(8) V. Zitko, ht. J. Environ. Anal. Chern., 2, 241 (1973). (9) A. A. Belisle, W. L. Reichel, and J. W. Spann, Bull. Environ. Contam.
roxicoi., 13, 129 (1975). (10) C. S. Giarn and M. K. Wong, J. Chromatogr. Scb, 7 2 , 283 (1972). (1 1) C. S. Giam, R. L. Richardson, M. K. Wong, and W. M. Sackett, Antarct. J. U.S., 8, 303 (1974). (12) C. S. Giam, R. L. Richardson, D. Taylor, and M. K. Wong, Bull. Environ. Contam. Toxicol., 11, 189 (1974). (13) c. s. Giam, M. K. Wong, A. R. Hanks, W. M. Sackett, and R. L. Richardson, Bull. Envifon. Contam. Toxicol.,9, 378 (1973). (14) C. S. Giam, A. R. Hanks, R. L. Richardson, W. M. Sackett, and M. K. Wong. Pestic. Monit. J., 6, 139 (1972).
(15) U.S. Department of Health, Education and Welfare, Food and Drug Administration, "Pesticide Analytical Manual", Vol. 1, Sect. 212.13, 1968. (16) D. F. Goerlitz and L. M. Law, Bull. Environ. Contam. Toxicol,, 6, 9 (1971).
RECEIVEDfor review May 12, 1975. Accepted July 30, 1975. We are grateful to the National Science FoundationInternational Decade for Ocean for the financial support of this study (NSF Grant No. 37349).
Hydroxylamine Determination of Cephalosporins D. L. Mays F. K. Bangert, W. C. Cantrell, and W. G. Evans Chemical Control Department, Bristol Laboratories Division, Bristol-Myers Company, Syracuse, N. Y. 1320 1
An Improved hydroxylamine determination is described for cephalosporins, offering adequate sensltlvlly, stable test and blank solutions, and good precision. Cephalosporins react more slowly with hydroxylamine than do penicillins, so that hydroxylamine methods used for penicillins suffer from incomplete reaction and poor reproducibllity when applied to cephalosporins. Work was undertaken to develop a suitable hydroxylamine method for process control of cephalosporins. The effects of reagent concentration and reaction conditions are discussed. Nickel( 11) was found to catalyze hydroxamlc acid formation of cephalosporins and to stabllize the color formed with iron(ll1). Optimized manual and automated procedures suitable for a variety of cephalosporins are presented. Comparisons with other procedures and precision data are included.
Hydroxylamine reacts with a number of carboxylic acid derivatives to form hydroxamic acids which yield colored complexes with iron(II1) in acidic solutions. These reactions, reported in 1899 by Bamberger ( I ) , were made popular in a spot test for carboxylic acid esters by Feigl (2, 3 ) in 1934. They have since been applied to the determination of acyl phosphates ( 4 ) ,esters (5, 6), amides ( 7 ) ,reducing sugars ( 8 ) , lactones (9, I O ) , and acid chlorides and anhydrides ( I 1 ). The hydroxylamine reaction-ferric iron complexation has also been applied to the determination of penicillins (12-1 7). Both manual and automated procedures have been used for several years to determine penicillin content in process samples and formulations. Although these procedures have also been applied to some cephalosporins (18-20), the procedures have not been generally satisfactory in our hands because of the greater stability of the cephalosporin nucleus (21). Near neutral pH, we found the reaction of hydroxylamine with cephalosporin C to be incomplete, resulting in poor sensitivity and reproducibility. At high pH, the reaction proceeded more nearly to completion, and sensitivity was improved, but reproducibility was poor, perhaps due to competition from alkaline hydrolysis of the P-lactam. This effect has been noted for some lactones (22). In addition, esters such as the 3-acetoxymethyl group of cephalosporins may react with hydroxylamine at high pH ( I 1 , 2 3 ) . A more concentrated hydroxylamine reagent a t neutral pH, as described by Flynn (21), increased the extent of reaction. In our hands, however, poor reproducibility was encountered; the wavelength recommended for measurement
was not the observed maximum for the ferric-hydroxamic acid complex; reaction with cephalosporin C was only about 80% complete in the time specified; and color stability of the complex was poor. We were unable to obtain agreement among absorbance measurements taken in immediate succession. Although these difficulties might be trivial for an automated application of the method, our aim was to develop a precise, sensitive procedure for manual or automated use. Recent work by Munson and others (2427), using nickel(I1) catalyst to form hydroxamic acids from carboxylic acids and acid hydrazides, encouraged us to evaluate hydroxylamine with nickel(I1) as a catalyst for the determination of cephalosporins. This report describes a simple and reproducible procedure for determining cephalosporins, using a hydroxylamine reagent containing nickel(I1). This procedure applies to a wide variety of cephalosporins, including 3-hydroxymethyl and 3-methyl substituted forms (Figure I), and eliminates the need for strict timing of absorbance measurements. Both manual and automated applications are described. The method shows great promise for high volume assays where good precision is required, such as for content uniformity testing.
