There are many ways to decrease the retention time in reversed-phase chromatography (RP-HPLC), you can decrease the retention by adjusting the polarity of the mobile phase, reducing the column length, increasing the column temperature, using a smaller particle size column, changing the pH value of the mobile phase.

Due to higher eluting strength, the use of ethanol–water mixtures also allows decreasing the amount of organic solvent required to achieve a separation, whereas, despite a higher viscosity, the chromatographic efficiencies are as good as those obtained with classical mobile phases.

All Answers (5) OH group in ethanol is polar, and C2H5 group is non-polar. Hence it can dissolve both.

Larger molecules take longer to move up the chromatography paper or TLC plate, whereas smaller molecules are more mobile….For example:

• the mobile phase moves more quickly through the stationary phase.
• the mobile phase moves more evenly through the stationary phase.
• there is a range of absorbencies for the stationary phase.

The cellulose paper support in paper chromatography is flexible whereas the adsorbent in TLC is coated onto a rigid metal, glass or plastic plate. This contributes to reproducibility of spots and faster development.

The more soluble a compound is in the liquid phase, the less time it will spend being carried along by the gas. High solubility in the liquid phase means a high retention time. The temperature of the column. A higher temperature will tend to excite molecules into the gas phase – because they evaporate more readily.

Retention time is the time that a solute spends in a column or it can be defined as the time spent in the stationary and mobile phases. The longer retention time depends on the interaction of the analyte with the stationary phase. The stronger the interaction, the more will be the interaction time.

As temperature is increased, retention will decrease. If the room experiences wide temperature fluctuations, the HPLC retention times will probably be affected. The best solution is to run analyses at a temperature that can be controlled by using an oven.

If the polarity of the stationary phase and compound are similar, the retention time increases because the compound interacts stronger with the stationary phase. As a result, polar compounds have long retention times on polar stationary phases and shorter retention times on non-polar columns using the same temperature.

Void volume is the volume of mobile phase (Vm or V0) in a column. In an ideal case, it is equal to the mobile phase hold-up volume. For example, if the stationary phase occupies 40% of the total column volume, the void volume would be 60% of the total column volume. The total column volume is 19.6 mL.

The HPLC column void volume denoted Vm or V0 is in simple terms the volume of the mobile phase in the column. It is the part of a fraction that when added to the volume of the stationary phase makes up a whole fraction or 100% volume.

Multiply the elution time of the unretained compound by the flow rate to get the actual void volume of the system and column. To determine the column void volume alone you would need to subtract the system void volume determined without the column attached.

When solvent is injected into a high-performance liquid chromatograph (HPLC), it will elute at the unretained peak position. The first item to check is if the peak coming before the void volume is resulting from a previous injection. This can be determined by varying the amount of time between injections.

Answer: Dead volume – Dead volume is extra volume experienced by solutes as they pass through the chromatographic system, in particular any unswept volume exposed to the mobile phase flow. Excessive dead volume causes additional peak broadening.

Relative retention time (RRT) is the ratio of the retention time of any extra peak in relative to that of principle peak reference obtained in same chromatography run under identical conditions.

Equilibration time is dependent on the flow rate and dimension of the column. In general, flushing the column with ten up to twenty column volumes is enough for equilibration. Therefore the flow rate has to be kept in mind. It is possible to increase the flow rate during equilibration to shorten the equilibration time.

Again, the void volume is important to establish the earliest possible retention time/volume. The (total) column volume is equal to the latest possible retention time (except for stuff that will stick on top because of precipitation etc).

Dead volumes are parts of the sample system where fluid can become stuck and won’t be able to move along with the main flow of the sample.

The dead volume can be calculated by the equation (inner diameter/2)²(π)(needle length). Add this to the dead volume of the needle hub to get the needle’s total dead volume. For a Metal or Kel-F Luer Lock Needle connected to a Hamilton syringe add 10 µL, and for a Removable Needle there is no additional dead volume.