Analysis of Saturated Aldehydes by Gas-Liquid Chromatography Using Methylolphthalimide for Regeneration of Their Girard=TDerivatives Donald F. Gadbois, P a u l G.Scheurer,' and Frederick J. King U.S. Bureau of Commercial Fisheries, Gloucester, Mass. 01930 SEVERAL METHODS have been used to isolate and identify micro amounts of carbonyls such as those found in food products. Identification has been accomplished by paper (1-3), column (4-6), thin layer (7, 8) and gas-liquid chromatography (9) of their 2,4-dinitrophenylhydrazonederivatives or by regeneration of carbonyls from their 2,4-dinitrophenylhydrazone derivatives followed by gas-liquid chromatography (10-12). Carbonyls have also been identified by gasliquid chromatography of their oximes (13, 14) or Girard-T (15) derivatives. To identify microquantities of carbonyls by such methods, a sample concentration step is often necessary. Dilute aqueous solutions of carbonyl mixtures have been concentrated by formation of their insoluble 2,4-dinitrophenylhydrazone derivatives in yields of 90% or greater (16). This precipitate is usually isolated either by filtration or extraction but in the case of dilute aqueous solutions, a significant amount of derivative material may remain in solution or colloidal suspension with respect t o the amount isolated (17). Dilute aqueous solutions of standard aldehydes (C2-C,), ketones (C,-C8) and other types of carbonyls have been concentrated by a modified Girard-T procedure which was described as semiquantitative (15). The object of the present investigation is t o study the quantitative aspects of a Girard-T procedure for concentrating carbonyls (15). The recommended procedure includes the use of methylolphthalimide (18) or paraformaldehyde (19) 1 Present address, R.F.D. $1, Spofford Road, Boxford, Mass. 01921
(1) D. A. Buyske, L. H. Owen, P. Wilder, and M. E. Hobbs, ANAL. CHEM., 28,910 (1956). (2) W. S. Lynn, Jr., L. A. Steele, and E. Staple, ibid., 28, 132 (1956). (3) R. G. Rice, G. J. Keller, and J. G. Kirchner, ibid., 23, 194 (1951). (4) E. A. Corbin, ibid., 34, 1244 (1962). (5) D. F. Meigh, Chem. hid, (Loiidori), 1956, 986. (6) D. P. Schwartz. J . Chromatog., 9, 187 (1962). (7) J. H. Dhont and C. de Rooy, Aiialyst, 86, 74 (1961). (8) J. Rosmus and Z. Deyl, J . Chromatog., 6, 187 (1961). (9) R. J . Soukup, R. J. Scarpellino, and E. Danielczip, ANAL. CHEM., 36, 2255 (1964). (10) T. P. Dornseifer and J. J. Powers, Food Teclinol., 17, 118 (1963). (11) J. W. Ralls, ANAL.CHEM., 32, 332(1960). (12) R. L. Stephens and A. P. Teszler, ibid., 32, 1047 (1960). (13) J. Cason and E. R. Harris, J . Org. Cliem., 24, 676 (1959). (14) L. J. Lohr and R. W. Warren, J . Chromafog., 8, 127 (1962). (15) D. F. Gadbois, J. M. Mendrlsohn, and L. J. Ronsivalli, ANAL. CHEM.,37, 1776 (1965). (16) J. H. Ross, ibid., 25, 1288 (1953). (17) J. M. Mendelsohn and M. A. Steinberg, Food Technol., 16, 113, (1962). (18) L. W. Kissinger and H. E. Ungnade, J . Org. Cliern., 23, 815 ( 1958). (19) L. F. Fieser and Mary Fieser, "Organic Chemistry," 3rd ed., Reinhold, New York, 1956, p 201. 1362
