Large Diameter Columns for Preparative Scale High Speed Liquid Chromatography John P. Wolf Ill E. 1. Du Pont de Nemours & Co., Inc., lnstrument Products Division, Wilmington, Del. 79898
Preparative scale columns for high speed liquid chromatography in the size range of 0.77- to 2.36-cm i.d. by 50 cm long are shown to be as much as a factor of 4 more efficient than the typical, small bore analytical column configuration. A linear relation is found between the square root of the column inner diameter and the number of theoretical plates for these columns when they are used at constant linear carrier velocity. Gaussian peak shapes are obtained with sample size proportional to column cross-sectional area. Very large samples result in flat-topped peaks but there is little or no tailing.
The potential of pressurized liquid chromatographic techniques has been, in general, only partially realized. The majority of current practice is concerned with its analytic capabilities and the use of long, narrow bore columns for the qualitative and quantitative characterization of less than microgram quantities of samples. The scale-up of chromatographic systems to rectify complex samples in gram sizes has not been investigated with the same vigor and detail as the analytical application. It has been shown ( I ) that columns can be enlarged to 10.9-mm i.d. with no loss and sometimes actually with an improvement in efficiency. In addition, the efficiencies of 7.9-mm i.d. columns were evaluated as a function of packing particle diameter ( 2 ) . Other workers have shown that large columns can be constructed that have efficiencies comparable to analytical columns (3, 4 ) . The purpose of this report is to show that large bore columns up to 23.6-mm i.d. do have improved operating performance relative t o analytical columns, theory and practice notwithstanding (5-8).
EXPERIMENTAL A Du Pont Liquid Chromatograph, Model 820, was used in this study with the standard UV photometric detector. Permaphase ODS packing was used as the test packing. A 1-kg batch was homogenized by extensive tumbling in a partially filled glass bottle. Particle diameter ranged from 10 to 40 l m (minus 400 mesh sieve). The carrier was 25% distilled water in methanol (Fisher Spectranalyzed) prepared by volume. All columns and fitting were made from 316 stainless steel. Column tubing, except for the analytical column, was seamless with wall thickness of 0.89 mm and 0.50 m long. Tubing diameters ( 1 ) J. J. DeStefano and H. C. Beachell. J. Chromatogr. Sci., 8, 434 (1970). (2) H . C . Beachell and J. J. DeStefano, J , Chromatogr. Sci., 10, 481 (1 970). (3) S. T. Sie and N. van denHoed, J. Chrornatogr. Sci., 7, 257 (1969). ( 4 ) J. H . Knoxand J. F. Parcher, Anal. Chem., 41, 1599 (1969). (5) L. R. Snyder, Anal. Chem., 39,698 (1967). (6) H. N. M . Stewart, R. Amos, and S. G . Perry, J. Chromatogr., 38, 209 (1968), ( 7 ) R. P. W . Scott, D. W. J. Blackburn. and T. Wilkins, "Advances in Gas Chromatography," A. Zlatkis, E d . , Preston Technical Abstracts Co., Evanston, Ill., 1967, p 164. (8) D. S. Horne. J. H . Knox, and L. McLaren. Separation Sci., 1, 531
(1966).
