Thermal Analysis and Photothermal Fourier-Transform Infrared

Anal. Chem. 2009, 81, 6612–6619

Thermal Probe Based Analytical Microscopy: Thermal Analysis and Photothermal Fourier-Transform Infrared Microspectroscopy Together with Thermally Assisted Nanosampling Coupled with Capillary Electrophoresis Xuan Dai,† Jonathan G. Moffat,† Andrew G. Mayes,† Mike Reading,*,† Duncan Q. M. Craig,† Peter S. Belton,† and David B. Grandy‡ School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk NR4 7TJ, U.K., and IPTME, Loughborough University, Loughborough, Leicestershire, LE11 3TU, U.K. In this study, we have demonstrated that a scanning probe microscope (SPM) can be used for thermally assisted nanosampling (TAN) with subsequent analysis by capillary electrophoresis (CE). Localized thermomechanical analysis (L-TMA) and photothermal Fourier-transform infrared (PT-FTIR) microspectroscopy can also be employed using the same probe, thus illustrating how a single instrument can carry out a number of different complementary analytical measurements. Benzoic acid and 4-hydroxybenzoic acid were manipulated with a heated Wollaston wire probe and successfully deposited onto the surface of a piece of CE capillary tubing. The deposited samples were then separated with CE. L-TMA and PT-FTIR were also used to characterize these materials. We have also demonstrated how a nanosample of a nonparticulate material can be taken and then deposited onto the surface of an inert matrix. TAN of a nonparticulate material was explored using polyethylene as the analyte and fluorene as the matrix. These examples show that thermal probe techniques provide a versatile “tool box” of modes of analysis with the potential to analyze a wide range of samples in a spatially resolved way. There is a growing need to analyze complex materials in a spatially resolved manner. This is usually undertaken with some form of analytical microscopy such as IR,1-3 Raman4-6 or * To whom correspondence should be addressed. E-mail: mike.reading@ uea.ac.uk. † University of East Anglia. ‡ Loughborough University. (1) Wolthuis, R.; Travo, A.; Nicolet, C.; Neuville, A.; Gaub, M. P.; Guenot, D.; Ly, E.; Manfait, M.; Jeannesson, P.; Piot, O. Anal. Chem. 2008, 80, 84618469. (2) Chan, A. K. L.; Kazarian, S. G. Appl. Spectrosc. 2008, 62, 1095–1101. (3) Stenlund, H.; Gorzsas, A.; Persson, P.; Sundberg, B.; Trygg, J. Anal. Chem. 2008, 80, 6898–6906. (4) Liu, Z.; Li, X.; Tabakman, S. M.; Jiang, K.; Fan, S.; Dai, H. J. Am. Chem. Soc. 2008, 130, 13540–13541. (5) Suzuki, M.; Maekita, W.; Wada, Y.; Nagai, K.; Nakajima, K.; Kimura, K.; Fukuoka, T.; Mori, Y. Nanotechnology 2008, 19, 265304/1–265304/7. (6) Jiang, Y.; Wang, A.; Ren, B.; Tian, Z. Q. Langmuir 2008, 24, 12054–12061. (7) Piehowski, P. D.; Carado, A. J.; Kurczy, M. E.; Ostrowski, S. G.; Heien, M. L.; Winograd, N.; Ewing, A. G. Anal. Chem. 2008, 80, 8662-8667.

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Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

