“It was the best of times, it was the worst of times” often applies to researchers faced with a challenging separation experiment. This early step (often multiple steps) within a workflow process sets the stage for success or failure of a project. High-performance liquid chromatography (HPLC) and medium- or low-pressure chromatography (sometimes referred to as fast protein liquid chromatography, or ‘FPLC’) are two widely used methods for analyzing and separating molecules, particularly proteins, from a heterogeneous solution. Borne of the same origins, HPLC and medium-pressure chromatography have many differences, making each of them more suited to achieving certain separation goals rather than others. Here, we review some of the differences between the two methodologies as well as the pros and cons that researchers should consider when determining which technique will address their separation needs.
Under pressure
First, let’s cover the main difference between the two techniques (a clue to which lies in their naming): their working pressures. Although HPLC operates under high-pressure conditions, medium-pressure chromatography uses lower pressures. “HPLC describes the instrumental implementation of liquid chromatography by the use of flow constant pumping devices that can support pressures of several tens of MPa (megapascals) for any possible analytical or preparative purpose,” explains Frank Steiner, manager and application development and scientific advisor in HPLC, chromatography and mass spectrometry at Thermo Fisher Scientific. “Medium-pressure chromatography is a subset of liquid-chromatography techniques dedicated to protein purification that apply some HPLC-like techniques but under much lower pressures,” he adds.
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Steiner lists as ‘HPLC-like’ techniques: ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC) and reversed-phase (RPC) chromatography. Typical medium-pressure chromatography separation techniques also use IEX (using salt gradients and pH gradients) and SEC, and HIC to a smaller extent. In addition, medium-pressure chromatography incorporates “affinity chromatography techniques like Protein A or immobilized metal affinity chromatography (IMAC),” Steiner explains.
Uncommon goals
Despite there being some crossover between terminologies, the two main chromatography types remain fundamentally different in terms of pressure and ultimate aims.
The separation, identification and quantitation of specific components in the mixture are the typical goal of HPLC.
Candice Cox, global product manager in protein purification at Bio-Rad Laboratories, provides a few examples: “Analysis of carbohydrates and alcohols using Bio-Rad Aminex® HPLC columns is a common HPLC separation technique.” She continues, “Furthermore, Aminex columns employ many of the common techniques, like ion exclusion, IEX, ligand exchange, SEC and both RPC and normal-phase partitioning at high pressure. These multiple modes of interaction offer a unique ability to separate compounds.”
“Often a particular molecule is being profiled, such as the clinical diagnostic test separating hemoglobin A1C, or glycosylated hemoglobin, from other hemoglobin species to determine a percentage of HbA1c in diabetes diagnosis and monitoring,” Steiner offers by way of further example. “At 45 seconds per sample, Thermo Fisher Scientific’s D-100™ System and reagents provide the fastest result for HbA1c testing while also detecting the presence of genetic hemoglobin variants.”
With HPLC, oftentimes, the sample is directly analyzed in-line with another technique (such as mass spectrometry) and not collected for further use.
“An example of how HPLC is applied is the Bio-Rad Aminex HPX-87P HPLC column, which is used in food testing and provides resolution of sucrose, lactose and fructose in dairy products, which go straight into further analyses,” Cox confirms.
On the other hand, the aim of most medium-pressure chromatographersis to acquire a purified biomolecule, like a protein, from a complex mixture for use downstream in various applications. To obtain adequate amounts of the desired product, larger sample volumes are required, compared with HPLC. Bio-Rad’s Life Science group offers resins and columns capable of separating complex mixtures of biomolecules, such as monoclonal antibody (mAb) or membrane proteins, by using either the NGC™ Medium-Pressure Chromatography System or Biologic LP low-pressure systems.
Material differences
Another difference between these two branches of chromatography lies in the makeup of their resins. “Generally, HPLC resins’ particle size is smaller and fits into a smaller column requiring a higher-pressure system for separation of the components in a sample,” highlights Steiner, adding that HPLC systems and columns usually are constructed from stainless steel to withstand high pressures. “With particles sizes generally falling between 2 and 10 µm, a smaller bead diameter results in higher-resolution separations,” shares Steiner.
Resin beads used in medium-pressure chromatography are generally larger than those used for HPLC columns, but the beads come in a variety of sizes. “Bead size varies widely, depending on the specific level of purity and yield desired of the purified sample,” says Cox. “Often, more than one column or type of resin or technique is used to achieve the level of purity required. For example, a therapeutic mAb requires a high level of purity with acceptable yield in order to meet the safety, potency and manufacturing requirements of the manufacturer … [and] meet FDA regulations,” she explains.
