What is liquid chromatography and how does it work?

Liquid chromatography principles

Liquid chromatography (LC) is a technique used to separate, identify, and quantitate components within complex mixtures. It works by passing a liquid sample through a solid stationary phase, typically small beads (resin) packed inside a column; other stationary phase options include membranes, monoliths, and fiber-based adsorbents. In all cases, a liquid mobile phase carries the sample through.

Different compounds interact with the stationary phase to different extents, causing each to travel at its own speed. As a result, they exit (elute) from the column at distinct times (called retention times). A detector monitors the output and produces a chart called a chromatogram, where each peak corresponds to a separated compound (Fig 1). This precise process makes liquid chromatography a key tool in science and industry.

Fig 1. Illustration of liquid chromatography using a chromatography column packed with resin beads. Specifically, it shows fast protein liquid chromatography, which is used to separate biological molecules (represented by different colors).

Liquid chromatography systems

Before getting into details, it's important to know the different types of systems for automated chromatography.

• Fast-performance liquid chromatography, later renamed fast protein liquid chromatography (FPLC), was developed in 1982 to purify and analyze biomolecules under gentle conditions, using water-based (aqueous) buffers. In general, it operates at lower pressures (< 50 bar, < 725 psi) compared with other systems, but some equipment can operate up to 200 bar (2900 psi). The particle sizes (resin beads) are typically 30–100 µm and made from materials such as agarose. Diameters down to 9 µm can be used to separate similarly sized molecules.
  
  FPLC continues to be used to separate and analyze proteins including antibodies. In recent years, it has been expanded to diverse molecule types—including mRNA, oligonucleotides, and viral vectors—in research, vaccine, and drug production.

• High-performance liquid chromatography (HPLC) operates at high pressures in the range of 50-400 bar (up to 6000 psi). It uses fine, tightly packed particles, such as 3-5 µm silica beads, to separate small-and mid-sized molecules for analysis. HPLC evolved to create ultra-high performance HPLC (UHPLC), which operates at very high pressures (up to ~1000–1200 bar, ~15 000–18 000 psi) with ultra-fine particles ((≤2 µm) and organic solvents. It's used in analyses where speed and very high resolution are needed.
  
  The high pressures and organic solvents in HPLC and UHPLC aren't well-suited for keeping biomolecules intact and active.

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What is liquid chromatography used for?

Liquid chromatography is widely used in both research and industry to analyze and purify chemical and biological samples. Major application areas include:

• Pharmaceutical and medical: In drug development and manufacturing, liquid chromatography (FPLC) is used to purify the target product. LC also checks the purity of active pharmaceutical ingredients (APIs) and measures impurities to make sure they're at acceptably low levels. HPLC/UHPLC are used in clinical diagnostics to measure biomarkers and drug levels in blood and other fluids.
• Food safety and environmental monitoring: LC (HPLC/UHPLC) detects contaminants in food to help meet safety standards. And it confirms nutrition to help prevent fraud. In environmental testing, it measures pollutants in air, water, and soil to control pollution and support compliance.
• Chemical and biological research: Liquid chromatography is widely used in labs to purify and analyze complex mixtures. In chemistry, HPLC/UHPLC separates reaction products and unknown compounds. In biology, FPLC is used to separate and study biological molecules such as proteins. Proteomics and metabolomics use HPLC/UHPLC to separate proteins, peptides, and metabolites – often combined with mass spectrometry (LC-MS), so they can be analyzed further.

How does liquid chromatography work?

Liquid chromatography separates parts of a mixture through a series of well-defined steps. Each step is key for good separation:

Equilibration

Before a sample is loaded onto a column packed with the stationary phase, the column is first washed with the desired buffer or solvent.

Sample loading

The sample, or part of it, is injected into the LC system. The sample enters the mobile phase and is carried to the column.

Interaction in the column (differential partitioning)

As the sample moves through the column, each compound (analyte) interacts with the stationary phase differently. Compounds that stick weakly move through quickly and exit first, while those that stick strongly move more slowly and exit later. This differential migration causes the mixture to separate into individual compounds.

