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European Biopharmaceutical Review

HPLC’s Golden Jubilee: Part 2

High-performance liquid chromatography (HPLC) has allowed researchers to successfully separate mixtures, identify features and quantify components. The technology has had a profound effect on pharma analysis, with new innovations continuing to further column lifetimes and keep the processes up-to-date.

HPLC Enhancements

Stationary Phase Chemistries
The earliest stationary phases used in HPLC with the pellicular packings were coated phases. The packing would be either coated externally and the particles dry-packed, or the coatings were applied in situ. Meanwhile, the dominant form of liquid chromatography was liquidliquid chromatography where analytes would partition into the coated phase. Unfortunately, if extreme care was not exercised, the coatings could easily be stripped from the packed bed and retention times would drift. The mobile phase had to be saturated within the stationary phase, gradient elution was not feasible, and the column temperature had to be strictly controlled to prevent retention changes. It was not long before researchers developed chemicallybonded phases that would negate some of these disadvantages.

Early chemical-bonded phases of the brush type with Si-O-C bonds were easily hydrolysed and could not tolerate any water or other proton-donating liquids. Siloxane (Si-O-Si-C) bonded phases proved to be more stable, and could be used with a wide variety of solvents and pH values. Even today, Si-O-Si-C phases are the most popular, and are available with an assortment of chemistries.

Reversed Phase

The availability of bonded Si-O-Si-C phases had a particularly favourable fallout – the development of reversedphase liquid chromatography (RPLC). The earliest stationary phases, developed for adsorption and normal phase chromatography, were polar (for example, silica gel and Carbowax™) and the mobile phase was non-polar (for example, isooctane or hexane). The elution order was non-polar compounds first, followed by compounds of increasing polarity. Initially, this was the ‘normal’ practice of HPLC.

The new mode of RPLC – never very popular in liquid-liquid chromatography – used the opposite combination of phases from the normal operation. In RPLC, the stationary phase was nonpolar (for example, octadecylsilane or octylsilane) and the mobile phase was polar (for example, water and watermiscible organic solvents like methanol and acetonitrile). Consequently, the name (coined by Csaba Horvath) was reversed-phase – a name still used today and by far the most popular mode of HPLC/ultrahigh-pressure liquid chromatography (UHPLC) (1).

RPLC exploded during the 1970s and has been widely used ever since. It can not only separate non-polar from polar compounds, but also has the ability to separate ionic and ionisable compounds by adjustment of the pH via buffers. Many compounds that show low solubility in water and higher solubility in water-miscible organic solvents are amenable to RPLC – over the years it has even increased in popularity.

Not only are popular alkylsilane C8 and C18 phases available, but a wide variety of other phases based on cyclic alkyl, aryl, mixed aryl-alkyl, fluorinated and polar-embedded phases, as well as aryl or alkyl ion exchange mixed mode phases, that have found some niche applications. Around 1,500 reversed-phase columns have been accounted for in over 170 different chemistries introduced between 1970 and 2010 (2). Over 93% of all liquid chromatographers use RPLC at some time in their laboratory (3).

New Bonding Phases
Over time, improvements have been made in the base silicas used for bonded phases, including the development of Type B low trace metal, non-acidic packings providing better separations and improved peak shapes. Exhaustive endcapping was another procedure to remove silanols which cause tailing. Polymeric packings provide a wider pH range but are also less efficient than the silica-based bonded phase materials. Silica-organic hybrids offer more extensive pH capability and can withstand the high pressures of UHPLC.

Despite the success of RPLC, other modes are widely used, and column improvements in those modes have been noted over the decades. Size exclusion chromatography (SEC), both in aqueous and non-aqueous mobile phases, is particularly useful in polymer characterisation and for the separation and purification of biomolecules. Smaller particle SEC columns have recently become available, allowing large molecule characterisations that used to take hours, to be performed in several minutes.

Polymer-based ion exchange phases with strong and weak ionic and ionisable functionalities are widely used in the proteomics and genomics separation area, as well as for the separation of other ionic and ionisable compounds, particularly in the area of ion chromatography. Separations of carbohydrates at high pH with pulsed amperometric detection makes use of polymeric ion chromatography packed columns.

Two of the most exciting areas of stationary phase development were the introduction of chiral phases and phases for hydrophilic interaction liquid chromatography (HILIC). The former were quickly accepted in the pharma industry since many of the drugs under development have one or more chiral centres, and regulatory bodies require the analysis of both enantiomers. In some cases, one of the enantiomers has detrimental side-effects for patients, while the other enantiomer provides the drug function. HILIC is useful for the analysis of polar compounds that eluted from RPLC columns quickly, or are even unretained. HILIC columns often have the opposite effect from RPLC in that polar analytes elute later than non-polars. Many pharma compounds contain polar functional groups such as amine and carboxylic acid, so HILIC has found widespread use in this marketplace.

