the tailing of high molecular weight fractions into lower molecular weight fractions. Figure 4 compares a molecular weight distribution curve (solid line) obtained by the present GPC method with corresponding data (squares) from preparative GPC separation and molecular weight determinations on individual fractions. The differences in these two sets of data arise mainly from errors in the corresponding molecular weight determination for the original sample is by VPO. If the VPO value of obtained from the composited GPC fractions (squares), a value of 837 is obtained, us. a value of 768 in the original sample. Use of the 837 value (rather than the 768 value) in the routine GPC calculation yields the dashed curve of Figure 4. This is seen to be in reasonable agreement with the data by preparative scale GPC. A series of fractions from the successive extraction of an asphalt at two temperatures with isopropanol are shown in Table 11. Comparison of the original feed analysis with the composited fraction data shows good agreement. These two examples indicate that the present method is reasonably reliable, despite the limitations of UV detection. The repeatability of the present GPC method is illustrated in Table 111, for replicate analyses of the same sample using a single experimental value of ATn. The resulting molecular weight distributions from the present GPC procedure furnish three useful parameters for characterizing the sample. The ratio of molecular weights at
90 and 10 wt % ( R W , ~is~a) good measure of the molecular weight spread of the sample. The quantity C/20 provides a measure of the deviation of the sample molecular weight from the calibration of Figure 3, and is an indicator of average molecular shape. Large values of C/20 correspond to relatively bulky molecules, while small values of C/20 mean long, narrow molecules. The number-average molecular weight is the third characteristic parameter of interest. These various parameters are tabulated in Table I and I1 for the samples shown there. The difference between these particular “good” and “bad” asphalts seems to be related to molecular weight spread (Roo,lo)and to mean molecular weight, but these may be accidental correlations based on different boiling ranges in the original crude oils. The molecular shape factor C/20 does not appear to be related to asphalt performance. Asphaltenes have higher average molecular weights and large values of C/20. As expected, asphaltene molecules are bulkier and more compact than other asphalt components. ACKNOWLEDGMENT
Several asphalt GPC fractions (triangles in Figure 3, sample C of Figure 2) were kindly supplied by S. W. Nicksic of the Chevron Research Corp., La Habra, Calif. RECEIVED for review April 4, 1969. Accepted June 2, 1969.
Rapid Analysis of Ribonucleosides and Bases at the Picomole Level Using Pellicular Cation Exchange Resin in Narrow Bore Columns Csaba Horvathl and S . R. Lipsky Section of Physical Sciences, Yale University School of Medicine, New Haven, Conn.
The separation of the four major bases and nucleosides of RNA has been investigated under conditions utilizing wide ranges of pH, temperature, and flow rates. Smallbore columns packed with pellicular cation-exchange resin were used in a high-pressure liquid chromatograph equipped with a micro UV detector. Dilute acidic potassium or ammonium phosphate solutions served as eluents. The four bases or nucleosides were analyzed at the subnanomole level in less than 6 minutes using 3-meter colunms and high-flow velocities. With a shorter column operated at relatively lowflow velocities, a few picomoles of ribonucleosides can be separated and determined without resorting to radioactive techniques.
THE DETERMINATION of the base composition of nucleic acids is one of the most important steps in sequence analysis. Further progress in this field largely depends on the development of highly sensitive and rapid techniques for quantitative analysis of nucleic acid constituents. High sensitivity is often required for the minute quantities of nucleic acids or nucleic acid fragments that may be available-e.g., by zonal centrifugation or single cell isolation. On the other hand, the need for a large number of analyses can only be overcome by using a fast and reliable method that is amenable to automation. Thus far, attempts to utilize gas chromatography to analyze nucleic acid hydrolyzates, although promising 1 Also Department of Engineering & Applied Science, Yale University.
