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Product Focus: Sample Preparation for HPLC/UHPLC

Most HPLC analyses employ some form of sample preparation, especially with low-concentration analytes in complex mixtures.

Angelo DePalma, PhD

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at

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Turning Samples into Results

According to Anil Kurup, senior marketing manager for pharmaceuticals and bioanalysis at Waters (Milford, MA), the type and extent of sample prep depend on the compound(s) under investigation (e.g., small or large molecule), sample matrix, required detection limits, and the instrumentation and detector. The Separation Science website, www., which is devoted to chemical analysis, expands on some of these considerations.

“Identifying the right sample preparation technique is critical for analyzing nanogram levels of a small-molecule drug with LC/ MS from a few microliters of biological sample,” Kurup says.

Analysts have many techniques to choose from: dilution, filtration, solid phase extraction, micro chromatography columns, affinity adsorption, enzymatic digestion, and solid phase extraction (SPE), to name a few.

Is UHPLC different?

One of the selling points for UHPLC has been a reduction in run times compared with standard HPLC. Three- to fivefold improvements are easily attainable, and tenfold not unheard of. But improved efficiency has shifted the workflow bottleneck from the run itself to sample prep.

“Users looking to reduce workloads and analysis times, at lower cost, often begin with sample preparation,” says Trisa Robarge, sample preparation product manager at Agilent Technologies (Santa Clara, CA). “Clearly, users are looking to reduce the amount of sample prep they do.”

A study published by Pall Corporation ( clearly demonstrates the value of 0.2-micron filtration in UHPLC. It compares injection of polymeric microspheres under three conditions: unfiltered, 0.45-micron filtration, and 0.20-micron filtration. After just 10 and 15 injections, respectively, columns clogged under the first two conditions as back pressures doubled from 2,000 psi to more than 4,000 psi. But back pressures remained constant under the 0.2-micron filtration condition, even after 1,000 injections.

The study concluded that 0.2-micron filtration “prolongs the lifespan of the UHPLC column at least fiftyfold relative to filtration using 0.45 μm syringe filters and over a hundredfold compared to use of unfiltered samples. The cost savings generated by this simple filtration step is thus likely to be very significant.”

Not all experts would agree on those cost savings.

According to Kurup, labs operating under GLP (Good Laboratory Practices) will routinely filter all samples, regardless of whether they use HPLC or UHPLC, through a 0.2-micrometer filter. Under these circumstances, UHPLC does not present any additional sample preparation burden. Nor does UPC2, Waters’ trademarked supercritical fluid chromatography (SFC) platform, according to Kurup.

So in the end, “To prep or not to prep?” becomes an economics question for which labs must weigh the time and cost involved in preparation against the cost of a column.

Rapid methods

The simplest sample prep routine involves “dilute and shoot”— simply adding buffer to bring the sample to an appropriate concentration, and injecting. Dilute and shoot has the advantages of speed and being practically free. It works well with pure compounds dissolved in pure HPLC-grade solvents, but many chromatographers avoid it because of the potential column damage.

As Jason Weisenseel, PhD, chromatography technical leader for aftermarkets at PerkinElmer (Orlando, FL), notes, “UHPLC operators need to be careful what they put into their system. Sample prep takes on even greater significance than in conventional HPLC.”

Weisenseel suggests, at the very least, passing samples through a 0.2-micron filter, even when a more thorough cleanup technique such as SPE is employed. “SPE removes particles, but I like to be on the safe side. You can get away without filtering up to a point, but particles will eventually build up within your system.”

Along with solid-supported liquid-liquid extraction (SLE), SPE has become a workhorse for HPLC sample prep. “Conventional liquid-liquid extraction is timeconsuming and uses a lot of glassware,” says Robarge. “The alternative, SLE, operates through the same principle as conventional liquid-liquid extraction, and is appropriate for automation.”

Depending on how they are used, SLE and SPE can address both matrix removal and analyte concentration. In ion exchange mode, SPE often removes all interfering matrix and achieves concentration. While SLE is not normally thought of as a concentration technique, depending on the reconstitution volume, analytes may be significantly more concentrated than they were originally.

Another consideration, particularly for biological samples analyzed by LC/MS, is lipid removal. “Lipids and phospholipids cause ion suppression in MS and negatively affect method ruggedness accuracy,” Robarge says. Of the lipid-depleting protocols and kits currently marketed, Agilent’s CaptivaNDLipids claims to remove particulates as well, according to the company, potentially saving a filtration step while sparing columns.

Lipid depletion is increasingly applied in the analysis of foods and samples outside the life sciences, which has led to various kits for removing this class of interfering species. In late 2013, Waters introduced the new Ostro™ sample preparation plate for removing phospholipids from biological samples. The product was designed to complement SPE. According to the company, Ostro removes 30 times more phospholipids than conventional extraction, and deals with multiple phospholipid subtypes as well.

QuEChERS update

Several preparative methodologies developed for one type of sample and analyte have shown to be useful, with modification, for other, seemingly unrelated workflows. For example, in the decade since its debut, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method of rapid sample prep for pesticide analysis has become a generalized method for both GC and LC. The technique uses centrifugation or homogenization, accompanied or followed by reagent addition, to render analytes suitable for chromatographic analysis.

Agilent, Waters, Phenomenex, SigmaAldrich, PerkinElmer, Restek, and Thermo Fisher Scientific (among others) sell QuEChERS sample prep kits for analyzing “everything under the sun,” according to Robarge. The proliferation of QuEChERS and similar approaches to sample prep leaves end users with more choices than for, say, columns. As a result, chromatographers need to be more cognizant than ever of sample characteristics and purification capabilities.

For additional resources on Sample Preparation for HPLC/UHPLC, including useful articles and a list of manufacturers, visit