olefins elute immediately after large saturate peaks; thus, separation from the saturates is favored by a low column tem- perature, which allows greater retardation of olefins relative to saturates. In the case of the CB hydrocarbons, the opposite is true, since most of the olefins in this range elute immediately preceding saturate peaks. Thus, to obtain optimum resolu- tion in both C4-C5 and C6 ranges, the column temperature is held at -5” C until the elution of isopentane, and then the column is warmed as rapidly as possible to 25” C. This is accomplished by activating the oven blower as soon as the dry ice is removed from the column coil. When significant deviations from the temperature program are allowed to occur, similar peak elution shifts are observed in several areas of the

(13) W. J. Youden, “Statistical Methods for Chemists,” Wiley, New York, 1951.

gasoline chromatograms. Longer squalane capillary columns were tried, and as expected, an increase in resolution was ob- served, However, with a 300-foot X 0.010-inch i.d. 10% wt squalane column, the analysis time was over 4 hours. Ob- viously, the chromatographic procedure used for routine GLC analyses of gasolines is a compromise which gives maximum information in a reasonable time period.

The capillary GLC method described in this article is also applicable to the analysis of other hydrocarbon mixtures such as catalytically cracked gasoline, catalytic reformate, alkylate, and light naphtha feedstocks. With modifications of present sampling procedures, the method could easily be applied in analysis of the individual hydrocarbons found in automotive exhaust gases.

RECEIVED for review October 16, 1967. Accepted January 2, 1968.

Apparatus Combining Gas Chromatography with Spectrophotofluorometry by Means of a Flowing Liquid Interface Malcolm C. Bowman and Morton Beroza Entomology Research Division, Agricultural Research Service, U. S . Department of Agriculture, Tifton, Ga. and B e l t s d e , Md . 20705

31 794

An apparatus has been devised to combine the high separative powers of the gas chromatograph with the high sensitivity and selective response of the spectro- photofluorometer. The solute in the effluent of the gas chromatograph is picked up by a slowly flowing stream of alcohol, and the alcohol solution is monitpred in a flow cell at the desired excitation and emission wave- lengths. The combination has been used to analyze pesticides, air pollutants, and methylenedioxyphenyl compounds. Analyses in the nanogram range were possible, and sensitivity frequently exceeded that of the flame ionization detector. The device appears to have general applicability to the analysis of fluorescing substances that can be determined by gas chromatog- raphy and captured by the solvent that travels to the flow cell. The eluate of the flow cell may be collected for retention of fractions or for spectral or other analyses.

GAS CHROMATOGRAPHY has been joined with a wide variety of instrumentation-e.g., the mass spectrometer ( I , 2 F t o ob- tain apparatus with enhanced analytical capabilities. Such combinations have improved the speed and sensitivity of analyses, circumvented the need to trap out minute amounts of pure substance, eliminated chemical change of substances un- stable subsequent to elution, provided identification more cer- tain than possible by retention time only, and elevated the selectivity of response to allow analyses of samples with mini- mum cleanup.

The combination of gas chromatography with spectrophoto- fluorometry (SPF) appeared to possess most of these virtues. SPF is widely used and well understood (3) ; the spectra of a

(1) R. Ryhage, J. Lipid Res., 5, 245 (1964). (2) J. T. Watson and K. Biemann, ANAL. CHEM., 37,844 (1965). (3) S. Udenfriend, “Fluorescence Assay in Biology and Medicine,”

Academic Press, New York, 1962.

great variety of compounds have been cataloged. SPF is also highly sensitive and its response can be made very selec- tive by appropriate choice of excitation and emission wave- lengths. Our own special interest in the union of the two techniques was for the analysis of such environmental con- taminants as pesticides and air pollutants, many of which fluoresce strongly ; however, the combination appears to have general applicability to fluorescent compounds that can be de- termined by gas chromatography.