06510
( I , 2), have not been wholly successful. Thin-layer and paper chromatography (3, 4) are widely used, but quantitative analysis is time consuming and generally inaccurate below the nanomole level (5). Using an elaborate radioactive tracer technique, however, the Randeraths (6, 7) recently achieved base composition determination at the picomole level. Higher sensitivity has been obtained only in the quantitation of ATP by the luciferin-luciferase enzyme system (8). Various column chromatographic methods with gels (9, IO), with a ligand exchange resin ( I I ) , with a liquid ion (1) C. W. Gehrke and C . D. Ruyle, J. Chromatog., 38, 473 (1968). (2) M. Jacobson, J. F. O’Brien, and C . Hedgcoth, Anal. Biochem., 25,363 (1968). (3) K. Randerath and E. Randerath, in “Methods in Enzymology,” Vol. XII, “Nucleic Acids,” Part A., L. Grossman and K. Moldave, Eds., Academic Press, New York, 1967, pp 323-50. (4) G. R. Wyatt, in “The Nucleic Acids Chemistry and Biology,” Vol. I, E. Chargaff and J. N. Davidson, Eds., Academic Press, New York, 1955, pp 243-65. (5) J. M. Gebicki and S . Freed, Anal. Biochem., 14,253 (1966). (6) K. Randerath and E. Randerath, ibid., in press. (7) E. Randerath, J. W. Ten Broeke, and K . Randerath, FEBS Letters, 2, 10 (1968). (8) G. E. Lyman and J. P. DeVincenzo, Anal. Biochem., 21, 435 (1967). (9) I. Mezzasoma and B. Farina, Boll. SOC.Ital. Biol. Sper., 42,1449 (1966). (10) M. Carrara and G. Bernardi, Biochim. Biophys. Acta, 155 (1968). (11) G. Goldstein, Anal. Biochem., 20,477 (1967). VOL. 41, NO. 10,AUGUST 1969
1227
exchanger (12), and with crosslinked poly-N-vinyl pyrrolidine (13), have also been developed for the analysis of nucleic acid hydrolyzates. Yet, ion exchange chromatography, introduced by Cohn ( I d ) , has retained its leading position in this field due to its versatility and reliability. Both anion and cation exchange resins have been employed successfully to separate various nucleic acid constituents. Utilizing the UV adsorbing properties of these compounds, Anderson et a1 (15) devised a continuous column effluent monitoring system which greatly simplified the procedure and gave impetus to a rapid development of the technique. Column efficiency and speed of analysis have been significantly increased by using very fine (particle diameters less than 20 microns) anion or cation exchange resins and high column inlet pressures (16-18). Despite these improvements, however, column chromatography had not been employed for the analysis of nucleic acid constituents at the subnanomole level. It has been shown recently that with small-inside-diameter columns and with a more sensitive detector system, the speed and sensitivity of analysis by ion exchange chromatography (19), can be further increased. Conventional ion exchange resins are, however, not suitable for packing long narrow-bore tubes (i.d. 1 mm or less) and no significant improvement in speed and column efficiency can be expected from such materials with respect to the previously described techniques. Therefore, a new type of ion exchange resin called pellicular ion exchange resin has been developed, which consists of thin shells of ion exchange resin bonded to the surface of glass microspheres. This material can not only be packed very easily into long narrow-bore tubes, but has a number of other favorable chromatographic properties. From chromatographic theory and practice it has been affirmed that high speed and efficiency are achieved at high column inlet pressures in liquid chromatography (20). This need arises from using long columns packed with small particles at high eluent velocities. Partly because of the relationship between pressure and performance, the term high pressure liquid chromatography is used to characterize a technique which is comparable to gas chromatography with respect to both the operation (sensitive detection system, temperature and flow control, long life column, convenience of operation) and the performance (high speed, high efficiency, reproducibility). In this paper we report on the utilization of pellicular cation exchange resins for rapid analysis of subnanomole quantities of the four major bases and nucleosides of RNA with a high pressure liquid chromatographic system. The separation is performed by elution with acidic salt solutions. This elution method for conventional cation exchange resins was developed (12) C. G. Horvath and S. R. Lipsky, Nature, 211, 748 (1966). (13) J. Lerner, T. M. Dougherty, and