1770
Anal. Chem. iga4, 56, 1770-1773
Experimental Achievement of One Million Theoretical Plates with Microbore Liquid Chromatographic Columns H. G. Menet, P. C. Gareil, and R. H. Ross&* Laboratoire de Chimie Analytique de I'Ecole Sup6rieure de Physique et de Chimie Industrielles de la Ville de Paris 10, rue Vauquelin, 75231 Paris Cedex 05, France
Followlng the theoretical predlctlons, the experimental achievement of 1 X 10' theoretical plates with concatenated packed microbore columns has been demonstrated. Stainless steel columns (1 m X 1 mm 1.d.) were packed with Zorbax slllca gel, nominal dlameter 7-8 pm, by a method described elsewhere. The columns selected for subsequent coupling In serles at least afforded 40 000 theoretlcal plates and a flow resistance parameter around 800. A 22 m long column was obtained which produced 10' plates at flow rates of 13-15 pL.mln-' under an Inlet pressure of 600-700 bar. The reduced plate height was 3.3, the flow resistance parameter was 820, and the separation Impedance was about 9700. To Illustrate the enormous separation power of this column, the chromatogram of a light gasoline sample Is glven, which affords resolution larger than unity for compounds having a selectlvlty coefficient of 1.01-1.02. This column now has been worklng for more than 2 months without performance alteration.
It has been experimentally shown since 1976 ( I ) that very high theoretical plate numbers, and thus, highly efficient liquid chromatographic separations, can be obtained in the exclusion mode with small diameter columns of great length. Subsequently, 1 X lo5 plates and more were also achieved in the normal or reversed phase mode by using concatenated packed stainless steel microbore (1mm i.d.) columns ( 2 , 3 ) ,packed Pyrex glass or fused silica microcolumns (0.2-0.3 mm id.), alone or concatenated (4-IO), and packed (11-13) or open tubular (14-18) capillary columns (30-70 pm i.d.) of several meters length. The main performances of these columns are analyzed in Table I. For the sake of comparison, the best efficiencies reported with classical columns (19,20)are also included in the table. It must be noted that classical columns fail to reach 1 X lo5 plates, unless using a slight mobile phase gradient effect (22),because these columns cannot be packed over 30 cm in length or coupled together without a critical increase in theoretical plate height. As theoretically predicted (23-25),the highest efficiencies experimentally attained up to now were performed with open tubular capillary columns (14, 16, 18), but with this class of columns, plate number is very dependent on capacity factor k '. In addition, an unexpected decrease in N when the linear velocity U increases can be noticed in ref 14: N is reportedly equal to 1.25 X lo6 for U = 0.18 cm-s-l and k' = 0 an falls down to 4.7 X lo6 for U = 0.3 cms-' and k ' = 0, and then to 3.2 X lo6for U = 0.3 c m d and k' = 0.15. The best packed capillary column (13) yields 390000 theoretical plates, but the separation impedance reported, 380, seems questionable since it would lead to a flow resistance parameter 4 of 13;the expected 4 value for this class of column lies between 100 and 300 (23). It is also worth noting the 650 000 plates produced with a 14 m long microbore column ( 2 ) ,but neither the pressure drop nor the separation impedance was cited. Microbore columns afford rather lower plates 0003-2700/84/0356-1770$0 1.50/0
per second than capillary columns but their efficiency remains roughly independent on capacity factors. As announced by Kucera and Manius (3) the plate number limit in this case should be around lo6 with a 18 m long column packed with 8-pm particles and working under 7000 psi (490 bar). The purpose of this paper is to report on the experimental achievement of 1 X lo6 theoretical plates generated by a 22 m long microbore column. EXPERIMENTAL SECTION Column Packing. One meter long microbore columns were made from '/I6 in. o.d., 1 mm i.d. stainless steel tubing. The bottom terminator was a Swagelok female zero dead volume in.