ANALYTICAL SCIENCES APRIL 2001, VOL.17 Special Issue 2001 © The Japan Society for Analytical Chemistry
s571
Characterization of Environmental Hydrocolloids by Asymmetric Flow Field-Flow Fractionation (AF4) and Thermal Lens Spectroscopy Andreas Exner, Ulrich Panne♣, Reinhard Niessner Institute of Hydrochemistry, Technical University of Munich, Marchioninistrasse 17, D-81377 Munich, Germany Hydrocolloids represent an important migration path for anthropogenic contaminants in natural aquatic systems. The utilized asymmetric flow field-flow fractionation (AF4) is a powerful tool for separation of colloids, however sensitive detection schemes are needed for the low particle concentrations encountered in natural systems. In this work, detection schemes based on conventional absorbance detection and thermal lens spectroscopy (TLS) were compared for hydrocolloids. Especially for particles below 20 nm TLS showed improved sensitivity. (Received on June 29, 2000 accepted on November 12, 2000) To reveal analytical information about the delicate structure and dynamics of environmental colloid systems, hyphenated techniques for separation and detection are needed. The characteristic dimensions of many environmental relevant colloids are between 1 nm and 500 nm, especially colloids with particle diameters < 100 nm are suspected to be of special importance for transport phenomena. The objective of our current work is to characterize environmental colloids such as humic substances in surface waters, respectively seepage waters from waste disposal areas. Especially, the correlation between the particle size distribution and the association of anthropogenic contaminants with colloids of different sizes is relevant for migration studies in natural aquifers1,2. In this work an asymmetric flow field-flow fractionation (AF4) system was combined with both a UV/vis-detector and a detection system based on thermal lens spectroscopy (TLS). Field-flow fractionation (FFF) is a liquid chromatography-like family of techniques for separation of particulate material in the range of 1 nm up to 100 µm as well as polymers and biopolymers (3 up to 100 kD). Instead of partitioning between phases the separation is based on the simultaneous action of a liquid carrier flow and a perpendicular external field (e.g. a second flow, an electric field), which induces a differential migration on unlike particles. In particular, an increased sensitivity for ultrafine colloids is envisioned with photothermal detection schemes3. A collinear pulsed TLS system (λ = 337 nm) was realized in combination with a flow cell. A set of yellow coloured polystyrene latex standard particles at seven different sizes between 17 nm and 440 nm were chosen to study the characteristics of TLS signal generation in colloidal systems.
Experimental AF 4 Principle and Set-up The AF4-System employed for this work is a modified system from Postnova Analytics (Munich, Germany). For AF4, a flat channel (100–500 µm) of 300 mm length is used together with a cross-flow, which is directed perpendicular to the laminar carrier
♣
flow as an external field. The cross-flow is maintained by splitting a defined percentage of the carrier flow through the lower part of the channel (see Fig. 1). The channel is designed as a two-layer sandwich, i.e. an ultrafiltration membrane, typically with a cut-off between 3 and 10 kD, and a 5 µm porous frit below the membrane, which allows the cross-flow to leave the channel. Field-flow fractionation is mainly based on an external field, which drives the particles against an accumulation wall, in this case the ultrafiltration membrane. Dependent upon the diffusion coefficient, which is indirect proportional to the particle diameter, a re-diffusion into the channel takes place4.
Injection valve Injection pump Autosampler
Solvent
Asymmetric channel Elution pump
Syringe pump Fig. 1:
Waste
Detector
The experimental set-up of the AF4 system.
In this ‘normal mode’ smaller particles, suspended in the fast regime of the laminar flow, are eluted before larger particles traveling along slower flow-lines. For particles larger than ≈ 400 nm, the asymmetric flow-FFF operates in the ‘steric- and hyperlayer-mode’ with inverted elution order5. The complete experimental set-up of the separation system, depicted in Fig. 1, consists of two conventional HPLC pumps for injection and sustaining of the laminar flow, a computer controlled valve- and flow-control unit to apply a defined cross-flow, the asymmetric
To whom correspondence should be addressed. E-mail:
[email protected]
ANALYTICAL SCIENCES APRIL 2001, VOL.17 Special Issue
separation unit with an exponential shaped channel, and the detector, typically a conventional UV/vis HPLC-detector. From the recorded fractograms not only information about the size distribution can be derived, but also secondary information related to other particle characteristics (diffusion coefficient, particle shape etc.).
Mirror
Energy monitor Pinhole Filter
Photodiode
Fig. 2:
1
1
1
0.1 0.08 0.06 0.04 0.02 0
Hydrodynamic diameter [nm]
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
E N TE R
1
ST
0.01 0.008 0.006 0.004 0.002 Colloid mass 0.001 0.0005 concentration 0.00025 [%(w/w)] 0
Digital Oscilloscope
Amplifier
Experimental set-up for the TLS detection scheme.
