1708
Anal. Chem. 1984, 56, 1708-1711
Semimicro Size Exclusion Chromatography for Polymers: Column Packing Procedure and Operational Variables Sadao Mori* and Masami Suzuki Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 514, Japan
Polystyrene gels of partlcle dlameter 10 i 2 pm were packed Into 1.5 mm 1.d. and 1.8 mm 1.d. columns by the balanceddensity slurry-packlng technique under a constant flow rate of 350 pL/min. The number of theoretical plates ( N ) of these columns was 30 000 plates/m for a 1.5 mm 1.d. column and 36 000 plates/m for a 1.8 mm 1.d. column at flow rates of 20 and 30 pUmln, respectlvely. These values were comparable to conventlonal SEC columns. The slurry solvent was a mixture of toluene and chloroform (52.7/47.3, v/v). Removlng flne fragments below 1 pm was the most Important factor for obtalnlng high-quallty columns. Optimum packlng flow rate lay between 300 and 350 pL/mln. Effect of a moblle phase velocity on N was observed and the moblle phase veloclty whlch produced the maxlmum value of N was about 0.4 mmh. The effects of lnjectlon volume and concentratlon on peak wldth at half helght and retentlon volume were examined and discussed.
In high-performance liquid chromatography (HPLC), procedures for producing high-efficiency columns have been published by several authors, among which the method by Siemion seems to be the newest one (I). However, these procedures are mostly for packing of rigid solids such as silica gels and silica-ODs. The procedures for packing hard gels such as polystyrene (PS) gels for size exclusion chromatography (SEC or GPC) are not well known. The only obvious method is the balanced-density slurry-packing technique used for preparing columns of hard gels (2), but details are not known. To know how to pack PS gels into a column was the first purpose of this work. Miniaturization of HPLC is useful for saving on solvents in addition to fabricating columns a t reasonable costs in lengths. One of the authors (S.M.) has already studied the preparation of high-resolution micro-SEC colunns and the application to separation of oligomers of epoxy and phenolformaldehyde resins (3). However, precise control of a mobile phase velocity in micro-SEC is not easy, due to lack of a reliable pumping system. In SEC for polymers, as molecular weight averages and distributions are calculated by knowing the relationship between molecular weight and retention volume from a calibration curve of the SEC column system, reproducible measurement of retention volumes is required in addition to improvement in resolution. The effects of operational variables and the optimum conditions in reduced column diameters might be different from those in 8 mm i.d. columns (conventional SEC). Therefore, semimicro-SEC for polymers with 1.5 mm i.d. or 1.8 mm i.d. columns should precede micro-SEC for establishing optimum procedures. This was the second purpose of this work.
EXPERIMENTAL SECTION Apparatus. Column packing and SEC measurements for operational variables were performed on a Jasco TRIROTAR-V high-performanceliquid chromatograph (Japan Spectroscopic Co., Ltd., Hachioji, Tokyo 192, Japan) which has a triple piston pump
with 50-pL cylinders for smooth delivery of 10 pL/min to 9.9 mL/min at a 10-pL interval in the semimicro mode and at a 0.1-mI. interval in the normal LC mode. An ultraviolet absorption detector Model UVIDEC-100 IV (Jasco) was used with a semimicro flow cell (0.5 mm i.d. X 5 mm length; cell volume 1 fiL). Sample injection was made by using a variable loop Model VL-611 and sample injection volume was regulated by time, Column dimensions for semimicro SEC were 1.5 mm i.d. X 25 cm length or 1.8 mm i.d. X 50 cm length and polystyrene gels were packed according to the procedure below. A differential refractometer Model SE-11 (cell volume, 8 pL) and a Shodex A80M high-performance SEC column (50 cm X 8 mm id.) were used for comparison purposes. Column Packing Procedure. Polystyrene (PS) gels used in this experimentwere taken from a Shcdex A80M HPSEC column, which was distributed by Shoko Co., Ltd., Minato-ku, Tokyo 105, Japan, packed with a mixture of PS gels of nominal exclusion limits of lo3,lo4, lo6, and lo6 A. A slurry reservoir (a packer) was an empty column of 7.2 mm i.d. X 25 cm (packer A) or 7.2 mm i.d. X 50 cm (packer B), which was attached by a stainless in. reducing union (Swagelock),followed by a steel 3/8 in.-1/8 in. reducer (Swagelock) to the inlet of a chromatographic column blank of 1.5 mm i.d. or 1.8 mm i.d. Packer A was used for the 1.5 mm i.d. column and packer B the 1.8mm i.d. column. The 1/8 in.