Chemistry Extended Response Chromatography Flashcards
Stationary Phase in TLC?
fine layer of adsorbent material e.g. alumina or silica, coated on an inert solid surface such as a glass plate
What does the stationary phase in TLC separate components by?
Polarity, Affinity; Physisorption; Strength and number of Intermolecular forces - more for polar, less for non-polar.
What is the mobile phase in TLC?
Suitable organic or water solvent
How does the mobile phase move up the TLC plate?
Capillary action
By what features does the mobile phase separate components in TLC?
Polarity, Affinity, Solubility
Retention Factor Definition
Retention factor is the distance travelled by an individual component along the stationary phase divided by the elution distance of the mobile phase.
Polarity interactions in TLC?
In normal phase:
Polar compounds are more attracted to the stationary phase and less attracted to the mobile phase; higher Rf
Non-polar compounds are less attracted to the stationary phase but more attracted to the mobile phase; lower Rf
and vice versa. for reverse-phase
Solubility and affinity for stationary and mobile phases
As solubility in and affinity for the mobile phase increases; the elution of the compounds becomes slower; lower Rf
As affinity for the stationary phase increases, slower elution of compounds; higher Rf
Steps of TLC
- The stationary phase is prepared with adsorbent
- The sample is applied towards the bottom of the place
- The plate is placed vertically in a developing chamber
- Separation takes place due to combined action of factors such as polarity, affinity, solubility, and adsorption levels for the stationary and mobile phases
- The plate is taken out of the developing chamber
- The samples resolution is visualized directly for colored samples or alternatively for invisible samples that have fluorescent properties, UV-light may be used for the visualisation.
What is the purpose of TLC
To qualify substances by visualisation; no quantification
Limitations of TLC
Requirement of pre-known Rf comparative values
Almost continuous possibility for separation by controlling plate length
Since TLC is generally conducted within an open system, environmental factors such as temperature and humidity can change the separation efficiency
Forensic and analytical application of TLC?
Forensic application:
- Drug Testing
Analytical applications:
- Identification of pesticides
- Lipid analysis
What is the stationary phase in gas chromatography?
The stationary phase is a very thin layer of inert high-boiling point liquid on an inert solid surface such as silica beads compressed in a long skinny tube. This tube is coiled multiple times inside a column oven which is kept at a constant temperature.
What is the mobile phase in gas chromatography
The mobile phase is an inert gas. This gas carries the sample.
Retention time definition GC?
Retention time refers to the time taken for a compound in a substance to pass through the thin tube called a column and reach the detector.
What is the purpose of GC? How is it done?
To identify and quantify the substances in a sample; by comparing to standard reference chromatograms
Features of the inert carrier gas
- Inert gas
- Low molecular weight to reduce resistance to components
Gas Chromatography (GC) Process
- Gaseous or liquid sample is injected into the vaporisation port
- The sample vaporisation port is then heated up for liquid samples.
- The vaporised sample is then introduced into the column by the action of the carrier gas from the cylinder.
- Separation occurs by polarity and other physical properties of components.
- Components exit column and enter into detector
- Detector outputs electrical signals, quantifying components
- Data processing unit plots a gas chromatogram (intensity of signal on vertical axis, retention time on horizontal axis). Qualifies and quantifies the sample components
How is the quantity of a substance read from a chromatogram
Area under substance peak
What factors does the stationary phase separate the components by in GC?
Volatility, Polarity, Solubility, and affinity
What factors does the mobile phase separate the components by in GC?
Contrasting volatility, solubility, polarity and affinity.
Analytical and Forensical application of GC
Forensic Applications:
- Forensic toxicology
- Drug Analysis
- Arson Investigation
What is the stationary phase in HPLC?
The stationary phase in HPLC in normal-phase is a polar solid, while in reverse-phase HPLC, it is a non-polar solid. The part of the solid packed in the column that reacts interacts with the target compounds are more accurately referred to as the stationary phase
What is the mobile phase in HPLC?
In normal-phase HPLC the mobile phase is a non-polar or relatively less polar liquid compared to the stationary phase. In reverse-phase HPLC, it is a polar liquid.
What is the purpose of HPLC?
To identify and quantify different components in a sample.
Compounds with the highest retention time in HPLC mainly due to polarity…
Elute last
Compounds with the lowest retention time in HPLC mainly due to polarity…
Elute first
Limitations of Gas Chromatography
- GC only has the ability to separate volatile (low boiling point) and thermally stable compounds. Highly sensitive equipment
- The sample retention times of a substance are usually required to be compared to a standard run with chemically pure substances of the compound suspected to be analysed in a chromatogram and signified.
HPLC process?
- A high pressure pump is attached to the column
- The column is filled with adsorbent material such as silica or alumina, that has a very small particle size, to increase surface area for interaction - The mobile liquid phase is taken from the solvent reservoir and pumped through the high-pressure pump and injected into the column by the sample injector, alongside the sample
- Separation occurs by polarity, affinity and molecular weight.
- The separated components are then introduced to the UV-Vis detector, where it exposed to UV light
- The absorbance values are recorded
- Detector outputs signals which quantify and provides the purity of components.
- Along with the retention times in the column, the absorbance values are recorded on the vertical axis on the chromatogram
Limitations of HPLC
- Lack of Universal detector
- Lower separation efficiency than capillary gas chromatography due to lower peak capacity
- Very expensive system and large quantities of expensive organic compounds are use
- Not suitable for gradient elution
In HPLC and GC, the detection of which types polarity of compounds are reverse phase, and normal phases suitable for respectively
Reverse phase is suitable for detecting polar compounds
Normal Phase is suitable for detecting polar compounds
Factors that lead to better of more efficient separation in HPLC
- Increased column temperature
- Wider and longer column
- Higher concentration of solvent
- Sample molecular size
HPLC forensic and analytical applications
Analytical Applications:
- BAC analysis
- Drug analysis
Forensic Applications:
- Trace analysis
