HPLC Explained

Every batch of paracetamol tablets, every vial of a vaccine component, and many of the blood tests run in hospitals depend on one instrument: the high-performance liquid chromatograph. HPLC separates, identifies and measures substances dissolved in a liquid, including large, fragile and non-volatile molecules that gas chromatography can’t handle. It’s probably the most widely used analytical technique in the pharmaceutical industry.

The basic idea

Like all chromatography, HPLC separates a mixture between a stationary phase and a mobile phase (see chromatography):

• The stationary phase is made of extremely small particles, typically 2–5 μm across, packed tightly into a steel column.
• The mobile phase is a liquid (often a mixture of water with methanol or acetonitrile) that’s pumped through the column at high pressure.

Why high pressure? Small particles give a huge surface area and very sharp separations, but they also resist flow. Pumps push the liquid through at pressures of typically 50–400 times atmospheric pressure (and over 1000 times in ultra-high-performance systems, UHPLC). The result is a separation in minutes that would take hours with gravity-driven column chromatography. The “H” originally stood for “high-pressure”; “high-performance” is now the standard term.

The instrument

1. Solvent reservoirs hold the mobile phase components, which are filtered and degassed.
2. High-pressure pump delivers the mobile phase at a precise, steady flow rate (often around 1 cm³ per minute).
3. Injector or autosampler introduces a small, precise volume of sample (often 1–20 μL) into the flowing mobile phase.
4. Column: a stainless-steel tube, typically 5–25 cm long and a few millimetres wide, packed with stationary phase particles. It’s often kept in a temperature-controlled oven.
5. Detector measures the components as they leave the column.
6. Data system plots the signal against time as a chromatogram.

Normal phase vs reversed phase

There are two main modes, depending on the polarity of the stationary and mobile phases.

Normal phase HPLC

• Stationary phase: polar (bare silica)
• Mobile phase: non-polar (e.g. hexane-based)
• Non-polar compounds elute first; polar compounds are held longer.
• This is the same logic as thin-layer chromatography on silica.

Reversed-phase HPLC (by far the most common)

• Stationary phase: non-polar. Silica particles are chemically bonded to long hydrocarbon chains, most often 18 carbon atoms long (called C18 or ODS).
• Mobile phase: polar, usually water mixed with methanol or acetonitrile.
• Polar compounds elute first; non-polar compounds dissolve into the hydrocarbon layer and are held longer.

Reversed phase is popular because water-based mobile phases suit most drugs, biological molecules and food components, and the columns are robust and reproducible.

Controlling the separation

• Mobile phase composition. In reversed phase, increasing the organic solvent (methanol or acetonitrile) makes the mobile phase less polar, so non-polar compounds come out faster.
• Isocratic vs gradient elution. In isocratic runs, the mobile phase composition stays constant. In gradient runs, the proportion of organic solvent increases during the run, which speeds up strongly retained compounds and sharpens their peaks.
• pH. For acids and bases, pH controls whether they’re ionised. Ionised forms are more polar and elute earlier in reversed phase. Buffers are added to keep pH constant (see buffer capacity).
• Temperature and flow rate fine-tune retention and peak shape.

Detectors

Detector	How it works	Good for
UV–visible absorbance	measures absorbance at a chosen wavelength	the most common; any compound that absorbs UV, including most drugs
Diode array (DAD/PDA)	records a full UV–visible spectrum for each peak	checking peak purity and identity
Fluorescence	measures emitted light	very sensitive for fluorescent compounds
Refractive index	detects changes in refractive index	sugars and compounds with no UV absorbance
Mass spectrometer (LC–MS)	measures molecular and fragment masses	identification and trace analysis
Electrochemical	measures current from oxidation or reduction	neurotransmitters, vitamins

Reading an HPLC chromatogram

As with gas chromatography:

• Each compound produces a peak.
• Retention time (the time from injection to the peak) helps identify a compound by comparison with a standard run under identical conditions.
• Peak area is proportional to the amount of compound.

Measuring concentration. A series of standard solutions is run to create a calibration curve of peak area against concentration; the unknown’s concentration is read from it. See calibration curves.

Worked example

A paracetamol tablet is dissolved in water and made up to 500 cm³. 1.00 cm³ of this solution is then diluted to 100 cm³ (a hundredfold dilution) and analysed by reversed-phase HPLC with UV detection. A calibration using paracetamol standards gives the relationship: peak area = 1520 × concentration (in μg/cm³). The diluted solution gives a peak area of 15,200 at paracetamol’s retention time. Find the mass of paracetamol in the tablet.

• Concentration in the diluted solution = 15,200 ÷ 1520 = 10.0 μg/cm³
• Before the hundredfold dilution: 10.0 × 100 = 1000 μg/cm³
• In 500 cm³: 1000 × 500 = 500,000 μg = 500 mg

That matches the typical label claim of 500 mg per tablet. Quality-control labs do exactly this kind of calculation for samples from every batch, and a result outside the permitted range (often around 95–105% of the label claim) would trigger an investigation.

HPLC vs GC

	HPLC	GC
Mobile phase	liquid	inert gas
Sample must be	soluble	volatile and heat-stable
Suits	large, polar, fragile molecules (drugs, proteins, sugars, vitamins)	small, volatile molecules (solvents, fuels, fragrances)
Temperature	usually near room temperature to about 60 °C	often up to 300 °C or more
Typical detector	UV absorbance, MS	FID, MS

See gas chromatography for comparison.

Where HPLC is used

• Pharmaceuticals: measuring the amount of active ingredient in medicines, checking for impurities and degradation products, and testing how quickly tablets dissolve.
• Clinical chemistry: measuring drugs and vitamins in blood; screening newborns for metabolic disorders (often by LC–MS/MS).
• Food and drink: caffeine content, sweeteners, preservatives, vitamins and contaminants such as mycotoxins.
• Environmental analysis: pesticides and pharmaceuticals in water.
• Biotechnology: purifying and characterising proteins, peptides and nucleic acids.
• Forensics: identifying drugs and toxins.

Looking after an HPLC system

HPLC columns are expensive and sensitive, so labs protect them carefully. Samples are filtered to remove particles that would block the column, solvents are high-purity and degassed so bubbles don’t form in the pump or detector, and a short guard column is often fitted in front of the main column to catch contaminants. After use, buffers are flushed out with water and then the column is stored in an organic solvent mixture, because salts left behind can crystallise and damage the pump seals.

Key takeaways

• HPLC pumps a liquid mobile phase at high pressure through a column packed with very small particles.
• Reversed-phase HPLC (non-polar C18 stationary phase, polar water–organic mobile phase) is the most common mode; polar compounds elute first.
• Mobile phase composition, gradients and pH control the separation.
• Retention time identifies compounds; peak area measures their amounts using calibration.
• HPLC handles non-volatile and heat-sensitive molecules that GC cannot.