168
Anal. Chem. 1981, 5 3 , 168-171
Determination of Nanogram Amounts of Carbonyls as 2,4-Dinitrophenylhydrazones by High-Performance Liquid Chromatography Kochy Fung and Daniel Grosjean” Environmental Chemistry Center, Environmental Research & Technology, Inc., 2625 Townsgate Road, Westlake Village, Caiifornk, 9 136 1
A slmple method has been developed for the separatlon and quantltation of nanogram amounts of carbonyl compounds includlng aliphatlc, unsaturated, and arometlc carbonyls as well as dlcarbonyls and other dlfunctlonal carbonyls. This method entails the Separation of the carbonyl compounds as thelr 2,4-dlnltrophenylhydrazone derlvatlves by high-performance liquid chromatography uslng lsocratlc solvent elution and their quantltatlon wlth appropriate internal standards uslng an ultraviolet detector. Separatlon of C,-C4 aliphatic carbonyls and benzaldehyde can be achieved In less than 10 mln. Analytical recovery and reproduclblllty have been determined for a number of carbonyls In the range 3-500 ng. Lowest quantifiable limits at a wavelength of 360 nm and a signal/ noise ratio of 4 range from 1 ng for formaldehyde to 4 3 ng for benzaldehyde and 5-hydroxy-2-pentanone, with RSDs of, for example, 4 % at the 20-ng level for formaldehyde and 2% at the 30-ng level for acetaldehyde.
of carbonyls of environmental interest including aliphatic, unsaturated, halogenated, aromatic, dicarbonyl, and hydroxycarbonyl compounds.
EXPERIMENTAL SECTION The method takes advantage of the well-known reaction of carbonyl compounds with 2,4-dinitrophenylhydrazine(DNPH) RR’C-0 + NHZNHCBH3(N02)2 HzO + RR’C=NNHC6Hs(N02)2 (1) -+
which proceeds by nucleophilic addition on the carbonyl followed by 1,2-elimination of water and the formation of the 2,4-dinitrophenylhydrazone. Because DNPH is a weak nucleophile, the coupling reaction is carried out in the presence of acid which promotes the protonation of the carbonyl. For ambient air sampling, we employ either microimpingerscontaining a mixture of organic solvent and acidic (2 N HC1) aqueous DNPH reagent or cartridges packed with glass beads coated with acidic (HBPOI) DNPH reagent. These sampling devices as well as detailed studies of their efficiency for the collection of microgram amounts of carbonyl compounds in air have been described elsewhere (6). After sampling of the carbonyls with DNPH reagent (Aldrich Chemical Co. or Eastman Organic Chemicalsfurther purified by five consecutiveextractions with 70%130% by volume mixtures of n-hexane and methylene chloride), extraction of the 2,4-dinitrophenylhydrazonesis performed by mechanical agitation with a mixture of methylene chloride and n-hexane (30%/70% by volume) for 15min. The 2,4-dinitrophenylhydrazoneof an internal standard (e.g., cyclohexanone or 3-methylbutanal)is added to each sample prior to extraction. The extract is washed with deionized water to remove the acid and most of the unreacted DNPH reagent and centrifuged, and the organic layer is transferred to a test tube and evaporated to dryness at 60 “C in a vacuum dmiccator. The residue is then dissolved in 200 FL of HPLC-grade methanol. Typically, 10-rL aliquots of this solution are employed for determination by HPLC. HPLC determinations are performed with an Altex Model 332 gradient liquid chromatograph, a Model 155-30 variable-wavelength UV-visible detector, a Model 210 sampling valve, and an Altex microprocessor. Separation of the hydrazones is performed on a 4.6 X 150 mm Ultrasphere ODS column (C18reversed phase) with isocratic elution using a 30%/70% by volume watermethanol solvent mixture and a mobile-phase flow rate of 1.5 mL/min.
