increasing the time allowed for signal integration would improve the signal-tonoise ratio a t an early stage; using a greater number of scans would also produce much the same effect. In summary, any method leading to increased time averaging results in an increased signal-to-noise ratio. The signal enhancement achieved here is not necessarily optimal or even typical. In particular a greater density of points would be desirable from the standpoint of resolution. The density of observations obtained in the present study was limited by the necessity of recording distinct lines.
With automated data collection devices, this restriction would be removed and any desired density of points could easily be obtained. Nevertheless, these results do suggest that the use of the integrator technique coupled with curve smoothing has the potential of dramatically improving the sensitivity of NMR spectrometers, and our belief is that application of this method merits further attention. ACKNOWLEDGMENT
The authors thank Roger W. Crecely for his assistance in several phases of this work.
LITERATURE CITED
(1) Guest, P. G., “Numerical Methods of Curve Fitting,” p. 349 ff., University Press, Cambridge, England, 1961. ( 2 ) Mayo, R. E., Goldstein, J. H., Rev. Sci. Znstr. 35, 1231 (1964). ( 3 ) Savitzky, A., Golay, M. J. E., ANAL. CHEM.33,1627 (1964).
(4) Whittaker, E., Robinso?
G., “The Calculus of Observations, p. 291 ff., Blackie and Son, Ltd., London and Glasgow, 1948. J. M. READ,JR. J. H. GOLDSTEIN Department of Chemistry Emory University Atlanta, Ga. WORKsupported by National Institute of Health Grant No. GM108 48-07.
A Device for Automatic Gradient or Stepwise Chromatography Application to the Separation of Peptide Mixtures by Gradient Chromatography on Dowex-1 SIR: The separation of peptide mixtures on Dowex-1 columns with a p H gradient made from volatile buffers has been previously described (for review see ref. 6). Schroeder and Robberson (7) have now improved their system by eliminating sharp drops in pH during the gradient from the starting basic medium of pH 9.4 to the acid medium of pH 2. The slightly sigmoid pH curve obtained is reproducible and has been shown to give excellent separation of peptides. Although complex mixtures can be separated using this resin ( 6 ) , its special value probably lies in its use to further fractionate simple mixtures of peptides obtained from initial separations on Dowex-50 columns. We have found it advantageous to rechromatograph many zones from Dowex-50 chro-
matograms on Dowex-1 and our experience has been that such a treatment results in peptides having virtually no contamination. To facilitate routine work of this type we have developed an automatic system for production of the new gradient described by Schroeder and Robberson (7) which includes automatic regeneration of the column after completion of the chromatogram. EXPERIMENTAL
The resin, Dowex-1 (200 to 400 mesh) was prepared as previously described (6). The buffers used were those described by Schroeder and Robberson ( 7 ) . Columns (0.6 X 60 cm.) were poured after equilibration of the resin with the starting buffer
of p H 9.4 and were thermostated a t 36” C. Table I shows the conditions used for chromatography. For the automatic production of the gradient a system of solenoid valves connected to a multiple sequence timer was used. Figure 1 depicts the system employed. The solenoid valves numbered 1, 2, 3, 4, and 5 in Figure 1 (Allied Control Co., Inc., New York, N. Y., Catalog No. 21384, 6-watt, 24volt 60 cycles, orifice 3/32, p.s.i. 80, stainless steel, normally closed) were connected a t the “In’, end to the buffer vessels A , B, C, D,and E. The rrOutJ’ ends were connected to the inlets of a mixing vessel. This mixer was a cylindrical borosilicate glass vessel (diameter 35 mm., height 40 mm.) with a male joint ST 40/35 having a borosilicate glass cover with a female joint
Table I. Conditions for Chromatography Column Temperature Flow rate Volume of mixer Fraction size Equilibrating buffer
0 . 6 X 60 cm. 36’ C. 40 ml./hour 40 ml. 2 . 0 ml. pH 9.4
Development
Time on sequence Volume, timer, ml. minutes
A. pH 9.4 buffera B. pH 8.4 buffer C. pH 6.5 buffer D. 0.5N acetic acid E. 2.0N acetic acid
10 30 40 60 100
15 45 60 90 150
Regeneration F. pH 9.4 buffer 80 120 a The composition, preparation, and storage of the buffers are as described by Schroeder and Robberson ( 7 ) .
