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Packed Microcapillary Columns in High Performance Liquid Chromatography Takao Tsuda’ and
Milos Novotny”
Department of Chemistry, Indiana University, Bloomington, Indiana 4740 1
New high-pressure LC (packed microcapillary) columns are described which consist of small adsorbent particles drawn Inside glass capillaries of 50-200 pm internal diameters. A typical ratio of the column inner diameter to particle size is 2, and a significant plate-height reduction is achieved with decreasing particle size. With typical flow rates of several pL/min through such columns, modlfied injection and detection techniques are necessary. The columns of different inner diameters and particle sizes were evaluated through the reduced plate height vs. velocity plots. The effect of column coiling diameter on chromatographlc performance was also studied. Whereas sample capacity of packed microcapillary columns is low, typical column efficiencies are significantly higher than those obtained with the hitherto available LC c oIumns
.
been made to its theoretical limit through t h e capillary column, there is a discrepancy in LC of many orders of magnitude. Giddings ( 4 ) derived a simple relationship between t h e limiting number of theoretical plates, Nlim,and certain parameters involved in the chromatographic separation (time of analysis disregarded):
where $ and y are geometrical constants, d, is the particle size, I p is pressure gradient, TJ is viscosity, and DM is solute diffusivity in the mobile phase. Thus, for the columns of t h e same geometrical characteristics used in GC and LC it applies that
T h e basic types of columns available in analytical gas chromatography (GC) are conventional packed columns, open tubular (capillary) columns, micropacked columns (with typical internal diameters around 1 mm), and packed capillary columns. These columns differ widely in terms of separation efficiency a n d sample capacity. Selection of a column type in liquid chromatography (LC) has been more restricted because of viscosities and solute diffusivities in the liquid phase which are orders of magnitude different from the values in t h e gas phase. T h e most efficient columns presently used in LC are those packed with totally porous small particles (with particle size down to several micrometers). T h e other column types, as known in GC, have not been sufficiently explored. This is primarily due t o t h e fact t h a t t h e mass transfer is a diffusion-controlled process. Thus, capillary LC is unlikely to give desired efficiencies under t h e conditions of laminar flow. Packed capillary columns studied extensively in GC by Halasz and Heine ( I ) a n d Landault and Guiochon (2) have rather unique characteristics. Their technology is different from other column types in t h a t t h e adsorptive material is drawn inside t h e glass capillaries, in a way t h a t is somewhat similar to t h e very common method for preparation of glass capillary helices (3). However, according to Halasz and Heine ( I ) , t h e most important feature of such columns is t h e ratio of particle size to t h e internal column diameter. Whereas packed capillaries possess usual values of this ratio between 0.2 a n d 0.5, t h e conventional packed columns are typically well below 0.1. Thus, packed capillaries have certain geometrical characteristics of their own which are reflected in their analytical performance. Considering a rather loose packing of these columns used in GC, their efficiencies are quite high. Halasz and Heine ( 1 ) attribute this to the mobile-phase mixing effect t h a t aids the radial diffusion and stress t h e importance of high column permeability. When comparing t h e theoretical separating power of GC a n d L C with t h e actual situation, Giddings ( 4 , 5 )and Golay (6) note that, unlike in GC where a reasonable approach has
When substituting typical values into Equation 1, N h for LC can be as much as lo8 theoretical plates. It also appears from the above considerations t h a t t h e pressure gradient Ap (and, subsequently, inlet pressure) is the most important variable in achieving higher column efficiencies. In modern LC using columns packed with micrometer-size particles, pressures up to several hundred atmospheres are typically employed. Although substantial reductions of t h e plate height are achieved while decreasing t h e particle size, there are some practical limits t o this procedure. As pointed out by Halasz e t al. (7), there are difficulties in uniform packing of very small particles, as well as t h e problem with t h e evolved heat of friction. T h e present investigation explores a version of packed capillary columns for high-performance LC. However, since both particle diameters and internal radii of such columns are typically an order of magnitude lower than those used in GC, we will refer to them as “packed microcapillaries” throughout the text. Special technology has been developed to achieve uniform packing of these columns. As demonstrated by t h e presented results, capacities of packed microcapillaries are significantly lower than those of typical LC analytical columns. However, column efficiencies are significantly higher. Since the injector and detector connections (split sampling and t h e use of a make-up liquid flow) are crucial to t h e column performance, this chromatographic technique is strongly reminiscent of t h e use of capillary columns in GC. In the present work, several column types with different particle size and t h e column internal diameter were investigated. Van Deemter plots obtained with t h e packed microcapillaries are presented and their shapes are discussed. Since the plate height, H , of such columns appears t o be strongly affected by hydrodynamic factors, a possible role of column coiling was briefly investigated. Evaluations were made for columns of identical internal diameter and particle size, b u t different column coil radii.