EXPERIMENTAL Materials and Apparatus. Cephalosporins, unless otherwise noted, were Bristol Laboratories' reference materials. Other chemicals and reagents were analytical grade, used as received. Technicon AutoAnalyzer Modules were used as follows: Sampler I1 with a 40/hour 1:l ratio cam; Pump 11; N-type colorimeter with 15-mm tubular flow cell; single pen recorder. Reagents for the Manual Determination. HydroxylamineNickel Reagent was prepared by dissolving 6.95 g of hydroxylamine hydrochloride (Mallinckrodt) and 2.39 g of nickel(I1) chloride hexahydrate (Fisher) in about 30 ml of water, adjusting to pH 6.20 f 0.05 with ION sodium hydroxide, and diluting to 50.0 ml with water. This solution is 2M in hydroxylamine and 0.2M in Ni. Iron(II1) reagent was prepared by dissolving 150 g of ferric ammonium sulfate hydrate (Allied Chemical), Fe(S04)3.(NK4)2S04.24 H20, in sufficient 1N sulfuric acid to make one liter. Reagents for t h e Automated Determination. Phthalate buffer, pH 6.0, 0.5M, was prepared by dissolving 10.2 g of potassium hydrogen phthalate (Fisher) in about 500 ml of water, adjusting to pH 6.0 with 10N sodium hydroxide, and diluting to 1 liter with water. Hydroxylamine-nickel reagent was prepared by dissolving 6.95 g of hydroxylamine hydrochloride and 2.38 g of nickel(I1) chloride hexahydrate in about 50 ml of water, adjusting the pH to 6.2 with 10N sodium hydroxide, adding 25 ml SD-32 ethanol (US. Industrial Chemicals Co.), and diluting to 100 ml with water. Stock iron(II1) solution was prepared by dissolving 214 g of ferric ammo-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
*
2229
@ MI
M2
M2
0 3
HOUR
ML/MIN
0 6 0 - SAMPLE AIR
kOOH 7 - Aminocephalos p o r a n i c acid ('I-ACA) 3-Hydroxymethyl'I-ACA 3-Methyl-7-ACA
DILUEYT. O.i%
R'
R
Cephalosporin
-OCOCH,
H-
-320$:+
IRON I N H RCElA G E h T
RESERVOIR
080-
@
I\
,OO
F L O W :E--
MI
h
F~LL
- R E S E R V O I R OVERFLOW
SO-
~
-OH
H Y D R O X Y L A M I N E - N I C K E L REAGENT
023-
' o , : , bM2 WASTE 4
H-
B R i J 35
023-
:
,
DOU8LE LENGTH M I X E R
M 2 : 2 D O U 8 i E LENGTH MIXERS
P
H-
-H
:
DULSE SUPPRESSOR - ONE L E N G T H OF ORANGE-BLACK M I C R O PUMPING T U B E
COOH
-OCOCH,
Cephalosporin C S H CH(CH ) CO-
3 -Hydr oxymethylcephalosporin C
COOH
-OH
I
1H CH(CH CO-
Figure 2. Flow system for automated hydroxylamine determination of cephalosporins
r
COOH
Cephalosporin D
rCH
)
1
CHCH OCOLHCtCH CO-
0.4
Cephapirin
S D S C H CO-
-OCOCH, -OCOCH,
Cephalothin
(SlrCH-CO-
-OCOCH,
Cephaloridine
&CH.CO-
49
Cephalexin
@CHCO-
I
-H
SH.
