shape (but of diminished size) is found a t the same position as the nitric oxide. If the gas eluted after adding nitrogen dioxide is trapped in liquid nitrogen, it is colorless after warming but becomes brown after being mixed with oxygen. This gas is unquestionably nitric oxide because nitrogen dioxide, with its high boiling point, would not be eluted a t room temperature from the Molecular Sieve. The lower curve of Figure 1 shows the calibration obtained with nitrogen dioxide in the sample bulbLe., the peak height of the resultant nitric oxide yield. A constant-volume sample bulb was used, and the pressure in the bulb is plotted us. peak height. The calibration of nitrogen dioxide is complicated by association.
2x02 .$ S*O(
(3)
The position of the equilibrium is a function of the total pressure in the sample tube. By utilizing data on the equilibrium constants (1) and taking into account the degree of association as a function of pressure, the points represented as crosses were plotted as the peak heights which would have been observed had the stoichiometry of the reaction of nitrogen dioxide and water been that predicted by Equations 1 and 2. The degree of association was determined a t the given pressure, and the theoretical equivalence of the mixture was calculated in terms of nitric oxide. From this procedure, the peak height which should have been observed was taken from the original nitric oxide
calibration. The agreement of the data is good and calibrations for nitrogen dioxide are accomplished conveniently with nitric oxide. It mas qualitatively established that the same process takes place on wet alumina and silica gel columns. LITERATURE CITED
(1) Glasstone, S., “Textbook of Physica Chemistry,” p. 837, Van Nostrand,
Sew Tork, 1946.
( 2 ) Greene, S. A , . Moberg, M.L., \Vilson, E. ll., ASAL. CHEM. 28, 1369
(1956).
(3) Szulczewski, R. B., Higuchi, T., Ibid., 29, 1541 (1957).
RECEIVED for review September 18, 1957. Accepted January 27, 1958. Division of Analytical Chemistry, 132nd Meeting, ACS, S e w York, X . Y., September 1957.
Gas Chromatographic Analysis of Engine Exhaust and Atmosphere Determination of C, to C, Hydrocarbons F. T. EGGERTSEN and F. M. NELSEN Shell Developmenf Co., Emeryville, Calif. )A sensitive gas chromatographic technique has been developed and applied to the determination of traces of individual CZ to C6 hydrocarbons in engine exhaust and city air. The threshold of detection i s 0.05 pap.m. or better, which brings the technique well within the concentration range of interest in urban air pollution studies. In this range the method i s simpler than spectrometric methods and provides much additional information. The Cs to C5 hydrocarbons are first trapped out in a short chromatographic column cooled in liquid oxygen, and then analyzed by elution with helium through a longer chromatographic column followed by a thermal conductivity detector operated at high sensitivity. Examples of application are given.
I
COKNECTIOS WITH air pollution studies there is a n urgent need for simple, accurate, and highly sensitive methods for determining traces of hydrocarbons. Although the available spectrometric methods are satisfactory in many respects, they often do not give a sufficiently detailed analysis, particularly with the low concentrations encountered in atmosphere samples, and the equipment required is usually elaborate and expensive. Gas chromatography offers the poten-
x
1040
ANALYTICAL CHEMISTRY
tial advantages of ready availability, relatively low cost, simplicity in operation, and a more detailed analysis. By adding a concentration step to a conventional gas chromatography apparatus and operating the detector cell a t high sensitivity, the useful range for C:! to Cshydrocarbons can be extended to or below the 0.05-p.p.m. level. This sensitivity is well within the range of interest in air pollution control. EXPERIMENTAL
Apparatus. As shown schematically in Figure 1, t h e apparatus consists essentially of a short trapping column, a longer separating column, and a thermal conductivity detector. The trapping column is made of 5/16inch (outside diameter) copper tubing, 12 inches long and bent into a U shape; it is partially filled with 6 ml. of dimethylsulfolane-crushed firebrick packing (40 grams of liquid per 100 grams of support, 20 to 30 mesh). During charging of the sample it is immersed in liquid oxygen in a Dewar jar. The separating column consists of 25 feet of */r-inch coiled copper tubing containing the same packing and maintained a t ice-water temperature. The thermal conductivity detection cell (Model 9285, Gow-)lac Instrument Co., Madison, N. J.) is a four-cell assembly in a circuit which is basically that described by Dimbat, Porter, and Stross ( I ) , with the t h i ~ m a lconduc-
