The Case for Upgrading Existing HPLC Assays to UPLC - Free Whitepaper

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For decades HPLC, high pressure liquid chromatography, has been the gold standard separations technique for biological compounds which cannot be analyzed in the gaseous phase. It is commonly used to assay drug compounds for impurities, derivatives, raw materials, and other components as part of the quality control process as well as during drug discovery and development. HPLC is commonly used in the pharmaceutical and biotechnology industries as well as other sectors that require high performance separation of compounds that cannot be analyzed using gas chromatography.

UPLC, ultra-pressure liquid chromatography, is the successor technology to HPLC techniques and assays that have been widely used since the 1970s. UPLC has emerged from the laboratory over the past few years and moved to the bench top. It offers similar analytic capabilities as the more familiar HPLC but operates at much higher pressures. This increased, or ultra, pressure provides the advantages of improved resolution, shorter run times, fewer consumables, and similar advances to separation science and those who depend on it to analyze and characterize vital compounds.

What is involved in converting from HPLC to UPLC?
Eric Hill: Conversion can be accomplished in as little as seven to nine hours of method development. UPLC hardware functions in the same way as HPLC hardware. Analytes are moved through separation columns under pressure, which allows the constituent components to separate out for deposition into the tetra one at a time for identification and characterization. But the hardware is not interchangeable. HPLC columns cannot be used for UPLC because of the significantly increased pressures used in UPLC. But instrument operation is nearly identical. All of the key method conditions, including temperature ranges and specific eluents such as methanol, water, acetonitrile, or other liquids, are identical. Because the key inputs and methods are the same in the two systems, conversion and UPLC method development are straightforward. And once the HPLC methodology has been converted for UPLC use, operation and data output methods are similar. The results, however, are dramatically different between the two systems. UPLC analysis results can be obtained at increased resolution compared to the HPLC results, in less time, and using lower eluent volumes.

What are the advantages of UPLC over HPLC?
Eric Hill: UPLC produces the same types of basic deliverables as HPLC, but with several distinct advantages over the older technology. One of the key advantages is resolution as defined by the peak shape. HPLC typically produces broad peaks that skilled operators can characterize very well, including peak heights and peak widths. In the hands of a skilled operator, HPLC can and does provide good data for quantitation and other uses.

Where HPLC produces good data, UPLC produces excellent data. The skilled operator who produces broad peaks using HPLC and moves to UPLC produces the kind of extremely sharp and extremely detailed peaks that are characteristic of gas chromatography technology. These sharp peaks allow researchers and operators to conduct more difficult separations using more complex mixtures that may have 10, 20, even 30 analytes. Traditional HPLC lacks the resolution to separate multiple peaks from similar components, which can make it difficult or impossible to adequately identify those peaks or to quantitate them. UPLC produces more peaks and sharper peaks to generate higher baseline resolution and more granular analysis.

Another important advantage is faster runtime. The typical HPLC run can take anywhere from 20 minutes to as long as 40 or 50 minutes. The higher pressure used with UPLC cuts that 20- to 50- minute runtime to between four and six minutes and almost certainly less than ten minutes. The increased resolution, the multiplicity of sharper peaks, come out in minutes versus tens of minutes. For many applications, particularly applications involving Good Manufacturing Practice or Good Laboratory Practice, the dramatic increase in throughput can be as important as the dramatic increase in resolution.

The significant reduction in solvent use is another important advantage for UPLC. Not only does the higher pressure system require less solvent, but shorter run times also require less solvent. The typical operation can expect to reduce solvent use by up to 80% after converting from HPLC to UPLC.

How do these advantages translate to the end user?
Eric Hill:
Moving from HPLC to UPLC means higher resolution coupled with increased throughput, decreased solvent use, decreased solvent disposal, and decreased cost. Take a typical HPLC run designed for batch use or method validation that takes 30 minutes and involves 40 to 50 injections. It will require about 15 hours to perform all of the injections and separations. Operators typically set the batch to run overnight and expect to read the results the next day.

Throughout that 15 hours, the typical analysis will pump approximately one liter of eluent through the HPLC unit. That involves two costs: First, the cost of the solvent; and second, the cost of appropriate and environmentally sound disposal of used solvent.

Using UPLC, that same 40 to 50 injections would take seven hours, possibly as little as five hours, depending on batch characteristics. During those five hours, the complete separation will require approximately 80 milliliters of solvent. That provides a double savings, first in the initial purchase of solvent and secondly in the ultimate disposal of used solvent.

A typical operation could easily conduct five 15-hour HPLC runs every week. Solvent alone would cost between $500 and $600, particularly if the operation requires acetonitrile. That same operation, using UPLC, could complete the same schedule of separations in half a week at a solvent cost of approximately $50. The advantage is increased resolution in half the run time and one-tenth the solvent cost. Users can expect UPLC disposal costs to be correspondingly lower based on the lower solvent volume to be disposed.

Are customers taking advantage of this technology?
Eric Hill:
It is moving into the market slowly as the established HPLC base learns of the new UPLC technology, compares resolution, throughput, and costs, and then creates their own pilot projects using their own baseline situation and data.

The improved resolution and the shorter runtimes have been well documented in the literature and in the marketplace. The connection to increased throughput and more granular data are clear. The dramatic difference in solvent use is equally well documented, but the cost advantage for UPLC is not always as well recognized. Historically, solvent cost has not been a key decision factor for many users. Consumables have been accepted as part of the inevitable cost of adopting a particular technology. But cost is increasingly entering into technology decisions. So are environmental considerations. As the world becomes more environmentally conscious, the prospect of disposing of 80 mL of used solvent as opposed to 1 L becomes increasingly attractive.

It is important to consider the advantages of UPLC, but it is equally important to consider the costs on an individual user basis. Many users have developed assays that were incorporated into good manufacturing practices when a drug product was originally developed, tested, and approved for marketing. Users who have a long history of HPLC data that has been collected and transmitted to the Food and Drug Administration are understandably hesitant to change methodology in ways that could affect GMP status of products that are on the market.

The reality is that FDA is more concerned that GMP compliance be established, be maintained, and be properly documented than in the specific analytic method used. Admittedly, there is some short-term cost to conversion from HPLC to UPLC. And the conversion itself must be documented as diligently as any other GMP process. But the medium-term and long-term benefits are enormous: Significantly increased resolution and more precise characterization combined with increased throughput, lower costs for solvent and solvent disposal, and a more environmentally friendly analytic process.

Are there any other technologies that offer similar advantages?
Eric Hill:
There are no competing technologies that offer a similar range of advantages to an equally broad range of users. Gas chromatography is a similar separation technology using the gas phase rather than the liquid phase. Some high-pressure gas chromatography units outperform UPLC, but gas separation has very limited and localized application in the pharmaceutical and biotechnology sectors. Once the appropriate conversion has been performed, UPLC can be applied to any HPLC assay. We are not aware of a single HPLC assay that cannot be improved significantly by conversion to UPLC.

Some of the newer inductively coupled plasma optical emission spectrometry units, ICP-OESs, are beginning to approach the detection limits of inductively coupled plasma mass spectrometry, ICPMS. The newest generation of ICP-OES offers users the advantage of near ICP-MS detection limits and quantitation limits at lower costs. But again, ICP has limited and localized applications in the pharmaceutical and biotechnology industries while UPLC is poised to become the next generation standard of separation science.