HPLC is a well-established separation technology that continues to evolve to meet the identification and purification challenges posed by pharma, biotech, and other life science applications. Some of the latest techniques and applications were discussed at “HPLC 2007,” held recently in Ghent, Belgium, and attended by market leaders and specialist companies alike.
In a seminar honoring the contributions to liquid chromatography of Maurits Verzele, professor emeritus from Ghent University, Ronald Majors, Ph.D., senior scientist, columns and supplies division of life sciences and chemical analysis, Agilent Technologies, commented that the pioneering researcher had been more prescient than most.
As long ago as 1983, Verzele reported on the use of spherical silica particles with a diameter of around two micrometers in packed columns. Such small particles are of great interest today because they mean higher efficiency. “We are now in the sub-2 micrometer world,” said Dr. Majors, “It saves time and money and enables you to get the same results with shorter columns.”
Accordingly, companies like Waters and Agilent are coming out with small-particle products. Agilent is also interested in adjusting the particle-size distribution of the stationary phase. Small particles tend to block the column and increase back pressure, while large ones broaden the peak. Getting the balance right is important in improving the performance of a stationary phase.
In 1985, Dr. Majors carried out a survey of experts, asking them to predict future trends in HPLC. They were correct in foreseeing that smaller particles would grow in importance, and also that silica would continue to be competitive. They also foresaw that LC would become important in preparative and process separations and that columns that can separate basic compounds with no tailing would also become available. Columns today also have longer lifetimes and there are biocompatible products for biotech applications, which was also predicted, along with the continued popularity of the C18 phase.
However, some expert predictions have been wrong, such as the increased use of radial flow columns, user-derivatized columns, open-tubular columns, and non-porous media for separation of biologics, and the increased popularity of polymer phases over silica. None of these developments has occurred to any significant extent, said Dr. Majors.
Recently, he carried out a survey of readers of LCGC magazine to further define trends. “Reverse phase is still king,” Dr. Majors noted. “But techniques such as hydrophilic interaction have become increasingly popular in the last ten years, along with chiral columns since the FDA began to demand chiral purity.”
Another recent survey, done for “Pittcon 2007,” showed that there has been a distinct downward trend in the size of particles used in HPLC columns.
In 1985, 38% of users were using 10-micrometer particles compared to only 6% in 2007. Those using particles of 3–4 micrometers diameter increased from 6% in 1985 to 38% today.
“The largest growth has been in those using particle sizes less than two micrometers,” said Dr. Majors. He also noted that there has been an increase in column lifetimes although, interestingly, there has been no change in column consumption. Users are tending to report the same problems in 2007 as they did in 1985—namely, column-to-column reproducibility and bonded-phase stability.
“This tells us that although columns and column packings have improved over the years, there is still room for continued innovation,” Dr. Majors concluded.
One of the new stationary phases appears in the Acclaim® column, which was introduced by Xiaodong Liu, Ph.D., group leader for HPLC consumables development, R & D, Dionex.
The Dionex Acclaim system scores by being applicable to the simultaneous separation of basic, acidic, and neutral pharmaceuticals, said Dr. Liu. It belongs to the mixed mode class of hydrophobic/ion-exchange materials and, as Dr Liu pointed out, there are various ways of mixing the components.
The Acclaim Mixed-Mode WAX-1 column has “tips” of ion-exchange material on “chains” of reverse-phase material. The selectivity of the column is adjustable by varying the ionic strength, pH, or organic content of the mobile phase so a mixture (1,2,3) that elutes in the order 1,2,3 under one set of conditions can elute in the order 3,2,1 in other conditions, if so required.
The Sigma-Aldrich Supelco® Ascentis® Express series of columns provides an alternative to small-particle HPLC technology, with its associated higher back pressures.
“In recent years, the driving force in HPLC has been for faster method development and analysis,” commented David Bell, Ph.D., manager of applications and technical services at Supelco, introducing the Ascentis Express system, which is based upon Fused-Core™ particle technology.
The particles within the system consist of a 1.7-micrometer diameter solid core surrounded by a 0.5-micrometer porous shell, making up a particle of 2.7-micrometer overall diameter. This structure gives a much shorter diffusion path and a better peak shape compared to fully porous particles on the same diameter, according to the company.
Together with a tight particle size distribution and high packing density, this means that Ascentis Express columns have an efficiency comparable to the sub-2 micrometer particle column and twice that of the 3-micrometer particle column, Dr. Bell said.
Since back pressure is proportional to the inverse square of particle size, the Ascentis Express column generates half the back pressure of the sub-2 micrometer column, which allows the use of longer columns for higher resolution and faster flow rates for higher throughput without compromising efficiency, explained Dr. Bell.
