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Liquid Chromatography Systems

Liquid Chromatography Systems

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Liquid chromatography diagram

Liquid chromatography systems are crucial in analytical laboratories, enabling the separation, identification, purification, and quantification of diverse biomolecules and small molecules.

Chromatography diagram

These versatile systems have evolved significantly and are essential in numerous analytical workflows.

The foundation of liquid chromatography (LC) lies in separating compounds within a sample based on their interactions with the mobile phase (solvent) and the stationary phase (chromatographic packing material). Higher pressures were adopted using smaller particles, leading to the development of high-performance liquid chromatography (HPLC).

In an HPLC system, a pump and mobile phase work together to move the sample mixture (analyte) through the column at high pressure. Compounds interact with the mobile and stationary phases, separating based on affinity or polarity. The retention time of each compound depends on its interactions.

HPLC’s remarkable sensitivity allows it to identify trace compounds at parts per trillion (ppt), making it indispensable in pharmaceuticals, environmental monitoring, forensics, and the chemical industry.

Now, let’s delve deeper into the world of liquid chromatography systems. In this blog post we’ll cover:

  • System components: Pumps, mobile and stationary phases, and more. We’ll also cover how they are used to achieve precise separation and analysis.
  • Different instrument and system types: HPLC, UHPLC, and FPLC and their applications in scientific and industrial fields.
  • The diverse uses of each system type: Applications of LC systems, from analyzing pharmaceutical compounds to characterizing complex protein structures, empowering researchers to choose the right tool for their needs.
  • Pricing ranges: The general costs of a basic setup and the prices of different liquid chromatography systems.

By the end of this blog post, you’ll gain valuable insights into liquid chromatography systems, allowing you to make informed decisions about the equipment you use and achieve exceptional results in your analytical endeavors, whether you’re an experienced chromatographer or a newcomer.

System Components, Types, Uses, & Price

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Learn about the major components that make up a liquid chromatography system. Discover the different types of systems used in laboratory research and their uses. Get an idea of what it costs to acquire an LC system.

Pump

Liquid chromatography pumps play a crucial role in the chromatographic process, as their pressure capacity and performance are key to achieving consistent and reproducible flow rates. Pressure levels can reach as high as 60 MPa (6000 lbf/in2), equivalent to about 600 atmospheres, providing the necessary force for efficient separations.

In HPLC, the pump is often referred to as a solvent delivery system, precisely controlling the flow of the mobile phase and eluent to the chromatographic column. HPLC pumps are known for delivering pulseless flows even against substantial backpressure, ensuring reproducible flow rates, regardless of changes in backpressure. These pumps are available in standalone models that are easy to operate and capable of generating gradient elutions—a valuable feature for various applications beyond HPLC.

Critical components of an HPLC pump include the piston, driver, pump head, high-pressure pump unit, check valve, proportioning valve, mixer, pulse damper, and degasser. These components work together to ensure efficient and reliable performance during chromatographic analysis.

A diverse range of available pumps in modern liquid chromatography systems include:

  • Constant pressure: Constant pressure pumps deliver a consistent pressure throughout the chromatographic process. These pumps maintain a stable pressure regardless of changes in backpressure, ensuring a steady flow rate for precise and reliable separations.
  • Constant flow: Constant flow pumps provide a consistent flow rate of the mobile phase throughout the analysis. They maintain a steady flow regardless of variations in backpressure, enabling accurate and reproducible chromatographic results.
  • Reciprocating: Reciprocating pumps use a reciprocating motion to deliver the mobile phase. They typically operate with a piston or diaphragm, which moves back and forth to generate the required flow, making them suitable for high-pressure applications.
  • Syringe: Syringe pumps are designed with a syringe-like mechanism to deliver the mobile phase. They offer precise and low-flow rates, making them suitable for applications requiring high accuracy, such as sample injections and microfluidic systems.
  • Dual-piston: Dual-piston pumps utilize two pistons working in tandem to deliver the mobile phase. This design provides smooth and pulseless flow, minimizing flow rate fluctuations and ensuring consistent separations.
  • High-pressure/low-pressure: These pumps combine the mobile phase from two different reservoirs—one with high pressure and one with low pressure—before delivering it to the column. The mixing process allows for precise gradient elutions, making them ideal for applications requiring varying mobile phase compositions.