techniques like imaging secondary ion mass spectrometry.7-9 All of these are very useful analytical tools but they struggle when the minimum volume that can be imaged consists of a complex mixture. A powerful approach to analyzing mixtures is chromatography, thus one approach to the spatially resolved analysis of complex systems is to find ways of combining microscopy with chromatography. This has been achieved in the case of gas chromatography coupled with mass spectroscopy (GC-MS)10 by capturing the gases evolved from local pyrolysis. In this technique a thermal probe used in a scanning probe microscope (SPM), such as is used for scanning thermal microscopy and localized thermomechanical analysis (L-TMA),11,12 is placed on a surface and rapidly heated to a sufficiently high temperature to cause a small volume to decompose. A capillary tube located immediately adjacent to the probe is placed under negative pressure so the evolved gases are sucked into a chamber containing a Tenax sorbent. This sorbent is then placed in a thermal desorption apparatus attached to a GC-MS. In this way a selected point on a surface (selected from a topography image obtained using the SPM) is subject to chemical analysis using chromatography. This approach has a range of applications but suffers from the disadvantage that molecules are broken up by the use of elevated temperatures. This complicates the interpretation of the analysis because the question, “was a component identified by GC-MS originally in the sample or was it created from the thermal decomposition of another compound”, often does not have a clear answer. Consequently, it would be beneficial to find alternative approaches that do not cause the sample to decompose or otherwise change its composition. (8) Debois, D.; Hamze, K.; Guerineau, V.; Le Caer, J. P.; Holland, I. B.; Lopes, P.; Ouazzani, J.; Seror, S. J.; Brunelle, A.; Laprevote, O. Proteomics 2008, 8, 3682–3691. (9) Baker, M. J.; Zheng, L.; Winograd, N.; Lockyer, N. P.; Vickerman, J. C. Langmuir 2008, 24, 11803–11810. (10) Price, D. M.; Reading, M.; Lever, T. J.; Hammiche, A.; Pollock, H. M. Thermochim. Acta 2001, 376, 95–97. (11) Price, D. M.; Reading, M.; Hammiche, A.; Pollock, H. M. J. Therm. Anal. Cal. 2000, 60, 723–733. (12) Reading, M.; Price, D. M.; Grandy, D. B.; Smith, R. M.; Bozec, L.; Conroy, M.; Hammiche, A.; Pollcok, H. Macromol. Symp. 2001, 167, 45–62. 10.1021/ac9004869 CCC: $40.75  2009 American Chemical Society Published on Web 07/21/2009

Another important development has been the introduction of thermally assisted nanosampling (TAN).13-16 In this technique, the thermal probe is placed on a sample and heated to a temperature at which it begins to soften or melt. The probe is then retracted; often material adheres to the probe and a very small sample is taken from a selected location. This can be applied to particles such that a whole particle is picked up and it has also been shown that it can then be put down.13 It has also been demonstrated that optical microscopy and topographic imaging can be combined with the probe-based manipulations described in this paper.13 This opens up the possibility of using this method to select an individual particle, remove it from a surface and present it for analysis by a separation based method. In this study we explore, for the first time, how this can be achieved using capillary electrophoresis (CE) with a UV detector. It should be noted that, although this approach does involve heating the sample, it is much less destructive than the high temperatures required for pyrolysis which implies that decomposition will occur. TAN will usually be possible without chemical modification of the sample, even compounds that decompose before or during melting can be thermally nanosampled because the heating and cooling rates used, up to 10 000 °C/s,17 are so fast that the material can be melted and cooled before significant decomposition can occur. Another aspect of TAN is its application to nonparticulate materials. The same basic approach is adopted, the tip is placed on a selected location, heated to a point at which the sample softens or melts and then the probe is retracted. Again a small sample often adheres to the tip. In this case the size of the sample is not governed by the size of the particle selected, as it is when particles are being sampled, but by the viscosity of the softened material, the adhesion between the sample and the tip, the size of the tip, the depth of penetration, and the speed of movement of the probe. Such nanosampling has already been demonstrated on a range of samples;14-16 however, subsequent deposition of the sample onto another surface as a precursor to possible further analysis has not been demonstrated. In this study, we explore for the first time, this possibility. The final aspect of this study is that a single instrument can be used for L-TMA and PT-FTIR,18-20 as well as TAN, with subsequent analysis by CE. We show, for the first time, the combination of all of these techniques. For the CE part of this study, benzoic acid and 4-hydroxybenzoic acid were selected as the model compounds. Benzoic acid and 4-hydroxybenzoic acid are simple aromatic carboxylic acids. (13) Harding, L.; Reading, M.; Craig, D. Q. M. J. Pharm. Sci. 2008, 97, 1551– 1563. (14) Harding, L.; Qi, S.; Hill, G.; Reading, M.; Craig, D. Q. M. Int. J. Pharm. 2008, 354, 149–157. (15) Reading, M.; Grandy, D.; Hammiche, A.; Bozec, L.; Pollock, H. M. Vib. Spectrosc. 2002, 29, 257–260. (16) Hammiche, A.; Bozec, L.; German, M. J.; Chalmers, J. M.; Everall, N. J.; Poulter, G.; Reading, M.; Grandy, D. B.; Martin, F. L.; Pollock, H. M. Spectroscopy 2004, 19, 20–42. (17) Gorbunov, V. V.; Grandy, G.; Reading, M.; Tsukruk, V. V. In Thermal Analysis of Polymers: Fundamentals and Applications; Menczel J. D., Prime, R. B., Eds.; John Wiley & Sons: New York, 2009, pp 615-649. (18) Moffat, J. G.; Mayes, A. G.; Belton, P. S.; Craig, D. Q. M.; Reading, M. Anal. Chem. In press. (19) Bozec, L.; Hammiche, A.; Pollock, H. M.; Conroy, M.; Chalmers, J. M.; Everall, N. J.; Turin, L. J. Appl. Phys. 2001, 90, 5159–5165. (20) Hammiche, A; Pollock, H. M.; Reading, M; Claybourn, M.; Turner, P. H.; Jewkes, K. Appl. Spectrosc. 1999, 53, 810–815.

