CORRESPONDENCE Heavy Loaded Columns in Liquid Chromatography SIR: It has been found experimentally that the porosity, e, of a given support is equivalent in gas and high speed liquid chromatography ( I ) . The porosity was determined from the measured flow rate and the hold-up time of a n inert peak. In addition, it has been found that Porasil A (Waters Associates, Framingham, Mass., specific surface area ca. 350 m2/g) can be coated with a nonvolatile liquid phase up to at least 5 0 z w/w (i.e. 1 gram of support coated with 1 gram of liquid phase) and still be free-flowing ( 2 ) . The peak broadening from such a column was not excessive in gas chromatography. At the present time, a major deterrent to the general acceptance of high speed liquid-liquid chromatography is the problem of bleeding of the column. This problem is especially severe if the coating is less than 5 %. Consequently, in the following, we describe some work with heavily loaded columns in high speed chromatography. The chromatographic apparatus has been previously described (3). Only the injection port was replaced with one for injection at pressures over 100 atmospheres ( 4 ) . The samples were injected with 5-pl syringe (Hamilton Company, Whittier, Calif. ; No. HP 305N). Porasil A (38-53 p) was coated with 5 0 z w/w P,P‘-oxydipropionitrile (ODPN) using methylene chloride as solvent. The column (i.d. 2 mm; 50 cm in length) was packed dry with the support. The recycled mobile phase was n-heptane, sat-
,b
ob
I
20
I
I
30 40 u [cm/sec]
I
I
I
50
60
70
Figure 2. Height equivalent to a theoretical plate as a function of linear velocity Column characteristics are identical as in Figure 1 A . Toluene (k’ = 0) B . Acetophenone (5) C. Dimethylphthalate (23) D. Benzyl alcohol (51) E. Phenol (180)
(1) G. Deininger and I. Halfisz, “Adounces in Chromatography, Miami 1970” A. Zlatkis, Ed., University of Houston, Texas, 1970, p 336. (To be published in J . Clzrornatogr. Sci.) (2) J. Asshauer, University of Frankfurt, unpublished results, 1970. (3) ~, B. L. Karger, K. Conroe. and H. Engelhardt, J . Chrornatogr. Sci., 8, 242(1970). (4) I. Hal& A. Kroneisen, H. Gerlach, and P. Walking, 2. Anal. Chem., 234,97 (1968).
Quinoline ( k ’ * 8)
TIME [minutes]
Figure 3. Separation on heavy loaded column Column characteristics are identical as in Figure 1 Linear velocity: 5.5 cm/sec Pressure drop: 53 atm Sample size: 50 pg
-
4I
I
I
I
I
I
Table I.
Empirical Constants for h = A
k’ A. Toluene
B. Acetophenone C. Dimethylphthalate D. Benzyl alcohol E. Phenol
1460
I
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
0 5
4- Cu
A , cm
c, sec
0.10 0.10
0.19 0.16
23
0.27
51
0.20
180
0.11
0.055
0.027 0.027
AcebDh8none
TIME [minutes]
Figure 4.
Separation on heavy loaded column
Column characteristics are identical as in Figure 1 Linear velocity: 5.8 cm/sec Sample size: 1 mg
urated with ODPN. The permeability of the column was cm2 and the porosity was 0.5. From this latter 2 X value it can be calculated that at least 7 0 x of the pore volume was filled with stationary liquid phase. As shown in Figure 1, the unusually high capacity ratios (k' = 5-180) are independent of the linear velocity, u, up to 5.4 cmisec. In Figure 2, the height equivalent to a theoretical plate, h, is plotted us. u for five compounds. The retention time of toluene and acetophenone (k' = 0 and 5 , respectively) was very short at high velocities; consequently, the error in measuring h and u are relatively high for these two compounds. If the velocity is greater than 1.6 cmisec, the h us. u plot is linear. The constants for the empirical equation h = A Cu are given in Table I. It is to be noted that the A term in this equation is large, and the C term is quite low. The value of the C constant decreases with increasing capacity ratio. The low values for the C term arise from the fact that stationary liquid phase replaces stagnant mobile phase in the pores. Since the diffusion coefficients of the sample components are to a first approximation equal in the mobile and stationary phases, mass transfer in and out of the pores can be considered to be roughly constant, independent of the relative amount of each phase in the pore. On the other hand, retention will occur in the stationary phase, thus slowing the travel of the sample components. Local nonequilibrium can be considered to be less in such a case, with a resulting smaller value for the C term. Since the equation is valid only over a restricted velocity range, the A term will not be discussed. Plotting h/u us. u, it can be shown that optimum velocity is about 2-2.5 cmjsec. Increasing the velocity over this optimum value results in increasing pressure drop without any commensurate decrease in the time of analysis. In Figures 3 and 4, the separations of two mixtures are shown using this heavily loaded column. Note that 5.8 cm/
+
sec linear velocity is achieved with only 54 atm pressure drop, because of the short column length. Because of the small porosity (E = 0.5) of the column packed with the heavily loaded porous support (a porosity comparable with that of columns packed with porous layer beads), higher linear velocities can be achieved with relatively low flow rates for a given column diameter. The retentions were reproducible, with recycling of the saturated mobile phase; consequently, bleeding is not a significant problem with the heavily loaded column and the retention times are reproducible. Another consequence of the heavy loading is that sample sizes up to at least 1 mg can be injected (i.d :2 mm) without affecting peak broadening. In addition because of the high capacity ratios, the peak capacity is great, Le., multicomponent samples can be resolved. A disadvantage of the described column is that in preparative scale work, the collected fractions contain not only the sample and the eluent, but a small amount of stationary phase as well. This disadvantage is typical, however, for liquidliquid chromatography. I. HALLSZ' H. ENGLEHARDT J. ASSHAUER' B. L. KARGER Department of Chemistry Northeastern University Boston, Mass. 02115 1 Permanent address, Institute of Physical Chemistry, The University of Frankfurt, Frankfurt/Main, Germany.
RECEIVED for review June 15, 1970. Accepted July 27, 1970. Work supported by N I H Grant 1 ROI G M 15847 and the Deutsche Forschungsgemeinschaft.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 12, OCTOBER 1970
1461
