Electrodes treated by methods (a) and (b) showed little response to Fe(III), and no perinanent current deflection was obtained until well past the end point. Method (c) led to improved electrode performance, but the cathodic currents after the end point changed with time, and the titrations were not reproducible. Best results were obtained when the electrode was conditioned by method (d). The base current was small and stable, and a slight excess of Fe(II1) produced a cathodic current. However, from f = 1.1 t o $ = 1.3, the cathodic currents increased with time, and current us. titer plots frequently showed serious scatter from a straight line. Equilibrium times at the R P E were in the range 10 to 15 seconds a t f = 1.00, and were not affected significantly by the pretreatment method. Biamperometric (Dead-Stop End Point). Twin platinum wire electrodes were used with a n applied voltage of 30 mv. With unconditioned electrodes, each addition of Fe(II1) gave rise to a current which decayed only slowly, making the titration rather slow (4). However when the electrodes were conditioned according to method (d), the electrode response was rapid. The titration curves were of conventional shape, and the end points were within 1% of the theoretical value.
Potentiometric. A platinum wire indicator electrode was used in conjunction with an Ag/AgCl reference electrode and a direct-reading p H meter. An indefinite end point was obtained with an unconditioned electrode, and the response time was very long. Conditioning the electrode by method (d) led to improved electrode response, with an equilibrium time of about one minute a t the end point. The potential break, however, was rather small and difficulty was experienced in accurate location of the end point. DiBerential Electrolytic Potentiometry. Twin platinum wire electrodes conditioned according t o method (d) were used with a 50-volt source and a 50 MQ ballast resistor. Potentials were measured on a directreading pH meter. No end point was obtained, the differential potential remaining almost constant from f = 0.5 to f = 1.5. Amperometry a t the D M E appears to be the best electrometric method for locating the end point in the titration of U(1V) with Fe(II1) a t room temperature. Methods using platinum electrodes were found to be less reliable and less precise. Sympson et aL’s (9) experience with the R P E differed from ours, even though the same con-
ditioning procedure was used. They reported good precision, but prolonged response times near the end point, whereas we found poor precision, but response times close to the theoretical value. This discrepancy is difficult to explain, unless it is connected with electrode surface area, or some slight difference in the pretreatment methods. LITERATURE CITED
(1) “Analytical Chemistry of the Manhattan Project,’’ C. J. Rodden, ed., p. 71, McGraw-Hill, New York, 1950. (2) Betts, R. H., Can. J. Chem. 33, 1780 (1955). (3) Feldberg,S. W., Enke, C. G., Bricker, C. E., J. Electrochem. Soc. 110. 826 (1963j. (4) Florence, T. M., Anal. Chim. Acta 23, 282 (1960). (5) Sagi, S., Rao, G. G., Tuhnta 5, 154 (1960). (6) Sewallc, W. J., Goward, G. W., U.S.A.E.C. Revt. WAPD-209 (Dec. (7) Shipley, E. D., U.S.A.E.C. Rept. AECD-2804 (Nov. 1949). ( 8 ) Smoler, I., J. Electroanal. Chem. 6 , 465 (1963). (9) Sympson, R. F., Larsen, R. P., Meyer, R. J., Oldham, R. D., ANAL. CHEM.37, 58 (1965). T. M. FLORENCE P. J. SHIRVINQTON Analytical Chemistry Section Australian Atomic Energy Commission Research Establishment Lucas Heights, N.S.W., Australia
Gas Liquid Chromatography of Alkaloids Using Capillary Columns and Four Packed Columns SIR: Analysis of alkaloids (or alkaloid-like substances, depending on the definition) using gas chromatography has been studied by a number of workers (1, 5-6). Lloyd and coworkers (4) demonstrated the feasibility of using gas chromatography for the isolation, separation, and identification of alkaloids by chromatographing 45 high molecular weight alkaloids on a packed column of S E 3 0 silicone polymer. Brochmann-Hanssen and BaerheimSvendsen (1) used $ E 3 0 in developing the gas chromatographic technique of direct analysis of alkaloidal salts employing temperature programming. They also discussed the catalytic effect of glass wool on thermal decomposition of alkaloids in the chromatograph. Parker, Fontan, and Kirk (6) presented retention data for 41 alkaloids chromatographed on an SE-30 column a t five different temperatures, and reported the use of Carbowax 2034 columns. Kazyak and Knoblock (3) compared the retention times of some alkaloids on SE-30 with those on QF-1 fluorinated silicone polymer. Grob (2) chromatographed the basic substances from 952
ANALYTICAL CHEMISTRY
cigarette smoke on capillary columns coated with basic polymers. We wish to report the extension of the analysis procedures of some 25 alkaloids to include capillary columns and four new packed columns. These columns are Epon 1001 Resin, a product of the reaction of bisphenol A and epichlorohydrin, XE60, a silicone copolymer consisting of 50% dimethylsiloxane and 50% cyanoethylmethylsiloxane, SE52, methylphenyl silicone polymer, and JXR, dimethylpolysiloxane. The capillary columns varied in length and had coatings of Apiezon L, QF-l , and SE30. EXPERIMENTAL
Apparatus. A Barber - Colman Model 5000 gas chromatograph equipped with a linear temperature programmer, a stream splitter, and a high temperature flame-ionization detector was used with a Leeds & Northrup Speedomax H Model S, 0 to 5 mv., recorder. The columns and conditions used are given in Table I. Nitrogen was used as the carrier gas. Flow rates were measured with a bubble flowmeter. Packed columns were made from 1/8-inch 0.d.
