Patent Application
20200246777
The present application relates to liquid chromatography apparatus, and particularly to a montmorillonite-based liquid chromatography column, especially for high-performance liquid chromatography (HPLC).
High-performance liquid chromatography (HPLC) is a powerful separation and analysis technique widely used to isolate and purify a wide range of chemicals, such as biological, pharmaceutical, environmental, food and petrochemical compounds. There is an everpresent and growing need to establish new HPLC methods, particularly methods that reduce analysis cost, time and waste while enhancing sensitivity and separation efficiency. In HPLC, separation of analyte mixtures takes place through a column, the separation efficiency of which relies mainly on the stationary phase materials contained therein.
Montmorillonite is a clay, specifically a subclass of smectites (2:1 clays). Montmorillonite consists of a central octahedral sheet of alumina surrounded by two tetrahedral sheets of silica. These silicate sheets have a plate particulate shape with an average thickness of about 10 Å. Bare montmorillonite is intrinsically hydrophilic. However, surface modification to increase hydrophobicity of the silicate layers is possible, making montmorillonite adaptable to a wide array of material applications.
Due to its considerable availability, low cost, good mechanical strength, excellent thermal stability, high solvent resistance, ease of functionalization and low toxicity, montmorillonite is commonly used, for example, as a sorbent for removing heavy metals and trace pollutants, a treatment for contact dermatitis, a component of drilling mud, an additive to hold soil water in drought-prone soils, a desiccant to remove moisture from air and gases, a component in foundry sand, an additive in catalytic processes, an annular seal or plug for water wells, a protective liner for landfills, a retention and drainage aid component, an anticaking agent in animal feed, an additive in cosmetics, a flocculant in ponds, an additive to minimize deposit formation in paper making, and many other applications. Many of the properties that make montmorillonite so useful in the above applications are commensurate with an effective stationary phase in a chromatography column.
Raw montmorillonite is not suitable as a stationary phase for reversed-phase liquid chromatography in the presence of water as a component of the mobile phase. Montmorillonite undergoes reversible expansion upon absorbing water, and would thereby be an unstable stationary phase material. Thus, a montmorillonite-based liquid chromatography column solving the aforementioned problems is desired.
The montmorillonite-based liquid chromatography column is a chromatography column, which may be steel, packed with unmodified montmorillonite for use in normal phase liquid chromatography, particularly high-performance liquid chromatography (HPLC). The column may be prepared by sieving montmorillonite to achieve a desired particle size range, preferably in the micrometer range, i.e., montmorillonite microparticles, and more preferably between 5-10 μm. The montmorillonite microparticles are suspended in a solvent, for example, ethanol, and packed into a column for use in HPLC. Before packing, the montmorillonite microparticles may be dried by, for example, heating for a period of time, e.g., by heating preferably at about 100° C. for at least 2 hours. The packing may be performed at a pressure of at least 5000 psi, and more preferably, between 5000-7000 psi.
The montmorillonite microparticles prepared as described above provide an effective stationary phase for use under low pressure conditions, e.g., in separating simple polar compounds, including some phenols and drugs, via normal-phase liquid chromatography mode. Alternatively, the montmorillonite could be used for functionalization of, or incorporation into, organic porous polymers, such as polymethacrylates, polyacrylates or polystyrenes, resulting in a composite material that could be applied as stationary phase for separations in reversed-phase mode, as well as use in normal-phase mode.
These and other features of the present disclosure will become readily apparent upon further review of the following specification and drawings.
Similar reference characters denote corresponding features consistently throughout the attached drawings.
Raw montmorillonite is usually present in a wide range of particle diameters. In order to prepare an efficient HPLC column with suitable particle size, shape and distribution, montmorillonite is sieved before being packed into the columns.
In preparing the exemplary montmorillonite column, the specific surface area of the montmorillonite before and after sieving was obtained using liquid N2 physisorption, analyzed according to the Langmuir and Brunauer-Emmett-Teller (BET) theory. Langmuir analysis of montmorillonite before and after sieving gave specific surface areas of 433.63 m2/g and 536.69 m2/g, respectively. BET analysis of the montmorillonite before and after sieving gave specific surface areas of 274.36 m2/g and 339.50 m2/g, respectively. These values confirm that the sieving process resulted in montmorillonite microparticles with significantly increased specific surface area, thereby providing more interaction sites and enhanced retention characteristics compared to raw montmorillonite.
To achieve and maintain a uniform stationary phase in the column, the sieved montmorillonite microparticles are dispersed in a solvent, such as ethanol. In an exemplary column preparation, 1.0 g of the sieved montmorillonite microparticles was dispersed by sonication in 10 mL ethanol for 10 min. The sieved montmorillonite microparticles were well suspended and stable in ethanol solvent. No deposition of montmorillonite microparticles was observed for at least 1 hour after dispersion. Other solvents, including methanol, isopropanol and cyclohexanol, could also used to obtain a homogenous montmorillonite microparticle suspension.
While maintaining a uniform and stable suspension, the montmorillonite microparticle suspension is poured, preferably immediately after mixing, into an empty stainless steel column. Preferably, the column has a height less than or equal to 10 cm, more preferably from 5-10 cm, and has an internal diameter preferably in the range from 2.1-4.6 mm. In the following exemplary applications, a steel column having approximate dimensions of 5 cm height×2.1 mm internal diameter was used. The suspension is packed under pressure, preferably above 5000 psi, more preferably in the range of 5000 psi to 7000 psi, and most preferably around 5000 psi (about 34.5 MPa), for an amount of packing time, preferably around 10 min. Prior to chromatographic evaluation and application, the montmorillonite packed column was washed with methanol and acetonitrile at a flow rate of 0.1 mL/min until a stable and constant column back-pressure was observed.