-1/16 in. reducer with the end fitting (with 2-pm frit) was attached to the outlet of the column blank. Before packing PS gels into a column, fines were first removed. This was accomplished by mixing 1.5 g of slightly dried PS gel and 100 mL of tetrahydrofuran (THF),allowing the mixture t o stand an hour until the main fraction was settled, and then pouring off the supernatant. This process was repeated twice and the remaining THF was evaporated at room temperature. The balanced-density slurry solvent was a mixture of toluene and chloroform (52.7/47.3, v/v), which has a similar density to PS gel. A 0.5-g portion of PS gel was swelled in 20 mL of the solvent for the 1.8 mm i.d. column and a 0.2-g portion of PS gel was swelled in 12 mL of the solvent for a 1.5 mm i.d. column. A 1.2-mL aliquot (for a 1.8 mm i.d. column) or a 0.5-mL aliquot (for a 1.5 mm i.d. column) of the slurry solvent was poured into the top of the reservoir-column assembly, followed by the dispersed and degassed slurry through a 300-mesh copper sieve in order to remove aggregates. The reservoir was then filled by the same solvent until filled. The reservoir was then attached to the pumping system by means of the 1 / 8 in.-1/16 in. reducer (Swagelock). The slurry solvent was pumped into the reservoir at the flow rate of 350 pL/min for 4 h (in the case of a 1.8 mm i.d. column) or 3 h (a 1.5 mm i.d. column) by the down-flow method. After being packed, the column was removed from the reservoir-column assembly, an inlet end fitting with a 2-pm frit was attached, and then the column was equilibrated by pumping THF at 40 fiL/min for 3 h. Gel weight packed into a 1.8mm i.d. column was about 270 mg at dry base. Determination of Column Efficiency and Operational Variables. The number of theoretical plates (N), which is the measure of the column efficiency, was determined by injecting 2 fiL (3 s) of 1%benzene solution at a flow rate of 40 pL/min. The mobile phase was THF. Detection was made at 254 nm and at 0.32 AUFS, which was enough to produce a 7040% deflection on the recorder. The value of N was calculated by measuring the width of the peak at half height. Samplesused for the measurementof operationalvariables were polystyrenes of narrow molecular weight distribution purchased from Pressure Chemical Co., in addition to benzene.
0003-2700/84/0356-1708$01.50/00 1984 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984
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Volume (mL) Flgure I. A semimicro-SEC chromatogram of benzene obtained on two 1.8 mm i.d. X 50 cm columns: flow rate, 40 pL/min; column pressure, 16 kg/cm2;sample volume, 2 pL of 1% benzene; attenuation, 0.32 AUFS.
100 200 300 400 500 600 Flow Rate (ULlrnin)
Retention
RESULTS AND DISCUSSION Packing Procedures. To establish the optimized column packing technique, the effects of fine particles in the mass of gels, packing flow rate, and packing pressure on the column efficiency were estimated. A small quantity of fine particles below 1pm remained in the gels which have been taken from a used Shodex A80M column and exerted a negative influence on the column efficiency. On inspection of the gel particles by microscopy, it was found that these fine particles resulted from fragmentation of main particles (particle diameter 10 f 2 pm) during packing and depacking processes. In order to sieve these fine fragments,several decantations are effective, as mentioned in the Experimental Section. To remove aggregates, filtration of a slurry by using a copper sieve is also valid. By these processes, a high-quality column such as 32 000 plates/m (HETP = 31.3 pm) has been obtained by using a 1.8 mm i.d. column under the conditions mentioned in the Experimental Section for measuring column efficiency. The value of N is comparable to conventional SEC (4). Figure 1 shows a chromatogram of benzene obtained by using two 1.8 mm i.d. X 50 cm columns. A symmetrical peak has been obtained with very small tailing. Without or with insufficient sieving of the fine powder, the value of N decreased and at an extreme case it was 4600 plates/m. For estimation of the optimum flow rate for packing, a 1.5 mm i.d. column was packed at different flow rates and the relationship between them and the values of N was plotted as shown in Figure 2. The most suitable flow rate for packing lies between 300 and 350 pL/min. Final column pressure is also shown in Figure 2. When a 1.5 mm i.d. column was packed at flow rate of 350 pL/min, the values of N lay between 24000 and 28000 plates/m and those for a 1.8 mm i.d. column were between 30000 and 32000 plates/m. The difference in N between the two is probably due to the difference in linear velocities of the mobile phase in two columns when the value of N is measured (this will be discussed later) and the difference of the ratios of the gel volume and the interstitial volume in them (this will be discussed elsewhere). When the column was packed under the constant pressure, e.g., 25 kg/cm2 for a 1.5 mm i.d. column (25 cm), the values of N were 6 0 4 0 % of those obtained under the constant flow. In order to estimate the effect of packing pressure under the constant flow, four 1.5 mm i.d. columns were connected in series and gels were packed into them at a flow rate of 350
1709
Flgure 2. Relationshlp between packing flow rate and the number of theoretical plates: column, 1.5 mm i.d. X 25 cm.