On leave from the Department of Applied Chemistry, Faculty of Engineering, Nagoya University, Nagoya, Japan.
Preparation of Packed Microcapillaries. A commercial glass drawing machine (Hupe and Busch, Groetzingen, West
0003-2700/78/0350-0271$01.OO/O
EXPERIMENTAL AND RESULTS
0 1978 American Chemical Society
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Figure 2. Microcapillary liquid chromatograph: 1, high-pressure syringe
pump; 2, injection point: 3, splitter: 3', metering valve to adjust split ratio; 4, packed microcapillary column: 5,make-up liquid mixing piece: 5', liquid reservoir: 5", flow restricting valve; 6, detector
Figure 1. Microphotograph of a section of alumina-packed microcapillary. Column inner diameter, 75 pm: average particle size, 30 fim
Germany) was used for the preparation of columns. The drawing apparatus was placed in the vertical position while the packing material was introduced uniformly into the drawn microcapillary by means of gravity. The packing uniformity of the original glass column (typically, 5.5-mm 0.d. and 0.25-mm id.) is very important; packing under the vibrator action aids this uniformity. The inner and outer column diameters can be varied through selection of the initial dimensions of glass tubing and drawing conditions. Because of its relatively low melting temperature, soft glass is a preferred material. Reproducible preparation of columns with internal diameters below 100 pm is relatively easy. A typical wall thickness of our columns is 250-300 pm. The uniformity of packing inside the microcapillaries will strongly affect column efficiency. Thus, selection of a uniform particle size fraction is initially desirable. Whereas it is technically feasible preparing packed microcapillaries from a variety of materials that do not undergo undesirable irreversible changes during the drawing process, such columns require some variations of technology. The present fundamental investigations of packed microcapillarieswere limited to the use of acidic and basic alumina packing materials. These were 30 pm and 10 pm activated alumina (LiChrosorb, AloxT, from E. Merck Reagents, Darmstadt, West Germany), 100 pm activated alumina (from M. Woelm, Eschwege, West Germany) as well as the correspondingparticle size fractions obtained from the coarse column chromatography materials after grinding, mechanical sieving, and sedimentation sizing process. Although various columns with different particle sizes and diameters were prepared and their analytical properties are described below, we presently consider columns with internal diameters between 50 to 80 pm, packed with particles around 30 pm, to be a good compromise between analytical performance and minimum difficulties of operating conditions (sample size, available inlet pressure, and sample introduction). The columns have a uniform appearance along their entire length. .4 microphotograph of a typical section of the alumina-packed microcapillary is shown in Figure l. Technological importance of the ratio of capillary inner diameter to particle size must be stressed. Although the columns of larger diameters packed with small particles (e.g., 140-pm i.d. and 30-pm particle size) have uniform appearance, their structure collapses under the operating conditions, and clogging occurs. Whereas the radius of most studied columns was 6 cm. the drawing apparatus had t o be provided with home-made coiling tubes for the preparation of columns coiled into greater or smaller radii. Analytical System. The high-pressure syringe pump and UV-detector of the Varian Model 4100 liquid chromatograph were used throughout the measurements. Since the problems of dead volume are considerablymore severe with these column types than
6
1 I
/*3 8 8
Flgure 3. (A) Injector/spiitter: 1, microsyringe needle: 2, column inlet: 3, 1.5-mm i.d. tube: 4, direction of discard flow; 5, injector flange; 6, polyimide ferrule. (B) Make-up liquid mixing piece: 1, entering make-up liquid; 2, column effluent: 3, liquid entering detector cell: 4,
