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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.