A. I. Schepartz,J. Chromatog., 37,453 (1968). (14) W. E. Cohn, J. Amer. Chem. SOC.,72, 1471 (1950). (15) N. G. Anderson, J. G. Green, M. L. Barber, and F. C. Ladd, Sr.,Anal. Biochem., 6 , 153 (1963). (16) J. G. Green, C. E. Nunley, and N. G. Anderson, Nut. Cancer Inst. Monogr., 21, 431 (1966). (17) C. D. Scott, J. E. Attril, and N. G. Anderson, Proc. SOC. Exptl. Biol. Med., 125, 181 (1967). (18) M. Uziel, C. K. Koh, and W. E. Cohn, Anal. Biochem., 25, 77 (1968). (19) C. Horvath and S. R. Lipsky, in “Advances in Chromatography 1969,” A. Zlatkis, Ed., Preston Technical Abstract Co., Evanston, 1969, pp 268-75. (20) C. Horvath, B. Preiss, and S. R. Lipsky, ANAL.CHEM.,39, 1422 (1967). (21) C. F. Crampton, F. R. Frankel, A. M. Benson, and A. Wade, Anal. Biochem., 1,249 (1960). 1228
ANALYTICAL CHEMISTRY
by Crampton et a1 (21) using gradient elution, as well as by Uziel et a1(18), by Katz and Comb (22), by Blattner and Erickson ( 2 3 ) , and by Busch (24), using a single eluent. Uziel et a1 (18) investigated a wide range of parameters, and their technique made possible the quantitative determination of the four ribonucleosides at the nanomole level within 1 hour. EXPERIMENTAL
Apparatus. A Picker LCS 1000 nucleic acid analyzer (Picker Nuclear Co., White Plains, N. Y . ) was used. The instrument can be operated at inlet pressures up to 3000 psi. The UV detector of this instrument operates at a 2 5 4 - m ~ wavelength and is equipped with a cylindrical flow cell having 1-mm diameter and 10-mm pathlength. The detector output is linear with respect to the solute concentration according to the manufacturer. A Texas Instrument Recorder with a Disc Integrator was used for obtaining the chromatograms. The recorder span was 0 to 10 mV or 0 to 1 mV; thus, full scale absorbancy ranges at the highest detector sensitivity were from 0 to 0.02 or from 0 to 0.002 absorbancy units, respectively. Columns. Pellicular cation exchanger was prepared by the method described previously (20). A sieve fraction U.S.S. No. 270-325 was used. The analysis of the resin showed 0.59% S, and 0.81% C, and 0.12% H, which indicates that the material contains approximately 1 % by weight of the dry resin. The ion exchange capacity calculated from the sulfur content is 0.185 meq per gram. Columns were made of I-mm i.d. #316 stainless steel tubing. Approximately 150-cm lengths were packed dry under vibration, then the deaerated eluent was pumped through the bed at 200 atm while the column was kept in a perpendicular position. A thin porous Teflon plug was used at the column outlet. Longer columns were assembled with Swagelok unions whose bores were also packed. Chemicals. Reagent grade KHzPO4 or NH4HP04 was used for the preparation of the eluents. The pH of the 0.02M solutions was adjusted with 0.1N KOH or NH40H, respectively, using a Leeds & Northrup pH meter. Samples were prepared with pure nucleosides and bases (Calbiochem), which were dissolved in water or in the eluent. The injected sample volume varied from 2 to 10 pl. Mode of Operation. All experiments were carried out by elution in the constant eluent strength mode. When the eluent was changed, the system was flushed and the column was conditioned for at least 1 hour at high flow rates before making a chromatographic run. Samples were introduced with a 10-pl Hamilton syringe in the intermittent flow mode as follows. The eluent flow was stopped and the pressure was released through the column outlet. The injection port was then opened and the sample was injected with a syringe onto the column. Thereafter, the port was closed and the pumping was started. Evaluation of Chromatograms. In the calculation of retention times and plate numbers, the error introduced by the transient flow regime of the chromatographic run was neglected, Adjusted retention volumes, V’R,(25) were calculated from the retention time, t ~from , the liquid hold up time, to, and from the steady flow rate, F,by the equation VIR = ( t R - t o ) F (1) In the case of asymmetrical peaks or doublets, t~ was calculated from the mode of the concentration curve which was obtained by halving the number of counts (Disc Integrator) related to the total peak area. The retention (22) S. Katz and D. G. Comb, J . Biol. Chem., 238, 3065 (1963). (23) F. R. Blattner and H. P. Erickson, Anal. Biochem., 18, 220 (1967). (24) E. W. Busch, J. Chromatog., 37, 518 (1968). (25) C. Horvath, in “The Practice of Gas Chromatography,’’ L. S. Ettre and A. Zlatkis, Eds., Interscience, New York, 1967, pp 152-61.