The present paper describes a device that proved to be a suit- able link between the two instruments. In essence, the fluorescent substances separate in the gas chromatograph and, upon emergence, are absorbed by a flowing stream of ethanol as the carrier gas escapes; the ethanol solution then passes through 8 flow cell that is monitored by a recording spectro- photofluorometer. The combination appears to have good sensitivity. The compounds checked could be determined in the nanogram range and some weredetectable at levels below 1 ng.

EXPERIMENTAL

Gas Chromatograph (GLC). An F & M Model 700 gas chromatograph (F & M Scientific Corp., Avondale, Pa,) was operated isothermally at oven temperatures between 130’ and 220” C. Two columns were used; both were 4-mm i.d., 6-mm 0.d. glass columns, 125 cm long. One contained 5 x w/w QF-1 on 80/100 mesh Gas-Chrom Q (Applied Science Labs., State College, Pa.), and the other 5z w/w DC 200 on the same support. The flow rate of the nitrogen car- rier gas was 100 ml/minute. Columns were conditioned over- night at 240” C before use. The injection port was maintained 20” C above oven temperature. A ’/*-inch 0.d. stainless steel line conducted the carrier gas from the column exit to the mixing vessel (Figure 1); it terminated in a male Luer

VOL. 40, NO. 3, MARCH 1968 e 535

n - I N J E C T SAMPLE

E3

C

-7.5 cm.----.I Figure 1. Mixing vessel

A , 1-liter separatory funnel with Teflon stopcock and stem cut perpendicular to its axis; B, 25-ml Erlenmeyer flask; C, liquid level-ca. 5-mm depth in flask; D, 15-mm length of 1.5-mm i.d. capillary tubing; E, male Luer; F, female Luer; G, 11-mm 0.d. tubing. Female Luer F attaches to male Luer H, which is the GLC outlet. Figure drawn to scale. All parts but Hare borosilicate glass

fitting (Becton, Dickinson, & Co., Rutherford, N.J., 426 A plug) which was drilled to accommodate a short length of a l/la-inch 0.d. stainless steel needlestock tube (bent as shown in Figure 1H) and silver-soldered to the line.

Mixing Vessel. The mixing vessel was fabricated as shown in Figure 1 from parts described in the legend. Luer fitting F was attached to fitting H (GLC exit port) to hold the mixing vessel in place. The height of the separatory funnel A was adjusted to maintain a 5-mm depth of liquid in the Erlen- meyer flask when the stopcock was open. Luer fitting E was connected to the bottom of the flow cell by means of a valve (Becton Dickinson M501), a hypodermic needle, and 1.5-mm i.d. Teflon tubing. The valve was used to stop and start the alcohol flow.

Spectrophotofluorometer and Accessory Equipment, An Aminco-Bowman spectrophotofluorometer (American In- strument Co., Silver Spring, Md.) was equipped with a xenon lamp, a 1P21 detector tube, and a flow cell (Aminco No. B-16- 63019, quartz, 3-mm id., 5-mm 0.d.). The exit (top) of the flow cell was connected to 1.5-mm i.d. Teflon tubing, and its outlet (end of liquid system) was set 27 cm below the liquid level in the Erlenmeyer flask of the mixing vessel. With this arrangement, the liquid flow rate was 3 ml/minute, and about 20 seconds (0.35 minute) were required for response after a fluorescent material entered the mixing vessel. The liquid flow rate was readily varied by raising or lowering the liquid outlet.

The slit arrangement was 5-54 mm (largest size), and no slits were used in the flow cell. The photomultiplier micro- photometer was operated at maximum sensitivity and was connected to a 1-mV Bristol recorder (chart speed 0.5 inch/ minute) by means of a center-tapped potentiometer which served as a voltage divider. The recorder was equipped with a Disc integrator. The potentiometer was adjusted to give the same reading on the recorder as appeared on the meter of the microphotometer. The dark current was zeroed with the shutter closed; then the shutter was locked open during operation of the apparatus. The zero knob of the micro- photometer adjusted the height of the base line, and the meter multiplier served as range selector (or attenuator). Range settings were 1.0, 0.3, 0.1, 0.03, and 0.01; the 0.003 and 0.001 settings were too sensitive.

