CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

Distribution Coefficient (Equilibrium Distribution ) Definition:   Different affinity of these 2 components to stationary phase causes the separation. Concentration of component A in stationary phase Concentration of component A in mobile phase

Retention Time The retention time of a solute is taken as the elapsed time between the time of injection of a solute and the time of elution of the peak maximum of that solute. The corrected retention time of a solute is the retention time minus the retention time of a completely unretained solute. By multiplying the corrected retention time of a solute by the exit flow rate then the corrected retention volume can be obtained. If the mobile phase is compressible (i.e. the mobile phase is a gas) a pressure correction must be applied which is a function of the column inlet-outlet pressure ratio.

Chromatogram – Basic Parameter tR = retention time tm = dead time H W1/2 1/2H unretained

Peak Shape The peak shape in chromatography is the name given to the type of curve resulting from plotting the concentration of solute in the mobile phase (or the mass of solute per unit time) eluted from a chromatographic column, against time. Its shape can be described mathematically or empirically. If the curve can be described by the error function then it is often described as a Gaussian or Error Function curve. If the adsorption isotherm is not linear the peak will be distorted. A non linear isotherm can cause different types of peak distortion: if the non-linearity results from column overload, then the peak will have a sloping front and a sharp tail. For obvious reasons this is called an overload peak. If the support in a packed column has adsorptive properties, the peak will exhibit a long tail and, again for obvious reasons, is called a tailing peak.

Peak Shape As the speed of migration of the peak is inversely proportional to the distribution coefficient, the front to the peak will migrate a little faster through the column than the rear of the peak and, thus, the front of the peak will be compressed and the rear of the peak extended, producing a slightly asymmetrical peak. Most chromatographic peaks will exhibit slight asymmetry resulting from thermal effects. In the front of the peak there is a net amount of the solute dissolving into the stationary phase which results in the heat of solution being continuously released. Breadth of a band increases as it moves down the column because more time is allowed for spreading to occur. Zone breadth is directly related to residence time in column and inversely related to velocity at which mobile phase flows.

Column efficiency The efficiency of a chromatographic column is a measure of the capacity of the column to restrain peak dispersion and thus, provide high resolution. The higher the efficiency, the more the peak dispersion is restrained, and the better the column. The column efficiency will vary with the retention of the peak The expression for calculating the column efficiency can be derived from the plate theory. The Height Equivalent to the Theoretical Plate (HETP) or the Variance per unit Length of a Column is calculated as the ratio of the column length to the column efficiency.

Variables that affect column efficiency Linear velocity of mobile phase Diffusion coefficient in mobile phase Diffusion coefficient in stationary phase Capacity factor Diameter of packing material Thickness of liquid coating on stationary phase

Theories of Chromotography Basic theories applicable to chromatography, Plate Theory Rate Theory The Plate theory describes, Mechanism of retention Calculation of the retention volume of a solute and the column efficiency. The Rate theory describes, Process of peak dispersion (band spreading) Calculation of the variance per unit length of a column (the height of the theoretical plate, HETP) Mobile phase velocity Other physical chemical properties of the solute Distribution system.

Plate Theory A number of different peak dispersion processes are proposed Describes the contribution of each process to the total variance of the eluted peak. The final equation gives an expression for the variance per unit length of the column. The processes proposed are Eddy diffusion, Longitudinal diffusion, Resistance to mass transfer in the mobile phase and Resistance to mass transfer in the stationary phase.

Rate theory The rate theory has been developed differently by a number of well established scientists in the field. This has resulted in a number of different equations; viz. Van Deemter Equation, Giddings Equation, Huber Equation, Horvath Equation and Knox Equation. All the equations give a type of hyperbolic function Predicts a minimum plate height , Optimum velocity, Maximum efficiency. At normal operating velocities it has been demonstrated that the Van Deemter equation gives the best fit to experimental data.

Rate Theory Based on a random walk mechanism for the migration of molecules through a column takes into account: band broadening effect of rate of elution on band shape availability of different paths for different solute molecules to follow diffusion of solute along length

Chromatography – van Deemter Plot Plate height (cm) Cu Mass transfer A Multipath effect B/u Diffusion (Longitudinal) Mobile phase velocity

Van Deemter factors: Conclusions: Minimum value for H is achieved when: stationery phase thickness is minimal column packed with the smallest particles capillary columns have the smallest internal diameter mobile and stationary phases have low viscosity and high diffusion coefficient

Chromatogram – Basic Parameter tR = retention time tm = dead time H W1/2 1/2H unretained

Chromatography - Resolution

Column Resolution The resolution Rs of a column provides a quantitative measure of its ability to separate two analytes. A chromatogram for species A and B on three columns having different resolutions. Column resolution can be defined as Rs = (tR)B - (tR)A/ WA/2 + WB/2 = 2[(tR)B – (tR)A]/WA + WB Effect of capacity and selectivity factor on resolution: Rs = N½/4 (α-1/α)(K'B / 1 + K'B ) N= 16 R2s (α-1/α)2(1 + K'B / K'B )

THANK YOU