Table IV.
Spectra of Polyphenols in Presence of Sodium Acetate/Boric Acid‘ Solution Amax mp Paper Amax mp
Reagent Plain Sprayed Substance Ethanol solution AA paper paper Morin 361 394 33 390 389 395 Quercetin 373 394 21 385 Umbelliferone 326 325 -1 324 324.5 366 351 371.5 20.5 351 Esculetin 338 338 3 334 Esculin 337 2’34 Boric acid and 0.27, anhydrous sodium acetate in ethanol.
small metallic ion impurity both in solution and more particularly on the paper, could give rise to a similar effect.
AX
ACKNOWLEDGMENT
-1 10 0.5 15 3
The author expresses appreciation to Fritszche Bros., New York, for the tangerine oil residue; t o Joseph Jairus for technical assistance and to H. H. Evers for permission to publish the results of this work.
5
LITERATURE CITED
coumarins, respectively (Table 111). Similar interpretations may be made by the two methods, with the leveling effect being noted for the flavonoids. Quercetin, having the 3,4’-dihydroxy structure, is unstable in the presence of sodium ethoxide. The sodium acetate/boric acid reagent causes a bathochromic shift with all ortho-dihydroxy phenols (Table IV). A solution of morin in the presence of this reagent results in a large bathochromic shift, suggesting the presence of adjacent hydroxyl groups, whereas
the correct result is obtained after absorption on paper. The position of maximum absorption observed for morin in ethanolic solution is in disagreement with previous workers (S). The cause of this discrepancy was elucidated when, by increasing the flavonol concentration, the position of maximum absorption shifted to a higher wavelength. This effect has only been observed with the 2-hydroxyflavone and may be caused by intramolecular hydrogen bonding of this hydroxyl function with the heterocyclic oxygen. Alternatively, a very
(1) Bradfield, A. E., Flood, A. E., J. Chem. SOC.1952,4740. (2) Geissman, T. A., “The Chemistry of Flavonoid Compounds,” pp. 119-27, Pergamon Press, New York, 1962. (3) Ibid., p. 112. (4) Horowitz, R. M., Gentili, B., J. Org. Chem. 25, 2183 (1960). (5) Nelson, E. K., J. Am. Chem. SOC.56, 1392 (1934). M. J. SAX BY^
Elis Salzman Rhodesia Tobacco
Science Institute Salisbury, Southern Rhodesia Present address, 15 Coppice Walk, Totteridge, London N. 20, England.
Automatic Method for Separation, Hydrolysis, and Detection of Peptides SIR: An automatic, continuous method for the alkaline hydrolysis and detection of peptides has been developed and will be described. By this technique the overall time of separation and subsequent hydrolysis has been dramatically reduced from days t o hours. A chromatogram of a tryptic hydrolyzate of oxidized bovine pancreatic ribonuclease has been completed in less than 30 hours. The presentation is such that information about the size of each peptide can be visualized and partially quantitated. EXPERIMENTAL
Apparatus. Chromatographic Column. 1 heavy-wall, jacketed, precision-bore glass tube was used. It was packed with a 4y0 cross-linked sulfonic acid type cation exchange resin which had a particle size of 20 to 22 microns (Technicon, Chromobeads-D). The dimensions of the column were 100 X 0.6 em. Hydrolysis Coil. A 130-ft. long, 0.085-inch wide, and 0.012-inch thick Teflon tube was used for the alkaline hydrolysis of peptides (Polymer Corp. of Pennsylvania). It was immersed in the form of a coil in a controlledtemperature oil bath. Variable Speed Pumps. They were similar to the proportioning pump first described by Skeggs (3) with the difference that their speed could be regulated by means of a built in rheostat. 1 146
ANALYTICAL CHEMISTRY
Photometers. The photometers which were used were of the dual beam type (1) with the substitution of a newer model flow cell having a 15-mm. light path and 2-mm. bore. I n this tubular cuvette the axes of liquid flow and of the light beam were the same. The absorbances of the liquid passing through the flow cell were measured a t 570 mfi. Reagents. Sodium hydroxide solution, 20% w./v. in distilled water. Acetate buffer (dN), 328 grams of anhydrous sodium acetate are dissolved in approximately 600 ml. of distilled water, 100 ml. of glacial acetic acid are then added slowly while stirring, and the mixture is diluted to 1 liter. The final pH of the solution should be adjusted to 5.51 0.03. Acetic acid solution, 4 N acetate buffer-acetic acid-water, 1:3: 3, respectively. Ninhydrin reagent, 20 grams of ninhydrin and 1.5 grams of hydrindantin are dissolved in 650 ml. of peroxidefree methylcellosolve. The solution is then transferred into a dark (amber) bottle and the air displaced by bubbling nitrogen (prepurified grade) through it for about 15 minutes. Three hundred fifty milliliters of 4147 acetate buffer, pH 5.5, are then added, and bubbling of nitrogen continues for approximately 30 minutes. The solution is then diluted 1:2 with a 50% v./v. aqueous solution of peroxide-free methylcellosolve and nitrogen bubbling continues for about 30 more minutes. The nin-
