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Educational - HPLC

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The main purpose of this experiment is to examine the effect of mobile phase, which was done with the different solvents and therefore to observer the effects on retention and selectivity chemistry in a reversed phase system.


 According to chromatographic theory, resolution between band pairs depends upon three parameters retention (K), theoretical plates (N) and selectivity (a ) as expressed in equation below:

 R = 1/4 N1/2 ( a - 1 ) [ k / ( k + 1 )]

 This equation tell us that by halving the column length while maintaining retention ( K ) and selectivity ( a = K2/k1 ), resolution decreases by

 30 %.
 This loss in resolution can be recovered without modifying K or a (without changing the stationary phase or the mobile phase), because N can be increased without increasing column length. In a given column length, N increases as packing particles size decreases.

 So, to alter the resolution of a HPLC system we can change the stationary phase through less or more polar columns or changing the solvent in the mobile phase, increasing or decreasing the size of column or changing the size of packing particles in the column.
 Change the chemistry of mobile phase in Reverse Phase Chromatography is a basic procedure to find the right resolution from two or more compounds. Such procedure is done using two solvents basically, methanol and acetonitrile. However the solvents should to have the same polarity with purpose to observer more accurate the interactions of solvent from the mobile phase with the compounds once each compound interact with different mobile phase in different ways because some compounds are more soluble in one or another solvent.
 In HPLC the interaction of the solute species with each substance in the column is known as relative distribution of a solute between two phases. The relative strengths of these interactions are determined by the variety and the strengths of the intermolecular forces that are present, or, in more general terms, by the polarity of the sample and that of the mobile and stationary phases.
 Intermolecular forces may be caused by a solute molecule having a dipole moment, whereby it can interact selectively with other dipoles, or if a molecule is a good proton donor or acceptor it can interact with other such molecules by hydrogen bonding. Molecules can also interact via much weaker dispersion forces, which rely on a given molecule being polarized by another molecule.
 The more polar a molecule, the more strongly it can interact with other molecules through the mechanisms above. If the polarities of stationary and mobile phases are similar then it is likely that the interactions of solutes with each phase may also be similar, leading to poor separations. Thus for hydrocarbon-type (non-polar) stationary phases we need a polar mobile phase, whereas unmodified silica, which is highly polar, needs a mobile phase with relatively low polarity.
 If we are concerned with the separation of solutes that are chemically very similar, we should try to choose a stationary phase that is chemically similar to our solutes. Changing mobile phase polarity usually alters the retention of solutes.

Materials and System Set-up:

Column: C18, 15 cm x 3.9 mm, 5 m m.
Flow rate: 1.0 ml/min.
Sample: 100 m g/ml methylparaben
              100 m g/ml propylparaben
   100 m g/ml hydrocortisone
              Mixture of the above.
   50 m g/ml Uracil
Injection volume: 20 m l
Detection: UV 254 nm
Detector Sens: 1 AUF.
Chart speed: 1cm/min.
Mobile phase:

Mobile Phase A: ACN:H2O (45:55)
Mobile Phase B: MeOH:H2O (52.5:47.5)
Mobile Phase C: MeOH:ACN:H2O (26.3:22.5:51.2)

Polarity of solvents:

Acetonitrile = 6.2
Methanol = 6.6
Water = 9.0

For 45% of acetonitrile how much is necessary for methanol to have the same polarity?
(6.2 x 45/100) + (9.0 x 55/100)=
2.79 + 4.95 = 7.74
7.74 = (6.6 MeOH x X/100) + (9.0 x (100 X/100)
774 = 6.6X + 900 9.0X
126 = 2.4X
X=52.5% Consequently, we should to use 52.5% methanol instead 49.2%.


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 or methanol, 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 and methanol were used as mobile phase in the HPLC experiment conducted. Each time that a mobile phase was changed the column was equilibrated with the new mobile phase after 5 minutes of purge with the new solution.

