Determination of Atmospheric Pollutants in the Part-per-Billon Range

Aromatic hydrocarbons in the atmosphere of the Los Angeles basin. William A. Lonneman , T. A. Bellar , and Aubrey P. Altshuller. Environmental Science...
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between the two groups. An illustration can be taken from the results reported by James (8) where he listed the relative retention times of the methyl esters of several fatty acids. Data for the cis-tram isomers of Ae-hexadecenoic and AQ-octadecenoicacids were given on Apiezon L and on polyethyleneglycol adipate columns. As shown in Table IV, Equation 7 is obeyed if the polyethyleneglycol adipate column is considered more polar. ACKNOWLEDGMENT

The author is indebted to Kenneth W. Greenlee of Ohio State Gniversity for special samples and for his encouragement. Roger E. Hinton of the Goodyear Research Laboratory spent 8 considerable amount of time in obtaining the retention date.

LITERATURE CITED

(1) Brown, Ian, Nature 188, 1021 (1960). (2) Cornforth, J. W., Cornforth, R. H., Mathew. K. K., J . Chem. Soc.. 1959. p. 112. (3) Evans, M. B., Smith, J. F., J . Chromatog. 5 , 300 (1961). (4) Zbid., 6, 293 (1961). (5) Zbid., 8 , 303 (1962). (6) Evans, M. B., Smith, J. F., Nature 190, 905 (1961). (7) Greenlee, K. W., Wiley, V. G., J . Org. Chem. 27, 2304 (1962). (8) James, A. T., J . C”hromutog. 2, 552 ( 1959). (9) James, A. T., Martin, A. J. P., Analyst 77, 915 (1952). (10) James. A. T.. Martin, A. J. P.. ’ Biochem.‘J. (London) 50, 679 (1952). ’ (11) Jame8, A. T., Martin, A. J. P., Smith, G. H., Ibid., 52, 238 (1952). (12) Knights, B. A., Thomas, G. Ii., Nature 194, 833 (1962). (13) Kovata, E., Helv. Chim. Acta 41, 1915 (1958).

(14) Kovati, E., 2. Anal. Chem. 181, 351 (1961). (15) Landowne, R. A., Lipsky, S. R., Biochim. Biophys. Acta 47, 589 (1961). (16) Lewis, J. S., Patton, H. W., Kaye, W. I., ANAL.CHEM.28, 1370 (1956). (17) Merritt, C., Jr., Walsh, J. T., Zbid., 34, YO3 (1962). (18) Miwa, T. K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A., Zbid., 32, 1739 (1960). (19) Onesta, R., Castelfranchi, G., Chim. Ind. (Milan) 42, 735 (1960). (20) Fierotti, G. J., Deal, C. H., Uerr, E. L., Porter, P. E., J . Am. Chem. SOC. 78, 2989 (1956). (21) Ray, N. II., J . Appl. Chem. 4, 21 (1954). (22) Smith, J. E., Chem. &- Znd. (London), (,,lp63 1024. e rli, E., Kovats, E., Helv. Chin. Acta 42, 2709 (1959). RECEIVEDfor review April 22, 1963. Accepted August 7, 1963. Presented at Eleventh Detroit Anachem Conference, Detroit, hfich., October 1963.

Determination of Atmospheric Pollutants in the Part-Per-BiI Iion Range by Gas Chromatography A Simple Trapping System for Use with Flame Ionization Detecfors THOMAS A. BELLAR, MARY F. BROWN, and JOHN E. SIGSBY, Jr. Division of Air Pollution, Robert A. raft Sanitary Engineering Center, Cincinnati 26, Ohio

b A simple procedure is described for the determination of atmospheric pollutants at concentrations as low as 0.1 p.p.b. Hydrocarbons in the part-perbillion range in 1 0 O - ~ m .gas ~ samples can easily be determined. A simple trapping system provides for high collection efficiencies over a wide range of flow rates. Most samples can be handled by this system in less than 10 minutes. This collection system is used in combination with analysis by flame ionization gas chromatography. Data are shown for analyses for atmospheric olefins and paraffins derived from automobile exhaust. The general utility of the method is evaluated and described.

