Make your own free website on
E-mail this page to a friend
Teory  | Project1 | Project2 | Progect3 | Project4|Project5|Project6|Project7| Home | Contact Me 
Educational - HPLC

Welcome to  HPLC site!

On this home page, I'll introduce my Tecnical Teory  and  Project in  my daily Work. 





The purpose in this experiment is to grasp knowledge and skill to measure the efficiency of chromatographic system and to calculate the resolution between two peaks.


The 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

    Where tR 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 / k1

    The degree 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 / w)2


N = 5.54 (tR / w1/2)2

    The more efficient the column, the smaller will be w at a given value of tR.

    The 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:

    a - 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.


Materials and System Set-up:

Column: C18 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 system.
    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.


Table 1. Raw Data. Retention and Peak Width Measurements. Detector l 254 nm.


tR (cm)

w1/2 (cm)










Table 2. Plates Counts.



N tangent



 2000 N

 2947 N



 4903 N

 4624 N


      The 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



    In the 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.


    The column 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.