Benzoic acid and its salts are used as food preservatives. 4-Hydoxybenzoic acid is a phenolic derivative of benzoic acid and is primarily known as the basis for the preparation of its esters, known as parabens, which are used as preservatives in cosmetics. Because of the structural similarity, benzoic acid and 4-hydroxybenzoic acid are often selected as the analytes to evaluate the performance of a CE system. The separation of benzoic acid and 4-hydroxybenzoic acid using capillary electrophoresis has been extensively studied.21-23 Calibration plots were obtained with the conventional vacuum sample solution injection. Subsequently a novel method was developed for solid sample introduction using thermal probes. L-TMA and PT-FTIR were also used to characterize these materials. We extend the original demonstration of TAN of a nonparticulate material15 by again using polyethylene (PE) as the material that was nanosampled, fluorene was chosen as the matrix material. These compounds were chosen purely as model samples because they have relatively simple IR spectra and there is no overlap in the region of the major absorption bands of PE. Thus, when PE is deposited on fluorene it is easily detected. In the original demonstration of TAN, the area sampled was shown to be of the order of a few micrometers and a fraction of a micrometer in depth, which gives an estimate of the spatial resolution that can be achieved with nonparticulate materials using the Wollaston probe (as in this case). Recent work by King et al.24 with smaller thermal probes suggests a spatial resolution of 500 nm can be achieved, however, when dealing with such small samples the use of chromatography is made difficult by the need for a highly sensitive detector. This issue is explored in more detail below. EXPERIMENTAL SECTION Reagents and Equipment. Benzoic acid (ACS reagent), 4-hydroxybenzoic acid (99%), hydrochloric acid (HCl, 37% purises) and sodium tetraborate decahydrate (Na2B4O7 · 10H2O, ACS reagent) were obtained from Sigma-Aldrich. Sodium hydroxide (NaOH, Pellets) was from BDH (Poole, U.K.). All chemicals were used without further purification. All solutions were prepared using analytical reagent grade water (Fisher Scientific, Loughborough, U.K.). Na2B4O7 · 10H2O was used to prepare a running buffer. The pH value of the buffer was adjusted to 10.0 using 1.0 M NaOH. Stock solutions of benzoic acid and 4-hydroxybenzoic acid were prepared by dissolving appropriate amounts of each acid in a 0.01 M NaOH solution. The mixtures of the acids in a concentration range of 1-25 µM were obtained by dilution of the stock solutions with water. The fluorene was supplied by Sigma-Aldrich (Steinheim, Germany), and the polyethylene came from BP Solvey. Localized thermomechanical analysis (L-TMA) was performed using a NanoTA Thermal Analyzer (Anasys instruments, Santa Barbara, CA) with Thermomicroscopes Explorer scanning probe microscope (SPM). Wollaston wire thermal probes (Veeco, Santa Barbara, CA) were used. The sensor signal (V) reflects the (21) van Pinxteren, D.; Herrmann, H. J. Chromatogr. A 2007, 1171, 112–123. (22) Fatemi, M. H.; Goudarzi, N. Electrophoresis 2005, 26, 2968–2973. (23) Deng, Y.; Wellons, A.; Bolla, D.; Krzyaniak, M.; Wylie, H. J. Chromatogr. A. 2003, 1013, 191–201. (24) Harding, L.; King, W. P.; Dai, X.; Craig, D. Q. M.; Reading, M. Pharm. Res. 2007, 24, 2048–2054. (25) Harding, L.; Wood, J.; Reading, M.; Craig, D. Q. M. Anal. Chem. 2007, 79, 129–139.


