Home
/
Blog
/
What Is Liquid Chromatography? Principles, Techniques, & Applications

What Is Liquid Chromatography? Principles, Techniques, & Applications

Chromatography is a powerful separation technique used in research to identify, quantify, and analyze the components of a mixture, providing valuable insights that can drive innovation in numerous industries.

It’s like a sorting process you can use to identify and measure the different substances present in a sample, providing researchers with a means to analyze complex mixtures, identify individual components, and quantify their concentrations. From solving intricate puzzles in chemical analysis to revolutionizing drug development, chromatography plays a crucial role in unraveling the mysteries of complex mixtures.

There are different types of chromatography, including gas chromatography, liquid chromatography, and thin-layer chromatography, each with specific applications and advantages.

This blog post will teach you about liquid chromatography, its working principles, instrumentation, various modes, and applications.

Need new or refurbished lab equipment? Excedr leases.

See our equipment list and browse a sample selection of what we can source. Or, if you’re ready, request an estimate.

Understanding Liquid Chromatography

Liquid chromatography (LC) is a specific type of chromatography technique that plays an essential role in research across multiple scientific disciplines. 

It involves the distribution of the mixture between two phases, one stationary, and one mobile, leading to differential migration and subsequent separation of the components.

What is liquid chromatography used for? Its primary uses include:

  • Separation of components: The primary purpose of liquid chromatography is to separate the individual components in a mixture. As the components interact differently with the stationary and mobile phases, they elute (come out) from the chromatographic column at different times. This results in a chromatogram, a graph representing the elution times of the components, allowing researchers to identify and quantify the various substances present in the sample.
  • Qualitative analysis: Liquid chromatography determines what components are present in a sample. Scientists can identify the substances in the mixture by comparing the retention times of the components with those of known standards or databases.
  • Quantitative analysis: LC is also employed for quantitative analysis to determine the concentration of specific components in a mixture. By comparing the peak areas or heights in the chromatogram with calibration standards, researchers can estimate the amount of each substance in the sample.
  • Sample purification: Liquid chromatography is widely used for sample purification. In preparative liquid chromatography, more of the mixture is separated, and specific components of interest are collected and purified for further analysis or use.
  • Chiral separations: LC is particularly valuable for separating chiral compounds, molecules that exist in two mirror-image forms (enantiomers). Chiral separation is crucial in pharmaceuticals, as only one enantiomer is often responsible for the desired therapeutic effect. At the same time, the other may cause unwanted side effects.

Separating complex mixtures allows scientists to study the composition of specific samples, investigate chemical reactions, analyze biological substances, monitor environmental pollutants, assess pharmaceutical purity, and perform countless other essential analyses.

The insights scientists can gain are often invaluable, offering a deeper understanding of various substances' properties, structures, and interactions. With numerous modes and advanced capabilities like HPLC, liquid chromatography is an indispensable tool for understanding the world at the molecular level.

Liquid Chromatography System Components

Generally speaking, a liquid chromatography system, or chromatograph, consists of several key components:

  • Mobile phase: The mobile phase is a liquid solvent in which the mixture to be analyzed is dissolved. It carries the sample through the chromatographic system, allowing the components to interact with the stationary phase within the column.
  • Stationary phase: The stationary phase is a specialized material packed into the column of the chromatography system. It remains fixed in place while the mobile phase flows through it. The stationary phase interacts differently with the mixture's components, separating them.
  • Column: The column is a long, thin tube where chromatographic separation occurs. It contains the stationary phase, and as the mobile phase passes through, the mixture's components undergo interactions that cause them to separate based on their properties.
  • Injector: The injector introduces the sample into the liquid chromatography system. It precisely and accurately injects the sample into the mobile phase stream before it enters the column for separation.
  • Pump: The pump is a crucial component that drives the mobile phase through the chromatographic system at a controlled flow rate. The pump ensures a consistent flow, enhancing the reproducibility and accuracy of the analysis.
  • Detector: The detector monitors the effluent from the column and identifies the separated components of the mixture. Depending on the type of detector used, it identifies specific properties of the analytes, such as absorbance, fluorescence, or mass.
  • Data system: The data system collects, processes, and interprets the signals from the detector, generating chromatograms that represent the separation profile of the mixture. This data is essential for analyzing and quantifying the components in the sample.

