HPLC

TN-1043

Bioethanol Production Monitoring using Ion Exclusion

HPLC with Rezex ROA Column
Michael McGinley* and Jim Mott**
*Phenomenex, Inc., Torrance, CA, USA
**Shimadzu Scientific Instruments Inc., Midwest Regional Office, Lenexa, KS, USA

Introduction
The dramatic increase in the price of fossil fuels as well as growing
concerns over greenhouse gas emissions has led to an enormous
interest in ethanol production from biological sources.1 While
there is considerable interest in generating ethanol from cellulose
based sources, the majority of current bioethanol manufacturing
is focused on generating ethanol from starch based foodstuff
such as corn or wheat. In the United States alone, there are 141
plants in operation as of 2007 with an additional 72 planned in
the next two years.2
The current process for generating ethanol from biological
sources relies on the use of amylase enzymes to break down
complex starches in foodstuff to simple sugars, followed by yeast
fermentation to convert the sugar to ethanol. Regular monitoring
of the process by HPLC allows operators to track breakdown of
starches to simple sugars as well as monitor ethanol production
after yeast has been introduced. In addition, organic acids are
also monitored in the HPLC run, allowing operators to assess
if microbial contamination is affecting the fermentation process
and, if additional remediation steps such as antibiotic addition
are necessary to maximize ethanol yield.3
The HPLC method used for bioethanol monitoring, ion exclusion
chromatography, is a method that uses several different separation
modes (gel filtration, ion exchange, and reversed phase) to
separate compounds of interest. This separation method has
been around for over 20 years, yet still remains the most popular
method for fermentation monitoring due to the ability to separate
different classes of compounds (sugars, organic acids, and
alcohols) all in one chromatographic separation.4 The method is
a simple and rugged isocratic method using a dilute acid mobile
phase, however some sample preparation of the fermentation
broth is required to ensure good chromatographic performance
and reasonable column lifetime.
In addition to the growing number of ethanol plants, many
plants are also expanding their operations by adding additional
fementers. Such additions require increasing throughput of the
current analytical methods for fermentation monitoring as groups

wish to monitor more fermentation runs with existing HPLC

equipment. Because the current ion exclusion HPLC method
is a multimodal separation, there are significant limitations on
increasing throughput of the method. However, some minor
changes can be implemented that can reduce the analysis time
by up to 50 % (from 24 minute to 12 minute run time). In this
technical note the basic principles of the ion exclusion HPLC
analysis will be discussed. Sample preparation methods to
ensure proper sample cleanup and minimize instrument down
time will be reviewed.
Materials and Methods
Analyses were performed using a Shimadzu LC-20AT LC system
(Shimadzu Scientific Instruments, Columbia, MD, USA) equipped
with a SIL-10AF autosampler, degasser, and a RID-10A RI
detector; data was collected using CLASS-VP Version 7 Software.
Various dimensions of Rezex ROA columns (Phenomenex Inc.,
Torrance, CA) were used (150 x 7.8 mm, 300 x 7.8 mm). Guard
columns were SecurityGuard Carbo-H+ 4 x 3.0 mm cartridges
(Phenomenex, Torrance, CA, USA). Aqueous mobile phase (0.005
N Sulfuric Acid in water) was purchased from CHATA (Ft. Collins
CO, USA). Several samples from various fermentation timepoints
were generously provided from ICM (Colwich, KS, USA). Ethanol
HPLC testing standard was obtained from Midland Scientific
(Omaha, NE, USA).
Crude samples from fermentation timepoints were filtered using
a 0.20 m Phenex-RC syringe tip filter (Phenomenex, Torrance,
CA). Filtered aliquots of 10 L were injected on HPLC operating
at a flow rate of 0.6 mL/min and the HPLC column was heated to
65 C. Run time was 24 minutes for the 300 mm length column
and 14 minutes for the 150 mm length column. SecurityGuard
cartridges were regularly changed every 100 runs or whenever
static backpressure increased 10 % above initial backpressure
values. 50 % methanol was used in the autosampler needle
wash to avoid bacterial contamination.

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HPLC

TN-1043

Figure 1: Bioethanol Standard

1

8
3

While the standard in figure 1 may show the idealized separation

of compounds, a more typical example of different timepoints
run on the 300 x 7.8 mm column is shown in figure 2. Early in the
fermentation, the oligosaccharide and saccharide peaks can be
so abundant that resolution between components is reduced;
later in the run, as sugars are converted into ethanol, resolution
of the saccharides is more in line with what is seen in the HPLC
standard. Conversely, early in the process, ethanol, glycerol, and
organic acid peaks often are below detection limits and increase
as fermentation progresses.

App ID 16650

2
6
5

10

20

(min)

Figure 1: A bioethanol fermentation standard was run on a 300 x 7.8 mm

Rezex ROA column. Note the excellent separation of all the components of
interest. 1. Dp4+, 2. Dp3, 3. Maltose, 4. Glucose, 5. Lactic Acid, 6. Glycerol, 7.
Acetic Acid, 8. Ethanol

Results and Discussion

HPLC run of a fermentation standard using the Rezex ROA 300
x 7.8 mm column is shown in figure 1. Note that the early eluting
peaks (Dp4+, Dp3, Maltose, Glucose) represent the different
degrees of polymerization of the various saccharides present in
the sample. Monitoring of these peaks during early time points
of the fermentation run gives operators a good indication as to
the progression of the various amylases used to break down
starches to simple sugars, and dictate when yeast is added
to the fermentor to start generating ethanol. The later eluting
peaks (Lactic acid, Glycerol, Acetic acid, Ethanol) represent the
organic acids and alcohols generated during the fermentation.
Monitoring of these peaks gives an operator an indication as
to the fermentation endpoint as well as indicate when bacterial
contamination is severe enough to warrant addition of an
antibiotic to limit bacterial by-products that may inhibit ethanol
production.

