Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics Juliana C. Franco, Grasiele Gonçalves, Monique S. Souza, Samantha B. C. Rosa, Larissa M. Thiegue, Teresa D. Z. Atvars, Paulo T. V. Rosa, and René A. Nome* Institute of Chemistry, State University of Campinas, Campinas, SP, 13083-970, Brazil * [email protected]

Abstract: We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laserscanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. ©2013 Optical Society of America OCIS codes: (070.6120) Spatial light modulators; (120.0280) Remote sensing and sensors; (180.1790) Confocal microscopy; (180.2520) Fluorescence microscopy; (300.2530) Fluorescence, laser-induced.

References and links 1.

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#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30874

13. A. F. Philip, R. A. Nome, G. A. Papadantonakis, N. F. Scherer, and W. D. Hoff, “Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface,” Proc. Natl. Acad. Sci. U. S. A. 107(13), 5821–5826 (2010). 14. S. Mukamel, Principles of Nonlinear Optical Spectroscopy (Oxford University, 1995). 15. C. M. Seifried, J. Crawshaw, and E. S. Boek, “Kinetics of asphaltene aggregation in crude oil studied by confocal laser-scanning micrsocopy,” Energy Fuels 27(4), 1865–1872 (2013). 16. W. Rothschild, Fractals in Chemistry (Wiley-Interscience, 1998). 17. C. Stockbridge, Y. Lu, J. Moore, S. Hoffman, R. Paxman, K. C. Toussaint, Jr., and T. Bifano, “Focusing through dynamic scattering media,” Opt. Express 20(14), 15086–15092 (2012). 18. D. L. Gonzalez, F. M. Vargas, G. J. Hirasaki, and W. G. Chapman, “Modeling study of CO2-induced asphaltene precipitation,” Energy Fuels 22(2), 757–762 (2008).

1. Introduction Petroleum is a complex fluid mixture of great technological importance. It is composed of several classes of compounds, and its complete characterization is a very difficult task. Petroleum components are usually specified in terms of four classes: saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes [1]. Asphaltenes are insoluble in light alkanes such as n-heptane and n-pentane, and soluble in aromatic solvents such as toluene [2]. The molar mass of asphaltenes varies from 500 g/mol (stable asphaltene form in oil) up to 50000 g/mol (asphaltene aggregates in oil), though the actual asphaltene composition also varies as a function of the n-alkane employed to induce precipitation [3]. In field environments and reservoirs, several different types of perturbation may provoke destabilization of asphaltene structures and, consequently, growth and precipitation of asphaltene aggregates from crude oil. Some of these perturbations include [4]: (i) changes in temperature and/or pressure, such as those that occur during oil production, (ii) changes in the composition of the system, as in the mixture of several types of oils or the injection of CO2 for the advanced recovery of oil, and (iii) intense shearing, as in some pumping operations. Major unwanted effects of asphaltene precipitation include oil flow interruption and duct ‘clogging’ during oil production or processing. Although the problems caused by asphaltene deposition are well documented, the precise knowledge of field-relevant conditions under which such deposition occurs remains a challenge. One approach involves downhole fluid analysis whereby near-infrared and fluorescence spectroscopies are implemented directly in reservoirs; this approach has been used only in selected applications due to the large scale nature of the experiments [5]. Usually, investigations of asphaltene precipitation in crude oils are performed by methods such as dynamic titration with n-heptane at room temperature and pressure. In such methods, precipitation is inferred either from optical microscopy or via filtration with 0.5 μm pore diameter filters. Onset determination is thus performed under conditions of temperature, pressure, and composition that are quite distinct from those of reservoirs or under flow. Moreover, kinetic retardation effects between the onset of asphalthene destabilization in n-heptane and the detection point based on traditional methods have been reported [6,7]. In the work of Khoshandam and Alamdari [6], it was observed that mean diameter of asphaltene aggregates on the order of 0.5 μm can be obtained only after nearly 40 minutes in n-heptane solution. Maqbool et al [7] have observed aggregate growth for weeks after addition of precipitating agent into oil. Recently, a quartz crystal resonator has been used to probe asphaltene flocculation, where it was noted that the measured quartz damping was more sensitive to flocculation than the corresponding resonance frequency [8]. Ideally, one would like to use methods that probe asphaltene precipitation under fieldrelevant temperature/pressure conditions. Working under such conditions may be accomplished by directly studying crude oil in the field as noted above; alternatively, highpressure cells operating at temperature/pressure conditions similar to those in the field have been employed. For example, conventional high-pressure cells can be used to investigate the thermodynamic behavior of oil at high pressures in the absence and presence of asphaltene precipitating agents such as CO2 [9]. In either case, in situ optical probes constitute a

