Liquid chromatography (LC) is essential in many laboratories for the separation, identification, purification and quantification of various compounds in different matrices. With so many different types and models of LC instruments to choose from, finding the right one could be a daunting task. This guide will walk you through the aspects you need consider when choosing the right LC system for your lab.
In liquid chromatography, the sample is carried across a stationary phase (the solid support in LC columns) by a moving liquid (the mobile phase or eluent). The components of the sample are separated based on their affinity with the stationary phase.
‘Classical’ or ‘traditional’ LC relied on gravity to get the mobile phase and sample through the column. But as smaller particles were used to improve separation power and higher pressures were needed to create the desired flow through the column, high pressure liquid chromatography, or HPLC, was developed. Eventually, as advances in instrumentation continued and performance improved, the acronym HPLC came to stand for high performance liquid chromatography. Systems that operate at pressures up to 35,000 psi are known as medium-pressure chromatography systems, and are typically used as part of a larger laboratory workflow to separate, prepare or quantitate samples.Every LC system has the following key components:
For applications that require close control of column temperature, column thermostats are usually included in the LC system. The other advantage of using a thermostat is that more reproducible chromatograms are usually obtained by maintaining column temperature. While organic-based chromatography usually involves column heating, the opposite is true for separating proteins. Most protein chromatography is either performed in a cold room (room temperature to 4°C) or, if the system is small enough, it can be kept in the fridge, such as Bio-Rad's NGC medium pressure chromatography system. The latter enables the researcher to work at room temperature and the system to stay cold, minimizing any protein degradation.
There are many detectors for liquid chromatography, some of which are: UV-vis, photodiode array (PDA), fluorescence, mass spectrometer (MS), refractive index, electrochemical, and evaporative light scattering (ELSD).
General-purpose LC systems are used in the separation of different types of molecules, depending on the column that is used (e.g. reverse phase, normal phase). There are different LC platforms, depending on the goal of the separation and the corresponding flow rate requirements. Below, we summarize the different LC systems in the market, and what each is typically used for:Preparative systems
A modular LC system is composed of separate modules (the modules are different components of the LC system), stacked and connected to function as one unit. In an integrated system, the modules/components are built inside a single housing. Compared to integrated systems, modular systems provide maximum flexibility and can be upgraded and expanded. Modular systems are easily serviceable, since internal components are more accessible (downtime is reduced during repair or maintenance service) and they enable you to continuously stay up-to-date with technology, rather than purchasing a whole new system every few years.
When analyzing proteins, whether you work in academia, industry or the pharmaceutical industry, it is important to find a system that works for you. Bio-Rad's NGC medium pressure chromatography system (Figure 1) is ideal for preparative life science and pharmaceutical applications, from discovery to early scale-up, as it is easy to use, robust and has the ability to be a scalable platform. The system is truly modular, enabling access to more sophisticated detection methods or higher throughput through the addition/switching out of modules. The ability to remotely control runs allows for less downtime and a continuous workflow.
LC-MS combines liquid chromatography (LC) and mass spectrometry (MS) for separation and analysis of complex mixtures. Thermo Scientific's Q Exactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer enables simultaneous quantitative and qualitative analysis of targeted and non-targeted compounds. This impressive technology is now accessible to environmental, food safety, clinical research, forensic toxicology, and pharmaceutical laboratories that require high throughput, improved sensitivity and increased selectivity at an affordable price. Read our exclusive webinar highlights (Figure 2) to learn how the Q Exactive Focus is used to quantitatively determine and screen pesticide residues in food samples.
Other analytical liquid chromatography systems include standard, microflow, capillary and nanoflow. These systems identify and quantify compounds with flow rates of up to 10mL/min (0.01-100µL/min for microflow, 0.01-1µL/min nano). Such flows are ideal for sample limited applications, such as proteomics.
