noise estimates show that, except for the previously discussed seventh interval, the detector is remaining quite consistent in its ability to measure peak areas. When automatic data handling equipment is available, the noise estimate can be obtained more easily than the peakto-peak estimates. The same length of base line must be recorded with either procedure to provide the same sample size, and reading a magnetic tape a t the computer is faster than scrutinizing and making measurements on a chromatogram. Repeatability. The statistical basis for this procedure (3) permits a definite
LITERATURE CITED
prediction of repeatability. Interpolation in Table I of Reference (3) for the present case of 50 Ni in the noise estimate calculation indicates that 19 out of 20 noise estimates should have relative values within 0.596 to 0.891 and, therefore, should not vary more than ~ = 2 0 7from ~ their mean. The values in Tables I, 11, and I11 support this conclusion. A section of base line 1200 times as long as the minimum interval was used for a set of noise estimates to help ensure a representative sample. I n fact, the data of Table I1 illustrate how multiple noise estimates can be used to detect variations in base line noise.
(1) Dimbat, M., Porter, P. E., Stross, F. H., ANAL.CHEM.28, 290 (1956). ( 2 ) Johnson, H. W., Jr., Ibid., 35, 521
(1963).
(3) Johnson, H. W., Jr., Stross, F. H.,
Ibid., 31, 1206 (1959). (4) Williams, A. J., Jr., Tarpley, R. E., Clark, W. R., Trans. Am. I n s t . Elec. Engrs. 67, 47 (1948). (5) Young, I. G., “Gas Chromatography,”
H. J. Noebels, R. F. Wall, N. Brenner, eds., p. 75, Academic Press, New York, 1961. H. W. JOHNSON, JR. Shell Develo ment Co. Emeryville, 8alif. PITTSBURGH Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1965.
An Improved Gradient for Ion Exchange Chromatography of Peptides on Dowex-1 SIR: Dowex-1 has proved to be a useful ion exchange resin for the separation of peptides. I n earlier application of this resin to the separation of peptides (9), three different developers were used. The choice of a particular developer was dictated to a considerable degree by the charge of the peptides to be separated. I n each of the three developers, a rather abrupt change in p H occurred a t some point in the gradient which was produced by the addition of acetic acid to a starting buffer. Because of the abrupt change, some peptides were incompletely separated and had to be rechromatographed with a more gradual gradient. A more generally applicable developer has now been devised; it begins a t a higher p H in order to retard basic peptides and has a gradual, slightly sigmoid pH gradient. EXPERIMENTAL
Materials. The preparation of Dowex-1 has been described in detail (9). Five developers are needed. Four liters of aqueous p H 9.4 buffer require 60 ml. of N-ethylmorpholine, 80 ml. of a-picoline, 40 ml. of pyridine, and sufficient glacial acetic acid (about 0.5 ml.) to give a p H of 9.4. Buffers of p H 8.4 and 6.5 are made up identically except that the quantity of acetic acid is approximately 3 and 37 ml., respectively. I n addition, 0.5N and 2147 acetic acid are required. The source and quality of the reagents have been described (9). All of them except the acetic acid were distilled without fractionation before use. N-Ethylmorpholine is best preserved in the deep freeze after distillation, Procedure. The procedure has not been altered appreciably from t h a t previously described in detail (9) except in the method of development, the size of the column, and the ninhydrin pro-
MIXER
A
HEATING BATH 2
I: 4
97.
MIXER
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mllmin
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c
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2;
Dummy
Dl SCA RD
Figure 1.
Revised system for ninhydrin analyses with Technicon AutoAnalyzer
cedure for detecting the position of zones. The use of smaller columns (0.6 X 60 em. instead of 1 X 100 em.) has been briefly outlined ( 3 ) . Prior to the pouring of the column, the regeneration of the resin (9) was completed with the pH 9.4 buffer described above, and the resin was suspended in it. The pouring of Dowex-1 columns can be troublesome because of the tendency of bubbles to be trapped or to form and grow. The following procedure usually resulted in a satisfactory column in the event that bubbles formed after a column was poured. The bottom of the tube was closed, a 1 X 35-em. extension was attached to the top of the chromatographic tube, buffer was added until the extension was about half full, and then the top of the extension was also closed. By carefully and repeatedly inverting the column and returning it to its normal position, the resin can eventually be suspended partially or completely in the supernatant buffer and then allowed
to resettle under gravity. If bubbles are present in the upper portion of the column, only this part need be suspended and resettled. After repeated use, the flow rate of a column a t a given pressure may decrease markedly. I t is usually simpler to suspend and resettle the column in the above way than to remove and repour the resin. The conditions of chromatography for two sizes of columns are listed in Table I. The columns differ by a factor of about 4 in volume and most quantities have been adjusted by about this ratio. The gradient was produced by a constant volume mixer of the indicated volume. The reservoir from which the various developers flow into the mixer should be attached directly to the mixer. If the changes were made manually, mixer and reservoir were filled initially with p H 9.4 buffer. After the indicated volume (Table I) had flowed through the column, p H 9.4 buffer in the reservoir was replaced VOL. 37, NO. 12, NOVEMBER 1965
1583
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0
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,
Effluent volume, mi.