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30875

promising approach to characterize the onset of asphaltene precipitation kinetics due to the rapid response and the possibility of remote sensing. Building new optical probes of the in situ onset of asphaltene precipitation from petroleum (crude oil) under field-relevant conditions is of major technological importance. Furthermore, when combined with laser excitation at optical wavelengths, structural and electronic chemical information may be obtained with high sensitivity [10]. Such information can be inferred by measuring spontaneous emission from the sample, both fluorescence and Raman. In particular, steadystate and fluorescence lifetime spectroscopies are sensitive to the formation of asphalthene aggregates since chromophore interactions modify the electronic structure. Similarly, Raman and FTIR spectroscopy intrinsically combine structural and energetic information at the molecular level. Given the goal of developing optical probes to characterize in situ asphalthene precipitation kinetics, one main difficulty is to design an instrument that satisfies several constraints that are uncommon in conventional linear spectroscopy. First, performing spectroscopy at high pressures and temperatures is challenging from the experimental point of view. In order to work in field-relevant conditions, we employ sapphire windows adapted for work in high-pressure cell to perform in situ spectroscopy of asphaltene precipitation kinetics; this is the main point of the paper. The high-pressure work will be realized by employing pressure cells as mentioned previously. It is important to note, however, that the optical window must have a large thickness in order to support the high pressures employed. Aberration corrections must be realized in order to obtain microscopic images with good contrast. In addition to aberrations, the large window thickness also introduces the necessity of working with standoff excitation and detection optical probes. Both aberration correction and standoff spectroscopy may be implemented using spatial light modulators. The present work provides extensive data on the characterization of the onset of light and heavy crude oils, as well as asphaltene precipitates by spectroscopy, confocal microscopy, real-time kinetic/morphology correlation studies, and simulations. The spectroscopy of asphaltenes and crude oils has been previously reported in the literature [5] To the best of our knowledge, the present work reports the first spectroscopic characterization of a new source of crude oil and asphaltene (see Experimental Section). 2. Materials and methods Crude oil samples containing saturates, aromatic, resins, and asphaltene fractions [1] were supplied by PETROBRAS (Brazil) and stored at 21 °C prior to analysis. To ensure sample quality, the American Society of Testing and Materials Standard Test Method D287 (ASTM D287) was employed to determine the API degree of the crude oil samples [11]. Based on the API degrees obtained from the ASTM D287 test, the crude oil samples were termed “light” and “heavy” [11]. The asphaltene fraction was extracted from crude oil using n-heptane following the procedure described in Standard Method ASTM D6560 [12] without modification. Steady-state emission spectra were obtained using a Cary Eclipse Varian spectrofluorimeter with a 450 W xenon arc lamp and a custom-built sample holder was employed in a back-face configuration for the opaque samples. Excitation wavelength ranged from 300 nm to 500 nm. Emission wavelengths were scanned from wavelengths longer than the excitation wavelength (e.g., begin at 330 nm for 300 nm excitation) up to 700 nm. Spectrophotometric measurements were carried out in a HP-8453 diode array spectrophotometer. Fourier-Transform Infrared images and spectra were recorded with a Nicolet model 520 spectrophometer using the attenuated total reflection (ATR) method at an angle of 45°; the samples were deposited on ZnS crystals. Spectra were recorded from 4000 to 650 cm−1 after 64 scans with 2 cm−1 resolution. Fluorescence decays were recorded using time-correlated single photon counting in an Edinburg Analytical Instruments FL 900 spectrofluorimeter with MCP-PMT in Peltier housing, featuring a Hamamatsu R3809U-50.

