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Abstract
We present a novel technique for capillary electrophoresis (CE) using terahertz (THz) waves, namely “THz-CE,” which enables us to sensitively detect separated substances in a solution flowing in a hollow of capillary whose inner diameter is smaller than 100 µm. Such THz detection could be achieved by utilizing the near-field interaction between a solution filled in a capillary and a point THz source that was locally generated by optical rectification in a nonlinear optical crystal irradiated with a femtosecond pulse laser. Here, we investigated the performance of THz-CE numerically and experimentally, and succeeded in observing the electrophoretic chromatogram for the separation between acetic acid and n-propionic acid by THz-CE.
© 2024 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Capillary electrophoresis (CE) [1] is a powerful separation technique which can provide high-resolution and high-speed separation with a few microliter volume of solutions, because high voltage can be applied to narrow open-tubular capillaries. The migration time and the intensity of the peak detected in the electrophoretic chromatogram correspond to the indices of electrophoretic mobility and concentration of the compounds, respectively. The separated compounds or their ratios are detected by various detectors such as ultraviolet-visible (UV-Vis) spectrometers [2–4], fluorometry [5,6], electroconductivity detection [7] and mass spectroscopy (MS) [8,9]. Although these are well-established methods as micro-volume separation analyses, there are still some difficulties. Indirect detection or chemical modification with pre- or post-separation column is often required, depending on analytes of interest.
The energy level of a terahertz (THz) wave region corresponds to intramolecular rotation or lattice vibration, which is rather low compared with that of the infrared region. THz time-domain spectroscopy (THz-TDS) [10–12] is an established tool to obtain amplitude spectra with phase information in THz region in a short time. Combining CE with THz techniques would be attractive for realizing a newly label-free detection technique for various compounds contained in solutions and bring novel and meaningful information, compared to the conventional on-line sensing methods. However, it is challenging due to the spatial resolution of THz waves and the low sensitivity against the polar solvent.
Our group has developed a novel THz technique called the scanning point THz source microscope (SPoTS microscope), which enables us to nondestructively and label-freely evaluate various micrometer scale samples [13–16]. In the SPoTS microscope, a nonlinear optical crystal (NLOC) was employed as a two-dimensional (2D) THz emitter, and THz waves were locally generated in the process of optical rectification at the irradiation spots of fs laser beams. Since the laser beams are focused at the output surface of the NLOC, the beam diameter of the generated THz waves is approximately the same size as that of the focused beam spot which overcomes the diffraction limit of THz waves. By setting samples in the vicinity of the THz generation spot on the NLOC, THz-TDS and THz-imaging have been successfully achieved on the samples of sub-THz wavelength scale. This technique has been applied to the measurements of human hairs [17], small metallic apertures [14], meta-atoms [18], nanoparticles [19], and biological samples including cancer tissue [20–22] and a pL volume of solutions flowing in a microfluidic channel [23–25]. Therefore, this technique can overcome problems on sensitivity and spatial resolution for the THz measurements and be applied to the micro-volume separation tools.
In this report, we tried to separate small molecular weight carboxylic acids such as acetic acid and n-propionic acid as representatives of biological compounds by CE and to detect by the THz-TDS technique. The new technique might be called as “THz-CE”. THz-CE enables the separation and detection of carboxylic acids without pretreatment of samples, including biological ones. Moreover, THz-detection may show the physical properties such as refractive index and permittivity of the analytes in addition to the quantitative analysis regarding peak intensity. If the physical properties are the characteristics of each carboxylic acid, frequency-domain spectra of the peaks possibly represent the individual information of the analytes such as an absorption spectrum caused by the chromophore.
