02 1.02 1.98 1.01
0,990 1.07 1.00
0.099 1.07
0.094
1.00
1.00
Recovery, Vo
Re1 std de@
101 98.8 100 101 96.6 101 103 101 101
2 ~ 2 . 5(5) h 3 . 9 (5) 1 4 . 4 (4) L 4 . 5 (5) i 5 . 8 (5) h 5 . 2 (5) h 5 . 8 (5) h 5 . 5 (5) 1 1 . 2 (3) C 3 . 0 (5)
100
95 97.3 100
4 ~ 4 . 2(3) h 5 . 7 (4) 1 3 . 2 (4)
25 25 120
100
1.04
Minimum reaction time, min
‘ Figures in parentheses indicate number of determinations. * 0.1M AgNO3 used in preparing reagent.
30b 135 120 25 30 45 90 120 6OC
0.25M AgN03 used in preparing reagent.
Analysis
carried out in the presence of 750 ppm KCl. e Samples in ethanol.
were found to be stable for 2-3 days, providing the substituents had positive Hammett constant values greater than +0.20. Aqueous solutions of aliphatic aldehydes and all other substituted benzaldehydes were found to deteriorate rapidly after 12 hours. A stock silver nitrate (Fisher) solution was prepared by weighing out 0.20-0.35 gram of AgN03 (dried at 100 “C for 1 hour), transferring to a 250.0-ml volumetric flask and diluting to volurhe with 1:l concd HN03:H20. Standard solutions were then prepared by diluting appropriate aliquots of this stock solution. The stock silver nitrate solution was found to be stable for about l week. and the standard solutions were prepared just prior to each determination. Procedure. A 0.100- to 1.00-ml sample of aldehyde in water is added to a 6-inch test tube. Depending upon the reactivity of the aldehyde, the concentration should be between 0.25-4.00 pmol/ ml. Under minimum lighting conditions, a volume of Tollen’s reagent equal to the volume of the sample is then added to the test tube. The contents of the test tube are mixed thoroughly and the test tube is placed in a light tight container and placed on a mechanical agitator for the specified time. After this time, the contents of the test tube are transferred to a fine fritted glass funnel and suction is applied. The test tube is rinsed with two 5-ml portions of 1:l NH3:H20 to dissolve any silver oxide present and two 5-ml portions of water; in both cases allowing the rinsings to pass through the filter. A clean 125-ml filter flask is placed below the filter and 6 ml of 1:l concd HN03:HzO is added to the test tube to dissolve the adhering silver mirror. This solution is then transferred to the filter and, upon dissolution of the silver precipitate, suction is applied. The test tube is rinsed with two 5-ml portions of water and these are also passed through the filter and collected. The contents of the suction flask are then transferred to a 50.0-ml volumetric flask and diluted to volume with water. In cases where less then 0.50 pmol of aldehyde is present, the acid and water volumes are adjusted so that the final volume of the sample is 10.0 ml. This solution is then analyzed by atomic absorption spectrophotometry for silver content. A calibration curve is prepared by taking aliquots of the stock silver nitrate solution and diluting to the same final volume as the sample. All rate data were obtained under the following conditions except where otherwise noted: [Ag+] = 0.156M: [RCHO] = 0.50 f 0.02 pmol/ml: temperature = 28 f 1 “C; [NaOH] = 0.19F; [NH3] = 0.94F. RESULTS AND DISCUSSION Choice of Silver Oxidant. The silver-tert-butylamine complex (6) which has been applied successfully on the millimolar scale was found to be unsuitable on the micromolar scale. The determination of 1.64 pmol of formaldehyde with 0.1M or 0.2M silver-tert-butylamine reagent resulted in low b u t reproducible recoveries. With reaction times of 0.5-2.0 hours, recoveries ranged from 72.5-78.0’70 using either the 0.1M or 0.2M reagent. Under the identical experimental conditions, using 0.1M AgIL’03 to prepare the Tollen’s reagent. quantitative recoveries were ob912
*
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tained. It is postulated t h a t in the presence of tert-butylamine, the following reaction may occur:
HCHO
+ H,N-C(CH,), * H?C=N-C(CH,), + H,O (1)
The imine formed in reaction 1 is not oxidizable by the silver reagent, and therefore, the consumption of aldehyde by tert-butylamine results in low recoveries. The use of Tollen’s reagent does not result in low recoveries since the reaction between ammonia and formaldehyde (to form hexamethylenetetramine) would be unfavorable a t low formaldehyde concentrations and, therefore, the oxidation reaction proceeds to completion. The extent of reaction of ammonia with any of the remaining aldehydes to form the benzylidenimine is apparently insignificant since quantitative recoveries were obtained. The reaction of tert-butylamine with aldehydes other than formaldehyde would most likely occur to various degrees; therefore, it was decided to adopt the Tollen’s reagent in this work. At the start of this study, very large and irreproducible blank values (equivalent to 0.3-0.5 pmol RCHO) were obtained using either the silver-ammonia or the silver-tertbutylamine complexes. It was found that in a n open vessel and also under the vacuum from the filtering operation, the following reactions occur:
Ag(NH,),f 2Ag’
+ 20H-
Ag’
+ 2NH,
+ Ag,O
+ H,O
(4)
Analogous reactions also occur with the silver-tert-butylamine complex. The precipitation of AgzO a5 illustrated in Equation 4 was found to be the source of the large blank value. Washing the precipitate with NH3:H20 1:l dissolves any Ag2O present and was found to reduce the blank value to the order of 0.06-0.08 pmol RCHO. Determination of Micromolar Quantities of Aldehydes w i t h the Modified Tollen’s Reagent. The results of the determination of aliphatic and aromatic aldehydes using the modified Tollen’s reagent appear in Table I. The determination of 0.099 pmol of p-nitrobenzaldehyde was carried out with a final sample volume of 10.0 ml. In all other cases, samples were diluted to 50.0 ml so t h a t the silver concentration would lie in the linear region of the calibration curve for silver. The reactivity of aliphatic aldehydes, other t h a n formaldehyde. was observed to be much less than the substituted benzaldehydes at the con-
+140t I
4201 I
50
1
+040. hi
.b
oI .020J 0
000.
