Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
Chromatography isn’t something people think of when talking about appendicitis. But in the near future, some physicians might. Turns out, a quick, simple and inexpensive test for the definitive diagnosis of that condition could someday become a reality, in part because of the separation afforded by column chromatography.
Using liquid chromatography to separate proteins from the urine of pediatric patients with abdominal pain, Boston researchers isolated leucine-rich α-2-glycoprotein. Patients who suffered from appendicitis had levels that were 100 times higher than those who didn’t [1].
The study is similar to the many proteomics-based approaches used in labs all over the world. They require separating and analyzing hundreds of samples to eventually pinpoint the exact proteins that are responsible in disease development or normal processes.
Central to these efforts is the chromatography column, with all its various forms. Keeping columns in tip-top condition will go a long way toward not only improving and standardizing your separations but also ensuring your columns’ longevity. Here are some things you can do to make that happen.
A little TLC
As with any investment, like a house or car, you can make your money go further with prepacked columns by applying some good old-fashioned TLC.
“If you take good care and clean regularly from the beginning, then [the column] lasts much longer,” says Paul Lynch, product manager for chromatography at Life Technologies. “Once it breaks down, then you’ll have a harder time recovering the performance.”
The type of solvents, acids and/or bases required to clean your columns depends on their design and the specific types of sorbents. Although some columns call for sodium hydroxide, the solution would damage the sorbents in other columns. Follow the instructions that come with your columns.
But it’s not just cleaning. Storage conditions matter, too.
“A lot of people don’t consider how they store their columns,” says Shawn Anderson, senior product manager of lab chromatography at Bio-Rad Laboratories.
Bacteria thrive in the neutral environment of buffers. Prevent bacterial growth by running water through the column. Then, place columns in bacteriostatic solutions, such as those containing 20% ethanol, and in the refrigerator at 4 oC to 8 oC, he says.
Anderson also recommends noting the back pressure of the column the first time you use it. If the back pressure goes up over time, you’re not cleaning it enough. Or, you can use one of Bio-Rad’s NGC chromatography systems, which automatically records the initial back pressure, he says, and prevents overpressuring the column.
Filters first
You can keep columns in pristine condition longer, if you keep foreign particles and contaminants at bay. That means using one or more of the various filters or membranes that minimize the amount of salts, fibers and other substances that don’t belong.
“Any particulates you can move out with certain filtration steps works very nicely in terms of prolonging the life of analytical columns,” says Dafydd Milton, product manager of liquid chromatography at Thermo Fisher Scientific.
Milton recommends running samples through cruder columns, such as trapping columns, prior to injecting them on to the more expensive analytical column itself. Doing so not only keeps the analytical column cleaner but also concentrates the sample, which means you can use more of it, yielding better sensitivity.
Companies offer various filtration solutions. Thermo Fisher Scientific, for example, offers a selection of trapping columns that retain peptides and proteins of different sizes. The company’s Titanium-Dioxide Nano-Trap Column concentrates phosphorylated peptides in your sample by retaining everything else and allowing only phosphopeptides to pass through in the eluate.
Phenomenex offers SecurityGuard guard cartridges for HPLC, UHPLC and preparative columns. Acting as filters or trapping columns, these cartridges are filled with sorbents that retain particulates and other contaminants.
Michael McGinley, bioseparations product manager at Phenomenex, also recommends staying within the suggested pH and temperature ranges of different columns.
“When you go to the edge of the range, then you could damage the silica resin,” McGinley says. “It’s just a matter of monitoring … the requirements.”
You also should consider filtering the buffers before you add them to the column during separation.
Bore necessities
For all the convenience of prepacked columns, many researchers refuse to pay the cost and insist on packing their own.
“The major problem of the prepacked columns is that they’re terribly expensive,” says Hanno Steen, associate professor of pathology at Harvard Medical School, who was a major contributor to the appendicitis studies mentioned above. “They cost $500 to $1,000, whereas the price for in-house columns is much, much less.”
Steen estimates that buying prepacked columns costs roughly 10 times more than packing his own. Larger and more specialized analytical columns can bear price tags of $1,000 to $2,000, and larger columns, such as those used commercially for purifying pharmaceuticals, go up from there.
In Steen’s lab, most column chromatography is performed with homemade columns, and prepacked column usage is closely monitored. The use of each column is logged with the user’s name. Lab members who use prepacked columns significantly more frequently than others could be flagged and examined for their treatment of the pricey equipment.
The cost isn’t really extravagant when you consider the high price of materials, the specialized machinery and the human power required to produce prepacked columns, McGinley says.
“Everyone thinks packing is just filling powder into a tube,” he says. “Actually, packing media to get high performance is a very complicated science that requires a lot of method development and design development.”
Packin’ ‘em in
Still, Steen tends to favor lab-packed columns—and not only for economic reasons.
“One of the problems with commercially packed columns is they have less flexibility. You’re stuck with what they have,” he says. Lab-packed columns, in contrast, can be customized. “That’s the beauty of in-house columns.”
Steen likes having the freedom to pack columns with personally selected materials—either novel or traditional—rather than being restricted to vendors’ catalog listings.
On the other hand, Steen does recognize the reproducibility that prepacked columns offer. Therefore, he tends to pack his own columns when conducting mass-spectrometry studies that yield qualitative results and to use prepacked columns for quantitative work.
Life Technologies’ Lynch agrees that the budget takes a hit when you first pay for prepacked columns. But, he argues, “consider all the time savings” that accrue because “you require fewer steps to get your research done with [prepacked columns]. That’s always lost on people.”
Whether your research budget is big or small, your protein separations will have the highest chance of succeeding when you choose columns—either prepacked or homemade—with the specifications that are most appropriate for your protein sample and your goals. Addressing all the details along the way, including the care and cleaning of your columns, will support your mission to make important discoveries.
Reference
[1] Kentsis, A, et al., “Detection and diagnostic value of urine leucine-rich α-2-glycoprotein in children with suspected acute appendicitis,” Ann Emerg Med, 60(1):78-83.e1, 2012. [PubMed]