HPLC analysis with Rezex Fast Acid H+
Standard Operational Procedure
Jagger
HPLC analysis with Rezex Fast Acid H+
Introduction
The Rezex column is suitable for analysis of short organic acids, mono- and disaccharides, certain
alcohols and furaldehydes. The column is typically used for fast screening of biomass based samples
after pretreatment and for analysis of monomer and dimers of chitin, but may be adapted to other
purposes.
The principle for separation is ion-exclusion where ionic and non-ionic analytes are separated. Ionic
species are repelled by the charged resin (excluded) and therefore elutes early whereas the non-ionic
species are more retained. This particular column is known for its low runtimes.
Depending on the type of analyte, either UV absorption or Refractive Index (RI) can be used as detection
principle. Monosaccharides and alcohols have low UV absorption and must therefore be detected by RI.
Organic acids and GlcNAcs absorb satisfactory at 210 and 195 nm respectively and is therefore detected
by UV, furaldehydes absorb with relatively high sensitivity at 278 nm.
The sensitivity greatly depends on the analyte in question. Remember also that, in general larger the
column dimensions the lower the sensitivity. This particular column has large column dimensions and
the sensitivity will therefore be poorer compared to other types of chromatography.
HPLC system
The analysis can be performed either on the Dionex Ultimate 3000 RSLC or Agilent Infinity 1290
chromatographs. See relevant instrumental SOP for operation of these systems. Both systems are set
up with UV .When RI-detection is needed the Dionex Ultimate 3000 RSLC is recommended (which can
fully control the RI-detector).
Analytical column
The analytical column is Rezex RFQ-Fast Acid H+ (8%) from Phenomenex, 100x7.8 mm. It consists of 8%
cross linked sulfonated styrene divinyl benzene. It is equipped with a guard column of the same
material. It is pH stable from 1-8 and the operating temperature lies between 60-85 °C. Organic
modifiers should be avoided and should not exceed 10%. This type of column material is especially
pressure sensitive and should never be operated above 69 bars (1000 psi).
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Edition 01
HPLC analysis with Rezex Fast Acid H+
Standard Operational Procedure
Jagger
Column precautions
The normal operating back pressure of the column incl. guard column at 85 °C is in the range of 56-60
bars at 1 mL/min, which is also maximum flow rate. The column can be stored in the eluent for
intermediate storage, but for longer storage, exchange the mobile phase with pure Milli-Q water.
Operating conditions
Column temperature: 85 °C
Flow conditions: 1 mL/min
Eluents: 5 mM H2SO4 (A)
Injection volume: 6-8 µL
Instrumental method: REZEX.
Detection: Organic acids (RI), GlcNAcs (195 nm), Furans (278 nm), Mono-/disaccharides (RI)
Note that the column always runs isocratically and therefore the only adjustable parameter is total run
time. This may be adjusted to suit different purposes.
Chromatographic run
12,00
11,25
µRIU
10,00
8,75
7,50
6,25
5,00
3,75
2,50
1,25
0,00
-1,25
-2,50
-3,75
-5,00
-6,25
-7,50
-8,00
0,00
min
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
9,00
10,00
Figure 1: Example of monosaccharide separation on Rezex. Poor/no resolution between xylose and mannose,
st
nd
glucose and galactose. 1 black: glucose, 2 black: mannose, green: galactose, brown: xylose, pink: arabinose.
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Edition 01
HPLC analysis with Rezex Fast Acid H+
Standard Operational Procedure
Jagger
Figure 2: Example of chitin mono- and dimer separation on Rezex.
Figure 3: Example of formic and acetic acid separation on Rezex
Sample preparation
Samples must be without any particulate/insoluble matter. This is obtained either by centrifugation or
by 0.22/0.45 µm filtration.
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Edition 01
HPLC analysis with Rezex Fast Acid H+
Standard Operational Procedure
Jagger
Calibration
The calibration range will be different for each analyte and if calibration is needed provide standards in
the relevant concentration range for each analyte. As a guideline organic acids may be calibrated in the
1-100mM range. Monosaccharides may be detected down to approx. 0.01 g/L (at 6 µl injection).
Always provide a new set of standards for the analytes of interest in every sequence and perform a
recalibration in the processing method. The calibration curves provided here are meant as inspiration.
Trouble shooting
Identify if any immediate precautions should be taken and then call instrument super users (currently
Jane W. A. or Bjørge W., IKBM-PEP).
Column regeneration
The column is cleaned by running pure Milli-Q water for 12 hours at 85 °C (0.6 mL/min) running in
reverse flow direction (remove guard column prior to cleaning). The producer recommends
regeneration with low flow (0.2 mL/min) with 25 mM H2SO4 for 4-16 hours at 85 °C (correct flow
direction, incl guard).
Column storage conditions
The column is stored in the 5 mM H2SO4 mobile phase for short storage. Longer storage is done in MilliQ water. Cap inlet/outlet to prevent drying.
Editorial log
First edition. 2012.04.16
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Edition 01