-'/l6 in. union fitting (Crawford Fittings) containing a stainless steel frit disk. The columns were packed by the method described in detail in ref 26. The stationary phase was spherical Zorbax B.P. Si1 7-8 pm from Du Pont. The slurry was made in methyl iodide. It was homogenized in an ultrasonic bath for 15 min and then poured into a homemade conical vessel connected to the column inlet. A 1800 bar pressure was applied within 15 s and maintained for 1h. After complete pressure dropping, the column was removed and a plug of porous poly(tetrafluoroethy1ene) (PTFE) was applied on its top. Column Testing. The 1 m long columns were tested by injecting a synthetic mixture of toluene, nitrobenzene, and 1,3dinitrobenzene successively at two flow rates, 100 pLemin-l and 20pL-min-l. Mobile phase was pure Spectrosol quality dichloromethane from S.D.S. (Peypin,France). The plate numbers were calculated by the classical method using the peak width measuremenb at half height, at the flow rate of 20 pl-min-', very close to the flow rate giving maximum efficiency (26). For a better accuracy the pressure drops through the columns were measured at a flow rate of 100 pLemin-l. The 1m long columns were considered to be suitable for further column coupling, if they produced between 40 000 and 50 000 plates and if their pressure drop lies in the range 120-150 bar at a flow rate of 100 pL-mid. The reduced plate height, flow resistance parameter, and separation impedance of the selected columns are given in Table 11. Liquid Chromatographic System. Throughout this work, the chromatograph used for column operation was composed of a Gilson 303 alternating pump having a maximum output pressure of 700 bar and a minimum flow rate of 0.5 pL-min-' (Gilson Biomedical Electronics, Villiers-le-Bel,France), a Rheodyne 7413 sampling valve fitted with 0.5 pL or 1 pL internal loop, and a Kratos 773 spectrophotometric detector fitted with a 0.5-pL micro flow cell. Column Coupling. All the 1m long columns were cut about 5 cm from their lower end. It was ascertained that permeability can thus be increased, probably because some fine particles appear during high pressure packing. Then the stainless steel frit disk in. union fitting were replaced, which, at the and the 1/16 expense of permeability, provides a better stationary phase stability and allows the disassembly of the component columns with little hazard of damage. The inlet of one column was then concatenated to the outlet of another one using a Swagelok male nut and ferrule. In this way, the upper end of the stationary phase bed of each column was only protected by the stainless steel frit disk of the preceding column. Porous PTFE plugs are not advised here, since they crush and partially clog under very high pressure, which decreases the overall permeability. Likewise 23 columns 0 1984 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
1771
Table I. Experimental Performances of Columns Generating More Than lo6 Theoretical Plates" column no. X length X i.d.
4, pm
phase system
P,
ref
N
bar
k'
to, h
N/to,* plates/s
0.14 0.28 3.3 9.0 3.3
20 17
E
h*
9
135 84
2.2
1110*
5400*
21
2.0 4.3 2.8
1400*
11000*
315* 990 650* 500*
2800* 7200* 3150* 6100*
Classical Columns 3 X 25 cm 9 X 15 cm
X X
0.46 cm 0.46 cm
5 5
R.P. R.P.
20 19
68000 85000
270
6 3
3.2
Packed Microbore Columns 10 X 1 m X 1 mm 14 X 1 m X 1 mm 9 X 0.5 m X 1 mm
20 5 8
E A R.P.
250 000 650000 200000
1 2
3
0 0
310
0-5
Packed Pyrex Glass or Fused Silica Microcolumns 1 m X 0.2 mm 1.1 m X 0.24 mm 5 X 1 m X 0.2 mm 8 X 0.5 m X 0.33 m
3 3 10
5
R.P. R.P. R.P. E
4,8 3 8 5-7
A R.P. A
12 11
110000 135000
200
230000 230000
100 80
6 0.55 0
0.29 0.5 2.7 6
104 11
3.0 2.7 2.2 3.5
0.75 1.50 4.07
37 28 27
8 2.6 6.8
50* 230* 13*
3200* 1600* 580
2.6 0.67 7.0 6.4 8.25 8.25
17 140 50 120 40 40
7.6 2.6 0.3 0.3 2.3 10
19*
1100*
75 24
Packed Capillary Columns 8 m X 40 pm 27 m X 70 pm 26.4m X 70 pm
10
30 10
100000
150000 390000
13
40 200 20*
0.56 0 0
Open Tubular Capillary Columns 15 17 14
N.P. N.P. N.P. A A
34.6m X 28.5 pm 30.6m X 34 pm 21 m X 60 pm 27.5m X 32 pm 105 m X 39 pm
16
18
160000 340 000 1250 000 2800000 1170000 270 000
10
1.2 0.02 0 0 0
3
d,, particle diameter; N , plate number; AP,pressure drop; k', capacity ratio; to,dead time; h, reduced plate height; 9, flow resistance parameter; E, separation impedance according to Bristow et al. (21). An asterisk indicates the values or the parameters are calculated from other reported data. Phase system: R.P., reversed phase; A, adsorption; E, exclusion; N.P., normal phase.
