Anal. Chem. 1984, 56,1249-1252
1249
Packed Microcapillary Columns with IO-pm Silica Gel for Liquid Chromatography Takao Tsuda,* Isao Tanaka, a n d Genkichi Nakagawa Laboratory of Analytical Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan
Packed mlcrocaplllary columns havlng a ratlo of 3-6 of column Inner dlameter to particle Size are observed to have good performance wlth q5 values of 80-120. Electron mlcroscoplc observatlons proved that some of the slllca gel particles were Inserted In the lnslde wall of mlcrocaplllary columns. The relatlon of helght equivalent to theoretlcal plate and llnear veloclty for these columns Is found to be same as that for conventional columns. An equatlon for the estimation of the real value of the column performance Is derived.
One of the recent topics in the field of liquid chromatography (LC) has been the development of microcolumns, because they have potentially higher resolution and lower flow rate than conventional columns (1-20). They are specially important in combination with new detectors for LC, such as flame photometric detection and mass spectrometry, where low flow rates of eluents are desirable (11-13, 21). There are currently three different types of microcolumns: slurry-packed microcolumns (1-6), packed microcapillary columns (7-15), and open-tubular microcapillary columns (16-22). The smallest inner diameter of the slurry packed microcolumns is around 0.1 mm. But, one of their optimum inner diameters is around 0.25-0.35 mm ( I , 3-6). However, the optimum inner diameter of microcapillary columns is less than 0.1 mm. Development of capillary liquid chromatographymight be one way to realize high efficiency liquid chromatography whose theoretical plate numbers per unit time are comparative to that of capillary gas chromatography. Theoretical studies of open-tubular capillary columns in LC have suggested that there are excellent prospects for capillary liquid chromatography if it is possible to reduce the inner diameter sufficiently and to solve the technical problem of detection and injection in the capillary LC system (10, 21-24). On this line, recent attention in the area of open-tubular liquid chromatography has been focused on the chromatographic behavior of microcolumns with 5-20-pm inner diameters (8-11,22). On the other hand, in packed microcapillary columns (PMC) it is essential to decrease the particle diameter (d,) of silica gel. The most favorable geometric design of PMC would be as follows: the silica gel has small d,; the ratio of column inner diameter (d,) to d, has a large value; any column length May be available. Packed microcapillary columns in LC has been proposed by Tsuda and Novotny (71, and their separation ability has been demonstrated (7-15).Most of above works were carried out by using a capillary column that was packed with 30-pm diameter silica gel and whose inner diameter was 60-90 pm (7-13,15). In this paper, we will discuss packed microcapillary liquid chromatography, using columns packed with silica gel of 10 pm particle diameter and with d,/d, = 3-12. EXPERIMENTAL SECTION Packed microcapillary columns were prepared by the drawing procedure reported earlier (7,14).All packing procedures should 0003-2700/84/0356-1249$01.50/0
be carried out very carefully to avoid moisture and to maintain the dryness of the packing materials and the original glass tubing. The packing material used was Develosil lO-60 (particle diameter 10 pm and pore size 60 A) made by Nomura Kagaku (Seto, Aichi-Pref., Japan). A LC-4A (Shimadzu)or FLC-A700 (Jasco) high-pressure pump was used with a constant pressure mode. Two samples introduction systems with or without sample splitting ( I , 11, 25) were used. For determination of the H-o relation, the former introduction system was used. A UVIDEC-100 I1 (Jasco)UV detector was used with a home-made microcell (0.1 mm i.d. and 2 mm length). The column outlet was interfaced with stainless steel tubing (0.13 mm i.d. and 7 mm length) and then PTFE tubing (0.07 mm i.d. and 20 mm length) or with a fused silica capillary tubing (50 pm i.d. and 12 cm length). The connection device was nearly same as that mentioned in a previous work (14). RESULTS AND DISCUSSION T h e Ratio of d,/d, and Column Performance. When silica gel of 10-pm particle diameter was used as packing materials, it was possible to produce packed microcapillary columns whose d,/d, ratios were from 3 to 12. Although the columns, whose d,/d, was more than 10, had been packed uniformally, they were not stable for use as a liquid chromatographic column. Some of silica gel flowed out from the column end when effluent was passed through the column. Therefore, microcolumns