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© 2021 MJH Life Sciences™ and Chromatography Online. all rights reserved.
Discussed the latest development of open-tube liquid chromatography (OTLC), focusing on the advancement of chromatographic column technology.
Liquid chromatography (LC) traditionally uses columns that are completely packed with discrete particles or built around a monolithic structure. The concept of open-tube columns (OTC) or narrow-diameter columns with a thin stationary phase coating has been known for many years. Limitations in sample capacity, column reproducibility, and instrumentation have prevented wider adoption of this technique. However, the latest developments in instrumentation, manufacturing, nanoparticle technology, and other new stationary phase carriers have reignited activity in this field. The interest in open-tube liquid chromatography (OTLC) stems from the potential for high efficiency, low back pressure, and the prospect of a more environmentally friendly and sustainable separation. This article discusses the latest developments in OTLC, with a focus on the advancement of chromatographic column technology.
Liquid chromatography (LC) is usually implemented using a column packed with discrete particles or using a monolithic structure. Open tube liquid chromatography (OTLC) describes the use of open capillaries in which the stationary phase is coated in a thin layer along the tube wall.
The concept of open-tube chromatography originated in the field of gas chromatography (GC). Golay predicted in 1958 that a capillary coated with a thin stationary phase layer could achieve high-efficiency separation (1,2). This discovery will transform GC from the packed column that previously dominated the laboratory environment to the open tube format that is prevalent today. According to Forster and colleagues, Tsuda originally transferred the concept of open-column chromatography to LC (3). OTLC is now considered a viable option in liquid chromatography and capillary electrochromatography (CEC).
OTLC initially attracted people's interest because the technology can provide efficient separation of complex mixtures, uses a small sample volume, consumes relatively little mobile phase, and operates at a relatively low back pressure. Unfortunately, the advantages of OTLC are offset by poor sample capacity and difficulties in column manufacturing and reproducibility. In addition, existing platforms lack instruments that can provide consistent low flow control, small volume operation, and detector sensitivity.
Some recent comments on OTLC indicate that there may be an emerging trend of renewed interest in this technology. Due to the complexity and limited volume of samples, the demand for chromatographic column technology continues to grow, especially in challenging biological applications such as proteomics and metabolomics and other scientific research. This, combined with advances in micro-manufacturing technology, instrument design, miniaturization, and improved detector sensitivity, has increased the efficacy of OTLC. This article aims to collect and refine current trends in OTLC, with a focus on new column development, motivation, and supporting technologies.
OTLC column type and preparation
OTLC columns are divided into wall-coated open tube (WCOT) or porous layer open tube (PLOT) (4). WCOT columns are characterized by a thin, non-porous coating of high molecular weight polymers (for example, polydimethylsiloxane [PDMS]). PDMS polymers can be modified with functional groups such as cyano and phenyl to change polarity and selectivity. Polyethylene glycol (PEG) and ionic liquid (IL) can also be used to produce WCOT columns (4).
In contrast, PLOT columns contain a thin layer of porous material on the capillary wall. The porous material may be in the form of an organic polymer (such as polystyrene) or an inorganic material (such as silica, etc.). Because PLOT columns have a higher sample capacity, they are generally more of interest in LC, while WCOT continues to dominate in GC due to its high efficiency and relative ease of manufacturing. Figure 1 shows the open-tube column form and its packed column and monolithic column form.
Although the WCOT OTLC column has the problems of low sample capacity and difficulty in preparation (especially small-diameter columns), some recent developments have aroused people's interest. In order to solve the problem of analyte loading capacity, Liu and Yang proposed that under very small diameters, due to the lack of dilution effect, the problem of low sample volume would be reduced (5). The team demonstrated that by using a high concentration of octadecyltrimethoxysilane (OTMS) solution in the coating process, a dense, non-porous coating can be achieved, resulting in a highly efficient chromatography column. However, the author does point out that the uncertainty of the absolute capillary inner diameter (id) may be an issue that hinders widespread acceptance of the technology.
Compared with inorganic chromatographic columns, organic polymer PLOT columns have the advantage of improving stability over a wider pH range and are generally considered easier to prepare (1). However, organic coatings will swell and shrink in the presence of organic solvents, so compared with inorganic-based PLOT columns, they may provide poorer reproducibility or overall lifetime of results.
The common technique for preparing organic PLOT columns is in-situ thermally initiated radical polymerization. Generally, fused silica capillaries are first activated by alkali treatment to produce silanol on the surface for anchoring. After neutralizing and drying the capillary, a silanizing agent, such as 3-(trimethoxysilyl)propyl methacrylate, is introduced to bind to the surface and act as a bridge to the polymer to be formed. Next, the mixture of monomer, crosslinker, porogen, and thermal initiator is pumped into the chromatographic column, and the end of the chromatographic column is sealed. Heating the chromatographic column will initiate a polymerization reaction, and finally, after a given time, the chromatographic column can be flushed and used. Styrene, divinylbenzene and various acrylate and methacrylate polymers are common. Obviously, the reaction conditions, choice of reagents and manufacturing technology at each step will affect the final chromatographic column. The polymer coating can be further modified to change the retention and selectivity of the chromatographic column.
For the preparation of inorganic PLOT columns, the sol-gel method is usually used. Likewise, the initial step is to treat the capillary and generate surface silanol. Silicon alkoxides, such as tetraethoxysilane (TEOS), are then hydrolyzed and condensed into a gel in the column, using acids or bases to catalyze the reaction. Subsequent aging and drying produce porous silica, which can then be further reacted with various functional groups in a manner similar to the modification of silica particles in the filled LC. As with the preparation of organic porous polymer coatings, the conditions of each step and the selected reagents will greatly affect the resulting chromatographic column and its characteristics.
