High Resolution Separation of Human Serum Albumin Tryptic Digest using Fused-Core HPLC Columns

By: Prof. Luigi Mondello, Reporter US Volume 26.4

Prof. Luigi Mondello
University of Messina, Italy



The last decades have witnessed the use of different analytical techniques to tackle such complex tasks as the analysis of biochemical systems like peptides and proteins. Liquid chromatography plays a central role in present-day proteome research because of its versatility as well as advances in LC column selectivity and resolution. Human Serum Albumin is a single-chain unglycosylated protein of 585 amino acids that act as a depot and transport site for fatty acids, drugs, bilirubin, heme, and hormones. It can undergo various modifications that significantly affect protein folding, stability, conformation, and function; characterization of the types and locations of such modifications is therefore of utmost importance in understanding HSA’s role in human pathologies. A typical procedure involves the preparation of a tryptic digest from the protein, then generation of reliable LC data as a preliminary separation step prior to subsequent characterization using complementary techniques such as MS or MS-MS and a database search.

In the case of highly complex samples with closely eluting components, the slow mass transfer of solute molecules inside the stationary phase particles can severely limit resolving power. The new Ascentis Express® columns, based on Fused-Core™ particle technology, ameliorate this limitation by providing a small (0.5 μm) path for diffusion of solutes into and out of the particles, thereby reducing the time that solute molecules spend inside the particles. As a result, these columns deliver more than twice the separating power of columns packed with 5 μm totally porous particles, which have been the workhorse of most HPLC laboratories for many years, and over 50% more separating power (theoretical plates) than columns of the same length packed with 3.5 μm particles, which often require higher pump pressures due to lower bed permeability. On the other hand, in cases when resolution is critical, smaller particle columns are not suitable for separating closely eluting peak pairs, as the use of a longer column to enhance separation is hampered by excessive backpressure above allowable limits. Thus, to fully exploit the higher efficiencies offered by the sub-2 μm particles, instrumentation beyond conventional HPLC is often required. The performance advantages that Ascentis Express columns packed with Fused-Core particles offer include efficiencies equal to sub-2 μm columns at half the backpressure due to their narrow particle size distribution, and a rugged design capable of high pressure operation and longer column lifetime in routine use.

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Results and Discussion

Initially, a conventional 5 μm C18 column was compared to the 2.7 μm Ascentis Express for the separation of a human serum albumin (HSA) tryptic digest (0.01 M in ammonium formate buffer), under gradient conditions, at a temperature of 35 °C. The higher efficiency of the 2.7 μm column versus the 5 μm column is clear in the chromatograms in Figure 1; almost twice the plates/ column were obtained with the Fused-Core stationary phase (i.e., 34,000 theoretical plates at 1.0 mL/min). The higher resolution and sensitivity of the Ascentis Express column allows for method optimization in the form of shorter runtime without sacrificing resolution, or by adding more column length at higher temperature to obtain higher resolution and/or higher speed, with no need to change the system configuration to overcome the high pressure drop generated by small particles.

Figure 1. Analysis of Human Serum Albumin Tryptic Digest on Ascentis Express (top) Compared to 5 μm Column (bottom)

Analyses were performed at 60 °C on a single column, two-column, and three-column set of the 2.7 μm particle size stationary phase, at a mobile phase flow rate of 1.0 mL/min, adapting the gradient on the serially coupled columns (5 cm length of 0.007” I.D. SS connectors) while keeping the relative retention constant by scaling to column length (chromatograms shown in Figure 2). Efficiency was increased three-fold by adding column length up to the system limits, while increasing the temperature allowed to reduce solvent viscosity and backpressure (pressure drop: 389 bar on the three-column set). Regarding retention times, two-fold and 1.5-fold increases were observed respectively when doubling and tripling column length, while resolution increased by a factor of √2, as predicted by the theory. To evaluate the performance of the gradient separation, peak capacities between the first and last peaks in the chromatograms were calculated from the average width of three peaks in the gradient run time. A peak capacity of 220 was obtained for the 15 cm column, increasing to 302 and to 367 respectively when doubling and tripling the length of stationary phases.

Figure 2. Gradient Elution of Human Serum Albumin Tryptic Digest: Comparison of an Ascentis Express C18 Columns

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Clearly, proper column choice and appropriate operating conditions eliminate the frustration and unnecessary time loss in method development and optimization of HPLC separations. The implementation of the LC analyses involves making good choices in stationary phase, resolution and peak capacity improvements, column selectivity, and particle size distribution, as well as full and proper control of critical system parameters such as mobile phase temperature and operating pressure. The unique performance of the Ascentis Express columns enabled faster method development for the separation of highly complex mixtures with closely eluting components, with a straightforward and scalable positive impact on turnaround times of existing analyses on standard LC equipment.

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