Langmuir 1993,9, 2297-2300
2297
Steric Exclusion Chromatography of Nanometer-Sized Gold Particles T. Siebrands, M. Giersig, P. Mulvaney, and Ch.-H. Fischer' Hahn-Meitner-Institut, Abteilung Photochemie, D-1O00,Berlin 39, Germany Received April 14,1993. In Final Form: June 14,1993
It is shown that steric exclusion chromatographycan be used to measure the size distribution of colloidal gold sols. Good separation can be achieved for particles ranging in size from 3.0 to 20 nm using columns containing 25-pm silica particles (pore size 50-100 nm) and with aqueous trisodium citrate (0.001 M)as the eluent.
Introduction Particle sized r a m a t i d y affects the catalytic, electronic, and optical properties of nanometer-sized metal particles. However comparison of experimental data with theory is continually hampered by the difficulty in preparing reasonably monodisperse samples in this size regime. The purpose of the present communication is to demonstrate the utility of steric exclusion chromatography as a means for measuring and improving the size distribution of metal sols in the nanometer size regime. Steric exclusion chromatography (SEC) has recently been used to prepare CdS sols with narrow size distributions.'s2 The standard deviation of the particle size in these sols after chromatographic separation was only 8%. However to date, SEC has not been applied to metal sols, although metal sols have recently been used for the characterization of chromatographic c o l ~ m n s . ~ SEC offers a number of advantages over other methods such as electron microscopy, UV/vis spectroscopy, or fluorescence spectroscopy. The size distribution can be determined within a few minutes when HPLC is used. In HPLC the same separation principle is used as in low pressure chromatography, except that the particles of the column are much smaller. Greater surface and smaller interstitial volume result in higher efficiency and speed. SEC is nondestructive, and when used in conjunction with diode array spectrophotometers or other detectors provides direct, in situ information on the properties of the particles. In the case of quantized semiconductor particles, the existence of preferred crystallite sizes ("magic numbers") could be inferred from the sequence of absorption spectra obtained during a SEC run.4 We elected to use gold sols to test the usefulness of SEC because they are readily prepared in colloidal form and have size-dependent optical ~ p e c t r a . ~The ' width of the surface plasmon band present in the optical absorption spectrum of these metal colloids is known to depend inversely on the particle size. In this paper it is demonstrated that good separation of sols is possible. Spectral evidence is also provided for the existence of nonmetallic, i.e. quantized, gold particles in the gold sols with the smallest particle size. (1) Fiecher,Ch.-H.;Lilie, J.; Weller, H.;Kataikae,L.;Henglein,A.Ber. Bunsen-Ges. Phys. Chem. 1989,93,61-64. (2) Fiecher, Ch.-H. Submitted for publication in Langmir. (3) Holtzhauer, M.; Rudolph, M. J. Chromatogr. 1992,605,193-198. (4) Fischer, Ch.-H.;Weller, H.; Kataikas, L.; Henglein, A. Langmuir 1989,5,429-432. (5) Kreibig, U.; Gemel, L. Surf. Sci. 1986,156, 678-700. (6) Kreibig, U. J. Phys. (Paris) 1977, 38, C2-97-103. (7) Doremus, R. H. J. Chem. Phys. 1964,40,2389-2396.
0743-7463/93/2409-2297$04.00/0
Experimental Section Gold sols with mean particle sizes in the range 14-20 nm were prepared according to the method of Turkevich et al.B Trisodium citrate15 mL (1% (w/v))was added to a boiling solutionof KAuCL (285 mL 0.1 % which was then refluxed for 20 min and allowed to cool. Particles with smaller sizes were prepared by using a mixture of trisodium citrate and tannic acid (Mahchrodt product no. 8835) as reducing agent? KAuC4 (85 d, 0.1%) was heated to 60 O C and stirred rapidly. A second reducing solution was prepared by mixing trisodium citrate (4 mL, 1%) tannic acid (0-5 mL, l%), and an equivalent amount of K&Os (0-5 mL, 0.01 M) and making up to 25 mL. This solution was also heated to 60 O C and then added rapidly to the chloroaurate solution. The color of these sols developed almost instantly. The solution was then boiled for several minutes and allowed to cool. Tannic acid increases the rate of nucleation, thereby generatingsmaller particles. The higher the tannic acidcitrate ratio, the smaller the particle size. The lowest size achievable was found to be about 2-3 nm. The sols so prepared were stable for months, although a slow sedimentation of tannic acid was obee~edover time. The particle size dietzibutionswere measured from electron micrographs taken with a Philips CM 12 electron microscope. All sizes refer to the average size determined from histograms of at least 200 particles. The standard deviationwas obtained by fitting Gaussian curves to the hiatograme. For all sols used the deviation was less than 12 % . The chromatography apparatus consisted of a Merck-Hitachi pump, type L 6oO0, and a Waters photodiode array detector, type 990. The diode-arrayspectrophotometerhaa the advantage that it allows not only editing of the absorption spectra of the separatedspeciea but ale0the editingof chromatograms at various wavelengths from a single run. A set of two 120 mm long Knauer columns (4 mm internal diameter) were used, the f i t packed with Nucleosil500 (15-25 pm) and the second with Nucleoeil lo00 (15-25 pm) from Macherey and Nagel. The mobile phaee was an aqueous solution of 1x 10-9 M trisodium citrate (Merck) and the sample volume 20 pL. Results and Discussion Steric exclusion chromatography (SEC) utilizes porous material as the stationary phase. The accessibility of the pores by diffusion is dependent on the size of the anal@. The smaller the analyte, the more readily it can penetrate into the pores, thereby hindering ita progress through the column, while larger species are transported forward more easily with the mobile phase. In SEC any interaction between the analyte and the stationary phase must be reduced as much as possible. Cs and CISmodified silica with pore widths varying between 30 and 100 nm were previously found to be superior over unmodified ones for inorganicallystabilizedsemiconductor particlesin aqueous (8) Enlleth, B. V.; Turkevich, J. J. Am. Chem. SOC.1963,86,33173328. (9) Slot, J. W.; Geuze, H. J. Eur. J. Cell. Chem. 1985, 38,8743.