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Figure 1. Solid sample introductions with thermal probes (a) schematic illustration of the principles of thermally assisted particle manipulation, (b) illustration of sample capillary attached to separation capillary, (c) particle approaching the surface of the capillary, (d) particle contact with the capillary, and (e) solid sample deposited on the capillary. The arrows indicate the position of the particle.

cantilever deflection. Temperature calibration was carried out using manufacturer supplied melting point standards, polycaprolatone, polyethylene and polyethyleneterepthalate. Benzoic acid or 4-hydroxybenzoic acid particles were freely dispersed on the surface of a piece of glass slide, which was attached to a magnetic stub using double-sided tape and mounted onto an X-Y translating microscope stage. During L-TMA scans, the particles were contacted with a Wollaston probe. Differential scanning calorimetry (DSC) studies were performed using a TA 2920 MDSC (TA Instruments, Leatherhead, U.K.). Sample sizes of 2-3 mg were used throughout in standard alumina pans (TA Instruments, Leatherhead, UK) with a heating rate of 10 °C/min. Calibration was performed using n-octadecane, indium, and tin as the standards and a purge gas of nitrogen was used with a flow rate of 50 mL/min. All photoacoustic Fourier-transform infrared spectroscopy measurements were carried out on an IFS/66 S spectrometer from Bruker Optics (Coventry, U.K.) fitted with a photoacoustic cell from MTEC Photoacoustics (Ames, IA). Background and sample measurements were taken at a resolution of 8 cm-1 for 100 scans. Carbon black was used to take the background measurements. Photothermal Fourier-transform infrared (PT-FTIR) microspectroscopy was setup by interfacing the same Bruker FTIR spectrometer with the Explorer SPM with a Wollaston wire thermal probe attached. The probes can be used to act as a temperature sensor. The SPM is placed on a custom designed optical interface which contains a flat mirror to reflect the IR beam to a mirror which condenses the beam to a diameter of ∼500 µm. An adjustable platform is employed to position the SPM so that the tip of the probe is located at the focal point of the beam. Samples can be loaded on an XYZ-controlled stage, and the probe is placed in contact with the sample using the SPM or can pick up the sample particle to be measured in air. 6614


For PT-FTIR measurements, background and sample measurements were taken at a resolution of 8 cm-1 for 200 scans. Particles can be picked up by heating up the thermal Wollaston wire probes to a certain temperature.13 The particle manipulation process is illustrated schematically in Figure 1a and b. In brief, the tip is placed on a particle of either benzoic acid or 4-hydroxybenzoic acid and heated to the lowest temperature that is found, by trial and error, to enable the particle to be picked up (80 °C for benzoic acid and 140 °C for 4-hydroxybenzoic acid) when the tip is subsequently cooled (Figure 1c). To avoid or minimize damage to the particle, the temperature of the tip can start at a low value then be progressively increased until adhesion occurs. While it cannot be guaranteed that this heating has not chemically altered the sample, most materials will soften at the glass transition temperature or melting point without decomposition provided the exposure to the high temperature is short, as is the case here where the tip need only be heated for a few seconds. Furthermore, photothermal spectroscopy can be used before and after picking up the particle to investigate whether any significant chemical changes have occurred (in this case there was no evidence of this). We noted that a particle would sometimes stick to the tip without the need for heating so it is sometimes the case that this first heating step can be omitted. After raising the tip, the particle was moved to the top of a section of capillary tube that was held in a micromanipulator (Figure 1d) and deposited via a second heating program (Figure 1e), during this heating program the sample was heated for only a few seconds and was then rapidly cooled. Since the melting temperature of 4-hydroxybenzoic acid is higher than benzoic acid, 4-hydroxybenzoic acid was deposited onto the capillary first. Then benzoic acid was deposited at the same place by repeating the manipulation process. In this way, benzoic acid and 4-hydroxybenzoic acid were presented at the same point to form a composite particle. The capillary with the

Figure 2. L-TMA (a) and DSC (b) results for benzoic acid (solid line) and 4-OH benzoic acid (dashed line). For L-TMA, the particles were held on the Wollaston probe and the heating rate was 10 °C/s with the scan range from 25 to 150 °C for benzoic acid and from 25 to 250 °C for 4-hydroxybenzoic acid. For DSC, the heating rate is 10 °C/min with the same temperature range as that in L-TMA.