.6
time of the small peak, obtained at maximum detector sensitivity by injecting a few microliters of 1 M KH2PO4 into the column, was considered as the liquid hold up time.
1
3.O
RESULTS AND DISCUSSION
The chemical composition of the pellicular resin used in this study was similar to that of the conventional (sulfonated crosslinked polystyrene type) strong cation exchange resins, such as Amberlite IR 120 or Dowex 50. Yet, its unique construction yields properties that are novel in liquid chromatography. In contrast to conventional soft ion-exchange resins, pellicular resins give uniform column packings that are stable at high pressure gradients (20) even with small-bore columns. In addition, changing temperature or eluent composition does not give rise to detrimental effects on column permeability or efficiency. Thus, the effect of these variables as well as that of flow velocity over a wide range can be studied with the same column. The lifetime of the column exceeds 6 months even if flow rate, eluent strength, and temperature are frequently varied. Columns of various diameters can be packed dry with pellicular ion exchange resins. This technique is not only very convenient for making long columns, but it is also superior with respect to column efficiency, as shown by Horne et a1 (26). Generally, columns packed with pellicular resins have higher column efficiency at high flow velocities then those packed with conventional resins (20). Small-bore columns (i.d. 5 1 mm) are particularly suitable for the analysis of minute samples because the high column efficiency and low flow rates make possible the elution of the sample components in a small volume of the effluent. Some of these advantages have been realized in this study involving the rapid determination of the major bases and nucleosides which arise from the hydrolysis of RNA. The bases are uracil (Ura), guanine (Gua), adenine (Ade), and cytosine (Cyt). The nucleosides are uridine (Urd), guanosine (Guo), adenosine (Ado), and cytidine (Cyd). The separation of nucleosides and bases can be achieved with pellicular cation exchange resins under a variety of conditions using the salt elution mode. The most important variables which determine the equilibrium and mass transfer phenomena are as follows: (a) The resin properties, such as shell thickness, structure of the polymer, degree of sulfonation, particle size, etc. (b) The chemical composition of the salt in the eluent. (c) The concentration of the salt solution. (d) The pH of the eluent. (e) The column temperature. (f) The length and diameter of the column. (g) The flow rate. (h) The elution mode-e.g., constant eluent strength or gradient elution. The effect of a number of these variables on the separation of nucleosides on conventional cation exchange resin column has been investigated recently by Uziel et a1 (18). This study was carried out with resin from one batch process and with acidic potassium and ammonium phosphate solutions, although preliminary experiments have shown that other salts, such as ammonium formate, ammonium acetate, and ammonium citrate, also give good separations. The phosphate solutions were selected for their low UV absorbance and because the stainless steel used for columns and for other wetted parts of the instrument was found to be resistant to them under the conditions used. Acidic ammonium formate appears to interact with #316 stainless steel. The pH of a 0.05M ammonium formate solution rose from 5.2 to 7.7 as it was pumped through the system. In addition, (26) D. S. Horne, J. H. Knox, and L. McLaren, Separation Sci.,1, 531 (1966).
L
01
n E