U
I
I
I I
I 1
I I
Fraction collector
1 Stirrer
L _ . - _ _ _ _ _ _ _ I_ - _ _ _ - _ - _ _ _ Figure 1 .
I
I
I
L
I I
--_-_____
I -I
Diagram of the gradient producing system VOL. 37, NO. 12, NOVEMBER 1965
1611
LEDEX SWITCH
SOLENOID VALVE CONTROL UNIT
I
I N.0.. NORMALLY OPEN NORMALLY CLOSED
N.C..
I I
I I I
I I I I
Figure 2.
Wiring diagram of the timing unit See text for details
ST 40/35. The cover had five narrow borosilicate glass inlet tubes (0.d. 3 mm.) which ended about 2 cm. above the buffer level (see Figure l), and one outlet tube which started a t the bottom of the mixer and was placed sideways to leave room for a magnetic stirring bar. The mixing vessel was filled a t the start of each run with 40 ml. of pH 9.4 buffer. The connections to and from the solenbid valves were made as follows. A stainless steel Swagelok “male adaptor tube to pipe” ending in a 1 4 mm. long tube of 3 mm. 0.d. (Cat. No. 201-A-2-316, Crawford Fittings Co., Cleveland, Ohio) was screwed in the In and Out orifices of the valves. Connection of each solenoid valve to the mixer and to the individual buffer vessels was then made according to Figure 1 with Intramedic Polyethylene Tubing P E 330, i.d. 0.115 inch X 0.d. 0.147 inch, Clay-Adams, Inc., New York, N. Y. The dir inlet tubes of the buffer vessels of pH 9.4, 8.6, 6.5 and of the regenerating buffer vessel were provided with a C 0 2 trap. The outlet tube of the mixing vessel was similarly connected to the In end of solenoid No. 7 (Allied Controls Co. Inc., Catalog No. 20482, 11-watt, 24-volt 60 cycles, orifice ‘/le, p s i . 200, stainless steel, normally open). This solenoid valve stays open during the whole chromatographic run and is therefore much larger than the former ones to prevent overheating. Its Out end was further connected to a pump (Milton Roy MiniPump, Philadelphia 18, Pa.) which carried the solvent to the column. Another solenoid, No. 6, similar to the first five, was connected a t the I n end to the regeneration reservoir F , and a t the Out end through a borosilicate glass Y-joint to the line going from solenoid No. 7 to the pump. Before the first operation, each solenoid valve should be put in the Open position, and a large volume of buffer 1612
ANALYTICAL CHEMISTRY
drawn through it very rapidly with a suction pump in order to eliminate all the air trapped in the interior of the valves and in the lines. The sequence timer is programmed to activate the solenoids Nos. 1-5 in sequence a t predetermined intervals based on the desired flow rate and the amount of each solvent to be used (see Table I) for production of the gradient. Only one impulse from the timer is necessary to close the operating valve and open the following one (see wiring diagram, Figure 2). During elution, 2-ml. fractions were collected. The peptide zones were detected by the ninhydrin procedure carried out automatically in a Technicon AutoAnalyzer according to Schroeder and Robberson (7). For the automatic regeneration, the timing impulse which closes solenoid No. 5 also opens No. 6 and closes No. 7. I n this way the regenerating buffer 1
I
1
I
C 0.5 N b C E T l C d C - - - - - Z . O N
C 9 . 4 4+8.4++#5-
2
of pH 9.4 bypasses the mixing vessel and is pumped immediately into the column. Only the void volume of 2.ON acetic acid present in the lines to the pump and in the column has to be neutralized before regeneration can start. The column itself was filled with resin until 2 cm. below the top, to keep the void volume as small as possible. After regeneration, the whole unit, including stirrer, pump, and fraction collector, shuts itself off. Before starting a new column, the only manipulations that must be carried out are: replacing 40 ml. of pH 9.4 buffer in the mixer, flushing the outlet line from the mixing vessel to the Yjoint (or to the column) with pH 9.4 buffer, applying the sample, and r e setting the Ledex switch manually to the starting position. The electronic part of the apparatus consists of (see Figure 2) the power supply and the actual switching assembly. The power supply follows a conventional circuitry using a power transformer type U.T.C. H95 (United Transformer Co., Culver City, Calif.) delivering 24-28 volts a.c. to the solenoid valves and the indicator lamps, and 24 volts d.c. through a rectifier (type GlB, International Rectifiers, El Segundo, Calif.) over a ripple suppressor condensor of 40 KpF 40 volts (type FD4010M-TA, Safe T-Mike Co., Oakland, Calif .) to a Ledex stepping switch (G. H. Leland Inc., Dayton, Ohio) having 4 switching wafers Ssa to Sad, with 12 positions each. This switch can be turned manually in any desired position; but it is normally powered by a timing motor (3 r.p. day, 110 volts ax., Bristol Motors, Old Laybrook, Conn.). On its axis is fixed a stainless steel circular cam (diameter 55 mm., thickness 3 mm.) which is divided in 32 sections, each corresponding to a 15-minute interval. Each of these positions carries a tapped screw hole in which a small circular bolt with an Allen wrench slot can be screwed, By varying the spacing and the number of these bolts, one can produce any timing interval between 15 I >
ACETIC
0.5-
I
I
I
I
I
50
100
I50
200
250
EFFLUENT (mi.)
