Chromatographic Process

Provides the analyte transport. Immobile phase. Mixture of components dispersed in the mobile phase.

 It is the column packing material that are the media producing the separation.  The properties of this media are important for successful separations.

chromatogram Use HPLC to separate the compounds physically Qualitatively by ? Quantitivly by ?

Total volume of eluent (in ml) require to elute certain substance. V R = t R * F

Time from injection point to maximum detector response for corresponding compound. Time require for non retaine substance to pass through the column. Use to describe the migration rate of solutes on columns. K=(tR -t ₀ )/t ₀ Recommended to be 2-10 Use to describe the separation of band centre ᾳ =K2/K1 should be > 1.2

It is measured in number of theoretical plates. N= 16 ( t R /w)². H=L/N * H plate height. *column length. R = 2(t R2 –t R1 ) / (w2 +w1) Should be 1.5

problems 1-On given 200 cm column the sample required 4.80 min. to emerge, an air bubble required 0.5 min.,and the time required for the sample pass the detector was 0.40min. Calculate N and platelet height? 2- the adjusted retention distance for a lipid sample on HPLC chromatogram with a 1.5m column was 59.6 cm. the recorder was calibrated at 3.60cm/min. the width of the peak at the base was 12.0 cm calculate N?

Width of peak base (w)(min) Retention time (min) -3.1Non retained A B C D

 Quantitative method.  Using external standard method (more basic and common).  AUC is directly relashion to the concentration.  It calculated by calibration curve or by matching point.