Ascentis Express is capable of resolving D6-benzene (where the hydrogen atoms have been replaced by deuterium or heavy hydrogen) from ordinary benzene which is, Dr. Bell said, “quite a feat.” This was done by coupling together four Ascentis Express columns to create one with dimensions of 55 cm by 4.6 mm.
Using moderate operating conditions of 1mL/min flow rate, 50º C column temperature, and an isocratic mobile-phase composition of acetonitrile/water, efficiencies of greater than 100,000 were measured on all peaks in a test mix of five compounds. Benzene and deuterobenzene were baseline resolved, and the column backpressure was 7,000 psi as measured on a commercial LC system.
Ascentis Express is also good for analyzing peptide fragments and, therefore for protein identification, because it gives more peaks and higher sensitivity. The Ascentis Express columns can be used in most conventional HPLC setups, thereby upgrading them to UPLC status, according to Dr. Bell. Due to the high efficiencies at low back pressures, Ascentis Express can provide high-resolution chromatography that was previously unattainable on commercial LC systems.
“With instruments capable of operating up to 15,000 psi, a quarter of a million plates per column may be possible,” concluded Wayne K. Way, market segment manager HPLC at Supelco. Plates, or theoretical plates, is an HPLC term referring to column performance in terms of separation power.
Hydrophilic interaction liquid chromatography (HILIC) is another separation technology increasing in importance in applications involving polar compounds. Carl Sanchez, research scientist, analytical support and development, R&D, at Phenomenex, discussed a structured approach to method development and optimization in HILIC. It was shown that in HILIC, a technique mainly applicable to polar compounds, selectivity is significantly different than in reverse phase.
Polar Drugs and Metabolites
In HILIC, for example, retention time increases with increasing concentration of organic modifier in the mobile phase. This allows polar compounds to be retained and eluted at high organic concentration, which is beneficial for mass spectrometric detection. HILIC is proving useful for the analysis of polar drugs and drug metabolites, since metabolites are typically more polar than their parent compounds. One example given by Sanchez was of nicotine and its metabolites where little to no retention was seen in reverse phase and elution reversal and increased MS sensitivity was seen.
Parameters shown to be of primary importance in optimizing HILIC separations were: column chemistry, type and concentration of organic modifier, and buffer pH, explained Sanchez. Ionic strength and temperature were generally less important. The method development approach described involved systematically varying the column chemistry, % organic, and pH according to a rationally designed approach. This approach allowed efficient determination of the applicability of HILIC to a given separation and a good approximation of the best separation conditions in the least possible time. These experiments also provided the basis for further optimization, Sanchez said.
In the Phenomenex study, nine water-soluble vitamins were used to demonstrate the approach. Three different column chemistries were screened using a 90–50% acetonitrile gradient at two different buffer pHs. The column chemistries recommended were two Phenomenex Luna® columns (Luna HILIC and Luna Si(2)) and a Zwitterion column, all showing very different selectivity, said Sanchez.
The best overall performance in this experiment was obtained using the Luna HILIC column at pH 5.8, according to Sanchez. Further optimization using alternate organic modifiers and higher ionic strength was demonstrated using the Luna HILIC column. This approach gave not only the best column chemistry, but also the optimum pH and organic solvent concentration in an automated operation.
Finally, Waters makes complete LC/MS systems and in 2004 introduced the Acquity UPLC® Systems, which combine small particle (1.7-micrometer) technology with a novel design using high-pressure fluidics that allows increased resolution, sensitivity, and speed. Now labs can get more peaks and faster run times, said Paul Rainville, senior applications chemist at Waters.
Rainville discussed the application of these systems to biomarker studies carried out in collaboration with Imperial College, London. “There are lots of challenges in collecting data for biomarkers from plasma and urine because there are so many peaks. We need to be able to detect all the components and quantitate and identify them.”
Their approach combined the advantages of small-particle and high-temperature (95ºC) work to get high resolution and sharp peaks. They were interested in the effect of peak width on MS data quality and found that accurate masses can be obtained for urine metabolites with a TOF system, which is able to keep up with the speed of the new UPLC systems.
Meanwhile, they have also developed a system called MSE, which is MS and MS/MS run together to detect both precursor and product in one run. These new techniques are highly applicable to metabolomics, which has been shown by various case studies that the Waters and Imperial team have been working on.
First, they used the axenic rat (which lacks the gut flora that play such an important role in normal metabolism) as a model for examining metabolic pathways. A second case involves the use of the Zucker rat, a model of type 2 diabetes, examining urine and plasma with the LC/MS Acquity UPLC MSE on the Q-TOF Premier™.
The researchers have also worked on lipid profiles. These are generally conventionally acquired with GC, GC/MS, or normal-phase HPLC, which are slow and of limited sensitivity. On the UPLC MSE set up, at high temperatures, they achieved faster separation and, due to the higher flow rate, greater efficiency too, said Rainville.