What it Takes to Run HPLC/UHPLC
Although by no means the only operational issue involved in HPLC, cost of ownership is something everyone considers and ultimately comes to grips with.
Although by no means the only operational issue involved in HPLC, cost of ownership is something everyone considers and ultimately comes to grips with. Cost of ownership for an HPLC system is complicated by several factors, for example operational robustness, minimal repairs and downtime, and rapid diagnostics for maintenance and repair.
It makes sense that cost of ownership increases with instrument complexity and usage. To manage operating costs, vendors vie to deliver systems that require less frequent maintenance and are easier to service. “If we look at throughput and quality of data, the cost of ownership has dropped,” says Shimadzu’s Simon Robinson. The key is ensuring that whether a set of runs is conducted in 20 minutes or 50 hours, cost per sample is roughly equivalent.
HPLC operators, regardless of technical level, require some degree of training in instrument use
and maintenance, and in keeping systems and columns in top condition. For example, sample prep
and mobile phase quality became serious issues for 5μ particle systems, and they are absolutely
critical below 2μ. Vendors do their part to ensure that users are properly trained, but this becomes
increasingly difficult after the unit ships.
Users of both UHPLC and HPLC can perform routine checks on valves, seals, pistons, and general
wear, with the higher-pressure instruments being somewhat more difficult and prone to problems.
Manufacturers have designed instruments for accessibility to critical components, but most large
companies, particularly in regulated industries, service their instruments through their vendor or a
third-party maintenance organization.
Because higher pressures stress instrument components, UHPLC systems operating above about
600 bar (about 9000 psi) require more frequent maintenance, usually at higher cost, than do standard HPLC systems. Additionally, the smaller column particles and narrower internal diameter
lines require that users filter all samples and mobile phase components to prevent clogging. “In my
experience, most users often skip these extra steps, resulting in more service calls,” notes Chiralizer Services’ Bill Letter. Additionally, the sub-2μ particles used in UHPLC columns contain more fines that can clog column frits and system lines.
Despite higher operating costs for UHPLC vs. HPLC, the former may return its investment rather quickly through improved throughput and reduced solvent usage, depending on workflows. “But users must run genuine UHPLC methods to attain these benefits,” cautions Elizabeth Hodgdon of Waters.
Core-Shell HPLC/UHPLC Columns | Kinetex®
Phenomenex | www.phenomenex.com
Planned maintenance is particularly important for longterm UHPLC performance. The quality and cleanliness of parts, design, manufacturing, and packaging all play into maximizing performance in instruments operating at up to 1000 bar (15,000 psi). At these pressures, fluid path components can fail or malfunction if part tolerances are not rigorously controlled.
Due to UHPLC’s higher sensitivity, service must follow exacting protocols that limit the introduction of contaminants such as chemicals, oils, or particulates. “What may be an acceptable level of particulate matter in HPLC is not acceptable for UHPLC,” Ms. Hodgdon explains.
COLUMNS: KEY VALUE DRIVERS
Column technology has been a rich area for HPLC R&D. As reasonably expensive consumables, columns are a significant factor in LC operating costs and performance—in other words, key value drivers.
Superficially porous particles (SPPs) represent a true breakthrough in column technology. SPPs go by different names depending on the vendor. The Kinetex® line of what Phenomenex (Torrance, CA) calls “core shell” particle columns competes with Agilent’s Poroshell, Advanced Materials Technology’s (Wilmington, DE) Fused-Core™ products, Thermo Scientific’s Accucore brand, and PerkinElmer’s Brownlee columns. Waters discovered the SPP phenomenon during the 1960s but abandoned the idea “in favor of fully porous particles and the benefits they bring,” according to a Waters web page.
SPPs consist of a solid silica core surrounded by a porous shell. Conventional particles are porous throughout. The basis for SPPs’ enhanced performance is more rapid mass transfer through the particle bed, which occurs at the expense of binding capacity. Most SPP sizes are in the 2.6μ range. Phenomenex also manufactures a sub-2μ SPP-based column that Philip J. Koerner, Ph.D., Senior Technical Manager, says provides the best of both technologies. “Superficially porous particles
provide the efficiency of sub-2μ without the immediate need to purchase a new UHPLC system.” SPPs enhance the capabilities of HPLC, while stressing the instrument less due to lower than-UHPLC pressure operation.