W

ITH THE ADVENT of the flame

ionization detector, direct analysis of the more abundant hydrocarbons in the at,mosphere is possible (2-4). The majority of the hydrocarbon contaminants, however, especially the reactive species, are present in concentrations well below the detection limit of even this detector. To extend the range of flame ionization detectors, a concentrating method similar to those used for thermistor analysis was sought. Various reported methods of concentrating were in1924

ANALYTICAL

CHEMISTRY

vestigated, but the results of these investigations were unsatisfactory. Reported trapping efficiencies vary between 80 and 100% (1, 5). In these methods the sample is generally pulled through the trap by a pump. The exhaust from the pump is measured for the volume of the air that has passed through the trap less the contaminants, COz,and water condensed by the trap. By this general method large volumes are concentrated for thermistor detector analyses. Such large volumes are not necessary for the flame ionization detector because of its greater sensitivity. Therefore a method was developed to concentrate a smaller volume (25 to 300 cc.) of polluted air. In this method a gas-sampling valve is used to inject accurately a calibrated volume into a trap. A slow flow of carrier gas (helium) is used to flush the sample from the calibrated volume into the trap. EXPERIMENTAL

Apparatus. P & E flame ionization detector with 0 to 1 mv. recorder. The sample trap (Figure 1) consists of a 10-inch section of No. 316 stainless steel tubing (3/16-inch o.d., 0.016-inch wall thickness). A piece of heavy-wall No. 316 stainless steel tubing (‘/*-inch 0.d.) is silver-soldered into each end of the 10-inch tube. The 10-inch section is

then packed with 10% Carbowax No. 1540 on 50- to 60-mesh firebrick. After both ends are plugged with glass wool, the trap is bent into the shape of a “U” to fit into a 1-liter Dewar flask. Toggle valves ( V , and V,) are then attached to both ends of the trap with swagelock fitt)ings, and the trap is installed on the chromatograph. For further protection a No. 316 stainless steel filter with a mean pore opening of 35 microns can be placed in the swagelock fitting to ensure that no glass wool or firebrick becomes lodged in the toggle valves. Before use the trap is conditioned by heating to approximately 150° C. while purging with helium for about an hour. Procedure. The two main valves (Vl and V,) are standard two-position, six-port, gas-sampling valves. V2 is the gas-sampling valve of the gas chromatograph. VI is used to inject the calibrated volume of sample into the trap. These valves have two Dossible positions : the fill position, a t which the calibrated volume or the trap is filled; and the inject position, at which calibrated volume or the trap is placed in the carrier gas stream. To start the trapping procedure VI and V 2 are placed in the fill position. A diaphragm pump is used t o pull a continuous sample through the calibrated volume; thus it is possible to monitor a changing system continuously.

151

O ~ l O O c c / m i nFLOW METER

Table 1. Carbowax Trap Di Butyl Maleatehifi-2(2-hIethoxyethy1)AdipateColumn FROM INSTRUMENT

-- -

Slope

min

cc .

I___

C-

Simple

2-Methyl- Butene2 349,349 56.3 326, 334 ... 331,332 56.4 342,339 56.4 Av. 338

fiize ( c c . ) I'ropnnc pentane 3.12 3.12 5.37 5.37

3 5 p M E A N POHE,U316 S S F I L I C I ?

14.1 13.6 16.1 15.0

DIAPHRAGM PUMP

-

ncttn

11 11

Std. dev. ~ I O % C A R B O W A NO X 1340 ON 50.60 MESH FiaE BRICK ACID WASHED

1

F N O . 316 SS-1O"r

1%

0 D x 016"

SCIIEUATIC I?= TEAPPllvG S Y S T E M

Figure 1 .

&

25 25 60 100 200 300

8 15.0 12.0 13.2 13.7

... ..

344, 321, 348, 329,

342 327 348 335

56.1 ,

.,

56.1 56.1 56.6 56.3

... ...

