2.6 RESULTS AND DISCUSSION
2.6.2 Central Composite Design
2.6.1.4. ANOVA: Resolution Factor
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The equation shows that the resolution factor was significantly affected by the antagonistic linear contributions of the model terms X1, X2, X1X2, X1X3 and antagonistic quadratic interaction effects of X12, X22 and X32. These relationships implied that increasing the concentration of the organic solvent, column temperature and mobile phase flow rate resulted in a decrease in the resolution factor.
2.6.1.4.2 Response Surface Model Plots for Resolution Factor
The relationship between the independent variables and the resolution factor was visualised using 3-dimentional Response Surface Plots (RSP). Increasing both the column temperature and the amount of organic solvent resulted in a slight decrease in the resolution factor as depicted in Figure 2.7. An increase in the concentration of the organic solvent while keeping the column temperature constant resulted in a decreased resolution. An increase in the column temperature while keeping the concentration of the organic solvent constant resulted in an increase in the resolution factor. The figure also shows that the highest values of resolution were achieved with high temperatures and low concentrations of the organic solvent compared to low temperatures and high concentrations of organic solvent.
Figure 2.7. RSM plot showing the effect of column temperature and amount of organic solvent on the resolution factor.
Design-Expert® Software Resolution factor
Design points above predicted value 6.9
1.26
X1 = A: Mobile phase composition X2 = B: Column temperature Actual Factor
C: Flow rate = 0.00
-1.00 -0.50
0.00 0.50
1.00
-1.00 -0.50 0.00 0.50 1.00
1.8 2.7 3.6 4.5 5.4
Resolution factor
A: Mobile phase composition B: Column temperature
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Increasing both the amount of the organic solvent in the mobile phase and flow rate significantly decreased the resolution factor, while an increase in the flow rate with constant organic solvent also resulted in a slightly decreased resolution factor as shown in Figure 2.8.
The figure also shows that increasing the concentration of the organic solvent while keeping the flow rate constant resulted in a decrease in the resolution factor and that highest values of resolution factor was achieved when low flow rates and high concentrations of organic solvent concentration in the mobile phase were used as compared to high flow rates and low organic solvent concentration.
Figure 2.8. RSM plot showing the effect of changes in mobile phase composition and flow rate on the resolution factor.
An increase in both temperature and flow rate resulted in a slight decrease in the resolution factor. An increase in flow rate did not have a significant effect on the resolution factor as shown in Figure 2.9. The figure also shows that increasing the column temperature up to approximately 30 °C while keeping the flow rate constant resulted in an increase in the resolution factor, thereafter an increase in the column temperature resulted in a decrease in the resolution factor. An increase in flow rate of mobile phase with constant column temperature did not have a significant effect on the resolution factor. The resolution factors for conditions of low temperatures and high flow rates were similar to those of conditions of low temperature and low flow rates. Furthermore, high temperatures and low flow rates
Design-Expert® Software Resolution factor
Design points above predicted value 6.9
1.26
X1 = A: Mobile phase composition X2 = C: Flow rate
Actual Factor
B: Column temperature = 0.00
-1.00 -0.50
0.00 0.50
1.00
-1.00 -0.50 0.00 0.50 1.00
1.6 2.55
3.5 4.45
5.4
Resolution factor
A: Mobile phase composition C: Flow rate
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resulted in resolution factors that were similar to those from high temperatures and high flow rates. These effects resulted in the formation of a ‘molehill’ shaped response surface plot shown in Figure 2.9. The figure also shows that highest values of resolution factor were achieved when medium low flow rates (approximately 1 mL/min) and medium temperatures (approximately 30 °C) were used.
Figure 2.9. RSM plot of the effect of flow rate and column temperature on the resolution factor.
2.6.1.5 Validation of Experimental Design
The optimised chromatographic conditions selected for the quantitation of NVP are summarised in Table 2.10.
Design-Expert® Software Resolution factor
Design points above predicted value 6.9
1.26
X1 = B: Column temperature X2 = C: Flow rate Actual Factor
A: Mobile phase composition = 0.00
-1.00 -0.50
0.00 0.50
1.00
-1.00 -0.50 0.00 0.50 1.00 2.73 2.9525 3.175 3.3975 3.62
Resolution factor
B: Column temperature C: Flow rate
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Table 2.10. Summary of chromatographic conditions
Parameter Setting
Integrator: Speed Input voltage Attenuation (AT) Offset (OF) Peak height (PH) Peak threshold (PT) Minimum area (MA) Peak width (PW)
0.25 cm/min 10 mV full scale 128 5
1 250 2500
Sensitivity 6 0.1 AUFS
Flow rate 1.0 mL/min
Injection volume 20 µL
Wavelength 284 nm
Temperature 30 °C
Mobile phase composition 44:56 % v/v acetonitrile: water
The optimised chromatographic conditions resulted in a separation for which the retention time of NVP and CBZ were 4.30 ± 0.014 min and 7.60 ± 0.014 min, respectively with a resolution factor of 3.82 ± 0.012 (n =5). A typical chromatogram of the separation of a standard solution of NVP and CBZ is depicted in Figure 2.10.
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Figure 2.10. Typical chromatogram of the separation of NVP (60 µg/mL) and CBZ (100 µg/mL).
The experimental design model was validated by comparing the predicted values and the actual experimental values obtained using calculated residual and percentage errors. The predicted retention times of NVP and CBZ, and the resolution factor in addition to the calculated residual and percent prediction errors (% P.E) are summarised in Table 2.11.
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Table 2.11. Validation of experimental model: comparison of predicted and actual responses Response Predicted Actual Residuals % Prediction error
RT NVP 4.00 4.30 0.30 6.98
RT CBZ 7.00 7.60 0.60 7.89
RS 4.00 3.82 0.12 3.00
The percent error between the predicted and actual retention times were 6.98 and 7.89 for NVP and CBZ, respectively while the percentage error for the resolution factor was 3%. It can be concluded that the empirical models that were developed were reasonably accurate, particularly for resolution that had a percentage error < 5%. Slightly lower prediction errors could have been obtained if transformation of the data was performed, however, the responses observed were within the set limits and the separation was rapid and produced reliable analyses.
The preliminary chromatographic conditions achieved using the traditional method of sequentially changing one variable at a time produced a mobile phase that was comprised of 40% acetonitrile and 60% water, and yielded retention times of 5.23 minutes and 9.25 minutes for NVP and CBZ, respectively whereas the RSM optimised mobile phase was comprised of 44% acetonitrile and 56% water with retetions times of 4.30 minutes and 7.6 minutes for NVP and CBZ, respectively. The use of RSM enabled achievement of conditions that resulted in much shorter retention times and adequate resolution between the peaks of interest.
2.6.2 HPLC Method Validation