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RECEIVEDfor review November 6, 1978. Accepted January 29, 1979. This work was accomplished a t the Pittsburgh Energy Technology Center during the summer of 1978 at which time M.L.L. and D.L.V. were Fossil Energy Summer
Research Participants under the administration of the Oak Ridge Associated Universities, University Programs. This work was partially aided by support from the National Science Foundation Grant No. M P S 75-04932 t o one of us (M.N.).
CORRESPONDENCE Reverse Phase Liquid Chromatography at Increased Temperature Sir: T h e recent article by Saner and associates ( I ) , concerning resolution of reverse phase columns a t increased temperature, sparked considerable interest in our laboratory, where we have been using this technique for drug analysis for some time. T h e authors' statement that "elevated temperatures are infrequently utilized as a means of improving resolution since the net improvement is small relative to the improvement gained by particle size reduction" must be investigated further, even though it is basically true. Particle size has for now reached a practical limit at 5 pm because of the tremendous pressure that would be required by smaller particles. Therefore, increasing column temperature is the next logical step toward increased resolution, since the pressure decrease that results will allow increased flow rate. Here we reach the point of interest because increased flow rate at lower mobile phase strength can give greatly enhanced resolution at the same or lower pressure than would be encountered in a room temperature system. However, in the application of temperature it is neither sufficient nor beneficial to heat only the analytical column. An obvious point that is usually neglected is that for maximum efficiency, the mobile phase must be heated to the system temperature before i t enters the column. EXPERIMENTAL Reagents. 3-Bromocinnamamide(I);its N-methyl (II),N-ethyl (111), and N-isopropyl (IV) homologues (Burroughs Wellcome Company, Research Triangle Park, N.C.); and clonazepam (V) (Applied Science Laboratories, Inc., State College, Pa.) were used as received. Apparatus. The liquid chromotograph consisted of a reciprocating piston pump with a pressurized tube pulse dampener, a fixed wavelength UV absorption detector (Laboratory Data Control, Riviera Beach, Fla.) operated at 254 nm, and a six-port sample injection valve with a 50-pL loop (Valco Instruments Company, Houston, Tex.). The columns were 250 mm X 4.6 mm i.d. tubes packed with a 10-pm microparticulate reverse phase (Partisil 10-ODs,Reeve-Angel,Liquid Chromatography Division, Clifton, N.J.) and a 5-pm totally porous spherical reverse phase (Spherisorb ODs, Laboratory Data Control). A 50 mm X 1.09 mm i.d. precolumn was connected directly to the main column inlet fitting and dry-packed in place with a pellicular C-18 phase (Vydac Reverse Phase, Applied Science Laboratories, Inc.) by vibration for 10 min. A 1/16-inchSwagelok union was attached, and the constricted portion was also packed. A small pad of glass wool was placed on top of the packing, and the entire system was connected to the sampling valve. The main columns were heated with a water jacket (Altex Scientific Inc., Berkeley, Calif.). A water jacket was made for the precolumns from a length of 9.5 mm i.d. X 6.4 mm wall Tygon tubing. Water ports were made by boring holes through the wall and inserting short lengths of glass tubing. The ends were sealed at the 1/16-inchnuts with O-rings. R E S U L T S AND D I S C U S S I O N The column-precolumn system described was designed for
maximum chromatographic efficiency. Precolumns that are commercially available a t the present time generally have the same internal diameter as the common analytical columns, 4.6 or 3.2 mm. These require a short length of tubing for connection to the main column. The '/,,-inch (1.09-mm i.d.) precolumn used here can be attached directly to the analytical column and dry-packed in place so that the inlet frit of the main column is the outlet frit of the precolumn. Heating the precolumn is a simple and efficient means of preheating mobile phase. Figure 1 shows chromatograms of a standard mixture containing approximately 0.1 pg/50 p L of compounds I and 11, and 0.2 pg/50 pL of compounds 111, IV, and V. Equivalent samples were injected in each case into the 10-Hm Part,isil ODS column, and chromatograms A, B, and C were obtained with the mobile phase, acetonitri1e:water (22:78), flowing at 120 mL/h. Sample A was run with the column a n d precolumn a t 60 "C, sample B with the column at 60 "C and precolumn a t 25 "C, and sample C with the column and precolumn a t 25 "C. Sample D was run with acetonitri1e:water (16:84) flowing at 160 mL/h and the column and precolumn at 60 "C. Pressures for systems A, B, C, and D were 10.8 M P a (1550 psi), 12.4 MPa (1800 psi), 18.6 M P a (2700 psi), and 14.1 M P a (2050 psi), respectively. Resolution of peaks I1 and V in systems A, C, and D are 0.57, 0.64, and 1.3, respectively, with R calculated simply as separation distance divided by peak width (assumed equal for peaks I1 and V). A similar experiment performed on the 5-pm Spherisorb ODS column with acetonitri1e:water (32:68) flowing at 160 mL/h gave resolution of peaks I1 and V of 1.15 and 1.07 in the room temperature (C) (29.1 MPa, 4230 psi) and the