Figure 1. Structure of cephalosporins nium sulfate dodecyl hydrate (Matheson, Coleman, and Bell) in 1 liter of 1 N sulfuric acid. The working reagent was prepared by diluting 60 ml of stock iron solution and 25 ml of SD-32 ethanol to 100 ml with water. All reagents were filtered, if necessary, to obtain sparkling clear solutions. Reagents were degassed briefly under 30 mm vacuum. Procedures. Manual Determination. Approximately mol of reference standard and sample cephalosporins were diluted to 50 ml with 1%sodium bicarbonate. Aliquots of reference solutions (1.00,2.00, and 3.00 ml) and sample solutions (2.00 ml) were transferred, in duplicate for test and blank pairs, to respective 20-ml test tubes and all volumes were made to 3.00 ml with 1%sodium bicarbonate. Hydroxylamine-nickel reagent, 2.00 ml, was added to each test solution and allowed to react at room temperature for exactly 20 minutes. Then 5.00 ml of iron reagent was added and mixed. Immediately the blank was prepared by adding first 5.00 ml of iron reagent, then 2.00 ml of hydroxylamine-nickel reagent to the respective blank solution. After 5 minutes, the absorbance of each test solution was measured at 470 nm vs. each respective blank solution in the reference beam. A working curve of absorbance vs. concentration of the reference cephalosporin was prepared and the sample concentration was obtained from the working curve. Automated Determination. Reference standard cephalosporins were prepared in phthalate buffer at concentrations of 0.8 X to 4.0 X 10-3M and applied to the sampler cups before and after each series of samples. Samples were prepared in phthalate buffer at concentrations in the mid-range of standards. The Technicon AutoAnalyzer flow system is shown in Figure 2. A working curve of peak-height absorbance vs. concentration of cephalosporin was prepared, and the sample concentration was calculated from the working curve. Alternatively, the recorder was fitted with a retransmitting slidewire to couple the colorimeter output t o an IBM 1800 computer where all calculations were carried out automatically.
RESULTS Except where otherwise s t a t e d , t h e following d a t a were based on Bristol Laboratories' reference S o d i u m Cephalosporin C using t h e manual procedure.
2230
0.1
k
k 20
0
5
10
15
Time, min
Figure 3. Absorbance of ferric-hydroxamic acid complex (2 pmol cephalosporin C and 2.5 mmol hydroxylamine) a s a function of time and as a function of time after after addition of hydroxylamine (O), addition of iron (0)
Investigation of Parameters, Nickel(I1) Absent. Att e m p t s were m a d e t o a d a p t s t a n d a r d procedures for d e t e r mining Cephalosporin C. T h e absorbance a t 470 n m , maxim u m of t h e broad hydroxamic acid-iron complex was studied as a function of hydroxylamine concentration. Maxim u m absorbance for 2 pmol of C e p h C was obtained using 2.5 m m o l of hydroxylamine in a final volume of 10 ml, in agreement with that reported b y Flynn (21). M a x i m u m absorbance was obtained after 15 minutes of reaction with hydroxylamine (Figure 3). Measurements were t a k e n 2 minutes after addition of t h e iron reagent, 0.7 m m o l Fe(II1). T h e colored complex was very unstable, however (Figure 3), a n d consecutive absorbance readings t a k e n without delay were different. Poor color stability h a s been a t t r i b u t e d t o reduction of Fe(II1) by excess hydroxylamine (6). According t o Notari (27), t h e most i m p o r t a n t factor for stability is t h e ratio of ferric iron to hydroxylamine, which should b e at least 4:l. In penicillin procedures ( 2 4 ) , t h e iron-hydroxylamine ratio is a b o u t 1 5 , a n d in t h e cephalo-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Hydroxylamine Concentration, 0.7
1.0
0
2.0
M
0.8 r-
4.0
3.0
I
I
0.6
0.1
I
pit'
1
0.2
I
0.1
t
~
0
.05
.IO
.I5
Nickel C o n c e n t r a t i o n .