HPLC systems can be classified into four basic types, depending on use, as illustrated in Table 1 below:
I. Basic Isocratic Systems
Simple, routine analysis (QA/QC)
II. Gradient Systems
Method development, complex analyses/separation, and dial-mix (autoblend) isocratics for routine analysis
IV. Fully automated gradient systems with state-of-the art detectors
Methods development, research
III. Fully automated, dedicated systems (gradient, isocratic); high-throughput
Cost-per-test, round-the-clock analyses (clinical and environmental laboratories)
Table 1: HPLC Classifications
The separation of unknown complex mixtures is time-consuming and a challenge for most scientists. Multi-dimensional chromatography provides a solution to this conundrum by combining fast and efficient separation, using different techniques in the same chromatographic run. Watch the video below to learn how 2D chromatography can help you with your separations (Figure 3).
Due to software limitations, many researchers find it difficult run tandem chromatography in the laboratory. Bio-Rad has developed the ChromaLab software, which works with the medium-pressure NGC and enables simplified method creation, set-up and data analysis for 2D chromatography analyses.
Other Application-Specific Liquid Chromatography
Applications include separation and quantitation of inorganic anions and cations, and low molecular weight water-soluble organic acids and bases. Ion chromatography is typically used in research and development labs for routine analyses.
Porous columns are used to separate, purify and characterize large biomolecules and synthetic polymers. Small particles diffuse into the porous beads and have a longer retention time compared to larger molecules, which flow around the beads and are expelled quicker. GPC and SEC are typically used in research and development laboratories. Read our exclusive webinar highlights (Figure 4) to learn how the Waters ACQUITY Advanced Polymer (APC™), a SEC system, enables efficient characterization and analysis of problematic polymers, such as low molecular weight polymeric materials.
Supercritical fluid chromatography (SFC), a hybrid of liquid and gas chromatography, is used in the separation of compounds that are difficult to analyze by either liquid or gas chromatography. SFC normally utilizes supercritical CO2 as a primary mobile phase and is used to analyze hydrophobic and chiral molecules such as lipids, fuels, natural products, surfactants and thermally labile analytes. UltraPerformance Convergence Chromatography (UPC2) combines SFC with UltraPerformance Liquid Chromatography (UPLC), enabling scientists to tackle routine and complex separation challenges that SFC can solve, while delivering reliability, robustness, sensitivity and throughput that the UPLC technology has demonstrated since its first introduction in 2004. Ultra high-performance liquid chromatography (UHPLC) is the general term given to LC systems that can withstand pressures up to 1000 bar. (UPC2 and UPLC are trademarks of Waters Corp.)
In order to get the most out of your purchasing power, you should ask yourself the following questions before speaking to a manufacturer:
By answering these questions, you equip yourself with the knowledge and understanding to enable you to make the right purchase for your lab and your experiments.System Performance
The overall performance of an LC system, and the quality of data generated, depends on a variety of components.
Consider the following performance attributes when purchasing an LC system:
Accuracy: This is the closeness of the values obtained by the LC analysis to the ‘true’ value. The accuracy of an LC system is influenced by the injector, pump and detector.
Accuracy of the pump: The pump must maintain accurate and consistent flow rate, necessary for stable and repeatable interaction between the analyte and the stationary phase.
Detector: Ideally, the detector should have low noise, low detection limits, high sensitivity and large dynamic range. Good stability and reproducibility (for publications) are important factors, as well as sensitivity towards solute over mobile phase.
Dynamic range: A performance parameter of the detector. There are two ‘dynamic range’ terms that are used: ‘dynamic range’ and ‘linear dynamic range’. ‘Dynamic range’ is the range of analyte concentration over which the detector continues to respond to changes in analyte concentration. The ‘linear dynamic range’ is the concentration over which the detector output is linearly related to the analyte solute concentration.
Injection, Autosampler: Samples may need high precision even at low volumes, accurate injection volumes or a range of injection volumes. Some samples may need to be heated or cooled and not all samples have this capability.
Precision: The closeness of the results of multiple LC analyses carried out under the same conditions, with the goal that repetitive analysis of the same sample gives similar results. The precision of an LC system often lies in the ability of the sample injection system, to introduce samples on to the column in a very reproducible manner. The injector should be able to draw the same amount of sample in replicate injections. Precision is also influenced in part by the pump, stability of the column oven (if used), and detector.
Precision of retention times: Excellent retention time precision is required, especially if this parameter is used for compound identification.
Pump: It is important to consider solvent compatibility, resistance to corrosion, accurate and precise flow rate, accurate and precise mixing of solvents, and availability and convenience of replacing worn out parts.
