A Review of the Challenges on the Practical Stage of the 4th Welch Materials Cup Chromatography Competition

The 4th Welch Materials Cup Chromatography Competition, concluded on August 19, 2026, evaluated participants' comprehensive capabilities in sample preparation, method development, chromatographic condition screening, instrument operation, and data interpretation.

Through competition, learning, and discussion, the event aimed to promote the exchange of practical experience across the industry, bridge the gap between theoretical knowledge and practical application for chromatography professionals and students, and strengthen communication between Welch Materials and chromatographic analysts.

In this article, we will review and analyze one of the tasks in the practical contest, Separation of Multiple Polar Nucleobases, highlighting its complexities, common pitfalls, and method development logic.

The competition included three different tasks, each with comparable difficulty and different technical emphasis. Participants were randomly chosen one of the tasks and must develop and complete the entire analysis for the given challenge. The tasks include:

• Separation of multiple aromatic structural isomers,
• Separation of multiple polar nucleobases, and
• Separation of natural products containing a polyhydroxy aromatic ring.

They cover common scenarios in routine laboratory analysis: retaining polar compounds, resolving isomers, and developing gradient methods for natural products.

Regardless of the task, participants were required to achieve three fundamental assessment criteria: resolution between adjacent peaks ≥ 1.5, theoretical plate number ≥ 3000, and a complete method development report.

For the Separation of Multiple Polar Nucleobases task, participants were required to resolve five fundamental nucleobases: Cytosine, Uracil, Guanine, Thymine, and Adenine. All of them are nitrogen-containing heteroaromatic molecules and constitute the fundamental bases found in DNA and RNA.

Challenges of the task occur at analyte structure analysis, column selection, mobile phase and buffer salt selection, sample preparation, and system suitability validation. Below are some of the key difficulties where participants may lose points.

The target analytes contain multiple amino, imino, and carbonyl groups, with multiple dissociation sites and distinct pKa values:

• Cytosine: pKa ~4.6 (amino), 12.2 (imino)
• Uracil: pKa ~9.5 (imino)
• Guanine: pKa ~3.3 (amino), 9.2 (imino), 12.3 (hydroxyl)
• Thymine: pKa ~9.7 (imino)
• Adenine: pKa ~4.2 (amino), 9.8 (imino)

As pH changes, the ionization state of each compound shifts, profoundly affecting:

• Molecular polarity: Ionized species become significantly more polar, reducing hydrophobic interactions with stationary phases
• Retention behavior: The degree of ionization directly influences retention factors (k') in reversed-phase chromatography
• Peak symmetry: Ionized analytes can interact with residual silanols on silica-based columns, causing peak tailing

Therefore, pH selection is the primary method development variable in this task. Generally, the pH of the mobile phase should be at least 1.5 units away from the pKa of the target analytes.

The high polarity and relatively good water solubility of the target analytes form the second major difficulty. In conventional C18 reversed-phase conditions, the hydrophobic interactions that normally drive retention are exceptionally weak. This results in very short retention times for the target analytes and high chances of co-elution between them.

To overcome this, columns that are compatible with highly aqueous conditions should be preferred in the method development, such as AQ-C18 or Polar RP. 

Guanine features a highly conjugated planar structure that promotes strong intermolecular π-π stacking, yet it exhibits an extremely low solubility, almost insoluble in water or common organic solvents, compared to the other analytes. An alkaline medium such as 0.25 M NaOH, combined with an appropriate buffer system, can be used to dissolve guanine.

Other analytes exhibit significantly better water solubility. Cytosine, uracil, and thymine can be dissolved directly in water, and adenine can be prepared using an appropriate aqueous-organic solvent mixture, such as 15% methanol in water.

Many analyses fail not because of chromatographic conditions, but because of sample preparation.

The competition required participants to prepare a mixed standard containing all five nucleobases. This seemingly straightforward step is an easy place to lose points, as the target analytes are dissolved in different solvents, where solvent effects and solubility must be taken into consideration. If participants were not careful when preparing the solutions, salt or analyte precipitation may occur unexpectedly.

The mobile phase composition is another major factor for participants. They need to decide whether to add methanol, acetonitrile, or neither as an organic modifier, since methanol and acetonitrile produce significantly different selectivity, even when their proportions are adjusted to achieve similar overall retention.

Whether to add trifluoroacetic acid (TFA) is also a concern. TFA improves peak shapes and resolutions in polar compound separation, but it also surpasses mass spectrometry, interferes with low-wavelength UV, and may shorten column lifespan.

All target analytes five nucleobases contain conjugated heteroaromatic structures and exhibit characteristic ultraviolet absorption. Therefore, UV detection provides a convenient and practical means of monitoring the separation.

The competition evaluated participants on two broad dimensions: operational performance and method development quality.

In operational performance assessment, the participants are evaluated their analytical habits and details in the use of analytical balance, mobile phase preparation (filtration, degassing, etc.), sample preparation, column installation and transition before injection, flushing and storage post-analysis, instrument operation, data recording, and report writing.

In method development quality, the participants are evaluated their rationality of method development, system suitability (which takes up the highest weight), and analysis efficiency. Simply achieving separation does not guarantee full points; resolution, column efficiency, peak shape, total run time, and single injection run time are all important.