Table 11. Main Chromatographic Parameters of the 1 m Long Microbore Columns Selected for Coupling in Series and of the Resulting 22 m Long Column" columns
h
4
E
1 m X 1 mm i.d. 22 m X 1 mm i.d.
2.7-3.3 3.2
550-680 820
5500 9700
"Experimental conditions are given in Figure 1. Key: h, reduced plate height; 9, flow resistance parameter; E = h24,sepamation imDedance. were coupled in series, which corresponds to a total length of 22 m.
RESULTS Achievement of Ultrahigh Efficiencies. The 22 m long microbore column was first operated with dichloromethane as mobile phase and a toluene sample was injected. The flow rate, measured a t the detector outlet with a 250-pL glass syringe, was 15 pl-min-l and the inlet pressure was 600 bar. The chromatographic peak shown in Figure 1exhibits 920000 theoretical plates. Its asymmetry fador, measured as the ratio of peak half widths at 10% peak height, was about 1.35. This slight asymmetry may come from the effects of pressure and temperature gradients on the viscosity and compressibility of the mobile phase and on the diffusivity and rate constant of the solute. The h value of 3.2 (Table 11) shows that there was no loss in the additivity of theoretical plates. The 4 value is higher than that of the component columns but the measurement at 15 pL.min-l is less favorable since the contribution of stainless steel frit disks to flow resistance seems higher at low flow rate. As a comparison, the $t and E values calculated from Scott's data (3)were 1400 and 11O00, respectively (Table 1). Figure 2 shows the test mixture separations obtained with the same phase system but with microbore columns 1m and
-
0
k
,
1080
1090
e time(min)
Flgure 1. Chromatographic peak of toluene on a 22 m X 1 mm microbore column: stationary phase, Zorbax B.P. Sil. 7-8 pm; mobile phase, dichloromethane; flow rate, 15 pL-min-'; Inlet pressure, 600 bar: N = 920000.
22 m in length. The plate numbers were around lo4and lo6, respectively. So, the increase in resolution was of 1order of magnitude. The sample dilution is expected to increase as the square root of column length. Owing to the high flow rate used with the 1m long column, dilution here increases by a factor of 2 only. The separation of a light gasoline sample previously studied on a 2 m long column producing 80 000 plates (26)(Figure 3) was achieved successively on columns of 8 m and 22 m in length. Figure 4 shows a significant part of the chromatograms obtained. I t can be noted that the major peak eluting from the 2 m long column at retention time 110 min (Figure 3) in fact was comprised of two compounds having a selectivity factor a = 1.016. The efficiency for these peaks was about 6.7 X lo6 plates and resolution was about 1.6. Likewise, the
1772
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
Y
J
* 10
11
time ihl
10
A
‘c;1.010
ci: 1.011
15
-
timi(hl
E
Flgure 4. Separatlon of a light gasoline sample on 1 mm i.d. microbore columns (phase system as In Figure 1): (A) column length, 8 m; flow rate, 20 pL-min-‘; (B) column length, 22 m; flow rate, 15 pL.min-’.
r p p r o x . 9 9
m
6
1
2
3
4
5 time (min)
5
0
10
A
15
20
25 time(h)
6
Flgure 2. Separation of a test mixture by adsorptlon chromatography on 1 mm i.d. microbore columns (phase system as in Figure 1): (A) column length, 1 m; flow rate, 150 pL.min-’; N = 10000; (B) column length, 22 m; flow rate, 15 pL.mln-’; N = 900000.
i T
3
Capacity ratio
N277000
‘j2
% ,
50
100
10
20
30
Tim.(h)
Figure 5. Separation of a synthetic mixture on the 22 m X 1 mm i.d. column working under an Inlet pressure of 600-700 bar for 2 months: mobile phase, dichloromethane-ethanol-water (98/2/0.1) (v/v); flow rate, 15 pL-min-’; inlet pressure, 700 bar; solutes (1) toluene, (2) nitrobenzene, (3) indole, (4) 1,3-dinitrobenzene, (5) methyl benzoate, (6) benzaldehyde, (7) impurity, (8) acetophenone.
600-700 bar. The plate numbers produced are indicated on the figure. It can be seen that 1 x IO6theoretical plates were reached for an impurity peak (capacity factor 1.3). To conclude, the overall column efficiency could be considered as constant and the stationary phase bed steady over this period of time. As theoretically predicted and then experimentally announced by Scott et al. (2, 3), microbore concatenated columns can actually cope with intricate separation problems requiring efficiencies around 1 X lo6 theoretical plates.