In summary, liquid chromatography instrumentation includes a mobile phase to carry the sample, a stationary phase for separation, a column where the separation takes place, an injector to introduce the sample, a pump to control the flow, a detector to identify components, and a data system to process and interpret the results.

Working Principles of Liquid Chromatography

The working principles of liquid chromatography rely on careful selection of the mobile phase, stationary phase, and column to create differential interactions between components of a mixture. The process generally involves the following key steps:

  1. Sample introduction: The mixture to be analyzed is dissolved in the mobile phase. The sample is then introduced into the chromatographic system using an injector.
  2. Mobile phase flow: The mobile phase, carrying the sample, flows through the column packed with a specialized material known as the stationary phase. The stationary phase is carefully chosen based on the type of separation required.
  3. Interactions with the stationary phase: As the mobile phase passes through the column, the mixture's components interact differently with the stationary phase based on their physicochemical properties. These interactions include adsorption, partitioning, and ion exchange, depending on the type of liquid chromatography being performed.
  4. Separating components: The differential interactions between the components and the stationary phase cause the components to move at different rates through the column. Components that interact more strongly with the stationary phase are retained longer in the column, while those with weaker interactions move faster.
  5. Detection and analysis: As the separated components elute from the column, they are detected by a detector, which may use UV-visible, fluorescence, or mass spectrometry detection, among others. The detector generates signals processed by a data system, producing chromatograms representing the separated components.
  6. Quantification and identification: The data obtained from the chromatograms allows researchers to quantify the concentrations of the components in the mixture and identify them based on their unique retention times and detector responses.

Liquid Chromatography Modes

In laboratories today, researchers use several types of liquid chromatography modes to perform different analyses. Depending on the choice of stationary and mobile phases, some standard modes are used to separate various compounds, including reversed phase, normal phase, ion exchange, and size exclusion.

Understanding these different liquid chromatography modes empowers researchers to choose the most suitable technique for their specific analytical needs, unlocking more accurate experimentation, reaching milestones more smoothly, and driving advancements in their field of research.

Reversed-Phase Chromatography

Reversed-phase chromatography, also known as RPC, employs a non-polar stationary phase and a polar mobile phase, contrary to traditional chromatography. Hydrophobic molecules in the sample tend to adsorb to the non-polar stationary phase. 

In contrast, hydrophilic molecules dissolve in the polar mobile phase and elute first. Reversed-phase chromatography is commonly used in High-Performance Liquid Chromatography (HPLC) for its broad applicability.

This technique offers excellent compatibility with diverse analytes, making it a versatile choice for various applications. In pharmaceutical analysis, food and beverage testing, environmental monitoring, and forensic analysis, reversed-phase chromatography is crucial in separating and analyzing complex mixtures.

The mobile phase composition in reversed-phase chromatography typically involves mixtures of water or aqueous buffers with organic solvents like acetonitrile or methanol to achieve optimal separation.

Additionally, gradient elution, where the water-solvent composition changes over time, is frequently utilized to enhance the resolution of samples with a wide range of components.

Normal-Phase Chromatography

Normal-phase chromatography follows the classic principle of liquid chromatography, where the stationary phase is polar, and the mobile phase is non-polar. In this mode, less polar compounds elute first, while more polar compounds elute later, providing effective separation based on their respective polarities.

Normal-phase chromatography is particularly useful for separating various compounds, including water-sensitive substances, geometric isomers, cis-trans isomers, and chiral compounds. Its ability to resolve complex mixtures of compounds with differing polarities makes it a valuable tool in various scientific fields, such as pharmaceutical research, natural product analysis, and environmental monitoring.

Researchers often rely on normal-phase chromatography to gain valuable insights into the structure and properties of these diverse compounds, further advancing our understanding of the molecular world.

Ion-Exchange Chromatography

Ion-exchange chromatography (IEX) is a versatile liquid chromatography technique that separates charged particles in a liquid and measures their concentration. It finds widespread applications in diverse industries, including environmental analysis, manufacturing, food quality control, pharmaceutical research, and chemical analysis. In this mode, the stationary phase contains many acid groups attached to a polymeric resin.

The mobile phase is typically an aqueous buffer, such as an inorganic salt dissolved in a suitable solvent. Ion-exchange chromatography has two variations: cation-exchange chromatography and anion-exchange chromatography. Cation-exchange chromatography retains positively charged cations, while anion-exchange chromatography retains negatively charged anions.