sample particles was cut (approximately 1 cm long) and attached to the end of the separation column using a small piece of carbon tape (Figure 1b). Electrophoretic analyses were carried out using an ABI 270A capillary electrophoresis system (Bioanasis), data acquisition was by a NI BNC-2090 A/D converter board (National Instruments, Hungary) to digitize the analogue detector output. Prior to the first use, the capillary column was conditioned by rinsing, in sequence, with 1 M HCl for 5 min, deionized water for 2 min, 1 M NaOH for 10 min, deionized water for 2 min, and finally running buffer for 10 min. The capillary column was rinsed by applying a vacuum pressure of 10 psi with 0.1 M NaOH for 1 min, deionized water for 1 min and the running buffer for 2 min before each injection. The samples were introduced by either vacuum sample injection (10 psi) for 1 s or solid sample with thermal probes. The direct UV detection was performed at a wavelength of 220 nm. The cathode was placed at the outlet buffer and the anode at the inlet buffer. Separation conditions for benzoic acid and 4-hydroxybenzoic acid were selected:23 10 mM sodium borate at pH 10.0, applied voltage 20 kV. The capillary dimensions were 50 cm × 50 µm I.D (Alltech no. 602035) with an effective length of 30 cm. The separation temperature was 25 °C.

RESULTS AND DISCUSSION Part 1: Localized Thermomechanical Analysis and Photothermal Fourier-Transform Microspectroscopy of Benzoic Acid and 4-Hydroxybenzoic Acid. L-TMA measurements on single particles of benzoic acid or 4-hydroxybenzoic acid were carried out with a heating rate of 10 °C/s. The particles were contacted with a Wollaston probe during L-TMA scans. The responses for benzoic acid and 4-hydroxybenzoic acid are shown in Figure 2a. For comparison, DSC curves of benzoic acid and 4-hydroxybenzoic acid (Figure 2b) were also presented. Ten L-TMA measurements were performed for each sample; three typical results are presented here. In Figure 1a, the thermal expansion followed by penetration into the sample occurs at ∼118 °C (±2 °C), which is close to the melting temperature (Tm) of benzoic acid from both DSC results (122.2 °C) and the literature value (122.4 °C). For 4-hydroxy benzoic acid, the onset of penetration is 224 °C (±3 °C), which is close to the Tm of 4-hydroxybenzoic acid from both DSC measurements (214.3 °C) and the literature (214 °C) (because the precision is good the discrepancy may arise from the polymer samples used for calibration, we are exploring the possibility of a set of pharmaceutical calibrants, further Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 3. Photothermal and photoacoustic Fourier-transform infrared spectra for benzoic acid (a) and 4-OH benzoic acid (b). For photothermal Fourier-transform infrared spectra, the particles were held in air with the Wollaston probe.

extensive discussion of calibration can be found in a recently published reference work17,25). These results showed that L-TMA has the ability to measure the melting point of a single particle with sufficient accuracy to enable a wide range of materials to be differentiated. PT-FTIR microspectroscopy of benzoic acid and 4-hydroxybenzoic acid were examined next with a single particle attached to the Wollaston wire thermal probe in air. The resulting spectra (interval from 2000 to 500 cm-1) are shown in Figure 3 for benzoic acid (a) and 4-hydroxybenzoic acid (b). In selecting a suitable background for PT-FTIR spectroscopy, spectra were taken 6616


with the thermal probe in air. For comparison, the spectra from photoacoustic Fourier-transform infrared spectroscopy (PAS) measurements are also presented in Figure 3 (benzoic acid (a), 4-hydroxy benzoic acid (b)). In comparing the two spectra it can be seen that, for the most part, the expected bands are present in the PT-FTIR spectrum although their relative heights are sometimes different; there is a general tendency for the relative peak size to diminish relative to the PAS peaks as the wavenumber increases. We believe this is because the thermal probe has a slower response time than the microphone used for PAS; this phenomenon will be the topic of future investigation. For the

Figure 4. Separation of 10 µM benzoic acid and 4-hydroxybenzoic acid in calibration (solid line) using CE conditions: 10 mM sodium borate at pH 10.0; applied voltage 30 kV; injection pressure 10 p.s.i.; injection time 1 s; UV detection 220 nm; operation temperature 25 °C. The dotted line shows the separation of thermally deposited particles of benzoic acid and 4-hydroxybenzoic acid using the same CE conditions.