In spite of the increasing interest concerning the levels and fate of aldehydes and other carbonyl compounds in the environment, there is a t the present time no simple method available for the determination of trace amounts (Le., nanograms) of carbonyls in air, water, and other environmental samples. In the area of atmospheric chemistry alone, measurements of parts-per-billion (ppb) levels of carbonyls in ambient air or simulated atmospheres (“smog chamber” studies) are critical to the development of a better understanding of hydrocarbon photochemistry, oxidant formation, and removal processes including scavenging by precipitation and atmospheric aerosols. Other important areas requiring measurements of trace levels of carbonyls in air include source and near-source sampling (engine exhaust, industrial emissions, etc.) and health-related studies (e.g., indoor pollution, work place environment, etc.), all of which have important regulatory implications. For these reasons, we have investigated the development of a sensitive and cost-effective method for the determination of aldehydes and other carbonyls. Taking advantage of the specific reaction between carbonyl compounds and nucleophiles including hydrazine derivatives, we have selected, on the basis of prior gas chromatographic (GC) studies of carbonyls in engine exhaust ( 1 , Z),to analyze trace levels of carbonyls as 2,4-dinitrophenylhydrazones. Papa and Turner (3) have reviewed the difficulties associated with GC analysis of these carbonyl derivatives and have reported ( 4 ) obtaining better results by using high-performance liquid chromatography (HPLC), while the work of Selim (5) suggests that HPLC is suitable for the separation and quantitation of carbonyl hydrazones at the low concentrations relevant to environmental applications. We report in this paper results concerning the development of a simple HPLC method and its application to the separation and quantitation, as 2,4-dinitrophenylhydrazones, of a variety 0003-2700/81/0353-0168$01 .OO/O
RESULTS AND DISCUSSION HPLC Separation of Hydrazones. In the experimental conditions described above, excellent and rapid separation has been achieved for a variety of carbonyl compounds. Examples are given in Figures 1 and 2, while retention times for a number of hydrazones of aliphatic, unsaturated, aromatic, and difunctional carbonyls are listed in Table I. As is shown in Figure 1,separation of formaldehyde, acetaldehyde, propanal, acetone, 1-butanal, 2-butanone, and benzaldehyde can be achieved in less than 10 min, while acetone and propanal, which are difficult to separate by GC (1-3), are well resolved in our system. Figure 2 illustrates the application of the method to the separation in less than 15 min of a mixture of six c& carbonyls including linear and branched-chain d-
B
1981 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL.
53, NO. 2, FEBRUARY 1981
Table I. HPLC Retention Values ( R f ,Minutes) for 2,4-Dinitrophenylhydrazonesof Selected Carbonyls monofunctional aliphatic carbonyls carbon --aldehydes ketones other carbonyls no. 1 formaldehyde 4.1 glyoxala 2 acetaldehyde 5.4 trichloroacetaldehy de methylglyoxal" acetone 9.3 3 propanal 8.2 methyl vinyl ketone 12.7 2-butanone 4 n-butanal 12.0 me thacrolein 2-methyl propanal 11.6 5-hydroxy-2-pentanone 2-pentanone 20.1 5 n-pentanal 19.0 3-pentanone 20.9 3-methylbutanal 17.1 3-methyl-2-butanone 20.0 cyclohexanone 6 n-hexanal 32.1 2-methyl-3-pentanone 33.1 4-methyl-2-pentanone 28.9 3,3-dimethyl-2-butanone 31.9 benzaldehyde
21.4 11.7
28.1 12.3 11.8 4.6 21.8 15.9
Dihydrazone derivative.
-
Table 11. Carbonyl (nanograms) in 10-pL Aliquots of Calibration Solutions calibration mixture A A Al2.5 AI5 carbonyl formaldehyde acetaldehyde propanal n-butanal benzaldehyde 2-butanone 5-hydroxy-2-pentanone 3
169 -
19.0 30.7 68.1 93.7 240
7.6 12.4 27.2 37.5 96.0
3.8 6.2 13.6 18.7 48.0
- calibration mixture B B
113
120.3 507
B/5
B/lO
22.6
11.3
24.1 101.4
12.0 50.7
4
7
5
0
I
0
m (0 Y
z 0
c
a
5:m
5 L 2 2
34
8 8 10 1 2 14 TIME, MINUTES
Flgure 1. HPLC separation of 2,4dinitrophenylhydrazonesof C,-C, carbonyls: formaldehyde (peak 11, acetaldehyde (2), propanal (3), acetone (4), n-butanal (5), 2-butanone (6), benzaldehyde (7), cyclohexanone (8).
dehydes, linear and cyclic ketones, and one hydroxycarbonyl compound. Obviously, the analysis of subsets of the compounds listed in Table I can be optimized by changing the analytical conditions (e.g., gradient elution, C8 instead of CI8 column 3% the higher molecular weight carbonyl%%&.). The effect of carbonyl. structure on retention tin@--nour experimental conditions is apparent from the examples shown in Figures 1 and 2 and from the data listed in Table I. For a given series (e.g., saturated aliphatic aldehydes), the compounds are eluted in order of increasing carbon number. Among aliphatic isomers, branched-chainaldehydes are eluted before linear (n-alkyl) aldehydes, which in turn are eluted before both linear and branched-chain ketones. Unsaturated
4
8
8 1 0 12 14
TIME. MINUTES
Figure 2. HPLC separation of 2,edlnitrophenylhydrazonesof selected C& carbonyls: 5-hydroxy-2-pentanone (peak l), acetaldehyde(:!), acetone (3),present as impurlty, 2-butanone (4), 3-methylbutanal (!5), cyclohexanone (6).