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30876

The samples investigated were crude oil and asphaltene precipitates obtained as described above. Measurements were performed with wavelength excitation of λexc = 335 nm (pulsed diode model EPLED-340 with spectral width = 14 nm, pulse width 815 ps) and the emission signals were collected at λem = 386 nm, which corresponds to the maximum of the emission band for the sample. The sample decay signal was deconvoluted from the laser pulse signal using a Ludox® scatterer. The experimental curves were treated using the F900 software from Edinburg Analytical Instruments, by fitting the decays with multiple exponential functions using non-linear least-squares routines minimizing χ2. Good fits were obtained when χ2 is close to 1. The experimental curves were treated using the F900 software by fitting the decays with multiple exponential functions as shown in Eq. (1) and using non-linear least-squares routines: N  t  F ( t ) =  Bi exp  −  i =1  τi 

(1)

In Eq. (1), Bi is a pre-exponential factor representing the fractional contribution to decay of the component with a lifetime τi and t is the time. The fitting parameters are displayed in Table 1. Confocal laser-scanning fluorescence microscopy was performed with a Leica SP5 microscope. Typically, measurements were performed in “xyz-λ” mode in which emission spectra and xy images were measured simultaneously. These measurements were then repeated at several different values of z, where “z” scans the full sample height. The measurements were performed at 488 nm and 514 nm excitation wavelengths; emission spectra were respectively collected from 508 to 700 nm and from 534 to 734 nm. For a quantitative analysis of the experimental data, the 8-bit grayscale images obtained with the confocal microscope were used as an input for further data analysis with ImageJ®. In order to eliminate the shadow effect it was necessary to calculate via software the two-dimensional Fourier transform of the image followed by use of the “Bandpass” filter option during the inverse transform operation. Image contrast was then maximized employing the ‘Brightness’ and ‘Threshold’ options. The particles considered in the statistical analysis were selected with the function “Show Outlines” from the software. The resulting information was used to calculate histograms of size distribution, which were then used to quantify particle number and particle size as a function of time for ‘reaction times’ of (i) 3 minutes, (ii) 18 minutes, and (iii) 32 minutes. For this study we have designed and built a sample compartment consisting of a microscope slide containing a cylindrical glass container of 1 cm radius and 2 cm height glued on the slide surface. With this design, we were able to (i) add crude oil to the sample, (ii) add precipitation-inducing solvent, and (iii) place the sample on the confocal microscope sample holder to obtain images and spectra of the sample surface as well as its interior. In the sample holder, approximately 0.01 g of light oil was added, followed by addition of approximately 1.00 g of heptane. The solution was stirred manually for three minutes and the sample holder cover was sealed with Teflon to avoid solvent loss. The resulting sample was then analyzed on the confocal microscope. Images were obtained at two spatial locations in the sample: (i) sample surface, and (ii) sample interior. Images were obtained every three minutes. The approach just described was employed to investigate the kinetics of asphalthene precipitation induced by solvent as measured by confocal microscopy. 3. Results and discussion 3.1. Steady-state spectroscopy As described in the Materials and Methods section, steady-state electronic and vibrational spectroscopies, together with fluorescence lifetime spectroscopy, were employed to

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30877

characterize both crude oil and asphaltene precipitate, so that a direct comparison between the samples can be made.

Fig. 1. Spectroscopic characterization of oil and asphaltene. (a): Plot of emission frequency as a function of excitation frequency for light oil diluted in n-heptane with error bars along the yaxis (black points), asphaltene solid (red and green points). The expected values for paraffin alkanes are shown as light and dark blue points. (b): Plot of fluorescence lifetime of asphaltene sample precipitated with n-heptane (black curve) and heavy oil (red curve) overlaid with the Ludox standard response (green curve).

