GC-Like Performance using HPLC Column Coupling

By: William Campbell, Wendy Roe, Wayne K. Way, Reporter EU Vol 29

High Resolution Liquid Chromatography with Convential HPLC Systems and Ascentis® Express HPLC Columns

William Campbell, Wendy Roe, Wayne K. Way wayne.way@sial.com


In this study, high resolution HPLC with effi ciencies greater than 100,000 plates/column was achieved under moderate conditions. Specifi cally, Ascentis Express columns were coupled and used on a standard Agilent® 1200 HPLC system. Both isocratic and gradient examples are illustrated.

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High resolution liquid chromatography is important in many areas, including pharmaceutical product development, natural product chemistry and synthetic peptide mapping, just to name a few. A common need is LC methods that provide optimum assurance to the purity of peaks. For instance, in stress studies of active pharmaceutical ingredients (API), the ability to unambiguously quantitate and subsequently identify and potentially purify degradants of the API are paramount to the determination of the effi cacy and safety of the pharmaceutical product. Modern hyphenated techniques such as LC-MS and LC-NMR often rely on the purity of a chromatographic peak for structural identifi cation and confi rmation.

To date, reducing the particle size of the packing in HPLC columns has been the strategy of many column manufacturers to provide higher resolution columns. Smaller particles have higher effi ciencies and, therefore, can provide higher resolution. Unfortunately, column back pressure increases at a greater rate than effi ciency as one decreases particle size. Therefore, high resolution LC with small particles (sub-2 μm) is diffi cult even with modern LC systems. For this reason, a particle with high effi ciencies yet low back pressure would be a more suitable candidate for high resolution LC.

Ascentis Express columns provide a breakthrough in high resolution LC performance. Based on Fused-Core™ particle technology, Ascentis Express provides the high effi ciency based benefi ts of sub-2 μm particles but at much lower back pressure. Due to the high effi ciencies at low back pressures, Ascentis Express can provide high resolution chromatography that was previously unattainable on commercial LC systems.

The Fused-Core™ particle consists of a 1.7 μm solid core and a 0.5 μm porous shell. A major benefi t of the Fused-Core™ particle is the small diffusion path (0.5 μm) compared to conventional fully porous particles. The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening. In fact, Ascentis Express columns are able to achieve effi ciencies of 240,000 N/m, which is similar to that obtained with sub-2 μm particle columns, even though the back pressures are only 50% of that achieved under similar conditions with sub-2 μm particles. This means that Ascentis Express can turn almost any LC system into a high resolution workhorse for your lab.

Column coupling in HPLC is gaining interest since LC systems are being designed to withstand column back pressures of up to 15,000 psi. Column coupling is a simple and practical way to increase resolution by simply increasing column length. Because Ascentis Express HPLC columns provide higher effi ciencies at any pressure compared to 3 μm and sub-2 μm particles, the coupling of Ascentis Express columns enables signifi cantly higher resolution than any other column on any commercial HPLC system.

In this study, coupled Ascentis Express C18 columns were used on a standard Agilent 1200 HPLC system. Effi ciencies greater than 100,000 plates/column are demonstrated in the isocratic separation of benzene and D6-benzene. Ascentis Express column coupling is further applied to the analysis of a synthetic hydrophilic peptide to separate the target peptide from its deletion side products.

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Separation of Benzene and Deuterobenzene

Figure 1 shows the efficiency obtained by coupling Ascentis Express columns together. Due to space limits in the column heater, three 15 cm x 4.6 mm columns and one 10 cm x 4.6 mm column were used to obtain a total column length of 55 cm.

Figure 1:Separation of Benzene from Deuterated Benzene using an Ascentis Express C18, 55 cm x 4.6 mm I.D.

Figure 2 shows a plot of efficiency as a function of column length. The linearity indicates that effi ciency is not sacrifi ced due to coupling hardware. It should be noted that effi ciencies of greater than 100,000 were achieved under isocratic conditions with a modest back pressure of 7,000 psi.

Figure 2: Efficiency as a Function of Column Length

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Analysis of Synthetic Hydrophilic Peptide

Amino acid deletion products frequently result during the preparation of synthetic peptides. Since deletion may happen at any stage of the synthesis, the deletion products are diverse yet very similar to the target peptide. This process provides a unique and diffi cult separation challenge. The following is a study carried out on a hydrophilic synthetic peptide. The 12-mer peptide is composed of cysteine, proline, lysine, serine, phenylalanine and aspartic acid residues. Any one of the amino acids may be deleted at any position, thus a large number of possible deletion impurities may exist in the end product. Separation and identifi cation of these side products is important.

Figure 3 demonstrates the effect of Ascentis Express column coupling on this separation. The column length was extended to 30 cm and compared to the 15 cm. The gradient rate was adjusted to account for the added column length. Comparison of the data shows the enhanced resolution obtained for several of the deletion products.

Figure 3: Gradient Elution of a Synthetic Peptide and its Deletion Products: Comparison of an Ascentis Express C18 at 15 and 30 cm Column Lengths 1

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This study illustrates the potential for high resolution LC using Ascentis Express HPLC columns (using commercial instrumentation) under moderate conditions. Dramatic improvements in resolving power beyond that shown in this study are possible with elevated temperature and ultra-high pressure instrumentation.

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