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Figure 3.
200
Separation of tryptic peptides of aminoethylated
y chain of bovine fetal hemoglobin (2) Column size 1 X 100 cm.
E f f l u e n t volume,ml.
Figure 2. Separation of peptides from a tryptic hydrolysate of bovine liver catalase See text for identification of peptides and for an explanation of pH curves. Column size, 0.6 X 60 cm.
by p H 8.4 buffer, it in turn by p H 6.5 buffer, etc. All of those changes may be made automatically if a device patterned after Brusca and Gawienowski (4) [see also Anderson, Bond, and Canning ( I ) ] is attached directly to the mixer. The procedure may be automated even to the extent of automatic regeneration of the column by the device of Rombauts and Raftery (8). The flow rates have been the maximum that can be achieved with a hydrostatic head of 6 to 8 feet of water. RThen a freshly resettled column was used, a chromatogram on a 0.6 X 60cm. column could be completed in 5 hours. With repeated use, the flow rate through the column decreases at a given hydrostatic head. Alternatively, a constant volume pump may be used until excessive pressure is produced and the column must be resettled or repoured. The examination of the effluent fractions now includes prior alkaline hydrolysis in the manner essentially that of Hirs, Moore, and Stein ( 6 ) except
that a boiling water bath was used and the duration of hydrolysis was 1.5 hours. Only alternate fractions were examined; 0.1-ml. or 0.2-ml. portions from fractions of the two sizes of column described here were routinely hydrolyzed unless the quantity of sample on the column was especially large or small. These hydrolyses are conveniently made in inexpensive and reusable polypropylene or polyallomer centrifuge tubes (Beckman Instruments, Inc., Palo Alto, Calif., No. 303958). After alkaline hydrolysis, the fractions were examined by the AutoAnalyzer system in Figure 1 which follows the suggestions of George Winter of the Technicon Corporation. The ninhydrin reagent was that of Catravas (,i)except that all reagents were bubbled thoroughly with prepurified nitrogen before mixing, and the 411.1 acetate buffer was made up with 294.5 grams of anhydrous KOAc, 136 grams of NaOAc~3Hz0,and 100 ml. of glacial HOAc per liter of solution (a small portion after dilution with 3 volumes of water should have a pH of 5.13) ( 7 ) . The diluent was 1:l watermethyl cellosolve by volume. RESULTS AND DISCUSSION
Table I. Conditions for Chromatography on Dowex-1 Size of column 0.6 X 1 X 100
60 em. cm. 60 ml. 100 ml. Equilibrating buffer (pH 9.4) 135 ml. Volume of Era40 ml. dient mixer 1 ml. 1 . 5 ml. Size of fractions Development" pH 9.4 buffer 10 ml. 40 ml. pH 8.4 buffer 30 ml. 120 ml. pH 6.5 buffer 40 ml. 160 ml. O.SN acetic 60 ml. 240 ml. acid 400 ml. 2N acetic acid 100 ml. Flow rate Maximumb Maximum* 75 ml. 150 ml. Reequilibrating volume = See text for further discussion. b With a hydrostatic head of 6 to 8 feet of water. See text. 1584
ANALYTICAL CHEMISTRY
Figure 2 is an example of the separation of a mixture of peptides on a 0.6 X 60-cm. column. The peptides derive from peaks equivalent to 16 and 17 of Figure 1 of Schroeder et al. (10) and have been given the numbers by which they are identified in Ref. 10. The p H curve is the composite from five chromatograms that were developed with the use of the automatic solvent changer (1, 4 ) . I t s width shows the extremes that have been observed. The variations have not had any obviously deleterious effect upon separations and probably result mainly from the rapid rate of solvent flow (see below), individual differences in mixing in the gradient vessel, and variations in chromatographic columns. Experience has shown that basic peptides emerge, in general, between fractions 10 and 90, neutral peptides