This powerful technique allows scientists to separate and quantify a wide range of ions and polar molecules, making it an essential tool for understanding the composition and concentration of charged species in complex samples across various scientific disciplines.

Size-Exclusion Chromatography

Size-exclusion chromatography, also known as gel-permeation chromatography (GPC) or gel-filtration chromatography (GF), is a specialized liquid chromatography technique that separates molecules based on their size relative to the pore size of the stationary phase.

In this mode, the largest molecules elute the fastest since they cannot penetrate the pores of the stationary phase. In contrast, smaller molecules take longer to elute as they can permeate the pores to a greater extent. This unique separation mechanism makes size-exclusion chromatography highly effective for analyzing polymers and proteins, where molecular size plays a crucial role in determining their properties and functions.

Scientists widely use this technique to characterize and purify macromolecules, gaining valuable insights into their molecular weight distribution, aggregation states, and structural characteristics.

Size-exclusion chromatography’s versatility and ability to handle a wide range of molecules make it an indispensable tool in fields like polymer chemistry, biotechnology, and biochemistry, contributing to advancements in materials science, pharmaceutical research, and biomolecular studies.

Flash Chromatography

Flash chromatography is a rapid and efficient liquid chromatography technique designed primarily for preparative-scale purifications.

This innovative method automates the purification process, reducing the need for extensive human involvement and significantly increasing productivity in process labs. Flash chromatography systems, categorized as low-pressure liquid chromatography (LPLC), are specifically engineered to handle complex mixtures, allowing researchers to separate and purify compounds quickly and effectively.

By automating the column chromatography process, which can be labor-intensive when performed manually, flash chromatography provides researchers with a reliable and time-saving solution. Its ability to deliver high-purity compounds makes it invaluable in pharmaceuticals, natural product isolation, and industrial organic synthesis.

With its streamlined approach to purification, flash chromatography is a powerful tool that accelerates scientific discoveries by enabling researchers to focus on other critical aspects of their work, ultimately advancing various scientific disciplines.

Unlike high-pressure systems, flash chromatography systems are designed to reduce human involvement in the purification process. By automating and simplifying the purification procedure, these systems offer an efficient alternative, striking a perfect balance between simplicity, affordability, and improved productivity.

Flash chromatography systems function at lower pressures, typically below 1000 psi, unlike the higher pressures utilized in high-performance liquid chromatography (HPLC) or FPLC. This lower pressure allows for more straightforward manipulation of the chromatographic media, making the process more cost-effective and convenient.

Fast Protein Liquid Chromatography

Fast Protein Liquid Chromatography (FPLC) is a specialized liquid chromatography primarily designed for protein analysis. Its widespread application is attributed to the diverse range of resins and buffers available, allowing for versatile separation and purification strategies for proteins of varying sizes and properties.

FPLC allows researchers to analyze complex protein mixtures with high resolution, providing valuable insights into protein structure, activity, and interactions. While FPLC is tailored for protein analysis, its broad utility stems from the flexibility to adapt to different purification scenarios and specific research needs.

By combining a relatively low buffer pressure with high flow rates, FPLC enables efficient protein separations, making it an indispensable tool in biopharmaceutical research, protein engineering, and numerous applications within the life sciences.

High-Performance Liquid Chromatography

What is high-performance liquid chromatography? Commonly referred to as HPLC, it is a type of liquid chromatography considered one of the most widely used modes.HPLC's popularity is mainly attributed to its high resolution, versatility, sensitivity, and ability to handle complex mixtures.

For example, the high pressure of HPLC systems allows for smaller particle sizes in the chromatography column, leading to higher resolution and faster separations. Smaller particle sizes (typically less than 10 μm) are used in the stationary phase of the chromatography column—smaller particles provide more efficient separations due to increased surface area and reduced diffusion of analytes. HPLC systems typically use higher flow rates compared to conventional liquid chromatography. The combination of higher pressure and flow rates contributes to faster analyses.

However, the field of chromatography is continuously evolving, and other liquid chromatography techniques, such as Ultra-High-Performance Liquid Chromatography (UHPLC), are gaining prominence due to their ability to achieve even higher resolution and faster separations. Additionally, specific applications and research requirements may dictate a particular type of liquid chromatography.

UHPLC (Ultra-High-Performance Liquid Chromatography)

Ultra-High-Performance Liquid Chromatography (UHPLC) systems represent a significant technological advancement in liquid chromatography. These state-of-the-art systems operate at substantially higher pressures, typically reaching up to 120 MPa (megapascals) or about 1200 atmospheres, compared to conventional HPLC systems.