ANALYTICAL CHEMISTRY
as the source for generation of formaldehyde which is used for regeneration of carbonyls from their Girard-T derivatives. Although ethanol is the preferred solvent in the literature for reaction of Girard-T reagent with carbonyls, it reacts with the Girard-T reagent (20). To reduce this interaction, we used t-butanol because it lacks alpha hydrogens and is, therefore, relatively inert. In addition, the reaction of Girard-T reagent with alkanals is reported to occur under the mildest of reaction conditions if t-butanol is the solvent (20). EXPERIMENTAL
Apparatus. An F & M gas chromatograph Model 810 ( F & M Scientific Co., Avondale, Pa.) was used with dual hydrogen flame detectors, an automatic column oven temperature programmer, a -0.2 to 1.0-mV Minneapolis Honeywell recorder and a Disc Chart Integrator Model 201-B. The retention column consisted of a IO-foot by */*-inch stainless-steel coiled tube packed with 10% (by weight) Carbowax 20M coated on 90jlOO mesh Diatoport-S. Initially, the column temperature was set at 90 "C and maintained at that temperature for 1 minute after sample injection. Then the temperature was programmed to increase a t the rate of 4 "C per minute until it reached the preset maximum of 175 "C. The operating conditions were: detector temperature, 255 "C, injection port temperature, 260 "C, nitrogen carrier gas flow, 15 ml per minute (measured at the column outlet by a soap bubble flowmeter); hydrogen flow, 40 ml per minute; air flow, 400 ml per minute; sample size, 2 pl for standard aldehyde mixture and 2 p1 for regenerated aldehydes; chart speed, '/z inch per minute; range l o 2 ; attenuation 2. Reagents. Reagent grade t-butanol was freed of residual carbonyls by shaking with concentrated sodium bisulfite solution (100 grams of sodium bisulfite in 300 ml of distilled water). The butanol layer was separated and shaken with 1 gram of sodium hydroxide pellets to remove sulfur dioxide and distilled to give an azeotrope (11.8% H 2 0 8 8 . 2 z t-butanol, b.p. 79.9 "C). Distilled water was purified to remove traces of carbonyls by distillation after refluxing a 1:l aqueous sulfuric acid solution. Reagent grade Girard-T reagent, 12.2 grams, was purified by crystallization from 17 ml of purified t-butanol azeotrope. The yield was 2.6 grams of pure crystalline reagent following two recrystallizations. Methylolphthalimide regenerant was prepared by reacting 0.28 mole of phthalimide with 0.29 mole of formaldehyde (18). The methylolphthalimide product was recrystallized three times from the t-butanol azeotrope and made up to 10 ml. Formaldehyde was prepared by depolymerizing 40 grams of paraformaldehyde dissolved in 60 grams of purified distilled water under mild reflux for 20 hours. The hot solution was then filtered through glass wool into a stoppered 125-ml
+
(20) A. M. Gaddis, Rex Ellis, and G. T. Currie, Nature, 19, 1391 (1961).
COLUMN TEMPERATURE
/;;90?--90’ I
114.
