The color was s t a b k and its aliiorbance ivas constant for about 4 hours not only in the dark but also in the light. It followed Bc'er's lam- when a concentration of chole3terol in a standard solution lay betw3en zero and 0.2 nig. per ml. Therefore, when serum has a higher cholesterol value than 600 nig. per 100 ml. it should he diluted with water before determination. Table 1V presents a result of comparison of the present method to Zak's and to Schoenheimcbr and Sperry's methods. Recovery tests of t'his method gave 100 i 2 7 , recovery as ihown in Table I-. Cholesteryl acetate and oleate alio developed colors, botE of xhich had an identical visible-light ab;.orption spectrum with that of c:holesterol. And 100 =t 1% and 97 =t1%; recoveries of equivalent cholesterol v:rlues were given by cholest'eryl acetat,. and oleate! respect'ively (Table TI). The relative standard deviation ~i this method a t a level of 225 mg. 1)er 100 nil. cholesterol in seri1.n n.a. O.itiyL:,.
Table VI.
Acetate Equivalent cholesterol values (mg./100 ml.) Added Recovered 180
181
180 161
Recovery Tests of Cholesteryl Esters
Recovery, r /O
101
100 101
Oleate Equivalent cholesterol values (mg./100 d . 1 Added Recovered 121
118
116 117
Recovery, % 98 96 97
271
ACKNOWLEDGMENT
The authors express their gratitude to Junji Nagai, Central Clinical Laboratory of the University Hospital, for the supply of sera. LITERATURE CITED
Ishii, T., Rinsho Byori, Special Issue S o . 10, p. 152 (1959). 12) Kitamura, hI., Arimatsu, Y., -Yisshin Igaku 48,456 (1961). 1 3 ) "Organic Synthesis,!' Vol. 35, p. 43, Wiley, S e w York, 1 9 ~ 0 . 14) Schoenheimer, R., Sperry, IV. X, (1)
J . Bid. Chem. 106,745 (1934); "Standard Methods of Clinical Chemistry," 5'01. 1, p. 43-54, Academic Press, New Y-ork, 1953. (5) Yoshikawa, H. et al., Igaku no Ayumi, 33, 375 (1960). (6) Zak, B., Ani. J . Clin. Path. 27, 583 (1967). TSUTOMU R~OMOSE Yo VEDA KYOKO YAMAMOTO TOSHIKO MASUMURA KIYOKO OHT.~ Institute of Pharmaceutical Sciences Faculty of Medicine, Kyushu University Fukuoka, Japan
Methyl Orange Spectrophotometric Method for Schiff Bases amineq by deposition on the glass surSIR:I n n rcJccnt issue of .IXALYTICAL
CHEMISTRY,Silverstfin (3) de-crihed the devc1ol)meiit of a methyl orange method for measurem1:nt of high molecular weight' amines in aqueous solution. It \vas I m c d on reaction of the ainiiics with methyl orange s t pH 3 tcJ 4 to form a yellon- coniljex soluble in an organic liquid. The color intensity o i the organic phase Jva:j used as a direct measure of tot'al amine concentration. This procedure was made specific for the detcct'ion and measurement of primary, secondary, arid tertiary amines. The basic methyl orange colorimetric method for arnincb >vas introduced by 13rodie and Udenfriend (1: in 1945 for the determination of cinchona alkaloids arid other organic bases in plasma and urinc.. Later. the writclr and Ellenboge.1 (wj applied this technique to t>he (.stirnation of damphetamine and dr'-amphetamine in blood and urine. I n the interim period this method ha, been u5ed by t'ht vvrit'er and,'or colle igues for many analyses involving det'ection and measurement of amines. Several Txriations hare been used compared to t'he method reported by Silverstein ( 3 ) . The methyl oracge technique ii made riiorc' reliable, especially a t the low coilcentration levels) by coating the glass equipment with a dicone film such as Desicote, which works very well. This prevents the loas of
faces and makes i t possible to obtain reproducible results under a variety of experimental conditions. The sensitivity of the methyl orange procedure iq increased for measurement of total amines by extracting the yellow methyl orange complex from the organic phase into aqueous acid solution. The abborbance of the resulting red solution at 508 m p is significantly higher than for the organic phase a t 430 mp. Other method variations mist including the use of benzene as a solvent instead of ethylene dichloride and the use of a different buffer system. This report describes a further application of the methyl orange procedure a j used in this laboratory for meaburernent of 100 p.p.m. of Schiff base3 in S o . 2 fuel oils. I n general, ,Schiff babes represent the class of compound3 derived by chemical reaction (condensation) of aldehydes or ketones with primary amines. The general formula for the lveakly basic Schiff base is RR'-C=S-R". The Schiff base for this work, I, is represented b y the formula tert-Cl,-lsH2,-31-T=CH2, hich is a mixture of highly branched primary amines ha1 ing a tertiary alkyl structure (nitrogen attached t o the tertiary carbon atom). The oil sample containing Schiff base I is dissolved in benzene and given a n alkaline wash. The benzene is shaken
u-ith methyl orange reagent at p H 5 and thcl excess methyl orange is removed. The methyl orange which dissolveb in the benzene through complex formatioii with Schiff baqe I is returned to an acid solution and measured spectrophotometrically. EXPERIMENTAL
Apparatus. Coat t h e inside of glass stoppered bottles, 100-ml. capacity, with a silicone product such as Desicott., produced b y Becknian Instrument Co. A Cary Model 11 spectrophotometer with 2 em. fused quartz cells was used for measurement i n t h e visible range. Reagents. Benzene Containing l.?iyOIsoamyl Alcohol. A technical grade of benzene is purified b y successive washings with 1N sodium hydroyide, 1 S hydrochloric acid, a n d distilled water. .4 reagent grade of isoamyl alcohol is mashed with 0.2 volunie of 1-1- hydrochloric acid followed b y several washes of distilled water. T h e absorbance of t h e reagent blank should not exceed o.ol"0. Methyl Orange Reagent. Dissolve 500 mg. of methyl orange crystals in 100 ml. of warm water. Immediately before use. mix eaual volumes of the methyl orange solition and saturated boric acid solution. Standard Solution of Schiff Base I. To prepare a stock solution, weigh VOL. 35, NO. 1 1, OCTOBER 1963
1753
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