Y R R I i L T ]

SOLVENT C A S CHROMA- TOGRAPH

M I X I N G V E S Y E L - I V O L T A G E 1

D l V l D E R

II I

Figure 2. Schematic diagram of GLC-SPF arrangement

Arrows show flow of fluid

Chemicals. The chemicals subjected to analysis were ob- tained from commercial sources. Four of the methylene- dioxyphenyl compounds are synergists for pyrethrins (an insecticide). Their trade or common names and chemical identities are: Bucarpolate, 2-(2-butoxyethoxy)ethyl piper- onylate; piperonyl butoxide, cu-[2-(2-butoxyethoxy)ethoxy]- 4,5-(methylenedioxy-2-propyItoluene; sesamex, 2-(2-ethoxy- ethoxy)ethyl 3,4-(methy1enedioxy)phenyl acetal of acetalde- hyde; and sulfoxide, 1,2-(methylenedioxy)-4-[2-(octylsul- finyl)propyl]benzene. Two insecticidal carbamates and their phenolic hydrolysis products that were analyzed are carbaryl, 1-naphthyl methylcarbamate, and its hydrolysis product, 1-naphthol; Niagara NIA-10242, 2,3-dihydro-2,2-dimethyl-7- benzofuranyl methylcarbamate, and its hydrolysis product NIA-10272, 2,3-dihydro-2,2-dimethyl-7-benzofuranol.

Flowing SoIvent. Ethanol (95 %) was used as received from U. S. Industrial Chemicals, New York, N. Y . Other solvents may be used as appropriate.

Principle of Operation. The path of a sample injected into the GLC may be followed through the system by referring to the schematic diagram of the GLC-SPF arrangement shown in Figure 2 and to the diagram of the mixing vessel in Figure 1. The carrier gas emerges from the exit port (Figure 1H) and escapes through tube G (Figure l), leaving the eluted solute in the alcohol. The alcohol solution flows by gravity down the Teflon tubing and passes through the flow cell. The appear- ance of the fluorescing solutes in the flow cell activates the recorder to produce a chromatogram of the products. The liquid effluent may be discarded or directed into a fraction collector. The fractions, if collected, may be selected for individual or combined examination or analysis in the spec- trophotofluorometer, can be examined in another analytical instrument, or can be recovered for bioassay or other pur- poses.

Procedure. Assemble the apparatus described, and start the gas chromatograph and spectrophotofluorometer. Fill the separatory funnel about 4/5 full with the 9 5 z ethanol, stopper, and position the funnel with the tip of its stem 5 mm above the bottom of the Erlenmeyer flask of the mixing vessel. Open the stopcock and the valve (B.D. M501), and make certain that all air bubbles are removed from the alcohol as it flows from the mixing vessel to the flow cell. Adjust the flow rate of the ethanol to 3 ml/minute by raising or lowering the liquid outlet, Set the desired excitation and emission wavelengths, adjust the dark current zero and then the height of the base line, and select the appropriate sensitivity with the meter multiplier. Inject the sample.

Calculations. Two methods were used to calculate re- sponse. Peak height, expressed as relative intensity (RI), was obtained by multiplying the recorder or meter response (meter) by the meter multiplier setting (MkO--i,e., RZ = meter X M M .

