There have been major advances in technology from separation columns to instrumentation to data analysis and reporting. Large-particle packing of 50 Âµm diameter has given way to micro particles with diameters smaller than 2 Âµm; HPLC systems have gone from constant gas pressure pumps operating at 1000 psi to pumps capable of 20,000 psi pressure; detectors have progressed from simple 254-nm UV detectors to several hundred thousand dollar high-resolution mass spectrometers; and data output has moved from strip-chart recorders to high-speed computers with ability to handle complex chromatograms.
Investment in column technology is bound to continue since the HPLC and ultrahigh-pressure liquid chromatography (UHPLC) columns market has been very strong; otherwise there would not be around 200 companies involved in some aspect of the business. An average system uses seven columns per year; so as the number of instruments grows, so does the columns market. The overall market for columns is estimated to be worth a billion dollars with a higher growth in the UHPLC segment in the coming years. Recently, the 2.6-2.7 Âµm superficially porous particle (SPP) columns have established themselves as the favored column type for new method development compared to the sub-2-Âµm totally porous particle (TPP) columns. Methods developed on older porous particle columns can be switched to these newer column types with minor adjustments. The new strain of SPP columns may actually break the 10-year cycle in adoption delay.
Strides have been made in chromatography technology over the years. However, user demand in industry for productivity and sensitivity improvement continues to push further developments in columns that are more efficient, faster, and more inert. These needs stretch from the research laboratory and through all phases of development up to manufacturing and quality control.
Biochromatography columns. As biopharmaceuticals, such as monoclonal antibodies and peptide-based compounds, continue to make inroads in the drug market, columns capable of providing high recovery separations of biologically derived compounds, oligonucleotides, and biosimilars, both neat and in biological fluids, will be in big demand. Column manufacturers are already responding with biocompatible columns that provide more selective separations with higher recovery. Bio-chromatographers will need to have columns that cover most of the HPLC modes. The main requirement is that the pore size of biochromatography packing must be large enough to accommodate the largest biomolecules encountered.
Column specifications and features. Columns that are more inert and provide symmetrical peak shape will always be in demand. In the past couple of decades, stationary phases have come a long way and there are seldom complaints about non-reproducibility. To realize equivalent performance with conventional analytical, narrow-bore, capillary, and nano-columns, a more systematic study on column-packing requirements will be needed. Approaches to increase and predict chromatographic resolution with improved stationary phases that show better control of selectivity for critical separations will be needed in the future. Although column lifetimes, even at higher pH values, are much longer nowadays than in yesteryear, many users consider columns expendable. Some laboratories actually take perfectly good columns out of service that have reached 1000 analytical injections. Similar procedures are used for preparative columns that cost much more than analytical columns because of the increased amount of packing.
Supercritical fluid chromatography columns. With its orthogonal separation power, supercritical fluid chromatography (SFC) has made a comeback in the rapid analysis of small pharmaceutical compounds. Initially, SFC made its contribution in the preparative arena for chiral drugs, but now it has been applied to more general small molecule applications. For some separations, SFC can be superior to HPLC and UHPLC, especially in the speed of analysis. Further systematic studies on new phases by SFC column suppliers would add to the knowledge of reliably selecting the best stationary phase for a given separation.
Columns for 2-D LC. In the research community, 2-D LC has been gaining momentum when extremely difficult separations are encountered or when every compound in a complex sample needs to be separated. Truly orthogonal stationary phases are desired; so phases that are specifically designed for multidimensional separations could be on the horizon. Fast columns in the second dimension will be in popular demand and specialty phases based on SPP or monolithic technology may be needed to fill the gap. The 2-D technique has not been accepted yet for routine pharmaceutical assays, but soon more complete characterizations required by regulatory bodies may require this degree of separation power. Major instrument companies are already assembling multidimensional instruments to respond to this potential market.
Future Trends in Instrumentation
Instruments have been trying to keep up with column developments. The lifecycle for instrument development is much longer than what is required for new packings and columns. An area where it is well recognized as a hindrance to exploiting further improvements in column efficiency is the instrument contribution to band dispersion attributed to the HPLC and UHPLC instruments and their column-instrument interface designs.
Instruments will see further improvement in lowering band dispersion to handle smaller SPPs. A closer integration of the column hardware and instrument connections is needed such that dead volumes may be almost nil, much like what has been achieved in some nano and chip instruments. The area of frit and end-fitting design needs attention since the column packing where the separation actually takes place should be located at or near the injector device and the detector measurement device. This may necessitate a new column design that not only cuts down on this extra column volume, but also can withstand the higher pressures anticipated with smaller SPPs. Such designs are within the capabilities of engineers at the instrument companies but may necessitate the development of proprietary interfaces that may rule out the ability of the end user to select a column of their choice. Getting universal agreement among the instrument companies on a standard zero-dead volume interface would probably be next to impossible given the competitive environment that currently exists within the community. Perhaps some sort of cassette system without the typical compression-end fittings, similar to what has been used in some commercial chip-based column configurations, might be used advantageously for closer coupling and easy, rapid column replacement.
As far as instrument pressure capability is concerned, UHPLC systems can be built to go to even higher pressures since pumps capable of thousands of bars are already available for industrial use and chromatography engineers would have to adapt some of the same operating principles to achieve pulseless flow control in the microliter- to milliliter-per-minute range at pressures up to 100,000 psi. If SPP columns continue to dominate in the future, there may not be a need to greatly exceed current pressure limits.