A ~ Im . rn. 1993, 65, 1476-1480
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Microcolumn Size Exclusion Chromatography with Polymeric Stationary Phases Hernan J. Cortes' and Curtis D. Pfeiffer The Dow Chemical Company, Analytical Sciences, 1897B Building, Midland, Michigan 48667
Miniaturization of the column used in liquid chromatographic separations to microcolumn dimensions (0.1-0.5-mm i.d.) offers various advantages to the technique, such as low eluent consumption, ability t o interface t o other chromatographic techniques and mass spectrometers, low cost per column, and reduced maintenance requirements, among others. This report documents studies conducted in the preparation of highly efficient microcolumns for size exclusion chromatography (micro SEC) with polymer packings of 50- and 1000-Aporesize. Studies conducted on the effects of slurry ratio and column packing pressure on efficiency indicate that higher packing pressures yield the lowest plate heights (higher efficiency) but at the expense of decreased specific column permeabilities, while slurry ratio only has a significant effect on plate height at low packing pressures (170 atm). Results obtained suggest that short columns (30 cm) should be prepared at 400 atm with high slurry concentrations while long columns (1 m) a r e best prepared a t low packing pressures with dilute slurries. Columns with -75 000 platedm can be routinely prepared. Resolution factors obtained (Du) were 0.033 for a conventional size column, 0.017 for 1-m microcolumns operated at the optimum linear velocity; and 0.021 for the microcolumns operated a t a linear velocity chosen to yield retention times equivalent to the conventional size column. The results obtained indicate better performance for the micro SEC system when compared to conventional size SEC.
In conventional size SEC, column segments are connected together to obtain better resolution; however,deviations from theoretical efficiency are observed due to the broadening caused by the eluting band's variable velocity experienced while emerging from the column in the tubing used to couple columns. Miniaturization of the system used for liquid chromatography offers a variety of advantages which have been previously demonstrated,2-4 such as low eluent consumption, low cost per column, greater ease in coupling to a mass spectrometer, and ability to prepare long columns, among others. Microcolumn SEC (micro SEC) was originally investigated by Takeuchi and co-workers,5where epoxy resin oligomerswere separated using manually packed short column segments connected in series. The technique has been applied to separation of proteins6 and steroid ester^.^ Studies on temperature effects on micro SEC were recently published.8 Kennedy and Jorgensong demonstrated that highly efficient microcolumns for aqueous SEC using silica-based packings on columns of 28-50-pm internal diameter could be prepared to yield more efficient separations than those obtained using conventional size SEC columns. Micro SEC has also found utility as a preliminary separation step in multidimensional separations.10-13 The preparation of efficient microcolumns packed with silica-based particles has been widely investigated,lP'6 but to our knowledge, no detailed studies using porous polymeric supports have been reported. In general, high packing pressures (>400 atm) are used to obtain efficient liquid chromatography columns; however, due to the wide range of pore sizes used in SEC, the use of high pressures may be disadvantageous, since particles become more fragile with increasing pore size and porous polymeric supports tend to be less rigid and swell to a larger extent. In this report, studies conducted on the preparation of highly efficient micro SEC columns with styrene/divinylbenzene-based,50- and lO00-A pore size packings are presented.
INTRODUCTION
(2)Novotny, M. Anal. Chem. 1988,60, 500A-510A. (3)Hirata, Y. J. Microcol. 1990,Sept 2,214-221. (4)Corks, H. J.;Larson, J. A.;McGowan, G. M.;J. Chromatogr. 1992, 607,131-134. (5) Takeuchi, T.; Ishii, D.; Mori, S. J. Chromatogr. 1983,257,327-335. (6)Takeuchi, T.; Saito, T.; Ishii, D. J. Chromatogr. 1986,351,295301. (7)Ghijs, M.; Dewaele, C.; Sandra, P. J. High Resolut. Chromatogr. 1990,13,651-653. (8) Takeuchi, T.; Matsuno, S.; Ishii, D. J.Liq. Chromatogr. 1989,12, 987-996. (9)Kennedy, R.T.; Jorgenson, J. W. J. Microcol. 1990,Sept 2,120-
Size exclusion chromatography (SEC) offers unique selectivity not available in other separation modes, as components are separated on the basis of their hydrodynamic volume in solution, which is related to the molecule's shape and molecular weight. Separations in SEC are governed by the components' ability to migrate into the stationary-phase pores, and other interactions with the stationary phase are usually not present or are intentionally minimized. Therefore, components elute in a volume which is less than the total liquid volume of the column. For this reason, the peak capacity' (the maximum number of peaks in the separation space that can be placed side by side with a given resolution) obtained in SEC is relatively low, and since modification of the mobile phase does not have a significant effect on selectivity in SEC, column efficiency becomes the primary means of increasing resolution. ~~
(1)Giddings, J. C. In Multidimensional Chromatography; Cortes, H. J., Ed.; Marcel Dekker: New York, 1990 p.1. 0003-2700/93/0365-1476$04.0010
126.
