The movement of the compound in the mobile phase through a column is called elution and the water-ethanol mobile phase is named eluent.
When it comes to a point where the least polar compound is separated and the most polar compound is left in the column; it will take a very long period of time to wash the most polar compound out of the column. In order to accomplish this at a faster rate, the mobile phase could be replaced with another eluent which has higher polarity. The greater attraction between a very polar eluent and the coloured compound will allow any other molecules of the coloured compound that had bonded with the stationary phase to be attracted back into the solution. Another way is to use a stationary phase which is less polar than the mobile phase. This way, the more polar compound will be attracted highly to the water-ethanol mobile phase and will run through the column at a faster rate.
Optimum separation is referred to the best separation possible of the compounds in the mixtures of substances under the current conditions; the peaks of the compounds in the mixture have no interference with each other, they are far apart
Optimum separation is dependent on the nature of the stationary phase, mobile phase, environmental temperature and the composition of the mixtures of substances that are being used.
The factors mentioned above can be varied in order to obtain an efficient chromatographic separation.
First of all, the stationary phase (adsorbent) is required to be suited to the separation process of a mixture of substances, in a way that there will be large differences in the rate of elution. This could be further explained as the strength of the adsorbent affecting the rate of elution negatively. For example, Alumina, being the “most strongly adsorbent” (chemwisc), may interact strongly with the more polar and less polar compounds in the mixture and allow them to have a close rate of elution to each other. Distinguishing the two different compounds in the mixture will not be done efficiently. Therefore, the optimum separation will not be obtained.
Similarly, with Silica, the “least strongly adsorbent” ,may not interact as much with both the compounds in the mixture. Thus, the two compounds in the mixture will have a close rate of elution with each other and the same situation will be seen as the optimum separation will not be reached.
Another factor being the mobile phase, the same principle in having a good selectivity also applies. The ability to dissolve the compound being separated by the mobile phase is important in how fast these compounds move up or down the stationary phase. The mobile phase has a more important property of being adsorbed on the adsorbent. Depending on how much interaction occurs between the mobile phase and the secondary phase, the mobile phase will push the compound being separated away and replace it. However, if the solvent phase is too strongly adsorbed, it may be able to displace all the compounds entirely, leading them to fuse and move to the solvent front without any separation at all.
On the other hand, if a too weakly adsorbed solvent is used, it may not sufficiently dissolve any compound to move fast enough in order to effect separation. For example, ethanol+ water were used as the mobile phase in the practicals with a ratio of 1:1. Water, alone has a very great eluting strength; therefore it was mixed with ethanol to decrease its polarity rate. Water’s polarity index is “9” (chemical-ecology) and ethanol’s is “5.2” (chemical-ecology), when the two is mixed the overall polarity of mobile phase will lower compared to water’s polarity individually. Say, if the ratio was altered and a bigger ratio of ethanol was used- such as 1:2 (water:ethanol)- that will make the solvent phase even less polar than the 1:1 ratio of water + ethanol. Additionally, if methanol was used to mix with water using a ratio of 1:1, the solvent phase will have a much greater polarity than the ethanol + water mobile phase. This is because “methanol is a stronger eluent than ethanol” (chemwisc); methanol has a stronger polarity than ethanol. The water and methanol mixture for the mobile phase may cause the compounds to move at a fast rate without being sufficiently separated.
High temperatures weaken any hydrogen bonds that form between polar stationary phases and strongly polar compounds in the mixtures due to kinetic energy disrupting the connections (chemistry). Another issue is that the compounds will dissolve in the solvent at high temperatures, without being separated. However, even if the chromatography procedure was done at very low temperatures, there wouldn’t be the exact amount of energy to trigger the bond formation and the samples would not develop. Moreover, an optimum temperature should be provided for correct separation of compounds. That is about 25°C-room temperature.