Figure 1(a) is a plot of emission frequency versus excitation frequency for light oil and asphaltene precipitate, as obtained from steady-state fluorescence spectroscopy. The black points indicate the position of the emission maximum at each excitation frequency investigated, whereas the vertical bars represent the full width at ¾ maximum. We have not used the more common full width at half maximum due to low signal-to-noise ratios, especially at longer excitation wavelength. The use of full width at ¾ maximum has been shown previously to provide useful information regarding spectroscopic linewidths [13]. The emission maxima can be well fit with two linear equations with correlation coefficients of r2 = 0.9989 and 1 respectively for high excitation frequencies and lower excitation frequencies employed. From the electronic spectroscopy data shown in Fig. 1(a), we sought to understand the relationship between incident and detected light frequencies. In addition to the experimental data, Fig. 1(a) also exhibits straight lines (with a slope of one) associated with four vibrational normal modes with frequencies of 1000 cm−1, 1458 cm−1, 1616 cm−1, and 2800 cm−1. We also have performed infrared spectroscopic characterization of asphaltene and crude oils. The main FTIR peaks and respective assignments are: 3445 cm−1(OH stretching), 2922 cm−1 and 2852 cm−1(sp3 CH stretching), 1602 cm−1(C = C stretching), 1455 cm−1 (CH2 bending), 1375 cm−1(CH3 bending), 867 cm−1, 798 cm−1, and 747 cm−1(out of plane = C-H bending). Both light and heavy crude oils exhibit similar spectroscopic features in the FTIR spectrum. A comparative analysis with Raman spectroscopic signals obtained from the same sample indicates that there are vibrational spectroscopic signatures of asphaltene even in the fluorescence emission spectrum of this sample. For visible wavelength excitation, we observe

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30878

an overlap between the experimental peak emission data of crude oil and the energies corresponding to specific vibrational frequencies of asphaltene. Specifically, the emission data fall fairly close to the 1458 cm−1 and 1616 cm−1 vibrational lines of asphaltene; accordingly, the excitation-emission data at lower frequencies shown in Fig. 1(a) can be fit to a straight line with a negative slope of 1.08. An experimental distinction between the two components (fluorescence and Raman) of the observed signal may be investigated by tuning the frequency of the incident light and recording the detected light frequency. Raman lines shift linearly in frequency as the excitation frequency is tuned whereas the fluorescence emission frequency will remain roughly in the same position as the excitation frequency is tuned [14]. As can be seen in Fig. 1(a), the experimental excitation/emission data at low frequencies lie within the spectral range associated with the 1616 cm−1 and 1458 cm−1 bands of asphaltene. The analysis shown in Fig. 1(a) suggests that the measured spectra are associated with vibrational structure of asphaltene, more specifically with the C = C stretching and CH2 bending modes. Even though the assignment is compelling, it is important to remark that at present we are unable to rule out a competing hypothesis of multiple species present in the sample. The data shown in Fig. 1(a) may be indicative of electronic and vibrational contributions to the observed spectrum, that is, fluorescence and Raman signals contribute to the detected emitted light frequency. We rule out vibronic contributions for two reasons: (i) the absence of progression in the observed emission spectra, (ii) the data plotted in Fig. 1 includes only the (0,0) mode. On the other hand, we are unable to rule out the possibility that the signal corresponds to fluorescence from a continuous progression of asphaltenes of different sizes. It is also of interest to note that under UV-excitation (higher frequencies) the emission frequency is nearly independent of excitation frequency, as shown in Fig. 1(a), quite different from a line with slope of one expected on the basis of a normal-mode analysis of the data; thus we believe the fluorescence signal is the major contribution to the observed emitted light frequency under UV excitation. 3.2. Time-resolved fluorescence spectroscopy We have performed time-correlated single-photon counting measurements to characterize the fluorescence lifetime of crude oils and asphaltene precipitates. Figure 1(b) shows the lifetime decay curves of crude oil and asphaltene precipitated from n-heptane. The decays shown in Fig. 1(b) are overlaid with the Ludox standard signal, which produces only scattered light and thus serves as a reference for the instrument response function. Table 1. TC-SPC fluorescence lifetime excitation and emission wavelengths, and nonlinear squares fitting parameters obtained for light oil, heavy oil, and asphaltene precipitate. Sample light oil heavy oil Asphaltene