tivity cells as the four arms of the Wheatstone bridge. As pointed out by these authors the sensitivity of the four-cell arrangement is about twice that obtained with the more common two-cell unit, To provide a further gain in sensitivity the bridge is operated a t high current, 300 ma., with a 12-volt battery as current source. K i t h a current of 200 ma., which is more commonly used, the response of the detector cells is only about one fifth as great, I n normal applications of gas chromatography, where milligram quantities of sample are employed, the thermal conductivity detection unit is operated far below the high sensitivity range of this work. Temperature stability of the detector cells is achieved by enclosing the cell block in an insulated chamber, where the block is maintained a t 75” C. by heating it electrically with a constant heat input, This heater is in the form of a brass rod containing two 50-watt cartridge heaters; the rod is ground flat on one side and bolted to the cell block. Preheat lines for the gas streams to the reference and measuring cells, each 1/8-inch by 2-foot copper coils, are wound around the brass rod and soldered to it. The helium flow scheme is designed to stabilize operation of the detectors; the flow control valve is placed at the outlet of the column where it helps to protect the measuring cells against flow upsets when the trapping column is heated. The reference cells are operated with a separate helium supply so
Cells
Figure 1. Schematic diagram of gas chromatographic apparatus for determination of hydrocarbons in exhaust gas and air
Therma!
VI
I
.is c a r it E
sample volumes charged were about 200 and 3000 ml., respectively. As Table I shows, the results were in fairly good agreement with the known values for this sample. Also, the total peak area for the lower concentration agreed \vel1 with that calculated by applying the dilution factor to the area for the higher concentration (not shown in the table). The results indicate essentially no losses in handling these gas mixtures by this procedure. I n terms of peak area a t full sensitivity, with a chart speed of 24 inches per hour (I em. per minute), the response was about 1.4 sq. cm. per microgram of hydrocarbons (1.36 and 1.48 for the concentrated and dilute sampks, respectively). Or, in terms of units suggested by Dimbat, Porter, and
Table I. Analysis of Standard Cz to Cs Hydrocarbon Mixture
that they are unaffected by f l o ~variations in the main system. I n the present study the helium (Air Reduction Go.) was used ~ i t h o u purit fication. However, i t was felt that this gas might sometimes contain contaminants which would interfere in the analysis. Accordingly, as a general practice, the helium is now purified by passing i t through a cold trap of activated carbon a t liquid nitrogen temperature. This trap is located directly ahead of the trapping column. Procedure. T h e sample iequirements may range from a few hundred milliliters of exhaust gas t o 5 t o 10 liters of air. T h e samples are collected in evacuated glass containers having stopcocks lubricated with Dow-Corning high vacuum silicone grease. T h e sample is first passed through a n Ascarite column, '/4 by 8 inches long, t o remove water and acid gases, and then through the trapping column immersed in liquid oxygen; this operation requires 10 to 45 minutes. (Because of the extreme explosion hazard, contact of organic material with liquid oxygen must be avoided.) A convenient method of charging is to dran- the sample from the container through the trapping column by house vacuum, removing about t n o thirds of the total xolume as determined by p r e s u r e measurements. (Some condenwte, chiefly water, \vas present in the sample tubes when exhaust gas samples nere run; no effort was made to vaporize this material before withdrawing the sample.) During the coldtrapping step the valve and inlet to the trapping column are kept warm mith a tape heater in order to prevent condensation at these points. After the sample is charged, the trapping column (still immersed in liquid oxygen) is connected t o the separating column (Figure 1) and is purged n i t h helium for 20 to 30 minutes. The helium inlet pressure is 12 p.s.i.g.