with inner diameter of more than 0.1 mm should be packed by the slurry method. But, if we kept the ratio d,/d, under 10, the packing state was stable enough for use as a separation column. Column efficiencies of PMC with d,/d, 3-6 were twice as good as those columns with ratios 8 and 9. It was not clear why column efficiencies depended on the d,/d, ratio. From the present experiments, the limitation of d,/d, has been enlarged about twice, as a ratio of 2-3 has hitherto been thought to be optimum (7, 8). With comparatively larger column inner diameters such as 4C-60 pm, which packed with 10-hm silica gel, it is possible to use about a 4 times larger amount of effluent per a certain liner velocity compared to the case of the column with 20-30 pm i.d. It is easier to construct the of injection device, the method of detection and the connections. And also it is possible to inject a little bit larger amount of sample, because the number of silica gel particles per unit column length is larger compared to the microcolumn with a small d,/d, ratio. Cross Sectional View of the Microcolumn. The electron micrographs of a cross section of PMC, ca. 50 pm inner diameter and packed with 10-pm silica gel, are shown in Figure 1, and the plane of cross section was inclined at 25O. The packing state of silica gels in the PMC has been thought to be loosely packed as compared with the packing state of the conventional columns. From the observation of the electron microscope, the packing state of PMC is rather like that of a slurry-packed conventional column. However, flow resistance parameters (23,24),4,of the present columns are about 100, which are very low as compared with those of conventional columns (usually 500-1000). So the present column has very desirable features, that is, well-packed but still highly permeable. 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL 56. NO 8. JULY 1984
-P
"9.
..
P 1 ,, "
150)-
0
0.5
"
1 .O
1.5
ICrn/IPCI
Relations of Hand linear flow velociiy. Lines 1 and 2 were packed miuocapilary columns. 48 Fm inner &meter. of 231 and 1030 cm length. respectively. Line 3 was calculated: see text. Lines 4 and 5 were for conventional columns. Flpure 2.
.-
r-
2 = Eo:,
where e,is the variance in time unit, is valid, ( c T , . ~ ) ~ is expressed as follows under a certain linear velocity: (0,,2)2
By use of eq 2-4, (UJ2
= L,-1L,(0,,,)2
(4)
becomes = L , ( L , - L,)-'(H*L, - H I L I )
(5)
Then the real value of H, H,eal, is
H,,, = (oc,1)2L,-' = ( H A - HiLJ(L2 - L J '
Figure 1. Electron micrographs of a cross section of a packed microcapillaly column. B was pad of A. Magnificationsof A and B were 1 5 0 0 X and 7500X. respectively. Some silica gel particles in Figure 1A are observed to have been inserted partly in the glass. For a more precise view of these conditions. an electron micrograph with a larger magnification, 75Wx. is shown in Figure 1R. Ahout one-third of the particles were in the glass wall. Thus, about one-tenth of the silica gel particles per cross section of PMC were held in the glass wall, stahilizing the other silica gel particles, that is why the packed microcapillary column was stable under a pressure drop of 200 atm per 1 m of column length. As silica gel is stabilized hy the column materials, no frit was necessary at both ends of the PMC. Estimation of Real HValues. Standard deviation of the peak, (rT. has always included the peak broadening due to extracolumn effects. So mr is expressed as "T2
=
er.2
+e9
(1)
where oc and oe are standard deviations of the peak due to column and extracolumn effects,respectively. It is necessary to estimate oCvalues for calculating real column performance. If two similar columns with different lengths are used under the same chromatographic conditions, apparent height equivalent to a theoretical plate, H . is expressed as
HILI= H,L2 =
+ + ee2
( o , , ~ ) ~ee2
(2)
(3)
where subscripts 1 and 2 mean column 1 and column 2, respectively, and L means column length. As the addition rule
(6)
Equation 6 is very simple, where H,., means that it does not contain any contribution of hand broadening of a peak due to extracolumn factors such as, injection, detection, and connection. T o utilize eq 6, it is necessary to get the same column conditions, except for column length. By the present exerimentation procedures a very long (up to 100 m) uniformaly packed microcolumn can he produced, and from this column several columns of the same character can he obtained, which is not possible by slurry packing. Lines 1and 2 in Figure 2 are obtained by using a column whose lengths are 231 and 1030 cm, respectively. Half height of the peak was used for measuring H and the theoretical plate number N. The chromatographic conditions for lines 1 and 2 were the sample was N-ethylaniline (k' = 0.61) and the eluent was a mixture of methanol, acetonitrile, dichloromethane, water, and hexane (1.59, 1.27, 1.59.0.01, and 95.23 VJV%). Line 2 is lower than line 1, because the effect of extracolumn to the apparent H value becomes relatively small at the longer column. H, was calculated from two experimental lines, lines 1 and 2, by using eq 6. Line 3 in Figure 2 shows the real relationship between H,,] and flow velocity for the present microcolumn. Knox (24) has proposed the following equation for the conventional column:
