A separation is a column, a solvent and a detector chosen together: the detector sets the solvent grade, the analyte sets the chemistry, and the instrument sets how small the particles can be. Most method problems are one of those three chosen without the other two in mind.
An hplc mobile phase filter and what it protects
An hplc mobile phase filter on the inlet keeps particulates out of the pump and the column, and it is a consumable rather than a fixture: a clogged frit shows as a falling flow rate, cavitation or a noisy baseline. Filtering and degassing the mobile phase are the two cheapest habits that extend a column's life.
hplc mobile phase solvents and how they are chosen
hplc mobile phase solvents are chosen for miscibility, viscosity, ultraviolet cutoff and selectivity: acetonitrile gives lower pressure and a lower cutoff while methanol changes the separation and costs less. Buffer choice follows the detector, since a non volatile salt cannot enter a mass spectrometer, and the pH has to sit inside the column's range.
A cyano hplc column and where that phase fits
A cyano hplc column has a moderately polar bonded phase that works in reversed and normal phase modes, which is why it is used for compounds poorly retained on alkyl phases and for isomer separations. It is less stable at extremes of pH than an alkyl phase, and that limit is what a method has to respect rather than test.
uplc chromatography and what smaller particles buy
uplc chromatography uses sub two micron particles, which raises efficiency and back pressure together, so the instrument rather than the column is usually the limit and the whole flow path needs low dispersion. A transferred method needs its gradient and injection scaled and the detector's time constant set faster.
An hplc to uplc method transfer calculator, and what it computes
An hplc to uplc method transfer calculator scales a method to the smaller particles and shorter column, holding linear velocity and gradient slope so the separation survives, and it predicts the pressure the new configuration needs. Injection volume scales with column volume, which is the step most often forgotten in a transfer.
chromatography column types and the axis that separates them
chromatography column types differ by mode, by particle and by dimensions, and each changes a different thing: mode changes selectivity, particle size and structure change efficiency and pressure, dimensions change load and run time. Swapping mode is a new method while swapping dimensions can usually be recalculated.
types of chromatography and what each separates by
The types of chromatography separate by partition, by charge, by size or by a specific interaction, in a gas or a liquid, and the technique follows the analyte: volatility for gas chromatography, hydrophobicity for reversed phase, charge for ion exchange, size for exclusion, and affinity where a specific ligand exists.
An hplc assay and what makes it one
An hplc assay is a validated method rather than a separation: specificity against degradants, a demonstrated linear range, accuracy and precision, a stated limit of quantification, and a demonstrated tolerance of small deliberate changes. Those experiments rather than the chromatogram are what make a number defensible in a report.
Questions people ask about chromatography solvent
Which solvent grade is needed?
The detector decides. Mass spectrometry needs low non volatile residue; ultraviolet needs transparency at the wavelength.
Can a method just be run on smaller particles?
Only with the gradient, injection and detector settings scaled. Otherwise the transfer loses resolution.
What separates an assay from a separation?
Validation: specificity, linearity, accuracy, precision, a quantification limit and tolerance of small changes.