introduction to chromatography - dr. hoyle · introduction to chromatography day 2 - read-only...
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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.