(10)Cortes, H. J.; Jewett, G. L.; Pfeiffer, C. D.; Martin, S. J.; Smith, C. G. Anal. Chem. 1989,61,961-965. (11)Cortes,H.J.;Bell,B.M.;Pfeiffer,C.D.;Graham,J.D.J.Microcol. 1989, Sept 1, 276-288. (12)Cortes, H. J.; Bormett, G. A.;Graham, J. D. J. Microcol. 1992, Sept 4,51-57. (13)Cortes, H.J.; Campbell, R. M.; Himes, R. P.; Pfeiffer, C. D. J. Microcol. 1992,Sept 4, 239-244. (14)Novotny, M.; McGuffin, V.; Hirose, A.; Gluckman, J. Chromatographia 1983,17,303-309. (15)Hoffman, S.; Blomberg, L. Chromatographia 1987,24,416-420. (16)Yang, F.Microbore Column Chromatography; Marcel Dekker: New York, 1989. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
EXPERIMENTAL SECTION Column Preparation. Fused-silica columns of 35- or 105cm X 250-pm i.d. (Polymicro Technologies, Phoenix, AZ) were equipped with a ceramic frit support cut to 0.5 mm prior to packing.17 The packing material used throughout the study was styrene/divinylbenzene PL-GEL of 5-pm particle diameter and 50- or lOOO-8, pore size (Polymer Industries Inc., Amherst, MA). Slurries were prepared in tetrahydrofuran (THF) and allowed to swell for at least 2 h prior to use. Slurry ratios (mL of THF/g of packing) of 10,20,and 40 were used in the study. The packing was suspended by ultrasonication for 5 min prior to introduction into the slurry reservoir, which consisted of a 10-cm X 2-mm i.d. x 6-mm0.d. stainlesssteel tube equipped with 1/4-1/lG-in.reducing fittings. An Isco Model pLC-500 solvent delivery system, (Isco Inc., Lincoln, NE) operated at constant pressure was used to pack the columns. After system decompression, columns were cut to a length of 30 or 100 cm. The results obtained were compared to those obtained on a commercially available conventional size column of 30-cm X 8-mm i.d. packed with the same material (Polymer Industries, Inc.). Column Evaluation. The systems used to evaluate the columnsconsisted of the syringe pump operated at constant flow rate, a Valco Model NI4W injection valve with a 60-nL internal loop volume (ValcoInstruments, Houston, TX) automated with an air-driven digital valve interface (Valco),and an AB1 Model 757 ultraviolet detector (Applied Biosystems Inc., CA) equipped with a modified detector cell of 6-nL volume. After the flow rate was varied; the system was allowed to equilibrate at each flow rate until the column pressure remained constant for at least 1 h, indicatingflow rate stability. Accurate flow rate measurements were obtained by connectinga 10-pLsyringeto the detector outlet and determining the time necessary to fill a given volume. For the conventional size column, a Spectra-Physics Model 8500 solvent delivery system (Spectra-Physics, San Jose, CA) was used, and detection was accomplished using an AB1 757 UV detector as described above. Data collectionwas performed using a Model 4270 integrator (Spectra-Physics) or a PE Nelson Access-Chrom data system (Perkin-ElmerNelson Systems Inc., Cupertino, CA). Chemicals used throughout the study were polystyrene standards of 2.2 million, 465 000,68 000,22 OOO, 12 500,5050, and 580 molecular weight (Polymer Labs), Irganox 1010 and Irganox 1076 (CibaGeigy,Basel,Switzerland),triphenylmethane, biphenyl (Aldrich, Milwaukee, WI), and toluene (Fisher, Fair Lawn, NJ).