Washing (for FPLC)

After the sample has finished loading, multiple column volumes (CV) of buffer are usually run through the system to remove poorly binding compounds, precipitates, and contaminants.

Elution (for FPLC)

As the mobile phase flows, it washes analytes off the column at different times. With isocratic elution, the mobile phase stays the same and compounds elute based on their natural retention time. With gradient elution, the mobile phase is gradually changed (such as adding more solvent or salt) to release strongly retained compounds.

Choosing the right elution method and conditions, such as flow rate and temperature, improves the resolution (degree of separation) between closely eluting compounds.

Detection and chromatogram generation

As each compound leaves the column, it enters a detector. In FPLC, this is usually a UV‑visible detector that measures light absorbance. Each compound creates a signal that appears as a peak on a time‑based graph called a chromatogram. Each peak represents one component. The peak's position (retention time) helps identify the compound, and the peak area shows how much is present. Calibration with standards allows accurate measurement.

A good separation produces a chromatogram with clear, well‑separated peaks. Figures 2 and 3 show poor and good resolution, respectively.

Fig 2. Chromatogram showing poor resolution of a bispecific antibody (bsAb) and kappa homodimer based on UV absorbance (green).

Fig 3. Chromatogram showing well-separated peaks.

Regeneration

If a column will be reused, it's cleaned with solvents to remove anything that might cross-contaminate future samples. Sodium hydroxide is typically used in FPLC. Then, the column is equilibrated with a suitable buffer or solution to store it or use it immediately for another run.

Key components of an LC system

A liquid chromatography system has several parts (Fig 4) that work together to separate compounds.

Fig 4. Parts of a liquid chromatography system. The image shows an FPLC system with UV-vis detector and conductivity monitor.

Here's a breakdown of each component and its function within the system:

Liquid reservoirs

Liquid reservoirs (sometimes glass bottles) hold the solvents that carry the sample through the system. In FPLC, these solvents are aqueous buffers with specific salt levels and pH. In HPLC and UHPLC, common solvents include water, methanol, and acetonitrile, often mixed to suit the analysis.

More advanced systems use multiple reservoirs to allow gradient elution, where the solvent composition changes during the run to improve separation. In FPLC systems, mixers are often used to ensure proper mixing.

Pump

The pump moves the mobile phase from the reservoirs to the column at a precise, steady flow rate. Different systems operate at different pressures, with UHPLC requiring the highest pressure, followed by HPLC, then FPLC, to push liquid through tightly packed columns (or other matrices for FPLC).

A consistent flow rate is essential for reliable results, including accurate retention times and good peak resolution. Modern HPLC and UHPLC pumps often use dual pistons to reduce pulsation and deliver smooth flow. FPLC systems usually use peristaltic pumps, which move liquid by using a rotor to pinch tubing and generate a vacuum that moves liquid forward.

Injector

The injector introduces the sample solution into the flowing mobile phase. This can be done manually using a syringe or automatically using an autosampler, which is very useful when purifying many samples. The injector must deliver a precise volume of sample in order to get consistent results.

In analytical LC, microliter volumes are typically injected, while preparative LC involves larger volumes.

Column

The column is where separation happens. It contains the stationary phase that interacts with the compounds in the sample. In HPLC and UHPLC, this is usually silica particles with specific chemical groups. In FPLC, agarose is commonly used instead.

The column's design—its size, particle type, and chemistry—directly affects how well compounds separate. Different columns are available for different purposes, ranging from small analytical separations to larger preparative ones.

Detector

Once the separated compounds exit the column, they pass through a detector, which identifies and quantitates them based on their physical or chemical properties. The most common detectors include:

• UV-visible (UV-vis): Measures absorbance of UV and visible light.
• Fluorescence: Detects compounds that emit light upon excitation.
• Refractive index (RI): Measures changes in refractive index of the eluent.
• Mass spectrometry (MS): Provides molecular weight and structural information.

A conductivity monitor may be included on FPLC systems to measure the ionic strength of the buffer in real time. It's especially useful to monitor salt concentration gradients in ion exchange chromatography.

