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Is HILIC in Your Future?Another option for separating polar components of complex mixtures» Angelo DePalma
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Figure 1: Melamine is not easily retained by reverse-phase LC. Most of the components in milk are hydrophobic. Melamine, therefore, may elute with matrix interferences and solvents when using reverse-phase chromatography. ESA Biosciences has developed and validated a simple method to determine melamine in milk using HILIC chromatography and the company's Corona charged aerosol detector (CAD). It separates melamine from matrix interferences and provides quantitative results as low as 1 µg/mL in milk. This figure illustrate the overlay of five replicate chromatograms of melamine standards (top) and spiked milk samples (bottom). | Hydrophilic interaction chromatography (HILIC) is rapidly catching on as a method for analyzing ionic or polar compounds, particularly biomolecules and drug metabolites that are poorly resolved by reverse-phase liquid chromatography.
The "book" on HILIC states that it is most suited to small polar molecules. Amos Heckendorf, Ph.D., President of the Nest Group (Southborough, MA), disagrees, arguing that "HILIC's sweet spot is molecules that differ by polarity."
HILIC columns consist of bare silica or a polar phase (amino, diol, cyano and others) bonded to silica. HILIC mobile phases are high in organic fraction—75% at least, and usually above 90%. The "strong" solvent in HILIC is either water, formic acid/formate buffer or acetic acid/acetate. Amino acids, pyrimidine bases and nucleotides/oligonucleotides, alkaloids, carbohydrates and other small polar or ionizable compounds are typical HILIC analytes.
In HILIC, water from the mobile phase forms a layer on the surface of the polar stationary phase. Part of the HILIC magic is liquid-liquid extraction that takes place between this layer and polar analytes.
HILIC was known since at least 1975, when it was used for separating small sugars and oligosaccharides. "It's been around for quite a while, we just didn't call it HILIC," observes John Waraska, president of ESA Biosciences (Chelmsford, MA). Early HILIC effects, observed on reverse-phase columns eluted at very high organic mobile phase, are believed to have arisen from unbonded silica jutting through the C18 bonded phase.
A seminal paper by Dr. Andrew Alpert, president of PolyLC (Columbia, MD), in 1990 coined the phrase "hydrophilic interaction liquid chromatography," established the HILIC acronym and defined it as a general-purpose method for separating polar and ionizable compounds.
As Waraska puts it, "add a little water, take a column surface that likes to hydrate, and a mobile phase the molecules don't care for, and you've got HILIC." But perhaps the most succinct summary of HILIC technology was provided by Maureen Joseph, Ph.D., LC Columns Product Manager at Agilent (Little Falls, DE): "HILIC is like normal-phase liquid chromatography but with reverse-phase solvents."Hedging your bets
Figure 2: Separation of "Group 4" analytes according to EPA method 1694, for analyzing common pharmaceuticals in water. Agilent developed this method on a Zorbax HILIC Plus column. Conditions: Column: Agilent ZORBAX HILIC Plus 2.1 × 100 mm, 3.5 µm (p/n 959793-901); Column Temperature: 25°C; Mobile Phase: 98% acetonitrile and 2% H2O with 10 mM ammonium acetate; pH: 6.7, Flow Rate: 0.25 mL/min. Click to enlarge | Reverse phase is the mode of choice in the life sciences. Chromatographers are comfortable with it because normal-phase separations are so demanding. But most metabolites and biomolecules are highly polar and are not retained by normal-phase chromatography.
Normal phase, that is to say bare silica or its close relatives, is not a natural LC format as it is in gas chromatography. Care must be taken that solvents are punctiliously dry. "Silica is a lot less forgiving in terms of mismatches in solvents and equilibration times," says Maureen Joseph of Agilent. Users must take care when switching from normal to reverse phase, since the apolar solvents used by the former are incompatible with polar aprotic solvent/water mobile phases of the latter. And HILIC demands tight control over pH because most analytes are charged, and separation depends on both absorption and ion exchange.
Agilent sells two HILIC columns. Zorbax HILIC Plus and Zorbax HILIC Sil use a bare silica stationary phase and come pre-equilibrated with acetonitrile-water. The company used HILIC Plus in its version of EPA method 1694 for drugs and drug metabolites in water; HILIC Sil is featured in a method for detecting melamine, a contaminant recently detected in some food imports. Agilent is also developing bonded-phase silica columns for HILIC.