and its flow rate, measured a t atmospheric pressure, is 60 ml. per minute. The flushing operation removes most of the material boiling below C p hydrocarbons which, if not removed, would obscure the early peaks for C p to C5 components, to be eluted later. After the flushing operation, the trapping column is quickly warmed to 0" C. by inimersion in ice m t e r . The material eluted is passed through a second Ascarite column (Figure 1) to ensure complete removal of carbon dioxide and water, and then through the separating column, which has been well purged beforehand (preferably backflushed). The chromatogram is recorded, using a 1-mv. potentiometer operated a t a chart speed of 24 inches per hour. The amount of each component is determined by measuring peak areas with a planimeter. The individual hydrocarbons are identified from their relative emergence times, as reported by Fredericks and Brooks ( 2 ) ; the peak area for each component is assumed t o be proportional to the weight present, which is appro\iniately true for the CZ t o Cs range. Sitrous oxide, which emerges IT ith propane and is determined with it, is assumed to have the same weight sensitivity as the hydrocarbons. Peak area is converted to neight by use of a sensitivity factor, determined by analysis of n standard Cz to Cs mixture. Concentrations are expressed as part5 per million by weight, assuming the density of the gas mixture to be that of air, 1.19 mg. per ml., a t ambient temperature and pressure. RESULTS
The accuracy and sensitivity of the method !\-ere first tested using a standard mixture of Cp t o Cs hydrocarbons (Phillips Petroleum Co., No. 40), diluted with nitrogen to concentrations of 500 and 2.5 p.p.m. by weight; the
(Phillips Petroleum Co., No. 40) Wt. %;: from Chromatogram AreaP.1".
p]iil]ips
values, K t . yo
Component Ethane Propane Propylene Iso66tane n-Butane 1-Butene Isobiitene '-Butene Isopentane 1-Ifent ene 2-Prntene Table
II.
of
Orig. Sample -
2 4
13 T 13.8 21.2 9.2 9.9
500
2.5
2 4 15 6 13 8
14 2 17 2
21.9 9.3 9.5 14.6
15.3
0.7' 7!
44 23.8 9.7 9.7 11.9
S o t obseivetl
Analysis of Cruise Engine Exhaust
Component Ethane Ethylene Propane + S20 Propylene Isobutane n-Butane Acetylene 1-Butene Isobutylene Propadiene trans-2-Butene Isopentane ,' &-%Butene n-Pentane 1 3-Methyl-1butene I 1,3-Butadien;! 1-Pentene' Prop)-ne Total CI t o C , , I
Wt. 70Eroni Chromatogram Area Diluted 7 5 t o 1 Tvitli Undiluted air" (2. i ' i b
(3.8Ih 4i.5
48.6' 67 . 73 0 0 25 0 0 1
7.8
i.5 0.G 2.2
5 6 8 5 5 5 ,3
24.;
0.6 1. 9 1.0
10
1.9 S o t observed
1 5
S o t observed
0 7
S o t observed Not observed
1 '3
p.p.,m.
(we1ght) 490 G 4c Corrected for blank determination of air. b Values in parentheses are considered less reliable than others. 6.5 calculated from dilution factor. a
VOL. 30, NO. 6, JUNE 1958
1041
"Air"
II 0.4
vi
,. .-
0.3
Propane + N20 SEPARATING C O L U M N ( D i m e t h y l s u l f o l a n e on F i r e b r i c k )
I TRAPPlNG COLUMN (Dimethylsulfolane on F i r e b r i c k )
-
.-Ethylene
I 0.2 M
+
$
Ethane '
d
Propylene
nButane Acetyl-
0.1
Isopent m e
nPentane
Propyne + I-Pentene
+ c I
0.0
E m e r g e n c e T i m e , hlinutes
Figure 2.
Table
111.
Analysis of Samples
Atmosphere
P.P.M. by Weight City air, Highv,axg high tunnel traffic air level (0 08'1" b 1 09 0 29 0 58 0 43 0 36 0 15 0 21 0 08 I 05 0 24 0 68 0 18 0 04 b 0 07 b 0 47 0.13 0 54 0 12 0 21 b
Component Ethane' Ethylene Propane S20 Propylene Isobutane n-Butane Acetylene 1-Butene Isobutylene Isopentanec n-Pentaned Butadiene 1-Pentene propyne (0 21)" (0 33)" Total 5 59 1 95 a Values in parentheses are considered less reliable than others. b Xot detected. c LIay include some 2-butene d May include some 3-methyl-1-butene.