Av. 337

Schematic of system

Std. dev. rt- 10

Silica Gel Column The mater manometer is used to maintain a constant pressure in the calibrated volume. A helium flow of 40 cc. per minute is adjusted by V g to flow through the trap with valves V4 and V S?pen: The trap is then immersed in liquid nitrogen. After the trap is cooled to liquid nitrogen temperature, VI is turned to the inject position. The helium is routed first through the calibrated volume and then through the trap. As the sample is condensed in the trap, the helium flow almost stops. No effort is made to maintain the 40-cc.per-minute flow rate. The valves are left in this position for approximately 4 minutes. The exact time is chosen so that two or more calitlrated volumes of helium may flush through the trap. The toggle valves ( V , and V,) are then closed, and Vl is returned to the fill position to condition and fill the calibrated volume for the next sample. The liquid nitrogen is then replaced by hot water to desorb the trapped sample. To inject the sample into the instrument the valves must be opened in this order : V n is turned to the inject position; in rapid succession, V Sand then Vr are opened. This helps l o ensure that the sample is introduced as a "slug" upon the column rather than as a slow "trickle". V 2 should be left in the inject position for E,pproximately l / t minute, depending upon the flow rate of the carrier gas, the trap volume, and the time required to desorb the sample from the trap. After V z is returned t o the fill position, the helium flow reconditions the trap. Sample liolumes can be varied by using dilyerent calibrated volumes or by injecting several samples in series from a calibrated volume into the trap. This svstem can be imwoved (Figure 2) by replacing toggle balves V d a n d V o with a seven-pxt, linear, gassampling valve produc.ed by Micro-? ek. This modification eliminates the stoppage of the carrier-gas flow during the time required to open Vd and V r in the sample introduction procedure. This stoppage usually causw a base line drift,

which may interfere with early peaks on the chromatogram. With the gas-sampling valve ( V S )in the fill position and the seven-port valve in the inject position, the regular trapping procedure is followed. After the sample is trapped, the seven-port valve should be placed in the fill position; thus both ends of the trap are sealed, and the trap's carrier gas is rerouted through the bypass and flushes out any air. The gas-sampling valve (V,) is then placed in the inject position to route the instrument's carrier gas through the bypass. By this time the trap is warmed up, and the sample is injected into the instrument by placing the seven-port valve in the inject position. Other types of gas-sampling valves and dual three-way selenoid valves viere used, but in every case most of the sample leaked out of the valve during heating. Valves containing rubber O-rings retained a portion of the samples, prob-

FROM V I

Samplc size (cc.) 1.20 2.00 3.73 5.28 25 50 75 100 125

mm Slope --

3

CC.

Ethylene 12.7. 12.5 12.4; 1 2 . 3 12.2, 1 2 . 3 12.0, 12.0 12.1 12.1 12.5 12.4 12.5

ably because of the internal geometry. With this arrangement, samples were contaminated by residue from the preceding samples. RESULTS AND DISCUSSION

To test the efficiency of the trap, several hydrocarbons were diluted with air in a Mylar bag. The concentrations

TO COLUMN

t'

TO

3 5 p MEAN PORE,#316

h', Y 316 SS

SWAGELOK UNION

SS FILTER

10% CARBOWAX e1540 ON 50-60 MESH FIRE ERlCK ACID WASHED

10"~b " 0 D I D16'WALL,1)316 SSTRAP-

FILL POSITION

INJECT POSITION

Figure 2.

Modiflcation of system VOL. 35, NO. 12, NOVEMBER 1963

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20.000

aromatic hydrocarbons by this trapping method. They could not be transferred quantitatively a t temperatures of hot water (approximately 70" C.), Higher temperatures would perhaps facilitate a more quantitative transfer. Sixtyto eighty-mesh glass beads were tried in place of the Carbowax packing, but in no case was the sample quantita-

18,000

16.000

2 METHYL PENTANE 14,000

12.000

E

a

:

The initial helium flow rate through the calibrated volume does not affect the trapping efficiency, as shown in Table 11. This flow does not remain constant throughout the trapping procedure because the condensing materials increase the restriction of the trap and cause the flow rate to approach

10,000

4

Y

=a

:0000 6000

4000

2000

1000

50

100

Figure 3.