45
90
t (min) Figure 3.
Reaction-time curve for p-tolualdehyde
1001
0
30
60
90
120
150
t (min)
02NQCH0
0
0
5
15
10
t Figure 2.
Figure 4.
Reaction-time curve for 3,5-dimethoxybenzaldehyde
L
20
25
(miri)
100.
Reaction-time curve for p-nitrobenzaldehyde 80-
centrations studied. For this reason, their determination is best accomplished by using smaller amounts of more concentrated reagent as described in a later section. The Hammett Relationship. From previous work involving funtional group analysis in dilute solutions ( I O ) , it was apparent t h a t the reactivity of a particular functional group and not the capability of the measuring device (AA, XRF, etc.) would ultimately determine the limit of detection. For this reason the existence of a Hammett relationship (Figure 1) among substituted benzaldehydes was considered as a means of systematizing their reactivity. The Hammett relationship is defined as follows:
log klk’
=
pu
(5)
where in this case, k is the rate constant of the substituted benzaldehyde, h’ is the rate constant of benzaldehyde, p is a measure of the sensitivity of the reaction rate to changes in the u value of the substituent, and u is the Hammett Substituent Constant. Since k is inversely proportional to the half life of the reaction, tl 2, Equation 5 may be rewritten: log l’l?’/tX = pa (6) Under the conditions employed in this study, p = +1.8 indicating t h a t substituents will greatly affect the reactivity of the substituted benzaldehyde. The practical significance of the large absolute value of the slope in Figure 1 is illustrated by a comparison of the rate curves for p-nitrobenzaldehyde ( u = +0.778) and p-tolualdehyde ( u = ( 1 0 ) P J Oles and S Siggia 45, 2150 (1973)
I $60.
Y 840-
8
20-
/ 1
OOCY
Figure 5.
30
60
90 t (rntn)
120
1
150
Reaction-time curve for p-chlorobenzaldehyde
-0.170) in Figures 2 and 3. A comparison of rate curves for 3,5-dimethoxybenzaldehydeand p-chlorobenzaldehyde (Figures 4 and 5 ) indicate that, in some cases. multiple substituents will have additive effects upon the reactivity since u = +0.227 for p-C1 and u = +0.113 x 2 = +0.230 for 3,s-dimethoxy. The fact that the Hammett relationship exists may be used to explain some observations concerning other compounds encountered in this study and other observations previously reported ( 2 ) .p-Methoxybenzaldehyde ( u = -0.268) does not undergo a quantitative reaction with the Tollen’s reagent, either on the micromolar or the millimolar level. This may be due to the fact that since the oxidation reaction is slow, the side reaction between ammonia and the aldehyde to form the substituted hydrobenzamide may occur to a significant extent (11). (11) Y Ogata A Kawasaki and N Okumura J Org Chem (1964)
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solvent for aldehydes in this procedure is not recommended a t aldehyde concentrations of the order of 1.00 pmol/ ml. Effect of Silver Concentration. The reaction rate data obtained with the Tollen's reagent described in the experimental section (0.3M Ag+ = final concentration) indicate that the reaction is approximately pseudo-first order with respect to aldehyde concentration for some of the aldehydes studied. Therefore, as the reagent is made more concentrated, the effect of adding smaller quantities of reagent (to avoid dilution of the aldehyde) was studied. The results shown in Table I1 indicate that the time required for the quantitative recovery of the aldehyde is drastically reduced by adding less reagent and thereby reducing the dilution of the aldehyde to a minimum. The Tollen's re-
T a b l e 11. Effect of D i l u t i o n of Aldehyde by Adding Various A m o u n t s of Reagent. Analysis of 1.00 pmol/ml p-Chlorobenzaldehyde Recovery, Time, min
1.00 ml reagent addeda
15 30 60 75
70 79 88 100
Yo 0.50 ml reagent addedb
76 100 100
100
'' Final approximate concentrations are: (Ag+l = 0.3F; (RCHOI = 0.5 @mol/ml; [OH-] = 0.37F; [NHIOH] = 1.9F.b Final approximate concentrations are: [Ag-I = 0.2F; [RCHO] = 0.67 pmol/ml; [OH-] = 0.25F; [NHPOH]= 1.3F.
