Building Blocks for the Development of an Interface for High

Technical Note pubs.acs.org/ac

Building Blocks for the Development of an Interface for HighThroughput Thin Layer Chromatography/Ambient Mass Spectrometric Analysis: A Green Methodology Sy-Chyi Cheng, Min-Zong Huang, Li-Chieh Wu, Chih-Chiang Chou, Chu-Nian Cheng, Siou-Sian Jhang, and Jentaie Shiea* Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan ABSTRACT: Interfacing thin layer chromatography (TLC) with ambient mass spectrometry (AMS) has been an important area of analytical chemistry because of its capability to rapidly separate and characterize the chemical compounds. In this study, we have developed a high-throughput TLC−AMS system using building blocks to deal, deliver, and collect the TLC plate through an electrospray-assisted laser desorption ionization (ELDI) source. This is the first demonstration of the use of building blocks to construct and test the TLC−MS interfacing system. With the advantages of being readily available, cheap, reusable, and extremely easy to modify without consuming any material or reagent, the use of building blocks to develop the TLC−AMS interface is undoubtedly a green methodology. The TLC plate delivery system consists of a storage box, plate dealing component, conveyer, light sensor, and plate collecting box. During a TLC−AMS analysis, the TLC plate was sent to the conveyer from a stack of TLC plates placed in the storage box. As the TLC plate passed through the ELDI source, the chemical compounds separated on the plate would be desorbed by laser desorption and subsequently postionized by electrospray ionization. The samples, including a mixture of synthetic dyes and extracts of pharmaceutical drugs, were analyzed to demonstrate the capability of this TLC−ELDI/MS system for high-throughput analysis.

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ques have been developed, the utilization of TLC−AMS for real sample analysis is still not as popular as high performance liquid chromatography mass spectrometry.31−42 One reason for this hindrance may be the lack of an automatic delivery system to continuously deliver the TLC plates to the AMS source for high-throughput analysis. To develop a high throughput TLCAMS system, the traditional methodology is to design and simulate the system using dedicated software, customizing or turning the components for fabrication, and testing the system for further modification or even redesign. However, this traditional model consumes considerable time, labor, and materials during the developing processes. Alternatively, a method using readily available components to develop a new system is another approach. Thus, the realization of the system can be efficiently achieved without further simulation and turning. In this study, we used a readily available material (LEGO building blocks) to design and construct an automatic TLC plate delivery system. The LEGO building blocks offer various sizes, shapes, and functions as well as several electronic parts, which allow a new system to be designed and constructed without any machinery on various materials. This approach greatly reduces the need of time, labor, and materials during the

reen chemistry is the trend of science that is recognized to be benign and friendly for environments.1,2 Several novel techniques and approaches in the field of analytical chemistry have been developed by reducing or eliminating the use of materials and by avoiding the generation of hazardous waste when the research is going.3,4 In general, replacing hazardous chemicals with safer reagents, reducing the number of sample pretreatment steps, and developing novel techniques for direct detection are typical approaches for developing green analytical methodologies.5−13 In the field of mass spectrometry, a green technique called ambient mass spectrometry (AMS) has been developed rapidly in recent years.14−18 AMS is capable of directly characterizing chemical compounds under ambient conditions, which provides the feature of minimal or no sample pretreatment. Several AMS techniques have been used to continuously monitor ongoing chemical reactions by observing a decrease of substrates accompanied with an increase of generated intermediates and products.19−22 This feature is useful to optimize the chemical reaction conditions, minimize the energy consumption, and inprocess monitor the formation of hazardous substances. To date, AMS has been applied to many areas to the detection of chemical and biological compounds on the sample surfaces and in solutions.15−18,23−28 Thin layer chromatography (TLC) is a commonly used technique because of its capability to purify, clean, and separate complex compounds.29,30 Although several TLC−AMS techni© 2012 American Chemical Society

Received: May 3, 2012 Accepted: June 15, 2012 Published: June 15, 2012 5864

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interface development processes. The TLC plate delivery system was then combined with electrospray-assisted laser desorption ionization (ELDI)/MS for high-throughput TLC− AMS analysis.

