Table 111. Analysis of Commercial Xylidine Standard deviation Component Wt o-Toluidine 2.8 0.06 0.07 o-Ethylaniline 1.8 0.17 2,5-DMAo 19.0 0.20 2,6-DMA 7.7 2,4-DMA 38.4 0.70 m-Toluidine 0.6 0.10 0.17 pToluidine 1.7 2,3-DMA 14.0 0.20 3,5-DMA 1 .o 0.02 0.05 rn-Ethylaniline 0.3 0.06 p-Ethylaniline 1.8 '0.13 3,4-DMA 11.6 Total 100.7 DMA is dimethylaniline.
z
(1
Relative reponse factors of several amines (weighed as amines and chromatographed as derivatives) us. n-dodecane are shown in Table 11. Amine isomers had identical responses. Although slight, there was an increase in relative response with increase in carbon content of the amine.
Peak symmetry of amine T F A derivatives was good. Anomalous peak shapes were noted in earlier work when aluminum columns were used. This was especially pronounced when solutions containing trifluoroacetic acid were chromatographed. Recourse to stainless steel columns completely eliminated this difficulty. Water, up to saturation levels for the amine mixture, did not interfere. Typical precision provided by the technique is illustrated in Table I11 where results are shown from the analysis of a distilled sample of a commercial mixed xylidine. Seven separate determinations of the 12-component mixture provided the data. Total material recovery had a mean of 100.7% and a standard deviation of 1.0 when using the internal standard approach. ACKNO WLEDGiMENT
The author thanks F. H. Wilson and D. Desillas, both of this laboratory, for the preparations of the purified amines and the amine TFA derivatives. RECEIVED for review February 10, 1967. Accepted May 11, 1967.
Water as an Internal Standard in Determination of Blood Ethanol by Gas Chromatography J. K. Colehour Biochemistry Branch, Naual Aerospace Medical Institute, Naval Aerospace Medical Center, Pensacola, Fla. 32512
THEUSE of porous, polymeric beads has made it possible to determine small amounts of water in volatile organic solutions by gas-liquid chromatography (I). The determination of small amounts of organic compounds in aqueous solutions has been less successful. This is especially true in the case of low molecular weight polar organics which elute with the water. Flame ionization is the detection system of choice for this purpose because of its insensitivity to water, high sensitivity to combustible organic compounds, and response linearity over a concentration range of 106 or more. However, with injection of samples in which the ratio of water to organic compound is high, the water not only tends to tail over into the other low molecular weight materials but also it affects detector response. These problems have been resolved so that flame ionization may be utilized without the disadvantage of a relatively large proportion of water, Indeed, it has been found that, in the method to be described, the water itself could be used as the internal standard for the microdetermination of blood ethanol. Although the procedure is specific for this alcohol, the details can be varied so that other polar organic compounds can be determined in aqueous solution in a similar way. EXPERIMENTAL
Chromatographic Equipment and Column Preparation. A n F & M Model 400 biomedical gas chromatograph equipped with a flame ionization detector was used for all (1) 0. L. Hollis and W. V. Hayes, J. Gas Chromatog., 4,235 (1966).
1 190
ANALYTICAL CHEMISTRY
determinations. The U-shaped glass column, 66 cm long and 4-mm i d . , was filled with 50- to 80-mesh Porapak Q by gentle tapping. Unless specified otherwise, all of the determinations were run under the conditions shown in Figure l , After the column was conditioned for 2 hours, 1.0 p1 of 40 methyl amine was injected, followed by 0.1 ~1 of 250 mg ethanol standard in water (see below). A t first only the ethanol peak appeared after the aqueous standard was injected. Injections of both were then repeated until, after a certain amount of loading, a second peak appeared early in the chromatogram caused by water. This peak, or rather the area under the peak, remained constant in relation t o the ethanol area even after several 0.1-wI injections of the standard; therefore, it was used as the internal reference. This reference area may be increased if necessary by additional injections of fractional microliter amounts of 4 0 z methyl amine or, if the column is excessively loaded, the area may be decreased by heating the column to 350" C as rapidly as possible with inert gas flow unchanged, and then similarly cooling it with the oven door open. Thus, aqueous response areas may be controlled if sensitivity settings cannot compensate for the variable loading that occurs. Quantification is accomplished by measuring the ratio of alcohol response area to water response area. Integration devices may be used for measuring areas but weighing cut-out sections is entirely adequate also. I n the latter case, it is preferable to increase chart speed and/or decrease the inert gas flow by 15 to 20 to improve weighing accuracy. All samples were injected on the column with a Hamilton 7101-N syringe or similar type in which the plunger extends to the tip of the needle. Although sample volumes d o not need to be precisely equal when internal standards are used, it is well t o use the same syringe for all injections.