λexc (nm) 335 320 320

λem (nm)

τ1 (ns)

τ2 (ns)

386 390 390

1.71 ± 0.01 1.18 ± 0.08 -

6.749 ± 0.003 7.14 ± 0.02 7.162 ± 0.005

Fitting range (channels) 180 - 1400 180 - 900 180 - 1000

χ2 1.096 1.048 1.021

The red curve in Fig. 1(b) shows the lifetime decay associated with crude oil. The data analysis was performed as described in the Experimental Section. The best fit was obtained with two values of lifetime, 1.18 ns and 7.14 ns, with relative amplitude contributions of 56% and 44%, respectively. A chi-2 value near 1 was obtained. The black curve in Fig. 1(b) shows the fluorescence lifetime decay observed for asphaltene precipitate obtained from heavy crude oil sample. We note that the signal did not decay to zero in the 50 ns window employed, thus indicating the need for a longer time window for the experiments. Data analysis shows that the best fit was obtained employing a single exponential decay time with a time of 7.162 ns. This is a plausible result since the sample consists of a single substance (asphaltene) with a single decay phenomenon.

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30879

A direct comparison of the lifetime data of asphaltene precipitate and heavy crude oil (from which asphaltene precipitate was obtained) indicates a near coincidence between the lifetime of asphaltene precipitate and one of the decay components of the heavy crude oil. Specifically, a lifetime of 7.16 ns was obtained for the precipitate whereas one of the decay components of heavy crude oil had a decay time of 7.14 ns. Thus, the 7.14 ns decay component in heavy crude oil presumably is associated with the asphalthene present in the crude oil. 4. Quantitative confocal microscopy and numerical simulations

Fig. 2. Quantitative confocal microscopy investigation of asphaltene precipitation kinetics. Sample: light crude oil in n-heptane. From left to right, we show data taken 3 minutes (left), 18 minutes (center), and 33 minutes (right) after sample injection into the confocal microscope. (A): raw data, (B): 8-bit grayscale image, (C): particle count, (D): statistical analysis of particle counts.

We have performed confocal microscopy measurements to characterize asphaltene precipitation from n-heptane solution in real time at two spatial positions along the optical axis: at the bottom of the sample and at the sample interior. Figure 2(a) shows images obtained by confocal microscopy for light oil in n-heptane at three instants in time after sample preparation: (i) 03 minutes (left); (ii) 18 minutes (center); and (iii) 32 minutes (right). From the images of Fig. 2(a) one can see that particle aggregates are visible even at the earliest time of three minutes after sample preparation. Qualitative visual inspection of the images shows that the aggregate size increases as time proceeds from 3 min. to 18 min. to #199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30880

