approximately 0.040 gram of Schiff base I accurately to ltO.0001 gram into a 100-ml. volumetric flask, dissolve in benzene containing 1,5y0isoamj 1 alcohol, and dilute to volume. To prepare a working solution, pipet 3 ml. of stock solution into a 100-ml. volumetric flask and dilute to volume with benzene containing 1.5% isonniyl alcohol.
Procedure. OIL CLEAN-UP. Weigh 4 t o 5 grams of fuel oil containing Schiff base I at t h e 0.01 % concentration level accurately t o =tO.O1 gram into a 50-ml. volumetric flask. Dissolve t h e oil in benzene containing 1.5% isoamyl alcohol and dilute t o volume. Transfer approximately 50 ml. of t h e benzene solution to a 125-ml. separatory funnel. K a s h the benzene with 10 ml. of 10% sodium hydroxide solution followed b y 10 ml. of distilled water with %minute shakings and discard the wash qolutions. Centrifuge approximately 25 ml. of the washed benzene phase for 2 to 3 minutes. COLORDEVELOPMENT. Transfer approximately 25 ml. of t h e centrifuged benzene t o a 100-ml. silicone-treated glass stoppered bottle. Pipet 1 nil. of methyl orange reagent into the bottle, stopper, and shake mechanically for 5 minutes. (The mechanical shaker should a t least simulate a very vigorous hand qhaking.) Decant approsimately 15 ml. of t h e supernatant benzene into a centrifuge tube, being careful to exclude any methyl orange reagent, and centrifuge for 3 minutes at 1500 to 2000 r.p.m. Pipet 20 ml. of 1N hydrochloric acid solution into a 60-ml. separatory funnel. Pipet 10
ml. of the centrifuged benzene into the separatory funnel, using special care not to pipet any methyl orange reagent from the bottom of the centrifuge tube, and shake by hand for 1 minute. Allow the phases to separate, isolate the acid phase, and centrifuge to clarify if necessary. Read the absorbance of the acid phase a t 508 n q (absorption peak) and 700 mp using a 2-cm. cell and water as a reference solvent. Determine the micrograms of Schiff base I from the reference curve and calculate the p . p m found. STANDARD REFERENCECURVE. b e pare a standard reference curve covering the range of 0 to 6 pg. of Schiff base per milliliter of final acid solution by pipetting 5-, IO-, 1 5 , 20-, and 25-m1. aliquots of Schiff base I working solution into 100-ml. glass stoppered bottles, diluting to exactly 25 ml. by pipetting in benzene containing 1.5% isoamyl alcohol, and analyzing according to the color development procedure. Plot (absorbance a t 508 nip minus absorbance a t 700 nip) as the ordinate and (micrograms of Schiff base I per milliliter of I S hydrochloric acid solution) as the abscissa. Once the reference curve has been established, one or two points suffice for checking. A slight deviation from Beer’s law a t the lower concentration levels necessitates the use of a reference curve.
applied to 11 out of 14 commercial base stock oils. These oils are representative of products from 14 companies and geographical locations in the I-nited States and Canada. Three of the oils have interfering constituents accounting for errors equivalent to 12 to 46% of the amount of Schiff base I at the 100-p.p.m. level. The oil colors ranged from light yellow to black with no correlation between color and amount of interfering constituents. The method has a degree of specificity in that common low molecular weight organic bases such as quinoline in fuel oils offer no interference. However, components with basic strength, structure, and molecular weight comparable to Schiff base I are likely to interfere. The molar absorptivity ( 6 ) of Schiff base I u i n g an average molecular weight of 218 is 36,200. Under the conditions of the method, 3.01 pg. of Schiff base I per milliliter of 1 X hydrochloric acid will give an absorbance of 1.0 using a 2-em. cell. The sensitivity limit based on a n absorbance of 0.1 is approximately 0.3 pg. per ml. LITERATURE CITED
(1) Brodie, B. B., Udenfriend, S., J . Bid. Chem. 158,705 (1945). ( 2 ) Keller, R. E., Ellenbogen, W. C., J. Pharniawl. Exptl. Therap. 106, 77 ( 1952).
DISCUSSION
(3) Silverstein, Ronald M., ANAL.CHEM. 35, 154 (1963).
Recoveries of Schiff base I a t the 100-p.p.m. level from fuel oil are 98 + 2%. The overall relative error is within 4% of the amount present n-hen
ROBERT E. KELLER Organic Div., Research Dept. Monsanto Chemical Co. St. Louis 77, hio.
