11 High-Error Questions from the 2026 Welch Materials Cup Chromatography Competition: Answers and Analysis

This question examines the effect of gradient delay volume (dwell volume) on chromatographic resolution.

When the gradient delay volume increases:

1. The arrival of the gradient at the column inlet is delayed, which is effectively equivalent to adding a period of isocratic elution before the gradient begins.
2. The absolute retention times of all components will increase, but the relative change of retention times depends on the properties of the individual components.
3. The relationship between the actual gradient slope and the time at which the components leave the column is altered.
4. The resulting change in resolution depends on several factors, including:
   • The retention characteristics of the sample components
   • The relationship between gradient slope and analyte properties
   • Column dimensions and other column parameters

Because these factors differ among separation systems, increasing the gradient delay volume does not necessarily cause resolution to increase, decrease, or remain unchanged.

Therefore, the direction of the change in resolution cannot be determined in general, making D the correct answer.

Note: This is one of the highest-error questions. 83.82% of participants chose C (decrease significantly), but the correct answer is A.

For a 3.0 × 100 mm, 3 µm ODS column transferred from a UPLC system to a conventional HPLC system:

1. UPLC systems generally have lower extra-column dead volume than conventional HPLC systems. Extra-column volume can have a particularly significant effect on the apparent efficiency of small-bore columns.
2. However, a particle size of 3 µm is not in the sub-2-µm range. Its sensitivity to extra-column volume is therefore substantially lower than that of columns packed with 1.7 or 1.8 µm particles.
3. In addition, a 3.0 mm internal diameter produces a larger column volume than a 2.1 mm I.D. column, making the separation less sensitive to differences in extra-column volume.
4. Under the combination of a 3.0 mm internal diameter and 3 µm particle size, the difference in extra-column volume between UPLC and conventional HPLC systems has a relatively small effect on apparent column efficiency.
5. Therefore, when this column is transferred from a UPLC system to a conventional HPLC system, the column efficiency is generally expected to remain essentially unchanged, making A the correct answer.

The key point is, while extra-column dead volume significantly affects column efficiency for columns of small particle sizes and small internal diameters, the effect is much less pronounced with a 3 µm particle size and 3.0 mm I.D.

Note: This question also has a high error rate. 59.34% of participants chose C (both detectors are affected), but the correct answer is D.

Changing the configuration from serial to parallel changes the flow-path arrangement:

• In a serial configuration, the mobile phase passes through one detector and then the other.
• In a parallel configuration, the flow is split and the two detectors receive separate streams.

The key concept is the relationship between detector response and flow rate.

The UV detector is concentration sensitive; its absorbance is described by:

A = ε × c × l

where ε is the molar absorptivity, c is analyte concentration, and l is the optical path length. The response intensity of a UV detector therefore depends on concentration, molar absorptivity, and path length; it is independent of flow rate.

A refractive index detector is also fundamentally concentration sensitive. Its response depends on the difference in refractive index between the mobile phase and the column effluent and is likewise independent of flow rate.

When the flow is split from a serial configuration into a parallel configuration:

• The flow rate through each detector decreases, assuming a 1:1 split.
• However, the change in flow rate does not change the detector response per unit analyte concentration for these concentration-sensitive detectors.

It is important to distinguish response intensity from peak area and peak shape. A change in flow rate may alter peak width and therefore affect peak area or other aspects of the chromatogram, but it does not inherently change the response intensity (response per unit analyte concentration) for a given analyte concentration.

Therefore, the response intensities of both detectors remain essentially unaffected, making D the correct answer.

For a compound with a pKa of 3:

• At pH 6, pH − pKa = 6 − 3 = 3, which is greater than 2. The compound is therefore essentially fully ionized (>99.9%) and exists as an anion.
• At pH 8, pH − pKa = 8 − 3 = 5, which is also greater than 2. The compound remains essentially fully ionized (>99.99%) and still exists as an anion.