Figure 3. Chromatography of a peptide mixture from bovine liver catalase on a Dowex-1 column (0.6 X 60 cm.) with the present gradient system Abscissa: effluent in mi.; left ordinate: absorbancy after ninhydrin color development; right ordinate pH. The p H curve is drawn as a combination of 4 similar curves from separate experiments. The sequence of the eluting buffers is indicated in the upper part of the figure. See text for further details
minutes and 8 hours. This system also allows changing of the timing intervals during a run. The precision of these intervals is within =tl’%. The bolts activate a microswitch SZ (Honeywell) which then closes the rectifier circuit to the Ledex switch. A thermal time delay relay (RF60NC28, G. V. Controls Inc., Livingstone, N. J.) is included in this circuit. It reopens the circuit a few seconds after activation of the Ledex switch. In this way, the solenoid in this switch is powered only for a few seconds during actual switching. Otherwise, overheating would occur during prolonged use. The Ledex switch in turn closes one of the solenoid circuits through its Sac wafer. The successive contacts on the timing motor cam will operate in this way all the solenoids in sequence, closing one as the next one opens. At the same time, the Sla wafer of the Ledex switch closes the circuits of the corresponding warning lights which indicate the operating solenoid, and, consequently, the eluting buffer. Each solenoid circuit has a switch (S4 to S9) which is normally closed. I n this way, the number of buffers can be decreased by switching the corresponding solenoids out of circuit and turning the Ledex switch manually to the next solenoid position. More solenoids can be added if needed. The two remaining wafers of the Ledex switch, Saa and S 3 d are used respectively to switch off the timing motor and the whole apparatus including pump, stirrer, and fraction collector after equilibration is completed. The whole unit is mounted on a single chassis, except for the solenoid valves which are mounted separately on a small rack just above the mixing vessel. The electric leads from the solenoids are connected to the chassis by microphone plugs. In this way, the sequence of operation of the solenoid valves can be changed a t any time. RESULTS AND DISCUSSION
I n Figure 3 is shown a separation of about 1 pmole of tryptic peptides from bovine liver catalase, which were eluted in the partially separated zones 16-17 on a Dowex-50 column as described by Schroeder et al. (8). I n order of emergence in Figure 3, the peptides were those having the numbers 17a, 17b, 17c, 16a and 16b following the nomenclature of Schroeder et al. (8). The separation of the peptides may be compared with the same mixture fractionated on the same type of column but using a different gradient producing device (7). The pH curve is drawn as a combination of the corresponding curves obtained from separations of 4 other mixtures. The p H gradient obtained with our present system is thus reproducible within a t most 0.25 p H unit a t any given position during elution, as shown by the width of the curve in Figure 3. Dowex-1 separates peptides almost
I
I
I
I
150
200
I 50
100
EFFLUENT (ml.)