h = Rv-' + + Cu (7) where h and yare reduced plate height and reduced velocity, respectively. B, A, and Care usually 2, 1, and 0.1, respectively. Line 4 was calculated by using eq 7 under the following conditions: particle diameter, 10 rm; diffusion coefficient of N-ethylaniline in hexane, 3 x cm2/s, which was calculated by using the Wilke-Change equation (26). The column efficiency of the PMC (line 3) is quite the same as the column efficiency of the conventional column which is given by line 4 in Figure 2. It is supposed here that the diffusion coefficient of the solute in hexane is approximately equal to that in the mixture used in line 1. The relation of line 5 was obtained experimentally by Reese and Scott (2)using a conventional column under the following conditions: the column, 4.6 mm
ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
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Table I. Typical Characteristic Values for Packed Microcapillary Columns no. dcr G J ~ dp, ~m dCld, hrnirl 0 la 70-90 30 2-3 3d 150-200 2b 60-100 30 2-3 3-4 70-100e 3c 30-60 10 3-6 3 80-120 Extrapolations of the h-v Reference 15. Present report and ref 14. References 7 and 23. e Calculated values from ref 15.
*
Table 11. Operational Conditions To Obtain 200000 Theoretical Plates by the Present Packed Microcapillary Columns V, H, Nlt, L, P, tm, cm/s pm S‘ ’ m kg/cm2 min E 74.2 1204 50 44.9 7.1 34.7 0.16 40.0 1383 90 7.4 83.4 37.2 0.31 27.8 2642 200 120 10.3 51.4 0.62 24.7 7569 640 17.5 135 87.0 1.18 inner diameter and 25 cm in length, was packed with Partisil-10 silica gel (mean particle diameter 7.9 pm). Benzyl acetate (k’ = 2.03) and the mixture of ethyl acetate and nheptane (1:19) were used as solute and eluent, respectively. The column efficiency of line 5 is also quite similar to that of line 3. 4 and E . General characteristic values for PMC are listed in Table I. Separation impedance, E, is defined by Knox and Gilbert (23,24).k’values of solutes used at 1,2, and 3 in Table I were 0 or 1.7,0.01, and 0.6-1.0, respectively. Flow resistance parameters, 4, obtained experimentally for PMC are about one-seventh of those for conventional columns and slurrypacked microcolumns, whose values are supposed to be around 500-1000 (4 values for most of conventional columns tested in our laboratory were 700-1000). The minimum value of reduced plate height, hmin,is 3 for PMC, while hminfor a conventional column is generally 2. So minimum separation impedance, which is given by for a packed microcapillary column would be from three-fourth to one-fourth of Emin for conventional columns (24) under the condition that both columns have been packed with 10-pm silica gel. Packed microcapillary liquid chromatography could perform with higher plate number (about 1.7 times) compared to that for a conventional column in the case of the same analysis period and inlet pressure. Column Performance in Terms of Plate Number per Unit Time. One factor concerning separation conditions of liquid chromatography involves the linear velocity which gives hmin,because it gives the best column performance. But, a chromatograph which shows very high plate numbers in a relatively short analysis priod would be one of the final goals. From this point, it is necessary to consider column performance in terms of plate number per unit time. Theoretical plate number, N , per time, t , is
Nt-’ = Dmv(d,2h)-’
(8)
D , and t are diffusion coefficient and retention time, respectively. As S(N/t)/Sv> 0, eq 8 increased when v becomes larger. But if we use a very large linear velocity, the reduced plate height will become larger according to eq 7. To overcome the latter problem, it is necessary to use a longer column. The experimental conditions to get a 200 000 plate number are shown in Table I1 using 4 100,q = 0.31 cP, H-u relation for PMC given in line 2 of Figure 2, and the equation
N = LR1 = (APdp2)(hu7p$)-l hp is the
(9)
difference between inlet and outlet pressure. t, in Table I1 is the elution time of the unretained solute peak.