Hara and colleagues explored the development of silica-based PLOT columns using tetramethoxysilane (TMOS) as the silica precursor (6). In a recent study, the team studied the use of a mixture of TMOS and methyltrimethoxysilane (MTMS) to produce what they call a hybrid PLOT column with an inner diameter of 5 µm (7). The term "hybrid" is intended to describe the incorporation of organic moieties into the silica structure of the porous material. Adding more methyl groups via MTMS results in a coating that is more hydrophobic and therefore more retentive than using TMOS alone. This also means that thinner coatings of mixed materials can provide the same retention as thicker TMOS-based coatings and have higher efficiency. The team pointed out that the layer thickness can be linearly controlled by the amount of TMOS/MTMS in the sol-gel mixture, and successfully prepared a column length of up to 1.3 m. An obvious limitation of this process is that the high viscosity of the sol-gel solution makes it difficult to fill longer capillaries.
PLOT column development is also interested in the research of new separation media such as metal organic framework (MOF) in chromatography. Zhu and colleagues published an example of incorporating MOF into OTLC (8). In this work, the researchers added iso-reticular metal-organic framework-3 (IRMOF-3) particles to the open-tube form of poly[2-(methacryloxy)ethyl]dimethyl-(3). -Sulfopropyl)ammonium hydroxide polymer. In addition, MOF is derivatized with vancomycin. Compare the chromatographic performance of the modified and unmodified versions with the base polymer. The vancomycin-modified MOF system has been shown to provide retention and separation for a set of neutral, acidic, and basic analytes, and exhibits many different retention mechanisms.
Current drivers and enabling technologies
Complex and limited sample: Some recent comments on OTLC suggest that the Karger team's interest in the technology has revived (1, 4). In an interesting paper, Karger’s group used a 10 µm inner diameter PLOT column in hydrophilic interaction liquid chromatography (HILIC) mode in combination with mass spectrometry (MS) to analyze the N-linked glycans released from ovalbumin. A comprehensive analysis was carried out (9). The PLOT column consists of an ethylenediamine modified poly(vinylbenzyl chloride-divinylbenzene) coating. In another example, the Karger group used a polystyrene/divinylbenzene 10 µm PLOT column coupled with MS to analyze protein digests originally sampled using laser capture microdissection (LCM) (10). According to the authors, LCM allows the separation of biologically different cell subtypes; however, obtaining large sample sizes is very laborious. In addition to the highly sensitive PLOT LC-MS/MS system, careful sample handling allowed the team to identify increased numbers of proteins. These examples highlight several driving factors—the need for efficient separation to process complex samples, and the ability to analyze precious or highly limited sample volumes. These examples also mention one of the main OTLC support technologies-mass spectrometry.
Instruments: The very small column volume associated with OTLC requires a large number of instruments from the surrounding instruments. Although it has long been known that this technology will provide high-efficiency chromatography, ordinary detectors lack the sensitivity to such small volumes and micro-sampling; in addition, nanofluidic technology and instruments are not common. With the development of high-sensitivity MS systems and electrospray ionization (ESI) interfaces, the detection sensitivity problem has been largely resolved. The direct coupling of OTLC and ESI-MS minimizes band broadening, and flow rate is usually the best choice for chromatographic efficiency and ionization efficiency. Improvements in micro-sampling equipment (usually integrated) and nano-flow instruments continue to further support technologies such as OTLC.
Two recent reviews on the trend of instrument miniaturization pointed out that the column is an important part of reducing the space occupied by the instrument (11,12). The promise of OTLC columns in terms of high efficiency, low back pressure, low mobile phase volume, and direct MS coupling is emphasized in both; however, the lack of commercially available columns and instruments is a universally accepted limiting factor.
There is still significant and new interest in open-tube liquid chromatography. This interest is largely motivated by the need to analyze complex samples, preserve materials in limited sample quantities, basic materials science and process research, and the ambitions of environmentally friendly separations. The development of instrument technology (such as improved low flow control, innovative micro-nano sampling equipment, especially mass spectrometry) continues to promote the progress of OTLC. As this article emphasizes, chromatographic column technology is also evolving in terms of preparation process and equipment design, as well as a basic understanding of the most suitable separation media for this type of separation.
For the wider acceptance and use of OTLC technology, there seems to be a lack of a complete collaborative system, including sampling, transmission, separation, and detection (and data analysis). As pointed out by Røberg-Larsen and colleagues, liquid chromatography is a key component of single-cell analysis, which is a complex and limited category of high-interest samples (13). The author goes on to point out that there are some commercial tools available (for example, packed nano-liquid chromatography columns) that can be used for this type of work, but other formats (such as OTLC, etc.) have great potential in further advancing single-cell omics analysis.
David S. Bell is the Director of Research and Development at Restek. He also serves on the editorial advisory board of LCGC and is the editor of "Column Watch". In the past 20 years, he has worked directly in the chromatography industry, focusing on the design, development and application of chromatographic stationary phases to advance gas chromatography, liquid chromatography and related combined technologies. His main goal is to create and promote new separation techniques, and to study molecular interactions that contribute to retention and selectivity in a series of chromatographic processes. Contact directly: amatheson@mjhlifesciences.com