Figure 4. Chromatography of the tryptic peptides of tobacco mosaic virus on a Dowex-1 column (0.6 X 60 cm.) with the present gradient system Abscissa: effluent in ml.; left ordinate absorbancy after ninhydrin calor development; right abscissa: pH. The sequence of the eluting buffers Is drawn in the upper part of the figure
exclusively on the basis of charge (6). I n the former manual procedures having a rather poor reproducibility of p H values with respect to elution volume, the p H of each 10th fraction or so had to be in measured in order t.0 determine the pH of elution of a peptide zone for comparison purposes. Because of the reproducibility of this gradient system, pH measurements after elution can now be dispensed with. Figure 4 depicts a separation made on 0.3 pmole of the soluble t,ryptic peptides from tobacco mosaic virus. This represents a separation of a complex mixture. Although the separation is sufficient for purification of some of the peptides, a larger column would effect improvement. This can easily be accomplished by merely increasing the size of the mixing vessel and employing the buffer volumes indicated by Schroeder and Robberson ( 7 ) for columns of 1 X 100 cm. The timing intervals on the multiple sequence timer must then be changed appropriately. The partial separation obtained with the present system should be compared with the complete separation obtained with the method of Funatsu (4) using a 0.9 X 150 cm. Dowex-1 column and a p H gradient generated by a “Varigrad” system. The present combination of timer and solenoid valves can be readily adapted to such large columns. The instruments presently used for gradient producing purposes have several disadvantages and limitations. The instruments of the “Varigrad” type (5) give a reproducible gradient but they must be filled rather precisely before each run and they cannot be used to regenerate the column automatically. Furthermore, they are fairly expensive. Total cost of the solenoid valves and
sequence timing unit described here is about $250.00. The system of Brusca and Gawienowski (1) adapted by Schroeder and Robberson (7) for peptide separations on Dowex-1 columns of 0.6 X 60 cm. gives reproducible gradients, is inexpensive to construct, and is operated by gravity flow so that no pump is needed. However, it also must be filled before each run, it cannot regenerate the column, and its flow rate has to be adjusted during the run by varying the height of the reservoirs above the column. A special buffer reservoir combination has to be designed for each specific gradient system. Recently a complete system for automatic peptide chromatography including detection has been described (2). illthough the system appears to be well engineered, it uses nonvolatile buffers which present severe disadvantages in peptide work (6) and the gradient producing unit is of the “Varigrad” type. Furthermore, whether very complex peptide mixtures, resulting from partial digests of large proteins, can be separated completely on one column type only is doubtful. The present apparatus, designed to offset some of these limitations, requires minimal manual labor, needs very little maintenance, makes a reproducible gradient, regenerates the column, and pumps the volatile buffers out of large storage vessels. With the present gradient and a flow rate of 40 ml./hour a complete run takes only 6 hours plus an additional 15 minutes for application of the sample and 2 hours for regeneration. Consequently, in a little over 8 hours, one run can be made completely and the second run started and left to run overnight with automatic shut-off. The VOL. 37, NO. 12, NOVEMBER 1965
0
1613
column is ready for another run the next morning. For less critical separations the flow rate can be speeded up, as discussed by Schroeder and Robberson ( 7 ) . The instrument can also be used to produce other types of gradients. The gradient generating system consists of a closed mixing vessel of constant size. Consequently, the formula of Drake (3) for such a system can be used to compute the various concentration parameters. The gradient can be calculated in successive portions, depending on its shape and the amount of solenoid valves available. If needed, stepwise elution can also be programmed. I n this case, the mixing vessel must be substituOed for a capillary tube having as many capillary inlet,s as there are to be steps in the elution proIf automatic regeneration gram. should also be desired, the regeneration buffer can t,hen be fed directly into the capillary collecting tube or a t some other point closer to the top of the column. Timers are commercially availablee.g. from Giannini Controls Co., Cramer Division, Centerbrook, Conn., Type 511 or 521, which could be adapted to this instrument whenever construction of the described timer should not be possible.
However, in most of these designs the sequence of timing impulses is not generated by a stepping switch, but by a battery of microswitches each activated by its own timing cam mounted on the timing motor axis. Therefore, considerable care must be taken to adjust the timing cams so that no overlap or dead time is produced between two buffers. Such timers are less readily adapted to produce other gradients because it is considerably more complicated to change all the timing cam settings. Changing the timing intervals during a run would also be difficult. ACKNOWLEDGMENTS
We thank W. A. Schroeder for a gift of the peptide mixture from bovine liver catalase, for the use of some of the facilities of his laboratory, especially the Technicon AutoAnalyzer, and for his advice during this investigation. We would also like t o thank Heinz FraenkelConrat for a gift of the tryptic peptides from tobacco mosaic virus. We are greatly indebted to E. Carver Jewett for his expert technical assistance in designing and constructing the timer.