Reagents and Chemicals The yellow, dye-modified polystyrene latex particle standards used for the TLS measurements were purchased from Prolabo, France, and had an absorption maximum at λ = 345 nm. Other, colourless, PS Latex standards for calibration were provided by Postnova Analytics (Munich, Germany). Procedure AF4 fractionation was carried out in 0.1 % buffer of sodium dodecylsulfate (Sigma, Munich, Germany) in ultrapure water. The employed ultrafiltration membrane was made of regenerated cellulose with a 5 kDa cut-off (Postnova Analytics, Munich, Germany). The eluting particles were detected by a variable wavelength UV/vis HPLC-detector (LAMBDA 1000, Bischoff, Leonberg, Germany); if not mentioned otherwise the absorbance was registered at λ = 210 nm, respectively at 337 nm. The flow rate at the outlet of the channel was fixed to 1 ml/min, while the cross-flow was varied through a linear gradient from 40 % to 0 %. For high resolution fractionations (see Fig. 7) an initial cross-flow of 55 % was used. For preliminary TLS and absorbance measurements in the flow cell (without the AF4 attached), the flow rates were adjusted to 1 ml/min. The TLS signal was averaged over a period of 600 laser pulses (i.e. 30 seconds) for each concentration and particle
the
Fig. 3 provides an overview of the entire data set used in this comparison, i.e. TLS signal vs. particle size and concentration. Fig. 4 illustrates that for TLS detection a maximum in intensity (and signal-to-noise ratio) is found between particle sizes of 100 and 200 nm, which is in good agreement to an earlier photoacoustic study9. Due to the pulsed nature of the excitation, the divergence of the probe beam by scattering does not result in a significant change in the TLS signal for moderate particle concentrations.
TLS Signal [a. u.]
Flow cell
0.12
440
x
0.14
298
N2 -Laser (λ = 337 nm)
0.16
Fig. 3: Dependence of the TLS signal upon hydrodynamic particle diameter and the concentration.
Computer
Filter Lens Trigger diode Mirror
y
0.2 0.18
246
Lens with xy-adjustment
The AF4 system is a versatile tool to determine the particle size distributions of colloidal samples. However, particle concentrations in many natural water samples are often too low for conventional absorbance detection. Additionally, particle diameters in these samples are well below 100 nm. For these sizes photothermal detection methods can be very sensitive in comparison to absorbance measurements. The TLS signal is dependent upon on the intensity of the exciting light source, the beam waist in the sample cell, the wavelength of the probe beam, the absorption of the sample itself, and the thermal-optical characteristics of the medium (liquid)6,7. For example, Georges8 calculated for a TLS system with a laser pulse energy of 100 µJ, a probe laser wavelength of 632.8 nm and a beam waist of 50 µm an enhancement factor for TLS versus absorbance detection between 1.8 (20 °C) and 2.4 (30 °C).
165
He/Ne-Probe-Laser (λ = 633 nm)
Results and Discussion
98 70 17
TLS set-up The thermal lens detector was set up with a collinear arrangement of the pump and probe laser (see Fig. 2). A pulsed nitrogen laser (337 nm, 20 Hz repetition rate, 200 µJ/pulse; VSL 337, Starna, Pfungstadt, Germany) was utilized for excitation, a stabilized He-Ne laser (632.8 nm, 3 mW, Laser 2000, Munich, Germany) for probing the thermal lens in the focus of the nitrogen laser. The excitation laser beam was directed by two dichroitic mirrors collinearely into the beam train of the probe laser. Absorbance at 337 nm and TLS detection were compared in a pulsed collinear set-up utilizing a quartz flow cell with 5 mm path length and a total volume of 16 µl. Signal acquisition was timed through a negatively biased photodiode (BPX 65, Laser Components Munich). The pulse energy of the nitrogen laser was recorded simultaneously via a beam splitter and a pulse energy monitor. The later was also utilized for the simultaneous registration of the absorbance. The deflection and divergence of the probe beam by the thermal lens were detected by a second photodiode (C 30807E, Laser Components, Munich, Germany) equipped with 50 µm pinhole. The pre-amplified (Mod 564, HMS, Leverkusen, Germany) signal was registered by a digital oscilloscope and further processed with a personal computer. After further treatment, the TLS system provided a signal, which could be read and recorded simultaneously by the AF4-system in combination with other detectors.
size.
