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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
A CHROMATOGRAPHIC SYSTEM AND RESOLUTION BETWEEN TWO PEAKS
purpose in this experiment is to grasp knowledge and skill to measure the
efficiency of chromatographic system and to calculate the resolution between
efficiency of a packed column increases as the size of the stationary-phase
particle decreases. In general the particles size in HPLC are between 3 to 10
m m. The small particles give better resolutions because they provide more
uniform flow through the column, therefore reducing the multiple path ways.
Another reason is that the distance through which solute must diffuse in the
mobile phase between particles is on the order of the particle size. The
smaller the particles, the less distance solute must diffuse in the mobile
phase. However, the small particle-size requires more pressure because it
gives more resistance to solvent flow.
In addition the efficiency of a chromatographic system
depends in our abilities to express the quality of a chromatographic
separation and the factors that affect the quality with the methods which can
be used to improve such factors using numbers. Such numbers can define
retention measurements, column performance and the resolution equation.
The retention measurements are the measurements that are
taken from the chromatogram in order to quantify the results. In general such
values are taking as retention times from the solutes and they are measured as
times, volume of solvent or distances on a recorder chart. However, a peak in
a chromatogram can be identified by its retention time, once this varies with
column length and mobile phase flow rate. The best way to identify a peak is
by the capacity factor (k’), which is given by the formula:
K' = tR –
t0 / t0
is the retention time of solute and t0 is retention time of the solute
travelling in the same speed of the mobile phase.
The separation of the two peaks relative to each other is
described by the selective or separation factor (?), which is defined for two
peaks as the ratio of the capacity factors.
a = k2’ /
of separation of one component from another is described by the resolution (Rs),
measured as the difference in retention time of the two solutes divided by
their average peak width.
Rs = tR2 –
tR1 / 0.5 (w1 + w2)
One of the
problems with any form of chromatography is that a band of solutes moves
through the system it becomes dispersed; the longer the solute spreads in the
system the more dispersed that becomes. So, the more efficient the
chromatography column the less of this band spreading will occur.
The column performance is measured by quantities called the
plate number (N), which is defined as follows:
N = 16 (tR /
N = 5.54 (tR
efficient the column, the smaller will be w at a given value of tR.
Resolution Equation shows the dependence of resolution on selectivity,
capacity factor and plate number for two peaks and is the key equation for the
optimization of resolution in chromatography.
Rs = 0.25 ( a
- 1 / a ) ( k / 1 + k) N1/2
Where k is the
average capacity factor for the two peaks and N is the average plate number.
The equation shows that, for a desired degree of resolution,
three conditions have to be met:
- The peaks must be separated from each other (a >1) b- The peaks must be retained on
the column (k >0) c- The column must develop some
minimum number of plates.
Another parameter that is
important in the calculation of the efficiency of a column is the calculation
of asymmetric factor known as tailing.
Tailing is calculated by the follow formula:
Tailing = W0.05
/ 2 F
W0.05 is the measure of peak at 5/100 height from the baseline.
F is the distance of the line that cut W0.05 around the middle to
the point where W0.05 start when the peak starts to be formed.
13.9 x 150 mm, 5 ?m. Part No: WAT 086344 Serial Number: T72971P 086
Mobile phase: 35% acetonitrile, 65% water
Flow rate: 1.5 ml/min.
Sample: 100 m g/ml methylparaben
100 m g/ml propylparaben
Mixture of the above.
Injection volume: 20 m l
Detection: UV 254 nm
Chart speed: N/A.
It was mixed
105 ml of acetonitrile with 195 ml of distillated water previous purified by
millipore water purification system with 0.22 m m pores filter. The
standards solutions were already prepared. However, around 1 ml of each
standard of 100 m g/ml methylparaben, 100 m g/ml propylparaben and mixture of
both standard were transferred to small vials and each vial was label with
respective name of the chemical standard that was transferred to it.
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. In addition the solutions were filtered by vacuum to avoid
formation of air bubbles which can cause problems at backpressure in the
The vials containing the standards were introduced in the
appropriate place in the HPLC and the apparatus was set as written above. The
software of the HPLC received the follow information. Vial # 1 is
methylparaben , vial # 2 is propylparaben and the vial # 3 is the mixture of
the both samples. The samples were injected by automatic injection in the HPLC
system under suitable conditions and two different peaks were observed.
The back pressure was kept by the system around. 2218 psi
once the back pressure can not be over 5000 psi in a pharmaceutical company or
the system shut down itself as self protection.
The peaks were identified in the final chromatogram by the
retention times. Since under the same HPLC conditions the retention time for
component is the same. Each peak was analyzed individually and the retention
time and the measures the widths of peaks at base and half height were done.
Raw Data. Retention and Peak Width Measurements. Detector l 254 nm.
resolution calculated from the two peaks, which was obtained from the mixture
chromatography injection, were 13.
Rs = 2 (11.90
cm – 4.75 cm) / 0.4 cm + 0.7 cm)
Rs = 13
table #2, one can observer that there is a certain difference in the values
for plate counts calculated by different method. Such difference is resulted
from the width measured that was used. In the case the tangent method, the
width was take from the base of the peaks at 5/100 height from the baseline.
Once the peaks show a small tailing, the width gives a large number that
decrease significantly the final values in the number of the plates. However,
in these cases the tailing 1.3 and 1.9 respectively to MP and PP are following
the regulations of USP because are less than 2.0. If the peaks are not
symmetric the column efficiency is better determined by the half width method.
If one wants to improve the efficiency in a chromatographic
separation, he or she should avoid that the solute slight interact with the
stationary phase, in this way may avoid tailing. Some additives such as
triethylamine and trifluoroacetic acid are often used to mask strong
adsorption sites to reduce tailing. Sometimes the tailing may indicate the
presence of an isomer or another compound. Others chemical and physical
phenomenon can cause the low efficiency in a chromatographic separation. Per
example: The dispersion due to flow through tubes. The speed of the liquid
varies over the cross-sectional area of the tube, and the solute molecules
near the center move faster than those near the walls.
The degree of the separation from the two components was
satisfactory, once the degree of separation was bigger than 10 inside the USP
regulations. If the resolution is high, the technician is going to waist more
time during each sample running. Since time is money, it is not desirable to
have a bigger resolution.
exhibited an efficiency of 2900 and 4600 plates to methylparaben and
propylparaben respectively by the half width method. This means that the
present column is in good conditions. If the measured column efficiency were
low, less than 75% of the original measurement for the column, the column
would be in bad conditions.