Choosing the most suitable pump and system for your laboratory will depend on several factors, with flow rate requirements being of utmost importance. Careful evaluation of these factors will enable researchers to make informed decisions and select the optimal pump to meet their analytical needs.

Sample Injector

The injector, a critical component in liquid chromatography systems, facilitates the introduction of the liquid sample, or analyte, into the chromatographic flow. Depending on the laboratory's requirements, the injector can be a single injection setup or an advanced automated injection system.

For high-performance liquid chromatography (HPLC) systems, it is essential that the injector allows injection of the liquid sample within the range of 0.1-100 mL with high reproducibility, even under high pressure conditions.

Modern HPLC systems offer various sample injectors, such as injection valves, sample pumps, and autosamplers, each with specific advantages. When selecting the ideal sample injector for a laboratory, precision, accuracy, and carryover are critical factors to consider. Precision ensures that the injected sample volume is consistent and reproducible, while accuracy guarantees the correct delivery of the desired sample volume. Minimizing carryover is essential to prevent contamination between injections, particularly in high-throughput applications.

While manual injection valves serve as a cost-effective option for introducing samples, automated injection methods are highly recommended when dealing with large sample volumes or requiring high throughput.

Autosamplers, a type of automated injection system, significantly enhance productivity by enabling the sequential analysis of multiple samples without manual intervention. This automation streamlines the analytical workflow, increasing sample throughput and improving overall efficiency in the laboratory.

Chromatographic Column

Deciding on the most suitable HPLC column for a separation relies on understanding the physical properties of the analyte to be separated. These properties encompass factors such as hydrophobicity/hydrophilicity, intermolecular forces (particularly dipole-dipole interactions), intramolecular forces (ionic interactions), and size.

HPLC column separations leverage these specific differences in molecular properties to achieve effective separations. The structure and chemistry of the HPLC column packing, known as the stationary phase, significantly influence the elution profile of the analytes.

HPLC columns come in various sizes, ranging from capillary to process scale. The internal diameter and volume of the column determine both the sample loading capacity and the sensitivity of the separation. Smaller internal diameters yield enhanced separation and detection sensitivity, especially when using gradient elution. For instance, capillary HPLC with small capillaries facilitates taller chromatographic peaks, leading to better detection limits for concentration-sensitive detectors like mass spectrometry.

While there is often a trade-off between sensitivity and the sample volume loaded onto a column, careful selection of the column size ensures optimal performance for specific analytical separations.

Modern HPLC columns are typically made of borosilicate glass, acrylic glass, or stainless steel, offering improved durability and chemical compatibility. These columns are further categorized based on their separation mechanisms, resulting in various types:

  • Ion exchange columns: Used for separating charged analytes based on their affinity for ionizable groups in the stationary phase. Positively charged analytes (cations) are retained by the negatively charged stationary phase, while negatively charged analytes (anions) are retained by the positively charged stationary phase.
  • Ion exclusion columns: Employed to separate ions and polar molecules from non-ionic species. These columns allow only neutral or non-polar compounds to pass through, while ions and polar molecules are excluded from the stationary phase.
  • Size exclusion columns: Separates analytes based on their size and molecular weight. Larger molecules elute faster as they cannot penetrate the pores of the stationary phase, while smaller molecules are retained longer in the column.
  • Reversed-phase columns: The most common in HPLC and are used for separating hydrophobic compounds. The stationary phase is non-polar, and analytes with greater hydrophobicity are retained longer, while more polar analytes elute first.
  • Normal phase columns: Used for separating polar compounds. The stationary phase is polar, and analytes with higher polarity are retained longer, while non-polar analytes elute first.

Each column type employs a different chromatography packing material to achieve distinct separation characteristics.