138‘
162’
1750-
6
12
18
22
(t)
_______________-_--_--------175’ 24
I
36
30
Figure 1. Standard aldehyde mixture in t-butanol diluted 1:lO
Erlenmeyer flask and stored in an oven held at 70 “C. The liberating reagent was prepared by diluting 2 ml of this solution to 10 ml, using the r-butanol azeotrope. Preparation of Standard Mixture. A standard mixture of 13 aldehydes (reagent grade) was dissolved in 50 ml of t-butanol azeotrope. A 1-ml aliquot of the standard mixture contained: 0.00008 mole of undecanal, 0.00005 mole of “citral” ( 4 0 x neral and 6 0 x geranial), 0.00008 mole of decanal, 0.00007 mole of nonanal, 0.00011 mole of octanal, 0,00011 mole of heptanal, 0.00010 mole of hexanal, 0.00017 mole of valeraldehyde, 0.00015 mole of isovaleraldehyde, 0.00016 mole of butyraldehyde, 0.00022 mole of isobutyraldehyde, 0.00021 mole of acrolein, and 0.00021 mole of propionaldehyde, The total concentration of aldehydes was 0.0017 moles per ml. Procedure. FORMATION OF GIRARD-TCOMPLEXES. The standard mixture of aldehydes was reacted with 300 mg (0,0018 mole) of Girard-T reagent in the presence of 250 mg of Rexyn 102(H), and 50 ml of r-butanol azeotrope. This mixture was refluxed for 1 hour at 80 “C filtered over glass wool and concentrated at room temperature to about 5 ml in a Buchler rotary evaporator. The concentrated solution was transferred quantitatively with t-butanol to a 25-ml volumetric flask and then diluted t o exactly 25 ml with the solvent. A 10-ml aliquot of this solution was pipetted into a 25-1111 Erlenmeyer flask and evaporated a t 40 “C t o near dryness using a stream of prepurified nitrogen. The
remaining solvent was removed by distilling the flask’s contents at room temperature under high vacuum. The residue, containing the Girard-T derivatives, was dissolved in approximately 0.5 ml of t-butanol. The solution was transferred quantitatively to a 1-ml volumetric tube and diluted to 1 ml. REGENERATION OF ALDEHYDES.A 5-111 aliquot of the Girard-T solution was injected into a capillary tube (0.8 to 1.2 by 90 mm), which was sealed at one end. Then 5 pl of the regenerant solution (either paraformaldehyde or methylolphthalimide) was injected into the capillary tube without contacting the other solution. After the open end had been sealed, the contents were mixed and the aldehydes in the tube were regenerated by placing the tube into the injector assembly and heating the assembly in the injector port of the gas chromatograph at 200 “C for 2 minutes, After complete reaction, the capillary tube was withdrawn and cooled to room temperature. The capillary was cleanly broken above the solution, and a 2 - 4 aliquot was withdrawn and injected into the injector port of the gas chromatograph. This procedure was more practical than the F & M solid sample injector technique because it avoided the difficulty of sealing an aldehyde mixture in a capillary tube without losing some of the more volatile aldehydes. CALCULATIONS. The concentration of a regenerated aldehyde was obtained by comparing its peak area on a chromatogram with that of a standard. Per cent recovery figures
COLUMN TEMPERATURE (‘C)
;90?--90’ ,140 1
i
6
138‘
I2
162‘ I8
-__-__----_---______------_-----175‘
1750,
I
,
I
22
24
!
I
30
36
Figure 2. Girard-T derivatives of standard aldehyde mixture liberated by formaldehyde VOL. 40, NO. 8, JULY 1968
e
1363
't w
8 + W
n
COLUMN TEMPERATURE ("C)
90'--9O0 114"
I
I
0
138'
162"
________175'
1750 I
1
I
I
I
I
6
12
18
22
27
Figure 3. Blank from purified t-butanol, Girard-T, and formaldehyde reagents were based on calculating the theoretical amount of regenerated aldehyde in a sample. The principal source of error in calculating recovery figures was a i5 error in measuring peak area.
DISCUSSION
RESULTS
The results demonstrate that the procedure is quantitative for most of the aldehydes studied (Table I). Figure 1 illustrates the efficiency of the gas chromatographic procedure for resolving the components of a mixture of standard aldehydes, while Figure 2 illustrates a similar efficiency after treating this mixture with Girard-T reagent and the methylolphthalimide regenerant. Comparison of Figures 1 and 2 shows that the derivatization-regeneration procedure did not introduce extraneous peaks on the chromatogram. However, when paraformaldehyde is used for regeneration, interfering peaks can result on a chromatogram because of inherent impurities in the reagents. For example, in Figure 3, peaks A
Table I. Gas Chromatographic Analysis of Aldehydes, after Regeneration from Their Girard-T Derivatives Retention Recovery time, obtained, Peak no. Aldehyde compound minutes Propionaldehyde 1.8 80.3 1 2.0 85.1 Isobutyraldehyde 2 Acrolein 2.4 33.4 3 2.8 See Notea 4 Butyraldehyde 3.5 93.5 5 Isovaleraldehyde 6 4.1 95.4 Valeraldehyde 1 Hexanal 7.5 96.7 10.9 100.0 Heptanal 8 Octanal 14.6 100.0 9 Nonanal 10 18.5 100.0 22.3 100.0 Decanal 11 Undecanal 12 26.8 100.0 Neral 13a 87.2 31.9 Geranial 13b 33.0 87.7 a Not calculated because the solvent peak (t-butanol) and the butyraldehyde peak were not sufficiently separated from each other on the chromatogram.