0 1993 American Chemical Society
2298 Langmuir, Vol. 9, No. 9, 1993
Siebrands et al.
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solution, because the organic surface diminished particle adsorption. lo However the metal particles investigated in this work are stabilized by organic anions. On modified silica this would result in a mixed size exclusion/reversed phase mechanism. Therefore an unmodified stationary phase was chosen, consisting of two columns in series with 50- and 100-nm pores, respectively, which previously gave good separation for semiconductor particles ranging in size from 1.3 to 20 nm. It was shown in previous studies that for the chromatography of nanosized particles the eluent has to contain a stabilizer, because weakly bound ligands desorb from the particles during the chromatographic process leading to colloid coagulation. Both tannin and citrate solutions were tested as eluents, and they displayed quite different (10) Fischer, Ch.-H.; Giersig, M.Langmuir 1992,8, 1475-1478.
behavior. Tannin, a nonuniform natural molecule with a molecular weight of about 1700, absorbs light strongly below 360 nm. Citrate is transparent right down to below 300 nm and showed better resolution as well as a more expandedretention range, but very large particles (20 nm) were partially eliminated by adsorption, which was evident from the much smaller peak height and the poorer signalto-noise ratio. Because the stabilizing effect of citrate was sufficient for most of the particles, it was used for all further studies. The chromatography functioned best with a stationary phase consisting of silica particles with sizes 15-25 pm, whereas most of the colloidal metal particles were irreversibly retained on 7-pm silica. The latter has a greater surface and maybe also a higher surface activity. Figure 1 shows electron micrographs of gold sols of different sizes, the resulting diameter histograms and the
Langmuir, Vol. 9,No. 9,1993 2299
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chromatograms, produced by monitoring the solution absorption as a function of time at 520 nm. The elution times varied from 2.30 to 2.95 min. In SEC of organic polymers there is a well-established semilogarithmic relation between molecular weight and elution volume or elution time." According to Fischer et al.,l a plot of the logarithm of the particle size as a function of elution time is linear if the particles are being separated purely by steric exclusion (the weight of a spherical particle is proportional to the cube of the diameter). A typical semilogarithmic calibration plot is shown in Figure 2. Excellent linearity is obtained over a wide range of particle sizes from 19.9 to 2.9nm. The size resolution of the method is not constant over the diameter range because of the logarithmicrelation. The elution time resolution is 0.03min, which corresponds to 1.4 nm or 18%, when a medium particle size of 8 nm is considered. However this value improves for smaller particles and the resolution can be further enhanced by the use of longer columns. The advantage of shorter columns is the high speed which is often necessary for unstable colloids. In order to get an idea about the band broadening due to factors other than particle size distribution, one can compare the peak widths of the colloidal samples, which lie in the range 0.28-0.6 min, with those of a small molecule or salt,e.g. acetone or potassium iodide, which are 0.18 min. The difference reflects the size distribution of the particles. Earlier work by Doremus' and KreibigG has shown convincinglythat the interband absorption below 460 nm should be independent of particle size,whereas the surface plasmon absorption band of smaller particles is damped by enhanced surfacescattering of the conduction electrons. This allows us to show spectroscopically whether smaller particles are retained on the column longer than bigger particles and also provides a sensitive test of the stability of the sols in the column. The results obtained on two gold sols are shown in Figure 3a. The upper curve shows the normalized elution curves for the two particle sizes. In the middle part, the ratio R1= E52o/EWis shown. During the elution itself, R1 is virtually constant demonstrating that both sols consist of gold particles with a very narrow size distribution. The ratio drops to zero abruptly as elution of the sol finishes. The smaller particles (diameter 40A) (dashed line) show a similar response but are eluted much later (2.58min). As can be seen, with the column length used, the separation of particles 145 A in size and 40 A in size is good; however longer columns would be needed to ensure complete separation of particles more similar in size. The ratio E520/Ew is only 1.07 for 40-A particles but is 1.73 for 145-Aparticles. This ratio is very sensitive to polydispersity in the sample. In the case of (11)Granath, K.;Flodin, P.Makromol. Chem. 1961,48,160-171.