ol5-mmid
s 18-mmi d x 8-mm I d
n60t
\
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Velocity ( m m / s )
Flgure 3. Effectof mobile phase velocity on plate height for different diameter of columns: sample concentratlon, 1 % benzene solution; sample volume and detector for 1.5 mm i.d. and 1.8 mm i.d. columns, 2 gL, and UV; for an 8 mm i.d. column, 50 pL and RI.
pL/min using packer B. The final pressure was 100 kg/cm2 and was obtained after 1 h. The total value of N was 25 000 plates, N of each column being 6000, 6400, 6800, and 6100 plates from the outlet of the packer in this order. On the other hand, when packed under a constant pressure of 100 kg/cm2, the value of N of each column was not the same and they were 6200, 3500, 6400, and 3400 in the same order, respectively. From these results, it may be stressed that much importance should be attached to the flow rate of packing solvent in order to get a high-quality column and that a constant-flow technique is preferable to a constant-pressuretechnique. Pressure monitoring in progress of packing is very effective to foresee the column efficiency. A smooth and rapid rise in pressure to the prescribed one and then holding the pressure constant result in producing a high-quality column. Operational Variables. Effect of mobile phase velocity on HETP (height equivalent to a theoretical plate) is shown in Figure 3 with the data of a conventional SEC column for comparison purpose. The linear velocity of mobile phase was calculated by dividing flow rate by an effective cross section area which was obtained as the product of a cross section area of a column and the ratio of interstitial volume Voin column and column volume V,. The value of Vo was assumed to be the retention volume of PS, having molecular weight 8500000, subtracted from the dead volume of the SEC system. The dead volume was estimated by injecting a specified volume of a benzene solution into the SEC system which disconnected a column from the system and connected it between the pump and injector. The effective cross section areas of columns
1710
ANALYTICAL CHEMISTRY, VOL. 56, NO. 9, AUGUST 1984
t
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is 2 4 6 8 10
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Injection Volume (uL) Flgure 4. Effect of injection volume on peak width at half height: column, four 1.5 mm 1.d. X 25 cm length; sample (1) benzene, (2) polystyrene (PS) (molecular welght (M) = 2100), (3) PS (M = 20400), (4) Ps (M = 1.8 X io5); (5) PS (M = 1.8 x io6);concentration, 0.1 %; flow rate, 40 pL/min. 0.1
employed were 0.00845 cm2for a 1.5 mm i.d. column, 0.012 12 cmz for a 1.8 mm i.d. column, and 0.2267 cm2 for an 8 mm i.d. column. The mobile phase velocity which produced the minimum value of HETP was around 0.4 mm/s and this velocity corresponds to 20 ML/min for a 1.5 mm i.d. column and 30 pL/min for a 1.8 mm i.d. column, respectively. At the minimum, the values of HETP and N were 32.75 pm and 30000 plates/m for a 1.5 mm i.d. column and 26.2 pm and 36000 plates/m for a 1.8 mm i.d. column. Contrary to data for semimicro SEC, an 8 mm i.d. column shows a decreased tendency in HETP with increasing mobile phase velocity over 1.47 mm/s (2.0 mL/min). The value of HETP at 0.4 mm/s (0.54 mL/min) was 35 pm (N = 28600 plates/m), that at 0.735 mm/s (1.0 mL/min) was 25.8 pm (N = 38800 plates/m), and that at 1.47 mm/s (2.0 mL/min) was 21.9 pm ( N = 45600 plates/m). A reduced plate height for an 8 mm i.d. column was minimum 2.2 particle diameters for totally permeating benzene and that for a 1.8 mm i.d. column was 2.6. The results obtained on a 7.8 mm i.d. column by Kirkland (5) were similar to our results and the minimum HETP was obtained a t around 1.5-2.5 mm/s by injecting toluene as a solute. Similar results were also obtained by Limpert et al. (6) on a 7.6 mm i.d. column. Though the reason for the difference of the linear velocity which produces the minimum HETP between semimicro SEC and conventional SEC is not clear, it can be said that the optimum mobile phase velocity decreases with