It is not just bead size that differs; the materials used for HPLC vs. medium-pressure chromatography vary, too.
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The stationary phases in HPLC are mainly silica-based, but some pressure-stable organic polymers (e.g., polystyrene crosslinked with divinyl benzene) are used, as well. The classic medium-pressure, stationary-phase materials are crosslinked agarose or dextran gels that are characterized by very limited pressure stability. Other organic polymer gels, like polyacrylamide and polyvinylpyrrolidone, for example, are also applied in medium-pressure chromatography. “There is ongoing improvement being made with bioprocess resins,” Steiner points out. “Hard resins made of polystyrene divinyl benzene are now available for IEX and HIC, and traditional medium-pressure applications can now be carried out at higher pressure, compared to the past. So at the moment there is a growing overlap between both techniques in terms of stationary phases.”
Overlapping boundaries
Medium-pressure chromatography is considered better suited to purifying biomolecules such as proteins or DNA, as the workflow is carried out under lower pressures. Often, biomolecules cannot stand the high temperatures, high pressures or solvents usually used in HPLC [1]. However, Steiner considers this out-moded thinking: “There are many perceptions of incompatibility of proteins with silica-based stationary phases, elevated pressures and elevated temperatures, which would prevent the application of HPLC to protein separations. However, these limitations mostly do not prove correct in practice, and proteins can be separated on silica phases—even being exposed to pressures several tens of MPa greater and temperatures of 60°C and higher without any obvious degradation or even denaturation.”
Pros and cons
According to Steiner, “HPLC is superior in efficiency. With modern UHPLC technologies, it can provide substantially higher theoretical plate numbers and can provide elevated separation speed,” yielding far more plates per unit time. He explains that theoretical plates are hypothetical zones or stages in which two phases, such as the liquid and stationary phases of a chromatography run, establish an equilibrium with each other—simply put, the more theoretical plates, the more efficient the separation. “This is facilitated by smaller particle sizes in the stationary phase, and instrumentation that can work at significantly higher pressures,” he adds.
“A challenge for HPLC is that the harsh conditions required to achieve separation can destroy the functionality of the end product,” shares Cox. If retaining the end product isn’t your main goal, she adds, then this negative is of little concern, especially if a high degree of separation is the aim. “HPLC has the ability to yield the high-resolution separations required for analytical chemistry,” Cox explains.
Another con for HPLC is its more limited degree of scalability, which is not the case for medium-pressure chromatography. “The key advantage of medium-pressure chromatography techniques is their scalability up to industrial-size protein purification, in addition to an excellent compatibility of columns with high salt contents, high pH values and corrosive salts, like chlorides,” explains Steiner. “In contrast, the efficiency advantage of HPLC is difficult to extend beyond semipreparative scale, albeit not totally impossible.
Cox stands by the superiority of medium-pressure chromatography methods for purifying biomolecules, stating, “Biomolecules are usually better purified using medium-pressure chromatography, because they need to remain intact to be functional or to maintain structural integrity. Functional groups often cannot withstand the higher temperature, pressure or the harsh solvents required in HPLC and are important aspects for mechanism of action studies for therapeutics or for structural biologists.” However, she admits that the biggest con of medium-pressure chromatography is its lower efficiency.
Continuing innovation
Yet, with continuing innovations in the field of medium-pressure chromatography, efficiencies are increasing steadily. “Bio-Rad’s NGC Chromatography System, with its intuitive ChromLab Software, increases efficiency, and [it] is flexible and can grow with the needs of a lab,” Cox offers as an example, hinting that other instrumental improvements are a way of getting around the lower theoretical plate numbers encountered with medium-pressure chromatography. “The system can be automated, with the amount of automation being determined by the end user. The system can be configured to meet the needs of discovery labs doing exploratory research or screening drug candidates through the process-development lab where optimization and scale-up are required.”
Steiner finishes by highlighting that the very limited capability for state-of-the-art analytical characterization of proteins is also a drawback for medium-pressure chromatography; however, he is of the opinion it’s hard to compare the two chromatography types directly. “Again, I would like to emphasize that both instrument types were designed to be fit-for-purpose for different goals,” he says.
The bottom line is that HPLC is a boon for analysis (e.g., component identity and quantity), and medium- or low-pressure chromatography is best suited for purification and production. Given the wide range of both systems and reagents (e.g., columns and media) available from tool providers, it is always best to explore the different systems and to test the performance of each to see which is best suited to your research needs.
Reference
[1] Stephanie Runde, FPLC versus Analytical HPLC: Two Methods, One Origin, Many Differences.
The Column, LCGC, chromatographyonline.com, Volume 12, Issue 15, pg 12–16, 2016.
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