z
OPERATING CONDITIONS
250
Ethanol
1
Packing-Porapok 9,50-80m Column-Glass"U" 66 cm x 4 mm id Carrier gas -He He flow-38 ml/min Column ternpemlura 121 C Range I Attenuation 32
i - U U U U L U
ETHANOL (mg%)
Figure 2. Typical standard curve used in determining blood ethanol
T I M E (MINUTES1
Figure 1. Chromatogram of blood samples containing various amounts of ethanol using water as the internal standard
Preparation of Standards. An ethanol in water standard was prepared which gave the same area ratios as the mean of 10 normal bloods containing exactly 250 mg of ethanol. The amount of ethanol necessary for this purpose was 3.50 ml of absolute ethanol per liter of solution. By using this solution as the stock standard, working standards containing lesser amounts were prepared as needed. All were stable for several weeks a t 3" C. Procedure. Capillary blood samples were collected in nonheparinized microhematocrit tubes, capped, and allowed to clot. The samples were centrifuged; 0.9 pl of air and then 0.1 p1 of serum were drawn into the syringe by putting the needle directly in the capillary. The sample was injected into the chromatograph as quickly as possible. Two o r more ethanol-water standard samples of similar percentage composition were also injected alternately with the serum. Swept areas were measured as described above; alcoholaqueous area ratios were plotted for the standards, the unknowns were similarly measured, and the percentage of ethanol read off the curve. If increased accuracy is required, duplicate determinations may be made. Serum samples may be stored without deterioration for a t least 1 week a t 3 O C if the tubes are tightly capped.
z
RESULTS AND DISCUSSION
Table I shows the recovery of absolute amounts of ethanol in blood a t various concentrations by the proposed method. A composite chromatogram of several sera prepared from bloods containing different amounts of ethanol is shown in Figure 1. Figure 2 shows a typical standard curve prepared by plotting area ratios of alcohol standards. The response is linear a t least u p t o 250 mg %. With this method there were no differences between the chromatograms of the standard solutions and those of the sera prepared from blood containing the same amounts of ethanol. However, if either was stored a t room temperature or for long periods of time, there was evidence that evaporation o r decomposition took place because some chromatograms became ragged or unreliable. The limitations of this type of determination are centered around two aspects of the method; the first is that concerned with the loading of the column. In this procedure the column
is partially loaded with strongly basic methyl amine and a portion is quantitatively eluted by the water in the standard o r sample depending on the load. In effect, this results in the amine being removed from the column, and since water response is dependent on the amount of amine present, over a period of time water response gradually diminishes. In practice, this diminution is kept below a significant level by the use of small samples (0.1 pl); the injection of samples and standards alternately on the same column compensates for the fact that n o two columns can be loaded in exactly the same degree. The standard curve shown in Figure 2 was prepared from analyses made concurrently, and since the response is linear, it is concluded that no significant decrease in reference area occurred. The other factor which would limit the reliability of the method is that the water content of injections would have t o be constant if comparative judgments were t o be made from analyses of samples from different subjects. As far as blood samples are concerned, Eisenman et al. ( 2 ) have shown that in 30 normals and 43 patients with various diseases, the water content of the sera could be defined as follows: W,
=
98.5 - 0.745 P,
where W , was the volume of water in 100 volumes of serum of protein. This would indicate that and P, was the gram sera water is relatively constant and varies mainly in gross disproteinemia.
z
(2) A. J. Eisenman, L. B. Mackenzie, and J. P. Peters, J . Biol. Chem., 116, 33 (1936).
Table I. Recovery of Absolute Amounts of Ethanol from Blood by Proposed Method Ethanol added, mg Ethanol found, mg % a 25.0 75.0 150.0
250.0 a
27.8 i 1.5 7 8 . 3 Ij, 3 . 3 147.2 Ij, 11.6 249.2 i 1 3 . 7
Each value is a mean of six single determinations.
VOL. 39, NO. 10, AUGUST 1967
1191
Similarly, the plasma water values determined by several investigators and tabulated by Davis et al. (.?) were shown to be quite consistent, the mean being 92.5 with extreme values Of and These Our ex-
perience that sera water is quantitatively adequate as a reference for the determination of blood ethanol.
94x.
(3) F. E. ~ ( 1953).
~Keith~K
i ~ and ~ Jack ~ , Kirk, ~
science, ~ 118,~276
,
RECEIVED for review April 28, 1967. Accepted June 12, 1967. Opinions o r conclusions contained in this report are those of the author and d o not necessarily reflect the views or endorsement of the Navy Department.