+
+
Stross ( I ) , the sensitivity is 2800 my.nil. per mg.; this is about nine times that reported by these authors for their tu-0-cell thermal conductivity detector operated a t 200 ma. A 300-ml. sample of cruise engine exhaust was then analyzed together with a sample of this exhaust diluted 75-fold with laboratory air (60 ml. in 4500 ml. total) in order to test the applicability of the method to atmosphere analysis. Table I1 shows the percentages of the individual Cz to Cs components found. I n general, the results for the whole exhaust and for the diluted sample agree well. Based on the calibration made with the standard hydrocarbon sample, the total Cz to Cs content of the whole exhaust mas 490 p.p.m. by weight, which is the order of magnitude reported for Cz+ hydrocarbons by other investigators employing other methods. The total Cz to Csfound in the diluted 1042
ANALYTICAL CHEMISTRY
Gas chromatogram of high traffic city air
sample, after making a blank correction for the diluent air, was 6.4 p.p.m., which is in good agreement with 6.5 p.p.m. calculated from the analysis of the undiluted sample and the dilution factor. However, five of the C4 and Cs components were present in concentrations too low to be distinguished in the diluted sample, and the values for some of the other minor components are of doubtul accuracy. Better values for minor components could, of course, be obtained with a larger gas sample. Table I11 shows representative data for analysis of tit-o atmosphere samples, and a typical chromatogram is reproduced in Figure 2. These samples contained only 2 to 6 p.p.m. of Cz to C5 hydrocarbons; even so, it vias possible to identify and measure 13 coml~onents in the tunnel air and nine in the city air. Some components n-ere determined below the 0.1- p.p.ni. level. Values for I-pentene plus propyne varied more than those of the other components and seemed to decrease markedly during several hours of storage prior to analysis. Ethane, as discussed in the following section, is determined only approximately. These values are therefore considered less reliable than the others and are so indicated in Table 111. DISCUSSION
According to results with the standard hydrocarbon mixtures, the limiting sensitivity for the apparatus appears to be about 0.2 y for a component emerging in about 10 minutes; this is comparable with the sensitivity' indicated by the results of Patton and Touhy (3)in their study on cigaret smoke, using a similar four-cell detector. IJ'ith a 10-liter (12-gram) sample of air, 0.2 y corresponds to about 0.02 p.p.m. by weight. This concentration is not the absolute limit; in fact, the only limiting factors would appear to be the capacity of the trap-out column and the amount of
sample charging time that can be allowed. Despite the precautions taken to ensure good detector stability, there is often a base-line drift (not shown in Figure 2 ) . However, with well-purged columns this drift is very gradual and does not have a significant effect on the analysis; the noise level is generally not more than 0.005 mv. The detector unit gave good service during 3 months of almost daily use, after which it became unstable and required a new set of thermal conductivity cells. To prevent unnecessary overheating of the cell filaments, the current through them should be turned off JT-hen helium is not flolying. When this current is off it is advantageous to maintain the desired cell block temperature by increasing the input of the heater. I n this Fi-ay temperature equilibrium can be more rendily established for the next experiment, In the procedure described, the trapping and charging of the sample is done in a single step, n-hich is more expeditious and less subject to manipulative errors than if the sample xere trapped, expanded, and then charged to t h e separating column in separate operations. Gas chromatographic packing n-as used in the trapping column because it initiates the separations, which map or may not be a significant advantage. It is likely that a trap containing an inert material such as glass n-001, which serves merely as a sample retainer, nould also be suitable, but this possibility was not extensively investigated. Elution by warming the trapping column is in effect a method of introducing the sample very sharply to the separating column. Liquid oxygen, rather than nitrogen, was selected as the coolant in order to ai-oid condensation or sorption of ouygen, because any oxygen retained may react with hydrocarbons, especially the olefins. However, it is possible that liquid nitrogen
could be employed if the sample is trapped at reduced pressure so that the saturation pressure of oxygen is never exceeded. Resolution by the dimethylsulfolane column is generally satisfactory for Cz to Cs hydrocarbons, as s h o m by Fredericks and Brooks ( 2 ) . However, under the conditions employed here, ethane and ethylene emerge too close together and too close to the “air” peak; thus, ethane appears as a small shoulder on either the air or the ethylene peak, and can only be approximated \Then relatively large amounts of ethylene are present. Nitrous oxide, which is generally present to some extent in exhaust gases and atmosphere, emerges with propane. Other possible coemerging compounds are indicated in Table 11. In later ndrk slightly better peak resolution was achieved with a longer (50-foot) column with less (207,) dimethylsulfolane on the firebrick. Also, a partial separation of nitrous oxide and propane was obtained by allowing the trapping column t o warm up slowly during the elution step. In all of the cruise exhaust gas analyses there was a low trailing peak for nitrogen dioxide, not evaluated, which reached its highest point just ahead of butadiene. Therefore, the butadiene peak, and any later ones, are superimposed on the tail of the nitrogen dioxide peak. Tests made with nitrogen dioxide alone indicated that this compound is not completely removed