536 ANALYTICAL CHEMISTRY

Table I. Data Describing the Gas Chromatography of Eight Methylenedioxyphenyl Compounds and Five Polynuclear Hydrocarbons on a QF.1 Column with SPF Detection

GLC _ _ - Wavelength Quantity oven settings, mp injected,~ temp, Response

RP Compound Ex Em Pug " C Meter MhP

Safrole Di hydrosafro Isosafrole Sesamex Sulfoxide

Piperonyl alcohol

Piperonyl butoxide

Bucarpolate F 1 u o r e n e

288 lle 290

312 297 292

285

295

300 292

334 332 348 345 333

333

333

350 320

0.5 0.5 0.5 5.0 0 .5

0.5

0.5

0.5 0.2

130 130 130 130 130 180 130 180 180

180 150

59 60 54 16 21 28 63 24 18

31 46

0.1 0.1 0.3 0.1 0.03 0.1 0.03 0.1 0.03

0.03 0.1

5.9 6.0

16.2 1.6 0.63 2.8 1.9 2.4 0.54

0.93 4.6

Anthracene 293 353 0.5 160 10 0.1 1 .o pTerpheny1 294 341 0.05 180 26 0.1 2.6 Chrysene 322 372 0.5 200 12 0.1 1.2 Benzo[a]pyrene 383 408 0.1 220 31 0.1 3.1

5 Injected in 5 pl 95% ethanol, chloroform, or ethyl acetate. b MM = meter multiplier setting; RI = relative intensity. c Estimated on the basis of the peak being twice the noise level of 0.01 RZ. d The short retention time indicates peak is caused by a decomposition product.

Smallest detectable quantity,e

ng 0.2 0.2 0.6 60 16 4 5 4

19

11 0.9

10 0.4 9 0.7

Retention time, min.

1.40 1.45 2.15 2. 5Od 2.706 1 . 0 9 3.15 1.10 5.50

6.30 2.05 4.65 3.90 5.15 6.35

Table 11. Data Describing the Gas Chromatography of Two Insecticidal Carbamates and Their Hydrolysis Products on a DC 200 Column with SPF Detection

GLC Smallest Wavelength Quantity oven detectable Retention settings, mfi injected,a temp, Response quantity," time,

Compound Ex Em fig " C Meter M W RP ng min.

1-Naphthol 306 362 0.5 150 41 0.1 4.1 2 2.55 Carbaryld 306 362 0.5 150 32 0.1 3.2 3 2.55 Carbaryle 285 340 0.5 1 50 19 0.1 1.9 5 2.55 NIA-10242 280 327 5.0 150 15 0.1 1.5 68 3.9Y NIA-10272 280 327 5.0 150 60 0.1 6.0 17 1.20

(1 Injected in 5 fil of chloroform or methylene chloride. b MM = meter multiplier setting; R I = relative intensity. c Estimated on the basis of the peak being twice the noise level of RZ = 0.01. d Completely hydrolyzed to 1-naphthol. 6 Read at wavelengths giving maximum excitation and emission for carbaryl.

Read at 1-naphthol wavelengths.

Partial hydrolysis, the extent of which is not reproducible, occurs.

Peak area was obtained by multiplying the Disc integrator units under the curve by MM. One traversal of the inte- grator pen was assigned a value of 100 units.

RESULTS AND DISCUSSION

Table I lists SPF wavelength settings giving maximum re- sponse, GLC oven temperatures, responses to given amounts injected, estimated lowest detectable quantities, and retention times in the chromatography of eight methylenedioxyphenyl compounds and five polynuclear hydrocarbons (air pollutants) on the QF-1 column. Table I1 lists similar data for four pes- ticides and pesticidal derivatives obtained on the DC 200 column. Less than 1 ng of some compounds was detectable based on the requirement that the peak be twice the noise level of 0.01 RZ. Because the fluorescent properties of compounds differ greatly and vary with the wavelengths monitored, sensi- tivities can be expected to parallel the intensity of fluorescence at the wavelengths chosen.