600-1600e 800-1100 relation.
2
1
0
Emi, 1300 -4 0 0 0
TIME
3
“our1
Flgure 3. Separation of N-alkylanllines with a long column: packed microcapillary column, 29.4 m X 42 pm; inlet pressure, 500 kg/cm2; sample, (1) N,Ndiethyl-,(2) N,Ndimethyl-, (3) N-butyl-, (4) N-propyl-, (5) N-ethyl-, and (6) N-methylanlline; linear velocity, 0.46 cm/s.
0
5
10
TIME
15
(min)
Flgure 4. Separation of dialkyl phthalates: column, 10.3 m X 47 pm; inlet pressure, 500 kg/crn2;sample, (1) didecyl, (2) dinonyl and dioctyl, (3)diheptyl, (4) dlcyclohexyl, (5) dibutyl, (6) dipropyl, (7) diethyl, and (8) dimethyl phthalate; linear velocity, 4.2 cm/s.
From Table 11, if we would like to reduce t , up to one-third without dropping down original plate number, it is necessary to use a 2.5 times longer microcolumn and a 15 times higher inlet pressure as compared with the original condition. As it is not difficult to prepare a long microcolumn, so the above operational conditions are feasible to capillry liquid chromatography for fast analysis if pump and injection systems are able to operate at very high pressure. The upper limits of column lengths with or without column coupling would be 2,10, and 200 m for conventional columns, slurry-packed columns, and capillary columns, respectively. Thus, maximum theoretical plate numbers would be supposed to be lo5, 5 X lo5,and 2 X lo6 for the columns of the above order, respectively. Although the maximum theoretical plate number for capillary columns is very high, the favorable region would be lo5to 5 x IO5 from a viewpoint of practical operational conditions. Capillary columns which have relatively small 4 and E values would show better performance in terms of plate number per unit time in comparison to those for conventional columns and slurry-packed microcolumns. Separations. Typical examples of separations are shown in Figures 3-5. The eluents of Figures 3 and 4 were the mixture of methanol, acetonitrile, dichloromethane,water, and hexane. The component ratio of Figure 3 was same as that for Figure 2, but that of Figure 4 was 1.3, 19.5, 1.04,0.08, and 78.1 v/v % in the above order. The eluant of Figure 5 was pure n-pentane. Theoreticalplate numbers for N-alkylanilines in Figure 3 are from 4 X lo5 to 8.8 X lo5. N for the last peak (k’ = 0.95 and retention time 198 min) is 6 X lo5. The sep-
1252
Anal. Chem. 1904, 56,1252-1257
2
3
4
5 hour
Flgure 5. Separation of heavy oil, class A: column, 29 m X 42 pm; inlet pressure, 200 kg/cm2.
aration at higher linear velocity is shown in Figure 4. N values for the last two peaks in Figure 4 are 68 000 and 53 000. The chromatogram of heavy oil, shown in Figure 5, looks nearly like that of the chromatogram obtained by capillary gas chromatography, although the former chromatogram still took a longer analysis period compared to that of the latter.
LITERATURE CITED (1) Yang, F. J. HRC CC, J. High Resoluf. Chromafogr. Chromatogr. Commun. 1983, 6 , 348-358.