Figure 5. Calibration plots for benzoic acid (×) and 4-OH benzoic acid (∆).

purposes of this study it is sufficient to observe that the correct absorption bands are present in the PT-FTIR spectra and so they can be used to identify and distinguish between materials in the same way as more conventional forms of IR spectroscopy. Part 2: Development of a Technique for Interfacing Thermally Assisted Nanosampling to Capillary Electrophoresis. Separation of Benzoic Acid and 4-Hydroxybenzoic Acid. Benzoic acid (25 µM) was injected with the selected CE separation conditions first to identify the peak. The retention times for benzoic acid and 4-hydroxy benzoic acid stabilized by injecting them singly under the selected CE separation conditions; the retention times were 1.5 and 1.8 min, respectively. Mixtures of benzoic acid and 4-hydroxybenzoic acid (1, 2, 5, 10, 15, 20, and 25 µM each) were then analyzed to obtain the calibration data. Typical separation peaks of benzoic acid and 4-hydroxybenzoic acid are presented in Figure 4 (solid line). Calibration plots where the peak areas are plotted versus the concentration of the compounds are shown in Figure 5. A linear relationship was

observed in a concentration range of 1-25 µM for both compounds tested. For benzoic acid, the linear equation is y(min × V) ) 1.618 × 10-5x (µm) + 1.581 × 10-5 (RSD ) 0.993) and for 4-hydroxybenzoic acid it is y ) 1.194 × 10-5x+2.506 × 10-5 (RSD ) 0.990). The limit of detection (LOD) was about 1 µM in both cases. The equation for calculating the injected volume is

V)

∆Pπr4t 8ηL

where ∆P is the pressure difference across the capillary (dyn · cm-2), r is the inner radius of the capillary (cm), t is the injection time (s), η is viscosity of the buffer (dyn · s · cm-2), and L is the total length of the capillary (cm). Considering the separation conditions used above, the injection volume of our experiments was 2.603 × 10-8 L. Thus the lowest detected mass was 3.176 pg for benzoic acid and 3.592 pg for 4-hydroxybenzoic Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 6. Photothermal spectra for polyethylene (0) and fluorene (9) (obtained with the unheated tip in contact with the surface of the sample), polyethylene nanosample on the tip (2), and polyethylene on the surface of the fluorene (b).

acid. This is limited by the available (UV) detector on the CE instrument since neither compound has a high extinction coefficient. Particle Manipulation and CE Separation of Particles. After the particle manipulation process with the thermal probe described in the experimental section, benzoic acid and 4-hydroxybenzoic acid were presented at the same point to form a composite particle. The capillary with the sample particles was cut (approximately 1 cm long) and attached to the end of the separation column using a small piece of carbon tape. The separation column was rinsed with 0.1 M NaOH, deionized water, and running buffer before the particle manipulation and was ready for the separation. After the short capillary with the sample deposited on to it was attached to the separation column, the separation voltage was applied. Three repeat measurements gave the relative peak areas of acid/hydroxyl acid ranging from 0.51 to 0.99 (corresponding to a sample size range of 5-10 pg) which were consistent in trend with the observed sizes of the particles (attempts to weight the particles by looking at the resonant frequency of the probe with and without the sample proved this method was too insensitive in this case), while the retention times were 1.712 min (sd 0.0018 min) and 2.013 min (sd 0.0267 min). The repeatability is comparable to that achieved with the standard CE technique which gives standard deviations of 0.0059 and 0.0069 min, respectively. This implies that, despite the fact that the method of sample introduction is less well controlled than with the standard method, fairly good repeatable separation is possible. Nevertheless, some form of spectroscopic detection would be advantageous, but this poses a challenge because of the very small sizes of the samples. The retention times are longer than for the standard calibration since the sample needed to dissolve and move over from the capillary onto which the sample had been placed over to the separation column. It is encouraging that reasonable repeatability of retention times could be achieved over a range of compositions and hence sample structures, despite the difficulties of controlling the exact details of the location of the sample relative to the separation column. This suggests this is a relatively robust method that could be more widely applied and this is the subject of ongoing work. 6618


