Liquid chromatography is an essential process in many laboratories for the separation, identification, purification and quantification of various compounds. When purchasing a liquid chromatography system, consider a more general purpose HPLC system versus an application-specific HPLC system, such as an ion chromatograph or FPLC. The new technology, UHPLC, is gaining rapid acceptance for its performance, speed of analysis and low consumption of eluent and is worth investigation if you are looking at HPLC. You’ll find that different vendors have different strengths, whether it’s the cost of operation or experience in the application for which you plan to use the system. Today, HPLC vendors are increasingly targeting system solutions to applications. An application-specific instrument can often be used for unplanned tasks in the future, but flexibility isn’t always an option.
HPLC is a chemical separation science in which a pump pushes the mobile phase with the sample through the column. This is similar to classical LC, in which the mobile phase and analyte are driven through the column by gravity alone. HPLC separation media is typically very dense, which creates a high back pressure, usually measured in hundreds to thousands of PSI. This allows for higher resolution and faster separation on columns of shorter length when compared to open column chromatograpgy. An HPLC system consists of a mobile phase, a pump, an injector, a column and a detector. Manufacturers typically offer these components as a system, however, there can be a mixing of the system components from various vendors.
In selecting an HPLC system, you want to look at identifying both operating and acquisition cost, service, support and training. Understanding your throughput requirements will let you know how much automation is required. Knowing your compounds of interest will allow you to select your column chemistry, which can be Ion Exchange, Gel Filtration, Affinity, Normal Phase or the most popular column, Reverse Phase, amongst others. You’ll also need to decide on your detector type, and there are as many detectors as column types. Your detector needs to be matched to identifying your compounds of interest. UV/Vis, a common detector, comes in a few flavors, including Diode Array. Other popular detectors include Fluorescence, Electrochemical, Refractive Index, Light Scattering and Radioactivity.
Ion Chromatography, which analyzes principally for inorganic anions and cations, can utilize the same hardware as HPLC systems and typically runs at a low to medium pressure, however, these systems are a little more complex than HPLC. Systems typically come with chemical and/or electronic suppression, not seen in HPLC systems. Perhaps the most important decision to make in the purchase of an IC is that the system you are looking at has a proven track record for the analysis you want to do. Dionex is the marketshare leader in IC, but as you can see below, there are a few other choices to consider as well.
An increasingly important application of HPLC is in the field of protein analysis and purification. Systems designed specifically for separating biomolecules are often the same as HPLC systems with some of the eluent and sample pathway reconfigured. Most vendors are capable of offering solutions for FPLC (Fast Protein Liquid Chromatography) and Bio HPLC, however, with any intricate application you will want to challenge a vendor’s experience to ensure the system you are looking at will deliver the results you seek. There are various decisions to be made with regard to scale up for purification, automation and analysis.
UHPLC, Ultra High Performance Liquid Chromatography, provides some significant benefits in performance and throughput. You can typically expect faster run times, higher resolution and reduced consumption of eluent. Cost of operation can be significantly reduced and more samples can be processed over time.
Many liquid chromatographic systems incorporate a detector that analyzes the different fractions. Common modes of detection include:
- Refractive index (RI)
- Evaporative light scanning device (ELSD)
- Mass spectrometry
However, as HPLC requires a high pressure and the use of an organic solvent, it is not ideal for biopolymers, including those proteins that are easily denatured.
Liquid chromatography can be used for analysis or for larger scale preparation. The appropriate pump must be selected.
- Analytical – flow rate required: 0.4-2 mL/min
- Semi-preparative – flow rate required: 2-10 mL/min
- Preparative – flow rate required: 10-150 mL/min
Some purchasers prefer an integrated chromatography system in which all components are supplied in a single unit. Other purchasers prefer a modular system, in which the individual components (including column, pump and detector) are purchased separately.