For applications requiring precise control of column temperature, column thermostats are commonly integrated into the LC system. Maintaining a consistent column temperature improves the reproducibility of chromatograms, which is especially beneficial in organic-based chromatography. Notably, column heating is often used for organic separations, while separating proteins typically involves column cooling.

Selecting the most appropriate HPLC column depends on a comprehensive understanding of the analyte's physical properties and the desired separation characteristics. By choosing the right column type and size, researchers can achieve efficient and reliable chromatographic separations tailored to their specific analytical needs.

Stationary Phase

The stationary phase is central in separating sample components during the chromatographic process. It is a solid or liquid material that remains fixed inside the chromatography column throughout the analysis.

The primary function of the stationary phase is to provide a surface with which the sample components can interact selectively. As the mobile phase (the solvent or mixture of solvents) moves through the column, it carries the sample. When the sample encounters the stationary phase, the different analytes within the sample interact with the stationary phase based on their chemical properties.

The interaction between the stationary phase and the sample components is crucial for separation. Depending on the nature of the stationary phase, certain sample components will adhere more strongly to it, causing them to move more slowly through the column. In contrast, others will have weaker interactions and will elute faster. This differential retention results in separating the sample components as they elute at different times, a phenomenon known as chromatographic peaks.

Various types of stationary phases are available for liquid chromatography, and their selection depends on the analytes being analyzed and the separation requirements. Some common types of stationary phases include:

  • Silica-Based Phases: Silica gel is a standard solid support material used in many liquid chromatography applications. It provides a versatile and widely applicable stationary phase.
  • Polymeric Phases: These stationary phases are based on synthetic polymers with specific chemical functionalities that can be tailored to interact selectively with specific analytes.
  • Ion-Exchange Phases: These phases, such as ions or polar molecules, contain ionizable groups that can attract and retain charged analytes.
  • Reversed-Phase Phases: Reversed-phase chromatography involves non-polar stationary phases that retain hydrophobic analytes, while polar analytes elute first.
  • Size-Exclusion Phases: Size-exclusion chromatography employs porous materials that separate analytes based on size, with larger molecules eluting first.

The choice of the stationary phase depends on the properties of the sample and the specific chromatographic method being used. Selecting the appropriate stationary phase is essential to achieving efficient separation and obtaining accurate and reliable results in liquid chromatography.

Detector

LC system detectors play a crucial role in identifying and monitoring components eluted from the chromatography column. These detectors generate electronic signals corresponding to specific compounds they are designed to respond to. There are various types of detectors, each employing different detection methods and offering varying results for purity screening:

  • UV/Vis: These detectors non-destructively measure UV absorption at fixed or variable wavelengths, making them popular for liquid chromatography. They quantify the amount of ultraviolet or visible light absorbed by the components in the eluent.
  • Photodiode array (PDA): A type of UV/Vis detection, PDA detectors simultaneously detect the entire spectrum of the eluent, providing more comprehensive information.
  • Refractive index: Non-destructive detectors continuously measure the refractive index of the eluent, offering excellent stability despite lower sensitivity.
  • Fluorescence: Utilizing fluorescence spectroscopy, these detectors irradiate the effluent with a specific wavelength of light and measure the resulting fluorescence, providing sufficient stability and ease of use.
  • Evaporative light scattering detectors (ELSD): A destructive detection method used for non-volatile analytes, ELSD detectors evaporate the effluent and measure light scattering of the aerosol, making them suitable for compounds unsuitable for absorbance detectors.
  • Conductivity: Used in ion chromatography, these detectors measure electronic resistance in ionic solutions, offering proportional readings to ion concentration.
  • Mass Spectrometers (part of LC/MS): Integrated with LC systems, mass spectrometers analyze molecular weight (mass-to-charge ratio) of compounds, providing valuable information for identifying unknown substances.

Each detector type has its unique strengths, and selection depends on the specific analytical requirements and nature of the analytes being analyzed. By employing the appropriate detector, researchers can obtain accurate and valuable data during liquid chromatography analyses.