.20
M
0
Figure 4. Absorbance of the ferric-hydroxamic acid complex (2 pmol cephalosporin C) as a function of hydroxylamine concentration (0) (upper ordinate),and nickel concentration (0)(lower ordinate).
r
o'6
0.4
EO r- 0.3 P
f
D L
:: 0.2
16
0.I
0 10 20 30 40 Time after oddition of hydroxylamine-nickel, min
Figure 5. Absorbance of the cephalosporin test solution as a function of time after addition of hydroxylamine-nickel reagent. Na cephapirin (0),cephalexin (O), cephalosporin D (A),7-ACA ( 0 ) .
sporin procedure (21), it is about 1:3. Therefore, increased concentrations of iron were evaluated. As the iron concentration was increased, the initial absorbance of the blank increased, and the absorbance loss with time became very rapid, more rapid than for the test solution. Therefore, other means were sought to provide color stability and improved sensitivity. Investigation of Parameters, Nickel(I1) Catalyst. The hydroxylamine reagent used by Connors (26) was 2M
2
4
6
8
10
Time ofter a d d i t i o n of iron, min
Figure 6. Absorbance of the ferric-hydroxamic acid complex as a function of time after addition of iron. Na cephalosporin C, 2 pmol, test (0)and blank ( 0 ) :Na cephapirin, 4.5 pmol, test (0)and blank (W): cephalexin, 5.8 pmol, test (A)and blank (A).
in hydroxylamine and 0.2M in Ni(II), adjusted to pH 6.2. A greater response was obtained for Cephalosporin C using this reagent than using a similar reagent without nickel, and the absorbance appeared to be stable. Therefore, several combinations of hydroxylamine and nickel were evaluated for Cephalosporin C. The reagent was limited by precipitation problems as the pH approached 7 and by a slower hydroxamic acid formation at lower pH. The absorbance of the test solution increases with nickel concentration up to 0.2M. Above that level, nickel salts begin to precipitate. The absorbance of the test solutions also increased as the hydroxylamine concentration was increased. However, above 2 M , the color stability of the complex began to worsen. Compensating increases in iron concentration caused high absorbances in the blank solution. Therefore, the reagent described by Connors was selected as the most practical for manual procedures (See Figure 4). The pH of the final acidified solution using iron reagent in 1N sulfuric acid was 1.20. Adjustments of the final pH showed higher absorbances for the test solution a t higher pH values, but an upper limit of pH 1.7 was imposed by solubility limitations. The pH of 1.2 obtained was well within that limit, reproducible, and seemed a good compromise. Application to Several Cephalosporins. The manual determination has been applied to a number of cephalosporins, including those shown in Figure 1. Reaction with hydroxylamine-nickel reagent was shown to plateau within 10-20 minutes for several cephalosporins tested, excepting cephalexin (Figure 5 ) . All cephalosporins tested showed similar color stability of test and blank solutions, the test absorbances attaining a stable plateau between 5 and 10 minutes after addition of the iron reagent (Figure 6 ) .