150

200

250

TRAP V O L U M E IN c c

3 50

300

Linearity of traD remonse

of the hydrocarbons were chosen so that it was just possible to measure the areas of the individual peaks accurately when an unconcentrated sample was injected directly into the instrument. Accurately calibrated volumes of 3.12 cc. and 5.37 cc. were used to analyze the samples in the bag. Then the trapping system was attached to the gas chromatograph, and a calibrated injection volume of 25.0 cc. was used. Samples of 25, 50, 100, 200, and 300 cc. were trapped and analyzed. The areas of the peaks were plotted against the total injection volumes. The resulting graph (Figure 3) shows excellent linearity of all points with respect to the origin. The slope was calculated from the origin for each individual point. For 2-methylpentane the average slope for the directly inmm jected samples is 338 -, with a cc. standard deviation of =tS. The trapped samples have an average slope of mm.* and a standard deviation of 337 1 1 0 . This is equal to an error of 10.01 p.p.b. in a 300-cc. sample. The differences between samples we due mainly to errors in area measurement. Among the samples shown in Table I, propane was the most difficult to measure because of its narrow peak width. The data for ethylene were obtained from another instrument with a silica gel column. The helium used to flush the sample through the calibrated loop was analyzed. Other than methane, no measurable amounts of hydrocarbons could be detected. At no time during the trap warmup and injection procedure were there any instances of high pressure,

.

I

an indication that no large volume of liquid 0 2 formed in the trap during the concentration step. Attempts were made to analyze

Table II.

Compound Isobutane n-Butane Butene-1 t~ans-Butene-2 cis-Butene-2 Butadiene 1-3

been made to extend the e past 300 cc., since all the atmospheric hydrocarbons of interest to this study mere detectable a t even lower volumes. The lower limit of quantitative detection a t these volumes is 0.1 p.p.b. Qualitatively, the lower limit is 0.01 p.p.b. Figure 4 is a chromatogram of urban Cincinnati air. The 200-cc. trapped sample, taken in the early afternoon, shows an abundance of olefins.

Effect of Carrier Flow Rate Area of Trapped Sample

30 20 10 50 100 200 300 cc./min. cc./min. cc./min. cc. /min. cc./min. ac./min. cc./min. for for for for for for for 2 min. 3 min. 6 min. 1 min. 1 min. 1 min. 1 min. 261 304 266 273 285 292 280 1910 1860 1920 1960 1880 1760 1840 1680 1710 1720 1720 1690 1740 1760 1360 1360 1330 1288 1360 1320 1340 1210 1220 1162 1150 1190 1200 1190 1062 1070 1150 1090 1110 1150 1150

cc.

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~

I

B

9

IO

/I

I2

13

14

Figure 4.

15

I6

17

I8

I9 20 MINUTES

?I

22

- ~ t L A - L u: 23

24

25

2E.

Chromatogram of Cincinnati air

27

28

I

-I_-!.-,

29

30

i 31

32

CONCLUSIONS

of the artificial smog mixture is no greater than 3 carbon p.p.m.

This system has provided an efficient method of trace analysis in the 0.1 p.p.b. range. It is now being used in routine analysis with an artific la1 smog chamber. Excellent results have been obtained in analyzing butenes rind higher-molecUlar-R-eight olefins and Paraffins, even when the total carbon concentration

LITERATURE CITED

(1) Altshuuer, A. p., Bellar, T. A., Clemons, C. A., Am. I n d . Hyg.Assoc.

J. 23, 164 (1962). ( 2 ) Altshuller, A. P., Bellar, T. A., J. Air Assoc. in press. (3) Altshuller, A. P., Clemons, C. A., ANAL.CHEM.34,466 (1962).

(4) Bellar, T-1SigsbY, J. E., Jr.1 Cle~nons, C. A., Altshuller, A. P., Ibid., 34, 763 (1962). (5) West, P. W., Sen, B., Gibson, N.A., Ibid., 30, 1390 (1958). RECEIVED for review January 21, 1963. Accepted July 22, 1963. Division of Water and Waste Chemistry, 142nd Meetr ing, ACS, Stlantic City, N. J., September 1962.

Determincrtion of Available Lysine in Oilseed Meal Proteins S. RAGHAVENDAR RAO', FAlRlE LYN CARTER, and VERNON L. FRAMPTON Southern Regional Research laboratory, Agricultural Research Service,

b A simple and rc8pid method for estimation of available lysine in plant proteins is described. The protein is dinitrophenylated, hydrolyzed in acidic media, and the epsilon-DNP-lysine produced is eluted ,From an ion exchange column with a solvent composed of three parts by volume of 3N aqueous HCI and one part methyl ethyl ketone, and is determined spectrophotometrically at 4 3 5 mp.