Dual-Chamber Micro Cross-Section Detector for Permanent Gas Analysis SIR: Recently Lovelock, Shoemake, and Zlatkis (3) reported on the design and response characteristics of a highly sensitive cross-section ionization detector. They were able to increase the sensitivity of this detector by simply decreasing its internal volume. For more than six months me have been using a dual-chamber micro crosssection detector based on Lovelock’s micro parallel plate design for the routine analysis of H2,02, N2, and COZin microbiological studies and for the nonroutine analysis of Hz, CH4, CO, CO2, C2Ha,C Z 4 , CzHz, and C a 8 produced during the pyrolytic decomposition of organometallics. The cross-section detector xas chosen for this application after we had verified that thermal conductivity detectors have insufficient sensitivity for the analysis of H, when using helium as a carrier and that 1754
ANALYTICAL CHEMISTRY
anomalous Hz peak reversal effects occur. The anomalous Hs responses can be eliminated by using a mixed carrier r----
1
U HE-
LM
Figure 1. Series column arrangment with dual-chamber detector
gas containing 60% helium and 40% hydrogen (1); however, we preferred t o use pure helium because of its availability. EXPERIMENTAL
A series arrangement of a silica gel column and a molecular sieve column as employed by Roxburgh ( 5 ) ,who used a thermal conductivity detector, was chosen, Preliminary experiments demonstrated that the parallel column, single detector method employed by Brenner and Cieplinski (2) for the analysis of mixtures of 0 2 , Sz, and CO, was not feasible when H2 was present; the relatively great mass difference between Hz and the remaining gases resulted in nonlinear stream splitting. The series arrangement utilized for our analysis is shown in Figure 1 with the dual-chamber cross-section detector shown in Figure 2. This detector
d-7
COLLECTOR
0
I
2
INCHES
METAL
-E'-3'1
-R
- -,.
c;
Figure 2.
Dual-chamber micro cross-section detector, quarter-section view
true with all the gases we have analyzed when utilizing helium as a carrier gas. The most apparent deviation from this is in the case of Hz, where peak reversal occurs similar to that found when mixtures of Hz and helium are analyzed by thermal conductivity detectors. This is indicated in Figure 3 which is a calibration curve used in the analysis of Hp,Oz,XZ,and Con. Although not shown in Figure 3, carbon monoxide and methane also show nonlinearity of response but do not exhibit peak reversal. The cause of peak reversal of Hzin helium in the cross-section detector is not clear; however, Otvos and Stevenson (4) reported that the value obtained for the apparent ionization cross-section of Hz in helium was different from that obtained when only Hz was present and speculated that this might be due to interaction of helium in its metastable state. This peak reversal was not serious for our analysis because under the conditions of analysis, the response was sufficiently linear and reproducible up to 15 pl, of Hz to allow quantitative analysis with a satisfactory accuracy. For our work a syringe transfer and
--
utilize- imir tritium titanate-coated foils, til o polarizing electrodes (+45 volts n i t h respect to ground), and a single collector electrode separating the h o chambers. With this arrangement, the response of gases eluting from both columns is positive (as opposed to the reversal of polarity obtained bp Rolburgh with conventional thermal conductivity detectors).
___---
DISCUSSION
~ l t h o u g hit has been stated that the responie of a cioss-sc.ction detector is linear to 100rc gas or vapor concentration (31, n e h a l e not found this to he
1' I ~
______~__
8I L
6
0
6
32
6'
28
ZC0
5'
2
SAMPLE S I Z E I N MICROLITERS
Figure 3.
Response curves for Hs, 0
Table 1.
Sample HP 0 2 N 2
CHI
co
CzHs
cos
CPHI
Figure 4.
Composite chromatogram
G r a y peaks are components eluted from the molecular sieve column: ( 1 ) unresolved components, (2) Hz, ( 3 ) 0 1 , (4) C2Hs, ( 5 ) Nz, ( 6 ) Cor, (7) CH4, ( 8 ) C z h , (9) CO, (10) CzHz, (1 1) C3H8
2,
Nz, and COz
Relative Retention Times
'Retention time (fin.) Molecular Silica gel aeve 0 35 0 5 0.5 0 6
;;5
4 2 5.1 14 0
1.0 1.75 2.7 44 6.2 25.5
... ...
... ...
CzHz CsHa 14.8 Flow rate = 4 ml /min., temperatwe = 25" C.
VOL. 3 5 NO. 1 1 . OCTOBER 1963
1755
injection was required. With syringe injections for a 100-unit response (10-pl. injection of 0 2 ) the standard deviation over a n 8-hour analysis period, sampling every 10 minutes, is 2.18 which corresponds to a relative error of =t2.2%. Better accuracy and reproducibility are obtained by using a gas sampling valve. The base line drift is less than 10% per 8-hour day when utilizing a commercial gas chromatographic electrometer-amplifier (Jarrellrlsh Model 26-770) at a sensitivity setting to provide a 2% noise level on a I-mv. recorder (about amp.).
