Chromatography General - PowerPoint PPT Presentation

ChromatographyGeneral

TLC/PC

PC-Paper Chrom

HPLC

GC/SFC

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

Sorption problems

ADsorption

ABsorption

Different sorptions explained

Partition

Ion exchange

Adsorption

Size Exclusion

Affinity

tR = retention time

tm = dead time

H

W1/2

1/2H

unretained

• 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

Chromatography – Peak Broadening

• 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

Amobile Astationary

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

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)

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

H

Plate height (cm)

Cu

Mass transfer

Multipath effect

A

Diffusion (Longitudinal)

B/u

Mobile phase velocity

DtR

tR1 tR2

R = 2(tR1 – tR2)/Wb1 – Wb2

Response

100%

Baseline resolution

for Gaussian

shape peaks = 1.5

Wb1

Wb2

• 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

• 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

tR = retention time

tm = dead time

H

W1/2

1/2H

unretained

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

Peaks Broadening