Chapter 26 An Introduction to Chromatographic Separations Chromatography 1
Chromatography-Model as Extraction Chromatography-Model as Extraction 2
Chromatography Planar Chromatography-Types paper chromatography thin layer chromatography (TLC) Gel electrophoresis Column chromatography Adsorption (SEC, IE Affinity) gas-liquid chromatography (GC) high-pressure liquid chromatography (HPLC - high-performance liquid chromatography) Capillary electrophoresis (CE) 3
Chromatography-Types Chromatography-Types 4
Chromatography-Types Chromatography-Types 5
Chromatography-General Process Chromatography-Output Chromatogram 6
Chromatography-Output Chromatogram Chromatography-Output Chromatogram 7
Chromatographic Theory Chromatographic Theory 8
Chromatographic Theory Chromatographic Theory 9
Chromatographic Theory Chromatographic Theory-Van Deemter Equation 10
Chromatographic Theory-Van Deemter Equation Chromatographic Theory-Van Deemter Equation 11
Chromatographic Theory- Longitudnal Diffusion Chromatographic Theory- Longitudnal Diffusion 12
Chromatographic Theory- Longitudnal Diffusion Chromatographic Theory-Van Deemter Equation 13
Chromatographic Theory-Slow Equilibration Chromatographic Theory-Van Deemter Equation 14
Chromatographic Theory-Zone Theory Chromatographic Theory- Column Efficiency 15
Chromatographic Theory- Column Efficiency Chromatographic Theory- Column Efficiency 16
Chromatographic Theory- Column Efficiency Chromatographic Theory- Column Efficiency 17
Chromatographic Theory- Column Efficiency Chromatographic Theory- Column Efficiency 18
Chromatographic Theory- Column Efficiency Chromatographic Theory-Non- Gaussian Peaks 19
Chromatographic Theory-Summary EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (a.) column resolution 2((t R ) y - (t R ) x ) 2(14.4-6.4) R s = ----------------- = ---------------- = 10.5 W x + W y (0.45 + 1.07) 20
EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (b.) the av. no. of plates in the column N = 16 * (t R /W) 2 for component A N A = 16 * (6.4/0.45) 2 = 3.2 x 10 3 plates EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (b.) the av. no. of plates in the column for component B N B = 16 * (14.4/1.07) 2 = 2.9 x 10 3 plates 21
EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (c.) the plate height H = L/N for component B H = L/N B = (22.6 cm)/(2.9 x 10 3 plates) = 7.8 x 10-3 cm/plate EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 N 1 (R s ) 1 2 --- = -------- = ((R s ) 1 /(R s ) 2 ) 2 N 2 (R s ) 2 2 22
EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 N 1 (R s ) 1 2 --- = -------- = ((R s ) 1 /(R s ) 2 ) 2 N 2 (R s ) 2 2 where N 1 = (N A + N B )/2 = (3.2 x 10 3 + 2.9 x 10 3 )/2 = 3.1 x 10 3 plates R 1 = 10.5 EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 N2 = ((Rs)2/(Rs)1)2 * N1 = (1.5/10.5)2 * 3.1 x 103 plates N2 = 63 plates 23
EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 Hav = (HA + HB)/2 = ((7.8 + 7.0)x 10-3)/2 = 7.4 x 10-3 cm/plate L = Hav * N2 = (7.4 x 10-3cm/plate)(63 plates) = 0.46 cm EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 (e) (t R ) 1 (R s ) 1 2 -------- = ---------- = ((R s ) 1 /(R s ) 2 ) 2 (t R ) 2 (R s ) 2 2 (t R ) 2 = ((R s ) 1 /(R s ) 2 ) 2 * (t R ) 1 24
EXAMPLE: Substances A and B were found to have retention times of 6.4 and 14.4 min, respectively, on a 22.6 cm column. An unretained sample of air passed through the column in 1.30 min. The widths of the peak bases were 0.45 and 1.07 min. Calculate the: (d.) the length of column required to achieve a resolution of 1.5 (e) (t R ) 2 = ((R s ) 1 /(R s ) 2 ) 2 * (t R ) 1 (t R ) 2 = (1.5/10.5) 2 * (10.4 min) = 0.13 min = 7.8 sec General Parts of Column Column copper tubing stainless steel tubing glass tubing Support finely divided solids (packed) ground firebrick alumina, specially treated walls of column for capillary columns 25
Parts of Column Stationary Phase stationary phase evenly dispersed on surface of support column chromatography non-volatile, viscous liquids dispersed evenly on surface of support Parts of Column Stationary Phase stationary phase evenly dispersed on surface of support column chromatography non-volatile, viscous liquids dispersed evenly on surface of support 26
Parts of Column Mobile Phase sample mixture carried through stationary phase by mobile phase non-reactive gas in glc (gas-liquid chromatography, gc) non-reactive liquid in llc (liquid-liquid chromatography, lc) Applications of Chromatography Qualitative Analysis Quantitative Analysis Analyses Based on Peak Height Analyses Based on Peak Areas Calibration and Standards The Internal Standard Method The Area Normalization Method 27