Figure 2A: 18HR Fermentation Sample

Resolution of key components will also tend to decrease over

time as sample contaminants build up on the column; key to
maintaining resolution and increasing column lifetime is using a
guard column system like the SecurityGuard guard cartridge
system. Regular replacement of the guard cartridge is indicated
when a significant decrease in resolution or increase in system
backpressure is noted. Filtration of sample timepoints with
a syringe tip filter can increase the lifetime of both the guard
and analytical column, as well as reduce chromatographic
interferences due to particulates.
As some groups have increased the scale of their ethanol
production and may now be operating several fermentors in
parallel, there is an interest in reducing the analysis time for
fermentation monitoring. Since the ion exclusion HPLC run is
isocratic, few parameters can be employed to reduce run time.
Mobile phase and temperature modifications have little effect
on the critical oligosaccharide peak separations early in the
chromatogram. Flow rate can be increased slightly to reduce run
time, however one must factor in the maximum backpressure of
the Rezex HPLC columns (600 psi) as well as reduced lifetime
when columns are run close to their maximum backpressure.

Figure 2B. 39HR Fermentation Sample

1
8

10

20

min

3
2

App ID 16652

App ID 16651

10

20

min

Figure 2: Different fermentation timepoints run on a Rezex 300 x 7.8 mm ROA column. Note the depletion in the early oligosaccharide peaks as fermentation
continues. The ethanol peak increases throughout the fermentation as sugar is converted into ethanol.

HPLC

Figure 3: Bioethanol Standard

Figure 4: 50HR Fermentation Timepoint

8

App ID 16648

6
7

10

12

14 min

Figure 3: The bioethanol fermentation standard was run on a 150 x 7.8 mm

Rezex ROA column. Note the limited resolution of the early eluting oligosaccharide peaks in the standard. In early fermentation monitoring such peaks
may not be resolved. 1. Dp4+, 2. Dp3, 3. Maltose, 4. Glucose, 5. Lactic Acid,
6. Glycerol, 7. Acetic Acid, 8. Ethanol

One method for reducing HPLC run time is by reducing the

length of the Rezex column used. An example is shown in Figure
3 where the fermentation standard is run on a 150 x 7.8 mm (half
the length of the typical 300 mm column used). As expected, the
run time using a shorter column is significantly reduced from 24
to 13 minutes. If one looks closely, while the resolution of the late
eluting organic acid and alcohol peaks are acceptable, the early
eluting oligosaccharide and saccharide peaks are significantly
reduced. Since the separation of oligosaccharides is based
primarily on a gel filtration mechanism, there is a limitation on
how much the column length can be shortened and still maintain
resolution of key saccharide peaks. Examples of a late timepoint
for a fermentation run using the 150 x 7.8 mm is shown in Figure
4. Quantitation of the Dp4+ and Dp3 peaks at early timepoints
may be compromised due to poor resolution of these abundant
peaks. However, in later timepoints the reduced levels of the
Dp4+ and Dp3 peaks may allow for accurate quantitation.
While the analysis of early timepoints from a fermentation run
may need the increased resolving power of the longer 300 x 7.8
mm Rezex ROA column, it may be practical to use a 150 x 7.8
mm Rezex ROA column for later timepoints, where saccharide
peaks are smaller. This is most practical in a larger operation
where multiple fermentors are used and multiple HPLCs might
be used for monitoring. Alternatively, for sites where only one
HPLC is used, a column-switching valve might be employed to
switch to the shorter column later in a fermentation run.

App ID 16649

TN-1043

1
3
5

10

12

14 min

Figure 4: A late fermentation timepoint run on a 150 x 7.8 mm Rezex ROA

column. Note that the low level of remaining oligosaccharide peaks allow for
some quantitation. The late eluting ethanol and organic acid peaks are well
resolved on the shorter column with reduced run time.

References
1. One Hard-hitting Year; H.Jessen, Ethanol Producers
Magazine, December 2006
2. American Coalition for Ethanol; www.ethanol.org
3. Using the HPLC System in a Fuel Grade Ethanol Production
Laboratory J.Mott, Shimadzu Scientific Application Note, 2006
4. HPLC of Carbohydrates with Cation- and Anion-Exchange
Silica and Resin-Based Stationary Phases C.G.Huber and G.K.
Bonn in Carbohydrate Analysis; Journal of Chromatography
Library, 1995, Vol. 58, 147-180.
Ordering Information
Part No.

Description

Unit

00F-0138-K0-TN Rezex ROA column; 150 x 7.8 mm

ea

00H-0138-K0-TN Rezex ROA column; 300 x 7.8 mm

ea

AJ0-4490-TN

SecurityGuard cartridges
Carbo-H+, 4 x 3.0 mm

AF0-3203-12-TN

Phenex-RC 0.20 m Syringe Filter

8/pk
100/pk

SecurityGuard Analytical Cartridges require universal holder Part No.: KJO-4282

Conclusions
The standard HPLC method used for fermentation monitoring
of bioethanol production is a simple yet powerful method for
monitoring saccharides, organic acids, and alcohols generated
during production. Simple steps such as using guard columns and
filtering samples can greatly increase the reliability of the method
as well as improve column lifetime (thus reducing analysis cost).
For larger operations, shorter columns can be used in some
circumstances to reduce analysis time for monitoring; potentially
reducing the need for additional analytical equipment.

TN-1043

5763_L

HPLC

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