At both pH 6 and pH 8, the compound is already overwhelmingly present in its deprotonated form. Its charge state therefore does not undergo a meaningful change when the pH is increased from 6 to 8. Consequently, its interaction with the reversed-phase stationary phase remains broadly similar, and its retention time should remain essentially unchanged, making C the correct answer.

Many participants may choose A because they assume that increasing the pH will cause the compound to become even more ionized and therefore reduce retention. However, that interpretation overlooks the fact that the compound is already essentially fully ionized at pH 6. Further increasing the pH produces little practical change in its ionization state.

Key note: When the pH differs from the pKa by more than 2 units, the ionization state of the compound is essentially unaffected by pH changes.

A. Correct. Ion-pair reagents work by adsorbing onto the stationary phase surface and forming a dynamic modification layer. When the concentration is changed, the original adsorption equilibrium is disrupted, and a new equilibrium must be established. The longer the carbon chain of the ion-pair reagent and the higher its concentration, the stronger its adsorption onto the stationary phase, and the longer the equilibration time.

B. Correct. The most reliable way to determine whether a column is sufficiently equilibrated is to perform repeated injections and observe whether the RSDs of retention time and peak area have become stable. If the RSD after three to five consecutive injections meets the requirement—typically RSD < 1%—the column can be considered sufficiently equilibrated.

C. Incorrect. Analytes with different charge numbers interact with ion-pair reagents through different mechanisms and with different strengths. For analytes carrying different charges, the ion-pair reagent concentration must be optimized separately; it cannot be assumed that a single concentration is suitable for all analytes.

D. Correct. When introducing a higher concentration of ion-pair reagent, the column needs to be re-equilibrated, but it is not necessary to flush the column thoroughly before re-equilibration.

All four statements represent incorrect understandings.

A. Incorrect. A ghost-peak trap column can greatly reduce ghost peaks, but it cannot guarantee complete elimination of all ghost peaks. Ghost peaks originate from a variety of sources, including mobile-phase impurities, system contamination, and sample matrix effects. Ghost-peak trap columns are mainly effective against mobile-phase impurities and have limited effect on ghost peaks from other sources.

B. Incorrect. A ghost-peak trap column adsorbs certain components in the mobile phase, especially hydrophobic impurities. Therefore, it changes the actual composition of the mobile phase to some extent. It cannot be said to leave the mobile phase composition unchanged.

C. Incorrect. The main function of a ghost-peak trap column is to trap impurities and thereby reduce ghost peaks. Its effect on baseline noise and baseline drift is not necessarily significant, so it cannot be generally described as significantly reducing baseline noise and drift.

D. Incorrect. Ghost-peak trap columns have an applicable range of mobile phases and are not suitable for every mobile phase. Different types of ghost-peak trap columns are suitable for different mobile-phase systems.

A. Correct. The corrected area normalization method requires that all components be completely separated and elute as peaks; otherwise, accurate quantification is not possible. This is a basic prerequisite of the normalization method.

B. Correct. The corrected area normalization method requires the relative correction factor (or mass response factor) of each component to convert peak areas into true component contents.

C. Incorrect. Normalization is a calculation method that makes the sum of the component contents equal to 100%. This is a mathematical treatment, not a prerequisite of the sample. In practice, the sum of component contents in a sample is not necessarily exactly 100%, especially when undetected components are present.

D. Incorrect. When trace components that do not elute as peaks are present, normalization results will be biased high because the mass of the undetected components is distributed among the detected components. Therefore, normalization cannot provide an accurate determination in this case.

E. Correct. A major advantage of the normalization method is that it does not require highly accurate injection because the component contents are calculated from ratios, so injection-volume errors do not affect the results. It is also simple to operate.

A. Incorrect. Ultisil AQ-C18 cannot simply be regarded as a “water-tolerant version” of Ultisil XB-C18. Although AQ-C18 does have tolerance to highly aqueous mobile phases, its design concept and technical characteristics differ from those of XB-C18. The difference is not limited to water tolerance.

