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Hemant A IIRBS Semester VII ID MS 09/05

Chromatography Separation using Chromatography Definition of standard chromatographhic terminology Types of Stationary Phase Types of Mobile Phase Normal Phase HPLC Reverse Phase HPLC

"A separation process that is achieved by the distribution of the substances to be separated between two phases, a stationary phase and a mobile phase. Those solutes, distributed preferentially in the mobile phase, will move more rapidly through the system than those distributed preferentially in the stationary phase.

Thus, the solutes will elute in order of their increasing distribution coefficients with respect to the stationary phase.

Three basic ways of classifying various Separation techniques : 1. Displacement Development it is effective if the stationary phase is a solid and the solutes are adsorbed on its surface. The solutes adsorb sequentially on the dist. surface and array themselves in the order of increasing adsorption strength. Only a substance more strongly held than the solutes(displacer) can elute the solute out in the order of decreasing adsorption strength 2. Frontal Analysis It separates part of the first compound in a relatively pure state, each subsequent component being mixed with those previously eluted. Consider a three component mixture, containing solutes (A), (B) and (C) as a dilute solution in the mobile phase that is fed continuously onto a column. The first component to elute, (A), will be that solute held least strongly in the stationary phase. Then the second solute, (B), will elute but it will be mixed with the first solute. Finally, the third solute (C), will elute in conjunction with (A) and (B). It is clear that only solute (A) is eluted in a pure form

Separation using Chromatography

3. Elution development Its a series of adsorption extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. Below a typical elution development is shown below :-

Plate theory Eq b/w mobile and statioary phases Prob b/w solute phase and stationary phase.

The baseline is any part of the chromatogram where only mobile phase is emerging from the column. The injection point is that point when the sample is placed on the column. If the sample has a finite volume, then the injection point corresponds the start of the sampling process. The deadpoint is the position of the peak maximum of an unretained solute. It is not the initial part of the dead volume peak as this represents a retarded portion of the peak that is caused by dispersion processes. The peak maximum is the highest point (the apex) of the peak and measurements as dead volume and retention volume. The dead time (t0) is the time elapsed between the injection point and the dead point. The retention time (tr) is the time elapsed between the injection point and the peak maximum. Each solute will have a characteristic retention time.

The retention volume (Vr) is the volume of mobile phase passed through the column between the injection point and the peak maximum. Vr = Qtr Each solute will also have a characteristic retention volume. The peak height (h) is the distance between the peak maximum and the baseline geometrically produced beneath the peak. The peak width (w) is the distance between each side of a peak measured at 0.6065 of the peak height.

It is the distance b/w 2 neighbouring peaks measured at the base.


where N is the plate number, a measure of the performance or the efficiency of the column. is the quotient factor b/w retention times k is a measure of the strength of the interaction of a given compound in a given chromatographic system. The resolution R the distance from peak base to peak base depends only on the following three factors: the strength of the interaction between the compound and the stationary phase(if the peak comes soon or late), i.e. on the k value, the ability of the chromatographic system to distinguish between the two components of interest, i.e. the value, if the relevant peaks are sharp or wide, i.e., the plate number.

Improving Resolution
Consequently, to improve resolution, there are in principle only three possibilities, namely a general increase in the interaction (k value increases), an analyte-specific change in the interaction ( value increases), or an increase in the efficiency of the separation (N value increases).

Non polar hydrophobic species attached by siloxane or ether bonds to the surface Inorganic silica forms the most common support Its mechanical strength Optimum pH 2-8 Silica pretreatment needed

Normal Phase Chromatography

Nominally used to separate non-ionic compounds with high molecular weight. Accounts for 1/5 th of all HPLC separations. SP > MP Silica used is porous and non crystalline and generally of the form :- SiO2-x/2(OH)X]n . H20p Silanols groups are responsible for the polar character of silica packings used in SP.

Reversed Phase Chromatography

Non polar SP Polar MP There is a certain non polar component added to the MP to lower the polarity The SP can also be varied using functional groups attached to it to vary factors such as affinity and retention factors. To make the column efficient high pressure is needed. As a result -> RP == HPLC,UPLC etc..

Optimization of RP-HPLC
Our aims must be : to separate better (higher resolution), to separate faster (shorter retention time), to see more (lower detection limit), to separate at lower cost (economic effort), to separate more (higher throughput).


Chemically bonded SP
They are synthesized by silica based SP by reacting an organochlorosilicane with the gel support material in an appropriate organic solvent. Two types of such phases :1. Monomeric SP :-

2. Polymeric SP :-

1. Monomeric SP :-

2. Polymeric SP :-

Isocratic reverse phase

Using a solvent system whose concentration does not vary with time. We use this method when the sample contains analytes that have similar properties and the hydrophibicity difference is very small. Else, The early eluted components may be unresolved and the final components elute out over a long period of time.

Gradient Reverse Phase

Vary the mobile phase solution continuously in a linear fashion from the beginning to the end of analysis period. The strength of the mobile phase is weak initially and increases gradually and then peaks when separation is completed This method encourages the use of tertiary and quarternary mixtures

The main disadvantage of the gradient method is that the time required to re-equilibriate the column takes few mins to several hours

Equipment's needed
Solvent reservoirs High pressure pump Mixer Injection device Column Detector PC

1. Chromatographic Theory jack Cazes 2. Handbook of HPLC Elena Katz 3. Encyclopaedia of Chromatography Jack Cazes 4. HPLC Solvent guide Paul C Sadek 5. Video courtesy of the Imad Haidar group at the university of minnesota.