Conclusions

Hyphenation with HTLC is one of the major recent trends in the usage of ELSD and C-CAD. Various aspects of HTLC separation and their benefits for specific applications have always remained a prime focus. Therefore, the central aim of the studies presented in chapter 3 was to examine the effects of HTLC conditions on the response of ELSD and C-CAD. The results from the present study showed that eluent temperature influence the response of these detectors only marginally and thus makes eluent cooling unnecessary. This suggested that the response homogeneity of ELSD and C-CAD can be improved by replacing solvent gradient with isocratic-HTLC separation. In chromatographic separations, temperature-induced alterations in elution bandwidth were found to influence the peak area produced by ELSD significantly. Further investigations revealed that an inverse relationship existed between elution bandwidth and ELSD response. The origin of this issue lies in the fact that the elution bandwidth dominates the size distribution of the tertiary aerosol reaching the optical unit and consequently alters the mechanism of light scattering. The ELSD response variability as a function of elution bandwidth becomes most pronounced in isocratic separations. Application of a temperature gradient to an isocratic separation reduced the elution bandwidth variation across the chromatographic separation and thus

128 improved the response uniformity of ELSD. In contrast to ELSD, a variation in elution bandwidth had little effect on peak area obtained with a C-CAD. Since C-CAD provided a uniform response independent of elution bandwidth, it was selected for further investigations. The primary aim of the studies described in chapter 4 was to assess the feasibility of replacing solvent gradient separation by isocratic separation methods employing a simultaneous variation in temperature and flow-rate, so that uniform C-CAD response could be achieved. Results from FIA studies showed that C-CAD was relatively insensitive to the changes in eluent temperature and flow- rate when water-rich eluents were used. Nevertheless, for optimum signal-to-noise ratio, it was necessary to consider the eluent composition while selecting the range of flow-rate variation. Based on these findings two separation approaches were developed. The first approach demonstrated that the temperature gradient applied to an isocratic water-rich mobile phase could be used to replace a solvent gradient separation. The sensitivity loss resulting from water-rich eluents in isocratic-temperature gradient separation can be compensated by post-separation addition of a secondary stream of pure organic solvent. The secondary eluent must be selected based on miscibility with the primary eluent and the solubility of the sample constituents.

The second approach involved simultaneous variation in temperature and flow- rate. Baseline disturbances resulting from the backpressure changes produced in a flow-rate gradient were compensated by post-column flow-rate make-up using an inverse flow-rate gradient of the eluent. This study has shown that temperature gradients suffered from low elutropic strength, resulting in broadening of late eluting peaks when elution was performed at a constant flow-rate, whereas a flow-rate gradient under isothermal conditions posed risk of high back-pressure. Combining a temperature gradient with a simultaneous flow-rate variation allowed use of the high flow-rate changes required for the flow-rate gradient, improved the separation speed and reduced the elution bandwidth variability across the separation. The proposed approaches, as well as use of an inverse gradient, allowed quantification at 5 mg/L level. Considering the wide dynamic range of the C-CAD, quantification of impurities present at lower concentration levels can be achieved by increasing the sample mass per injection. The approaches described in the present study minimise the necessity of solvent gradients and thus reduce the response deviation across the separation.

129 Moreover, these approaches offer flexibility in the use of C-CAD for relatively unconventional separation modes and thereby contribute to extend its universality. The proposed approaches do have some limitations, such as a limited choice of thermo- stable stationary phases, the difficulty in applying a high temperature ramp, slow thermal equilibration due to the high thermal mass of the analytical scale columns, and the risk of high backpressure in maintaining a sufficiently high flow-rate range in flow- rate gradients. These limitations are particularly characteristic of conventional analytical scale separations. It should be noted this is proof-of-concept work and potential future work in instrument development could resolve these issues. Moreover, these approaches could be further extended to micro and capillary scale separations.

The studies in chapter 5 investigated different strategies to overcome response irregularities of the ELSD. It was shown that real time gas flow-rate programming can be used to compensate partially for response variations resulting from solvent gradient effects. However, significant loss in sensitivity is one of the major concerns in the implementation of this approach. A lack of commercially available software to precisely control gas flow-rate is another constraint, as Agilent Technologies Inc. is the only provider of the real time gas programming software for ELSD and has recently discontinued the development and sale of this software. Although use of an inverse solvent gradient helped to maintain constant composition of the eluent entering into the ELSD, bandwidth variability across the separation still contributed to response deviation. The temperature and flow-rate gradient approaches discussed earlier can help alleviate this issue, but are not recommended for ELSD as they employ water-rich eluents and cause sample dilution because of post-column solvent mixing. These findings reiterated the urgent need to reduce bandwidth variability for uniform response using ELSD.

The possibility of using valve-based post-separation flow-rate modulation to control the width of the analyte band entering the ELSD was investigated. Although flow-rate modulation causes a decrease in peak area, the amplitude of the modulated peaks remained comparable to those obtained without modulation. The results from a FIA study showed that the slope and intercept values of the ELSD response curves (logarithmic scale) obtained by flow-rate modulation remained relatively unaltered irrespective of elution bandwidth. This implied that flow-rate modulation improved the

130 analyte mass transport and thus could be used to overcome the bandwidth variability issue. However, when applied to isocratic separation, flow-modulation showed only a marginal improvement in response uniformity. Although flow-modulation is a conceptually interesting approach, it has some limitations. It requires a high flow-rate differential for optimum modulation, but at the same time, the resultant sample dilution significantly lowered the ELSD response. Experiments showed that the performance of flow-rate modulation depended strongly on the characteristics of the principal peak. Because of the wide molar mass distribution across the broad chromatographic peaks, response correction was required for individual peak slices generated by modulation. Considering the complexity of the light scattering mechanisms in ELSD and its high detection limit, flow-rate modulation appears not, at this stage of its development, to be a practical solution to response irregularities of ELSD.