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EXPERIMENTAL SECTION Materials. Synthetic dye standards, rhodamine 6G, crystal violet, and methylene blue, were purchased from Sigma− Aldrich (St Louis, MO, USA). Over-the-counter drugs including Chyrtongdan (Min Tong Pharmaceutical, Taiwan), Panadol (Glaxo Smith Kline), and Noharegen (KINKI) were obtained from the local pharmacy. Methanol, chloroform, and ethyl acetate were purchased from Merck (Darmstadt, Germany), and acetic acid was purchased from Fluka (Milwaukee, WI, USA). Distilled deionized water (produced by a Milli-Q plus, Millipore system; Molsheim, France) was used for sample preparation or pretreatment. The building blocks (8547 mindstorms NXT 2.0, Billund, Denmark) were purchased from LEGO, which contain 612 elements and several electric parts (three motors, an ultrasonic sensor, two touch sensors, and a color sensor). Normal-phase TLC plates (silica 60 F254, aluminum based, 300 μm thick) purchased from Merck were cut to the size of 4 × 2.5 cm (L × W) for analysis. TLC Separation. The mixture of synthetic dye standards was prepared in methanol solution. The concentration of each dye standard in the sample solution was 10−2 M. The sample solution (5 μL) was spotted on a TLC plate and dried at 80 °C for 5 min; then the dye standards were developed by methanol/ water solution (1:1, v/v). For the analysis of the chemical compounds in the over-thecounter cold formula, methanol (100 μL) was mixed with the drug powder (10 mg), and the solution was sonicated for 30 min. After centrifugation, the supernatant was reserved for further analysis. The extracted solution (10 μL) was spotted on the TLC plate and then placed in the oven for drying (80 °C, 5 min). A mixture of ethyl acetate, acetic acid, and chloroform (98:1:1, v/v/v) was used to develop the analytes. Automatic TLC Plates Delivery System. To develop the interface for TLC−ELDI/MS, a conveyor system is built for continuous transport of the TLC plates into the ambient ion source. A dealing system is also necessary to deliver the TLC plate stored in a box to the conveyor (Figure 1). The materials required for building such a system include bricks of different sizes and shapes, motor, light sensor, rubber band, and control box. The acrylic plates were used to brace the delivery system. The photos in Figure 2 display three parts of the TLC plate delivery system including a dealing system (Figure 2a), a

Figure 2. Photographs of three TLC delivery parts: (a) TLC plate collecting box, (b) dealing component, and (c) conveyor component used to deliver the TLC plate.

conveyor (Figure 2b), and a collecting box (Figure 2c). The automatic TLC-AMS interfacing system was assembled by combining the three components together (Figure 3). ELDI/MS System. Details of the ELDI source have been described in our previous publications.34,43,44 As shown in Figure 3, the ELDI system contains an electrospray source and a pulsed UV or IR laser beam (266 or 1064 nm; Minilite I, Continuum Electro-Optics Inc., USA and LS-2130, LOTIS TII, Belarus, Russia). As the TLC plate passed through the ELDI source, its surface was irradiated by the pulsed laser beam to desorb the analytes on the plate. A methanol/water solution (1:1, v/v) containing 0.1% acetic acid was used to generate an electrospray plume through a fused-silica capillary (located approximately 3 mm above the irradiation spot) at a flow rate of 2.5 μL/min. A voltage of 5 kV was applied to the ESI source to induce the ESI plume. The analytes generated by UV or IR laser desorption were postionized by reacting with the charged species (e.g., proton, charged solvent species, and charged droplets). Analyte ions were interrogated by the entrance into the inlet of mass analyzer either an ion trap (Bruker Esquire 3000 plus) or a single quadrupole (Shimadzu LCMS 2010).

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RESULTS AND DISCUSSION In our previous study, ELDI/MS was demonstrated to be useful in characterizing the mixture of dyes, amines, and drug extracts separated on the TLC plate.34 The TLC plate was placed on an acrylic sample holder and was moved through the ELDI source by a syringe pump. The detection limit of 10−6 M and a linear range within 10−3 to 10−6 M (R2 = 0.9886) for FD&C red dye on a C18 TLC plate have been reported. However, this technique is labor intensive and time-consuming because the TLC plate has to be manually delivered to the sample holder before analysis. In this study, we used the popular building blocks by LEGO to design, assemble, and test a system to interface TLC with ELDI/MS for high-throughput sample analysis. Figure 2 shows photographs of three main components of the system: the dealing, conveying, and collecting parts. The collecting box comprised 42 bricks of different sizes and dimensions (Figure 2a). The function of the collecting box was to collect the TLC

Figure 1. Schematic illustration of the automatic TLC−ELDI/MS system for high-throughput analysis. 5865