2. Experimental setup of THz-CE
Figure 1(a) shows a schematic of the experimental geometry of homemade THz-CE. A quartz capillary was set directly on the surface of a 500μm-thick-(110)-oriented gallium arsenide (GaAs) substrate, a THz emitter (Crystal Base Co., Ltd., Osaka, Japan: Undoped and semi-insulting GaAs with a resistivity of 0.8∼4.8 × 108 Ω·cm). The GaAs substrate can be slid on X- and Y-axes on a manual stage and be rotated manually. Untreated fused silica hollow capillaries made from quartz were purchased from GL Science Inc. (Tokyo, Japan). Their outer surface was coated with polyimide to prevent the breakdown. We used two kinds of capillaries: 0.1 mm inner diameter, 0.2 mm outer diameter for the THz detection and 0.1 mm inner diameter, 0.375 mm outer diameter for the conventional contactless electroconductivity detection. The polyimide surface membrane needs to be removed in the conventional optical detection [2–4], but not in THz-CE owing to the transparency in THz frequency region, which can be simplified the experiments.
Fig. 1. Schematics of (a)terahertz (THz) capillary electrophoresis (THz-CE) setup, (b) the localized THz-TDS system, (c) an enlarged cross-sectional drawing of the THz emitter, GaAs with a capillary, and (d) experiment flow chart of THz-CE.
A localized THz-TDS system (near-field configuration for samples) as illustrated in Fig. 1(b) with a fiber-coupled fs laser source (TOPTICA FemtoFiber pro IR: center wavelength of 1.56 µm, maximum power of 350 mW, pulse width of 100 fs, and repetition rate of 80 MHz) for capillary detection was essentially the same one as described elsewhere [13]. THz waves transmitted through the sample filled in the capillary were detected at a bowtie-shaped photoconductive antenna (PCA) fabricated on a low-temperature (LT-) grown GaAs (Hamamatsu Photonics KK, Shizuoka, Japan). Phase modulation detection was held by a home-made electronic circuit with a phase detector, CD-552R4 (NF Corporation, Kanagawa, JAPAN), and an optical chopper, 300CD (Scitec Instruments Ltd, United Kingdom), at the modulation frequency of 11 kHz. To compare with the THz data, a conventional contactless electroconductivity detection method using a C4D detector, ER815 (eDAQ Pty Ltd., NSW, Australia) that equipped with its special head stage for a capillary was also employed.
As shown in Fig. 1(c), when the GaAs is excited with fs pulse laser beam, a point THz source is created in the process of optical rectification at the laser irradiation spot whose beam diameter is approximately 10μm. The center of the capillary is set at the vicinity of the THz source.
Figure 1(d) shows an experiment flow chart of the THz-CE. In the measurements, a Good’s buffer, 50 mM MES at pH 5.5 (Sigma-Aldrich Japan K.K.) was used as the electrolyte for capillary electrophoresis. An aliquot of ∼25% in methanol solution of hexadecyltrimethylammonium hydroxide (HTAOH) (Sigma-Aldrich Japan K.K.) was added to be 5 mM in order to reverse the electroosmotic flow (EOF). The analytes separated were acetic acid (CH3COOH, MW 60.05) and n-propionic acid (CH3CH2COOH, MW 74.08) in this study, which were purchased from Nakalai Tesque Inc. and Sigma-Aldrich Japan K.K., respectively. Firstly, the electrolyte was manually injected into the capillary with a microsylinge, and then a mixed solution sample of acetic acid and n-propionic acid was injected into the capillary by a gravity method, 50 mm for 10 s or 15 s. Then, minus twenty kV was applied by a high voltage power supply, HCZE-30PN0.25 (Matsusada Precision Inc., Shiga, Japan).