Future of HPLC/UHPLC Columns

The HPLC/UHPLC columns market has been very strong, and columns are considered to be a consumable. An average instrument uses 6-8 columns per year, so as the number of instruments grows, so does this columns’ market. The overall market for columns (analytical, preparative, capillary/nano and accessories) is now estimated to be $1.3 billion, with an overall growth of 3.5% and an even higher growth in the UHPLC segment (4).

Tremendous strides have been made in particle and stationary phase technology. However, user demands in the drug industry for productivity improvements continue to push further development in columns that are faster, more efficient and more inert. These needs stretch from the drug discovery phase and all stages of development up to manufacturing and quality control accuracy.

In the laboratory, the superficially porous particle (SPP) columns have now firmly established themselves as the favoured column type (lower pressures, higher or equivalent efficiency, same loadability) over smaller completely porous particles for new method development. It has been shown that methods developed on older porous particle columns can be switched to these newer column types.

Over the next few years, expectations are that column manufacturers will continue to exploit the technology by filling out their SPP offerings with stationary phases that chromatographers apply to their everyday separations, as well as for specialised separations such as chiral compounds and biological pharmaceuticals. Even smaller SPP particles are envisioned in the future.

No doubt instruments will see further improvement in lowering band dispersion to handle smaller SPPs. There may be closer integration of the column hardware and instrument connections such that dead volumes may be almost nil. Although systems can be built to go to even higher pressure, if SPP continues to dominate there may not be a need to greatly exceed today’s pressure limits, but in chromatography, pressure is always a useful commodity.

Monolithic Columns
Monolithic columns still hold great promise if researchers can figure out how to improve the efficiency, without great increases in backpressure, and make them in longer lengths needed for difficult separations. For the most difficult separations requiring longer separation times, theoretical models have shown that monoliths have the greatest degree of promise (5). Polymeric monoliths could be quite attractive since their wider operating range gives them some advantages, but now that certain silicabased monoliths are coming off patent, more columns’ companies may desire to invest into this technology, thereby moving the field forward. Monoliths may become the favoured approach for laboratory-on-a-chip systems, since they can be synthesised in situ inside the narrow channels where efficient packing of particulates may prove exceedingly difficult.

As biopharmaceuticals, such as monoclonal antibodies and peptidebased 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. Oligonucleotide purity requires columns that separate a wide range of oligomers, sometimes at high pH, so chromatographers in that field are always on the lookout for high-efficiency, high-pH tolerant columns.

New Shapes

Columns that are more inert and provide symmetrical peak shape will always be in demand. Stationary phases have come a long way and seldom are complaints heard about non-reproducibility. 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. Small changes in selectivity provide the biggest changes in overall resolution – much bigger than particle size effects alone.

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 contributions in the preparative arena for chiral drugs, but it has now been applied to more general small molecule applications. For many separations, SFC can be superior to HPLC/UHPLC, especially in the speed of analysis. The phases used for SFC are different than those used for liquid chromatography, so additional polar phases are required to exploit this technology.

In the research community, twodimensional (2D) liquid chromatography has been gaining momentum when extremely difficult separations are encountered or when every compound in a complex sample needs to be separated. Here, truly orthogonal stationary phases are desired; so phases that are specifically designed for multidimensional separations could be on the horizon. The 2D technique has not been accepted yet for routine pharmaceutical assays, but the day may come when more complete characterisations required by regulatory bodies entail this degree of separation power. Already, the major instrument companies are assembling multi-dimensional instruments to respond to this potential marketplace.

Concluding Thoughts

Although column lifetimes are much longer nowadays, many users in the pharma environment consider columns expendable. When dealing with precious, high-activity, high-value pharmaceuticals, compound purity and accuracy of analysis are of utmost importance, and a column that has been used for thousands of injections may have some degree of contamination that may affect retention and peak shape, as well as compound purity, not worth risking in quantitative analysis. Some labs actually take perfectly good columns out of service that have reached a thousand analytical injections. Similar procedures are used for preparative columns that cost much more than analytical columns due to the increased amount of contained packing.

Note: To read Part 1, see EBR July 2014 or visit our online archive at:


1. Karger BL, Snyder LR and Horvath CS, An introduction to separation science, John Wiley and Sons, p586, 1973
2. Majors R, Developments in LC column technology, LCGC North America Special Supplement 28S: pp8-17, 2010
3. Majors R, LCGC No America 30: pp20-34, 2012
4. Strategic Directions International, Global assessment report, 2012-2016, published October 2012
5. Desmet G and Cabooter D, J Chromatogr A 1228: p20, 2012

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Recently retired as a Senior Scientist and currently a consultant for Agilent Technologies, Ronald Majors has been active in the field of liquid chromatography columns and sample preparation for over 40 years. He received his BS in Chemistry at California State University and completed his PhD in Analytical Chemistry at Purdue University. Ronald is the author of more than 150 publications in HPLC, gas chromatography, sample preparation and surface chemistry. He has received many chromatography awards, including the Chromatographic Society’s 2007 Martin Gold Medal.
Ronald Majors
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