aliphatic carbonyls elute before saturated ones (e.g., methyl vinyl ketone before 2-butanone). In the same way, retention times for hydroxycarbonyls (e.g., 5-hydroxy-2-pentanone, which is eluted before acetaldehyde in our experimental conditions) and aromatic aldehydes (e.g., benzaldehyde) are consistent with polarity considerationsand substituent effects. Although no attempt is made here to further discuss relationships between retention times and other physicochemical properties, the data in Table I may serve as a guide when optimizing HPLC conditions for a given mixture of carbamyl compounds. Quantitative Analysis. Quantitation of the hydrazones is performed on the basis of measured absorbance a t a wavelength of 360 nm, using an internal standard (e.g., cy-
170
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
701
Table IV. Analytical Detection Limits
carbonyl formaldehyde acetaldehyde
equiv lowest detectn quantifiable limit in limit: ambient ng air,b pg m-3 1.1
2.OC l.gd
MA,
Flgure 3. Calibration curves: amount of carbonyl (nanograms)vs. carbonyl/internal standard absorbance ratios ( A /A,,): A = formaldehyde, B = acetaldehyde, C = propanal, D = n-butanal, E =
benzaldehyde, F = 2-butanone, G = 5hydroxy-2-pentanone. Internal standards are cyclohexanone (A, B, C, D, and E) and 3-methylbutanal (F and G). Full scale ordinate = 70 (A, B, C), 140 (D), 280 (E, F), and 700 ng (G).
Table 111. Analytical Recoveries for Selected 2,4-Dinitrophenylhydra~ones~ hydrazone . aldehyde recovery,b % HCHO 96.0 CH3CH0 97.6 C,H,CHO 99.9 C,H,CHO 94.4 C,H,CHO 90.8 a Following the overall analytical protocol described in text. Measured in the range of concentrations listed in Table 11.
clohexanone-2,4-dinitrophenylhydrazone)and calibration solutions containing known amounts of the carbonyl-2,4-dinitrophenylhydrazone of interest (Table 11). In the range of concentrations listed in Table 11, all carbonyl-2,4-dinitrophenylhydrazonesfollowed the Beer-Lambert law at 360 nm. This wavelength was selected on the basis of existing UVvisible spectra of the 2,4-dinitrophenylhydrazonesof formaldehyde (X, = 354 nm), acetaldehyde (358 nm), propanal (358 nm), n-butanal (362 nm), and benzaldehyde (375 nm), which have molar absorptivities of ~ 2 0 0 0 0at or near 360 nm. The calibration mixtures are analyzed by using at least three hydrazone/solvent dilution ratios. Calibration curves relating the concentration of a hydrazone to the ratio of the peak area of the compound to that of the internal standard are constructed by regression analysis and are shown in Figure 3. These calibrations are performed along with each batch of samples analyzed and cover the range of concentrations listed in Table I1 (-3 X to 0.5 pg). Hydrazone Recovery. Recoveries of microgram amounts of hydrazones were determined by HPLC of known amounts of calibration mixture A in Table I1 subjected to the entire analytical protocol and comparison with direct HPLC analysis of the same calibration mixture. The results are listed in Table I11 and indicate good recoveries for the five carbonyls studied. Reproducibility. In our experimental conditions, absolute retention times were reproducible within 6% (or within 3% for retention times relative to that of the internal standard). For example, a set of seven injections using mixture A in Table I1 and cyclohexanone as internal standard gave relative standard deviations (RSD) of 2.8% for the absolute retention time of HCHO, 4.2% for CH3CH0, 5.3% for C2H6CH0,3.5% for n-C3H7CH0, 3.3% for C6H6CH0,and 2.3% for cyclohexanone. The correspondingRSDs for the relative retention times were 0.6%, 2.0%, 3.4%, 1.3%, and 1.2% for form-
1.8 X lo-' 3.3 x 103.2 X 3.8 X
