1. Created with MindGenius Business 2005® Performance parameters (Advanced) Theoretical Plate Model Performance parameters (Advanced) Theoretical Plate Model Mathematical performance assessment Based on a series of “snapshots” to mimic continuous behaviour Retention time of analyte: t R = t S + t M where t S is the time spent in the stationary phase and rate of movement = 0 t M is the time spent in the mobile phase, rate of movement = mobile phase rate Each molecule has a chance of being mobile or stationary during any “snapshot” Distance travelled between periods in the stationary phase = one theoretical plate Assumes no diffusion in the mobile phase This gives us simple equations describing chromatographic performance

2. Created with MindGenius Business 2005® Performance parameters (Advanced) Chromatographic Efficiency Column Efficiency (measured by plate number: N) N = (t R /s) 2 t R is total retention time, and s is the standard deviation of a gaussian peak BUT s requires accurate determination of points of inflection, so we use N= 5.54 x (t R / peak width at 50% height) 2 Large N indicates good column performance (should be ~10,000 for HPLC) N is increased by: increased temperature, column length decreased stationary phase particle size, flow rate, mobile phase viscosity

3. Created with MindGenius Business 2005® Other variants of chromatographic efficiency Effective N (N eff ): If t R is low, then t 0 affects apparent efficiency N eff = 5.54 x [(t R - t 0 ) / peak width at half height] 2 Plate Height (H): Used to compare columns of different lengths: H= L / N L = column length, N = number of plates H is a measure of plate size, the smaller (lower H) the better (HPLC ~ 10mm) Effective plate height: takes account of columns with different dead spaces H eff = L / N eff Reduced plate height: allows comparison of columns with different particle sizes h = H / dp where dP is the particle diameter (same units as L); (Good HPLC column: h = 3) Performance parameters (Advanced) Chromatographic Efficiency

4. Created with MindGenius Business 2005® The ratio of mass in the stationary phase (mS) to that in the mobile phase (mM) where total mass: m T = m M + m S Capacity Factor: k = m S / m M = K. (V S / V M ) where K is the equilibrium constant, V S is the volume of the stationary phase, and V M is the volume of the mobile phase (dead volume). Determining k Assume V R : V M = m T : m M Then: V R / V M = m T / m M = (m M + m S ) / m M = 1 + k So: V R = V M (1 + k) And: t R = t M (1 + k) Since t M = t 0 k = (t R - t 0 ) / t 0 K should be between 1 and 5 Performance parameters (Advanced) Capacity Factor (k)

5. Created with MindGenius Business 2005® Selectivity Factor ( α ): Comparison of interaction with stationary phase Ratio of capacity factors α = k (B) / k (A) Primarily affected by changing the stationary or mobile phases Larger α means better separation (but little gain in resolution beyond α=3) Resolution (R S ): A measure of how well separated two peaks are: R S = 2(t R peak A - t R peak B) / (wA + wB) Since measuring w is difficult, can use: R S = 0.25 x [( α - 1) / α ] x [ k(B) / (1 + k(B)) x  N where B is the last eluting peak, and N is the plate number for B High Rs is better: should be at least 1.5 for baseline separation Performance parameters (Advanced) Selectivity ( α ) and Resolution (R s )

6. Created with MindGenius Business 2005® Performance parameters (Optimisation) Optimisation depends on type of chromatography, usually involves changes in: Stationary Phase: Hundreds of kinds on offer, choice based on analytes to be separated, cost Differences based on chemical structure, particle size, column bore and length, compressibility of packing Mobile Phase: Use changes in polarity, pH, viscosity Detector: Use best sensitivity available for analytes Flow rate: affects retention time and diffusion and thus performance Amount of sample: Too much will overload the column, too little will be difficult to detect accurately Sample Matrix: Avoid incompatible contaminants, use minimum injection volume, preferable similar to the mobile phase (HPLC) Temperature: increases improve performance (must avoid decomposition of sample) by increasing solubility and reducing viscosity. Very important in GC.

7. Created with MindGenius Business 2005® Performance parameters (Summary) C Chromatographic systems must be optimised to give: Good Peak Shape α, R s ) Good separation/resolution (N, k, α, R s ) Flat and horizontal baseline No “artifactual” peaks Shortest possible analysis times Resolution and Retention time are key