B. Correct. Ultisil AQ-C18 is prepared with hydrophilic end-capping groups. This prevents hydrophobic phase collapse under 100% aqueous conditions and helps maintain stable retention behavior.

C. Incorrect. Ultisil AQ-C18 is not only suitable for highly aqueous mobile phases; it is also suitable for conventional reversed-phase separations. It is a general-purpose C18 column that performs well under highly aqueous conditions but is not limited to such applications.

D. Correct. Ultisil AQ-C18 and Ultisil XB-C18 use the same silica support, which provides consistent mechanical strength and physical properties. The main differences lie in the bonding and end-capping processes.

A. Incorrect. The mobile-phase inlet frit is located at the pump inlet, before the pump. Blockage at this point causes insufficient solvent intake, which typically appears as a pressure decrease, pressure fluctuation, or a solvent-intake alarm—not as an increase in system pressure.

B. Correct. The injector or autosampler is located in the high-pressure flow path after the pump. Sample residues, particulate matter, or other contaminants in this part of the system can cause a blockage and increase system pressure.

C. Correct. The chromatographic column is one of the most common sites of blockage. Particulates from samples, precipitates from the mobile phase, or accumulated strongly retained components can block the column and increase system pressure.

D. Correct. The detector is located in the flow path after the pump. Although the pressure immediately before the detector is generally lower than that in the column inlet, blockage of the detector flow cell or connecting tubing can increase backpressure and raise the system pressure.

Key point: System pressure reflects the resistance of the post-pump flow path. Only blockages in the post-pump flow path cause an increase in system pressure. Pre-pump blockages do not cause increased system pressure.

A. Correct. Trifluoroacetic acid participates in ion-pairing interactions. In the mobile phase, TFA dissociates to form trifluoroacetate ions (CF₃COO⁻), which can interact with protonated basic compounds (BH⁺) to form ion pairs (BH⁺·CF₃COO⁻). The resulting ion pair is more hydrophobic, which enhances retention on a reversed-phase stationary phase.

B. Correct. Trifluoroacetic acid has a chaotropic effect. TFA is a chaotropic agent that disrupts the ordered water layer surrounding the analyte, lowers the free energy of hydration, and makes it easier for the analyte to interact with the stationary phase. This enhances retention and improves peak shape.

C. Incorrect. Trifluoroacetic acid actually has stronger UV absorption at low wavelengths than formic acid. TFA absorbs strongly in the region around 210–220 nm, which is a disadvantage because it can affect baseline stability and detection sensitivity at low wavelengths. Moreover, this property is not directly related to improvements in peak shape or retention.

D. Correct. Trifluoroacetic acid is more acidic. The pKa of TFA is approximately 0.23, whereas the pKa of formic acid is approximately 3.75. The stronger acidity of TFA produces a lower mobile-phase pH, which more effectively suppresses the dissociation of residual silanol groups. This reduces secondary interactions between silanols and basic compounds—a major cause of poor peak shape—and thereby improves peak shape.

The 11 questions discussed above are not necessarily difficult because they cover obscure chromatographic knowledge. Rather, they are challenging because they require a deeper understanding of the principles underlying chromatographic behavior.

A strong chromatographer must be able to move beyond memorized rules and ask why a system behaves in a particular way: how dwell volume influences a gradient, when extra-column volume actually matters, how detector response relates to flow rate, why pH changes sometimes have little practical effect on retention, and how solvent compatibility affects column handling, etc.

We hope this collection of high-error questions and their detailed explanations will help readers identify gaps in their knowledge, correct common misconceptions, and strengthen their ability to apply chromatographic theory to real-world analytical problems.

If you have a different interpretation of any of these questions, or an explanation of a particular point that you believe more appropriate, feel free to share your thoughts and leave them in the comments.