propanal 2.3 n-butanal 5.0 8.3 X lo-' 3-methylbutanal 3.2 5.4 x lo-' benzaldehyde 5.9 9.8 X lo-* 2-butanone 3.3 5.5 x lo-' cyclohexanone 3.7 6.2 X lo-' &hydroxy-2-pentanone 5.7 9.5 x 10-2 a At an integrator-microprocessor signal/noise ratio of 4. On the basis of a 60-L sample (e.g., 1-h at a flow rate of 1 L/min). Derived from data for CH,CHO in mixture A in Table 11. Derived from data for CH3CH0in mixture B in Table 11. aldehyde, acetaldehyde, propanal, n-butanal, and benzaldehyde, respectively. In the same way, a set of 12 injections of mixture B in Table I1 using 3-methylbutanal and cyclohexanone as internal standard yielded RSDs of 2.6%, 1,870, 1.3%, 1.4%, and 1.3% for the absolute retentibn times of 5-hydroxy-2-pentanone, acetaldehyde, 2-butanone, 3methylbutanal, and cyclohexanone,respectively. The corresponding RSDs for the relative retention times were 2.2% for 5-hydroxy-2-butanone, 1.4% for acetaldehyde, and 0.5% for 2-butanone with 3-methylbutanal as the interrial standard. Reproducibility was also determined for quantitative analysis of calibration mixtures, samples generated in the laboratory, and field samples. Triplicate injections of Calibration mixtures A and B in Table 11,with cyclohexanone and 3-methylbutanal as internal standards, respectively, yielded RSDs of 4.2% for formaldehyde, 1.1% and 1.9% for acetaldehyde (mixturesA and B, respectively), 0.8% for propailal, 2.3% for n-butanal, 0.3% for benzaldehyde, 1.6% for 2-butanone, and 1.2% for 5-hydroxy-2-pentanone. Good overall reproducibilities (HPLC method and sampling method) were also achieved for samples generated in the laboratory and actual field samples. In the laboratory, two sets of six acetaldehyde samples were collected and analyzed once each, with RSDs of 5.5% and 8.3% at concentra$ions of 82 and 54 ng, respectively. These amounts are equivalent to ambient air concentrations of 0.75 and 0.50 gpb, respectively, collected as a 60-L sample (e.g., 1h X 1 L min-*). A set of 15 replicate analyses of ambient air samples yielded RSDs of 2.9% and 5.9% for formaldehyde and acetaldehyde, respectively (6),for ambient air concentrations of 2.4-7.9 ppb (HCHO) and 0.5-2.3 ppb (CH3CHO). Detection Limits. Detection limits afforded by the HPLC method are of the order of a few nanograms. Lowest quantifiable limits (LQL) have been determined for 10-pLaliquots of calibration mixture samples using (conservatively) a peak area integrator signal-to-noiseratio of 4. These LQL are listed in Table IV,along with the cortespondingcarbonyl levels that could be detected in air on the basis of a 60-L sample (e.g., a 1-h sample at a sampling flow rate of 1 L m i d ) . These pg m-3 for analytical detection limits range from 18 X formaldehyde (or -15 ppt) to -98 X loe3pg m-3 for benzaldehyde (or -30 ppt). In practice, it is obviously extremely difficult to eliminate all carbonyl impurities in the reagent, solvents, glassware, laboratory or sampling protocol, etc. at levels corresponding to these very low analytical detection limits (6). While further work is in progress in our laboratory to achieve better sensitivity, the method is now suitable for measurements of formaldehyde and a variety of other carbonyl N
Anal. Chem. 1981, 53, 171-174
compounds at the parts-per-billion levels relevant to atmospheric and other environmental applications.
ACKNOWLEDGMENT Mr. Itsvan Ary of ERT participated in the early phase of the method development.
LITERATURE CITED (1) Fracchia, Mario F.; Schuette, F. J.; Mueiler, Peter K. Enviion. S d . Techno/. 1967, 7 , 915-922. (2) "Procedures for Determining Exhaust Carbonyl as 2,CDinitro~hen~lhydrazones", Bartlesville Petroleum Research Center, Bureau of Mines, Final Report for Project CRC-APRAC No. CAPE-1 1-68, Revised Aprll 1969; available as Natlonai Technical Information Service Report
(3) (4) (5) (6)
171
No. PB-200-883; National Technical Information Service: Sprlngflekl, VA, 1971. Papa, L. J.; Turner, L. P. J. Chromatogr. Sci. 1972, 70, 744--747. Papa, L. J.; Turner, L. P. J. Chromatogr. Sci. 1972, 70, 747-750. Seiim, Sami J. Chromatogr. 1977, 736,271-277. Grosjean, Daniel; Fung, Kochy; Atkinson, Roger Paper No. 80.50-4, 73rd Annual Meeting of the Alr Pollution Control Assoclation, Montreal, Canada. June 23-27. 1980: Air Pollution Control Assoclation: Plttsburgh, PA.