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Table I. Physical and Chromatographic Properties of Test Columns Packing Colweight, Flow, ml/ u m n o.d., in. i.d., cm Area, cm2 grams mina A 0 C
'/d
3/a '12
D E
?/a
F
1.0
3/4
0.21 0.77 1.09 1.41 1.71 2.36
0.036 0.475 0.938 1.57 2.52 4.39
1.4 38.2 74.5 120.4 184.6 338.7
0.52 6.82 16.3 22.6 32.0 65.0
nb
600 1325 1550 1800 1900 2350
=Measured at constant carrier velocity of 1.4 cm sec-' using inlet pressures from 500-660 psig. *Taken from the phenanthrene peak (k' = 0.81) at a carrier velocity of 1.4 cm sec-'. Length of packing bed in column was 0.5 m in all cases. were selected that gave roughly a doubling of cross sectional area with each increase in internal diameter. The end fittings were fabricated from Swagelok caps (Crawford Fitting Co., Solon, Ohio). The cap portion was machined on the inside to give a constant i.d. terminated by a flat surface. The cap was then bored through axially to an interference fit with a short (extension of 25 mm) length of 2.1-mm i.d. by 6.35-mm 0.d. tubing. The terminal tubing was pressed in place such that its end was flush with the machined inner face of the cap. The assembly was then welded together. Another cut 0.12 mm deep having the i.d. of the column was taken off the fitting inner face to provide a small volume for carrier distribution across the entire column cross section. A cross-section of the fitting design is shown in Figure 1. The outlet end of the column was assembled by placing a sintered disk of 316 stainless steel 1.6 mm thick, 2-micron porosity and diameter of the column tubing (Mott Metallurgical Corp., Farmington, Conn.) in the machined recess, then inserting the tubing and tightening up the assembly as described by the Swagelok catalogs. Columns were loaded from the inlet end by adding packing in small increments while tapping the assembly on a block to consolidate the packing bed. When the tube was full, the bed was wiped flush with the tubing end using a straight-edge. The inlet fitting was then installed, made u p with a 10-micron porosity sintered disk. The end fittings provided a transition from the large column to the 0.d. of standard analytical columns and thus allowed for normal installation of the large diameter columns in the Du Pont 820 Liquid Chromatograph. Column performance was evaluated with a standard sample of mixed polynuclear hydrocarbous dissolved in methanol. The components were naphthalene, phenanthrene, fluoranthene, pyrene, and chrysene which had k' (s) of 0.28, 0.31, 1.33, 1.62, and 2.78, respectively, when the mixture was chromatogrammed on the analytical column.
RESULTS AND DISCUSSION The two factors of principal concern when the subject of large diameter chromatographic columns is considered are efficiency and capacity. They are not independent aspects of column performance but, for convenience, they will be discussed separately. The physical and chromatographic properties of the test columns are given in Table I. Column A is a commonly used size and configuration for analytical applications and
1
Figure 1. Schematic cross section of column end fitting with
tubing and sintered disk in place
i A
G
c
F
1
Figure 2. Chromatograms of test mixture on columns A , 6,and F with sample sizes of 1 , 10, and 100 PI, respectively.
has been included to provide a reference point for discussing the performance of the large diameter columns. Linear regression analysis of the relation between column areas and packing weights for all six sizes shows an excellent straight line fit having a correlation coefficient of 0.999 and per cent deviation from linearity of 4.2 X 10-2. The relation between flows and cross sectional areas is also linear with a correlation coefficient of 0.998 and per cent deviation from linearity of 0.35. These correlations require that the packing beds in the various columns have the same average densities and permeabilities. The column to column correspondence implies uniform and reproducible loading of the packing beds. This was verified by emptying columns C and D after they had been evaluated and repeating the experiment with them. The results differed from the original data by less than 2.3% for Column C and 1.1% for Column D. Figure 2 is a representation of chromatograms of the test mixture obtained with columns A, B, and F. There is an improvement in efficiency with increase in column diameter. Sample volumes were increased in approximate proportion with flow. All columns were then tested with what could be called a flow-proportional analytical sample and the number of theoretical plates per column, n, was determined by measurements on the phenanthrene peak 0.81). These values are tabulated in Table I. (K’ Figure 2 suggests that there might be a relation between column area and n. A plot of log ( n ) us. log (area) was made and found to be reasonably well fit by a straight line having a slope of 0.25. This requires that there be a simple relation between n and the square root of the column i.d. A linear regression analysis of n as a function of (i.d.)1’2 gave a straight line fit having a correlation coefficient of 0.998 and a per cent deviation from linearity of 0.41. The best fit is given by Equation 1with i.d. in mm. n = 500 (i.d.)1’2 - 107