between fractions 90 and 130, and acidic peptides between 130 and 250. Under these conditions of chromatography, histidine behaves as a neutral amino acid, and peptides that contain histidine, phenylalanine, or tyrosine emerge more slowly than one would predict on the basis of charge alone. By altering the volume of the mixer and/or the volume and type of the various developers, the entire gradient or individual portions of i t may be altered if it is desired to do so for special separations. The dashed p H curve in Figure 2 shows the p H curve when the sequence of developers was 10 ml. of p H 9.4 buffer, 30 ml. of p H 8.4 buffer, 80 ml. of 1 N acetic acid, and 120 ml. of 2N acetic acid. Obviously, if the volumes of the first developers were increased, the portion of the chromatogram in the high p H range would be extended. The procedure has been applied for the most part to the separation of relatively simple mixtures that viere isolated from chromatograms on Dowex-50 (9). However, more complex mixtures may also be separated as shown in Figure 3 where a slightly different gradient was used on a 1 x 100-cm. column. The gradient was produced with a 135-ml. mixer and 40 ml. p H 9.4 buffer, 125 ml. p H 8.4 buffer, 160 ml. each of 0.1N, 0.5N, and 1N acetic acid, and 240 ml. of 2N acetic acid. Few of the zones in Figure 3 contain a single peptide, but the mixtures could be separated by chromatography on Dowex-50 ( 9 ) . The gradient in Figure 3 actually is very similar to that in Figure 2 and can be scaled down for smaller columns. However, it is somewhat less reproducible on smaller columns, and, therefore, the other gradient in Table I is recommended. The flow rate of liquid through the 0.6 x 60-cm. columns can be as great as 180 ml. per sq. cm. of cross-sectional area per hour. The rate in the chromatogram of Figure 3 was only about 40 ml. per sq. cm. per hour. This factor has not been systematically investigated. Most of the zones in the simple mixtures that have been chromato-
graphed on Dowex-1 have been so well separated that flow rate has seemed to be a n inconsequential factor. Accordingly, whereas some of the initial chromatograms on 0.6 X 60-cm. columns were made with flow rates of only about 35 ml. per sq. cm. per hour, experience with increasing rates has not been accompanied by any evident broadening of the zones or decrease in the quality of the separations. However, it may be that the separation of complex mixtures as in Figure 3 would be less satisfactory if the flow rate were increased to 180 ml. per sq. cm. per hour. The mixture whose separation is pictured in Figure 2 contained a total of approximately 7 pmoles. Most of the separations are so good that a greater quantity could no doubt have been chromatographed without difficulty. The soluble tryptic peptides from about
110 mg. (or approximately 7pmoles) of aminoethylated bovine y chain were the sample for the chromatogram of Figure 3. An important factor in deciding the quantity to be chromatographed clearly is the degree of separation-a factor that usually is unknown until the chromatogram has been completed.
(4) Brusca, D. R., Gawienowski, A. M., J . Chromatog. 14, 502 (1964). (5) Catravas, G. N., ANAL. CHEM.36, 1146 (1964). ( 6 ) Hirs, C. H. W., Moore, S., Stein, W. H., J . Biol. Chem. 219, 623 (1956). ( 7 ) Moore, S., The Rockefeller Institute, Xew York, N.Y., (personal communication). (8) Rombauts, W. A,, Raftery, M. A., ANAL.CHEM.37, 1611 (1965). ~~(9) Schroeder, W. A., Jones, R. T., Cormick, J., McCalla , K., Ibid., 34, 1570 (1962). (10) Schroeder, W. A., Shelton, J. R., Shelton. J. B.. Olson. B. M.. Biochim. Biophys. Acta’89, 47 11964). ’ W. A. SCHROEDER BARBlRA ROBBERSON Division of Chemistry and Chemical Engineering California Institute of Technology Pasadena. Calif. 91109 INVESTIG.~TION supported in part by a grant (HE-02558) from the National Institutes of Health, United States Public Health Service. \
ACKNOWLEDGMENT
Parts of this investigation have had the assistance of Philip Lieberman. LITERATURE CITED
(1) Anderson,
N. G., Bond, H. E., Canning, R. E., Anal. Biochem. 3, 472 (1962). (2) Babin, D. R., Schroeder, W. A., el al. (unpublished). (3) Babin, D. R., Jones, R. T., Schroeder, W. A., Biochim. Biophys. Acta 86, 136 (1964). ~
Ion Exchange Separation and Spectrophotometric Determination of Cerium in Cast Iron SIR: Addition of small quantities of cerium to cast iron t o improve the nodular graphite structure in the iron is well known (2) and in 1948 Westwood and Mayer (4)developed a method for the quantitative determination of cerium in cast iron. The use of high concentrations of cyanides and the evolution of hydrogen cyanide a t one stage of the process are undesirable features of the method for routine control applications. I n 1950, Moore and Kraus ( 1 ) showed that iron(II1) was readily held by anion exchange resins in strong hydrochloric acid solution and that the rare earth elements were practically not adsorbed. The quantitative separation of iron(II1) from micro amounts of cerium by anion exchange in hydrochloric acid solution was confirmed in this laboratory in 1953 ( 3 ) . It appeared that this separation, which eliminated the highly toxic cyanide system, would be an improvement over the Westwood and Mayer procedure if it could be adapted to the analysis of cast iron. EXPERIMENTAL