Typical chromatograms are shown in Figures 3 and 4. In general, the peak shape showed that excessive spreading did not occur in passage from the GLC to the SPF. Because

there was no immediate detector response to sample injection, the time of injection was marked by quickly turning the chart drive off and on; the vertical lines at 0 minute in Figures 3 and 4 were produced in this manner. The peaks that began to emerge at about 0.35 minute resulted from the injection; they did not appear to have any bearing on the peaks that emerged subsequently, and their origin or cause is unknown. They could be caused by impurities in the compounds, flash-back against the septum on sample injection, or some other condi- tion. A few of these injection peaks have been included in the figures to show their appearance; they are not necessarily re- producible and vary with amplification. If these injection peaks interfere with a compound of interest, the column tem- perature may be lowered to increase the retention time of the compound beyond that of the interfering peak.

In Figure 5, the response of 1-naphthol in terms of peak height and area is plotted against concentration. Although neither response was perfectly linear, peak area was close to linearity. (For true linearity in a log-log plot, the slope should be 1.0.) In the log-log plot shown in Figure 5, peak area gave a straight-line relationship over the three decades of

VOL. 40, NO. 3, MARCH 1968 537

IK

i I t I L

6 4 2 0

.A

M I N U T E S

Figure 3. Chromatograms of polynuclear hydrocarbons and methylenedioxy- phenyl compounds

Peak identification, compound, amount injected, meter multiplier setting, and oven tem- perature were: A, fluorene, 0.2 pg, 0.1, 150" C; B, p-terphenyl, 0.05 pg, 0.1, 180" C; C, anthracene, 0.5 pg, 0.1,160" C; D, chrysene, 0.5 pg, 0.1,200" C; E, benzo[a]pyrene, 0.1 pg, 0.1, 220" C; F, sulfoxide, 0.5 pg, 0.1, 180' C; G, piperonyl alcohol, 0.5 pg, 0.1, 180" C; H, dihydrosafrole, 0.5 pg, 0.1, 130" C; I , piperonyl butoxide, 0.5 pg, 0.03, 180" C; J, Bucarpolate, 0.5 pg, 0.03, 180" C; K, safrole, 0.5 pg, 0.1, 130" C; L, isosafrole, 0.5 pg, 0.1, 130" C; M, sulfoxide, 0.5 pg, 0.1, 130" C. Unmarked peaks at 0.35 min. are injection peaks

concentration plotted, and the plot of peak height was a straight line over about two decades of concentration (up to the 1-pg level of 1-naphthol). Because the peak area response was proportionately less at lesser concentrations than at higher concentrations, the departure from linearity may have arisen from less efficient absorption of solute by the alcohol as a re- sult of relatively greater volatility losses with lesser amounts of compound. Regardless of these deviations, accurate quanti- tation of 1-naphthol was possible by reference to a standard curve such as the one shown in Figure 5 .

Insecticide Analysis. The possibility of analyzing car- baryl and its hydrolysis product, 1-naphthol, with the GLC- SPF arrangement was investigated. Milk was fortified with 0.5 ppm each of carbaryl and 1-naphthol, which fluoresce strongly (4) . This sample and an unfortified one were ex- tracted, and the insecticide and its hydrolysis product were separated on alumina by a method described previously for determining Mobil MC-A-600 (benzo[b]thien-4-yl methylcar- bamate) and its hydrolysis product in milk (5). The carbaryl fraction (in chloroform) was concentrated to 5 ml, and 5 pl of the raw concentrate (equivalent to 100 mg of milk) were in- jected. Recovery was 95-1OOx based on peak area. The phenol fraction (in methanol) was extracted with methylene chloride, concentrated to 5 ml, and a 5-pl aliquot (equivalent to 100 mg of milk) injected. Recovery of the 1-naphthol was 73- 78 %, a recovery that compares favorably with that obtained in analyses of the phenolic products of insecticidal carbamates (6). The chromatograms of the unfortified milk contained no peaks that interfered with the analyses. Analyses were rapid and reliable.