024
Nz85000
0
l
ACKNOWLEDGMENT J. M. Colin, S. Thiault, J. Goupy, J. Grosmangin, and P. Vercier from the Compagnie FranCaise de Raffinage, are gratefully acknowledged for their interest in this work.
LITERATURE CITED *
I50
Flgure 3. Separatlon of a llght gasoline sample on a 2 m X 1 mm 1.d. column by adsorption chromatography. The phase system used Is given in Figure 1. Flow rate is 25 pL-min-’. The vertical dashed lines bound the chromatogram reglon detailed In Figure 4.
pair of peaks having a selectivity factor of 1.024 in Figure 3 appeared to include three major compounds, whose selectivity factors are 1.011 and 1.020. Finally, Figure 5 shows the separation of a synthetic mixture after 2 months of operation of the 22 m long column a t
(1) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1976, 125, 251-263. (2) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1979, 169, 51-72. (3) Kucera, P.; Manius, G. J. Chromatogr. 1981, 216, 9-21. (4) Yang, F. J. J. Chromatogr. 1982, 236, 265-277. (5) Takeuchi, T.; Ishii, D.J. Chromatogr. 1982, 238, 409-418. (6) Ishii, D.;Takeuchi, T. J. Chromatogr. 1983, 255, 349-358. (7) Takeuchi, T.; Ishii, D.;Mori, S. J. Chromatogr. 1983, 257, 327-335. (8) Hirata, Y.; Jinno, K. HRC CC, J . Hlgh Resolut. Chromatogr. Chroma-
togr. Common. 1983, 6 , 196-199. (9) Gluckman, J. C.;Hirose, A.; Mc Guffin, V. L.; Novotny, M. Chromatographla 1983. 17, 303-309. (IO) Yang, F. J. J. Chromatcgr. Scl., in press. (11) Hirata, Y.; Novotny, M.; Tsuda, T.; Ishii, D. Anal. Chem. 1979, 51, 1807-1809. (12) Tsuda, T.; Tanaka, I . ; Nakagawa, G. J. Chromatogr. 1982, 239, 507-513. (13) Mc Guffin, V. L.; Novotny, M. J. Chromafogr. 1983, 255, 381-393. (14) Krejci, M.; Tesarik. K.; Pajurek, J. J. Chromatogr. 1980, 791, 17-23. (15) Tsuda, T.; Nakagawa, G. J. Chromatogr. 1980, 199, 249-258.
1773
Anal. Chem. 1984, 56,1773-1777 (18) (17) (18) (19) (20) (21) (22) (23)
Tijssen, R.; Bleumer, J.
P. A,; Smlt, A. L. C.; Van Kreveld, M. E. J . Chromatogr. 1981, 278, 137-185. Takeuchi, T.; Ishll, D. J . Chrometogr. 1982, 240, 51-80. Kucera, P.; Gulochon, G. J . Chromatogr. 1984, 283, 1-20. Snyder, L. R.; Dolan, J. W.; Van Der Wall, S. J . Chromatogr. 1981, 203,3-17. Verzele, M.; Dewaele, C. HRC CC, J . H7gh Resdut. Chromtogr. Chromatogr. Commun. 1982, 5 , 245-249. Bristow, P. A,; Knox, J. H. Chromatographia 1977, IO, 279-289. Cooke, N.; Olsen, K. J . Chromatogr. Sci. 1980, 18, 512-524. Knox, J. H. J . Chromatogr. S d . 1980, 78, 453-481.
(24) Guiochon, G. Anal. Chem. 1981, 53,1318-1325. (25) Gulochon, 0. J . Chromatogr. 1979, 785,3-26. (28) Menet, H.; Gareii, P.; Caude, M.; Rosset, R. Chromatographla 1984, 18, 73-80.
RECEIVED for review February 14,1984. Accepted May 1,1984. The authors acknowledge financial support from Compagnie Franqaise de Raffinage (Paris et Le Havre, France).