Additionally, UHPLC employs chromatography columns packed with smaller particles, often less than 2 μm in size, which significantly enhances the separation efficiency.

By utilizing smaller particles and higher pressure, UHPLC achieves faster separations and higher resolution than traditional HPLC. The increased pressure allows for more efficient packing of the column, reducing band broadening and minimizing the effects of diffusion, resulting in improved peak sharpness and sensitivity.

UHPLC's exceptional performance makes it a valuable tool for various applications, including analyzing complex mixtures and high-throughput screening. Its superior sensitivity enables the detection of even trace components, making it invaluable in pharmaceutical research, environmental monitoring, and biomolecular studies.

With its ability to expedite separations and provide more accurate results, UHPLC has transformed liquid chromatography, empowering scientists with a cutting-edge technique that accelerates research and enhances productivity across diverse scientific domains.

Types of HPLC Columns

HPLC columns come in various types, each designed to achieve specific separation goals. C18 and C8 columns are used in reversed-phase chromatography for hydrophobic compounds, while ion-exchange columns specialize in analyzing charged species—size-exclusion columns efficiently separate molecules based on their size.

By carefully selecting the appropriate column, scientists can optimize separations and enhance the accuracy and efficiency of their HPLC analyses across diverse research and industrial applications.

The Advantages of HPLC & UHPLC

HPLC and UHPLC offer several advantages that contribute to their popularity:

  • High sensitivity: HPLC can detect and quantify trace amounts of compounds in a sample, even at low concentrations.
  • Versatility: It can be applied to a wide range of compounds, from small molecules to large biomolecules, providing flexibility for diverse analytical needs.
  • Automated and reproducible: HPLC systems are highly automated, allowing for consistent and reproducible results, reducing human error.
  • High resolution: HPLC provides excellent resolution, separating closely related compounds with similar properties.

Advanced Techniques

Advanced techniques in liquid chromatography encompass various methods that enhance separation efficiency, sensitivity, and specificity. Let’s review some of the most common.

Liquid Chromatography-Mass Spectrometry (LC-MS)

So what is liquid chromatography-mass spectrometry? Mass spectrometry (MS) is another powerful analytical technique. It identifies and characterizes molecules based on their mass-to-charge ratio (m/z). 

It involves ionizing the molecules and measuring the mass-to-charge ratio of the resulting ions, providing valuable information about the elemental composition and structure of the compounds.

When combined with liquid chromatography, the LC-MS technique becomes even more potent. The liquid chromatograph is the front-end separation method, where complex mixtures are separated into individual components before entering the mass spectrometer. The combination offers several advantages, the most important of which include:

  • Enhanced analysis of complex samples: LC-MS allows the analysis of complex samples containing numerous compounds. Liquid chromatography separates the mixture into distinct components, reducing the complexity of the sample entering the mass spectrometer. This enhances the sensitivity and specificity of the analysis, making it easier to detect and quantify trace amounts of compounds.
  • Structural information about compounds: Mass spectrometry provides structural information about the analyzed compounds. The mass spectra generated by the mass spectrometer offer insights into the molecular formula, fragmentation pattern, and presence of specific functional groups in the compounds. This structural information is crucial for identifying unknown compounds and elucidating their properties.

There are other advanced techniques, two of which we’ll discuss next: high-resolution and multidimensional chromatography.

High-Resolution Chromatography

This technique provides superior separation and detection capabilities compared to conventional chromatography. In high-resolution chromatography, narrow peaks are achieved, allowing for better resolution of closely eluting components in a mixture.

One of the critical advancements in high-resolution chromatography is modern instruments that can handle smaller particle sizes in the chromatography column. These smaller particles enable better peak separation and improved resolution. High-resolution chromatography is especially valuable when analyzing complex samples with numerous overlapping components.

Multidimensional Chromatography

This is a sophisticated technique that involves the coupling of two or more chromatographic separation methods to achieve superior separation capabilities. It is particularly useful for complex samples with many components that a single chromatographic technique cannot fully separate and identify itself.

The idea behind multidimensional chromatography is to use multiple orthogonal separation mechanisms. Each dimension of the separation (e.g., size-exclusion, reversed-phase, ion-exchange) provides different selectivity, allowing for enhanced resolution and separation of components. This results in a highly efficient separation with improved peak capacity and increased sensitivity.