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Figure 3. Photograph of the combination of LEGO-made TLC plate delivery system and ELDI/MS. This system contains several parts including (a) a control box, (b) TLC plate storage box and dealing system, (c) light sensor, (d) conveyer, (e) collecting box, (f) pulsed laser, (g) reflector, (h) focusing lens, (i) electrospray, (j) inlet of MS, and (k) CCD.

plates after each had passed through the ELDI source. The light sensor, a small conveyor, and a motor as well as 115 bricks were used to assemble the plate-dealing component containing a TLC storage box (Figure 2b). The TLC storage box, with internal dimension of 5 × 3 × 2 cm (L × W × H), could store up to 50 TLC plates. At the very bottom of the box, there was an exit for each TLC plate to emerge. The height of storage box exit was adjusted, so only one TLC plate was allowed to pass during each dealing process. The function of the dealing component was to dispatch the TLC plate stored in the box to the conveyor component. A small conveyor controlled by a step motor was used to dispatch one TLC plate out of the box at a time. A light sensor placed between the storage box and ELDI source was connected to the motor to control the movement of the small conveyor. As a TLC plate was delivered to the ELDI source, this would trigger the light sensor and the step motor to dispatch the next TLC plate out of the dealing box. The plate conveyor component, delivering the TLC plate from the dealing component to the ELDI source, was made of six parallel rubber bands (30 × 4.2 × 4 cm, L × W × H) and a motor (Figure 2c). Two acrylic plates were used to brace the conveyor. A control box obtained from LEGO was set on the acrylic plate to control the moving speed of the rubber bands. After assembling these three components, a TLC plate delivery system was completed. This system was integrated with an ELDI/MS system by setting it in front of the inlet of a mass analyzer. An ESI emitter was then set 4 mm above the rubber band. A pulsed laser beam was introduced and focused at the center of the rubber band using the reflector and focusing lens. As the TLC plate moved through, the surface of the TLC plate was laser-irradiated. Although some TLC gel particles would be desorbed by laser irradiation if proper laser energy was used (e.g., 12 mJ), only a trace amount of the particles

would be produced. This would not interfere with the detection of the analyte ion by ELDI/MS. To examine the capability of this LEGO-made TLC−AMS system for high-throughput analysis, four TLC plates used to separate a mixture containing three dye standards, rhodamine 6G, crystal violet, and methylene blue, were placed in the TLC plate storage box in the dealing system. The analytical procedures started by dispatching the first TLC plates out of the storage box. The plate was then transported by the conveyor to the ionization region of the ELDI source. The function of a light sensor installed before the ELDI source was to trigger the dispatch of the second TLC plate while the first plate passed through the sensor. After TLC−ELDI/MS analysis, the detected TLC plates were collected in the collecting box. Figure 4 shows the results of the use of the LEGO-made TLC−ELDI/MS system to repetitively characterize the dye standards on the TLC plates. Three dye standards were all detected by ELDI/MS, and their extracted ion chromatograms (EICs) (methylene blue (m/z 284), crystal violet (m/z 372), and rhodamine 6G (m/z 443)) are shown in Figure 4a−c. Figure 4d−f displays the ELDI mass spectra of three dyes recorded at 1.91, 2.04, and 2.09 min, respectively. The repeatability [relative standard deviation (RSD), n = 4 ] of rhodamine 6G was calculated to be 33.3%. The experimental results presented here have demonstrated the feasibility of the use of building blocks for the development of an analytical instrument, especially in the early stage. This methodology greatly reduces the consumption of time, labor, and materials during development. Because the TLC plate was swaying gently as it passed through the ELDI source, this influenced the stability of analyte signal. Furthermore, several problems were found in the use of the LEGO-made TLC−ELDI/MS system for routine sample 5866

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Figure 4. Extracted ion chromatograms of (a) methylene blue (m/z 284), (b) crystal violet (m/z 372), and (c) rhodamine 6G (m/z 443) detected by TLC−ELDI/MS. TLC−ELDI/MS mass spectra of (d) methylene blue, (e) crystal violet, and (f) rhodamine 6G recorded at 1.91, 2.04, and 2.09 min. (g) Structures of methylene blue, crystal violet, and rhodamine 6G.