Materials. All chemicals used were of analytical reagent grade. Standa r d cerium(II1) solutions were prepared from a solution of primary standard grade ammonium hexanitratocerate by precipitation with ammonium hydroxide and by dissolving t h e hydrous oxide precipitate in dilute hydrochloric acid to which a little hydrogen peroxide had been added. The solution was boiled to remove excess peroxide and then diluted to volume to give a final concentration of 0.100 mg. of cerium per ml. of solution. Spectrophotometric Measurements. T h e spectrophotometric method of
Westwood and Mayer (4) in which the 8-quinolinol complex of cerium is extracted into chloroform and measured spectrophotometrically was used for final determination of cerium. A Model B Beckmann spectrophotometer with 1-cm. cells was used. The broad absorption peak of the extracted 8-quinolinol solution had a maximum absorption a t 480 mp and this wavelength was used for all subsequent measurements. A’Beer’s law test showed linearity between 0.02 mg. and 0.4 mg. of cerium per 50 ml. of solution which exceeded the expected concentration range of actual samples. The p H dependence and other conditions of this extraction were studied by T$7estwood and hfayer and were not repeated here. While the exact nature of the extracted species is not yet known, the accurate and reproducible results found by Westwood and Mayer justify the adoption of their procedure. Rlinor changes such as extraction time are noted in the appropriate sections. Ion Exchange Separations. Conditions for t h e ion exchange separation of iron and cerium were established using a solution of iron(II1) chloride t o which measured amounts of standard cerium chloride solution were added. This solution was applied t o t h e t o p of a Dowex 2- X 10 chloride form resin column and eluted with aqueous hydrochloric acid. Separations using 50- to 100-mesh resin were generally unsatisfactory. With 100- to 200-mesh resin a column 9.0 cm. high and 3.5 cm. in diameter gave satisfactory separations of 5 mg. of cerium from 1.0 gram of iron. Effect of Hydrochloric Acid Concentration a n d Flow Rate. Experiments with 6J1, 9111, and 1 2 N HC1 as eluant showed t h a t in each case t h e iron was efficiently retained on t h e column and t h e cerium quantitatively
and rapidly eluted, 100 ml. of eluant easily sufficing for 5 mg. of cerium and 1 gram of iron. T h e 9 M concentration finally adopted was t h e result of tests on the interference of manganese and other elements in the actual samples. Relatively large variations in flow rate caused no appreciable change in the separation. A flow rate of 5 to 10 ml. per minute was convenient and satisfactory. Interferences. M a n y possible interferences were investigated by M7estwood and Rfayer. Manganese was t h e most serious offender and its effect on t h e present method was therefore considered in detail. As little as 1 pg. of manganese in t h e extracted solution caused a serious increase in absorbance. Attempts t o find conditions under which manganese would be retained quantitatively with iron on the ion exchange column failed. RIanganese is held to a considerably greater extent a t 931 HC1 concentration than a t 6 M , and somewhat better still a t 12111. However, in all cases appreciable manganese was eluted causing spectrophotometric interference and the large concentration of acid was inconvenient for subsequent removal before the extraction step. Furthermore, traces of nickel and chromium are more effectively retained on the column in 9 J i HC1. Manganese interference could be eliminated by precipitation of manganous ferrocyanide. Following the elution from the column, 2 ml. of 10% potassium ferrocyanide were added to the eluate followed by citric acid and ammonia to p H 8. Manganous ferrocyanide precipitated whenever the manganese content exceeded 0.2 mg. With smaller amounts of manganese no precipitate was observed. These smaller amounts of manganese were removed only after the addition of a few seed VOL 37, NO. 12, NOVEMBER 1965
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