10/11/2013

B. Chromatography Basics
Theory and principles

a)Partitioning and adsorption b)Theoretical plates c)An overview of the types of chromatography

Beginnings
Michael Tswett
1903 Separated plant pigments on Chalk

JP Martin
Gas Chromatography Plate Theory 1941 Practical application 1952

JJ Van Deemter
Rate theory 1956 kinetics/ dynamics

Early 60s Liquid Chromatography detectors

Refractive index UV

10/11/2013

http://www.chromatography-online.org/ion-chromatography/Column-Efficiency.html

Basic Theory
Equilibrium process
Stationary phase Mobile phase

Plates
Derived from distillation Greater number of plates the greater the separation. (More later)

10/11/2013

Adsorption vs Partition
partition chromatography Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography) IUPAC Compendium of Chemical Terminology 2nd Edition (1997)

Adsorption vs Partition
Adsorption Chromatography --. Analytes are separated due to their varying degree of adsorption onto the solid surfaces.

1997 Kevin Yip - Introduction to Biochemical Engineering

10/11/2013

Adsorption vs Partition
Partition Chromatography -- In partition chromatography the stationary phase is bonded to inert particles. Analytes separate due to the differences in their partitioning between the mobile phase and the stationary phase. 1997 Kevin Yip - Introduction
to Biochemical Engineering

Adsorption vs Partition
It is concluded that a clear distinction between partition and adsorption in RPC of non-polar elites is not apparent from thermodynamic analysis. Vailaya A, Horvath C. 1998 J Chromatogr A.;829(1-2):1-27.

10/11/2013

Plate Theory
A plate represents a single equilibrium exchange between the stationary and mobile phases

Insert or draw obligatory Pchem slide here

Lots of cool pChem we will ignore for the most part. Equilibrium is key to function Smaller particles result in reaching equilibrium quickly Failure to reach Keq results in peak abnormalities

10/11/2013

Fractional distillation a distillation in which the vaporization and condensation cycle are repeated successively Theoretical plate the number of effective cycles of vaporization and condensation in a fractional distillation.

Fractional distillation

10/11/2013

Theoretical plates
Math Take home: in situations where you do not count plates you can observe them and calculate them.

Theoretical plates
Math
N = Plates VR= retention volume 2= variance in terms of volume sV st2= variance in terms of time s2= variance in terms of distance W= peak width =4 s expressed in any dimision L = column length

10/11/2013

Theoretical plates

VR L2 N= 2 = 2 sV s

Chromatograms

10/11/2013

Chromatograms

More Chromatograms

10/11/2013

More Chromatograms

Peak shape as a measure of separation efficacy.

Peaks should be symmetric. Peaks should have a Gaussian shape.
Non symmetric peaks and their meaning
Tailing: non-Ideal interaction with the stationary phase Fronting = Overload: just what it says Asymmetry; undefined Unresolved peaks: Just what is says

10

10/11/2013

Types of partition chromatography

Normal Phase Reverse phase Chiral Ion exchange/Affinity Gas/Liquid

Ion exchange/Affinity
+ +

-+ -+
+ + + + +- + +

`+ ` `

++

+-

11

10/11/2013

Ion exchange/Affinity

1997 Kevin Yip - Introduction to Biochemical Engineering

Adsorbtion
TLC
Silica Gel Alumina

Column Chromotography
Silica Gel Alumina

12

10/11/2013

Other types of Chromotography

Gel filtration

Frontal analysis

13