*
hydrin reagent is always kept under nitrogen. Diluent. One volume of 4N acetate buffer, pH 5.5, is diluted with 3 volumes of water and the p H of the solu0.05 with tion is adiusted to 5.4 4 N NaOH, Buffers. Citrate, pH 3.10, 0.2N Na. Dissolve 21.00 grams of citric acid.HzO and 8.30 gra&s of NaOH in 900 ml. of distilled water. Titrate with 6N HCl to pH 3.1, dilute to 1 liter, and make final adjustment of pH. Citrate-acetate, p H 5.10, 2.0N Ka. Dissolve, with cooling, 105.0 grams of citric acid.HzO, 21.47 ml. of glacial acetic acid and 47.06 grams of NaOH in 850 ml. of distilled water. Titrate with 50% acetic acid to p H 5.10, dilute to 1 liter, and make h a 1 adjustment of PH. Acetate, pH 6.8, 2.ON Na. Dissolve 272.17 grams of sodium acetate.3 HzO in 800 ml. of HzO, titrate to pH 6.8 with 50% v./v. acetic acid, make to 1 liter, and make final adjustment. Analytical System and Operational Procedure. Three to four milligrams of performic-acid-oxidized bovine pancreatic ribonuclease (Mann Research Laboratories, Lot N o . H2723) hydrolyzed by trypsin (C. F. Boeringer 8: Soehne, Control No. 6053513) were employed per experiment. The digestion was carried out a t 27’ C. in 0.2M phosphate buffer a t pH 7.3 and lasted 6 hours. The reaction was then terminated by adjusting the pH of the solution to approximately 2.2 with I N
*
FLOW SCHEWTIC
Table 1. Buffer Composition of Autograd Chambers
COLUMN
-
Citrate CitrateCham- (ml.), acetate Acetate ber pH (d.), (ml.), Water I
I
KO. 3.1 p H 5 . 1 p H 6 . 8 1 2
02 92 92 92
Figure 1. Flow scheme for apparatus for automatic separation, hydrolysis, and detection of DeDtides
. .
HCl and the sample added immediately to the column, the temperature of which was maintained a t 38' C. throughout the experiment, The eluting liquid was supplied t o the column by means cf a high-pressure piston type pump (Milton Roy Co., Philadelphia, Pa.) frcm a 9-chamber Autograd developed after the model of Peterson and Sober ( 2 ) . The pump was adjusted to deliTer 0.42 ml. per minute. The composition of the gradient employed is listed in Table I. The effluent from the column was divided into three streams by means of a three-arm glass cactus fitting attached to the bottom of the column. The diameters of the Tygon tubings used on the first pro2ortioning pump and hence the rates of flow were such that the column effluent was divided into approximately three equal portions, one third each going to the fraction collector and the two flow circuits. The portion of the effluent to be hydrolyzed mas mixed with the aqueous solution of sodium hydroxide and then segmented with nitrogen (1, 3 ) . After passing through a mixing coil it was introduced by means of the first proportioning pump into the alkaline hydrolysis coil which was immersed in an oil bath heated E d t 105' C. The flow ratio of the sample and alkali were such that the concentration of alkali in the hydrolysis coil was approximately 3.3"V The speed of the pump was adjusted tc allow hydrolysis time of approximately 70 minutes. Because glass is not compatible with alkali, a tube made of Teflon (fluorinated hydrocarbon) was chosen which is alkali-resistant even a t elevated temperatures. Surging developed in the tube because of the hydrophobic nature of this materid. It was eliminated by inserting a length of Tygon polyvinyl chloride tuking as a vent on the high pressure side of the circuit. This was approximatdy 6 inches long and had an internal diameter 0.005 to 0.01 inch. The vent acts as a pressure regulator by allowing a limited amount of alkali to pass through to waste. To eliminate the possibility of losing any peptide solution during the rLn, the vent tube was connected to the alkali supply line at approximately 2 inches before the alkali was mixed with the sample. The diameters of the pump tubings through which the column effluent,
57 4 9
...
