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Chromatography Separation using Chromatography Definition of standard chromatographhic terminology Types of Stationary Phase Types of Mobile Phase Normal Phase HPLC Reverse Phase HPLC
Chromatography
"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
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 :-
Theory
Plate theory Eq b/w mobile and statioary phases Prob b/w solute phase and stationary phase.
Chromatogram
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.
Resolution
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).
STATIONARY PHASE
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
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).
VIDEO
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 :-
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
HPLC
Solvent reservoirs High pressure pump Mixer Injection device Column Detector PC
References
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.