copper tubing. Packings were prepared by the conventional slurry and evaporation technique using a rotary vacuum evaporator. The solid support was SO/lOO mesh Diaport S; 1% J X R on 100/120 mesh Gas Chrom P was obtained from Applied Science Laboratories, State College, Pa. One end of the empty columns was plugged with a small amount of glass wool, suction was then applied t o the blocked end and the packing was poured in the open end and settled firmly by vibration with an electrical vibrator. The packed columns were conditioned with no carrier gas flow for 30 minutes a t their maximum recommended temperatures, cooled to 200” C., and then conditioned with carrier gas flow under maximum operating temperatures for 12 hours. The SE-30 stainless steel capillary column was obtained from PerkinElmer Corp., Norwalk, Conn. The Apiezon L S.S. capillary column and a 300 foot QF-1 S.S. capillary column were obtained from Barber-Colman CO., Rockford, Ill. Alkaloids were obtained from Aldrich Chemical Co. and K & K Laboratories. Procedure. Temperature programming was started from isothermal conditions 5” to 10’ below the given
~~
~
Table 1.
Operating Conditions
Packed columns
% Liquid phase Column dimensions
1% JXR 6 feet x '/a inch 0.d.
1% SE-52 6 feet x '/a
Column temperature,=C."
100-300° 12" /min. 58 ml./min. 40 0.1-1 d.
100-300" 12" /min. 70 ml./min. 40 0.1-1 pl.
mch 0.d.
6 feet x inch 0.d.
0.5% Epon 1001 2 feet x inch 0.d.
100-250 " 12"/min. 70 ml./min. 40 0.1-1 pl.
100-250" 12"/min. 91 ml./min. 40 0.1-1 pl.
1%XE-60
Flow rate Inlet pressure, b p.s.i. Sample sizec Split ratio ... ... ... 0 Flash heater temperature, 290" C.; detector temperature, 310" C. b Outlet pressure, atmospheric. Sample concentration, 1 to 5 pg. alkaloidjpl. solvent (usually acetone).
Capillary columns QF-1 100 feet x 0.01 inch i.d. 100-200" 12O/min. 15 ml./min. 80 0.5 p1. 1/10
...
200 feet X 0.01 inch i.d. 100-250° 12'/min. 8 . 5 ml./min. 80 1 pl.
Apiezon L100 feet X 0.01 inch i.d. 100-250O 12"/min. 15 ml./min. 80 0.5 pl.
1/10
1/10
SE-30
0
initial temperature. Injection of 0.1to l-pl, samples (containing -1 pg. alkaloid in acetone or alcohol plus nicotine or caffeine as internal standard) was made as the initial temperature was approached. Individual retention times were measured from the emergence of the solvent front to the top of the alkaloid peak. I n cases where several peaks were eluted, the largest peak was taken as the designated alkaloid. Whether the multiple peaks were evidence for thermal decomposition or impure alkaloids was not determined. Generally, all the alkaloids showed one main peak with smaller satellites and the patterns were perfectly reproducible. Special attention to the size of the sample injected on the capillary columns was necessary. Loss of resolution was noted when sample sizes were too small or too large. (Table I gives the sample sizes used.)
RESULTS AND DISCUSSION
molecular weight alkaloids, and the program rate was adjusted to give the best compromise between resolution and analysis speed for a sample containing mixed alkaloids such as might be found in natural products. The flow rate was adjusted to give the best compromise between resolution, speed of analysis, and sharpness of peaks for high molecular weight compounds. This was done because our primary interest is in the rapid survey of plants and microorganisms for new alkaloids. The Apiezon L capillary column was useful only for alkaloids with molecular weights below about 175. At 125' C. (isothermal) it separated nicotine and neonicotine by six minutes, but tailing was pronounced. The SE30 capillary column resolved the lower molecular weight (