Figure 5. Extracted ion chromatograms of (a) acetaminophen (m/z 152), (b) sodiated acetaminophen (m/z 174), (c) chlorpheniramine (m/z 275), (d) sodiated ethenzamide (m/z 188), and (e) ethenzamide (m/z 166) detected by TLC−ELDI/MS. (f) Structures of acetaminophen, chlorpheniramine, and ethenzamide. The extract of Chyrtongdan was detected in the 1st, 4th, 7th, and 10th analysis. The extract of Noharegen was detected in the 2nd, 5th, 8th, and 11th analysis. The extract of Panadol was detected in the 3rd, 6th, 9th, and 12th analysis.

analysis, including: (1) the lack of inherent strength and stability in the system meant that the system shakes somewhat during operation and the joints may be apart after a long time of use; (2) the size of the light sensor was kind of large; therefore, it is difficult to install the sensor underneath the conveyor; (3) the operating friction of the motor acquired from LEGO was too high, making it unsuitable for long-term operation; (4) the laser beam was continuously irradiated; this would cause a waste of energy and decrease the service life of the pulsed laser. To fulfill the requirement of stable and long-term operation, appropriate materials and electronic components were obtained to replace the building blocks and unsuitable components in the LEGO-made mode. However, the concept of building the system remained the same. In the new system, the light sensor was replaced by an optical fiber to trigger the dispatch of the TLC plate from the storage box and the irradiation of the pulsed laser in the ELDI source. The plastic steel was used to replace the building blocks and acrylic plates. An appropriate motor with much less friction as the one from LEGO was used to power the conveyor for long-term operation. To examine the usability of the new TLC−ELDI/MS system, the extracts of three over-the-counter drugs (Chyrtongdan, Noharege, and Panadol) were prepared for the test. Each sample was subjected to repetitive analysis on four TLC plates. The separated TLC plates were then placed in the storage box for further analysis. Figure 5 displays the EICs of five selected analyte ions detected on the TLC plates. Acetaminophen (MH+, m/z 152), its sodium-adduct ions (MNa+, m/z 174), and chlorpheniramine (MH+, m/z 275) were detected in all three drugs (Figure 5a−c). Ethenzamide (MH+, m/z 166) and its sodium-adduct ions (MNa+, m/z 188) were only detected in Noharegen (Figure 5d,e). The repeatability (RSD, n = 4) of chlorpheniramine was calculated to be 12.8%. Since the duration of the ion signal on TLC−ELDI/MS analysis is

longer than 15 s and the ion intensity is usually high enough (>105 counts), performing MS/MS analysis to characterize the structure of analyte ions would be easily achieved. Since the analysis of a TLC plate with a length of 4 cm was completed within 2.5 min (the speed of the conveyer was set at 0.05 cm/ s), this means that, if the TLC−ELDI/MS system was continuously run for 24 h, more than 400 TLC plates could be screened. This feature makes TLC/AMS particularly useful in many fields such as phytochemistry, pharmaceutics, and combinatorial chemistry where a large number of TLC plates are used in a day.

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CONCLUSIONS We have demonstrated a green methodology of designing, constructing, and testing the dealing, delivery, and collecting systems for integrating the TLC plate with AMS using commercially available LEGO building blocks and electronic parts. The LEGO-made TLC plate dealing and delivery system was successfully combined with ELDI/MS to detect the dye standards separated on TLC plates. Since the building blocks offer various sizes, shapes, and functions, which are costeffective and easily assembled, disassembled, and modified, this property greatly minimized the consumption of testing materials and reduced the generation of waste during the instrument developing processes. However, the swaying and shaking structure as well as high friction motor limited the LEGO-made system to be used for long-term operation and decreased the repeatability of the analyte signals. Therefore, firm materials and appropriate electronic components, including plastic steel, optical fiber, 5867

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and motor, were used to replace the flimsy components. This effort certainly enhanced the performance of the TLC−ELDI/ MS system, improving the repeatability of analyte signal and making the system firm enough for long-term operation and high-throughput analysis. In this study, we only demonstrated the integration of the LEGO-made TLC plate dealing and delivery system with ELDI/MS for high-throughput analysis; however, the system can also be easily combined with other ambient ionization techniques such as laser-induced acoustic desorption/electrospray ionization, desorption electrospray ionization, easy ambient sonic-spray ionization, direct analysis in real time, plasma assisted multiwavelength laser desorption ionization, laser ablation inductively coupled plasma, and surface sampling probe.35−41 In addition to a TLC plate, other planar plates such as a glass slide, stainless steel plate, wood section, and paperboard may also be adapted to the system.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax:+886-7-5253933. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Science Council of Taiwan and National Sun Yat-Sen University-Kaohsiung Medical University Research Project (#NSYSUKMU 101-012) for financial support of this study.

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