heated (A) (16.1 MPa, 2330 psi) systems, respectively. A run with the precolumn a t 25 "C and the main column a t 60 "C also resulted in almost complete loss of resolution of peaks I1 and V. Retention volumes for the final peaks in chromatograms C and D (10-pm Partisil ODS) are 31.6 and 30.4 mL, respectively. The advantages of the application of temperature in reverse phase liquid chromatography are clearly seen. However, chromatogram B, obtained without preheating t h e mobile phase, is seriously lacking in resolution and peak symmetry. Actually, the peaks are not simply broadened or skewed, but each component elutes as two unresolved bands. This is seen more clearly in Figure 2, which shows the difference in peak shape and retention of a single component (111) injected into the 5-pm Spherisorb ODS column with acetonitri1e:water (32:68) flowing a t 120 mL/h. Peak A was obtained with a heated precolumn and peak B with an unheated precolumn. Symmetry appears to be maintained in this case; however, the splitting of the single component would lead one to think that each compound travels through the analytical column under at least two different sets of conditions. In a very gross sense, a t a particular flow rate, an equilibrium exists in which there
This article not subject to US. Copyright. Published 1979 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 6, MAY 1979
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fFigure 1. Chromatograms of a standard mixture obtained with ackoniblkxwater (22:78/flowing at 120 mL/h, and column (10-pm Partisil ODs) and precolumn at 60 O C (A), column at 60 O C and precolumn at 25 O C (B), and column and precolumn at 25 O C (C). Chromatogram obtained with acetonttri1e:water (16:84)flowing at 160 mL/h, and column and precolumn at 60 O C (D). Compounds are 3-bromocinnamamide (I) and 3-bromo-N-methyl cinnamamide (11) at 0.1 pg/50 pL, and 3-bromo-N4hyl cinnamamide (HI),3-bromo-N-isopropyl cinnamamide (IV), and clonazepam (V) at 0.2 pg/50 pL
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la Ib 2a 2b 30 3 b Figure 3. Chromatograms of identical samples, containing 3bromo-N-methyl cinnamamide (11) and clonazepam (V), run at 40 mL/h ( l ) , 80 mL/h (2), and 120 mL/h (3) with unheated (a) and heated (b) precolumns on 5-pm Spherisorb ODS column
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Flgure 2. Chromatograms of identical samples of 3-bromo-N-ethyl cinnamamide (111) indicating changes in peak shape and retention with heated (A) and unheated (B) precolumn. Analytical column was 5-pm Spherisorb ODs with flow rate of 120 mL/h (acetonitri1e:water(32:68). Retention time for peak A is 9.42 min is a cone of room temperature mobile phase surrounded by cones of higher temperature mobile phase that extend into the column to the point where the mobile phase temperature all the way across the column equals the set temperature. The reduced rates of mobile phase and stationary phase mass transfer in this low temperature area account for the variation in peak shape and retention. The depth of this temperature differential increases with flow rate and column temperature so that at low flow or low temperature or both, the effect may be difficult to detect. Figures 3 and 4 illustrate this flow dependence for the 5-pm Spherisorb ODS column. As shown in Figure 3, identical samples containing components I1 and V were injected at 40, 80, and 120 m L / h with unheated (a) and heated (b) precolumns. Resolution decreases dramatically for the former samples while it is maintained for the latter. Figure 4 shows the variation in peak shape and size with flow rate. Curves A, B, and C were obtained after injections of identical samples of I11 onto the 5-pm Spherisorb column with an unheated precolumn at flow rates of 58,115, and 230 mL/h, respectively. Although retention volume (to center of band) remains approximately the same in the three cases, elution volume increases from 0.76 mL (A) to 1.65 mL (B)to 3.34 mL (C), accounting for the decreased response. In the usual case, as in the b chromatograms of Figure 3, both retention volume
Flgure 4. Chromatograms of identical samples of 3-bromo-N-ethyl cinnamamide (111) run at 58 mL/h (A), 115 mL/h (B), and 230 mL/h (C) on 5-pm Spherisorb ODS wlth unheated precolumn and elution volume are constant with increasing flow rate, so that peak area decreases while peak height remains relatively constant. These data show that, in reverse phase chromatography, the effects of using high temperature with unequilibrated and equilibrated mobile phase are similar for both 5- and 10-pm