column outlet; 5, detector connecting capillary: 6, mixing tee with the conventional LC columns, both injection and detection techniques were modified. In order to overcome these problems, we resorted t o the commonly practiced techniques of capillary GC (sample splitting and the use of additional liquid flow at the column outlet). Figure 2 shows the schematic diagram of the system used, whereas Figure 3 details the parts of injector/splitter and the make-up solvent assembly. Typical split ratios around 1:lOOO resulted in the use of maximum detector sensitivity. However, the detector response is decreased by the use of an extra liquid flow to overcome the detector cell dead volume. Whereas such flow was around 20-50 pL/min, typical flow rates through 70-pm i.d. packed microcapillaries are less than 10 pL/min. As judged from reproducibility of retention times, flow appears constant among individual runs, once the inlet pressure is set to a desired value. It should be emphasized that this work primarily addresses itself to fundamental column performance investigations. Thus, better sample introduction techniques and detectors with small internal volumes and greater sensitivities are needed for future work. Column Efficiency Studies. Three different ratios of particle size and inner diameter were used in preparation of packed microcapillaries: 100/200,30/70, and 10/50 pm. Van Deemter plots were measured for standard UV-absorbing solutes, introduced onto the columns in the amounts close to lo-*g. As ex-
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1478 H‘mm’
I
273
h
6t
t
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t
I
I
I
P
5
50
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iic
‘CR
5m
V
0
0.6
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Figure 4. Plate height vs. velocity curves for standard solutes run on 100-pm (broken line) and 3 0 - ~ m(solid line) particle size columns. 0 = benzene; x = methyl benzoate (retention relative to benzene, 1.34 for 100-pm column and 1.14 for 30-pm column): A = quinoline (retention relative to benzene, 1.71). To adjust approximately the same relative retention on both column, the mobile phase was hexane with 0.03% and 0.05% methanol, respectively, for a 30-pm and 100-pm column. Column lengths were 14 m and 40 m for 30+m and 100-pm columns, respectively
Figure 5. Reduced plate height vs. reduced veloclty curves for benzene measured on 0 = 10-pm particle size column (length, 3.6 m), H = 30 p m (length, 14 m), and 0 = 100 pm (length, 40 m) Mobile phase: same as Figure 4
h s
t
I
I
,
pected, values of the height equivalent to a theoretical plate, H , were reduced with decreasing particle size, d,. Figure 4 demonstrates differences in H vs. u (average linear velocity of the mobile phase) curves for a 100-pm compared to a 30-pm particle column. Whereas the minima of these H vs. u plots apparently lie at considerably lower velocity values than measurable, the plate heights at the lowest velocity values of Figure 4 roughly correspond to those theoretically predicted ( 7 ) :
H,,
=
A
,/
+ 2 dBc.=3.6 d,
Shapes of H vs. u curves are important for both understanding the physical phenomena occurring in the columns as well as their analytical utility. Obviously, the measured curves fail to fit the usually proposed equations of liquid chromatography ( 7 , 19). Whereas the initial nonlinearity of the measured curves is not unusual for some LC columns and can be explained by the coupling theory of Giddings (81,there appears no simple explanation for the further rise of H with flow rate. I t should be stressed that this general curve shape (rise, a plateau, and a second steeper rise) is highly reproducible with sequentially prepared columns of the same particle size. As indicated below, the curve shape holds also for other d, values, although particle size or packing geometry appears to determine the second onset of the curves. It is also unlikely that mechanical changes of the column bed due to high pressure and/or flow shear are implicated; repeatedly measured curves gave identical shapes. Plots of reduced values (8) are generally considered as good indication of the column’s chromatographic quality across a range of particle sizes. Figure 5 shows the reduced plots for three columns of different particle size. The plots were obtained using a nonretained (or only slightly retained) solute, benzene, in order to dissociate mass-transfer processes from those of hydrodynamic nature. The initial h values of the smallest-particle column (10 pm) are somewhat higher than those of other two columns. This is most likely a result of our present insufficient packing technology at this particle size and less homogeneous column bed. Figure 5 further demonstrates the effect of particle size on band broadening under the dynamic conditions. The shapes of curves obtained with retained solutes on the columns with different particles are nearly identical (Figure 6). With the current column technology, less than 10% column-to-column variation in H at a given velocity and retention time was observed.