1477
5
0
a
4
Time (min)
Flgue 1. Representatbechromatogramon miaocolumsire exduakn column: column 30cm X 250-pm 1.d. fus8d-sllica packed wlth PLGEL, 5-pm, 50-A pore size; eluent, THF; flow rate, 1.85 pL/mln; detectlon, UV at 254 nm; Injection size, 60 nL. Peaks: (1) polystyrene (MW 2.2 mlllion), (2) Irganox 1010 (MW 1178), (3) Irganox 1078 (MW 530); (4) Cyanosorb UV-531 (MW 326), (5) trlphenylmethane (MW 244), (8) biphenyl (MW 154), and (7) toluene (MW 92). 7
t
I
4.5
5
5.5
6.5
6
7
7.5
ELUTION VOLUME (uL)
RESULTS AND DISCUSSION Efficiency studies were conducted using polystyrene of 2.2 million molecular weight to determine the exclusion volume (to), and toluene was used as the totally permeated probe. Column efficiencywas determined using reduced parameters18 according to the following equations:
h = Hld, = L/N(d,) where h is the reduced plate height, H the plate height, d , the particle size,L the column length, and N the plate number, measured for toluene, and v = pddD,
where Y is the reduced velocity, p the linear velocity, and D, the diffusion coefficient in the mobile phase, estimated for toluene as 8.33 X 10-6cm2/susing the Wilke-Chang equation.lg (17) Corks, H.J.; Richter, B. E.; Pfeiffer, C. D.; Stevens,T. S.HRC & CC, J. High Resolut. Chromatogr. Chrornatogr. Common. 1987,IO, 446-448. (18) Giddings, J. C.Dynamics of Chromatography; Marcel-Dekker: New York, 1965. (19) Wilke, C.R.;Chang,P. Am. Inst. Chem. Eng. J . 1955,I , 264-269.
Flgwr 2. Plot of log molecular we microcolumns packed with PLQEL, 50
'ET
t vs elution volume for
Column performance was also evaluated using the flow resistance factor, 4, specific column permeability, KO,and separation impedance,E, defied by the followingequation@
where
hp
4 = d,2hplcJltl (3) is the pressure drop, 9 the eluent viscosity, and
= d;/@
(4)
E = h24
(5)
KO
Figure 1 represents a typical chromatogram obtained on a series of compounds with various molecular weights, while Figure 2 is a calibration plot of elution volume v8 log molecular weight for the 5 0 4 pore size packing microcolumn. Table I summarizes the results obtained on column performance, while Figures 3-5 represent plots of reduced plate height vs reduced velocity obtained at 170,270, and 400 atm packing pressure as a function of slurry ratio (SR). (20) Knox, J. J . Chromatogr. Sci. 1980,18, 453-461.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993
Table I. Performance of Size Exclusion Chromatography Columns packing measure (atm)
8000
1000 -
KO
SRa h ( m i n )
170
40 20 10 40 20 10 40 20 10
270 400 conventional size column 1-m microcolumnb 50-A pore size 1ooo-A pore size
(X1010cm2) E
6
2.7 2.9 2.1 2.4 2.2 2.2 2.4 2.1 2.1 3.2
280 300 750 510 700 800 870 1250 1400 490
8.93 8.33 3.33 4.90 3.57 3.12 2.87 2.00 1.78 5.13
2040 2530 3210 2940 3330 3550 5010 5500 6820 4980
2.6 2.7
480 370
5.81 6.74
2950 2780
-
i: :/& 3000
2000 -
'
1000
l
'
l
'
l
'
i
'
l IO
20 20
SR,slurry ratio. Packed at 170 atm.
Flgure 6. Separation impedance (E) vs packing pressure as a functlon of slurry ratio for micro SEC columns: (0)SR 10, ( 6 )SR 20, (R) SR 40.
..