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particle columns. From Figure 1 for the 10-pm Partisil ODS column and from the similar data presented for the 5-pm Spherisorb ODS column, we see that in going from a room temperature system to an equilibrated (heated precolumn) high temperature system, there is a slight loss in resolution but a significant reduction in system pressure and retention volume. This allows modification of mobile phase strength and flow rate to give complete resolution (Figure 1D). In the unequilibrated systems (unheated precolumn, Figure l B ) , there is almost complete loss of resolution a t high flow rates. T h e exact flow rate at which these effects become evident would be determined by the mobile phase composition, column temperature, and column configuration (length and diameter) of each system. In the unequilibrated systems used here, the variation in peak shape and resolution appears between 40 and 80 mL/h. In Figure 3, parts l a and b, obtained a t 40 m L / h , resolution are actually greater in the unequilibrated system, indicating that flow is low enough that the mobile phase comes to temperature almost immediately in the main column and that the additional time the solutes spend in the room temperature precolumn and the main column contributes to the separation. However, at 80 m L / h (Figure 2a), the peaks are distorted enough to decrease resolution. Since most reverse phase work is done at flow rates at or above 60 m L / h , it is important that a totally equilibrated system be used. Reverse phase chromatography is a powerful tool; however, in room temperature systems, in which the operational constraints of pressure and time are maximized, much of the power of the medium is wasted and instrumentation can unnecessarily be pushed to its limits. In most cases, selectivity is achieved with a reverse phase column by decreasing mobile
phase strength. The exception occurs infrequently when there is some specific solute-solvent intsraction that contributes to the separation. Barring this occurrence, operation of these columns above room temperature with equilibrated, lowstrength mobile phase at high flow rate will allow separations that are impossible at room temperature because of instrumental or temporal restraints. Any separation that is already possible a t room temperature can be done a t higher temperature in a shorter time, with less consumption of mobile phase, at a lower pressure, and with greater sensitivity. ACKNOWLEDGMENT The authors thank D. M. Mitchell for technical assistance. LITERATURE CITED (1) Saner, W. A.: Jadamic, J. R.; Sager, R. W. Anal. Chem. 1978,50, 749.
Robert J. P e r c h a l s k i * Research Chemist, Research Service (151) Veterans Administration Medical Center Gainesville, Florida 32602 B. J. Wilder Neurology Service Veterans Administration Medical Center Department of Neurology, College of Medicine University of Florida Gainesville, Florida 32602
RECEIVED for review November 6, 1978. Accepted February 5, 1979. This work was supported in part by the Medical Research Service of the Veterans Administration, the Epilepsy Research Foundation of Florida, Inc., and Burroughs-Wellcome Co.
Gas Chromatographic Determination of Glycols at the Parts-per-Million Level in Water by Graphitized Carbon Black Sir: The analysis of very polar compounds, such as glycols and glycerol in particular, is still a problem due to the limited availability of thermally stable, highly polar stationary phases and inert solid supports. Poly(ethy1ene glycol) (PEG) 400 ( I 1, poly(ethy1ene glycol) pentaerythritol adipate (LAC-2R-446) (2),and poly(viny1formal-propionitrile) (3) have been used for the analysis of free glycols. However, in the first two cases no results were reported concerning very dilute solutions of glycols and, in the third case, tailed peaks for glycols were reported. Porous polymers ( 4 , 5 ) have been used in the analysis of diols but, also in this case, the reported procedures were not concerned with t h e determination of part-per-million quantities. An original approach for the analysis of glycols was suggested by Phifer and Plummer (6) who used water as the stationary phase and water vapor as the carrier gas. More recently, tetrahydroxyethylenediamine (THEED)coated Chromosorb W (7) has been used generally for separating light molecular weight diols and specifically for determining traces of 2,3-butanediol in 1,2-propanediolSSevere drawbacks of this method are that a long analysis time is necessary and the column deteriorates rapidly a t the temperature of operation. The object of this paper is to show that the use of THEED as a tailing reducer and selectivity modifier on an inert adsorbing medium such as Carbopack C yields untailed peaks even for a few nanograms of diols and glycerol. The separation 0003-2700/79/0351-0776$01 .OO/O
Table I. Capacity Ratio Data at 115 C THEED percentages compound 0.45 0.60 0.70 0.80 1.0 1,4-butanediol 250 1 4 8 160 185 235 glycerol 850 810 900 1050 1700 of seven diols from 1,2-ethanediolto 1,4-butanediol, including diethylene glycol, in aqueous solution at the level of 10 ppm has been achieved in 1 2 min a t 115 OC by Carbopack C modified with 0.8% THEED. At the column operating temperature, the packing had a good stability. By raising the column temperature to 125 "C, it was possible to chromatograph a water solution containing 50 ppm of glycerol and triethylene glycol with an analysis time of about 30 min. At this column temperature, however, a slow deterioration of the packing occurs. The separating ability of the THEED + Carbopack C system has been investigated by varying the relative amount of THEED. EXPERIMENTAL Carbopack C, which is an example of Graphitized Carbon Black, was supplied in the 80-100 mesh range by Supelco, Bellefonte, Pa. It was ground and sieved to obtain the 1OC-120 mesh range. The procedure of coating Carbopack C was similar to that reported previously (8). Glass columns (55 cm X 0.2 cm id.) were filed with the packing material by following a procedure described elsewhere (8). After this. the columns were conditioned under flow at 115 "C for 24 0 1979 American Chemical Society
