Last Updated on
August 1, 2023
By
Excedr
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.
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:
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.
Generally speaking, a liquid chromatography system, or chromatograph, consists of several key components:
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.
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:
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, 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 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 (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, 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 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 (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.
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.
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.
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.
HPLC and UHPLC offer several advantages that contribute to their popularity:
Advanced techniques in liquid chromatography encompass various methods that enhance separation efficiency, sensitivity, and specificity. Let’s review some of the most common.
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:
There are other advanced techniques, two of which we’ll discuss next: high-resolution and multidimensional 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.
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.
LC has several applications across various industries and research fields. Some of the key areas where liquid chromatography finds extensive use include:
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.
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.
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.
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.
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.
LC is employed in clinical laboratories to quantify biomarkers, drugs, and endogenous compounds in patient samples, assisting in disease diagnosis and monitoring.
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.
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:
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.