TLS-Signal [a. u.]
s572
0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0
Concentrations [%] 0.01 0.008 0.006 0.004 0.002 0.001 0.0005 0.00025 Blank
0
100 200 300 400 Hydrodynamic diameter [nm]
500
Fig. 4: TLS signal dependence upon particle size for different concentrations. For comparison with Fig. 4, Fig. 5 shows the relationship between the simultaneously recorded absorbance and particle size, resp. concentration. The absorbance signal is increasing with particle size, due to the increased scattering cross section of larger particles10. The concentration of the 165 nm colloid
ANALYTICAL SCIENCES APRIL 2001, VOL.17 Special Issue
1.2 1 0.8
Absorbance at 210 nm
Concentrations [%] 0.01 0.008 0.006 0.004 0.002 0.001 0.0005 0.00025 0Blank
1.4
0.6 0.4 0.2 100
200
300
400
0.10 0.08 0.06 0.04 0.02 0.00
b
2-10 nm
0.8 0.6 0.4
17 nm
0.2
Fig. 5: UV-absorbance vs. hydrodynamic particle diameter for different colloid concentrations. 98 nm
246 nm
0.17
0.7
0.15
0.6
0.13
0.5
0.11
0.4
0.09
0.3
0.07
0.2
0.05
0.1
0.03
0 0
200
400 600 800 Retention Time [s]
1000
0.08
2-10 nm
0.06 0.04 0.02 0.00
TLS-Signal at 337 nm [a. u.]
17 nm
c
500
Hydrodynamic diameter [nm]
Absorbance at 337 nm
0
Absorbance at 337 nm
a
2-10 nm
0.0 0.10
0
0.8
0.12
1.0
1.6 Absorbance at 337 nm
0.14
TLS Signal at 337 nm [a. u.]
standard was too low, due to an artefact by coagulation as verified later by means of AF4 and scanning electron microscopy. Fig. 6 exhibits a comparison of the performance of TLS vs. absorbance detection in combination with the AF4. Shown is the fractogram for separation of three different particle sizes (yellow latex particles 17 nm, 98 nm and 246 nm). As expected from Fig. 4 and Fig. 5, the conventional UV/vis-detector (used at 337 nm) based on absorbance performs better at larger particle sizes, while TLS has its merits for small particle sizes.
s573
0.01 1200
Fig. 6: Comparison of TLS (dashed line) and absorbance detection (solid line) in combination with the AF4-system. This observation is supported by Fig. 7, which shows in a highresolution fractogram the elution of the smallest particles (17 nm diameter). Obviously, this particle standard contains smaller particles (possibly dye-latex aggregates in the range between 2 nm and 10 nm) besides the expected particles with a hydrodynamic diameter of 17 nm. Absorbance measurements at 210 nm and 337 nm, as well as TLS measurements, revealed that only a small part of the signal recorded from both detectors in Fig. 6 is actually from particles with a diameter of 17 nm, but from the much smaller aggregates. Hence, Fig. 7 strongly supports our earlier findings from Fig. 4 and Fig. 5 that for particles below 30 nm TLS detection is superior to absorbance detection schemes (at the same wavelength) and is a useful addition to an AF4-system.
0
100
200
300
400
500
Retention Time [s]
Fig. 7: AF4-fractionation of ultrafine colloids with TLS detection (dashed line) at 337 m (a) and absorbance detection (solid line) at 210 nm (a) and 337 nm (c).
Acknowledgement We thank the Bayerische Staatsministerium für Landesentwicklung und Umweltfragen (BayFORREST program) for financial support. In addition, we would like to thank W. Faubel and B.S. Seidel for their initial help in setting up the TLS system.
References J. Buffle and G.G. Leppard, Environ. Sci. Technol., 1995, 29, 2169. 2. A. Exner, M. Theisen, U. Panne and R. Niessner, Fresenius J Anal Chem, 2000, 366, 254. 3. J. F. Power and C. H. Langford, Anal. Chem., 1988, 60, 842. 4. J. F. McCarthy and C. Delgueldre, in "Environmental Particles ", ed. J. Buffle and H.P. van Leeuven, 1993, Lewis Publishers, Boca Raton, Vol. 2, 247. 5. J. C. Giddings, Analyst, 1993, 118, 1487. 6. Q. He, R. Vyas and R. Gupta, Appl. Opt., 1997, 36, 7046. 7. M. Franko and C. D. Tran, Rev. Sci. Instrum., 1996, 67, 1. 8. J. Georges, Talanta, 1999, 48, 501. 9. T. Sekine, S. Naito, Y. Kino and H. Kudo, Radiochim. Acta, 1998, 82, 135. 10. P. Schurtenberger and M. in "Environmental Particles ", ed. J. Buffle and H.P. van Leeuven, 1993, Lewis Publishers, Boca Raton, Vol. 2, 37.
1.
