A surprising new application for a traditional HPLC column has created
a new market
segment for the separations industry.
click to enlarge
The production process for biofuels is complex, and necessitates different steps to
convert varieties of starches. Source: Phenomenex |
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The biofuels industry has weathered two storms. First, the industry was criticized for using food
crops to meet the transportation requirements of a SUV-hungry nation. Then, in 2008, the price of
oil spiked and then plummeted, sending some high-profile ethanol producers out of business. But in
those few years of heady growth, developments took place that could help ethanol recover its
former stature. These include a better understanding of biofuel chemistries and a better use of
analytical instrumentation.
"About 4 or 5 years ago I was started noticing that a lot of customers were ordering one of our
HPLC columns. There were all sorts of companies from the midwest," says Michael McGinley,
bioseparations product manager, Phenomenex, Torrance, Calif. Specifically, they
were buying the company's Rezex, an ion-exclusion column for high-performance liquid
chromatography (HPLC) that is typically used for analyzing sugars.
The company names were unfamiliar and included ICM, Colwich, S.D., and
POET, Sioux Falls, S.D., the world's largest producer of ethanol. Jim Mott, a
bioethanol expert at Shimadzu, Kansas City, Mo., had been working with these
companies on a rapid HPLC method to help them monitor productivity. McGinley met with a few people
at ICM and a few others at Poet. These discussions led to technical improvements by McGinley and
Mott, as well as a new biofuels market focus at Phenomenex.
"Ion exclusion is a very old separations technology, dating back to the 1970s and 1980s. It's
been mostly used for looking at carbohydrates and organic acids. Some people used it a long time
ago for fermentation monitoring,” says McGinley.
Monitoring production
The production of ethanol is not much different than beer or wine making. First, enzymes are
digested to release sugars from stored starches. The current process for generating ethanol from
biological sources relies on the use of amylase enzymes to break down complex starches into simple
sugars, which are fermented with yeast. After the alcohols are recovered, the product is distilled
and dried. Ultimately, the process is much the same for any feedstock, says McGinley "What
[producers] do is add enzymes and heat. The carbohydrates get broken down to simple sugars, and at
a certain point in the process they throw in some yeast. The yeast generates ethanol," says
McGinley.
Regular monitoring of the process by HPLC allows operators to track the breakdown of starches
to simple sugars, as well as monitor ethanol. Producers like to use the ion-exclusion
chromatography method because it monitors carbohydrate size and ethanol. It also keeps tabs on
organic acids. Ion exclusion uses several different separation modes (gel filtration, ion
exchange, and reversed phase) to separate compounds of interest.
The carbohydrates get broken down to glucose and maltose, and when the yeast is added,
producers can monitor the fermentation process throughout. They also look for lactic acid, which
is also detected by these columns. This acid is a marker for bacterial contamination, and, if
enough of it gets into the fermentation vat, the conversion is diverted to lactic acid, which cuts
into ethanol recovery.
"Everything these guys do is about maximizing their ethanol recovery," he says. "They can
actually throw in antibiotics and knock down the bacteria and get a higher ethanol yield."
The fermentations typically take between 30 and 60 hours, and producers usually run a column
every two hours. There are often up to eight large fermenters all active at once, which means the
HPLC runs can add up. The current ion exclusion HPLC method is a multimodal separation, so there
are significant limitations on increasing throughput of the method.
However, says McGinley, some minor changes can be implemented that can reduce the analysis time
by up to 50%. He and Mott published an application note after conducting numerous tests using
Phenomenex columns and a Shimadzu LC-20AT instrument. They found that simple steps such as using
guard columns and filtering samples can greatly increase the reliability of the method as well as
improve column lifetime. This, in turn, reduces analysis cost. For larger operations, they wrote,
shorter columns can be used in some circumstances to reduce analysis time for monitoring,
potentially reducing the need for additional analytical equipment.