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Figure3. (a,top) Chromatogramsof two gold solswith different particle sizes: upper part, normalized chromatograms; center, lower part, ratio chromatoratio chromatogram R1= Esm/Ec(~; (b, bottom) The ratios R1 and Ra are a gram RS = Err~lE~m. function of the retention time.
the smaller particles, a slight decrease in R1 during elution can be seen in Figure 3a. This may indicate that some smaller particles are indeed present, which are eluting slightly later. In both cases R2 = E d E w is constant at about 0.95. This is in accord with the earlier results on gold sols' which also showed that there was virtually no size dependence of the interband transitions in gold particles in this size regime. More importantly, it demonstrates that the ratio R1 can be used to determine the size of gold particles eluting at any time from the column, even in the absence of a calibration curve based on electron microscopy. In Figure 3b, the ratio R1 and R2 are shown as a function of the retention time. On the upper abscissa, the actual particle size is shown using the calibration data from Figure 2. As can be seen, the chromatographic separation separates larger particles first (high R1) and smaller particles later. Good linearity is obtained. This confirms that the gold colloids can be eluted without undergoing any coagulation in the chromatographic column. Kreibig has previously compared the literature values of R1 as a function of particle size for nanosized gold particles? A comparison of his data and ours is shown in Figure 4. The agreement is good, although our values show a little more scatter than his.
2300 Langmuir, Vol. 9, No. 9, 1993
Siebrands et al.
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Figure 5. (Inset) Chromatogram of 2.9 nm diameter gold sol measured at 520nm, showingelution of twospecies. (A) Spectrum of a gold sol at 2.58 min. (B)Spectrum obtained at 3.05min. (C) Spectrum obtained from a 0.1 7% tannin solution at 3.07 min. The samples eluting at the longest time (2.62 min) and containing the largest tannin concentrations showed evidence for another species eluting after the sol particles (which had a mean diameter of 2.9 nm). A chromatogram at 520 nm is shown in Figure 5 (inset). At 3.05 min a species is eluted with the spectrum B in Figure 5. It has an absorption maximum at 360 nm but does not possess a plasmon absorption band at 520 nm. Tannin itself is yellow and does not absorb at 520 nm although it too elutes at almost the same time (3.07 min). The spectrum of
tannin as it elutes is also shown in Figure 5 (spectrum C) for comparison. Electron microscopy of this sample prior to chromatographic separation did not show any significant population of particles other than the 2.9-nm gold sol. The species eluting at 3.05 min was separated by micropreparative SEC and reexamined by electron microscopy. Gold particles with a size of about 2 nm were seen. EDAX analyses of these ultrafiie particles confiimed the presence of gold. However the elution time corresponds to particles only a few tenths of a nanometer in diameter. One possibility is that some of the primary gold nuclei become encased in tannin and are unable to grow. These nuclei then elute with the tannin. However at the present time we are unable to identify the species with spectrum B. The results presented here demonstrate the complementary uses of SEC and electron microscopy. The SEC needs the electron microscope data in order to establish the calibration curve, otherwise only relative size distributions can be measured. But SEC gives a more statistical view of the size distribution because it measures the distribution of all the particles in the probe in situ. The clumping that often occurs during electron microscope grid preparation is avoided. The last experiment also demonstrates another useful feature of SEC coupled with DAD. During the chromatography the excess tannin stabilizer is separated (it is smaller than the particles and therefore elutes later). Only after this separation was it possible to observe the presence of the species eluting after the gold sol particles. In the bulk spectrum it was hidden under the strong absorption of the tannin.
Conclusions Colloidal gold sols with a narrow size distribution have been used to determine the suitability of steric exclusion chromatography for measuring the size distribution of nanosized metal particles. Good clear separation has been achieved with conventional silica columns using citrate as the eluent. The optical properties of colloidal gold (2.920 nm) prepared by citrate or tannin reduction were shown to be in accord with the predictions of the surface scattering model. Evidence for the presence of ultrafiie gold nuclei in solutions containing tannin were obtained after separation of the reducing agent by SEC. Acknowledgment. The authors thank Professor A. Henglein and Professor U. Kreibig for useful discussions and Ms. U. Michalczik for helpful assistance with the laboratory work.