by the Ascarite absorber. Nitric oxide emerges with the air peak and does not interfere. I n the procedure described, the trapping and flushing steps take 0.5 t o 1.5 hours, the analysis step 1 to 2 hours, and the peak area measurements and calculations 0.5 to 0.75 hour. The capacity of a single unit is, therefore, two to three analyses per 8-hour day, The time per analysis could be reduced by increasing the rate of charging the sample to the trapping column; however, this possibility was not investigated. An automatic integrator for measuring peak areas would save operator time. The possibility of reactions in the sampling vessel prior to analysis was not studied, but is a problem which will have to be considered in applying the method. Alqo, the technique used here for sampling exhaust gas should be modified to prevent possible losses by condensation in the container. Complete vaporization might be ensured by warming the sample container or by dilution with an inert gas. Adsorption of various hydrocarbons in the Ascarite tube probably needs further study. However, the good results obtained with the standard C2 to Cs hydrocarbon mixture indicate that this is not a serious problem, a t least not with the C5 and lighter components. Any hydrocarbons which might be retained in the Ascarite during charging could probably be swept into
the trapping column with helium. This could be done conveniently by merely connecting the Ascarite tube ahead of the trapping column during the helium flushing operation. The technique appears to have promise for determination of other components in air and exhaust gases. Different columns or combinations of columns would enable methane and hydrocarbons above CS to be determined, and perhaps certain oxygenated hydrocarbons. However, some of the latter, notably the aldehydes, may be removed by the Ascarite absorber. If only total hydrocarbons are desired, rapid estimation should be possible by using the short trapping column and heating it sufficiently for complete elution. ACKNOWLEDGMENT
The authors are indebted t o E. D. Peters, who initiated the study and helped plan the scheme of analysis. Special acknowledgment is also given to Sigurd Groennings for valuable counsel and encouragement. LITERATURE CITED
(1) Dimbat, M., Porter, P. E., Stross, F. H., ANAL.CHEM.28, 290 (1956). (2) Fredericks, E. M., Brooks, F. R., Zbid., 28, 297 (1956).
(3) Pat,ton, H. W., Touhy, G. P., Zbid.,
28, 1685 (1956). RECEIVEDfor review September 26, 1957. Accepted January 27, 1958.
Automatic Equipment for Determination of Amino Acids Separated on Columns of Ion Exchange Resins D. H. SIMMONDS‘ Biochemistry Unit, Wool Textile Research laboratories, Commonwealth Scientific and Industrial Research Organization, Melbourne, Australia
b Development of automatic equipment for determination of amino acids was undertaken to reduce the manual labor and systematic variability of these analyses, and to increase the output. Analytical variations have been reduced b y a factor of 3 over the manual method. No manual work is involved beyond loading the columns, changing buffers, and finally calculating results from the graphical presentation. Because it can work 7 days a week, the machine approximately doubles the output of results obtained by the manual method. Besides its use in determination of amino acids, it may b e applied directly to estimation of any groups of substances separable b y column chromatography, provided a
common colorimetric, absorptiometric, or turbidimetric assay method is available. With minor modifications it could b e used for the routine assay of large numbers of individual samples.
S
Stein, and Moore (11) have recently described their equipment for the automatic determination of amino acids in protein hydrolyzates. I n these laboratories, progress has also been made toTvard automation of the excellent but somewhat tedious colorimetric assays involved in the manual ion exchange procedures developed by Moore and Stein (4-‘7). This paper describes the equipment developed and report5 some results obtained Tvith it. PACKMAX,
The following sequence of manipulations is concerned in manual estimation of amino acids by the ion exchange chromatographic procedures of Moore and Stein (5, 6).
-4. Collection of fractions from chromatographic column effluent. B. Adjustment of each fraction to pH 5.0, if the reagent of Moore and Stein ( 4 ) is used. If their other reagent (7) is used, this step is omitted. C. *4ddition of ninhydrin reagent. D. Heating t o develop color. E. Dilution with ethyl alcohol-water (1t o 1,v./v.). F. Spectrophotometric estimation of diluted color. 1 Present address, Waite Agricultural Research Institute, Adelaide, South rlustralia.
VOL. 30, NO. 6, JUNE 1958
0
1043