LITERATURE CITED
(1) Brusca, D. R., Gawienowski, A. M., J. Chromatog. 14, 502 (1964). (2) Catravas, G. N., ANAL. CHEM.36,
1146 (1964). (3) Drake, B., Arkiv Kemi 8 , 1 (1955). (4) Funatsu, G., Biochemistru 3, 1351 (1964). (5) Peterson, E. A., Sober, H. A,, “A Laboratory Manual of Analytical Methods of Protein Chemistry,” Vol. I, p. 88, P. Alexander, R. J. Block, Eds., Pergamon Press, New York, 1960. (6) Schroeder, W. A,, Jones, R. T., Cormick, J., McCalla, K., ANAL.CHEM. 34, 1570 (1962). (7) Schroeder, W. A,, Robberson, B. ANAL.CHEM.37, 1583 (1965). (8) Schroeder, W. A., Shelton, J. R., Shelton, J. B., Olson, B. M., Biochim. Biophys. Acta 89,47 (1964). WILFRIED A. ROMBAUTS A. RAFTERY MICHAEL Division of Chemistry and Chemical Engineering California Institute of Technology Pasadena, Calif. 91109 INVESTIGATION supported in part by the Arthur Amos Noyes Research Fund and in part by a grant (HE-02558) from the National Institutes of Health, United States Public Health Service.
Analysis of Cigarette Smoke Fraction by Combined Gas Chromatography-Infrared Spectrophotometry SIR: Recently, an instrument was described (4, 7 ) which is claimed to eliminate the need for manual transfer of collected gas chromatographic peaks to infrared absorption cells. The instrument permits the shunting of an eluting peak directly into a gas absorption cell and provides a rapid (45 seconds) scanning of the infrared spectrum of the sample from 2.5 to 15.0 microns. The utility of the equipment was demonstrated experimentally by separation and spectral analysis of components in simple synthetic mixtures of compounds ( 7 ) . As with many instruments, such separations do not prove the value of the equipment when used in studies on complex mixtures such as natural products. T o determine such value, we have used the instrument to study components in an ether codistillate of cigarette smoke condensate. Details of the performance of the equipment and on the possible identities of some components in the codistil1at.e are presented herein. EXPERIMENTAL
Sample Preparation. Smoke condensate (980 grams) was partitioned between 4 liters of ether and 4 liters of 1N aqueous NaOH. The alkaline layer was washed with an additional 1614
ANALYTICAL CHEMISTRY
4 liters of ether, and all ether solutions were combined. Bases were removed from the combined ether solutions with two successive extractions (3.6 liters each of 0.2N HC1). The resulting ether solution of neutrals was washed free of mineral acid with small portions of water, dried over Na2S04, and the ether slowly distilled. The distillate, which gradually assumed a light yellow color during collection, was then concentrated on Stedman and spinning band columns to a volume of 5 ml. as previously described (6). Gas Chromatography. Usually, 100-pl. aliquots of the concentrated distillate were sufficient for both gas chromatography and infrared spectral analysis of individual peaks. The gas chromatographic portion of the instrument was a Loenco Model 70 (no endorsement implied) equipped with a thermal conductivity detector and fitted with dual columns (10 feet X 0.25-inch 0.d.) packed with 20% Apiezon L on Chromosorb W. Operating conditions were as follows: column temperature, 55’ C. for 20 minutes, then programmed a t 2’ per minute to 100’ C.; flow rate, 60 ml. per minute of helium; injector temperature, 200’ C.; detector temperature, 250’ C. Infrared Spectral Analysis. Using the Wilks chromatograph spectrograph, selected gas chromatographic peaks were shunted into the infrared absorption cell heated to 200’ C.
-
Between successive samples the cell was flushed with carrier gas by opening the entrance and exit valves. Infrared spectra of authentic compounds were obtained in the same manner as the unknowns in the mixture. RESULTS AND DISCUSSION
The gas chromatogram of the ether codistillate and tentative identifications of some components are included in Figure 1. Peaks were identified by infrared spectral characteristics and gas chromatographic retention times. The infrared spectra of some peaks indicated the presence of more than one component, as would be expected in a complex mixture of this type. Although the infrared spectra were somewhat lacking in resolution, they were of sufficient value when used in conjunction with retention data to provide further evidence of peak identities. For example, the spectra of peaks 4 and 5 were indicative of simple aldehydes. A study of the elution pattern of a homologous series of authentic normal aldehydes showed that the peaks in question elute between n-butyraldehyde and n-valeraldehyde. Therefore, peaks 4 and 5 appeared to be branched chain aldehydes and probably CS compounds.