(1)
Photometric detector at A X32, carrier velocity of 1.4 c m s e c - ’ and operation at room temperature
An interesting aspect of Equation 1 is that it predicts that a column having an i.d. approximately equal to 2 packing particle diameters or less will have no theoretical plates. It is obvious that Equation 1 cannot be valid for indefinite extrapolation to larger values of (i.d.)I 2 since experience indicates that there is an upper limit to the efficiency of a chromatographic column. Because Equation 1 is empirical, it is very likely to give meaningless results outside the range of the original data, and should be used and discussed only with the appropriate caueat. Within the range of the data, Equation, 1 suggests that there is a relation between the HETP and a physical characteristic of the packing bed which is yet to be identified. One hypothesis which has been advanced to account for this effect ( 1 ) is that these large diameter columns are “infinite diameter” and thus wall effects which tend to degrade performance are attenuated with increase in column diameter. This theory cannot account for the data reported here; the reason being that for a column to be operable in the “infinite diameter” mode, it is necessary that the sample be injected a t a point on the column axis. The column end-fitting was designed to transpose a slug of solute in the carrier stream into a uniform disk over the entire cross section of the packing and vice rersa. The actual sample distribution and flow pattern were determined by the following experiment. After reevaluating column D, it was opened a t the inlet end, the packing removed to a depth of 3 cm, the column refilled with Zipax chromatographic support, and reassembled. The column was conditioned by pumping with distilled water at 500 psig. When a stable base line was obtained, a 40-111 sample of 0.5% crystal violet in distilled water was introduced by syringe through the injection port. The carrier flow was ANALYTICAL CHEMISTRY, VOL. 4 5 , NO. 7 , JUNE 1973
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maintained for 5 minutes, then the column was removed from the chromatograph and the inlet end reopened. The packing was dissected to expose a cross-section of the packing to a depth of 2.5 cm. It was colored to a deep blue because of the adsorbed crystal violet sample. The colored band extended uniformly through the bed to a depth of about 1 cm and uniformly along the diameter of the column. This form of sample application excludes the “infinite diameter” effect as an admissible explanation for the results obtained with these columns. Values of H E T P us. linear carrier velocity were obtained for columns A, B, and F, using phenanthrene as the test solute. The data, when plotted, gave the usual smooth, concave downward curves and did not show any unusual characteristics. The maximum sample size that a given column can handle would be a valuable measure of column performance. Unfortunately, it is impossible to specify such a quantity based only on column size or packing bed volume. It does seem reasonable to expect that if a small column with a packing bed of n cm3 and having y plates could rectify a sample of z grams, then an equally efficient column of n2x cm3 and similar configuration should be able to rectify a sample of nz grams. This hypothesis was tested and found to be valid for columns A, B, and F. A difficulty was encountered that precludes giving quantitative data for the upper limits of sample capacity study. The detector cell in the Du Pont 820 Liquid Chromatograph was the standard analytical size with an 8-mm
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optical path length. All of the components in the test mixture have molar absorbtivities greater than 15,000 and drove the photometer to full response a t fairly dilute concentration levels. As concentrations were increased, the shapes of the component peaks remained symmetrical but were truncated a t ca. 35% of full scale a t maximum attenuation. Another effect was observed which restricted the amount of sample that could be put on the column. At a sample concentration of 2.0% phenanthrene in methanol, the flow through column B was greatly reduced when an injection of 50 p1 was applied to the column. In addition, the sample peak shape degraded markedly into an irregular, very tailed form. This effect was caused by precipitation of the sample a t the inlet of the column. Peak symmetry and constant flow were regained when the sample was diluted to 500 gl with methanol and then injected onto the column. In this system, it is apparent that the amount of sample that can be applied to the column is controlled by the solubility of the sample in the carrier. In spite of the very low solubility limits of the compounds in the aqueous methanol used in this study, it is still possible by enlarging the column diameter to substantially increase the sample size that can be accommodated per chromatogram. Received for review November 29, 1972. Accepted February 15, 1973.