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2231
Table I . Replication of the Manual H,vdroxylamine Determination of Cephalosporin C among Analysts Cephalosporin content, uglmg Analyst 4 Sample KO.
Analyst 1
Analyst 2
Analyst 3
Day 1
Day 2
*Mean
Re1 std dev, 9;
1 2 3 4
694 778 591 72 0
699 764 591 7 04
694 751 594 716
717 743 603
695 744 608 714
700 756 597 714
1.4 2 .o 1.3 1.o
Table 11. Precision of .Manual and Automated H,vdroxylamine Determination of Several Cephalosporins Mean result,
R e 1 std
replications
u glmg
dev,
6 6 9 10 12
922 965 865 571 919
N O . oi
Cephalosporin formulation
Cephalothin, Na,' powder for injection Cephaloridine,' powder for injection Cephalexin, ' experimental tablets Cephalosporin D, DCHA,* reference material Cephalosporin D , Na,' reference material
::
0.9 1.o 1.5 0.6 0.7d
a Manual determination. * Bis(dicyclohexy1)amine salt, automated procedure, 10 replicate samples within a single run. Disodium salt, automated procedure, sample applied at 4 levels in each of 3 separate runs for a total of 12 replicates. 0.71 across levels, 0.68 across runs. 0.71% overall.
Table 111. Comparison of Methods for Determination of 7 -Aminocephalosporanic Acid (7-ACA) 7-ACA content, uglmg
0.8 O'g[
~~
High pressure liquid
0.7 E
Hi droxylamine
lodometric
:hromatographic
method
method
met hod
977 938 946 969 998 956 977 952 962 963 917 991
980 985 980 990 990 990 1000 920 980 980 980 990
964 962 980 975 984 972 980 986 861 907 912 961
962
980
9 54
Sample h o .
1 2 3 4 5 6 7 8 9 10 11 12
$ 0.6 0 9
- 0.5 Y)
'y
: 2 e
0.4
w
0 C
n 0 0
0.3
.
a
Mean 0.2
0.1
0
100
200
300
Cephalosporin concentration, p g / m l
Figure 7. Reference working curves for several cephalosporins. Cephalosporin C (0),Na cephapirin (U), cephalexin (A), 3-hydroxymethyl-7-ACA ( O ) ,3-methyl-7-ACA (H), 7-ACA (A).
Blanks exhibited less than 5% decrease in absorbance over the same time interval. Desacetyl (3-hydroxymethyl) and desacetoxy (3-methyl) cephalosporins also reacted with hydroxylamine under the same conditions; Beer's law was followed in each case as indicated by linear working curves (Figure 7). Working 2232
curves are also shown for cephalosporin C, cephalexin, and cephapirin. Reproducibility. Replication among analysts determining cephalosporin C is shown in Table I. The variability between runs and days is also included in the overall variability since each analyst made individual runs on separate days. Precision within a manual run is demonstrated in Table I1 for 3 cephalosporins, commercially obtained sodium cephalothin and cephaloridine powder for injection and an experimental batch of cephalexin tablets. Cephalosporin D is an intermediate in the production of 7-aminocephalosporanic acid (7-ACA), which in turn is a precursor for many cephalosporins. The automated procedure has been used for in-process control of both the disodium and the bis(dicyclohexy1)amine salt forms of cephalosporin D. Precision within a run and across runs and levels is shown in Table 11. Comparison of Methods. Samples of 7-ACA were assayed by the alkaline inactivation-iodometric procedure,
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Table IV. Comparison of Methods for Determination of N a Cephapirin Cephapirin Content (rrglmq) Biological Hydmxylamine
turbidimetric
Iodometric
method
method
method
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
830 890 960 94 0 940 900 920 940 880 930 920 900 860 920 840 920 930 890 880 870 760 770
890 920 870 920 930 850 930 930 930 920 880 860 850 900 830 840 910 920 890 850 870 800
920 910 880 910 900 890 930 890 830 920 880 860 8 50 890 840 840 930 900 880 890 810 790
Mean
886
886
879
Sample KO.
