Application Note AN061
Acetone in UV-based Gradient Flash Chromatography | Page 1
Using Acetone in UV-based
Gradient Flash Chromatography
Bob Bickler, Senior Product Manager, Biotage
Panagiotis Ioannidis & Christel Ellström, Application Developers, Biotage
Acetone is successfully used as solvent in normal-phase flash chromatography
when used with an Isolera™ Spektra flash purification system. The new λ-All
detection and baseline correction features provide compound detection at all
wavelengths in the detector’s range while minimizing any baseline drift due to
solvent UV absorption.
Background
Acetone is an excellent solvent for many organic compounds.
It is miscible in both polar and non-polar solvents. Acetone is
inexpensive, has low toxicity, low viscosity, and a low boiling
point – all properties desired for chromatography. However, it
is rarely used because of one major drawback: detection and
collection interference from its strong UV absorption between
220 and 330 nm – the primary wavelength range where
aromatic and medicinal compounds absorb UV light. Because
of this issue, ethyl acetate is instead the usual polar solvent
chosen for normal-phase flash chromatography.
When using gradient elution with UV-triggered fraction
collection, this is a problem. During an acetone gradient
the chromatographic baseline will rise with the percentage
of acetone, Figure 1. Acetone’s absorption interferes with
compound-triggered fractionation and increases both the
solvent volume collected and the number of collection vessels
needed for purification. Increased collected solvent increases
fraction evaporation time.
Eliminating this baseline rise during a gradient removes
the issues associated with acetone and other UV absorbing
solvents. A new flash purification system from Biotage called
Isolera™ Spektra provides this baseline correction capability as
well as an all wavelength detection feature, λ-All.
In this application we will show how acetone is successfully
used as solvent in normal-phase flash chromatography when
used with an Isolera Spektra flash purification system. The
new λ-All detection and baseline correction features provide
compound detection at all wavelengths in the detector’s
range while minimizing any baseline drift due to solvent UV
absorption.
Figure 1: Ethyl acetate (left) and acetone (right) UV absorption in a gradient. Ethyl acetate is the usual solvent of choice because of its UV transparency
above 240 nm, an important detection range for many aromatic compounds. Acetone’s strong UV absorption from 220 nm to 320 nm will interfere with
compound fractionation unless the absorption is corrected.
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Application Note AN061 ©2012 Biotage
Acetone in UV-based Gradient Flash Chromatography | Page 2
Experimental
Results
With ethyl acetate (EtOAc) and acetone sharing the same
selectivity class, they provide similar separation selectivity
(elution order and spacing) for the same compounds, Table 1.
Acetone is slightly more polar than EtOAc so compounds will
usually elute a little earlier than when using EtOAc.
Though both solvent systems absorb UV light in the
wavelength range used (200 nm–400 nm), the separations and
fraction collection were achieved without interference from
a rising baseline. Acetone’s higher polarity provides sharper,
more concentrated fractions as well helping to reduce solvent
evaporation time.
In this application a mixture of four compounds was purified
using flash chromatography with both a hexane/EtOAc
gradient and a hexane/acetone gradient using an Isolera™
Spektra Four.
Because of the flat baseline each eluting peak was collected
into a single test tube saving fraction capacity and maximizing
fraction concentration, fraction 2.
Sample
• 0.1 g of each compound in 1-mL of acetone
• Naphthalene
• 2-Nitroaniline
• Methyl paraben
• 4-Nitroaniline
Equipment
Flash system:
Isolera™ Spektra Four with variable UV
detector
Flash cartridge:
SNAP Ultra 10 g
Solvents:
n-Hexane, Ethyl acetate, Acetone
Equilibration:
7% B at 100 mL/min for 3 CV
Gradient:
7% B for 1 CV
7% B to 60% B in 10 CV
60% B for 2 CV
Flow-rate:
12 mL/min
Detection:
λ-All, monitor 254 nm and 340 nm
Baseline correction:
On
Wavelength range:
200 nm–400 nm
Threshold:
20 mAU
Sample load:
50 mg
TLC:
Biotage 2.5 cm x 7.5 cm KP-Sil plates
Hexane/Ethyl
acetate
TLC data
Strong solvent %
Hexane/Acetone
30%
30%
Naphthalene
0.81
0.81
2-Nitroaniline
0.48
0.45
Methyl paraben
0.30
0.30
4-Nitroaniline
0.19
0.21
Solvents:
Experiment 1:
Experiment 2:
A. Hexane
A. Hexane
B. Ethyl acetate
B. Acetone
Solvent
Selectivity
group1
Solvent strength
vs. SiO22
Figure 2: The Isolera™ Spektra flash system used with λ-All and baseline
correction with both hexane/ethyl acetate (top) and hexane/acetone
(bottom) gradients. The baseline correction feature eliminates a baseline
rise due to solvent UV light absorption which eliminates excessive fraction
volumes while minimizing the number of collection vessels needed.
As seen in Figure 1 the use of UV absorbing solvents in flash
chromatography can present issues with UV-based fraction
collection. In Figure 3, we see the impact of acetone’s UV
absorption with the 4-component test sample: increased
solvent use (272 mL vs. 221 mL), fraction volumes, and number
of collected fractions (16 vs. 4).
Boiling
point
UV max
Absorbance range
Acetone
VIa
0.50
56 °C
270 nm
210–330 nm
Ethyl acetate
VIa
0.43
77 °C
210 nm
< 200–255 nm
Table 1: Solvent properties
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Application Note AN061 ©2012 Biotage
Acetone in UV-based Gradient Flash Chromatography | Page 3
Conclusion
The Isolera Spektra baseline correction feature now allows UV
absorbing solvents like acetone to be used for flash chromatography without increasing solvent use, fraction volumes or
runtime. Using acetone provides multiple benefits including
lower solvent costs, sharper peaks, and faster fraction
evaporation.
References
1. ”Introduction to Modern Liquid Chromatography” by L. R.
Snyder and J. J. Kirkland (Wiley: New York, 1979).
2. Sanderkok.com
Figure 3: (Top) Without the Isolera Spektra baseline correction
capability, Acetone’s strong UV absorption increases the number of
fractions collected as well as their volume and the total amount of solvent
required for the purification compared to the results where baseline
correction is used to eliminate acetone’s impact.
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©2012 Biotage. All rights reserved. All brand and product names are trademarks or registered trademarks of their respective companies.
Part Number: AN061
Application Note AN061 ©2012 Biotage
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