33 min. For a quantitative analysis of the experimental data, the images were analyzed with ImageJ® as described in the Experimental Section. After contrast adjustment of the original images, we obtain the images shown in Fig. 2(b). The images presented in Fig. 2(b) were employed for statistical analysis of aggregate particles formed. The particles considered in the statistical analysis were selected with the function “Show Outlines” from the software. The resulting selections are shown in Fig. 2(c). The information shown in Fig. 2(c) was then transformed onto histograms of size distribution, as shown in Fig. 2(d). The images shown in Fig. 2(c) and the histograms shown in Fig. 2(d) can be used to investigate and quantify particle number and particle size as a function of time for (i) 3 min., (ii) 18 min., and (iii) 33 min. As shown in Fig. 2, one can see that initially there is a relatively small number of aggregates with smaller size. As the precipitation reaction proceeds, one can observe a larger number of aggregates, which are also larger in size. We have not observed significant changes in particle size between the intermediate and final observation times (18 min. and 33 min. respectively). Quantitative analysis of the data shows that the total number of particles increases from 68 to 379, the mean area increases from 38 μm2 to 465 μm2, and the mean particle size increases from 3 μm to approximately 10 μm. In order to observe smaller particles, one may use a higher numerical aperture objective. In order to obtain higher quality images, one may work with n-heptane concentrations which are closer to the onset point (the concentrations employed in the present work are well above the onset point) thereby allowing better image averaging. Recently, optical microscopy has been used to investigate the kinetics of asphaltene aggregation from solution [15]. Image analysis as a function of time was contrasted with aggregation theories; good agreement was found between diffusion-limited aggregation (DLA) and experimental data on average particle size as a function of time. The resulting fractal coefficient was consistent with both particle-cluster and cluster-cluster aggregation mechanisms. We sought to compare our experimental results on the kinetics of asphaltene aggregation (shown in Fig. 2) with stochastic simulations of particle-cluster and cluster-cluster aggregation. Thus, we have performed stochastic simulations of two-dimensional diffusionlimited aggregation (DLA). DLA is commonly used to describe aggregation when colloidal particles bind to one another irreversibly upon contact (cluster growth limited by diffusion). Briefly, we modeled cluster growth and aggregation as diffusion in a two-dimensional square lattice by analyzing the probability distribution of random particles on a square lattice [16]. The probability P(x,t) of finding a particle at a given position and instant of time is proportional to the density of particles as a function of time, ρ(x,t), which in turn obeys the diffusion equation in the continuum limit: ∂P ( x, t ) = D∇ 2 P ( x , t ) ∂t

(2)

Fig. 3. Stochastic numerical simulations of two-dimensional diffusion and aggregation on a square lattice as a model for diffusion-limited aggregation.

The numerical simulation investigated here started with five seeds for growth, and the result shown in Fig. 3 indicates a fractal growth for each seed as a function of time.