Table 111. D e t e r m i n a t i o n of Total Aldehyde C o n c e n t r a t i o n in Mixtures of Aldehydes Concentration, pmol ArCHO/ml
Taken, pmol
Found, rmol
Recovery, R
Re1 std devb
+
0.963
0.963
0.97
101
h 4 . 6 (5)
+
1.655
0.828
0.82
100
+ 5 . 5 (4)
Mixture"
0.535 pmol m-nitrobenzaldehyde 0.428 pmol p-nitrobenzaldehyde 0.299 pmol m-cyanobenzaldehyde 0.529 pmol 3,5-dimethoxybenzaldehyde "
Analysis accomplished with reagent prepared from 1M AgNO3. Figures in parentheses indicate number of determinations.
p-Acetamidobenzaldehyde ( a = +O.OO) should react a t approximately the same rate as benzaldehyde. However, quantitative recoveries were not obtained, presumably since p-acetamidobenzaldehyde will hydrolyze under the experimental conditions to p-aminobenzaldehyde which may then condense with another molecule of p-aminobenzaldehyde to form the resultant imine. Further, p aminobenzaldehyde (a = -0.66) would be quite unreactive toward the silver oxidant. Vanillin (4-hydroxy-3methoxybenzaldehyde) did not react under the experimental conditions after a period of 3 hours. The additive effects of p - 0 - ( a = -1.00) and m-OCH3 (a = fO.115) may be used to predict that overall vanillin should be quite unreactive a t the concentration employed in this work (1.00 pmol/ml). Effect of Chloride, A solution of p-nitrobenzaldehyde (1.07 pmol/ml) was prepared containing 750 ppm KCl to determine the effects, if any, upon the analysis by the chloride ion. The results of this analysis appear in Table I. It was observed that sufficient ammonia is present in the reagent t o just dissolve any silver chloride which precipitates from the solution. Larger amounts of chloride present in a sample would probably require additions of ammonia which has been reported (2) to decrease the rate of reaction. Effect of Solvent, T o determine the effect of the solvent upon reactivity of the aldehydes, a solution of p-nitrobenzaldehyde (1.00 pmol/ml) in pure ethanol was prepared and subsequently analyzed for aldehyde content. Blank values were somewhat higher in this solvent (equivalent to ca. 0.4 pmol RCHO); however, they were reproducible and were not a function of time. When 1.00 ml of the Tollen's reagent is added to a 1.00-ml sample of ethanol, a precipitate of silver oxide is immediately observed. The addition of approximately 0.2 ml of NH3:HzO 1:l is required to dissolve the silver oxide precipitate. The results of this determination appear in Table I. Two hours are required to obtain a quantitative recovery of 1.00 pmol of p-nitrobenzaldehyde. Therefore, the use of ethanol as a 914
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 7 , JUNE 1974
agent used in this study was prepared by pipetting 5.00 ml of 1.OM AgN03 into a beaker, adding 1.00 ml of 6M NaOH, and 2.00 ml of concd "3. The volume of this reagent which was found to produce the optimum time required for the quantitative recovery of p-chlorobenzaldehyde was found to be 0.50 ml. Theoretically, a more concentrated reagent could be prepared by adding solid NaOH to the silver nitrate solution and passing NH3 gas through the resultant solution to dissolve the AgzO formed, however, this procedure was not investigated. Concentration R a n g e of Aldehyde. The oxidation of aldehydes with Tollen's reagent proceeds by the following reaction: RCHO
-
+ 2Ag(YH,),+ + 2 0 H 2Ag + RCOONH, + 3XHl + HjO
(7)
The optimum working range for silver under the instrumental conditions employed is reported to be 2-20 pg/ml (22). This corresponds to 0.10-1.0 pmol of aldehyde or 0.5-5.0 pmol of aldehyde depending upon whether the final volume of the samples is 10.0 ml or 50.0 ml. Of course, larger amounts of aldehyde may be determined by this method by diluting to a larger final volume, or by choosing a less sensitive line for the analysis of silver, for example, the 338.3-nm line. Analysis of Mixtures. The results of the analysis of some mixtures of aldehydes appear in Table 111. It was found that each aldehyde in a mixture reacts independently, and the reaction time required for the quantitative recovery of the total aldehyde content of a sample is approximately the same time required for the quantitative recovery of the slowest reacting component in a mixture. Received for review October 9, 1973. Accepted January 23, 1974. This work was supported by Grant No. GP-28054 and GP 37493X from the Kational Science Foundation. ( 1 2 ) 'Analytical Methods for Atomic Absorption Spectrophotometry Perkin-Elmer Corporation. Norwalk, Conn , 1968
