HPLC LC/MS Troubleshooting

To optimize HPLC selectivity, work through the levers in order and track the critical pair: scout mobile-phase pH against the analytes’ pKa with a buffer matched to each pH, compare acetonitrile, methanol and tetrahydrofuran at similar retention, screen temperature with gradient time, then confirm the chosen conditions in a robustness check.1,2,3 How do you optimize …

How to Optimize HPLC Selectivity: A Systematic pH, Solvent and Temperature Screen Read More »

Extra-column dispersion is the peak broadening that the injector, connecting tubing, fittings and detector add outside the column; their variances, not their widths, add to the column variance.1,2 Small, early-eluting peaks from short, narrow, small-particle columns lose the most efficiency.3,2 Measure it with a zero-dead-volume union, then change contributors one at a time.1,4 What is …

Extra-Column Dispersion: Where Efficiency Is Lost Outside the Column Read More »

A sample diluent stronger than the mobile phase carries the analyte band through the column inlet inside the injected plug until that plug is diluted, so peaks front, broaden or split, early eluters worst.1,2 The damage scales with injection volume and strength mismatch together; diluting the strong solvent or injecting less improves peak shape.3,4 What …

Injection Solvent Effects: How Diluent Strength and Injection Volume Distort Peaks Read More »

A USP L-designation names a packing type, not a column: many dissimilar C18 columns are all L1. To replace the column in a USP monograph method, keep the same bonded substituent, rank candidates by their USP–PQRI similarity value F (≤ 3 suggests equivalence), stay within the ⟨621⟩ adjustments and pass system suitability. Other changes need …

Choosing an HPLC Column for a Regulated Method: USP L-Designations, Equivalency and Revalidation Read More »

To scale an HPLC method to a new column, keep column chemistry unchanged, hold the length-to-particle-size ratio (L/dp) within −25% to +50%, scale flow rate with the square of internal diameter (under ⟨621⟩, also particle size) and scale injection volume and gradient time with column volume so the gradient volume, in column volumes, stays constant.1,2,3,4 …

HPLC Column Dimensions and Particle Size: How to Scale a Method Correctly Read More »

Use HILIC instead of reversed phase when polar or ionized analytes stay weakly retained after pH adjustment and aqueous-stable reversed-phase columns have been tried, and when sample solubility, diluent, detection and method intent allow a mode change.1,2 Very low k is a warning sign, not a universal switch threshold. Why does reversed-phase retention collapse for …

When Should You Switch from Reversed-Phase HPLC to HILIC? Read More »

Choose an orthogonal HPLC column for impurity profiling by its selectivity, not its label. Pick a phase whose interactions differ from the primary column, change pH or organic modifier where the impurities allow, and test both methods on the same stressed and spiked samples. A useful orthogonal method finds peaks the primary method hides.1,2 What …

How to Choose an Orthogonal HPLC Column for Impurity Profiling Read More »

Basic analytes can tail differently on nominally similar HPLC columns because their silica surfaces expose different secondary interaction sites. Older Type A silica carries metal impurities that make residual silanols more acidic than on high-purity Type B silica. Endcapping reduces accessible silanols but does not remove them. Compare a neutral marker, mobile-phase pH and column …

Type A vs Type B Silica and Endcapping: Why Basic Analytes Tail Differently Read More »

C18 and C8 primarily retain analytes through hydrophobic interactions, while phenyl and pentafluorophenyl (PFP) phases can alter selectivity through aromatic, dipolar and other interactions. HILIC instead favors retention of many polar solutes under high-organic conditions. Choose the phase by the critical analyte pair, not by a stationary-phase label alone. How does stationary-phase chemistry change HPLC …

C18 vs C8 vs Phenyl vs PFP vs HILIC: How Stationary-Phase Chemistry Changes Selectivity Read More »

To localize LC-MS/MS carryover, first establish whether a clean blank acquires analyte-associated response after a high sample. Then compare an injected blank with a true no-injection run, test column involvement and isolate fluidic sections one at a time. A blank peak alone does not identify the contaminated component. Mobile-phase contamination, vial contamination, interference and late …

LC-MS/MS Carryover Localization: Autosampler, Column, Tubing or Source? Read More »