In order to identify the primary organic functional groups of the stationary phase material, a sample of the montmorillonite microparticles used to pack the column was examined by Fourier-transform infrared (FT-IR) spectroscopy. The FT-IR spectrum of the montmorillonite microparticles prepared as described above is shown in
The stability of the montmorillonite microparticle column prepared as described above was also investigated. Different common chromatographic solvents were selected and passed through the columns in order to measure the column back-pressure at different flow rates. In particular, back-pressure flow rates ranging from 0.1 to 1.0 mL/min were tested for hexane, acetonitrile and methanol, and from 0.1 to 0.5 mL/min for ethanol and isopropanol.
The pressure drop of the exemplary prepared columns increased linearly over the applied flow rate ranges; 0.1-1.0 mL/min for hexane, acetonitrile and methanol, and 0.1-0.5 mL/min for ethanol and isopropanol, at a constant column temperature of 25° C. A linear fit of the column back-pressure vs solvent flow rate has regression factors R2 between 0.9994 and 0.9998, indicating good permeability and mechanical stability of the prepared montmorillonite columns.
The stability of the exemplary columns prepared as above was evaluated over 6 successive days.
Exemplary HPLC columns were prepared as above with unmodified montmorillonite as the stationary phase and used in the following HPLC separation applications, although use of the montmorillonite-based liquid chromatography column is not limited to the particular polar compounds mentioned in the examples.
The montmorillonite-based liquid chromatography column was used to separate a mixture of phenolic compounds (i.e., phenol, resorcinol and phloroglucinol) under different conditions. Under optimized conditions, the three phenols were completely separated in less than 7.5 min, as shown in
The prepared column was evaluated in terms of plate numbers, capacity factors, peak asymmetry, and chromatographic resolution for each standard. The performance in terms of the column plate number was between 26,000 plates per meter for phenol and 28,900 plates per meter for resorcinol under optimum conditions. The capacity factors for phenol, resorcinol and phloroglucinol solutes were 0.73, 1.07 and 1.47, respectively, while the chromatographic resolution between the peaks was more than 1.84 in all cases. Peak asymmetry factors were 1.26, 1.29 and 1.44 for phenol, resorcinol and phloroglucinol, respectively.
Exemplary montmorillonite-based liquid chromatography columns were applied for the separation of caffeine and ibuprofen drugs extracted from Profinal-XP tablets, labeled at 400 mg ibuprofen and 65 mg caffeine per tablet (manufactured by Julphar, Gulf Pharmaceutical Industries, Ras Al Khaimah, UAE), under different experimental conditions. As an example,
At optimum separation conditions, the column exhibited an efficiency of 4,200 plates per meter for caffeine and 5,300 plates per meter for ibuprofen, while a higher plate number was obtained at lower applied flow rates. The average tailing factor for caffeine and ibuprofen was 1.52 and 1.60, respectively. All parameters obtained after validation are in agreement with the criteria as per International Council for Harmonisation (ICH) guidelines.
Exemplary montmorillonite-based liquid chromatography columns were applied to separate vitamin C and aspirin compounds extracted from Aspirin-C tablets, labeled 400 mg aspirin and 240 mg vitamin C per tablet (produced by Bayer pharmaceutical company, Aktiengesellschaft AG, Germany), under different chromatographic conditions. At optimum chromatographic conditions, the two active ingredients were totally separated, as presented in
Under the above conditions, the two extracted compounds were separated in 4 min with a chromatographic resolution of 2.17. The calculated efficiency values of the column were 2,100 plates per meter for aspirin and 3,600 plates per meter for vitamin C. However, much higher plate number values were obtained at smaller flow rates. The average asymmetry factors were 1.23 for aspirin and 1.37 for vitamin C. All separation and efficiency parameters are in agreement with the criteria as per ICH documents.
The prepared montmorillonite-based liquid chromatography columns were used to separate paracetamol and chlorzoxazone active ingredients extracted from Relaxon capsules, labeled 300 mg paracetamol and 250 mg chlorzoxazone per capsule (manufactured by Jamjoom Pharma, Jeddah, KSA), under different experimental conditions. As shown in
The column exhibited a good efficiency in terms of the number of theoretical plates with 4,400 plates per meter for chlorzoxazone and 7,100 plates per meter for paracetamol. The tailing factor for the detected peaks was 1.36 for chlorzoxazone and 1.41 for paracetamol. The analytical performance and validation parameters are in agreement with the criteria as per ICH guidelines.
The exemplary montmorillonite-based liquid chromatography columns, prepared and applied as in the above examples, proved to be stable, reproducible and efficient for separation of drug compounds under normal-phase liquid chromatography conditions. However, the montmorillonite-based liquid chromatography columns prepared as described above should be understood to be applicable to a wide range of other research and industrial areas. This could be achieved by specific functionalization of the surface of the montmorillonite microparticles (e.g., silylation, alkylation, acylation) to allow their use as stationary phase in either normal or reversed liquid chromatography modes. The montmorillonite microparticles prepared according to the present specification, and the HPLC columns prepared with the montmorillonite microparticles, provide a novel separation media that may open up promising avenues for food, environmental and pharmaceutical analysis.
It is to be understood that the montmorillonite-based liquid chromatography column is not limited to the specific embodiments described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter.
