In normal-phase chromatography, it is used a polar stationary phase and a less polar solvent. A more polar solvent has higher eluent strength.  However, in reversed-phase chromatography, the stationary phase is non-polar or weakly polar and the solvent are more polar. A less polar solvent has higher eluent strength. The reversed-phase chromatography eliminates peak tailing because the stationary phase has few sites that can strongly adsorb a solute to cause tailing. Reversed-phase chromatography is also less sensitive to polar impurities in the eluent, such as water. The reversed-phase chromatography uses steel or plastic columns that are 5-30 cm in length, with an inner diameter of 1-5mm and the column is packed with small particles sizes that are from 3 to 10 m m. The small the particles higher is the efficiency but require a high pressure. Such particles form the stationary phase that consist of microporous particles of silica that are permeable to solvent and have a surface are of several hundred square meters per gram.
    There are two kinds of elution that can be done in reversed-phase chromatography. Isocratic and Gradient elution. In Isocratic system, the elution is performed with a single solvent (or constant solvent mixture). If one solvent does not provide sufficiently elution of all components, then the Gradient elution can be used. In this case, increasing amounts of solvent are added to another solvent to create a continuous gradient. In general the two kinds of solvents generally used in preparing the mobile phase in a reversed phase system is acetonitrile and methanol.
    The eluent strength is a measure of the solvent adsorption energy, with the value for pentane defined as 0 for adsorption on bare silica. The more polar the solvent, the greater the eluent strength for adsorption chromatography with bare silica. The greater the eluent strength, the more rapidly will solutes be eluted from the column.
    The retention time is the time required elute the corresponding band from the column and such band is the zone of each sample component from a mixture. The retention volume, Vt, is the volume of mobile phase required to elute a particular solute from the column:
Retention Volume: V_t = t_r x u_v
    Where u_v is the volume flow rate (volume per unit time) of the mobile phase. The retention volume of a particular solute is constant over a wide range of flow rates.
    The void volume of a system in terms of volume, is a measure of system volume from the injector to the detector. In reality, most systems are designed such that the volume of the transport tubes which connect the injector to the column and the column to the detector is negligible compared to the internal void volume of the column. Thus, void volume as measured from a chromatogram in terms of volume (retention time multiplied by flow rate), is generally assumed to be equal to the total internal volume of the bed, that is, that volume of the column which is not being occupied by packing material. In most cases this include the volume inside as well as outside the packing particles. Void volume is usually easy to measure as a distance on the chromatogram since any component, which does not retain on the packing, will elute at the void volume.
    The t₀ is the void volume of a system in terms of time and it is important to calculate the capacity (K’), which is one parameter of resolution.
The mathematical expression for K’ is:

K’ = T/ T₀ – 1 or (V_s –V₀) / V₀

    The K’ is a numerical value given to the partition coefficient of a compound in a particular separation. For a given substance, K’ represents the ratio of the distribution of the compound in the mobile phase.

Column: C18 13.9 x 150 mm, 3 ?m.
Flow rate: 1.5 ml/min.
Sample: 100 m g/ml methylparaben
              100 m g/ml propylparaben
              Mixture of the above.
   50 m g/ml Uracil
Injection volume: 20 m l
Detection: UV 254 nm
Chart speed: 1cm/min.
Mobile phase:  300 ml each:
  50% acetonitrile in water (150 ml ACN + 150 ml water)
  45% acetonitrile in water (135 ml ACN + 165 ml water)
  35% acetonitrile in water (105 ml ACN + 195 ml water)
  25% acetonitrile in water (75 ml ACN + 225 ml water)
 

It was prepared 300 ml of each the following solutions: 50% acetonitrile in water (150 ml ACN + 150 ml water), 45% acetonitrile in water (135 ml ACN + 165 ml water), 35% acetonitrile in water (105 ml ACN + 195 ml water), 25% acetonitrile in water (75 ml ACN + 225 ml water). The distilled water used was previous purified by Millipore water purification system with 0.22 m m pores filter.
All solutions to the mobile phase were prepared in the high standard of purity to avoid to damage the column and consequently to lose the efficiency of the column by contaminating the column with solutions containing big particles.
After each solution was prepared with the right concentration of acetonitrile, the sample was stirred with magnetic bar. Finally the solution was filtered again through filter in paper by vacuum to avoid formation of bubbles of air and to do the filtration faster and in addition to purify against a particles that could contaminated the solution.
The solutions with different concentration of acetronitrile were used as mobile phase in the HPLC experiment conducted. Each time that a mobile phase was changed the column was equilibrated with a new mobile phase after 5 minutes of purge with the new solution.
The pressure reading was 2148, 1800, 2277 and 2434 psi with the follow composition changes 50, 45, 35 and 25% respectively.
Each peak was identified by the retention time. Once under the same HPLC conditions the retention time for component is the same.

Table # 1.
Retention Time (minutes) for Compounds Injected as a Mixture. Detector l 254 nm.

Table # 2.  Retention Time (minutes) for Compounds Injected Individually.
 

Table 1B:
Retention Times (minutes) of the Compounds in Each Mobile Phase.
 

The mathematical expression for K’ is:
K’ = T/ T₀ – 1 or (V_s –V₀) / V₀

The graph #1 about Relative Retention Map shows that the best mobile phase composition is 45% of acetonitrile. Once such composition yield capacity factors between 1 and 10. Since if k’ values are too low it is likely that the solutes may not be adequately resolved, and for high k’ values the analysis time may be too long. The same graph shows that the co-elution mobile phase is around 57.5% of acetonitrile.

In these chromatography the parameter used to describe retention was the retention time from each peak. The uracil in the experiment has the function to show the void volume from the column because uracil is a polar chemical substance and the reverse-phase column the polarity is almost zero. Consequently, uracil does not retain in the column.
In the table 2B one can see that when the concentration of acetonitrile the retention time elutes faster than at low concentrations of acetonitrile.
The elution order in all composition of mobile phase is methyl paraben and after it, the propyl paraben. The reason is that methyl paraben is a small molecule when compared with propyl paraben therefore methyl paraben will elute first.
There is some change in elution order caused by changing mobile phase strength. In general composition of mobile phase used in this experiment the hydrocortisone eluted after the uracil. However, when the composition of mobile phase was 25% acetonitrile the hydrocortisone eluted after uracil and methyl paraben. The reason for such behavior was that hydrocortisone is organic, for such reason hydrocortisone can do interactions with acetonitrile more easy special dipole-dipole interactions, as results when the acetonitrile is more concentrate the hydrocortisone dilute fast and elute before the methyl paraben and propyl paraben. However, when the concentration of acetonitrile is 25% in the solution of mobile phase, the hydrogen bonds between water and methyl paraben will make the methyl paraben dissolve faster and elute first in the series.
The graph Relative Retention Map can be use as a useful tool to determine the best composition of mobile phase. So, it is recommend that one looks for a composition that yield a capacity factor between 1 and 10.
The way to identify the %ACN where no separation occurs between the compounds is only extrapolating the values in graph and in this case the approximately %ACN is57.5%,
Finally the most desirable retention time is a retention time that is short in time of elution but with a good resolution accepted by the USP.

 Index