3. THz-TDS measurements with conventional system with a thin-gap cell
To take advantage of THz-CE, we preliminary examined the extraction of physical properties of the samples, acetic acid aqueous solutions. Here we used a conventional THz-TDS system equipped with a thin-gap cell for the measurements of liquid samples. Conventional THz-TDS measurements were held with a commercial system, TR-1000, Otsuka Electronics Co. Ltd. A femtosecond pulse laser, Femtolite CS-20 (IMRA, Aisin, Aichi, Japan) was equipped to the instrument, with an oscillation frequency 50 MHz, an average output higher than 20 mW and a pulse width shorter than 100 fs at a center wavelength of 780 nm. A THz source was a photoconductive switch made of a low-temperature GaAs substrate on which a dipole-shaped electrode was formed (Hamamatsu Photonics KK, Shizuoka, Japan), and the detector was the same one. A delay stage slid for the moving distance of 10 mm at 1 mm/s with the positioning accuracy of 0.01 μm. The time waveforms were acquired by a phase modulation circuit with a frequency of 10 kHz. The data acquisition time for each time waveform was 150 s with ten times accumulation (e.g., Fig. 2(a), Fig. 3(b) and (c), Fig. 6(a)). The complex refractive index and complex permittivity were estimated from time waveforms by the calculation method proposed by Usami (Usami, M., Japan patent JP2002-277393A (2002)). A thin-gap quartz cell was custom-made by Kadomi Optical Industry (Tokyo, Japan). The gap thickness and diameter of the cell are 0.1 mm and 8 mm, respectively, with 1 mm base plate and 0.5 mm roof plate.
Fig. 2. (a) Terahertz time-domain waveforms of acetic acid aqueous solutions at various concentration measured by a conventional THz-TDS system with a thin-gap cell for liquid. (The light path length of 0.1 mm.) (b) Concentration dependence of complex refractive index (refractive index, n, and extinction coefficient, κ) at 1 THz.
Fig. 3. (a) Schematic (not to scale) showing the location of a capillary under measurement by a point terahertz source at different positions Typical THz-time-domain waveforms for a capillary filled with (b) air and (c) water.
Figure 2(a) represents the measured THz time-domain waveforms of acetic acid aqueous solutions at various concentrations. Figure 2(b) shows the concentration dependence of complex refractive indices at 1 THz calculated from the time waveforms. The broad band frequency-domain spectra were obtained by Fourier transform of the time-domain waveforms. We chose the values at 1 THz because they were generally stable and highly reliable with GaAs as a THz-emitter. The frequency-domain spectra in the frequency region below 0.3 THz and above 2 THz show lower S/N ratio with larger dispersion. Though the extinction coefficient, κ, shows the linear relationship, the refractive index, n, shows the nonlinearity against the concentration of acetic acid. It means THz detection will provide totally new multidimensional informations. It might reflect the intermolecular interaction such as hydrogen bonds among acetic acid and water molecules. Hydrogen bonds are formed between water and acetic acid molecules, i.e., water-water, water-acetic acid, acetic acid-acetic acid. The abundance ratio and configuration of the various hydrogen bonds may possibly induce the convex-shape nonlinear dependence of refractive index upon acetic acid concentration.
4. Search of the best position of capillary
Prior to the measurements, we investigated the optimal location of a capillary tube and a point THz source as shown in Fig. 3(a). As the capillary on the GaAs substrate was shifted by every 62.5 µm in the direction perpendicular to the capillary tubing, THz time waveforms were measured to determine the best position where most of the THz beams were crossing over the hollow of the capillary. Figures 3(b) and 3(c) represent the time waveforms for a capillary filled with air and water, respectively. Position 1 of two figures showed the waveforms similar to that of no capillary and may correspond to the position where no THz waves were passing through the capillary. At position 8, as indicating time waveforms with light-colored blue lines in Figs. 3(b) and 3(c), THz waves passing outside of the capillary were figured to be the least. The position 8 was supposed to be the best position to observe the filling materials of the hollow capillary.
5. Results and discussion
5.1 FDTD simulation of THz time waveforms passing through a capillary
The THz wave propagation through the capillary and its time-domain waveform were numerically calculated by using commercial FDTD software (ANSYS Lumerical Software ULC: ANSYS, Inc). Figure 4 shows a setting of the calculation. For the refractive indices in hollow capillaries used for the simulation are 1.0, 2.2, and 1.9 for air, water, and acetic acid, respectively. That of quartz, the material of capillaries, employed is 1.95. All complex refractive indices, refractive indices and extinction coefficients used for the simulation were estimated values at 1.0 THz by the measurements with the conventional THz-TDS. THz waves emitted from a GaAs substrate mainly contain the frequency components in the range between 0.5 THz and 1.5 THz where the complex refractive indices show rather small frequency dependence. The values at 1 THz, consequently, were considered to be average one over the frequency range when GaAs was used as a THz-emitter.