Fraction collector

This is an optional part often used in FPLC, to automatically collect fractions of liquid that exit the chromatography system.

Data system

This is the software that runs the chromatography system and processes the output from the detector. It collects the raw data, turns it into a chromatogram, and provides tools for analyzing peaks, calculating results, developing methods, and creating reports.

More advanced systems also support real-time monitoring, automated calibration, and compliance features required in regulated environments (e.g., 21 CFR Part 11 in pharmaceuticals).

Common liquid chromatography modes

Liquid chromatography uses different techniques ("modes") to separate molecules based on their chemical properties. Learn what they are, how they work, and where they're used:

Type	Description	Typical applications
Affinity chromatography	Uses specific binding interactions between a target molecule and a biologically specific ligand to achieve purity of up to 95% in a single step. Often used as the first step in purification because of its efficiency.	Antibody purification, enzyme isolation, purification of recombinant proteins with an affinity tag in FPLC
Ion exchange chromatography (IEX)	Separates molecules based on their charge using charged stationary phases. Includes cation and anion exchange.	Protein purification, purification of other biological molecules in FPLC, water quality analysis in HPLC and UHPLC
Hydrophobic interaction chromatography (HIC)	Separates molecules based on their hydrophobicity. Begins with a high salt concentration. Decreasing the salt gradually elutes molecules starting with the least hydrophobic.	Protein purification, especially antibodies in FPLC
Multimodal (mixed-mode) chromatography	Uses ligands with multiple interaction types, such as charge and hydrophobicity. Results in high selectivity.	Biologic purification in FPLC
Size exclusion chromatography (SEC)	Also known as gel filtration. Separates molecules based on size. Large molecules travel quickly around the beads, eluting earlier than smaller molecules that can enter pores.	Polymer analysis, sizing of proteins and other biologics in FPLC, desalting and buffer exchange in FPLC
Normal phase LC (NPC)	Uses a polar stationary phase and a nonpolar mobile phase that contains mostly water.	Lipid analysis, separation of isomers in HPLC and UHPLC
Reversed phase LC (RPC)	Uses a nonpolar (hydrophobic) stationary phase and a polar mobile phase, reversed from the polarity in NPC.	Metabolomics, drug analysis, environmental testing in HPLC and UHPLC
Hydrophilic interaction chromatography (HILIC)	Similar to normal phase except the mobile phase is mostly organic instead of aqueous.	Separates sugars, amino acids, and polar pharmaceuticals In HPLC and UHPLC

Conclusion

Liquid chromatography (LC) separates mixtures with high precision. It can be used in many ways – for example, checking drug purity in a quality control lab, identifying metabolites in blood, or purifying therapeutic proteins. By understanding how LC works and the different types available, scientists can use it to solve many analytical and purification problems. In FPLC, several chromatography steps are often combined to either analyze a mixture in detail or isolate a single compound at high purity. This flexibility is why LC is one of the most widely used techniques in chemistry and biology.

Frequently asked questions (FAQs)

1. What is meant by liquid chromatography?

Liquid chromatography is a technique used to separate, identify, and quantitate components in a liquid mixture. Used in laboratories and pharmaceutical manufacturing, it works by passing the mixture through a column or other container, such as a capsule, filled with a stationary phase. A liquid mobile phase carries the sample through the system.

2. What is a liquid chromatogram?

A liquid chromatogram is a graphical output of a chromatography run. It displays peaks that represent different compounds, with peak height or area indicating their concentration.

3. What are the main types of liquid chromatography?

The main types are high-performance liquid chromatography (HPLC), ultra-high-performance LC (UHPLC), and fast protein LC (FPLC).

4. What instruments are used in liquid chromatography?

Typically, an automated chromatography system is used. System parts generally include liquid reservoirs, a pump that delivers the required pressure, an injector, a chromatographic column, a detector, and software for analysis.

5. In which industry is liquid chromatography used most?

Liquid chromatography is most widely used in the pharmaceutical industry for drug development, quality control, and manufacturing. It's used both to purify the target product and to analyze its purity.