... ,.. ... ... 15 45
88 67
...
... ... ...
...
...
...
...
20
87
(ml.)
... ... ... ... 20 40
... ...
GAUGE
COLLECTOR
Figure 2. Detailed flow diagram of apparatus for automatic separation, hydrolysis, and detection of peptides
sodium hydroxide, and nitrogen were introduced into the Teflon hydrolysis coil, and of the tubing through n-hich the solution was pumped out of it, were such that a positive pressure was constantly maintained in the hydrolysis system. No disturbance in the segmentation pattern or of any other kind was therefore observed a t the temperature of hydrolysis (105' C.). The hydrolyzed peptides were then transmitted in succession to a second proportioning pump a t which point the p H of the solution was brought t o about 5.4 by the addition of the acetic acid reagent. The solution was then resegmented with nitrogen, mixed with ninhydrin reagent, and after development of the color its absorbance was recorded. The third line from the chromatography column was also connected to the first pump. After its exit from the pump, it passed through an approximately 50-ft. long time delay coil made of capillary polyethylene tubing (0.035 inch id.). The peptide solution was then mixed with ninhydrin, diluent, segmented with nitrogen, and sent to the second glass coil of the ninhydrin color development bath ( 1 ) which mas maintained a t 95' C. The liquid finally passed through a second photometer, and the ninhydrin color value was recorded. A two-pen recorder was
used in these experiments (Technicon Instruments Corp.). The length of the capillary polyethylene tubing which was inserted in the control line was adjusted so that the overall time delay of the nonhydrolyzed stream was the same as that of the one going through the alkaline hydrolysis bath. Thus the hydrolyzed and nonhydrolyzed effluents entered their respective photometers simultaneously. Consequently, the ninhydrin peaks which were obtained on the two-pen recorder appeared a t the same time, and could therefore be conveniently compared. The concentration of the peptides in the control line mas the same as that in the hydrolysis line; therefore, the two colorimeter tracings on the recorder represented the same initial proportion of samples. Figure 1 shows a flow schematic and Figure 2 a detailed diagram of the apparatus. The sizes of the Tygon tubes on the proportioning pumps and hence the relative flow rates of reagents, samples, etc., are also indicated. Figure 3 shows one of the chromatograms obtained under our experimental conditions. DISCUSSION
Peptides generally react with ninhydrin only a t the free amino groups. However, if a peptide is hydrolyzed, VOL 36, NO. 6, M Y 1964
1147
20-
Yz 0068:
HOURS 0
A
b
I
8
I
12
I
15
I
18
I
dl
217
24
Figure 3. Chromatogram of a tryptic hydrolyzate of performic acid-oxidized bovine pancreatic ribonuclease before and after alkaline hydrolysis
amino groups), could be due to a small amount of a single amino acid present. Also it is quite obvious from this chromatogram that without alkaline hydrolysis many peaks would have been missed. The above information is obtained in just over 27 hours. This represents a savings in time of days over conventional methods. LITERATURE CITED
then all the liberated amino acids will react with ninhydrin. Therefore, if one were to examine ninhydrin color development before and after hydrolysis one could get an idea of the number of amino acids present in the peptides. An examination of the peaks in Figure 3 readily gives one this information. For instance, peak A shows little
or no color development before hydrolysis and a large amount after. Therefore one can deduce that it must have been large peptides. Peak B , however, could not be a large peptide since the ninhydrin color difference before and after hydrolysis is not very large. Peak C, unchanged before and after hydrolysis (no liberation of free
(1) Ferrari, A., Russo-Alesi, F. M., Kell, J. M., ANAL. CHEM.31, 1710 (1959). (2) 31, Peterson, 857 (1959). E. A., Sober, H. A., Ibid., (3) Skeggs, L.T., Am. J . Clin. Pathol. 28, 311 (1957).