Multidimensional chromatography is commonly used in advanced research applications such as proteomics, metabolomics, and complex pharmaceutical analysis. By combining multiple separation dimensions, scientists can gain deeper insights into complex mixtures, identify low-abundance compounds, and perform comprehensive structural analyses of biomolecules.

Applications of Liquid Chromatography

LC has several applications across various industries and research fields. Some of the key areas where liquid chromatography finds extensive use include:

Pharmaceuticals

Liquid chromatography is crucial in drug development, quality control, and pharmacokinetic studies. It is employed to analyze drug compounds, metabolites, impurities, and drug concentrations in pharmaceutical formulations and biological samples.

This involves quantifying drugs and their metabolites in blood, plasma, or urine to understand their absorption, distribution, metabolism, and excretion in the body. Accurate pharmacokinetic data helps optimize drug dosages, assess drug interactions, and ensure drug safety and efficacy.

Biotechnology & Biopharmaceuticals

LC is used to analyze biomolecules, such as proteins, peptides, nucleic acids, carbohydrates, antibodies, and vaccines, contributing to biopharmaceutical research and protein characterization. Specifically, it is used to characterize these various molecules' purity, identity, and structural integrity.

Environmental Analysis

LC is used to detect and quantify environmental contaminants, pesticides, and pollutants in soil, water, and air samples, aiding in environmental monitoring and regulatory compliance. For example, LC is employed to detect pesticide residues in agricultural runoff, trace organic pollutants in drinking water sources, and industrial pollutants in wastewater.

Food & Beverage Industry

LC ensures food safety and quality by detecting food additives, pesticide residues, mycotoxins, and other contaminants in food and beverage products. For instance, LC detects and quantifies mycotoxins in grains, pesticide residues in fruits and vegetables, and additives in processed foods.

Forensics

Like pharmaceuticals, LC is applied in forensic science to analyze drugs, toxins, and metabolites in biological samples. However, its use in forensics is to aid in criminal investigations and toxicological assessments.

Clinical Diagnostics

LC is employed in clinical laboratories to quantify biomarkers, drugs, and endogenous compounds in patient samples, assisting in disease diagnosis and monitoring.

In Summary

Liquid chromatography is a versatile analytical technique widely used in research and industry, as the ability to separate, identify, and analyze is essential in pharmaceutical development, environmental monitoring, food safety, forensics, biotechnology, and clinical diagnostics.

LC's high resolution and sensitivity enable the detection of trace compounds in complex samples, making it invaluable for precise analysis, and automation and continuous advancements continue to drive scientific progress and innovation across various fields and sectors.

Leasing a Liquid Chromatography System

Liquid chromatography systems are often expensive due to their sophisticated technology, precision engineering, and high-quality components. A basic HPLC system's price range can vary significantly, from around $20,000 to $100,000 or more.

However, it's important to note that these prices are general estimates and can vary based on system specifications, brand reputation, and additional features.

Leasing a liquid chromatography system offers several advantages, particularly for laboratories conducting research and utilizing chromatographic techniques:

  • Lower initial investment: Leasing allows laboratories to acquire advanced liquid chromatography systems without a significant upfront capital investment. This is especially beneficial for smaller or newly established labs with budget constraints.
  • Cost management and flexibility: Fixed monthly lease payments enable better financial planning and budget management. Labs can allocate resources more efficiently and adapt to changing research needs without the burden of a lump-sum purchase.
  • Access to latest technology: Leasing provides access to state-of-the-art liquid chromatography systems with cutting-edge features and improved performance. Regular upgrades can be made at the end of the lease term, ensuring labs stay at the forefront of scientific advancements.
  • Maintenance and repairs: Leasing often includes comprehensive maintenance and technical support from the leasing company, ensuring optimal system performance and minimizing downtime.
  • Potential tax benefits: Lease payments may be tax-deductible as operating expenses, providing potential tax advantages for the laboratory.
  • Rapid implementation: Leasing allows labs to quickly acquire and deploy liquid chromatography systems, streamlining research processes and accelerating scientific discoveries.

By leasing rather than purchasing, laboratories can optimize their research capabilities, stay up-to-date with the latest technology, and efficiently manage their resources, contributing to more productive and cost-effective research endeavors.

If you’re interested in leasing, let us know. Excedr’s team of experts is ready to walk you through our leasing program and create a chromatography system lease estimate for you and your lab.