decreasing column diameter. Therefore, the value of HETP on a column of a smaller diameter becomes higher at the optimum velocity from a column of a higher diameter. These results agreed with those obtained by Kirkland (5). The effect of injection volume on peak width at half height ( WllZ) was shown in Figure 4. The peak width of benzene was unchanged below 6 p L and increased gradually with increasing injection volume over this point. These data were obtained on 1.5 mm i.d. columns. Also, no differences in peak width of benzene were found below 10 pL on 1.8 mm i.d. columns. Peak widths at half height of polystyrene samples increased as injection volume increased. These findings have been reported on conventionalSEC columns, where the effect depended on also sample concentration and flow rate (7). Though the increase in peak width with injection volume was found for PS samples, the injection volume up to 10 pL will be acceptable without serious loss of resolution except for the case of PS, molecular weight 1.8 X 106. As PS samples have molecular weight distributions, the observed peak width
0.2
0.3
0.0
Concentration ("lo) Flgure 5. Effect of concentration on peak width at half height: column, two 1.8 mm i.d. X 50 cm length; sample (1) benzene, (2) PS (M = 2100), (3) PS (M = 20 ~ o o )(4) , PS (M = i.Ex (5) PS (M = 4.11 X lo6), (6) PS (M = 6.7 X lo5), (9) PS (M = 1.8 X 10'); injection volume, 8 pL; flow rate, 40 pL/min.
107,
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16
-
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I
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I
20
30
40
Injection Volume (pL) Figure 6. Effect of injection volume on retentlon volume. Conditions are same as those in Figure 4.
is the sum of peak broadening due to the spreading of PS along the column and the molecular weight distribution. The decreased amount of peak width of PS with decreasing injection volume can, therefore, be regarded as the part of the normal peak broadening within the column. It can be stressed, especially to the higher molecular weight samples, that the less injection volume will give a better result if it does not result in the serious loss of peak response. Figure 5 shows the effect of concentration on peak width at half height which was found for benzene to be independent of concentrations studied. Similarly, peak widths of PS samples below molecular weight 4.11 X lo6were almost unchanged below 0.2%. However, those of PS over molecular weight 6.7 X lo6increased with increasing concentration from 0.05%. The concentration effect on peak width of a polystyrene sample has been reported by several workers (7-9). The effect of concentration on peak width at half height has also depended on injection volume and flow rate. When four 8 mm i.d. x 30 cm columns were used, the concentrationdependence of peak width was observed over a 0.25-mLinjection, whereas the peak width at injection volume of 0.1 mL was found to be independent of concentration (7). Peak width was also independent of concentration at high flow rate (8). The relationships of retention volume as a function of injection volume and concentration are shown in Figures 6 and 7, respectively. Retention volume increased with increasing
1711
Anal. Chem. 1984, 56, 1711-1715
Retention volume increased with increasing sample concentration, which was similar to that reported previously using conventional SEC columns (7, 10, 11).
ACKNOWLEDGMENT The authors wish to express their appreciation to Yasuhiko Nishimura for technical assistance. Registry No. Polystyrene (homopolymer), 9003-53-6.
l5I-
LITERATURE CITED 1
- 41
01
02 0.3 04 Concentration (%)
Figure 7. Effect of concentration on retention volume. Conditions are the same as those in Figure 5.
injection volume and the amount of variation is significant below 10 pL. This observation is similar to the result on the conventional SEC columns, where the amount of increase in retention volume when the injection volume increased from 0.1 to 0.25 mL was 15-20 times larger than that from 0.25 to 0.5 mL (7).
