Simple Guidelines for Choosing the Right
Injection Solvent for UltraPerformance
Convergence Chromatography(UPC2)
Jacob N. Fairchild
GOAL
To optimize UPC2 separations, choosing the
Provide general guidelines regarding the
choice of sample injection solvents based
on the analyte and stationary phase properties
right injection solvent can tremendously impact
peak shape.
when performing separations on an
ACQUITY UPC 2™ System.
T H E S O LU T I O N
Two very different stationary phases were
selected for comparison. A polar stationary
phase, ACQUITY UPC 2 BEH Column, consisting
only of polar surface groups (silanols), and
d-Tocopherol
in heptane
9 µL
7.5 µL
6 µL
4 µL
2 µL
1 µL
AU
0.60
0.40
0.20
0.00
0.80
0.40
0.48
0.56
0.64
2.80
d-Tocopherol
in isopropanol
2.80
0.52
0.60
0.68
0.76
0.60
0.68
0.76
0.68
0.76
Butylparaben
in isopropanol
AU
AU
0.44
2.10
1.40
0.70
0.40
0.48
0.56
0.64
0.72
0.00
d-Tocopherol
in acetonitrile
2.80
0.60
0.44
0.52
Butylparaben
in methanol
2.10
AU
0.40
0.20
0.00
1.40
0.00
0.72
0.20
0.80
9 µL
7.5 µL
6 µL
4 µL
2 µL
1 µL
0.70
0.40
0.00
Butylparaben
in heptane
2.10
0.60
AU
Diluent effects are extremely important to
consider when developing a UPC 2® method.
First and foremost, the sample must be soluble
in a solvent compatible with the mobile phase.
In general, if a sample can be dissolved in
methanol (with up to a few percent of water) or
any other organic solvent, it can be successfully
injected into supercritical CO2, notwithstanding
any adverse effects to the column. Sample
solvent effects of some common diluents were
previously investigated on 2-ethylpyridine
bonded phases.1 We found that nonpolar sample
solvents such as heptane and THF should be
used to minimize peak distortion and it is
always advantageous to use low dispersion
instruments, even when ideal sample solvents
are not used. In this work, we examined the
former point for ACQUITY UPC 2 BEH and HSS
C18 SB columns.
0.80
AU
BA C K G RO U N D
1.40
0.70
0.40
0.48
0.56
Minutes
0.64
0.72
0.00
0.44
0.52
0.60
Minutes
Figure 1. Injections made on an ACQUITY UPC2 BEH Column. (Left): d-tocopherol injections of
1, 2, 4, 6, 7.5, and 9 µL, dissolved in heptane, isopropanol, and acetonitrile. (Right): 1-, 2-, 4-,
6-, 7.5-, and 9-μL injections of butylparaben dissolved in heptane, isopropanol, and methanol.
The orange stars indicate the best solvents to minimize peak distortion.
what can be considered a mixed polarity stationary phase, ACQUITY UPC 2 HSS C18 SB Column, consisting of
nonpolar, trifunctional C18, ligands, and silanols. Three sample diluents were chosen for each to demonstrate
proper selection of an injection solvent based on the stationary phase and analyte. Two analytes, butylparaben
(polar) and d-tocopherol (nonpolar), were chosen. Both are easily soluble in the three sample solvents chosen for
each column at sufficient concentrations for this study (0.4 g/L). The two analytes were dissolved individually
in each of the sample solvents and injected at volumes between 1.0 and 9.0 µL. Notice the evolution of the peak
profile of butylparaben injected onto the ACQUITY UPC 2 BEH Column shown in Figure 1. The flow rate was set to
1.2 mL/min of 95:5 CO2/MeOH at 40 °C and 2175 psi (ABPR). The peak height of the heptane-dissolved sample
increases linearly to the point of detector saturation. Injections of d-tocopherol above 2.0 µL begin to show peak
distortion, but heptane is clearly the best choice of the solvents tested. For the ACQUITY UPC 2 HSS C18 SB Column,
heptane also gives the best peak shape for butylparaben (Figure 2). However, peak shape for d-tocopherol is best
with acetonitrile as the injection solvent instead of heptane, which was the best injection solvent for this compound
on the BEH column.