Solvent Selectivity in the Liquid Chromatographic Separation of Polystyrene Oligomers on Silica T.H.Mourey* and G. A. Smith Research Laboratories, Eastman Kodak Company, Rochester, New York 14650
L. R. Snyder 2281 William Court, Yorktown Heights, New York 10598
Narrow-molecular-welght polystyrene standards wlth welght-average molecular welghts of 800, 2100, and 4800 were separated on 6 and 50 nm pore diameter slllca wlth n-hexaneltetrahydrofuran, n-hexanehthyl acetate, and nhexane/dlchloromethane gradients. Tetrahydrofuran and ethyl acetate eluents gave separatlons accordlng to the number of ollgomer unlfs, and dlchloromethane eluents further separated the stereoisomers of Individual ollgo-mers. Selectlvlty dlfferences In the oligomer separatlons are dlscussed In terms of solvent localization and preferred solute conlormatlon.
liquid chromatography. Solvent selectivity in reversed-phase separation of oligostyrene stereoisomers has been explained in terms of oligomer solubility in the mobile phase and solvent-induced changes in the conformations of stereoisomers and long-chain hydrocarbon bonded phases (6);however, the subtleties of solvent selectivity in normal phase separations of oligomers cannot be unambiguously elucidated from the reversed-phase case. Our objective is to help identify the sources of solvent selectivity in the adsorption chromatography of oligostyrenes and thus optimize oligomer separations.
EXPERIMENTAL SECTION Oligomers are loosely defined by the polymer chemist as compounds consisting of a series of repeat units whose molecular weights total less than -10000. This definition is broad enough to describe the molecular weight region in which small-organic-molecule character disappears and measurable polymer physical properties become evident. Analysis of oligomer molecular weight, constitutional, and configurational distributions has been a long-standing challenge to separation scientists, primarily because of limited molecular weight ranges of traditional separation techniques. Gas chromatography is restricted to volatile oligomers with few repeat units, and the limited peak capacity and resolution in size-exclusion chromatography (SEC) is usually insufficient to adequately separate oligomers on the basis of compositional, constitutional, or configurational differences. Numerous papers on the separation of oligomers have clearly demonstrated the difficulties encountered in this transition region. Low-molecular-weightpolystyrene has been fractionated by recycle SEC, and the isolated oligomers have been characterized by nuclear magnetic resonance (NMR) spectrometry (1-3). This technique is time-consuming and inefficient. Until recently, supercritical fluid chromatography suffered from long separation times as well as complicated, expensive equipment; however, separation of up to 42 styrene oligomers has been reported (4, 5). Several workers have investigated reversed-phase (6-10) and adsorption chromatography (9-12) on microparticulate media as time-saving alternatives. Rapid, efficient separations of several oligomers have been obtained from both modes of high-performance 0003-2700/84/0356-1773$01.50/0
Narrow-molecular-weightpolystyrene oligomer samples with weight-average molecular weights of 800, 2100, and 4800 were obtained from Pressure Chemicals (Pittsburgh, PA). Samples were dissolved in 3:l n-hexane/B solvent, where solvent B was dichloromethane, tetrahydrofuran (THF), or ethyl acetate. Sample concentrations were 3.3-100 mg/mL. The samples (10 rL) were injected onto a 4.6 mm i.d. X 250 mm column packed with either LiChrosorb Si60 silica (E. Merck, 5-km particle diameter) or Hibar I1 LiChrospher Si500 (10-pm particle diameter). Nominal pore diameters are 6 and 50 nm, respectively, for Si60 and Si500. The LiChrosorb Si60 column was packed by the stirred-slurry method, and the Hibar I1 column was obtained commercially. Both columns were thermostated at 30.00 k 0.05 OC. Polystyrene oligomers were gradient eluted by use of either two Waters Associates M6000A pumps with a 720 system controller or a Varian 5060 liquid chromatograph. Ultraviolet absorbance of the eluent was monitored at 265 nm with a PerkinElmer LC-55 variable-wavelength detector. Stereoisomerswere semipreparatively separated in two steps. Oligomers 1-6 of polystyrene-800were isolated individually by separations on a 10 mm i d . X 500 mm column of 15-25 pm LiChroprep Si60 (E. Merck). A dichloromethane solvent gradient programmed beginning at 92/8 (v/v) n-hexane/dichloromethane, at a 0.3%/mL dichloromethane rate of increase, gave base line separation of the first six oligomers. Three injections of 500 mg in 1mL were made, and each oligomer from 1to 6 was collected, and the fractions of each of the three separations were combined and blown to dryness with nitrogen. Stereoisomers in each oligomer fraction were then separated isocratically on a Partisil M-9 (Whatman) 10 mm i.d. X 250 mm column by using n-hexane/ dichloromethane binary eluents at a flow rate of 4.2 mL/min. Oligomer injections of 20 mg/100 pL were repeated until -20 mg of each separable isomer was collected. 0 1984 Arnerlcan Chemical Soclety
