Impacted by advancing instrumentation and techniques, column technology must evolve to keep up in the UHPLC and biopharma world.
Chromatography columns are used for preparative applications on scales from micrograms up to kilograms—in other words, they have a foothold in almost every laboratory using any type of chromatographic technique. While columns do not evolve nearly as often as other technologies, they are still impacted by the overall advancement of chromatographic technology as a whole.
For the last three decades, columns packed with 5 µm particles have done the trick. There are many validated methods using this particle size and researchers became accustomed to the technology. However, the use of smaller particle sizes—eg. 2 to 4 µm—has gained traction in recent years and finally supplanted 5 µm particles as the gold standard in most laboratories. The reduction in particle size leads to improved efficiency, which in turn leads to a reduction in column length. In keeping with this improved efficiency and overall advancement, many researchers are already turning toward the next big trend: sub-2 µm particles.
Sub-2 µm particle columns sprang up in the industry out of necessity for a column that could handle UHPLC pressures.
“The primary challenges in designing products for UHPLC as compared to conventional LC is the need for media that is capable of withstanding the very high pressures that are enabled by UHPLC systems,” says Jeff Laye, Product Manager, Phenomenex (Torrance, Calif.) “This means that the individual particles must be mechanically stable. In addition, extremely stable beds must be formed during the packing process to ensure the media does not form voids or channels under UHPLC pressures.”
Previously, most work was done using HPLC techniques and therefore corresponding HPLC columns (with the then-common 5 µm particle size). As more and more researchers move toward UHPLC technology, the adoption of sub-2 µm particle size columns is expected to increase rapidly.
Megan Kerr, Column Marketing Manager at Tosoh Bioscience (King of Prussia, Penn.) agrees with the expected UHPLC emergence. “As the cost of UHPLC instrumentation continues to go down and its acceptance by regulatory agencies continues to increase, I expect fully porous UHPLC columns to replace fully porous HPLC columns,” she says.
Another recent column trend is the adoption of core-shell technology, or superficially porous particles (SPP). Unlike standard columns, these superficially porous particles have a solid silica core and porous outer shell that generate high plate counts compared to fully porous particles. According to Phenomenex, using sol-gel processing techniques that incorporate nanostructuring technology, a durable, homogeneous porous shell is grown on a solid silica core to create a core-shell particle. This particle morphology results in less band broadening compared to fully porous particles, which allows the column to deliver high efficiencies.
SPP columns are considered a partial substitute for sub-2 µm particles due to similar efficiency at half the pressure drop. And while SPP columns are boasted for their improved speed and performance with UHPLC instrumentation, core-shell technology can also be utilized in HPLC and preparative LC systems alike.
Phenomenex’s Kintex product line features a range of particle sizes from 1.3 µm for the highest UHPLC efficiency possible, up to 5 µm for the improvement of common chromatographic methods. With efficiency levels more than 400,000 plates/meter, the Kinetex 1.3 µm allows UHPLC users to reach previously unavailable levels of sensitivity and performance, with the added benefit of easy method transferability and scalability.
The biopharmaceutical industry has been one of the driving forces behind recent column technology and evolution. Most work in the industry is done on large molecules, such as proteins and oligonucleotides, which illustrate different kinetics than small molecules. To achieve highly resolved peaks with good peak shape, most columns designed for this work must utilize wider pore sizes than that found in traditional columns.
“Column efficiency in the separation of biomolecules tends to be significantly lower than that found with small molecules, so there is always a drive to design columns that can maximize the number of theoretical plates without generating excessive back pressure or long run times,” says Kerr.
Within the biopharmaceutical industry, there is an additional push toward single-use, pre-packed columns. At the analytical level, there are virtually no disadvantages to pre-packed columns—they provide a higher level of column-to-column reproducibility, are easy to handle, exhibit excellent longevity and durability and do not require a high level of capital investment. At the realistic level, though, pre-packed technology runs into a wall when it comes to larger-sized columns, especially for large preparative purification. While that technology does not exist yet, pre-packed columns are considered an ideal fit for small- to mid-sized preclinical and clinical stage monoclonal antibody (mAb) production and vaccine validation.
For example, Tosoh Bioscience released the TSKgel SuperSW mAb columns this year, a line of three size exclusion columns optimized for mAb analysis. These columns use a pore-controlled technology that produces a shallow calibration curve in the molecular weight region of a typical monoclonal antibody, about 150 kDa, which allows for high-resolution separations. The TSKgel SuperSW mAb HTP can even be used on an UHPLC system due to its smaller dimensions and ability to withstand high flow rates and low back pressure.
Millipore also recently released Chromabolt, a family of columns pre-packed with EMD Millipore chromatography resins that is specifically designed for early clinical stage manufacturing. Available in 3 sizes—10, 20 and 32 cm ID—the columns were designed with customer input to ensure they could be used ergonomically and easily within manufacturing facilities.
Additionally, Sigma-Aldrich Corp.’s Titan columns contain sub-2 micrometer porous silica that is monodisperse and high-performance, capable of more than 250,000 plates/meter. This technology combines the advantages of solid-core and fully porous particles in one. According to the Paul Ross, Director of Marketing and R&D, Analytical Separations at Sigma-Aldrich, Titan columns reduce the cost to perform UHPLC, leading to improved productivity for pharmaceutical and contract research organizations currently employing the technology for medicinal chemistry, toxicology and clinical manufacturing.
The importance of column life cannot be understated—it is often one of the most highly ranked operating parameters when it comes to the selection of a column. To that end, we asked the experts to let us in on the secret to long column life:
• “Everybody thinks the key to extending the life of a column lies in the column itself, but the truth is the secret to long column lifetime is in sample clean-up,” says Tosoh’s Kerr. “Every column manufacturer will suggest that a guard column be used to extend the life of an analytical column, and of course this is true as the guard column is essentially ‘catching’ all of the particulates and sample matrix impurities that will eventually foul the analytical column. But if more sample preparation work is performed (eg. centrifugation, decanting, filtering, etc.) before anything is injected into the HPLC system, the lifetime of the column will be greatly extended, guard column included.
• According to Phenomenex’s Laye, “long column lifetime is best accomplished by (1) adequate sample preparation, whether by using techniques such as solid phase extraction or simple filtration, to ensure that few particulates enter the column; (2) operating within the acceptable range of mobile phase parameters, such as pH or solvent composition, as dictated by the manufacturer; and (3) flushing/cleaning the columns thoroughly, again following the manufacturer’s recommendation, before storing the column.
• “Garbage in, garbage out,” says Richard Lee, Lab Chromatography Marketing Manager at Bio-Rad Laboratories (Hercules, Calif.). “Clarify the starting material/load, regularly strip and clean the column and observe manufacturer recommendation concerning operation conditions, especially with flow rate and pressure.”
In the immediate future, we can expect to see a continued decrease in particle size and the use of core-shell technology, in addition to an increase in UHPLC column use, as well as modified column use in the biopharmaceutical industry. Although not prolific yet, long-term trends to look out for include monolithic columns, which are now benefiting from improved materials and manufacturing procedures that allow the generation of high numbers of plates with low back pressure.