I
5
‘0
‘SC
5:
55,’
‘:x.
Y
Figure 6. Reduced plate height vs. reduced velocty curves for retained solutes on same columns as indicated in f:igure 5, under the same conditions. Solutes of similar relative retention were used: pyridirie (10-pm column); quinoline (30-pm column); and methyl benzoate (100-pm column)
If hydrodynamic factors are involved in the zone broadening phenomena, it is of some interest to investigate whether the column coil radius can influence the plate height as well. This situation received some attention previoudy ($1 I ) with different column types. The results obtained with the columns of identical inner diameter and particle size. but different column coil radii, are shown in Figure 7. Although analytical applications of packed microcapillaries will be a matter of future research, two important questions are of immediate interest: first, what are the typical column efficiencies to be expected, and second, what sample sizes can be tolerated. A chromatogram of a standard mixture obtained on a “typical” packed microcapillary is shown in Figure 8. Whereas the total number of theoretical plates obtained on this run is not identical with the so-called effective plates (it is estimated that some 70% of the column space is occupied by the mobile phase), there is, every indication of achieving column efficiencies approximately an order of magnitude higher than those currently available in practice of modern LC. Sample capacity of a 10-pm particle column was investigated using benzene, methyl benzoate, and dimethyl phthalate introduced through the splitter in varying amounts (Figure 9;.
274
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
I 1.0
1
1 0
10
0.6
1 5
v
( C r n sec- )
Flgure 7. Effect of column coil diameter on plate height vs velocity curves. Solute: benzene; all columns were 12 m X 75 pm i.d , packed with 30-pm alumina. Mobile phase: hexane with 0.05% methanol. Column coiling diameters: 0, 30 cm; 0 ,12 cm; and R, 5.7 cm
0-
'
10'~
10"
10"
IO-$
sample size ( g l
Figure 9. Plate height vs. sample size for a 10-pm particle size (3.6 m long) microcapillary. Mobile phase: hexane with 0.05 % methanol,
flow linear velocity, 0.4 cm/s. Solutes: 0, benzene: X, methyl benzoate; 0 , dimethyl phthalate; UVdetector sensitivity, 0.02-0.16 OD, full scale
0
20
40
60
80
100 TIME (MlN.)
Figure 8. Chromatogram of the standard mixture on a 29 m X 75 pm
i.d. (30-pm particle size) alumina-packed microcapillary column. Mobile phase: n-hexane with 0.05% methanol, flow linear velocity, 0.65 cm/s. Solutes: benzene, methyl benzoate, and quinoline (in the order of elution); number of theoretical plates for quinoline = 85000; inlet pressure, 1500 psi; column permeability, 6.1 X 10-O
Obviously, the plate height increases with sample size Microcapillaries of somewhat wider diameters and greater lengths will be more tolerant to slightly larger samples. Nevertheless, a need for sensitive detection is clearly suggested for these columns.