z
1364
ANALYTICAL CHEMISTRY
and B represent less than 0.2 pg of material, and the two peaks before the t-butanol peak were related to the amount of excess formaldehyde present.
The results demonstrate the improvements in the accuracy over a previous procedure (15) for analysis of saturated aldehydes present in dilute aqueous solution. The new procedure has three main steps. First the aldehyde mixture is reacted with Girard-T reagent, and the derivative mixture is concentrated to dryness. The aldehydes are then liberated from the derivative mixture by heating with an excess of pure formaldehyde generated from methylolphthalimide. Finally, the aldehyde mixture is analyzed by gas-liquid chromatography. Obviously, the derivatization step has to go to completion if the entire analysis is to be quantitatively successful. This step was checked by using a relatively large quantity of propionaldehyde (0.0017 mole) and nonanal (0.009 mole) in two separate experiments. After forming the Girard-T derivative for each aldehyde, the solution was examined for the presence of unreacted aldehyde. For this examination, the electrometer was operated at high sensitivity and could easily detect less than 1 l g of propionaldehyde or nonanal. Under these of the original concentration of each conditions, less than aldehyde remained unreacted. From these results, it was assumed that the derivatization procedure was quantitatively satisfactory for the other analyses. F o r the regeneration step, methylolphthalimide was the preferred source of pure formaldehyde. A reagent blank determination based on using methylolphthalimide yielded a chromatogram which had no evidence of impurities. This freedom from interfering impurities is a distinct advantage over using paraformaldehyde as the regenerant. Attempts to repurify the paraformaldehyde by sublimation, followed by depolymerization (heating with distilled water) and repolymerization (cooling the hot solution) were unsuccessful. I t is possible to reduce the amount of excess paraformaldehyde to a minimum and increase the sensitivity of the procedure to allow detection of as little as gram of aldehyde per microliter of solution. However, it is more convenient to use
2z
methylolphthalimide in order to achieve this level of sensitivity Several time-and-temperature conditions were studied for satisfactory regeneration of the aldehydes using either of the regenerants. Intervals of 1, 2, 5 , 10, and 60 minutes were studied a t temperatures of 250, 225, 200, 180, 100, 70 "C and room temperature. Two minutes at 200 "C was selected t o minimize the time required t o complete this reaction and yet minimize the amount of extraneous peaks o n the chromatogram. The results obtained in this investigation indicate that this procedure is suitable for quantitative or highly semiquantitative determination of saturated aldehydes u p t o CU. The applicability of this method to quantitative determination of
polyunsaturated or polyfunctional carbonyls or methyl ketones is being investigated. For example, the method may require modification in the case of acrolein (Table I) because this carbonyl presumably undergoes Claisen and Michael type condensations (21).
RECEIVED for review August 2, 1967. Resubmitted October 30, 1967. Accepted April 8, 1968. Work supported by the Division of Biology and Medicine, U. S. Atomic Energy Commission, under Contract No. AT(49-7)-2443. Trade names referred t o in this publication d o not imply endorsement of commercial products. ~~
(21) C . W. Smith, "Acrolein," Wiley, New York, 1962, pp 110-13.