Resolution: A measure of how well peaks are separated; largely dependent on the stationary phase (column) and the mobile phase.
Robustness: The LC system is robust if it is able to perform difficult analyses while still maintaining quality results.
Selectivity: The ability of the LC method to separate two analytes from each other. It is largely dependent on the characteristics/properties of the mobile and stationary phases.
Sensitivity: The ability of the system to discriminate between small differences in analyte concentration. The sensitivity of an LC system depends a lot on the detector, but is also influenced by the column parameters, the performance of pump, choice of eluent, and electronic noise.
Software/Computer: This is important for controlling the system, enabling easy data interpretation and storage. Some systems have the ability to be controlled remotely and send emails alerts if a problem occurs.
Speed, Throughput: Fast separations can be achieved in several ways: by using fast systems that use sub-2µm particles (ultra-high performance chromatography, UHPLC), high temperature, using other column platforms (core-shell or superficially porous particles, monolith), shorter columns, and increasing flow rate.
Automation: Will the system allow you to set up a run and walk away? How many columns can you run or set up at in one experiment? Does the level of automation meet your needs?
Ease of Use: A new LC system needs to be fairly easy to use/understand. Some vendors include technical training for lab personnel in the price of the LC system. This will prove beneficial, since you can ask questions in person, instead of having to call or find answers in the instrument’s manual.
Maintenance: Beyond the simplicity of operation and ease of use, consider the LC system’s ease of maintenance. If special maintenance is required, do you have lab personnel trained to do it? Can you afford the downtime required for the maintenance?
Availability of supplies and accessories: Make sure supplies and accessories for the system are easily accessible.
Cost: Consider not only the initial investment, but also the long-term cost of ownership. When you think about the return on your investment, consider aspects such as productivity (higher throughput with the same or higher quality results) and savings on solvents.
Compatibility with consumables - make sure that your system is compatible with consumables not only from the system provider but from other vendors.
Space: How much space does the system require? Do you have this space in the lab?
Service and SupportIt is important that the LC system manufacturer can provide not only excellent hardware, but also a responsive and knowledgeable service.
Make sure the company provides excellent customer support. A company’s support reputation may change over time; check out a company’s customer support reputation from current users. Good local sales and service representatives can help you interface with the company to make sure that problems are solved at a reasonable time. Also, a good support means the company is able and willing to help you, should you decide to venture into new uses/applications for your LC system.
LC systems are almost ubiquitous in any laboratory, since they can be used to analyze a broad range of analytes in various matrices. The use of mass spectrometers as detectors in LC systems is becoming more common. LC-MS has found applications in clinical diagnostics, and is expected to continue to be used for new applications. Since the introduction of the first system about a decade ago, UHPLC is becoming increasingly popular and is being adapted in many laboratories across the world. Future LC systems that can withstand even higher pressures than current UHPLC systems will enable the use of even smaller particles, yielding even faster and better separations.
In ion chromatography, IC-MS is becoming more common. An IC system operates at higher pressures, allowing for the use of columns packed with smaller particles. The smaller particles provide better performance than conventional columns.
Liquid chromatography will continue to be an essential analytical instrument. Modular systems are proving more popular, as they enable scientists to have access the latest technology by switching and adding modules.
Software plays a key role moving forward with innovations in LC systems. Manufacturers are developing software that is easy to use and does not require one to be an expert to run the chromatography system. This opens up applications of LC Systems to a wider purchasing audience with a variety of applications.
As novel applications for LC emerge, and users require better system performance and increased automation, we will continue to see improvements and new developments in LC instrumentation.
Visit the SelectScience Separations product and user product reviews directory for an overview of the latest UHPLC/UHPLC, SEC, IC products from leading manufacturers.
Keep up-to-date with the latest techniques and advances in liquid chromatography by visiting the SelectScience Separations pages for application notes, videos and the latest news.
Additionally, watch on-demand webinars and attend future events.
Other Liquid Chromatography Webinars:
"I currently use AKTA pure for my affinity purification and it saves lots of time. The software interface is now more user-friendly and valves are made really simple and accessible for all. It is easy to use, even for a beginner."
Dinesh Dhurvas Chandrasekaran, Leibniz Institute of Plant Biochemistry