DISCUSSION T h e novel LC columns described in this paper may have utilization in three analytically important directions: (1) availability of greater resolving power; (2) the overall miniaturization of LC systems; and (3) increased compatibility of LC with mass spectrometry or other ancillary techniques that require low mobile-phase flow rates. The obtained results are discussed in view of these trends below. Small-particle column technology has drastically improved the state of modern LC since several years ago. H values lower than 20 pm have been reported ( 7 ) for short columns packed with 4-pm particles. Whereas difficult-to-resolve pairs of solutes (e.g., various isomers) can frequently be separated through mobile-phase selectivity adjustments in LC, similar degree of resolution in GC may necessitate a "brute force" approach, Le., a great number of theoretical plates. However, it is the overall efficiency of a given chromatographic column which is instrumental in separating complex mixtures. Alternatively, it is often of advantage to combine high plate numbers with selectivity. J u s t as with GC where the plate height values of capillary and best packed columns are roughly comparable, the column length that can be practically utilized for a given separation problem becomes crucially important. Since the inlet pressure remains a single most important parameter in gaining higher efficiencies ( 5 ) ,longer columns are likely to be more emphasized in future LC separations. Present pressure limitations of the chromatographic equipment are less severe than is commonly believed. On the other hand, t h e approach of decreasing particle size with a simultaneous increase of column length in a usual way (slur-
ry-packed columns) has its limitations. Namely, these include the evolved heat of friction, difficulties in packing extremely small particles, and joining shorter column sections together. The packed microcapillary columns described in this paper may provide an alternative solution. Their column technology is relatively easy. The described columns are quite unique, both structurally and analytically. Whereas they are a microversion of packed capillaries used previously in GC, their analytical characteristics differ significantly from t h e irregularly packed columns investigated by Halasz and Walkling (12). These authors observed a turbulence-caused decrease of H. Their plate height values typically ranged from 2 to 10 mm. Van Deemter plots constructed for various solutes and measured on the packed microcapillaries of varying internal diameter and particle size show quite unusual behavior. Whereas H values as low as 100 pm were achieved at very low flow rates and the curves remained flat over an analytically useful range of flow velocities, a sudden increase of H is observed at higher velocities. Interestingly enough, this increase demonstrates itself in the areas of velocities roughly comparable to those of decreasing H of the irregularly packed columns of Halasz and Walkling (12). Thus, some secondary flow effects are suggested. However, their interpretation may be a difficult matter. Reduced plate heights are comparatively low for microcapillaries filled with different particles. Furthermore, such columns can be prepared with good reproducibility. T h e preparation of columns with 10-pm particles packed into 50-pm capillaries is somewhat exceptional, since they yield consistently less satisfactory results. The most likely reason for this is insufficient uniformity of 10-pm packing in t h e original (0.25-mm id.) glass tube. Indeed, particles of this size are known to be packed with difficulties in a dry state. In addition, the higher ratio of column diameter to particle size may also play some role. Although the packing technology for small particles may still be improved in the future, we presently consider columns with 70-pm i.d., packed with 30-pm particles, to be a good compromise. Without excessive inlet pressures, good efficiencies are obtained. Adequate sample capacity is yet another consideration, Since the packed microcapillaries of great lengths are coiled into helices, it has been of some interest finding whether there is some effect of column coiling on their performance. Long
ANALYTICAL CHEMISTRY, VOL. 50,
ago, such a possibility was considered in chromatography by Giddings (9, I O ) . A simple relation was derived (10) for the plate height contribution due to coiling:
H=
7vr4 12R2yDM
(3)
