The instrumental method of determining the Scoville Heat Value (SHV) of various capsanoid substances have several advantages. The first one is the sample can be tested immediately without any pre-treatment. Another advantage is that the results can be obtained and analyzed in about four hours, which is a relatively short amount of time. Finally, the instruments used for the experiment has an adequate sensitivity for determining the SHV capsanoid in common food products. However, this method is not without its limitations.
Background noises may appear in the graphical result near the 280-nm wavelength which can be hard to be distinguished from the capsanoid peaks if the sample is not strong enough. Pressure is also a limitation of this method if particle based columns are used. Using a column packed with larger diameter particles reduce the limitation of column length and flow rates but cannot handle large pressures. Because the mobile phase has to travel through larger particles, the flow rate can be slowed down to allow better separation on the retention time.
On the other hand, using smaller particles columns would limit column length and flow rates despite the ability to handle higher pressures. The adverse effect is that it could be harder to separate the retention time of the mobile phase because of the low column length and high flow rate. The effect of the composition of the mobile phase on the retention time is what makes liquid chromatography works. The retention time of a solution is unique to that solution and therefore can be used as an identification.
The reason why the mobile phase composition affects the retention time is because the mobile phase interacts physically and chemically with the stationary phase. The more interaction it has, the higher the retention time. Knowing this, an improvement can be made to the experiment to improve separations of compounds. By using a column packed with larger particle size, the flow rate of the mobile phase can be lowered and thus, a better separation of the retention time can be obtained.
The difference between normal and reverse phase chromatography is in the polarity of their mobile and stationary phases. In normal phase chromatography, the mobile phase is non-polar and the stationary phase is polar. Reverse phase chromatography is the opposite. The mobile phase is polar while the stationary phase is non-polar. Reverse phase chromatography is now the more common phase used in experiments because non-polar mobile phase like water can damage the silicon component of the stationary phase.
Sources:
Batchelor, J & Jones, B. Determination of the Scoville Heat Value for Hot Sauces and Chilies: An HPLC Experiment. Journal of Chemical Education. Wake Forest University. Vol. 77 No. 2. February 2002. Isimer, A. et al. Effects of Mobile Phase Composition on Separation of Cathecolamines by Liquid Chromatography with Electrochemical Detection. Journal of Islamic Academy of Science. 4:2. 1991.