In a similar manner, carbaryl was determined in corn plants after extraction and separation of carbaryl and its phenolic hydrolysis product (1-naphthol) on alumina as described for Mobil MC-A-600 in grass (5). Recoveries of carbaryl from

(4) M. C. Bowman and M. Beroza, Residue Rev., 17,23 (1967). (5) M. C. Bowman and M. Beroza, J. Agr. Food Chem., 15, 894

(6) M. C. Bowman and M. Beroza, J. Assoc. Ogic. Anal. Chemists, (1967).

50,926 (1967).

I 7 I

I 1 I I

6 4 2 0 I t

6 4 2 0

m m v) -0 0 z v) m

M I N U T E S

Figure 4. Chromatograms of insecticidal carba- mates and phenolic derivatives

Peak identification, compound, amount injected, meter multiplier setting, and oven temperature were: N, NIA-10272, 5 pg, 0.1, 150" C; 0, MA-10242 (de- composes to 2 peaks), 5 pg, 0.1,150" C; P, l-naphthol, 0.5 pg, 0.1, 150" C; Q, carbaryl, 0.5 pg, 0.1, 150" C (1-naphthol max wavelengths used); R, same as Q but carbaryl max wavelengths used. Unmarked peaks beginning at 0.35 min. are injection peaks

samples fortified at the 1- to 5-ppm level were 89-93 Z. Re- coveries of the phenol were unsatisfactory (16-34 %) because of incomplete extraction, not the instrumentation; however, this is not important since the phenol is not toxic in trace amounts. The interference of the corn extract limited the detection of carbaryl to 10 ng. With an injection of extract equivalent to 100 mg of corn plant, sensitivity was 0.1 ppm.

Ebing (7) reported that N-monomethylcarbamates were un- stable during gas chromatography on two liquid phases.

(7) W. Ebing, Chimiu, 19, 501 (1965).

538 ANALYTICAL CHEMISTRY

t

t 7 I,' I

O ~ O ~ ~ ~ d 0.10 ID MICROGRAMS I -NAPHTHOL INJECTED

Figure 5. Standard curves of 1-naphthol us. response by peak height and by peak area

Carbaryl, which falls in this category, was degraded completely to 1-naphthol during our gas chromatography. The preferred excitation and emission wavelengths should, therefore, be those that are maximal for 1-naphthol. Niagara NIA-10242, also a monomethylcarbamate, broke down too, but only partially (see curve 0 of Figure 4). A short plug containing 85 phosphoric acid can be inserted at the head of the GLC column as previously described (5 ) to convert all the carba- mate to the phenol (NIA-10272), and in this way results may be readily quantitated. As with carbaryl, the wavelength maxima of the phenol are preferred.

Effect of Flow Rate. Figure 6 summarizes experiments conducted to study the effect of the flow rate of ethanol on response (both peak height and peak area), retention time, peak width, and trapping efficiency. 1-Naphthol, '/z pg, in 5 pl of ethanol was injected in each test except that 5 pg were used to determine efficiency. As may be observed in Figure 6, the parameters were markedly influenced by the flow rate of alcohol. This rate may be adjusted to give maximum performance for the 1-naphthol analysis depending on which parameter (or parameters) is assigned the greatest importance. It is desirable to maintain the loss in resolution of substances eluting from the gas chromatographic column at a minimum because the interface arrangement between the instruments, with the attendant diffusion occurring in the transfer, necessar- ily entails some loss of resolution. The peak width at half height was selected as a measure of resolution; the smaller the value obtained with a given set of conditions, the sharper the peak will be and the less the loss of resolution. Flow rates of alcohol between 0.5 and 2 ml/minute were considered unsatis- factory because they broadened the peaks excessively and thereby sacrificed resolution. Quantitation with Disc integra- tor units (area) also was less reliable because the longer base line of the peak at lesser flow rates makes integration less accurate. (Peak height remained fairly constant for flow rates between 0.5 and 2.5 ml/minute.) Flow rates above 3 ml/min- Ute may be desirable if resolution is a primary consideration, but they produce a loss in sensitivity in terms of peak height and peak area. If sensitivity is the prime consideration and significant interference is absent, flow rates in the range of

00-60 4 4

ALCOHOL FLOW RATE M L / M I N OO

Figure 6. Effect of flow rate of alcohol on peak height, peak area, retention time, and trapping efficiency of 1-naphthol

2.0-2.5 mi/minute appear to be desirable, especially if peak height is the mode of quantitation.