Data Acquisition System

Chromatography data systems (CDS) are essential components in liquid chromatography equipment, including HPLC, GC, and IC analyses. These systems play a pivotal role in instrument control, data processing, archiving, and report generation, ensuring efficient and accurate handling of chromatographic data.

There are three primary setups for utilizing data acquisition systems in chromatography:

  • Standalone system for multiple chromatographs: In this configuration, the data handling and instrument control system operate independently, overseeing and managing multiple chromatographs simultaneously. This setup is particularly beneficial in laboratories with several chromatographic instruments, allowing seamless coordination and analysis.
  • Standalone system for a single chromatograph and LC/MS instruments: In this scenario, the data acquisition system functions as a self-contained unit dedicated to controlling a single chromatograph or even LC/MS instruments. This arrangement ensures precise control and focused data processing for individual analyses.
  • Networked system for multiple instruments in one or more labs: This setup involves connecting the data acquisition system to multiple chromatographic instruments across one or more laboratories through a network. This networked approach allows efficient sharing of data and centralized control over various instruments, streamlining data management in larger analytical facilities.

The data acquisition system, supported by its specialized software, seamlessly handles instrument control, data acquisition, and analysis, ensuring reliable and accurate results for chromatographic analyses.

By employing the most suitable configuration based on the laboratory's requirements, researchers can maximize the capabilities of their liquid chromatography equipment and streamline their data management processes.

Fraction Collector

Fraction collectors automate the collection of separated compounds from the chromatographic column. 

As the sample mixture passes through the column and is separated into its components, the fraction collector collects and stores each component in separate containers or test tubes based on specific criteria, such as elution time or detector signals. The automation ensures accuracy, consistency, and reproducibility in sample collection.

Fraction collectors also significantly increase the efficiency of liquid chromatography experiments, especially when dealing with complex mixtures. They allow researchers to conveniently collect and analyze individual fractions, saving time and effort.

By promptly collecting each fraction, this component prevents the potential degradation or loss of analytes, especially when dealing with sensitive or labile compounds. Collecting individual fractions simplifies the analysis of each component, enabling more detailed investigations and identification of target compounds.

The need for fraction collectors in liquid chromatography systems depends on the specific application and experimental requirements. In some cases, fraction collectors are essential, mainly when dealing with:

  • Purification and isolation: When isolating specific compounds from complex mixtures, fraction collectors are necessary to collect and concentrate the target components.
  • Pharmacokinetics studies: Fraction collectors are vital for accurately determining analyte elution time and concentration profile during pharmacokinetic studies.
  • Protein and biomolecule analysis: In protein purification and biomolecule analysis, fraction collectors facilitate the collection of individual protein fractions for further characterization.

However, in specific applications where simple detection and identification of analytes are sufficient, fraction collectors may be optional. Researchers may choose to manually collect and analyze the fractions for routine analyses or when the cost is a significant factor.

Mobile Phase Reservoirs

Mobile phase reservoirs are essential components in a liquid chromatography system responsible for holding and delivering the mobile phase, the solvent or mixture of solvents used to transport the sample through the chromatographic column.

These reservoirs are typically constructed from high-quality materials chemically compatible with the mobile phase and prevent any interactions or contamination with the sample.

In a typical liquid chromatography setup, there are two mobile phase reservoirs—one for the aqueous mobile phase and the other for the organic mobile phase, when gradient elution is employed. The selection of appropriate mobile phase solvents and their proportions is crucial for achieving efficient separation of analytes in the sample.

Mobile phase reservoirs have solvent level sensors to monitor and maintain a consistent mobile phase level throughout the chromatographic run. This ensures a continuous and reproducible mobile phase flow to the chromatographic system.

Additionally, the reservoirs may have built-in degassing mechanisms to remove dissolved gases from the solvents, enhancing system stability and reducing the risk of bubble formation, which could adversely affect chromatographic performance.