SR 20
SR 10
0
1
/
1
/
l
I
l 8
6
4
1
~
~
10
i
Flgure 3. Reduced plate height (h) vs reduced velocity (v) for microcolumns packed at 170 atm.
5208
{
li'
20'
30' Time
.SR
10
O
I
L
SR 20 SR 10
I
1 2
1
I 4
i
1 6
50'
KO
'0'
Flgure 7. Micro SEC chromatogram of standards: column, lOOcm X 250-pm 1.d. fused-silica packed with PL-GEL, 5 pm, 50-1(pore size; packing Conditions, 170 atm, slurry ratio 20; other conditions as In Figure 1.
1 -
0
40' (mi")
1
i
a
L Figure 5. Reduced plate height (h) vs reduced velocity (v) for microcolumns packed at 400 atm.
In all cases, minimum values of h below 3 were obtained. At the lower packing pressure (170 atm), best results were obtained using the most concentrated slurry (SR lo), while increases in the packing pressure to 270 and 400 atm minimized the effect of slurry ratio on reduced plate heights. Examination of the specific column permeability indicated a trend of decreasing permeability with increasing packing pressure and increasing slurry ratio, which may suggest
packing deformation (or crushing) during high-pressure column packing. Figure 6 represents a plot of separation impedance vs packing pressure e~ a function of slurry ratio. Results presented suggest that the best possible columns are those prepared a t 170 atm with SR 40, when all factors are taken into account. The data obtained suggest that long column segments are best prepared using these conditions, while short columns should be prepared at high packing pressure and high slurry concentration. Figures 7 and 8 represent chromatograms of standards obtained on long columns (1 m) packed at 170 atm with SR 20, using 50- and 1000-Apore size packings, respectively. Figure 9 represents the separations of a sample of polystyrene of 580 molecular weight obtained on the 50-A pore size columns. Column efficiencieswere calculated using the Foley-Dorsey approximation,2l which yielded 74 500 plates/m for the 50-1( pore size column and 73 000 plates/m for the 1000-A pore size column. Table I summarizes the results obtained on column performance. The results obtained correlate well with predictions made using Figure 6 and demonstrate that using the conditions described, columns prepared with large-pore packings have performance equivalent to those columns prepared with small-pore packings. It should be noted that to obtain equivalent efficiency using conventional size columns, relatively expensive systems with four columns would be necessary. (21) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983,55, 730-737.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1003
1470
5
Tlmi
lmlnl
Flguro 8. Micro SEC chromatogram of standards: column, lOOsm X 250-pm1.d. fused-silica pecked with PL-GEL, 5 pm, 1000 A; condtlonsas In Figure 7. Peaks: (1) polystyrene (PS) (MW 2.2 mllllon), (2) PS 485 000, (3) PS 68 000, (4) PS 22 000, (5) PS 12 500, (6) PS 5050, (7) Irganox 1010 (MW 1178), (8) Irganox 1076 (MW 530), (0) Cyanosorb UV-531 (MW826), (10) triphenylmethane (MW 244), (1 1) impurity, (12) blphenyl (MW 154), and (73) toluene (MW 02). 12046
1
I
T i m (.in)
Flguro 10. Comparison of resolutlon obtained for poly8tyreno 680 on (A, top) a conventional size column (3Osm X 8-mm 1.d. PL-GEL 50-A pore size, 5 pm; eluent, THF; flow rate, 0.8 mL/min) and (e, bottom) a micro SEC column (flow rate, 0 pL/mln). Other condtlons as in Figure 7.
mYII
=%Flgurr 0. Mlcrocolumn SEC chromatogram of polystyrene (MW 580). CondMns as In Figure 7.
In contrast to the results obtained using micro SEC columns, the conventional size column evaluated yielded a minimum value of h of 3.2 and separation impedance of 4980 (Table I). Although in theory it has been suggested that the column diameter does not play a role in column performance unless the dimensions are within the Knox-Parcher ratio?* in practice, fluctuations in packing densityacross the column1S and temperature gradients due to viscous friction23 are detrimental effects which can be minimized by using microcolumns. More importantly, however, we believe that the differences observed are not so much an issue of initially packing larger diameter columns of low h and low E, but rather a problem of maintaining these properties, even immediately after the initial column packing. We believe that the fundamental advantage of microcolumns is the narrowness of the column, which allows the walls to fully support and maintain even a nondensely packed bed structure. This effective wall support prevents the small particle shifta that occur in larger diameter (22) Knox, J.; Parcher, J. F. Anal. Chern. 1969,41, 1599-1602. (23) Halaez, I.; Endele, R.;Asshouer, J. J. Chrornatogr. 1976,112,37-
44.