As Waters points out, SPPs will not provide significant performance enhancements unless one addresses the system contributions to band broadening. Dr. Koerner agrees, but notes that “this is done relatively easily.”
HPLC Columns | AccucoreTM
Thermo Fisher Scientific | www.thermo.com
Manufacturers provide kits for minimizing band broadening, or dispersion, through modifications to connecting tubing or UV detector cells.
UHPLC columns, with their minimized volumes and high theoretical plate numbers, are demanding with respect to system effects on peak broadening. “Your separation can be destroyed between the column and detector, or within the detector itself,” notes Frank Steiner of Thermo Fisher. “This requires adapting detector flow cell volume to a minimum of one-tenth the expected peak volume. Otherwise you cannot exploit the column’s theoretical resolution.” Other trouble spots include tubing, pre-heaters, column thermostat, and autosampler.
Almost any standard HPLC system with a pressure maximum of less than 600 bar (8500 to 10,000 psi) can utilize columns packed with 2.1μ-diameter to 5μ-diameter particles. These provide rapid resolution analysis, often with no further system modification, according to Bill Letter. “Columns containing 2.1μ to sub-5μ particles in smaller formats can often provide many of the same benefits of UHPLC systems, at lower pressures, and still reduce throughput times and increase solvent savings.”
These columns, he says, are “more reliable and rugged” than sub-2μ columns since the latter are more difficult to pack uniformly and reproducibly. Furthermore, standard HPLC systems provide adequate means to adjust the internal delay volume sufficiently to accommodate narrow columns with smaller internal void volumes. “These savings have been available for decades to anyone who wishes to utilize them.”
Despite the value of SPP columns, and the fact that even UHPLC manufacturers are eager to sell them, their eventual effect on the course of HPLC/UHPLC is subject to debate. Tom Jupille, President of LC Resources (Walnut Creek, CA), who is not unfriendly to older HPLC technology and likes SPPs, says the extent to which they extend the life of conventional HPLC systems will not be dramatic. “I’m not certain they will be a game changer.”
PerkinElmer’s April DeAtley disagrees. “As column technologies catch up, I believe more people will choose lower-pressure systems simply due to their convenience and lower maintenance. Most users will probably own a UHPLC pump, and they will prefer to obtain results using column technology in conjunction with their instrument, while avoiding issues encountered at ultra-high pressures.”
BEYOND THE COMMON COLUMN
Specialty columns are an oft-overlooked avenue to higher performance. How many readers have heard of Jordi Labs, Sepax Technologies, Nacalai Tesque, Dikma, Chromenta, or Sepax Technologies? In addition to manufacturing conventional columns (often at significant discounts compared with better-known manufacturers), these small column companies specialize in stationary phases that can only be described as exotic. For example Jordi's (Bellingham, MA) stationary phases are based on polymer particles, not silica, and are claimed to be stable from pH 0 to 14 and 100 percent aqueous to 100 percent organic mobile phases. Nacalai Tesque’s (Tokyo, Japan) columns include cholesteryl chemistry, pyrenylethyl (which separates based on pi-pi electron interactions), and pyrenylpropyl (for fullerenes).
“Not too many people know these chemistries even exist,” says Columnex President Ken Tseng, Ph.D. Columnex sells columns from 13 boutique manufacturers.
Also in the niche category are mixed-mode columns that combine ion exchange with reverse phase or HILIC. Large manufacturers like Agilent, Dionex, and GE Healthcare offer mixed-mode columns, as do several firms in the Columnex stable, particularly Sielc (Prospect Heights, IL) and Intakt (Tokyo, Japan).
Dr. Tseng explains that mixed-mode HPLC has always been around but not recognized as such. “People tried to isolate one mode or the other.” For example, depending on the pH, an amino column works through either the – NH2 functional group (cation exchanger) or the –NH3+ (anion exchanger). “Now that we have better control over stationary phases, we can put those modes back in and make them work for us.”
Evaluating alternatives to the hard upgrade from HPLC to UHPLC has become an interesting and instructive exercise, but not all options are considered equal, and not every solution is for everyone.