Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
High-pressure liquid chromatography (HPLC) is one of the most popular methods for separating biomolecules in complex, heterogeneous samples. The sample is applied to a column of tightly packed particles (also called packing material, resin or the stationary phase). The molecules in the sample travel through the column along with the solvent (also called the mobile phase). Because of competing interactions with both the mobile and stationary phases, different sample molecules exit the column at different times—a property that is specific to each analyte.
The key to the process, of course, is the column itself. But there are so many types, choosing a column can be bewildering. These guidelines should help you make the right choice.
Separation modes
Your first task in picking a column is selecting a separation chemistry. There are many ways to separate molecules, the most widely used of which include reverse-phase, normal-phase, hydrophilic-interaction, ion-exchange and size-exclusion HPLC. There are also less common and more specialized separation modes, such as specific chirality chromatography, which uses either a chiral stationary or mobile phase.
Reverse-phase columns, frequently used for non- or mildly polar organic compounds, contain a nonpolar stationary phase (such as C18) and typically are paired with a polar mobile phase, says Jason Weisenseel, chromatography technical leader at PerkinElmer. The method should not be used for proteins, Weisenseel cautions: “The mobile phase, often containing acetonitrile or methanol at low pH, could denature proteins and hence either give rise to artifactual peaks or lead to protein aggregation and column clogging.”
Normal-phase HPLC—in which the column packing is a polar material such as silica, and the mobile phase is nonpolar—is “the opposite of reverse-phase,” says Weisenseel. It usually is used to separate polar molecules.
Other popular modes include ion-exchange HPLC, used to separate ionic compounds, and size-exclusion chromatography (SEC), which separates molecules by their size or shape. Users might choose the latter to separate protein aggregates from protein monomers but opt for the former to separate a phosphorylated protein from its nonphosphorylated form, says Åke Danielsson, staff scientist in research and applied markets at GE Healthcare Life Sciences.
Column length
After you’ve decided on a column type, consider its length. The ideal length will depend to some extent on your particular samples and separation mode. But, a general rule of thumb (and trade-off) is that longer columns take longer to run but yield better separations. The key is to “choose a column length that achieves a good balance between resolution and analysis time,” says Tony Edge, scientific advisor at Thermo Fisher Scientific. A common mistake, Edge says, is using a type of column you’re familiar with from previous experiments and applying it to a new type of experiment without considering whether it’s the appropriate length.
Column length (sometimes referred to as “bed height”) is particularly important for separating proteins according to size, according to Danielsson. “A 300-mm bed height is a good choice for high-resolution SEC of proteins,” he says. “For the other separation modes that are relevant for proteins, much shorter columns can be used, such as around 50-mm bed height.”
Size and type of column particles
HPLC column beds are built of spherical, functional particles that are usually 2 to 10 μm in diameter. The most common sizes for standard HPLC systems, according to Weisenseel, are 3 and 5 μm. “For more complex samples, the resolution is improved by going to a 3-μm particle-size column,” he says. Particle diameters smaller than 2 μm are used in ultra HPLC (UHPLC), a specialized form of HPLC that uses smaller particles and higher pressures than HPLC.
As with everything in HPLC, column properties represent a trade-off. In general, smaller particle sizes give greater resolution—with the caveat that back pressures are also greater, to pump the mobile phase through the denser column. Not surprisingly, “columns packed with smaller beads are more prone to clogging,” says Danielsson.
In addition to bead size, users can often select the material from which they are made. Popular choices include silica, hydroxyapatite and cross-linked polymeric resins. Common hydrophobic alkyl chains come in such lengths as C4, C8 and C18. Usually the material has some degree of selectivity for the analytes of interest—and making this choice is important to optimize resolution. For instance, C18 is more often used for separating peptides or small molecules, while C4 is better suited for proteins. “Selectivity has a greater impact on resolution than bead size has,” says Danielsson. “Resolution can thus be dramatically enhanced by changing the selectivity,” for example, by using different pore sizes in the particles used for SEC or by using “a quaternary amine ligand instead of DEAE [diethylaminoethyl] in ion-exchange chromatography.”
The chemical nature of the particles also can make a difference in protein separations. Danielsson says that silanol groups on silica, a common column-packing material, can adsorb proteins nonspecifically. Surface-coated silica alleviates this somewhat, but damaged particles can still adsorb proteins. In addition, silica packing cannot tolerate the standard, high-pH cleaning protocols often used in protein separation, says Danielsson, who adds that “agarose-based stationary phases exhibit low, nonspecific protein adsorption and tolerate high-pH column-cleaning protocols.”
Technological tweaks are yielding new particle classes, as well. A new silica-based material called superficially porous particles (SPPs), or core-shell particles, promise resolution normally derived only from sub-2-μm particles but without the higher pressures. Usually, HPLC columns with conventional particles smaller than 2 μm would require specialized UHPLC pumps and/or systems to handle the higher back pressure. Unlike conventional silica particles, which are spherical and fully porous, “SPP particles are composed of a solid silica core coated with a porous silica shell,” says Weisenseel. “The advantage of these particles is they exhibit efficiency like sub-2-μm particles, in particle sizes from 2.6 μm and up, but operate at a little more than half the back pressure.”
Throughput
If throughput is an issue, pay attention to column design. “Using a shorter column will reduce the total analysis time but will also affect the resolution,” says Edge. For high-resolution SEC separations, 300-mm columns are recommended, says Danielsson, but one can increase overall throughput by using a 150-mm column for screening purposes.
Still, HPLC usually isn’t the bottleneck in separation studies, Weisenseel notes—it’s sample preparation. “You can scrimp on the sample prep to improve throughput, but dirty samples lead to much more HPLC maintenance and downtime, which also adversely affect your throughput,” he says. “Good sample prep is a key factor in obtaining consistent and reliable results.” Ultimately, matching your best-prepped sample with the most appropriate HPLC column will yield the highest quality results.