Chromatography Midterm 2 - HPLC

Pump

sets flow and gradient composition.

Injector

adds a narrow, reproducible sample plug.

Column

creates selectivity and efficiency.

Detector

converts eluting concentration into signal.

5.0 µm particle

Low pressure

lower efficiency

longer run.

3.0 µm particle

Higher efficiency and speed

clear pressure cost.

1.5 µm particle

fast and efficient

only a high-pressure system can operate

Symptom: Pressure rises and falls with each stroke

First check: the inlet and outlet check valves.

symptom: solvent appears near piston

First check: the piston seal and wash system.

symptom: pressure collapses and erratic flow

First check: solvent supply and trapped air.

Dual piston HPLC pump

A delivers, B refills

Then B delivers and A refills

Degassing prevents what failure

Removes dissolved gas before it forms bubbles in pump or detector.

Filtration prevents what failure

Removes particles before they reach narrow tubing, valves, frits, and the packed bed

If sharp baseline spikes:

Inspect degassing and the detector flow cell first.

if pressure rises over several runs

Inspect sample and solvent filtration first.

if pump loses prime…

Inspect: solvent inlet, degassing

Steps to loading injection valve:

Draw sample → fill loop → switch valve → carry to column

5 µL x 1 concentration

Small volume and low mass

inlet can focus sample, and stationary phase remains within working capacity.

25 µL x 1 concentration

Large volume, moderate mass

Mass overload unlikely

but large solvent plug may cause broadening or splitting

5 µL x 8 concentration

Small volume, high mass

The solvent plug small, but stationary phase may overload.

Fronting becomes more likely

25 µL x 8 concentration

Large volume and high mass

Both mechanisms act together.

Reduce concentration or volume, then test one change at a time

If carryover appears:

Inject concentrated sample

Inject blank

Run needle wash

Inject another blank

Matched or weaker diluent

The analyte focuses near the column head before separation begins

so band stays narrow

Stronger diluent than the starting mobile phase

The sample plug begins eluting the analyte before the inlet can focus it. Broadening, fronting, or splitting can result.

Isocratic elution

Composition stays constant.

Choose when solvent strength resolves sample in reasonable time.

Gradient elution

Elution strength increases during run.

Use it when retention spans a wide range or late compounds elute too slowly.

Changing solvent strength: isocratic

Use a fixed composition when the analytes have a modest retention range.

It is simple to transfer and needs little composition recovery between runs.

Changing solvent strength: linear gradient

Increase solvent strength steadily.

This is a strong default for mixtures with a wide polarity range because late compounds elute sooner and often more sharply.

Changing solvent strength: step gradient

Jump to a stronger composition for a fast wash or a defined fraction.

Abrupt changes can compress or distort bands, so step timing requires care.

Simple routine wants what kind of elution

Isocratic

Mobile phase