Pyrolysis-Gas Chromatographic Determination of Organics in Aqueous Solutions Ihor Lysyj and Kurt H. Nelson Research Dicision, Rocketdyne, 6633 Canoga Acenue, Canoga Park, Calq. 91304 GASCHROMATOGRAPHY has been used for the determination of volatile organics in aqueous solutions o n numerous occasions. As early as 1960, attempts were made to remove or chemically change the water in a sample so as to reduce its effect on thermoconductivity detection. For example, drying towers were incorporated into gas chromatographic instrumentation in order to remove water ( I ) , and conversion of water to acetylene was investigated in a procedure for the determination of short chain alcohols, aldehydes, and esters (2). With the advent and wide commercial availability of hydrogen flame ionization detectors, it became possible to analyze directly any volatile organic species present in aqueous solutions. None of these techniques, however, is applicable to the analysis of nonvolatile organics dissolved in water. These organics constitute a group of materials important in industry and commerce-i.e., foods, drugs, polymers, etc. They also figure prominently in biological systems. The prior gas chromatographic approach to the determination of such materials is based principally on syntheses of higher vapor pressure derivatives, which can be analyzed in the vapor phase. One example of this approach is the esterification of amino acids followed by subsequent gas chromatographic analysis of the derivatives (3). Such indirect methods, however, are often tedious, and may require a number of complex procedural steps and relatively large samples. In this study, an effort was made to combine the direct pyrolysis of water samples containing organic matter with gas chromatography for separation of the pyrolytic fragments so as to permit qualitative and quantitative characterization of a n aqueous sample. Steam was used as the carrier gas. The addition of water vapor from the sample to the steam carrier has little or no effect on the performance of the hydro(1) I. R. Hunter, V. H. Ortegren, and J. W. Pence, ANAL.CHEM., 32,682 (1960). (2) J. T. Kung, J. E. Whitney, and J. C. Cavagnol, ibid.,33, 1505 (1961). (3) S. Makisumi and H. A. Saroff, J. Gas Cliromak~gr.,3,21 (1965).
gen flame ionization detector, and this permits the use of relatively large samples. Use of sample sizes as large as 0.25 cc permitted detection of dissolved organics at trace concentration levels. EXPERIMENTAL
Apparatus. The instrumental system consists of a sample injector, a pyrolytic unit, a carrier gas source, and a gas chromatograph with potentiometric readout. The sample injector is mounted horizontally and is connected through a Swagelok reducer t o the pyrolysis tube. It consists of a three-way valve, a glass syringe of appropriate size, and a sample intake tube. The pyrolytic unit consists of a Lindberg Hevi-Duty combustion furnace, and a 60-inch long by 3Il6-inch Monel pyrolysis tube filled with granular nickel which is retained by a small plug of quartz wool at each end. A check valve is connected to the carrier gas inlet of the pyrolysis tube to prevent pressure surges into the custom-built steam generator which is the source of carrier gas. The check valve, the carrier gas line, and the portion of the pyrolysis tube outside the furnace are maintained a t 125 "C with heating tapes to prevent condensation of the steam. An Aerograph Model 600-C gas chromatograph equipped with a hydrogen flame ionization detector is used for separation and measurement of the pyrolytic fragments. A potentiometric recorder with a I-mV full scale response completes the instrumentation. Procedure. The operating conditions of the instrumentation are 21 cc/min for the steam carrier gas, and 25 and 250 cc/min for the hydrogen and air, respectively. The temperatures are 700 "C for the pyrolysis tube and 120 "C for the gas chromatographic column. This column is a 6-foot long by 3/lG-in~h column with a filling of 20 per cent Carbowax 20M on 60 to 80 mesh Chromosorb W (AW-DMCS). The instrument is operated under standard conditions for at least 24 hours before the analysis of water samples is attempted. The sample sizes are from 0.1 to 0.25 cc. The injection technique consists of drawing the sample from a foil-closed flask into the syringe, turning the three-way valve, and injecting the measured quantity of sample into the pyrolysis tube. VOL. 40, NO. 8, JULY 1968
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