where u is flow velocity, r is column radius, R is coil radius, y is the obstruction factor of the B term of the van Deemter equation, and DMis the solute diffusion coefficient in the mobile phase. This contribution to the plate height primarily arises from the different velocities of t h e molecules encountered in different distances from t h e column wall. Whereas in gas chromatography, this contribution can be significant perhaps only in preparative-scale work, greater caution must be exercised in high-pressure liquid chromatography because of t h e considerably smaller values of DM. The effect of column shape in LC was experimentally explored by Bart,h et al. (11). Significant H increases were observed with columns of conventional diameters, but no particular changes were observed for a 0.76-mm i.d. tube. Even though the microcapillary radius is very small to amount to an appreciable increase of H through the coil effect (9, I O ) or the limitations by flow of the lateral mass transfer (13), some secondary flow phenomena could occur in t h e complex structure of packed microcapillaries. Because of the complexity of hydrodynamic conditions inside the column, Reynolds number would probably be a useless criterion. Whereas mass transfer is increased because of the secondary flow in open tubular columns (14), coiling may have only a minor effect on the performance of packed microcapillaries. Certainly, Figure 7 seems to indicate that. T h e differences in measurements with the columns of different coil radii are indeed very small and within the reproducibility range of column technology. Miniaturization of equipment without sacrificing its resolving power is a n important trend in analytical separation methods. As recently shown by Ishii ( I s ) ,a miniature LC equipment is feasible that employs detector cells of very small volume. T h e packed microcapillaries described here naturally lend themselves to miniaturization, with typical flows of a few microliters per minute. Admittedly, t h e instrumental conditions used in this work are less than ideal and the necessary instrumentation has yet to be designed. In particular, smaller detection cells are needed. In order to utilize more effectively the separating power of this technique, longer columns and correspondingly higher pressures are needed. T h e columns with diameters and lengths as described in this work seldom required inlet pressures over 200 atm. However, with the typical liquid flow
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volumes of these columns, there should be no major technological problems to utilize pressures around 1000 atm. Chromatography carried out under the extreme pressure conditions was demonstrated for both dense gases (16) and liquids ( 1 7 ) . Possibilities of combining LC with MS have now been under focus for several years. I t appears that some technical problems of a successful coupling of these methods arise from excessive column liquid flows. Again, the described columns (or any other small-diameter columns) can potentially reduce these problems. Whereas the present study is confined to one particular type of chromatography (the liquidsolid separation principle) and the alumina adsorbent, other column materials are worth exploring. Thus, in situ modifications of alumina with either salts (18)or liquids are clearly feasible, as is the preparation of bonded phases with this material (19). As described by Halasz and Heine ( I ) , particles of graphitized carbon black can also be drawn inside the glass capillaries if special precautions are made. Obviously, a choike of sorption materials is limited to those of sufficient thermal stability. Finally, if the very useful siliceous materials are to be used, special techniques must be developed to regenerate the thermally lost surface hydrated structures. The surface silanol groups are needed for the preparation of chemically bonded stationary phases of different selectivities.
LITERATURE CITED (1) I. Halasz and E. Heine. Adv. Chromafogr., 4, 207 (1967). (2) C. Landauk and G. Guiochon, in "Gas Chromatography 1964", A. Goldup, Ed., British Petroleum Institute, London, 1965, p 121. (3) D. H. Desty, J. N. Haresnape, and E. H. F. Whyman, Anal. Chem., 32, 302 (1960). (4) J. C. Giddings, Anal. Chem., 36, 1890 (1964). (5) J. C. Giddings, Ref. 2 , p 3. (6) M. J. E. Golay, Chromatographia, 6, 242 (1973). (7) I . Halasz, R Endele, and J. Asshauer, J . Chromatogr., 112, 37 (1975). (8)J. C. Gddings. "Dynamics of Chromatcgra?hy", Marcel Dekker, New Yofk, N.Y., 1965. (9) J. C. Giddings, J . Chromatogr., 3, 520 (1960). (10) J. C. Giddings, J . Chromatogr., 16, 444 (1964). (11) H. Barth, E. Dallmeier, and B. L. Karger, Anal. Chem., 44, 1726 (1972) (12) I. Halasz and P. Walkling, J . Chromatogr. Sci.. 7, 129 (1969). (13) J. C. Gddings, "Qnarnics of Chromatography", Marcel Dekker, New Yo&, N.Y., 1965, p 52. (14) R. Tijssen, Chromatographia, 3, 525 (1970). (15) D. Ishii, K. Mochizuki. and Y. Mochida. Abstracts of the 1977 Pittsburgh Conference on Anatytical Chemisby and Applied Specboscopy, Cleveland Ohio, No. 385. (16) L. McLaren, M. N. Myers, and J. C. Giddings, Science, 159, 197 (1968). (17) E. A. Bidlingmeyer, R. P. Hooker, C. H. Lochmuller, and L. E. Rogers. Sep. Sci., 4, 439 (1969). (18) C. G. Scotl and C. S. G. Phillips, Ref. 2, p 226. (19) J. H. Knox and A. Pryde, J . Chromatogr., 112, 171 (1975).
RECEIVED for review July 20, 1977. Accepted November 7, 1977.
