Liquid Chromatography Questions and Answers

Answer: C

The first peak leaves at the interstitial volume and the last at the total permeation volume, so the entire chromatogram is squeezed into the difference between them. Pore size sets the range rather than the capacity, the detector is not the limit, and the eluent is held constant. A separation bounded at both ends by fixed volumes can never spread out, which is why the mode is used for molecular weight distributions rather than for resolving mixtures. Checking the units of each quantity catches a wrong formula before the arithmetic begins.

Answer: D

An antibody, an enzyme substrate or a metal chelate is bonded to the support and binds one particular analyte while everything else passes through. Charge, partition and size define the other modes. Selectivity approaching a single compound, rather than a class, is what makes the mode so powerful for purification, and the elution step must then be harsh enough to break a deliberately strong interaction. Checking the units of each quantity catches a wrong formula before the arithmetic begins.

Answer: A

Polar analytes such as sugars and metabolites elute near the void in reversed phase, and a polar stationary phase with a mostly organic eluent retains them properly. Non-polar compounds, charged species and large molecules are handled by other modes. Reaching for a different mode when an analyte falls outside reversed phase's comfortable range is more effective than straining the dominant mode to cover everything. The shared properties explain why these belong to one family, and the differences explain why more than one exists.

Answer: B

A thin aqueous layer is held on the polar surface, and analytes partition into it from the largely acetonitrile mobile phase - so retention rises as the water content falls, the reverse of reversed phase. Solubility, phase stability and detector requirements are not the mechanism. That the elution order and the gradient direction both invert compared with reversed phase is what catches people out when they first use it. A limit that survives making every external component ideal is an intrinsic one, and worth knowing for that reason.

Answer: C

Ion exchange retains proteins by their net charge, which varies with pH, and elutes them with a salt gradient under conditions gentle enough to preserve activity. Reversed phase would denature them, size exclusion separates by size instead, and normal phase is unsuitable for aqueous samples. Choosing a mode means naming the property the separation is to exploit, and for proteins charge and size are the two usually available. A single measurement chosen to respond to the one asymmetry beats several that respond to shared behaviour.

Answer: D

Because selectivity gives by far the largest return, trying a few phases and organic modifiers at a few pH values reveals whether the separation is possible at all. Gradient slope, wavelength and injection volume are refinements applied once a promising system is found. Establishing feasibility before optimising detail is what stops days being spent perfecting a separation that a different column would have made trivial. Naming the cost of a choice alongside its benefit is what turns a fact into a design decision.

Answer: A

Near an analyte's pKa a fraction of a pH unit can change retention several-fold, so pH is one of the most powerful selectivity levers available. Efficiency, detector response and pump capability are largely indifferent. That a variable with such leverage must also be controlled tightly in the final method is why a buffer rather than a bare acid is specified.

Answer: B

An injected sample can shift the local pH enough to change retention, and a buffer of adequate capacity resists that. Transparency is usually worsened, pressure is unaffected, and adsorption is a phase property. Controlling a variable that has great leverage is worth more than merely setting it once, which is the difference between a buffer and an acid.

Answer: C

Validation demonstrates linearity, accuracy, precision, specificity, range and robustness with documented evidence, so that a result can be relied upon and defended. Servicing, training and column provenance support it without constituting it. Establishing what a method actually does, rather than what it was intended to do, is what separates a validated procedure from a working one. A limit that survives making every external component ideal is an intrinsic one, and worth knowing for that reason.

Answer: D

Specificity is demonstrated by showing that degradation products, excipients and matrix components do not contribute to the analyte's peak, often with a peak purity check from a diode array detector. Instrument, column and operator restrictions would be limitations rather than specificity. Proving that a measurement responds to the analyte and nothing else is the claim on which every other validation parameter rests. Most errors here come from carrying a result across from a neighbouring case where it does not hold.