by an unpublished high pressure liquid chromatographic (LC) procedure, and also by the hydroxylamine-nickel procedure (Table 111). Overall, the lowest results were obtained by the LC method, as might be expected since the LC method discriminates against the desacetyl form whereas the other two methods do not. This difference between methods is particularly obvious in samples 9,10, and 11 where some desacetyl impurity is known to be present. Although the hydroxylamine-nickel procedure does measure both forms, overall results are close to the LC results, presumed to be correct. Even though the iodometric results were generally higher than those of the other two methods, an analysis of variance did not reject the hypothesis that the three methods were equivalent, a t the 95% confidence level. A series of sodium cephapirin samples which had been subjected to adverse environmental conditions to accelerate degradation were assayed for cephapirin content by biological turbidimetric, alkaline inactivation-iodometric, and the hydroxylamine-nickel procedures. Results (Table IV) show overall good agreement among the methods. Again, analysis of variance was unable to reject the hypothesis of equivalence of methods a t the 95% confidence level. DISCUSSION As in the case with penicillins (28), different cephalosporins exhibit different molar responses to the hydroxylamine test. Hydroxamic acid formation is not contingent upon the substitution in the C-3 position: 3-methyl, 3-hydroxymethyl, and other 3-methyl substituted cephalosporin derivatives can be determined by this test. This is advantageous in that many types of cephalosporins including those shown in Figure 1 are amenable to the same procedure. It is disadvantageous in that the 3-hydroxymethyl forms which may exist as impurities in 3-acetoxymethylcephalosporins
are included in the measurement of the parent compound and, to that extent, the method is not selective. Furthermore, under acidic conditions, desacetyl forms may lactonize through the carboxyl group, giving rise to a second functional group capable of forming a hydroxamic acid upon reaction with hydroxylamine. Fortunately these lactones, which give a high positive bias, are present in only trace amounts in most cephalosporin samples. Other common analytical methods being used or developed for cephalosporin determinations include the biological turbidimetric and agar plate assays, the alkaline inactivation-iodometric method, and various chromatographic techniques. In biological methods, a test response is obtained for any material present which is sufficiently toxic to the test organism. Thus, metabolites, some degradation products, and other antibiotics may interfere. The iodometric test responds to all components present which, upon alkali treatment, produce iodine-absorbing substances. The hydroxylamine test is more selective in that a functional group capable of forming a hydroxamic acid a t neutral pH is required. Therefore, a cephalosporin degradation product arising through hydrolysis of the P-lactam should not interfere. Whether degradation products having an open P-lactam are common in cephalosporin samples and respond to the iodometric test has not been reported. The iodometric test measures various 3-methyl substituted forms as does the hydroxylamine test. Gas chromatography of even the most simple forms of cephalosporins has not been successful in this laboratory, and no such methods have been reported to our knowledge. On the other hand, high pressure liquid chromatographic techniques may prove quite successful in cephalosporin determinations. Such methods are already in use in this laboratory and elsewhere (29). Liquid chromatographic procedures have the potential for high selectivity among compounds, including degradation products and impurities. Chromatographic procedures may likely, as we have found, suffer the inconvenience of different conditions for each individual cephalosporin to be determined. The hydroxylamine procedure described, however, provides the same procedure for a variety of types within the cephalosporin class. It is even practical to mix types within a run if standards of each type are included. Furthermore, it is easier than other chemical methods to automate, which makes it excellent for large volume, high precision work. The procedure described for cephalosporins may also apply to penicillins, with similar advantages. Penicillin response is considerably higner when nickel is present in the hydroxylamine reagent, improving sensitivity. Since standard hydroxylamine procedures require strict timing control to obtain reproducibility, improved precision could be expected from color-stable test and blank solutions. CONCLUSIONS The hydroxylamine procedure described for cephalosporins using nickel(I1) as a catalyst is a significant improvement over current hydroxylamine methods. This procedure provides a rapid reaction between hydroxylamine and the cephalosporin nucleus, which reaches a stable plateau in 15 to 20 minutes for most cephalosporins tested. Furthermore, the color developed in both blank and test solutions is stable with time, allowing accurate and reproducible measurements to be made. The net result is a procedure which offers greater sensitivity and much better precision than its predecessors. The test method is very simple, using a minimum number of reagents and reaction sequences. It is more selective than iodometric or biological methods but less so than high pressure liquid chromatographic methods.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2233
The method can be used for in-process control and single unit dose testing. It should find wide application in content uniformity testing because of the precise nature of the assay. For 3-methyl substituted cephalosporins the method should be stability indicating. The general method shows further promise for application to penicillins.