In order to compare the THz waveforms between by the simulation and by the experiment, covering of THz waves taking a roundabout path outside the capillary was attempted with a precision air slit with the slit size of 200 ± 5 μm × 3 mm (#39-731, Edmund Optics Japan Ltd., Tokyo, Japan). It was positioned and fixed at the center of a capillary as shown in Fig. 5(a). The waveforms observed with the conventional THz-TDS system (Fig. 5(b)) show good agreements with those calculated by numerical simulation (Fig. 5(c)). As the optical simulation and experiments reveal comparable results, we succeed in obtaining the first attempt to obtain the spectral information in THz region of filling materials in the hollow capillary.
Fig. 5. (a) Schematic of cross-section around the terahertz (THz) emitter with an air slit (b) typical THz waveforms (c) Numerical calculation of the THz waveforms
5.2 THz time waveforms observed with a capillary
To obtain THz electropherograms, the delay stage of THz-TDS system was fixed at the position where the amplitude change was largest over time during the CE. To determine this position, THz time waveforms were measured with the system presented in Figs. 1(a) and 1(b), by filling a capillary with air (empty), water, acetic acid, or acetic acid aqueous solutions. The time waveforms observed are shown in Fig. 6(a). Since a shoulder peak around 14.8 ps of the time waveforms with a capillary possibly corresponds to the THz waves which take a roundabout path outside the capillary by comparing them with the waveforms of Fig. 5(b). (Note: the starting times of the vertical axis (delay time = 0) of the two series of time waveforms are different, depending upon the series of the measurements. Remark their shapes with time course.) When the time waveform of water (blue line) was subtracted from that of acetic acid (red line), the shoulder peak derived from the THz waves taking a roundabout path outside disappeared (orange line, AA-water). The peaks and troughs shift, depending upon the refractive index of filling materials in the capillary. Therefore, the informative intensity values were evaluated by the differences of the maximum and minimum of amplitude intensities after 15 ps of the time waveforms as indicated by double-headed arrows in Fig. 6(a).
Fig. 6. (a) THz time waveforms of a capillary filled with air in hollow (green line), that with water (blue line), that with acetic acid (AA) (red line), and difference time waveform between acetic acid and water (orange line). Vertical double arrows correspond to the maximal amplitude intensity changes for the capillaries with each filling. The brown arrow indicated the fixed position of a delay stage when the electrophoresis was achieved. (b) THz time waveforms of the AA aqueous solutions at various concentrations. (c) Calibration curve of acetic acid in water as for the maximal change of the time waveform. The amplitude intensities at certain concentrations were estimated as shown in (a) at various concentrations of acetic acid in water and corrected as the maximal intensity change of the time waveform obtained with a capillary filled with air (as a green line in (a)) was unity for each of measurements. Black circles and error bars represent average and standard deviation of four different measurements, respectively. (d) Calibration curve of acetic acid in water at the stage position fixed.