The efficiency of the apparatus was determined by collecting the peak effluent in 10 ml and comparing its fluorescence with that of the injected solution (5 pg of 1-naphthol in 5 pl) made up to 10 ml. At first, some efficiencies exceeded 100%. How- ever, an investigation disclosed that the contents of the needle of the syringe (ca. 0.55 p1) had been expelled into the gas chromatograph by the heat of the injection port, but had not been expelled in adding the solution to the 10 ml of ethanol at room temperature. Accordingly, the contents of the needle were washed into the 10 ml to make the amounts added com- parable, Efficiencies at the 2-3 ml/minute ethanol flow rate were about 80 %. Unquestionably, efficiency was affected by the amount analyzed because response was not exactly linear with concentration.

Our selection of the 3 ml/minute flow rate was a compromise between the desired values of response and resolution (peak width). This rate may be altered in accordance with the needs of the individual analysis.

Apparatus Design. Proper operation of the apparatus required minimum spread of peaks and attainment of maxi- mum sensitivity. The spread of peaks was minimized by mixing the emerging carrier gas and its solute with a minimum volume of ethanol and using small-bore Teflon tubing to transfer the ethanol from the mixing vessel to the flow cell. The capillary tube between the Erlenmeyer flask and mixing section prevented back flow of the alcohol to the reservoir. Although the liquid dead volume was kept small, about a 1-ml volume at 3 ml/minute was required for transfer of the solute, and the appearance of a peak on the chromatogram was thus delayed 20 seconds. Figure 6 shows that this delay was re- duced very little when the flow rates were faster than 3 ml/ minute (see curve of retention time us. flow rate). A better means of reducing the delay, which will also improve resolu- tion, is to shorten the distance between the mixing vessel and the flow cell.

A pump could be used to add the alcohol, but it would intro- duce the possibility of contamination and would not be con- sistent with the idea of keeping the apparatus simple. The flow rate was therefore regulated by the gravity drop of the alcohol, an arrangement that proved to be both trouble-free and flexible. The volume of ethanol in the separatory funnel was maintained between 400 and 850 ml for best operation. An Erlenmeyer flask was used in the mixing vessel to hold changes in the liquid level to a minimum; when the level dropped sufficiently, air rushed into the funnel and released

VOL. 40, NO. 3, MARCH 1968 539

liquid into the flask. Because the liquid level was located at the largest diameter of the Erlenmeyer flask, this level varied very little during the entire operation.

The exit port was kept at 200" C to avoid solute retention. Cooling of the port or transfer of heat to the flowing alcohol was not excessive, probably because the exit port was made of a poor thermal conductor (stainless steel) and very little of its surface was exposed to the liquid.

Discussion. When SPF is used to detect an unknown substance, the maximum excitation and emission wavelengths are not known. The need to use different wavelengths for each compound has both advantages and drawbacks. Thus, certain wavelengths may produce a maximum response to one compound of a mixture and a negligible response to the re- maining ingredients, which might ordinarily interfere. Also, by appropriate selection of wavelengths, the response of sev- eral compounds in a mixture may each be enhanced enough to permit them to be analyzed individually. Should sensitivity be no problem, specificity may be improved by narrowing the slit widths of the spectrophotofluorometer. However, mix- tures in a GLC effluent will probably not be overly complex owing to the high resolving power of GLC. The simplest way to determine the desired wavelengths with the present setup is to collect the liquid effluent from the flow cell-e.g., with a fraction collector-and select wavelengths based on the com- plete excitation and emission spectra of the liquid effluent. The collected fractions may be used to assess the purity of a substance of a peak because the spectra of the front and rear sections may be checked for identity. With a completely un- known GLC effluent, the checking of several sets of wave- lengths will help prevent overlooking an important ingredient. Drushel and Sommers (8) stressed the advantages of using fluorescence and phosphorescence with gas chromatography in the analysis of petroleum fractions. The present apparatus simplifies the collection of fractions for this purpose.