(15) (16) (17) (18)
LITERATURE CITED
(19)
(1) E. Bamberger. Ber., 32, 1805 (1899). (2) F. Feigl and V. Anger, Mikrochemie, 15, 23 (1934). (3) F. Feigl, V. Anger, and 0. Frehden, Mikrochemie, 15, 9 (1934). (4) F. Lipmann and L. C. Tuttle, J. Biol. Chem., 159, 21 (1945). (5) S. Hestrin, J. Biol. Chem., 180, 249 (1949). (6) U. T. Hill, lnd. Eng. Chem., Anal. Ed., 19, 932 (1947). (7) F. Bergmann, Anal. Chem., 24, 1367 (1952). (8) R. Hilf and F. F. Castano, Anal. Chem., 30, 1538 (1958). (9) A. F. Brodie and F. Lipmann. J. Biol. Chem., 212, 677 (1955). (10) 0. Cori and F. Lipmann, J. Biol. Chem., 194, 417 (1952). (11) R. F. Goddu. N. F. LeBlanc. and C. M. Wright, Anal. Chem., 27, 1251 (1955). (12) F. W. Staab, E. A. Regan, and S.E. Binkley, Presentedat the 109th National Meeting, ACS. Atlantic City, N.J., April 1946. (13) J. H. Ford, lnd. Eng. Chem., Anal. Ed., 19, 1004(1947). (14) G. E. Boxer and P. M. Everett, Anal. Chern., 21, 670 (1949).
(20) (21) (22) (23) (24) (25) (26) (27) (28) (29)
D. J. McLaughlin, J. Wilkie, and J. M. Kelly, Presented at the 138th National Meeting, ACS, New York. N.Y., September 1960. A. 0. Niedermayer, F. M. Russo-Alesi, C. A. Lendzian, and J. M. Kelly, Anal. Chem., 32, 664 (1960). J. R . Lane and P. J. Weiss, Presented at the Technicon Symposium, "Automation in Analytical Chemistry", New York, N.Y., October 17, 1966. R. S . Santoro in "Analytical Profiles of Drug Substances", Vol. 2, K . Florey, Ed., Academic Press, New Brunswick. N.J., 1972, p 334. H. E. Roudebush, Presented at the Technicon International Congress on Automated Analysis, Chicago, Ill.,June 6, 1969. Code of Federal Regulation, Title 21, Paragraph 436.205, May 30, 1974. E. H. Flynn, Ed., "Cephalosporins and Penicillins," Academic Press, New York, N.Y., 1972, pp 537, 680. 0. G. Lien, Jr., Anal. Chem., 31, 1363 (1959). V. Goidenberg and P. E. Spoerri, Anal. Chem., 30, 1327 (1958). J. W. Munson and K. A. Connors, J. Am. Chem. SOC.,94, 1979 (1972). J. W. Munson and K. A. Connors, J. Pharm. Sci., 61, 211 (1972). K. A. Connors and J. W. Munson, Anal. Chem., 44, 336 (1972). R. E. Notari and J. W. Munson, J. Pharm. Sci., 56, 1060 (1969). J. M. T. Hamiiton-Miller et al.. J. Pharm. Pharmacol., 15, 81 (1963). J. Konecny et al.. J. Antibiot., 26, 135 (1973).