RECEIVED for review July 15,1980. Accepted October 23,1980. This work was supported by Environmental Researclh & Research and funds part Of Project NO. M-0171-001.
Liquid Chromatograph/Mass Spectrometer Interface with Continuous Sample Preconcentration Richard G. Christensen," Harry S. Hertz, Stanley Melselman, and Edward Whlte V Center for Analytical Chemistry, National Bureau of Standards, Washington, D.C. 20234
A liquid chromatographhnass spectrometer system whlch performs enrichment of the sample In the effluent of a conventional llquid chromatograph prior to Its being introduced into a dlfferentlaiiy pumped quadrupole mass spectrometer is descrlbed. The effluent from the liquid chromatograph is concentrated by evaporation of most of the solvent. Solvent evaporation is accomplished by allowing the effluent to flow down an electrically heated wire with the current to the wire controlled by a feedback loop from a volume-sensing photocell. The concentrated effluent flows through a very small needle valve which regulates the flow into and thereby the pressure inside the mass spectrometer. The valve Is constructed such that llquld Is sprayed into the Ion source of the mass spectrometer. The appllcatlon of the system to polynuclear aromatlc hydrocarbon characterization and to quantltatlon of phenolic compounds In alternate fuels is shown.
The great potential of combined liquid chromatographyand mass spectrometry (LC/MS) as an analytical tool has fostered research by a considerable number of laboratories. In addition to the collection, evaporation, and transfer of LC fractions to the mass spectrometer, a number of means have been used to combine LC and MS. Recent reviews by Arpino and Guiochon ( I ) , Zerilli (Z),and McFadden (a review which contains examples of applications of LC/MS contributed by other authors) (3) describe the operating principles, construction, and performance of these devices. Two methods, direct liquid injection (DLI) and evaporation onto a moving wire or belt, are now offered commercially. Although the DLI technique is intrinsically simple to implement, the moving helt interface has the advantages of concentrating the solute, thereby introducing a greater proportion of the sample into the ion source of the mass spectrometer and of permitting both electron impact and chemical ionization modes of operation. A system which could preconcentrate the liquid stream and introduce the concentrate by DLI would combine some of the advantages of both or-
dinary DLI and the moving belt technique. The interface described in thispaper was designed and built with this aim in mind. The interface device concentrates a liquid stream by allowing it to flow down a resistance-heated stationary wire. The residual liquid is drawn into the mass spectrometer through a capillary tube with a needle valve at the ion source end. The liquid sprays from the needle valve into the ion source of a conventional, differentially pumped, quadrupole mass spectrometer.
EXPERIMENTAL SECTION Concentrator Wire. The construction of the concentrator wire is shown in Figure 1. The concentrator wire consists of three segments, the active lengths of which are 15, 7.5, and 15 om, respectively. The segments are connected by silver-soldering in butt joints which are then filed to present a short, symmetrical taper between segments. The concentrator wire is held taut and straight by a spring at its lower end through which the lower electrical connection is made. The wire passes across a gap in the DLI probe and the residual stream forms a drop in the gap. When the drop becomes too large, it flows down the wire or along the outer surfacer3 of the DLI probe and is lost. A light emitting diode and photocell are fitted at the gap and sense the size of the drop. Feedback from the photocell controls the current through the wire to hold the drop size constant. The circuit for the photocell sensor and power supply to the wire is shown in Figure 2. An envelope of glass is provided to exclude dust and air currents from the stream on the wire, and a TFE manifold is set into the top of the glass envelope to direct a stream of filtered air or inert gas downward through the tube to carry away solvent vapors. 'I% cap has passages to conduct the liquid chromatographic effluent to an opening concentric with the concentrator wire. DLI Probe. The construction of the DLI probe is shown in Figure 3. The tip of the probe is formed into a needle valve in order to assure that the pressure drop occurs at the ion source and to control the rate of flow of liquid into the vacuum. The stem of the valve is a 0.10-mm tungsten wire ground to a pencil-point at the tip. The probe itself is formed from a hard-drawn nickel tube with 1mm 0.d. and 0.12 mm i.d. The valve seat at the end of the probe is formed by peening the opening of the tube until it is almost entirely closed (-0.01 mm i.d.). When the seat and stem pieces
This article not subject to U.S. Copyright. Published 1981 by me Amerlcan Chemical Society
