1

Chapter 26

An Introduction to

Chromatographic Separations

Chromatography

2

Chromatography-Model as

Extraction

Chromatography-Model as

Extraction

3

Chromatography

Chromatography-Types

Planar

• 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)

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Chromatography-Types

Chromatography-Types

5

Chromatography-Types

Chromatography-Types

6

Chromatography-General

Process

Chromatography-Output

Chromatogram

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Chromatography-Output

Chromatogram

Chromatography-Output

Chromatogram

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Chromatographic Theory

Chromatographic Theory

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Chromatographic Theory

Chromatographic Theory

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Chromatographic Theory

Chromatographic Theory-Van

Deemter Equation

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Chromatographic Theory-Van

Deemter Equation

Chromatographic Theory-Van

Deemter Equation

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Chromatographic Theory-

Longitudnal Diffusion

Chromatographic Theory-

Longitudnal Diffusion

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Chromatographic Theory-

Longitudnal Diffusion

Chromatographic Theory-Van

Deemter Equation

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Chromatographic Theory-Slow

Equilibration

Chromatographic Theory-Van

Deemter Equation

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Chromatographic Theory-Zone

Theory

Chromatographic Theory-

Column Efficiency

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Chromatographic Theory-

Column Efficiency

Chromatographic Theory-

Column Efficiency

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Chromatographic Theory-

Column Efficiency

Chromatographic Theory-

Column Efficiency

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Chromatographic Theory-

Column Efficiency

Chromatographic Theory-

Column Efficiency

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Chromatographic Theory-

Column Efficiency

Chromatographic Theory-Non-

Gaussian Peaks

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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((tR)y - (tR)x) 2(14.4 - 6.4)Rs = ----------------- = ---------------- = 10.5

Wx + Wy (0.45 + 1.07)

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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 * (tR/W)2

for component A

NA = 16 * (6.4/0.45)2 = 3.2 x 103 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

NB = 16 * (14.4/1.07)2 = 2.9 x 103 plates

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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/NB = (22.6 cm)/(2.9 x 103 plates)

= 7.8 x 10-3cm/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

N1 (Rs)12

--- = -------- = ((Rs)1/(Rs)2)2

N2 (Rs)22

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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

N1 (Rs)12

--- = -------- = ((Rs)1/(Rs)2)2

N2 (Rs)22

where

N1 = (NA + NB)/2 = (3.2 x 103 + 2.9 x 103)/2

= 3.1 x 103 plates

R1 = 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

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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)

(tR)1 (Rs)12

-------- = ---------- = ((Rs)1/(Rs)2)2

(tR)2 (Rs)22

(tR)2 = ((Rs)1/(Rs)2)2 * (tR)1

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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)

(tR)2 = ((Rs)1/(Rs)2)2 * (tR)1

(tR)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

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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

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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