by Laura Lane
Chromatography has certainly had a long run—in a good way. Not in the sense of the long lasting chromatographic runs of yesteryear, when aggravatingly slow separations were the only option. It is the protocol’s staying power that is worth attention. For nearly 80 years, life scientists have depended upon the technique to pull apart and isolate both organic and inorganic molecules. Now just as relevant, the separation prowess of ion exchange chromatography instruments and materials has never been better suited to serving the needs of today’s researchers.
The technique grew—in 1938—out of a need to separate potassium and lithium ions.
Soon after, nuclear scientists of the Manhattan Project harnessed ion exchange chromatography to aid in efforts to purify radioactive elements. Stanford Moore and William H. Stein of Rockefeller University in New York continued to advance chromatography to separate amino acids. Their efforts gave a substantial boost to the technique and earned the two scientists the 1972 Nobel Prize in chemistry.
While various industries continue to employ ion exchange chromatography, it has found a solid home in the life and biomedical sciences. Separating proteins, carbohydrates, nucleic acids, peptides, amino acids, and just about any other charged molecule, the latest columns and consumables clearly bear the signs of today’s need for total specificity, absolute reproducibility, and complete reliability. All the while, the technique’s basic premise has remained the same: harness the laws of attraction to segregate target molecules. The straightforward nature of this approach is perhaps what has given chromatography so many fans and followers.
“Ion exchange chromatography is beautiful because it’s simple, predictable, and cost-effective,” says Gunter Jagschies, senior director of strategic customer relations for GE Healthcare. “It’s the best way to go.”
Samples of all kinds—from cell lysates to blood samples—begin their purifying journey at the top of a column. Much as with a filter, the contents of the column—such as resin beads, gel, or membranes—divide the multiple components of the sample. Usually, researchers design the column to grab on to target molecules, while allowing everything else to flow through. For example, to isolate proteins or any other molecules that bear a net negative charge, the column requires a positively charged matrix that will latch on to proteins. Collecting the proteins requires the addition of a solution that alters the pH or otherwise weakens the electrostatic bonds between the matrix and target molecules.
A focus on the bottom line has, in part, increased the popularity of the reverse: capture the impurities while allowing the target molecule to flow through. Proven to yield more product per column, this flow-through strategy fits in with companies that are trying to keep pace with productivity of cell culture, which has over the years increased by 1,000-fold, says Jagschies. “The downstream has to catch up with that,” he says. “That’s where the new developments of ion exchangers are coming from.”
So far, companies seem to be offering some very clever solutions for meeting efficiency goals and reducing production costs. Bio-Rad Laboratories has introduced its UNOsphere Polymer Technology based on continuous-bed technology.
“Take the monolith chemistry and turn it into sphere,” says Larry Cummings, a consulting scientist at Bio-Rad, referring to an alternative chromatographic material that is made in a one-shot process that insures product reproducibility. “It’s like a conventional spherical particle, but retains continuous bed characteristics that can provide high capacity at high flow rates and has excellent capture kinetics.”
GE Healthcare’s answer to efficiency involves reducing the number of separation steps required to purify target products. With ion exchange chromatography, the purification process usually requires several separations that successively remove different types of impurities and contaminants. “If you want to do that, then the steps need to be more powerful in removing impurities with better selectivity,” Jagschies says, pointing to the method’s limited selectivity.
Affinity chromatography offers optimal selectivity by attaching ligands to the matrix designed to capture specific molecules. But, for the purposes of manufacturing quantity, some want to avoid the higher expense of the affinity approach. GE Healthcare’s multimodal strategy tries to integrate a higher level of selectivity while retaining the efficiency of ion exchangers, Jagschies says. “That’s the future challenge: the balance of cost and performance,” he adds.
Shielding any possibility of hydrophobic interactions is the key to selectivity for Dionex. The company starts with polymer beads of polydivinylbenzene and covers them with a hydrophilic film that they call a “hyper-branched ion exchange polymer,” says Chris Pohl, Dionex’s chief scientific and technology officer. “It’s a tree-like structure growing off the surface” of the bead.
Growing the hydrophilic polymer, which crosslinks within itself, the film achieves a density that shields any hydrophobic influences of the core. The company currently uses the technology for separating small ions and metabolites. Making modifications to separate macromolecules, such as proteins and carbohydrates, the company will also offer applications in the form of both bead-based and polymer monolith-based materials.
“We believe that it should have advantages over the current technology,” Pohl says, explaining that the hydrophilic film overcomes the challenge of molecules being retained unintentionally. “The more hydrophilic you can make the material, then the better the performance in terms of transport,” which results in improved chromatographic performance.
However, efficiency is less of a priority for basic science and/or drug discovery where the primary task is to characterize and analyze target molecules. For these researchers, low capacity ion exchange columns, such as Thermo Fisher Scientific’s BioBasic columns, prove more suited to isolate sufficient quantities of molecules while also preserving their characteristic integrities.
“Very often, the larger the ion exchange capacity, the greater the amount of salt that will be needed to elute ionic species,” says Dafydd Milton, product manager for liquid chromatography columns at Thermo Fisher. “That can be harmful for sensitive mass spectrometry applications.”
Sensitivity is the key for the Biochrom 30, which clinical labs use to separate and identify amino acids in blood, urine, and other samples of bodily fluids. With a 30-year history, the instrument has proven reliable for achieving fine separations for medical diagnoses. The original model required a full day to complete separation and detection, says Wendy Rasmussen, product manager at Hoefer Inc., which distributes the instrument. The latest model, which will be released around March 2008, can complete the process in 90 minutes.
Continued modification of the column’s packing material, the size of the column, the pressure applied for flow and other parameters may result in shorter times. However, Rasmussen points out that many researchers have pulled back the reins, in favor of higher resolution rather than slightly faster run times. “Faster separations can sometimes compromise the quality of separation,” she says. “So they go back to slower rates for better separation.”
One of the biggest advantages of the Biochrom 30 is its ability to manage the complex mixtures of unprocessed bodily fluids, Rasmussen says. And, succeeding runs can process samples of different forms without any adjustments. “You can just load and go,” she says.
