a diluent study investigating a wide range of...a set of polar diluents were compared to the...
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A DILUENT STUDY INVESTIGATING A WIDE RANGE OF ORGANIC SOLVENTS COMPATIBILITY WITH UPC2 FOR A
MEDICINAL CHEMISTRY LABORATORY
Janet Hammond1, Michael D. Jones2 and Sean M. McCarthy2 1Waters Corporation, Manchester, UK; 2Waters Corporation, Milford, MA, USA
INTRODUCTION In the medicinal chemistry laboratory,
synthetic reactions are carried out using a
wide range of solvents. To analyze reaction
mixtures, the chemist would favor injecting
the reaction mixture directly onto a
chromatographic instrument without any
dilution. However; this is not practical when
considering the high concentration of a
reaction mixture, therefore simple dilutions
are necessary.
The impact of sample diluents on reversed
phase separations in relation to peak shape,
retention and other factors are well
understood. It is generally understood to
dissolve the analyte in a solvent with an
elutropic strength inversely proportional to
the strength of the chromatographic elution
solvent. By this definition, non-polar solvents
such as hexanes would be optimal diluents
for Convergence Chromatography (CC) due to
their equivalent elutropic strength to that of
CO2. However when using UltraPerformance
Convergence Chromatography (UPC2), this
can be challenging for the analysis of polar
compounds due to insolubility with the
‘optimal’ choice of a non-polar solvent.
Presently, knowledge of the appropriate
diluent choice is under-developed
In this study, we investigated the effect
of different sample diluents on a probe
analyte with a low retention factor (k’) when
injected on the UltraPerformance
Convergence Chromatography (UPC2) system.
Our study included a range of solvents which
span a range of polarities and relative
elutropic strengths. The goal of this work was
to provide recommendations for diluent
selection for the analysis of reaction mixtures.
Figure 6: A comparison of peak distortion of polar solvents
DMF diluted with non-polar solvent heptane/IPA 9:1 using CSH Fluoro-Phenyl 3.0 mm x 100mm; 1.7µm column
Figure 5: A comparison of peak distortion with the polar
solvent NMP diluted with non-polar solvent heptane/IPA 9:1 using CSH Fluoro-Phenyl 3.0 mm x 100mm; 1.7µm column
Methanol Dimethyl sulphoxide
(DMSO)
Heptane/IPA 90:10 N-methyl pyrrolidone
(NMP)
Heptane/IPA 70:30 Dimethylformamide (DMF)
2-Propanol (IPA) Dimethyl Acetamide (DMA)
Toluene Ethyl Acetate
Acetonitrile Methanol/
Dichloromethane1:1
Dichloromethane THF/Heptane 1:1
Dioxane
Tetrahydrofuran
(THF)
Methyl-t-butylether
(MTBE)
Table 1: Solvents used in the study
RESULTS AND DISCUSSION
Analyte Probe:
4-Hydroxy butyl-benzoate was the chosen analyte
and prepared as 0.2mg/ml solution in a range of
polar and non-polar solvents as shown in table 1.
EXPERIMENTAL UPC2
Instrument: ACQUITY UPC2 w/ PDA detection
Mobile Phase: 97% Methanol: 3% CO2 (medical grade) Columns: UPC2 BEH
UPC2 CSH Fluoro-Phenyl UPC2 BEH 2-Ethyl Pyridine
UPC2 HSS C18 SB
Dimensions: 3.0 x 100mm; 1.7um
Injection Vol.: see figure captions
Run time: 3 minutes
Column Temp.: 45 °C
Flow Rate: 2.0 ml/min
ABPR pressure: 2000 psi
Wavelength: 254 nm
CDS: MassLynx 4.1
A comparison between the non-polar solvent of
heptane/IPA 9:1 and DMSO when varying injection
volumes (Figure 1). In the case of DMSO, peak
distortion is observed when the injection is increased
to 2µl, however peak shape is Gaussian for the
heptane/IPA 9:1 diluent. Once the injection volume
was increase to 5ul, peak distortion occurred
irrespective diluent.