Tubing & Connectors

Tubing and connectors are vital components that facilitate the flow of the mobile phase and the sample through the liquid chromatography system. These tubes are carefully selected to be inert and chemically compatible with the mobile phase and the sample to avoid unwanted interactions or analyte adsorption during the chromatographic process.

High-quality tubing connects various parts of the chromatography system, such as the mobile phase reservoirs, the pump, the injector, and the detector. Using the appropriate tubing material and dimensions is crucial to minimize extra-column band broadening and ensure efficient chromatographic separation.

Connectors are critical in ensuring leak-free connections between different system parts, maintaining a consistent and uninterrupted flow of the mobile phase and the sample. They are designed to withstand the high pressures typically encountered in high-performance liquid chromatography (HPLC) and other techniques.

LC System Price Range

Liquid chromatography equipment prices can vary widely depending on the complexity and features of the system. However, in general, basic HPLC setups with a simple detector, injector, and pump are relatively affordable compared to more advanced configurations. Opting for HPLC over UHPLC may also lead to cost savings.

However, as you add additional detection modules, include an autosampler, or upgrade to a high-pressure pump, the overall cost of the system will increase significantly.

The general price range for liquid chromatography equipment can start from around $10,000 to $30,000. More advanced liquid chromatography systems with additional detection modules, autosamplers, and high-pressure pumps can range from $40,000 to $100,000 or more.

Specialized or cutting-edge systems with advanced features and technologies may exceed several hundred thousand dollars. For example, UHPLC systems tend to be more expensive compared to traditional HPLC systems due to the higher pressures and improved performance they offer.

Here’s a breakdown of price by equipment type:

  • Basic HPLC systems: Basic HPLC systems with a simple detector, injector, and pump can range from $10,000 to $30,000. These systems are suitable for routine analyses and are commonly used in academic and industrial laboratories.
  • HPLC systems with added detection modules: As you add multiple detection modules to the basic HPLC setup, such as UV/Vis, PDA, fluorescence, or mass spectrometers, the price can increase to $40,000 to $100,000 or more. These systems offer enhanced sensitivity and specificity for a wide range of applications.
  • Autosamplers and high-pressure pumps: Integrating an autosampler into the HPLC system allows for automated sample injection, increasing efficiency and throughput. The addition of an autosampler can add an additional cost of $15,000 to $30,000 to the system. Upgrading to a high-pressure pump for improved separation performance can also add $10,000 to $20,000 to the total cost.
  • UHPLC systems: UHPLC systems, which provide higher resolution and faster separations, can range from $50,000 to $150,000 or more, depending on the included features and detection modules.

It's essential to consider the specific analytical needs and budget constraints when selecting liquid chromatography equipment. Investing in the right system tailored to the laboratory's requirements will ensure accurate and efficient analyses and maximize the value of the equipment.

Lease Liquid Chromatography Systems from Any Brand

Liquid chromatography systems are powerful analytical tools used in various scientific and industrial applications to separate, identify, and quantify compounds. There are several types of systems available, all of which rely on similar components to generate accurate results. However, the high costs of purchasing this equipment can pose a challenge for many laboratories.

While purchasing a new or refurbished liquid chromatography system outright may not be feasible within your budget, there is an excellent alternative—leasing the equipment at an affordable rate. At Excedr, we offer cost-effective lease options that can be tailored to your laboratory's needs, providing a flexible solution without the burden of large upfront expenses.

Leasing an HPLC system through Excedr allows you to significantly reduce upfront costs and manage your laboratory's cash flow more efficiently. With affordable, predictable lease payments, you can access the cutting-edge equipment your research requires without straining your financial resources.

Whether you need a liquid chromatography, gas chromatography, or thin-layer chromatography system, our lease program is designed to accommodate your specific requirements. Experience the benefits of top-quality chromatography instruments without compromising your budget.

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Popular Liquid Chromatography Equipment Manufacturers

Agilent
Bio-Rad
Cytiva
Danaher
Gilson
GL Sciences
Jasco
Novasep
Shimadzu
Perkin Elmer
Thermo Fisher Scientific
Waters