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30881

Accordingly, the growth kinetics – quantified by calculating the average aggregate size as a function of time – was well described by a diffusion-limited aggregation model (DLA): the average aggregate size increases with a fractional exponent of approximately 0.8 as a function of time. Interestingly, both experiment and simulation are consistent with this mechanism, and thus we tentatively attribute the confocal microscopy kinetic data as arising from diffusionlimited aggregation. Our results are consistent with previous work [15,16]. Thus, we believe that laser scanning fluorescence confocal microscopy measurements employing 470 nm excitation and the 490 – 600 nm emission window is a suitable probe of asphaltene aggregation and precipitation kinetics from n-heptane. 5. Towards an in situ optical probe of asphaltene precipitation kinetics inside highpressure cells On the basis of our experimental fluorescence spectroscopy and microscopy results shown above, we sought to build laser-based optical probes of asphaltene precipitation from crude oils inside high-pressure cells that mimic field temperature and pressure conditions. In agreement with previous work reported in the literature [5], the data shown in Figs. 1 and 2 indicated that asphaltene fluorescence is a suitable probe for detecting asphaltene in crude oil. Figure 4 shows a schematic of the experimental apparatus we have designed to probe asphaltene aggregation from crude oils by fluorescence spectroscopy and microscopy. An excitation laser (L in Fig. 4; PicoQuant) is sent to the sample by two approaches: (i) focusing with a long-working distance (LWD) objective; and (ii) employing a fiber optic and collimator, both of which were adapted to a mechanical support that can be connected to a high-pressure cell (labeled “in-situ probe” in Fig. 4). The main difference between the use of a long-working distance objective (approach (i)) and an optical fiber (approach (ii)) is that the long-working distance objective focuses excitation light through the thick optical window thus obviating the need for a mechanical support for the high-pressure cell optical probe, as employed in approach (ii). The design and construction of the optical probe shown schematically in Fig. 4 employed a sapphire cell to be used in conjunction with a high pressure cell. Specifically, the sample was placed in a sample container made of sapphire windows held firmly in place with a thick teflon ring container (Fig. 4). The thickness (1 cm) and diameter (2.54 cm) of the sapphire windows were chosen to fit into our high-pressure cell while at the same time supporting high mechanical stresses. In a typical experiment, a droplet of the sample of interest is sandwiched between microscope slides, and the slides are placed between the sapphire windows; the estimated path length is less than 100 µm. In both approaches described in Fig. 4, an epi-fluorescence geometry is employed to direct light emitted from the sample placed between the sapphire windows through the dichroic mirrors and to characterization with a CCD camera (CCD), spectrometer (SP), and single-photon avalanche photodiode (APD) detector. With such a design, it is in principle possible to simultaneously obtain images, spectra, and photon intensity correlations. Employing the apparatus described in Fig. 4(a), we show in Figs. 4(b) and 4(c) fluorescence spectra of two samples: DMSO solution of fluorescein (black curve), and light crude oil (red curve). Fluorescein is commonly employed as an optical probe in fluorescence spectroscopy, and it was used in the present work for instrument calibration purposes. Light crude oil was chosen because it will be employed in our own work using high-pressure cells. In both Figs. 4(b) and 4(c), the same layout was employed except for the excitation conditions: Fig. 4(b) shows the emission spectra obtained using a long-working distance focusing objective (approach (i)) whereas Fig. 4(c) shows the emission spectra obtained using our custom-built fiber-optic probe (approach (ii)). In order to obtain the spectra shown in Figs. 4(b) and 4(c), the same excitation laser power and overall alignment was used, that is, for each sample we sequentially performed measurements using approach (i) and then approach (ii). As expected, we have observed that a higher signal-to-noise ratio was obtained when focusing the excitation light into the sample and employing the same objective to

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30882

collect emitted light (approach (ii)). Tight focusing also increases the ratio of fluorescence to Raman contributions to the spontaneously emitted signal. By contrast, approach (ii) sends a collimated excitation beam to the sample, collecting a much smaller angular field of view. Although the signal-to-noise ratio is much smaller in approach (ii), we note that the use of monochromators couplied to single-photon APDs allow measurement of both spectra and lifetimes even when using fiber-optic excitation schemes.

Fig. 4. (A) Schematic design of a fluorescence-based optical probe for high-pressure cells. L: laser; LWD: long-working distance objective; F: optical fiber; CCD: CCD camera; SP: spectrometer; APD: single-photon avalanche photodiode. (B) Fluorescence spectra measured employing excitation approach (i); (C) Fluorescence spectra measured employing excitation approach (ii) (see Main Text for details).

Fig. 5. (A) Adaptive optic setup for aberration correction. Two Shack-Hartmann wavefront sensor screen captures are shown: (B) the spot field obtained before aberration correction, and (C) after aberration correction.

The long-working distance objective employed in approach (i) of the present work is a 50x air objective. Therefore, this objective focuses light optimally when air is used as the index-matching medium. On the other hand, the refractive index of sapphire is 1.77 at our excitation wavelength. The thick sapphire windows placed between objective and sample thus lead to optical aberrations with two main effects: (1) a decrease in fluorescent light focusing and collection efficiency, and (2) distortion in the images obtained. Effect (1) can be partially overcome by increasing the excitation laser intensity and employing single-photon APDs. On the other hand, image aberration due to refractive index mismatch is not easily corrected using conventional approaches. In the present work, we aim to correct for such distortions with a micro-electromechanical mirror system-based spatial light modulator (MEMS-SLM) [17].