Part 3: Thermally Assisted Nanosampling of a Nonparticulate Sample with Subsequent Deposition. TAN of nonparticulate materials has already been reported including the specific example of polyethylene.13 The novel feature that we report here for the first time is the ability to deposit the nanosample onto another material so that it is available for further analysis by a variety of means. The compound fluorene was chosen because its spectrum has only one small peak in the region 2800-3000 cm-1,26 which is the region where the largest absorption peaks of polyethylene (the CH stretch peaks) are found, and this fluorene peak is lost in the noise in our PT-FTIR measurement, see Figure 6. Consequently it is easy to detect the presence of polyethylene in the presence of fluorene. Furthermore fluorene is inert, and so, no chemical reaction occurs between the two materials. In Figure 6 the contact spectra (obtained with the unheated tip in contact with the surface of the sample) for polyethylene (0) and fluorene are (9) shown. The spectrum of the polyethylene nanosample on the tip is also shown (2). To obtain this nanosample the tip was heated up to 120 °C, the melting point, then retracted; the spectrum that is shown proves that some polyethylene was retained on the tip as it was withdrawn. This contaminated tip was then placed on the surface of the fluorene sample, and its temperature was increased again to 120 °C; then the tip was withdrawn. We note that this temperature is above the melting point of fluorene, as well as that of the polyethylene; whether during this part of the transfer process it is advantageous to melt the matrix the nanosample is deposited onto or whether this is not optimal is an open question that will be addressed in future work. In this case it did not, as we shall see, hinder the transfer. The tip was then cleaned by heating it to a high temperature, sufficient for it to glow red hot, then a PT-FTIR spectrum was taken (not shown), and as expected, no material was detected on the tip. The clean tip was then placed back onto the fluorene surface at the same point it was previously placed and a spectrum was taken. This final spectrum, the uppermost in Figure 6, clearly shows the presence of polyethylene on the surface of the fluorene as the CH stretch peaks of (26) Thormann, T.; Rogojerov, M.; Jordanov, B.; Thulstrup, E. W. J. Mol. Struct. 1999, 509, 93–104.

polyethylene are clearly present. The significance of this is that the fluorene crystal could be removed and dissolved in a suitable solvent then analyzed using a variety of forms of chromatography. Provided the fluorene is pure, or its contaminants well characterized, then anything other than that arising from the fluorene would be the analytes contained in the nanosample. It can easily be envisaged how a number of nanosamples might be taken from a range of sites of interest on the surface of a sample, imaged by the SPM and then deposited onto a matrix like fluorene thereby providing detailed information about the chemical composition on the surface. It can, in fact, be envisaged that a very large number of nanosamples could be taken automatically in an array on the surface of a sample for subsequent automated analysis by, for example, high performance liquid chromatography (HPLC)MS. In this way a map of the surface chemistry could be built up based on a chromatographic technique. However, one potential problem is that, although nanosamples taken from solid surfaces can be as big as 500 pg;16 they can also be and more commonly are as small as 500 fg,13 which then poses a problem for many detectors (as it does in this case). This underlines the need to match the size of the sample taken to the sensitivity of the subsequent analytical method. One solution to detecting the analytes is to use the probe itself as the detector and details of this approach will be the subject of a future article. CONCLUSIONS Benzoic acid and 4-hydroxybenzoic acid were manipulated with a heated Wollaston wire probe and successfully deposited onto the surface of piece of CE capillary tubing. The deposited samples were separated with capillary electrophoresis via applied voltage. This study shows, for the first time how particles in the size range

of micrometers to tens of micrometers can be characterized by L-TMA, PT-FTIR, and then CE. To make analysis by CE possible, we have demonstrated a novel means of sample introduction based on thermally assisted nanosampling. Although we have demonstrated the principle by sampling and depositing two separate particles of different materials to form a composite particle in situ, in practice it would typically be the case that an individual particle containing a mixture of materials would be analyzed. We have also demonstrated for the first time how a nanosample of a nonparticulate material can be taken and then deposited onto the surface of an inert matrix. The matrix particle could then be dissolved in a suitable solvent and the solution analyzed. In this way local chemical analysis of complex mixtures could be performed. It is possible to envisage that both of these nanosampling methods could be automated and high throughput analysis of small (particulate in form) samples and chemical mapping of the surface of nonparticulate materials would be possible. Finally, it can be seen that using a thermal probe provides for a versatile form of analytical microscopy that has the functionality and spatial resolution of an atomic force microscope and a scanning thermal microscope together with the possibility of performing local analysis by PT-FTIR, L-TMA, and TAN coupled to capillary electrophoresis. ACKNOWLEDGMENT We acknowledge EPSRC (EP/D038448/1) for financial support.

Received for review March 5, 2009. Accepted June 30, 2009. AC9004869


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Thermal Analysis and Photothermal Fourier-Transform Infrared

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