BASIC
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
EFFECTS OF SOLVENT
CHEMISTRY ON RETENTION AND SELECTIVITY IN HPLC
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.
Introduction:
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%.
Procedure:
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:
Results:
CALCULATED RESULTS:
CAPACITY FACTOR OF COMPOUNDS
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
Discussion:
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.
Index |