'
2.3
10
I
20
30 ELUTION TIME (rW)
40
50
Flguro 11. Callbration curves for PL-Qel, 50 A: (0)conventlonakize column, (+) 1 0 h m microcolumn operated at 0 pL/min, and (). 100cm microcolumn operated at 0.65 pL/min.
columns, resulting in a loss of efficiency and the generation of higher back pressure. In the case of very large columns, (Le., process scale of 5-80-cm i.d.1 it is not possible to maintain low h and low E for even short periods of time without using some form of external column compression to maintain bed integrity. Upon the basis of these observations, we would expect to observe both higher h and E values in the larger 8-mm-diametercolumn than in the 250-pm-diameter microcolumns. It is interesting to note that the values of 4 obtained using long microcolumns are similar to those obtained using the conventional size column of one-third the length, an often overlooked advantage of packed fused-silica capillaries. In order to further demonstrate experimentallythe better performance of microcolumns for SEC,the 1-m small pore
1480 ANALYTICAL CHEMISTRY, VOL. 65. NO. IO. MAY 15. I993
0.021 for the microcolumn operated a t high linear velocity, and 0.033 for the conventional size column. Thew results further confirm the better performance of microcolumnsfor SEC when compared to conventional size systems. Because the polymeric packings used in SEC swell to a degree dependent on the solvent used, columns must not be allowed to dry out in order to maintain their performance. Figure 12 represents a photomicrograph obtained on a micro SEC column which w a ~purposely allowed to dry. Voids within the column are apparent and were caused by packing shrinkage upon drying. Attempts to restore the column performance by extended solvent flushingwere unsuccessful.
CONCLUSIONS
Flgure 12. Photomicrograph of micro SEC column which was allowed Io dry. indica1 ng column voids dde 10 packing shrinkage.
size column described above was operated at high linear velocity, and the efficiency and resolution obtained were comparedtovaluesobtainedon theconventional column using polystyrene 580. It should be noted that both systems were operated M yield equivalent retention times. Figure 10 represents the chromatogramsobtained. The plate numbers obtained for the last peak in the chromatogram were 56 500 forthemicrocolumnand 18 800for theconventionalcolumn, a 3000b increase in efficiency for microcolumns without any increase in analysis time. This increase in efficiency is also observed for the higher oligomers, although the gain is not as dramatic (50% gain). Resolution in SEC is typically describedusingtheresolutionfactor,D~,whereDistheslope ofthelinearportionofthecalibrationcurveandaisthepeak standard deviation, measured for a small molecule and represented by the following equation:
Do = d log M/dV
(6)
Calibration curves obtained using polystyrene 580 are included in Figure 11. The values of Du obtained were 0.017 for the microcolumn operated at optimum linear velocity,
The conditions for the preparation of highly efficient mien, SEC columns with 50-A pore size packings have been determined. Said conditions were also applicable to the preparation of microcolumns with large pore size packings (loo0 A). Higher packing pressures yielded lower plate heights, hut with decreased specific column permeabilities. The effect of slurry ratio on plate height was found to be significant only a t the lowest packing pressure (170a h ) . However, a correlation between K" with decreasing slurry ratio was found for each packing pressure. Flow resistance factors and separation impedances calculated suggest that long column segments (100 em) are best prepared a t low packing pressures with dilute slurries. Comparison of the results obtained indicates better overall performance for the micro SEC columns prepared by packing a t 170and 270 atm when compared to a conventional size column. The optical transparency of fused-silicacolumnsallowsvisual observation of the column packing, which indicated that columns must not be allowed to dry to maintain their original performance.
ACKNOWLEDGMENT The authors thank Steve Erskine, Duane Krueger for the photomicrograph, and Tom Mffiee for his support.
RECEIVEDfor review September 15, 1992. Accepted February 4,1993.