Part of HILIC's attraction lies in its orthogonality to reverse-phase LC without the hassles of traditional normal-phase chromatography.
Other selling points include very high sensitivity as a front end to LC-MS detection, and its reduced sample preparation requirements. Because it uses no ion pairing agents and mostly organic solvents, HILIC reduces the need for sample drying or solid-phase or liquid-liquid extraction.
The Zic-HILIC columns from Merck Sequant (Umea, Sweden), touted as "truly orthogonal" to reverse-phase LC, sport a zwitterionic stationary phase bonded to silica that analyzes both charged species. Using this column, John Waraska of ESA Biosciences quantifies anions and cations simultaneously. "Because you have these other interactions going on, we've been able to analyze an active pharmaceutical ingredient at the same time as its counter-ion."
Figure 3: A comparison between the ordinary silica surface and the silica hydride surface. Click to enlarge
| Human pharmacology studies have, not surprisingly, been a major driver of HILIC's popularity, says Michael McGinley, LC Product Manager at Phenomenex (Torrance, CA). Reverse phase retains many intact pharmaceutical molecules isolated from body fluids, but a drug's metabolites tend to be quite polar, and hence "fly through" a C18 column. HILIC, while lacking the resolution of reverse phase, holds up these polar materials. Mr. McGinley says the eluent from his company's Luna HILIC column (diol bonded to silica) is suitable for direct injection into a mass detector following solid phase extraction or protein precipitation
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HILIC is known to address many problems that normal-phase or polar-embedded columns pose for mass detectors, specifically the need for ion pairing agents and a highly aqueous mobile phase, both of which ruin MS sensitivity. Because HILIC's mostly organic mobile phases are easier to volatilize than water-based buffers, the technique provides as much as one hundred times the sensitivity of reverse phase in electrospray ionization MS.
HILIC does not provide a similar direct advantage for non-mass detection modes, but analysts who do not rely on MS have begun to take notice. Many of them have adopted HILIC, which Dr. Joseph finds intriguing. "It's not often that mass spec drives the rest of the LC business, but in this case it does because HILIC and MS add another dimension."All mixed up
Figure 4: Examples of two bonded groups on a silica hydride surface. Click to enlarge | Over the last 15 years, HILIC has progressed into second- and third-generation embodiments, most of which involve mixed or multiple-interaction solid phases. Many column vendors sell both traditional HILIC and its more sophisticated relatives. For example Waters (Milford, MA) offers three HILIC column platforms: Atlantis underivatized silica in HPLC format, a 1.7-µm Bridged Ethyl Hybrid (BEH) particle that is UPLC-compatible, and a larger particle size for conventional HPLC HILIC. UPLC is Waters' proprietary sub-two micron particle technology. BEH provides silica's selectivity for polar molecules like amino acids, oligonucleotides, carbohydrates and proteins/peptides, but with higher chemical and pH resistance. "BEH offers better quantitation of polar metabolites from plasma, urine and other tissues through mass analysis," says Eric Grumbach, marketing manager.
Prof. Joseph Pesek, Ph.D., at the Chemistry Department at San Jose State University (San Jose, CA), has developed a HILIC-like stationary phase capable of retaining both polar and non-polar analytes. Pesek replaces about 95% of the active sililoxy (-S-OH) groups on silica with a silane-like phase in which the active group becomes –SiH. The idea for this unusual stationary phase arose from the need to analyze drug metabolites, which are generally polar alongside nonpolar compounds. This bonded material is marketed as the Cogent type C stationary-phase column, distributed by Microsolve Technology (Eatontown, NJ).
Silane-capped silica resembles HILIC most closely in its separation of small polar molecules, but Dr. Pesek prefers to call the method "aqueous normal-phase" chromatography.
Contrary to what chemists might guess from its chemical structure, –SiH groups retain polar or hydrophilic compounds under elution by high-organic mobile phases. Hydrophobic materials are also retained slightly, but that interaction requires a 90% or higher aqueous component."We don't quite understand the phenomenon" admits Dr. Pesek, who is investigating it with NMR and other techniques.