From the injections of butylparaben and d-tocopherol, it can be concluded that polar compounds are separated
by the polar substituents of the stationary phases. These data suggest that polar compounds should be dissolved/
diluted in nonpolar solvents for UPC 2 separations. For nonpolar compounds (i.e., d-tocopherol), separated on a
non-polar stationary phase, a polar injection solvent (i.e., acetonitrile or methanol) should be used. We considered
the maximum solubility of d-tocopherol and found it to be ~4.2 g/L in pure acetonitrile with copious sonication.
Figure 3 demonstrates a linear response for peak area to d-tocopherol (in acetonitrile) concentration obtained
using a 2-μL injection volume. Using solvents of opposite polarity from the solute can be difficult or impractical.
When a specific analyte cannot be dissolved in detectable quantities in a very weak solvent, an effective option
is to dissolve an analyte in relatively high concentrations in a stronger solvent, such as methanol or isopropanol,
then diluting with a weaker solvent, e.g., heptane in Figure 1 or acetonitrile for d-tocopherol in Figure 2.
0.40
d-Tocopherol
in heptane
9 µL
7.5 µL
6 µL
4 µL
2 µL
1 µL
0.20
0.10
0.00
0.40
1.24
1.32
1.40
1.48
d-Tocopherol
in isopropranol
2.40
AU
AU
0.52
0.60
0.68
0.76
0.52
0.60
0.68
0.76
Butylparaben
in isopropranol
1.20
0.60
1.24
1.32
1.40
1.48
0.44
1.56
d-Tocopherol
in acetonitrile
2.40
1.80
0.20
1.20
AU
0.30
0.10
0.00
0.44
1.80
0.10
AU
1.20
0.00
1.56
0.20
0.40
9 µL
7.5 µL
6 µL
4 µL
2 µL
1 µL
0.60
0.30
0.00
Butylparaben
in heptane
1.80
AU
AU
0.30
2.40
Butylparaben
in methanol
0.60
1.24
1.32
1.40
Minutes
1.48
1.56
0.00
0.44
0.52
0.60
Minutes
0.68
0.76
Figure 2. Injections made
on an ACQUITY UPC2 HSS
C18 SB Column, results for
d-tocopherol dissolved in
acetonitrile (bottom left) are
opposite to the observations
made on an ACQUITY UPC2
BEH Column. The orange stars
indicate the best solvents to
minimize peak distortion.
40000
y = 8929.6x - 116.21
R2 = 0.99985
35000
30000
Peak area
25000
20000
15000
10000
5000
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Concentration (g/L)
Figure 3. d-Tocopherol
dissolved in acetonitrile,
up to 4.2 g/L, concentration
linearity on an ACQUITY
UPC2 HSS C18 SB Column
(2-μL injections). Similar
results were found for
1-, 4-, 6-, 7.5-, and 9-μL
injection volumes.
C O N C LU S I O N
Choosing the best solvent for a particular sample can be challenging and often results in a pragmatic choice.
However, better chromatographic results can be obtained when properly chosen solvents are used. Making
a poor selection of sample solvent can lead to very distorted peak shapes and under-informed decisions
concerning the chromatographic results. Two basic guidelines are useful for UPC 2 separations: 1) Polar analytes
should be dissolved in nonpolar solvents when possible, independent of the stationary phase. 2) Based on the
stationary phase, nonpolar analytes should be dissolved in solvents of the opposite polarity. Nonpolar solvents
should be used for polar stationary phases and polar solvents for nonpolar (or mixed) stationary phases.
References
1. Fairchild JN, Hill JF, Iraneta PC. Influence of Sample Solvent Composition for SFC Separations.
LC GC North America, 31:4 (2013), 326-333.
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©2014 Waters Corporation. Produced in the U.S.A. March 2014 720004981EN TC- PDF
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