GEORGE N. CATRAVAS Research Laboratories Technicon Chromatography Corp. Chauncey, N. Y.
Thermometric Determination of Copper by Iodometry SIR: Thermometric titration techniques have been adapted to what is believed to be the first successful application of this methodology to the iodometric determination of copper. While the literature reports that thermometric titrations have been applied to numerous types of reactions, little work has been done with redox reactions (6). The method offers the advantage of rapid, convenient, and precise automatic determination of copper by iodometry. The titration of iodine, generated by the addition of excess iodide ion to a copper(I1) sohtion, with standard thiosulfate results in a temperature rise of the reaction mixture yielding the typical rectilinear ascendingtype curve generally obtainrd by thermometric techniques. The equivalence point is characterized by a sharp break in the curve followed by a straight negatively sloped excess reagent line. Accuracy and precision on the order of 1 to 3% were achieved.
iodide was added to the copper titrate and the lid placed on the Dewar. Kater-saturated nitrogen was passed into the flask a t a rate of 5 to 10 cc. per minute to prevent air oxidation of the iodine. After thermal equilibrium was reached titration was commenced with standard thiosulfate. The thiosulfate was supplied to the reaction vessel as a solution of Na2S20a.5H20 with 0.1 gram per liter of sodium carbonate added as a preservative. These solutions were standardized by conventional methods ( 3 ) . To obviate volume corrections, the concentration of titrant was maintained a t 50 to 200 times the concentration of the titrate. The concentration of the titrants employed varied from 1.2 to 2.5M. The titrant solutions were stable for periods of 3 to 4 weeks. The titrations were designed to reach the end point approximately 1 minute after the first addition of titrant. All titrations were carried out a t 25 =k 1' C. The methods of calibration, extrapolation, and interpretation of thermometric titration curves have recently been outlined ( 2 ) .
The enthalpy of the iodide-thiosulfate reaction under the experimental conditions listed above was estimated at -3.9 =t0.4 kcal. per mole. The free energy of Reaction 1 a t 25" C. has previously been reported as -16.5 kcal. (4). The equilibrium constant of the reaction is then 1012. If these were the only thermodynamic parameters involved it should be possible to achieve satisfactory results a t concentrations close to 10-411.1 using present instrumentation. However, since the thermistor only detects the overall heat effect, the concomitant endothermic dilution of thiosulfate, estimated a t 2.1 f 0.5 kcal. per mole from titration curves, tends to mask the end point in the lower concentration ranges. In most runs, except for those at the lowest concentrations, the results tended to be low. This is probably because of the sublimation of iodine from the reaction mixture before attainment of thermal equilibrium. This may be minimized by adjusting the temperatures of
EXPERIMENTAL
Instrumentation. The apparatus and calibration techniques used were similar to those previously described by Jordan and hlleman (1) except that a commercially availab!e multispeed horizontal constant flow automatic syringe buret, supplied by Harvard Apparatus Go. Inc., Dover, Mass., was employed to deliver the titrant. A 2-mv. recording potentiometer was used to plot the curves. Procedure. Fifty milliliters of copper(I1) solution, prepared gravimetrically from copper and nitric acid, with a concentration between 10 -2 and 10-331 wag added t o a 500-ml. Dewar flask. One gram of sodium 1148
ANALYTICAL CHEMISTRY
DISCUSSION
From Table I it can be seen that copper in the concentration range between 5 X lob2and 2 X 10-3M can be determined with 1 to 3% relative error by means of thermometric titrimetry using standard thiosulfate solutions. The direct enthalpimetric determination of copper employing the enthalpy of Reaction 1 below, is limited by the appreciable endothermic heat of dilution of thiosulfate.
+
2 SzOs-2(aq.) 18-(aq.).
S108-Yaq.)
..
+ 3 I-(aq.)
(1)
Table I. Typical Results Obtained in Thermometric Determination of Copper by lodometry
51.8 10.4 5.18 2.07 1.04
52.2 10.1 5.03 2.05 1.19
+1 -3 -3
0 1 2
+14
1 2
+I
Average relative error baaed on a t least dualicate trials. b Relative standard deviation of the mean. 5