The use of 100% methanol as the diluent was
investigated for the probe analyte on three different
columns. Each column resulted in a difference in
retention (k’). The comparison of the peak shape
from the three columns is shown in figure 2. This
example suggests that analytes eluting with a lower
k’ is more susceptible to peak distortion when using
100% MeOH.
A set of polar diluents were compared to the non-
polar diluent; used in Figure 1, by injection of the
analyte probe on the UPC2 BEH column (Figure 3).
Each injection of the diluent study showed acceptable
peak shape. It should be noted that retention of the
DMA and DMF were observed and would interfere with
analytes with lower k’. NMP retention was also
observed, but exhibits low absorbance at 254nm
which is a typical monitoring wavelength.
The same experiment performed for Figure 3 was
repeated by injection of the probe analyte on the
UPC2 CSH Fluoro-Phenyl column. The extent of the
peak distortion was observed to be much more
severe for this column when compare to the UPC2
BEH results. Further investigations to explaining this
phenomena would have to consider loadability studies
such as those performed in Figure 1 for the different
columns. *Diluent retention does not appear to be the major issue, but should be confirmed as part of the further investigation.
Figure 3: A comparison of peak distortion of polar solvents
DMSO, NMP, DMF, DMA and heptane/IPA 9:1 using a UPC2 BEH 3mm x 100mm; 1.7µm column
DMA
DMF
NMP
DMSO
Hep/IPA
Figure 4: A comparison of peak distortion of polar solvents
DMSO, NMP, DMF, DMA, and heptane/IPA 9:1 using a UPC2 CSH Fluoro-Phenyl 3mm x100mm; 1.7µm column
DMA
DMF
NMP
DMSO
Hep/IPA
In many cases, a mixture of these solvents may be used for solubility or reaction rate purposes. The
experiments from Figure 4 were taken further to explore varying percentages of the NMP and DMF solvent
combined with heptane/IPA 9:1. Compositional mixtures of 2%, 5%, 10%, 20%, and 50% of each of the
NMP and DMF were combined with the heptane/IPA mixture. The NMP results (Figure 5) show Gaussian peak
shapes for the probe analyte up to a compositional mixture of [10% NMP : 90% heptane/IPA] on the Fluoro-
Phenyl column. The varying compositional mixtures of DMF and heptane/IPA diluent (Figure 6) showed
peak distortion with each compositional diluent mixture suggesting alternative solvents or an alternative
column which provides a greater k’ to minimize the strong solvent effects should be considered when using
DMF.
Figure 2: Comparison of peak distortion when 3 different col-
umns were used A) UPC2 CSH Fluoro-Phenyl, B) UPC2 BEH,
and C) UPC2 BEH 2-EP
UPC2 CSH Fluoro-Phenyl
UPC2 BEH
UPC2 BEH 2-EP
Figure 1: Comparison of peak distortion between DMSO and
Heptane/IPA 9:1 at various injection volumes
5µl DMSO
2µl DMSO
0.5µl DMSO
5µl Hep/IPA
2µl Hep/IPA
0.5µl Hep/IPA
+2% DMF
Hep/IPA
+5% DMF
+10% DMF
+20% DMF
+50% DMF
Neat DMF
+2% NMP
Hep/IPA
+5% NMP
+10% NMP
+20% NMP
+50% NMP
Neat NMP
CONCLUSIONS Many of the solvents in Table 1 were highly
compatible for use as diluents when using UPC2, with
some precautionary exceptions...
Strong diluent effects can be observed for some
solvents on specific columns, such as the case with
DMF and UPC2 Fluoro-Phenyl combinations.
Lower k’ analytes are more susceptible to peak
distortion.
Dilution with a weaker elution solvent (such as 9:1
heptane:IPA) can minimize chromatographic band-
spreading of the analyte caused by strong solvent effects.