Introduction to Chromatography

General Chemistry (CHMA1001)March 6, 2019

Thin Layer Chromatography (TLC)

• The TLC plate is placed in a reservoir of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid.

• The components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others.

http://www.coleparmer.co.uk/products/chromatography/Chromatography.asp

Thin Layer Chromatography

http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html

Thin Layer Chromatography

• The Rf (retention factor) can provide corroborative evidence as to the identity of a compound.- (Qualitative information – “what is present”)

• If two substances have the same Rfvalue, they are likely (but not necessarily) the same compound.

Thin Layer Chromatography

• Rf is dependent on– The solvent system– Adsorbent– Thickness of the adsorbent– Amount of material spotted– Temperature

Factors are difficult to keep constant from experiment to experiment – considered relative (compared to standards run on same plate)

Thin Layer Chromatography• Some samples are colored.

Simply mark by circling the spots before fading.

• Others colorless samples may require UV to visualize samples or to spray with an appropriate reagent.

Thin Layer Chromatography• If a set of standards that cover the concentration

range of interest are separated on the same plate as the sample and the spots simultaneously developed with an appropriate coloring reagent, an approximate estimation of the concentration of the unknown can be made by comparing its intensity with those of the standards.(semi-quantitative analysis)

• For more accurate work, other methods of spot evaluation must be used and this will involve the use of scanning instrumentation (spot scanning techniques).

Gas Chromatography

ØThe stationary phase is usually a high-boiling point liquid. The stationary phase is held permanently in a metal or glass column that is coiled to conserve space.

ØThe mobile phase is an inert gas (such as Helium, nitrogen or argon) which continuously flows through the column.

Schematic Diagram of GC

http://pharmaresearchdevelopment.blogspot.com/2010/12/gas-chromatography-general-introduction.html

Gas Chromatography

• Involves a sample being vaporised and injected onto the head of the chromatographic column.

• The sample is transported through the column by the flow of inert, gaseous mobile phase.

• The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Gas Chromatography

ØThe components of the mixture distribute between the liquid stationary phase and the mobile gas (vapor) phase. The separation mechanism is ABSORPTION.

ØThe components of the mixture distribute between the solid stationary phase and the mobile gas(vapor) phase. The separation mechanism is ADSORPTION.

Gas Chromatography

ØThe gaseous mixture flows through a detector (for example -TCD – thermal conductivity detector, FID – flame ionization detector, ECD electron capture detector, etc.) at the end of the column and individual components show as different peaks on a recorder.

Chromatogram

http://www.wcaslab.com/gif/perm.gif

Qualitative Analysis

• Retention time (tR) is used for identification in comparison with the retention time of a standard.

http://www.clu-in.org/characterization/technologies/gc.cfm

Quantitative Analysis

• The size (area; height) of the peak is used for quantitative information.

• Comparison to standards (standard curve).

http://online.cit.edu.au/toolboxes/labtech/Laboratory/StudyNotes/snGCRetentTimePeaks.htm

Common Uses for GC

ØPesticidesØFatty acidsØAntioxidantsØEnvironmental contaminants

High Pressure/Performance Liquid Chromatography(HPLC)• HPLC is a type of chromatography in which

the mobile phase is a liquid which is forced under high pressure through a stainless steel column packed with a sorbent.

• The stationary phase (sorbent) is a liquid that is coated to solid particles

• The mobile phase is a liquid (usually mixture of organic solvents, water and/or buffers).

• The separation Mechanism is Absorption.

High Pressure/Performance Liquid Chromatography(HPLC)• The separated sample exits the column

and individual components are detected by a detector (for example UV- Vis, Fluorencence, refractive index, etc.).

• Individual components show as different peaks on a recorder.

Schematic Diagram of HPLC

http://www.comsol.com/stories/waters_corp_hplc_systems/full/

Quantitative and Qualitative Analysis

• Retention time (tR) is used for identification in comparison with the retention time of a standard.

• The size (area; height) of the peak is used for quantitative information. Comparison to standards (standard curve).

Chromatography

Type of

Chromato-

graphy

Separation

Mechanism

Mobile

Phase

Stationary

Phase

Quantitative

Analysis

Qualitaitve

Analysis

Column Adsorption Liquid

(organic)

Solid

(alumina)

*Intensity/

hue

*Color and

order

TLC Adsorption Liquid

(organic)

Solid

(alumina)

Intensity/

hue

Rf Value

GC – liquid

GC - Solid

Absorption

Adsorption

gas Liquid

Solid

Area/height

(std. Curve)

tR Value

HPLC Absorption Liquid

(organic/

water/

buffer)

Solid

coated

with a

liquid

Area/height

(std. Curve)

tR Value

Efficiency of the Chromatographic Process

• The efficiency of a chromatographic column is a measure of its ability to separate the components of a given mixture.

• It is important that the components do not spread out in the column and overlap each other.

Column Efficiency• The unit of efficiency of a

chromatographic column is expressed as a theoretical plate.

• A Theoretical plate is the length of a column in which an equilibrium distribution of solute between mobile phase and stationary phase is established.

http://chromatographyonline.findanalytichem.com/lcgc/article/articleDetail.jsp?id=714660

Factors Affecting Column Separation Efficiency

• Flow Rate - There is a certain velocity, the so-called optimum flow, at which the plate number is highest (and H is lowest). A lower or a higher flow rate provides less plates (higher H).

• Column Length – Increased length of column can lead to an increase in total theoretical plates. With great increases in length there will be a greater diffusion of the sample.

• Temperature – Decreases in temperature will lead to poorer separations or increased plate heights.

Factors Affecting Column Separation Efficiency

• Particle Size - the plate number is inversely related to the particle size of the column packing. The smaller the particles, the higher the plate number and the separation power.

• Particle Size Distribution - The column packing consists of particles with flow channels in between. Due to the difference in packing and particle shape, the speed of the mobile phase in the various flow channels differs and analyte molecules travel along different flow paths through the channels.