(2) Reese, C. E.; Scott, R. P. W. J . Chromafogr. Sci. 1980, 18, 479-486. (3) Takeuchi, T.; Ishil, D. J. Chromafogr. 1981, 218, 199-208. (4) Ishii, D.; Takeuchi, T. J. Chromafogr. 1983, 255, 349-358. (5) Hirata, Y.; Jinno, K. HRC CC,J . High Resoluf. Chromafogr. Chromatogr. Commun. 1983, 6 , 196-199. (6) Gluckman, J. C.; Hirose, A.; McGuffin, V. L.; Novotny, M. Chromafographia 1983, 77, 303-309. (7) Tsuda, T.; Novotny, M. Anal. Chem. 1978, 5 0 , 271-275. (8) Hirata, Y.; Novotny, M.; Tsuda, T.; Ishii, D. Anal. Chem. 1979, 51, 1807- 1809. (9) Hirata, Y.; Novotny, M. J . Chromafogr. 1979, 186, 521-528. (10) Novotny, M. J. Chromafogr. Scl. 1980, 18, 473-478. (11) McGuffin, V. L.; Novotny, M. Anal. Chem. 1981, 5 3 , 946-951. (12) McGuffln, V. L.; Novotny, M. J. Chromafogr. 1981, 218, 179-187. (13) McGuffin, V. L.; Novotny, M. Anal. Chem. 1983, 5 5 , 2296-2302. (14) Tsuda, T.; Tanaka, I.;Nakagawa, G. J. Chromafogr. 1982, 239, 507-513. (15) McGuffin, V. L.; Novotny, M. J. Chromafogr. 1983, 255, 381-393. (16) Tsuda, T.; Tsuboi, K.; Nakagawa, G. J . Chromafogr. 1981, 214, 283-290. (17) TIJssen, R.; Bleumer, J. P. A.; Smit, A. C. C.; van Kreveld, M. E. J. Chromatogr. 1981, 218, 137-165. (18) Krejci, M.; Tesarik, K.; Rusek, M.; Pajurek, J. J. Chromafogr. 1981, 218, 167-178. (19) Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. 1983, 255, 335-346. (20) Takeuchi, T.; Ishii, D. J. Chromatogr. 1983, 279, 439-448. (21) Ishii, D.; Takeuchi, T. J. Chromafogr. Sci. 1980, 18, 462-472. (22) Tsuda, T.; Nakagawa, G. J. Chromafogr. 1983, 268, 368-374. (23) Knox, J. H.; Gilbert, M. T. J. Chromatogr. 1979, 186, 405-418. (24) Knox, J. H. J. Chromafogr. Scl. 1980, 18, 453-461. (25) Tsuda, T.; Ishii, D. The 22nd Symposium on Liquid Chromatography, Kyoto, Japan, Feb 16, 1979. (26) Giddlings, J. C. "Dynamics of Chromatograpy"; Marcel Dekker: New York, 1965.
RECEIVED for review December 6, 1983. Accepted February 27, 1984. Part of this paper was presented at the 7th International Symposium on Column Liquid Chromatography, Baden-Baden, F.R.G., May 2-6, 1983. This work was supported by a grand-in-aid from the Toyota Foundation (No. 82-1-111-031) and the Ministry of Education of Japan (No. 58390013).
Open Tubular Liquid Chromatography with Thermal Lens Detection Michael J. Sepaniak,*' John D. Vargo,2Charles N. Kettler, and Michael P. Maskarinec
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, and Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
Thermal lens detection is demonstrated for use In open tubular liquid chromatography. An argon ion laser beam Is used to both create and probe thermal lenses formed in 100- and 200-pm flow cells. Base line noise levels of approximately 3 X 10" absorbance units are obtained. Chemically bonded, reverse-phase, open tubular columns with Inner dlameters of 20 pm are used to provide efficient separations of derivatlred amines and nltroaniiine mixtures. Laser fluorometric detection Is employed to evaluate the columns and study the effect of flow cell design on chromatographlc efficiency.
Several theoretical discussions regarding the relative merits of open tubular and packed liquid chromatography columns have appeared in the literature (1-3). Conclusions regarding ' A u t h o r t o w h o m correspondence should be addressed a t t h e U n i v e r s i t y of Tennessee. *Present address: General M o t o r s Research Laboratories, Anal y t i c a l Chemistry Division, Warren, MI 48090-9055.
which column type has the greater potential for high separating efficiency vary, depending largely on the author's choice of realistic operating parameters. However, there is general agreement that open tubular liquid chromatography (OTLC), using columns with inner diameters (i.d.) less than 10 pm and employing detectors with high sensitivity and low nanoliter volumes, will yield superior performance. Recent success in the chemical bonding of substrates to the inside surfaces of narrow-bore open tubular columns ( 4 , 5 )has greatly increased the analytical utility of OTLC. However, there still remains a need to develop detectors that satisfy the extremely low volume and high sensitivity requirements of OTLC. Electrochemical (6), mass spectrometric (7), and spectrophotometric detection have been used in the development of OTLC. Novotny and McGuffin used flame photometric detection to monitor organophosphorus compounds separated by packed microcapillarycolumns (8). Commercial absorbance and fluorescence liquid chromatography detectors can be used in OTLC if a makeup flow is employed at the detector inlet (9). However, the large amount of sample dilution that occurs
0003-2700/84/0356-1252$01.50/00 1984 American Chemical Society