RECEIVEDfor review June 9, 1975. Accepted July 21, 1975.
Continuous Monitoring Instrument for Reactive Hydrocarbons in Ambient Air Bernard E. Saltzman, William R. Burg, and John E. Cuddeback Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267
This modification of a total hydrocarbon analyzer made it capable of simultaneously monitoring ambient air for reactive hydrocarbons with the potential for producing photochemical smog. Metered sample air flows were contlnuously pumped through an empty column and an absorbent column arranged in parallel. The air stream exiting from each column was directed alternately in a 5-minute cycle by a switching valve to the flame ionization detector for quantltative measurements. A chromium trioxide-sulfuric acid packing was optimal to absorb olefinic and higher aromatic hydrocarbons, and suitable column dimensions, flow rates, and temperatures were determined to obtain desired separations. Reactive hydrocarbon concentrations, obtained by difference from the instrument recordings, are reported for Cincinnati. These measurements should be more relevant than those of total or non-methane hydrocarbons.
Individual hydrocarbons vary widely in their participation in the complex kinetic processes of smog formation (1-6). The relative importance of each of the approximately one hundred hydrocarbons in the atmosphere results from its individual reactivity and concentration as well as ability to form strong oxidants and lachrymators (7). The current federal ambient air quality standard and reference method are for non-methane hydrocarbons (8). However, neither total hydrocarbons nor total hydrocarbons minus methane are accurate measures of smog potential. Because of the substantial economic and legal significance, more specific and valid measurements are urgently desirable. Differential instruments have been developed for monitoring ambient air for total hydrocarbons and methane (9, IO), for selectively combusting some hydrocarbons (111, or for exhaust gas analyses (12-14). Chemical absorbents for the exhaust concentrations, however, were ineffective for 2234
the concentrations present in ambient air. The purpose of this study was to further develop a monitoring instrument for ambient air that measured as specifically as possible only the olefinic and aromatic hydrocarbons that are reactive in the processes producing photochemical oxidants. The basic work was the selection and characterization of a solid absorbent capable of absorbing each type of hydrocarbon, such as olefins and aromatics, in proportion to its degree of reactivity in the smog forming processes. Continuous monitoring of both total and reactive hydrocarbons at ambient air concentrations was accomplished by relatively simple adaptations of available instrumentation.
EXPERIMENTAL Monitoring System. The instrument, shown schematically in Figure 1, was assembled from commercially available components: a continuous FID total hydrocarbon analyzer (Beckman Model 109A), a thermostated valve oven (Carle Model 4301) which contained a two-section absorbent column and an empty column in parallel, and a microvalve (Carle Model 2012, two-position, two stops a t 4 5 O ) which was turned by a valve actuator (Carle Model 4201) operated by a timer (Carle Model 4102 thirty-minute cycle valve minder). The Beckman instrument was slightly modified by disconnecting the sample air line from the flame detector just upstream from the capillary restrictor. This flow was diverted through the parallel columns in the oven. The exiting flow from each column was connected alternately by the microvalve back to the sample capillary tube at the base of the burner. The needle valve, 3, was adjusted to vent the exiting sample flow that was not directed to the flame at the identical rate (11 mlimin for a span of 3 ppm C). Event markers placed in the valve minder controlled the valve actuator switches a t the desired intervals. A 5-minute cycle time was chosen to correspond with the sampling frequency established for air quality data banks; although shorter cycle times are feasible, the requirements for data handling would be more complex. Thus, the flow from each column alternately was directed to the flame ionization detector for a 2.5-minute interval. In order to protect the switching valve from dust, an all-metal 13-mm diameter Millipore Swinny filter holder with a Whatman No. 41 paper
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
