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Chromatography General. Chromatographic Process. Chromatographic Systems. Chromatographic Techniques. TLC/PC. PC-Paper Chrom. HPLC. GC/SFC. Chromatography – Separation Mechanism. Adsorption Partition Ion - Exchange & Ion - Interaction Size Exclusion

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chromatographic techniques
Chromatographic Techniques

TLC/PC

PC-Paper Chrom

HPLC

GC/SFC

chromatography separation mechanism
Chromatography – Separation Mechanism
  • Adsorption
  • Partition
  • Ion - Exchange & Ion - Interaction
  • Size Exclusion
  • Affinity (antibody-antigen interactions; chemical interaction; attraction)
  • Complexation - Chelation
  • Ion – exclusion (Separation of weak acids)
slide6

Sorption problems

ADsorption

ABsorption

Different sorptions explained

chromatograhy mechanism of separation
Chromatograhy – Mechanism of Separation

Partition

Ion exchange

Adsorption

chromatogram basic parameter
Chromatogram – Basic Parameter

tR = retention time

tm = dead time

H

W1/2

1/2H

unretained

chromatographic theories
Chromatographic Theories
  • Adjusted retention time: tR’ = tR – tM
  • Plate theory – distillation – plate number

N = 5.54[(tR – tM)/w1/2]2

  • Plate height H = L/N
  • This theory did not include interaction of analytes with stationery phase
chromatographic theories14
Chromatographic Theories
  • Rate Theory – kinetic factors – van Deemter

H = B/u + Cu (+ A)

Where: u – velocity of mobile phase

B – effect of molecular diffusion

C – Resistance to mass transfer

A – Spreading related to different distance traveled by molecules in packed columns

chromatography equilibrium
Chromatography - Equilibrium

Amobile Astationary

van deemter factors
Van Deemter factors:

Molecular diffusion (B)– in mobile phase

  • proportional to time analyte spends in a column
  • affected by diffusion coefficient of analyte in mobile phase
  • affected by temperature and pressure
  • not important in LC – low diffusion coefficient
  • inversely affected by mobile phase velocity
van deemter factors18
Van Deemter factors:

Resistance to mass transfer (C):

  • Mass transfer in mobile and stationary phase
  • Lack of equilibrium – moving phase
  • Affected by thickness of liquid phase
  • Affected inversely by the diameter of particles or inner diameter of capillary column
  • Lower at higher temperatures (viscosity)
van deemter factors19
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
chromatography van deemter plot
Chromatography – van Deemter Plot

H

Plate height (cm)

Cu

Mass transfer

Multipath effect

A

Diffusion (Longitudinal)

B/u

Mobile phase velocity

chromatography resolution
Chromatography - Resolution

DtR

tR1 tR2

R = 2(tR1 – tR2)/Wb1 – Wb2

Response

100%

Baseline resolution

for Gaussian

shape peaks = 1.5

Wb1

Wb2

chromatography resolution22
Chromatography - Resolution
  • Resolution equation where separation parameters are included:

Rs = ½ x (a-1/a+1) x k’2/1+k’2x (L/h)1/2

Where: a – selectivity factor (separation) a = tR1/tR1

k’ – migration term, capacity factor;

k’ = ms/mm

L – column length

h – plate height

chromatography
Chromatography
  • Qualitative Analysis
      • Retention data – RT; Rf; RRT; Kovacs Index
  • Quantitative Analysis
      • Peak area and height usually proportional to the amount of component
      • Calibration
      • Internal Standard method
      • External Standard method
      • Area Normalization method
chromatogram basic parameter25
Chromatogram – Basic Parameter

tR = retention time

tm = dead time

H

W1/2

1/2H

unretained

slide26

4

RRT1 = RT1/RTIS

RRT2 = RT2/RTIS

RRT3 = RT3/RTIS

1

3

Cholesterol

Accurate to e few

digits (2) at fourth

Decimal Point

IS α - Cholestane

2

5