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30883

The layout of the SLM apparatus is shown in Fig. 5(a). A laser diode is spatially filtered and focused on the sample holder (same sample holder employed in the experiments described in Fig. 4). Two telescopes are then employed to image transmitted light onto the SLM and then to a wavefront sensor to characterize optical aberrations induced by the sapphire windows. Table 2. Adaptive optics-based correction of aberration induced by high-pressure cell optical windows.

Without sapphire window, no cancel tilt (no wavefront correction) With wavefront correction With sapphire window, no “cancel tilt” (no wavefront correction) With wavefront correction With sapphire window, with cancel tilt (no wavefront correction) With sapphire window, with cancel tilt, with wavefront correction

Amplitude RMS (μm)

Amplitude P-V (μm)

0.374

2.283

0.144 0.609

0.524 1.522

0.846 0.292

5.009 1.248

0.194

0.896

Figures 5(b) and 5(c) respectively show spot field distributions obtained before and after aberration correction with the SLM. Qualitative analysis of the field distributions obtained with the wavefront sensor indicate that the major effect of the introduction of non-indexmatched optics between objective and sample is an overall tilt of the wavefront along the optical axis of propagation, which can be calculated using the spot field distribution information. As shown in Table 2, Quantitative analysis of the measured wavefronts indicate that aberration correction with the SLM lead to a significant decrease in both wavefront amplitude RMS (from 0.85 µm to 0.2 µm) and wavefront resolution (from 5 µm to 0.9 µm). 6. Conclusions In summary, we have employed optical spectroscopy to the investigation of asphaltene in crude oils and asphaltene precipitation kinetics induced by n-heptane. Fluorescence lifetime measurements showed that both crude oil samples exhibited bi-exponential decay with lifetime constants of approximately 1.5 ns and 7 ns, whereas asphaltene precipitate exhibited a single lifetime decay component of approximately 7 ns. Thus, it was possible to spectroscopically identify asphaltene inside opaque crude oil samples. Confocal laserscanning microscopy was employed to study asphaltene precipitation kinetics induced by n-heptane. Quantitative analysis of the confocal microscopy data was employed to calculate particle-size distributions as a function of time, thereby allowing a direct comparison with results from stochastic numerical simulations of diffusion-limited aggregation. Overall, these results are consistent with previous work on crude oils and asphalthenes obtained from other sources. Moreover, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene from crude oil. The design enables studies inside high pressure cells. The current design employs 470 nm excitation, fluorescence detection, and remote-sensing by (i) fiber optics and (ii) long-working distance objectives. In both cases, the collected light was employed to obtain fluorescence spectra, images, and photon-counting traces. Finally, a spatial light modulator was employed for aberration correction due to the optical window used in the high-pressure cell. Finally, we comment on the behavior of asphaltene under high-pressure, which is relevant for our design as well as for oil field management in general. At reservoir conditions of temperature and pressure, the asphaltenes are soluble in crude oil. The pressure-temperature phase diagram for asphaltenes in crude oils shows an envelope where the asphaltenes are soluble. This envelope is located at pressures larger than the bubble point of the system. The section comprised between this envelope and the bubble point curve represents the region where the asphaltenes can precipitate in the system. Furthermore, asphaltenes can be #199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30884

precipitated at pressures slightly lower than the bubble point [18]. Thus, during oil production the crude oil will experience a decrease of both pressure and temperature and asphaltene precipitation can happen. If the precipitated asphaltene is deposited on the tubing surface, the oil production will decrease or even be interrupted. Employing in situ fluorescence spectroscopy and microscopy, asphaltene precipitation at the tubing surface may be readily identified and quantified with the approach presented herein. Acknowledgments We gratefully acknowledge financial support from FAPESP (fellowships 2011/20143-5, 2011/20144-1, 2012/14987-9, 2012/15018-0, research grant 2008/10593-0), ProFIS/CNPq for a fellowship and Petrobras (research grant P-02180).

#199266 - $15.00 USD Received 11 Oct 2013; revised 21 Nov 2013; accepted 24 Nov 2013; published 6 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.030874 | OPTICS EXPRESS 30885

Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation...
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