I

is being given to plant materials especially oilseed meals, as sources of proteins of high quality for human beings and domestic animals. Because of the serious nutritional problem in other parts of the world, and our own dcmestic problem of supplying adequate proteins of suitable quality to the swine and poultry industry, interest has devel3ped in analytical means of determining the nutritive quality of proteins of .iegetable origin. The view that the nutritive value of vegetable proteins, arid of animal protein of inferior qualiLy, is directly related to the lysine in these proteins with the epsilon amino gi-oup free is well established (4, 8, 7, 16, 18). Lysine is not used unless the epsilon amino groups are free (16). Correlations between growth response of young animals and the free epsilon amino groups of lysine in the proteins they receive are invariably high, and, moreover, analyses of variance of the data indicate that a substantial portion of the variance of growth response obtained with different sources of most proteins of plant origin can tit? accounted for l ~ ythe variance of the free epsiloii amino groups of the lysine in these proteins. I n the case of commercial cottonseed meals, for example, the contribution of lysine in NCREASING ATTENT [ON

Prcsent address, Regional Research Laboratory, Hyderabad 9 A.P., India.

U. S. Departmenf o f Agriculture, New Orleans 7 9, l a .

the proteins with the epsilon amino group free to the total variance in growth of broilers and swine is so overwhelming that contributions of other seed constituents such as gossypol, carbohydrates, crude fiber, fat, etc., to the total variance in growth are completely masked (12). Several methods have been proposed for determining the free epsilon amino groups of lysine (available lysine) in foodstuffs (1, 60). The more promising methods are those involving the use of 2,4-dinitrofluorobenzene (DNFB). Lea and Hannan (15) and Schober and Prinz (20) utilized dinitrophenylation to study the reduction of epsilon amino groups during processing of milk products. Carpenter and his associates applied this procedure (5) to study the available lysine of a wide variety of animal and plant products. In the earlier methods, Carpenter et al. (8) directly measured the absorbance of the yellow, ether-extracted acid hydrolyzates of dinitrophenylated proteins in their estimation of the available lysine. I n a later modification (6), Carpenter reduced some of the error in the analyses by reacting the epsilon-dinitrophenylhydrazone-lysine with methyl chloroformate to produce an ether-soluble lysine derivative. The difference in color intensity of the hydrolyzate before reaction with methyl chloroformate and after reaction and extraction with ether was taken as a measure of available lysine. Dinitrophenol is the major by-product of the dinitrophenylation of proteins. Conkerton and Frampton (IO),by taking advantage of the difference in absorbance of dinitrophenol a t 380 nip in acid and alkaline media, were able to correct for the quantity of dinitrophenol present in the protein hydrolyzate. Other yellow substances might occur in the hytlrolyzates of some dinitrophenylated proteins (13) and introduce errors in a colorimetric estimation of

available lysine. I n addition, the brown humin pigments that invariably occur in acid hydrolyzates of proteins may also be expected to contribute to the error in some of the earlier procedures. I n a more recent study of dinitrophenylated cottonseed meal proteins, Frampton (12) found differences in the lysine content of the oilseed proteins before and after dinitrophenylation. The lysine lost on dinitrophenylation was recovered as epsilon-DNP-lysine. Di-DYP-lysine was not detected in any of the hydrolyzates. Presumably the free epsilon amino groups of lysine in the oilseed proteins will react with DXFB, while those that are bound chemically will not. This difference in the lysine content, as determined by the ion exchange technique of Spackman, Stein, and Moore (22) is taken in this paper as the reference in establishing the validity of the procedure reported here for available lysine. Many of the sources of error which are found in the earlier methods are eliminated in the procedure described herein where epsilon-DXP-lysine is separated from dinitrophenol and other yellow derivatives of the reaction between DNFB and the meal proteins, as well as from the brown humin products of acid hydrolysis, through the use of an ion exchange column that is developed with a mixture of methyl ethyl ketone and aqueous HCl(91). EXPERIMENTAL

Apparatus and Reagents. All absorbance determinations were made at 380 or 45.5 mp (unleis otherwise stated) with 3 Beckriian spectrophotometer, Model B. Ten-millimeter cuvettes were 1ised . The chromatographic column was constructed from IO-mm. i.d. glass iribiiig 1.5 to 16 rrn. i i i lcfiiigtli,\ k i t h ~1 medium-porosity sintercd glass disk and a Teflon stopcock fused in the VOL. 35, NO. 12, NOVEMBER 1963

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