Table #1. Approximate back pressure with each mobile phase:
Mobile Phase A Mobile Phase C Mobile Phase B
  916~930 psi  1265~1272 psi 1758~1772 psi

The pressure increase due to viscosity of the organic compounds. The viscosity of methanol is higher than the acetonitrile for this reason the backpressure for methanol solution is higher than the acetonitrile. However, the volume of water decreasing in the order Mobile Phase A, Mobile Phase C and Mobile Phase B shows  that water has the highest viscosity among the three.
Each peak was identified by the retention time. Once under the same HPLC conditions the retention time for component is the same.

Part A:

Raw Data:

Table # 2. Retention Time (minutes) for Compounds Injected as a Mixture and Individual. Detector ? 254 nm.

tR (minutes) Mobile Phase A Mobile Phase B Mobile Phase C
uracil 1.013 1.075 1.070
HC 1.504 4.580 3.079
MP 1.665 2.235 2.002
PP 2.905 6.044 4.939
Mix-Peak1 1.486 (HC) 2.215 (MP) 2.022 (MP)
Mix-Peak2 1.632 (MP) 4.578 (HC) 3.091 (HC)
Mix-Peak3 2.882 (PP) 6.036 (PP) 4.938 (PP)

MP = Methylparaben, HC = Hydrocorstisone, PP = Propylparaben


Part B:




The mathematical expression for K is:
K = T/ T0 1 or (Vs V0) / V0

Table #3. K' Value for the Compounds in the Mixture
K' Value Formula HC MP PP
Mobile Phase A (tR/1.013)-1 0.467 0.611 1.845
Mobile Phase B (tR/1.075)-1 3.259 1.060 4.615
Mobile Phase C (tR/1.070)-1 1.889 0.890 3.615

MP = Methylparaben, HC = Hydrocorstisone, PP = Propylparaben

  Table #4. Alpha Value For All Pairs of Compounds in the Mixture

        alpha = K'2 / K'1 = tR2' / tR1' (tR' = tR - tO)
Mobile Phase A Mobile Phase B Mobile Phase C
PP/MP = 3.020 PP/HC = 1.308 PP/HC = 1.416 HC/MP = 3.075 PP/HC = 4.602 HC/MP = 2.122

MP = Methylparaben, HC = Hydrocorstisone, PP = Propylparaben


The elution order in ACN mobile phase was hydrocortisone, methylparaben and propylparaben. The elution order in MeOH mobile phase was methylparaben, hydrocortisone and propylparaben. Once in the mobile phase, which contains MeOH, the degree of hydrogen bond is bigger because MeOH is polar. Therefore, the methylparaben will elute first than hydrocortisone. However, in the ACN mobile phase the dipole-dipole charge between hydrocortisone and ACN will result in a faster dilution of hydrocortisone than methylparaben.
 There is a huge difference in retention time between acetonitrile mobile phase and methanol mobile phase. The acetonitrile mobile phase elute faster than the methanol mobile phase, such behavior is due in consequence of rheologic properties of the solutions. In this case methanol is more viscous than acetonitrile.
 The predominant interacting forces between acetonitrile molecules and between methanol molecules are due in the interactions that each phase has with the compounds. In this experiment we can see that the interactions between acetonitrile and hydrocortisone due the dipole-dipole charges make the hydrocortisone elute first. When methanol is used in the mobile phase the hydrogen bonds make the methylparaben elute first than hydrocortisone. Consequently, since the both mobile phases have equal polarity all effects are because of solubility of the compound in one or in the other mobile phase.
 The relative retention (a ) is always expressed as a number greater than unity. The relative retention ( a ) can not be smaller than the unity.
 Each organic solvent had a different effect in the backpressure of the HPLC column system. The pressure reading was 916~930 psi to 45% acetonitrile and 1758~1772 psi to methanol at 52.5%. The viscosity of methanol is higher than the acetonitrile for this reason the backpressure for methanol solution is higher than the acetonitrile.

 An organic solvent for to use in HPLC must elute the compounds in a short time and should to have a relative retention (a ) higher than 1 allowing this way a good separation between each compound.