Figure 5: Separation of leucine and isoleucine on the Diamond Hydride column, detection by MS. Click to enlarge | It also is possible to bond hydrocarbons to approximately 30% of the free sililoxy groups before capping the remainder with silane, thus increasing retention of hydrophobic compounds as well as polar materials. Mixed-mode separation of both polar and non-polar molecules occurs at about 40% aqueous mobile phase composition. Amphiphilic compounds, which incorporate hydrophilic and hydrophobic regions, are retained through a combination of reverse- and normal-phase interactions. "You have two opportunities for optimizing a method for these compounds," Dr. Pesek notes.
HILIC pioneer Dr. Andrew Alpert has invented a related technique, electrostatic repulsion hydrophilic interaction chromatography (ERLIC). ERLIC makes possible, with the right mobile phase, the retention of molecules with charges similar to those on the stationary phase. Think of ERLIC as having an immobilized salt gradient on an ion-exchange column.
Rather than trying to minimize secondary or mixed-mode effects, ERLIC embraces and exploits them and does so isocratically, even for mixtures of compounds that differ significantly in polarity.
Where HILIC is performed on a neutral column, ERLIC separations use a stationary phase with the same charge as the most highly retained analyte. For example, a cation exchanger might be used to separate the nucleotides AMP, ADP and ATP, which contain one, two and three phosphate groups, respectively. Normally, eluting these three compounds within a reasonable time would require a steep gradient. Here, it can be done isocratically because the anionic stationary phase chemistry repels the negatively charged phosphate groups. "This effect throws the best-retained nucleotides back into the elution timeframe of compounds containing one or two phosphates," notes Dr. Alpert.
Figure 6: Separation of seven compounds all with the same MW in a urine sample on the Diamond Hydride column with MS detection. Click to enlarge | ERLIC has produced some startlingly efficient separations, but Alpert has no illusions about its rapid uptake among analytical scientists. While LC moves rapidly scientifically and technologically, the practice of chromatography is highly protocol- and method-driven. Industries like pharmaceuticals, in particular, are slow to adopt new technologies when old ones are already validated and accepted by regulators.
"ERLIC may be too complex for some people," notes Dr. Heckendorf of the Nest Group. "It forces you to look at the molecule and ask which characteristics you need to optimize to perform the separation." Raising the pH well above alkaline, for example, can cause repulsion between the phosphate group on the analyte and a sulfonate on the mobile phase, subsequently causing re-orientation of the molecule such that the side opposite the phosphate interacts with the stationary phase. By allowing chromatographers to exploit these secondary effects, ERLIC makes unique separations possible, "which is why ERLIC is the frontier, the next point beyond HILIC," Heckendorf adds.
Like PolyLC, SIELC Technologies (Prospect Heights, IL) embraces mixed-mode separations. "Most chromatography tries to eliminate secondary interactions; we enhance and control them," says company president Vlad Orlovsky. All SIELC's major products, which have a silica base, incorporate at least two chromatographic interactions that affect retention time, selectivity and/or separation. SIELC's commercial columns include reverse phase or HILIC, plus cation or anion exchange.
Mr. Orlovsky says mixed modes can streamline method development. "You don't need to know the molecule's precise chemical structure, only whether it's hydrophobic or hydrophilic, and whether it ionizes." By allowing chemistry to determine retention mechanisms, one can elute peaks in different orders by adjusting percent organic phase, buffer concentration and pH, usually under isocratic conditions and without the need for ion pairing agents. "Our phases have ion pairing agents attached to the surface."ConclusionDespite its many plusses, HILIC can be demanding of its practitioners. Columns, particularly bare silica, take time to re-equilibrate, one reason why early HILIC implementations suffered from poor reproducibility. Failure to control ionic strength also leads to inconsistent separations. HILIC is often run isocratically or over a narrow (compared with reverse-phase) gradient, but separations are quite sensitive to pH, as protonation state can markedly affect HILIC-type interactions.
HILIC and its many mixed-mode offspring have become topical in the world of LC, particularly among academic groups and companies that specialize in stationary-phase development. As mentioned earlier, HILIC has real-world significance as well, in that several companies interviewed for this article have published HILIC methods for detecting melamine. Yet HILIC remains a specialized LC separation mode. While it will never replace reverse phase for non-polar compounds, pure HILIC and its variants will continue to find niche applications, particularly for separating mixtures of polar and non-polar materials. Angelo DePalma is a chemist-turned-freelance writer based in Newton, NJ.
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