Blog · HPLC Tutorials
Practical guides written by analytical chemists. No filler theory, the key concepts, the method decisions, and the common problems that show up in a real lab.
LC-MS detection: how to read the m/z and adducts
What m/z is, how ionization (ESI+, ESI-, APCI) sets the polarity, what the adducts [M+H]+, [M-H]-, [M+Na]+ are, how to compute the m/z from the formula, and the difference between full scan, SIM and MRM.
Read article →How to read an HPLC chromatogram
A beginner-friendly walkthrough: the axes, dead time, retention time and factor (k), area vs height for quantitation, resolution, plate count and the tailing factor, the five questions that turn a plot into data.
Read article →Negative peaks in HPLC: causes and fixes
A negative peak is not always a fault. Tell apart the four causes by their fingerprint, refractive index (often normal), sample diluent mismatch, the detection wavelength, and vacancy peaks, and apply the specific fix.
Read article →How to calculate concentration from peak area
Turn a peak area into a number: external-standard calibration curves, single-point response factors and the internal-standard method, with the formulas, a worked example and the mistakes that ruin a good chromatogram.
Read article →The resolution map: why the highest Rs is rarely where you should run
A resolution map sweeps two method variables and paints the resolution of the hardest pair across your whole window at once. The trap: the maximum-Rs cell usually sits on a cliff that normal drift breaks. Learn to read the design space (QbD/ICH Q8), the robust working point, and critical-pair migration.
Read article →The S parameter in the LSS model: why solvent strength depends on the analyte
The S parameter in log k = log kw − S·φ is not
constant: it grows with analyte size (S ≈ 0.25·√M). What it
means, how it's estimated, the exceptions (bases, steroids), and why modeling
it per analyte is what makes a prediction faithful at high % organic.
Why do HPLC peaks tail? Causes and fixes
Peak tailing is the #1 problem in liquid chromatography. Learn to pin down its cause, active silanols, column overload, dead volume, the wrong pH, and the specific fix for each, with the USP tailing factor as your diagnostic.
Read article →Gradient vs isocratic: when to use each
Snyder's 2× rule, a full decision tree, and gradient-slope guidance. When a simple isocratic method is enough, and when a gradient is the only way to elute a wide-polarity mixture.
Read article →Comparing C18 columns: the Snyder-Dolan HSM model
Not all C18 columns are equal. Read the Hydrophobic Subtraction Model (H, S*, A, B, C) and use the Fs factor to pick an equivalent replacement, or an orthogonal column to confirm peak purity.
Read article →Vitamin analysis by hybrid SFC/UHPLC
Fat-soluble and water-soluble vitamins are too different for a single method. See how a hybrid SFC/UHPLC system separates both, plus the column and MS takeaways from a real Agilent application note.
Read article →More English tutorials coming soon, method-development walkthroughs and HILIC for polar compounds.