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May 1, 2014 (Vol. 34, No. 9)

GEN’s HPLC Roundup

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    Unlike conventional packed HPLC columns, Chromolith® HPLC Columns from EMD Millipore are not filled with minute silica particles. Instead, they consist of a single rod of high-purity monolithic silica with a bimodal pore structure—macropores reduce column back pressure, enabling faster flow rates; mesopores form a fine porous structure, providing a large active surface area.

    Any analytical method must strike a balance between its intrinsic scientific potential its capacity for robustness and reliability, particularly in regulated industries.

    In the case of HPLC, this balancing act is part of an initiative by the U.S. Pharmacopeia (USP), which is modernizing its pharmaceutical monographs.

    A monograph is “the book” on a drug product. Besides specifying the active ingredient’s chemical composition and the product’s ingredients, packaging, storage, and labeling, a monograph describes the analytical methods required to ensure strength, quality, and purity.

    “These tests need to be very selective, sensitive, and precise,” says Petra Lewitz, product manager for analytical chromatography at EMD Millipore. “In that context, HPLC is preeminent, but it is not the only method.”

    In many USP monographs, the specified analytical methods are completely out of date. The USP is therefore recruiting industrial collaborators to provide sensitive, robust, current methods that improve overall understanding about pharmaceuticals and enable labs to operate more efficiently.

    For example, many monographs rely on C18 columns, but as Lewitz notes many pharmaceuticals are polar or hydrophilic: “This stationary phase lacks selectivity and sensitivity for many pharmaceutical compounds unless ion-pairing agents are employed. But then, they become unsuitable for mass spectrometric analysis.”

    In this case switching to a more selective column, such as a hydrophilic interaction liquid chromatography (HILIC) column, or a slightly smaller particle size, might be sufficient for method modernization. “You don’t have to change the instrument,” Lewitz adds.

    Interestingly, Lewitz does not categorically endorse such modern alternatives as UHPLC or core shell columns. Column clogging, she observes, is too likely using these technologies, especially with complex matrices such as formulated drugs.

    For that reason, Lewitz favors monolithic columns, which provide the backpressure advantages of core shell (and HPLC) but will stand up to harsh, complex samples. “It’s important to find the best compromise between speed, resolution, and robustness.”

  • Solving Niche Problems

    Instrument vendors are constantly alert to their products’ potential for solving narrow scientific problems. For example, some vendors are using HPLC to detect aflatoxins in foods.

    Aflatoxin B1 occurs most commonly in corn and peanuts, but aflatoxin M1 occurs in products derived from animals that consume tainted feed. European regulations require an upper aflatoxin M1 concentration of 50 parts per trillion. Analyzing the toxin at this concentration requires matrix cleanup, post-column derivatization, and highly sensitive fluorescence detection.

    “Aflatoxin is difficult to detect at those levels, and milk is a difficult matrix,” says Jason Weisenseel, Ph.D., chromatography technical leader at PerkinElmer.

    Post-column derivatization involves running eluents and reagents through a loop and activating linkage with pulses of light. The fluorescence method involves mixing reagent with column effluent, which increases dead volumes and reduces sensitivity. Reagents and derivatization kits add to assay costs and complexity. Pre-column derivatization, by which reagent is added to the entire sample through the autosampler, could potentially eliminate dispersion. “But we don’t have that luxury with aflatoxins,” Dr. Weisenseel explains.

    Dr. Weisenseel and co-workers have developed a rapid, sensitive ultraviolet detection method that requires no derivatization. The keys are immunoaffinity solid-phase extraction to remove matrix, a photodiode array UV detector, a long fiberoptic flow cell, and fused core column technology. Fiber optics efficiently convey light to and from the flow cell while reducing noise. Together, instrument innovations reduce limits of quantitation to about 100 parts per trillion. Matrix removal gets detection limits down to the 10–20 parts per trillion range.

  • Mixed-Mode Analysis

    GEN has reported on the potential for mixed-mode chromatography to crack key downstream purification issues in large-scale protein purification, particularly for capture. Typical mixed-mode resins include cation or anion exchangers plus reverse-phase or hydrophobic interactions. In biomanufacturing, however, mixed-mode HPLC has remained experimental for the usual reason: risk avoidance.

    Fewer barriers exist for adopting mixed-mode analytical chromatography, a specialty of scientist Xiaodong Liu, Ph.D., manager of R&D, chromatography consumables at Thermo Fisher Scientific. A recent presentation by Dr. Liu notes the shortcomings of conventional HPLC for analytes that are diverse in terms of size, charge, hydrophobicity, etc.

    Reverse-phase HPLC shows poor retention for hydrophilic analytes, is incompatible with some aqueous mobile phases, and has a limited selectivity range. HILIC suffers from analyte solubility and matrix effects and is limited to hydrophilic analytes.

    Dr. Liu’s mixed-mode resins consist of hydrophilic or hydrophobic interaction combined with cationic or anionic exchange, plus a zwitterionic resin.

    “Mixed-mode columns provide adjustable selectivity, where separations are optimized on a single stationary phase by adjusting the chromatographic conditions without the use of ion-pairing reagents,” Dr. Liu observes. This allows retention of hydrophilic analytes that reverse-phase HPLC fails to separate, which addresses what Dr. Liu calls “a critical gap in reverse-phase capabilities.”

    Resolution in HPLC depends on selectivity, efficiency, and retention time, with selectivity having the greatest impact. Selectivity range in reverse-phase HPLC is limited, and relatively unaffected by pH and ionic strength. In mixed-mode HPLC, these variables allow fine-tuning selectivity for optimal results.

    For example, in a separation of penicillin G potassium on Thermo Fisher’s Acclaim Trinity P1 column, elution of drug and counterion were reversed simply by switching the acetonitrile:buffer ratio from 80:20 to 60:40, at constant pH. “The column is switching from HILIC mode to ion exchange,” Dr. Liu explains.

    Mixed-mode HPLC has been around for 30 years, but it has experienced a renaissance during the last decade. Yet adoption, as in bioprocessing, has been slow. “Part of the reason is that reverse-phase HPLC is so easy to use,” Dr. Liu says. Mixed-mode HPLC is perceived to be more difficult because there are at least two separation modes to deal with, and relatively minor mobile-phase alterations significantly affect selectivity.

    “We realize that reverse-phase HPLC is the norm, and recommend that people begin with that,” Dr. Liu admits. “But where reverse-phase doesn’t do the job, mixed-mode can be a good alternative.”

    On this basis, Dr. Liu is developing a technique for specific applications that remain problematic for conventional HPLC. Such applications include the analysis of glycans and surfactants and the detection of contaminants in drinking water.


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