A possible weak point in the analysis is the uncertainty in transferring the solute from the GLC effluent to the flowing solvent phase. With substances of high volatility or with those that have poor solubility in the flowing liquid, excessive amounts may be lost with the carrier gas. However, most organic substances are soluble in ethanol, and it is probably the most widely used SPF solvent. Its use is therefore recom- mended as a first choice, especially because it is inexpensive and readily available in pure form. Although recoveries of substances are not stoichiometric, they have been found to be reproducible. Thus, the preparation of standard curves will be necessary.

(8) H. V. Drushel and A. L. Sommers, ANAL. CHEM., 38,lO (1966).

Even though SPF was the mode of detection in the present study, other means of analyses or detection-e.g., ultraviolet absorption, coulometry, or colorimetry-may be adapted for use in a similar manner. For example, the flowing liquid may be a chromogenic agent and the color developed may be measured. Several combinations of the gas chromatograph and ultraviolet absorption detectors have been described (9-11). Unlike the present apparatus, these devices main- tain the GLC effluent in a vapor state by heating the detector cell and connecting lines; the need to maintain this vapor state appears to limit analyses to low boiling compounds. The present apparatus can extend the use of ultraviolet spec- trometry with GLC to higher boiling compounds. Obviously the flowing alcohol interface may also be used for preparative purposes.

A number of other methylenedioxyphenyl compounds that fluoresce strongly were tried in the GLC-SPF setup, but they gave no peaks. Presumably this occurred because they did not come through the gas chromatograph under our conditions of analysis. These were propyl isome, sesamolin, asarinin, tetrahydrosesamin, isosesamin, and piperonylic acid. These chemicals are identified in a recent publication (12). As noted by Zielinski and Fishbein (13), sulfoxide breaks down during gas chromatography. In our tests, its excitation and emission maxima shifted from 292 and 333 mp in the original compound to 304 and 345 mp in the eluted product. The retention time of the peak from sesamex is much less than would be expected on the basis of a comparison of its structure with that of the closely related piperonyl butoxide; the peak obtained is un- doubtedly caused by a decomposition product though the exci- tation and emission maxima of the product were the same as those of the original compound. Other fluorescing chemicals that were checked but were not detected were quinine, indole- 3-acetic acid, 1 -naphthaleneacetic acid, dibenz[a,h]anthracene, and Bay 22408 (0,O-diethyl phosphorothioate 0-ester with N- hydroxy naphthalimide).

RECEIVED for review November 2, 1967. Accepted December 20, 1967. Mention of proprietary products is for identifica- tion only and does not constitute endorsement of these prod- ucts by the U. s. Department of Agriculture.

(9) W. Kaye, ANAL. CHEM., 34, 287 (1962). (10) T. J. Klayder, J. Assoc. Ofic. Agr. Chemists, 47, 1146 (1964). (11) J. Merritt, F. Cornendant, S . T. Abrams, and V. N. Smith,

(12) M. C. Bowman and M. Beroza, Residue Reo., 17, 1 (1967). (13) W. L. Zielinski, Jr., and L. Fishbein, ANAL. CHEM., 38, 41

ANAL. C H E M . , ~ ~ , 1461 (1963).

(1966).

540 ANALYTICAL CHEMISTRY