Figure 6(b) shows the THz time waveforms of the acetic acid aqueous solutions at various concentrations. Each measurement was repeated four times for reliability and the informative amplitude intensity was calculated as
5.3 CE separation of carboxylic acids with THz detection
The separation of acetic acid and n-propionic acid by capillary electrophoresis was tried with localized THz detection at room temperature. The delay stage for the THz detection was fixed at the position where the difference between the amplitude intensity of acetic acid and that of water is maximum (the fixed position is shown in Fig. 6(a)), and this intensity was recorded during the CE measurement as a function of the migration time of the sample solutions. Figure 7(a) shows a capillary electrophoretic chromatogram (electropherogram) of the separation of acetic acid and n-propionic acid at pH 5.5 with THz detection with the capillary, whose effective length and total length were 1200 mm and 1800 mm, respectively. The peak of acetic acid was separated completely from that of n-propionic acid. As anticipated, the electropherograms with THz detection were relatively noisy with the baseline containing irregular angularities. We assigned the peaks of target compounds, by comparing them to the electropherograms with THz detection of the solutions which contain only one analyte and to those with conductivity detection. The measurements at the same conditions repeated at least 5 times in different days, presented in Table 1. The detailed processes for identification and quantification of the analytes in CE measurements with conventional detection technique have been explained in the publications [2,3,5]. The migration times of acetic acid and n-propionic acid peaks of the electropherogram were close to those observed by contactless electroconductivity detection, shown in Fig. 7(b). The asymmetric peaks recognized by electroconductivity detection might be caused by the difference in mobilities between the sample ions and the buffer electrolyte ions [26].
Fig. 7. Electropherograms of the separation of acetic acid (AA) and n-propionic acid (PA) measured by (a) THz-CE; (b) electroconductive-CE. Effective and total lengths of the capillary were 1200 mm and 1800 mm, respectively. Applied voltage: -20 kV.
Table 1. Comparison of migration time and mobilities of acetic acid, n-propionic acid, and electroosmotic flow for the THz-CE and electroconductive-CE.
The migration times of acetic acid, n-propionic acid and electroosmotic flow peaks, as well as their electrophoretic mobilities for both THz-CE and contactless electroconductivity detection are shown in Table 1. Here, the mobilities are described as
6. Conclusions
A novel THz-CE technique which enables us to detect separated substances in a solution flowing in a hollow of capillary has been proposed and developed. The time waveforms brought by the photonics simulation software were quite similar to those observed by the experiments with the capillary. The separation between acetic acid and n-propionic acid at pH 5.5 was achieved and the electrophoretic chromatograms were successfully obtained. The identification and quantification in this study were achieved comparing with the results using standard samples. The processes are the same as those with conventional on-line detection techniques. However, when the sensitivity improves, complex refractive index or complex permittivity will be acquired and new informations at the low-energy level will be brought about for the analytes separated.
The complex optical parameters of aqueous acetic acid solution obtained with a conventional THz-TDS system indicated that the multiple data set acquired with THz-CE possibly help us understand the molecular dynamics in addition to the qualitative and quantitative analysis of mixture solutions without post or pre column labeling. For the acquisition of frequency-domain spectra through capillary electrophoresis, a time waveform should be measured in time scale of seconds. At present, however, the data acquisition time is more than one minute for each time waveform, typically 150 s. The sensitivity improvement, therefore, will be inevitable. We have some ideas to improve the sensitivity and to obtain time waveforms. For examples, the employment of a THz emitter with a higher output, an organic non-linear optical crystal, e.g., 4-N,N-dimethylamino-4'-N'-methylstilbazoliumtosylate (DAST) [27,28], optimization of the THz optics, e.g., investigation of wall thickness of the capillary and/or the laser light irradiation method, etc.. These techniques will enable us to conduct a more comprehensive analysis, including the hydration state of the samples [29].
We believe to succeed in measurements of THz time waveforms over time by THz-CE in the near future. Localized THz detection will be able to be used for the samples containing many kinds of chemical components, which will be separated, identified and quantified for each component. Additionally, this technique will be applied to the connection with various chromatographic or separation techniques.
Funding
Japan Society for the Promotion of Science (JP16K14255, JP17H03252, JP20H00247, JP21H01392, JP21H05092); Adaptable and Seamless Technology Transfer Program through Target-Driven R and D (JPMJTM20QB); Fusion Oriented REsearch for disruptive Science and Technology (JPMJFR2029).
Acknowledgments
K. Kitagishi thanks to Dr. Toshio Takagi, professor emeritus of Osaka University for his encouragements and suggestions to arrange and complete this report.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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