(1) Siemion, C. C. J. Liq. Chromafogr;,1983, 6 , 765-775. (2) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography”, 2nd ed.; Wlley: New York, 1979;pp 217-219. (3) Takeuchi, T.; Ishii, D.; Morl, S. J. Chromafogr. 1983, 257, 327-335. (4) Mori, S. J. Cbromafogr. 1979, 174, 23-33. (5) Kirkland, J. J. J. Chromafogr. 1976, 125, 231-250. (6) Limpert, R. J.; Cotter, R. L.; Dark, W. A. Am. Lab. (Fairfield, Conn.) 1974, May. 63-69. (7) Mori, S.J. Appl. Polym. Sci. 1977, 2 1 , 1921-1932. (8) Little, J. N.; Waters, J. L.; Bombaugh, K. J.; Paupils, W. J. J. folym. Sci., Pari A-2 1969, 7 , 1775-1783. (9) Chuang, J.-Y.; Johnson, J. F. Sep. Sci. 1975, 10, 161-165. (IO) Boni, K. A.; Siiemers, F. A.; Stickney, P. B. J. Polym. Sci., fart A-2 1968, 6,1567-1578. (11) Spatorico, A. L. J. Appl. Polym. Sci. 1975, 1 9 , 1601-1610. ’
RECEIVED for review March 6, 1984. Accepted April 18, 1984.
Some Fundamental Aspects of Surface-Film Analysis with Variable Angle Ultrasoft X-ray Fluorescence Spectrometry George Andermann* and Francis Fujiwara
Department of Chemistry, University of Hawaii at M a m a , Honolulu, Hawaii 96822
The use of varlabie angle ultrasoft X-ray fluorescence spectrometry Is suggested as a nondestructive method for “real world” surface-flim analysis. The fundamental aspects of the film emlssion, substrate line attenuatlon, and ultrasoft scattered X-rays are evaluated. I t Is shown that In the ultrasoft X-ray region the fluorescent radiation escape depth reaches values around lo2 A at low exlt angles and lo3 A at conventional exit angles, thus providing the possibility of sample depth proflling wlth the film emission method. The substrate line attenuatlon technique is proposed for thln film structural and thickness studies. The use of ultrasofl scattered X-rays Is suggested as an internal standard In surface-film analysis as well as for correcting for surface lrreguiaritles at grazing angles.
In discussing surface analysis Hercules (1) has recently pointed out a number of pertinent features. Most importantly he cited that “true” surface analysis involves the outermost 1-5 atomic layers of a solid. According to Hercules, of the large number of spectroscopic techniques XPS (X-ray photoelectron spectrometry),AES (Auger electron spectrometery), SIMS (secondary-ion mass spectrometry), and ISS (ionscattering spectrometry) appear to be most useful for such analysis. All of these methods (and UPS (ultraviolet photoelectron spectrometry) also) are based on the quantitative manipulation of ejected particles (electrons or ions). All of 0003-2700/84/0356-1711$01.50/0
the above are associated with escape, Le., critical depths, d,, which are in the range of about 5-25 A, thus fulfilling the requirement of a “true” surface analytical tool, and, therefore, automatically requiring the use of UHV (ultrahigh vacuum) conditions at least in the sample chamber. Photon methods were indicated by Hercules to involve d, values of lo3 8, in the IR region and lo4 8, using 103-eV X-rays. Clearly, therefore, according to the viewpoint suggested by Hercules none of the photon methods could be considered to be “true” surface-thin film analytical tools. Aside from the importance of clearly delineating which spectroscopic techniques fulfill the role of “true” surface characterization, Hercules’ review is useful also because it shows that none of the techniques reviewed by him have d, values in the range of 50 8, to lo3 A, indicating a serious gap in analytical capability. As indicated by Hercules there are at least two other fundamental capabilities that need to be looked at in comparing the various techniques, and these are spatial resolution and chemical speciation capabilities. Only AES has microprobe capabilities and only XPS, UPS, and AES provide chemical speciation capabilities. Not reviewed by Hercules specifically is the Rutherford backscattering (RBS) method which has d, values in the range of 50 to lo3 8, but which provides only atomic information and cannot be used as a microprobe. Hercules’ comments on “true”surface analysis are especially noteworthy for solid samples for which there is a reasonably sharp distinction between bulk and surface properties and where surface properties are present for layers which are 25 0 1984 American Chemical Society