The sensitivity of response at this amplification level is sufficient t o provide a signal twice the noise level for 1X gram of COZ (about 0.05 p1.j. I n Figure 4, a composite chromatogram is shown of all the gases we have analyzed in this system to date, while the relative retention times for each component are given in Table I.
( 2 ) Brenner, IT., Cieplinski, E., -4n~z. Y..4curl. scz. 72, 705 (1959 1. (3) Lovelock, J. E., Shoemake, C. R., Zlatkis, A., A s . 4 ~ .CHEM.35,460 I 1963). (4) Otvos, J. R.,Stevenson, D. P., J . Am. ( h e m . SOC.78, 546 (1956). (5) Roxburgh. J. 31., C'an. J . Jlzciobzol. 8 , 221 (1962).
KEXUETH ABEL~ H ~ A \ IL B L DESCHVERTZISG
Research Division hlelpar, Inc. Falls Church, \-a.
LITERATURE CITED
(1) Aznavourian, W., McIntyre, E. A., Pittsburgh Conf. on Anal. Chem. and Applied Spectroscopy, Abstracts, p. 50 (1963 ).
1 Present address, Laboratory of Technical Development, Sationa! Heart Institute, Hethesda, .\Id.
Effect of Pectinol 100 D on the Spectrophotometric Determination of Pectic Substances c ' STANDARD 050 -
(NO PiCTlNOLl D - S T A N D A R D t PECTIUOL Q 13 E , STANDARD t P K T ' Y C L K S K
2
L
SIR: Work on the determination of pectins by the procedure of Postlmayr, Luh, and Leonard (6) indicate. that use of the pectic enzyme preparation Pectinol 100 D (Rohm and Haas Co., Philadelphia, Pa.) in the amounts specified may lead to erroneously high values for protopectin and correspondingly low values for mter-soluble pectin. The Postlmayr procedure is an adaptation of the method of 1lcCready and McComb (6) which makes use of Pectinol 100 D , and makes possible the determination of protopectin as well as total pectin. Since water-soluble pectin is assumed to be the difference betryeen total pectin and protopectin, the Postlmayr procedure has found wide w e as a means of determination of the various pectin components. The determination i i based on Dische's sulfuric acid-carbazole reaction with hexuronic acids ( I , 2 ) which produces a pink color. Potter and McComb ( 7 ) h a > e found that Pectinol 100 D also reacts with sulfuric acid and carbazole to produce a pink color. They state, however, that for the analysis of fruits by the method of McCready and LIcComb ( 5 ) the color produced by pectinol is insignificant. 1756
ANALYTICAL CHEMISTRY
I n our laboratory we have found the color contribution due to Pectinol 100 D to be insignificant only a t very low concentrations, Figure 1. The interfering color has an absorption spectrum identical to that produced by the reaction of galacturonic acid with the carbazole reagent and the intensity of the color is directly proportional to the concentration of pectinol. The amounts used by hIcCready and McComb (5) for total pectin determinations resulted in a pectinol concentration of 16 pg. per 2-ml. aliquot, which produced no significant interfering color. Postlmayr et al., in their protopectin determinations, used an amount which gives a final concentration of 400 pg. of pectinol per 2-ml. aliquot. I n this laboratory, use of the larger quantity of pectinol in the determination of protopectin in fresh peaches gave values which averaged 207, higher than the actual values. Figure 2 illustrates the effect of 400 pg, of pectinol in increasing the intensity of color produced by reaction of galacturonic acid and carbazole reagent, It can be seen that 400 fig. of Pectinol 100 D gives a significant amount of color, and that the amount varies among samples from different lots. Therefore, if Pectinol 100 D is
used, each lot should be cl7.eckeil to determine 11 hether a correction muit 'le made for the color produced by the pectinol. Figure 2 also indicates that Pectinol R-10 (Rohm and Haas Co.) which has been used by Esau, Joslyn, and Claypool ( 3 ) and by Jnqlyn and Deuel (.$)> and Pectinol (K 6r E( Laboratoiies, Jamaica, S . T.1 do not interfere nith the color reaction. LITERATURE CITED
(1) Dische, Z., J . Biol. Che?, . 167, lS9
(1947).
( 2 ) Ibid., 183, 489 (1950).
(3) Esau, P., Joslyn, 31. A., Claypool, L. L., J . Food Sei. 27, 509 (1962). (4) Joslyn, AI. 1., Deuel, H., Ibid., 28, 65 (1963).
( 5 ) McCready, R. M.,McComb, E. A , -4~3.~. CHEM.24, 1966 (1952). ( 6 ) Postlmavr, H. L., Luh, B. S., Leonard, S. J., FooJ Tech. 10, 618 (1956). ( 7 ) Potter, A. L., NcComb, E. A, -4m. Potato J . 3 4 , 342 (1957).
United States Dept. of Agriculture Agricultural Research Service Beltsville, hld.