Kinetic Study of Cerium(1V)-Vanadium(IV) Titration Reaction G . A. Rechnitz and G . N. Rao Department of Chemistry, State Uniuersity of New York, Buffalo, N . Y . 14214 USEOF THE CERIUM(IV)-VANADIUM(IV) reaction for the estimation of vanadium(1V) was first studied by Furman (I) who reported that, while potentiometric titrations in sulfuric acid media could be satisfactorily carried out a t room temperature, the attainment of steady potentials near the end point requires considerable time. Elevated temperatures (50"-60" C) facilitate the rapid attainment of steady potentials and permit satisfactory titrations in sulfuric, hydrochloric, and perchloric acid media (2). More recently, the beneficial effects of orthophosphoric acid (3) and of acetic acid, for the titration with ferroin indicator (4), in producing rapid end point attainment, even at room temperature, have been demonstrated. These observations and empirical studies have led to the impression that the sluggishness of the titration is due to the slow reaction between cerium(1V) and vanadium(1V) at room temperature. However, we found the cerium(1Vbvanadium (IV) reaction to be quite rapid ( k N 1350 M-l sec-I), even a t 25" C in molar H2S04, and, therefore, undertook to study in detail the kinetics of the cerium(IVbvanadium(1V) reaction as well as the reversible vanadium(V)-ferroin reaction in a n effort t o identify the cause of sluggishness in the analytical titration and to provide information useful for the selection of optimum reaction conditions. EXPERIMENTAL
The experimental arrangement and the procedure for the kinetic runs were as described earlier (5). Stoppered 4-cm silica cells were employed so that cerium(1V) concentrations in the range of 2 t o 10 X 10-5M could be determined spectrophotometrically a t 350 mp (E in 1M H2SO4a t 25" iv_ 4000). Cerium, vanadium, and ferroin solutions were prepared from reagent grade materials and were standardized by conventional methods (5, 6). Cerium salts and ferroin were obtained from the G. F. Smith Chemical Co. Potentiometric titrations were performed using standard procedures. The reactions involving ferric and ferrous o-phenanthroline complexes were followed by monitoring the ab(1) N. H. Furrnan, J. Am. Cl7em. Soc., 50,1675 (1928). (2) H. H. Willard and P. Young, Zrid. Eng. Chem., 20, 972 (1928). (3) G. Gopala Rao and L. S. A. Dikshitulu, Tu/un/a,9, 289 (1962). (4) K. Sriramarn and G. Gopala Rao, Tulurzfa, 13. 1468 (1966). 38, (5) G. A. Rechnitz, G. N. Rao, and G. P. Rao, ANAL.CHEM.,
1900 (1966). (6) G. F. Smith and F. P. Richter, "Phenanthroline and Substituted Phenanthroline Indicators," G. F. Smith Chemical Co., Columbus, Ohio, 1944. 1 192
rn
ANALYTICAL CHEMISTRY
sorbance at 510 mp (molar absorptivity of ferrous o-phenanthroline a t 510 m p N 11000). RESULTS AND DISCUSSION
Several kinetic experiments performed with different initial concentrations of cerium(1V) and vanadium(IV), followed the second order rate law In
6 (a - x ) a (b - x)
=
kt (a
- b)
where a and b are the initial concentrations of the reactants and x the Ce(II1) concentration a t time, t . The rate constant was found to be 1350 =t100 M-l sec-l in 1 M H 2 S 0 4at 25" C with initial concentration of Ce(1V) varying from 2.5 to 9.0 X 10-5M and of V(1V) from 4.0 to 8.0 X 10-jM. Further experiments, performed in presence of 0 to 2.5 x lO-3M cerium(II1) and 0 to 2 X 10-4M vanadium(V), employing 5 X 10-jM each of cerium(1V) and vanadium(1V) in one molar sulfuric acid, indicated that the products d o not have any appreciable effect on either the form of the rate law or the rate constant. The absorbance values of cerium(1V) were corrected for the contribution from vanadium(V) in these experiments. The second order rate constant decreases slightly with increasing sulfuric acid concentration. Addition of different amount of sodium perchlorate at constant sulfuric acid concentration also results in a n increase in the value of rate constant, but the effect of solvent species upon the kinetics of the primary redox reaction was not studied in detail, because of the many possible reactant and product species present in these media (7-9). The temperature dependence of the rate constant gave the following thermodynamic values in M H2S04: activation energy = 13.0 + 1 Kcal., AH* = 12.4 i 1 Kcal., AS* = 2e.u. In view of the fairly high rate constant observed for the cerium(1V)-vanadium(1V) reaction, the need for elevated temperatures in the potentiometric titration cannot be due to the sluggishness of the primary reaction. On the other hand, the sluggishness of the titration could be due to slow attainment of potentiometric equilibrium. This possibility was examined by carrying out the titration (platinum indicator electrode GS. saturated calomel reference electrode)
+
(7) L. A. Blatz, J . Phys. Cliem., 66, 160 (1962). (8) T,J. Hardwick and E. Robertson, Cur?.J . Chem., 29, 828 (1951). (9) J. S. Littler and W. A Waters, J . Clzem. SOC.,1959,4046.
