FE 315 INSTRUMENTAL ANALYSIS

TLC/FID
(Thin Layer Chromatography/Flame Ionization
Detector
Dr. Hasene KESKİN ÇAVDAR
2024
Contents
• Information about experiment report/data
• General Information about Chromatography
• General Information about TLC/FID
• Working Principle of TLC/FID
• Experimental Section
Experimental Section
Experimental Procedure
TLC/FID Instrument Flaming using hydrogen Blank scan for activation

Drying of the chromarod First development Spotting of the sample

Second development Drying at 120 C Scanning

Monitorizing
DATA

Identify the peaks and their

percentages that were obtained after
the experiment considering the
chromatographic separation
principle.
Chromatography
• Chromatography is a technique for the analysis and separation of chemical mixtures.
• Individual components of a chemical mixture can be analyzed by chromatographic methods.
• The separation principle is based on the polarity difference between the sample components,
stationary phase and the mobile phase.
• Different affinities of the different components of the sample towards the stationary and mobile
phases results in the differential separation of the components.
Stationary Phase and Mobile Phase
• Stationary phase is solid and mobile phase is liquid or gas.
• Mobile phase (carrier) is the solvent moving through the stationary phase.
• Stationary phase (adsorbent) is the substance that stays fixed inside the column
• Stationary phase does not move, while the mobile phase flows across, or through the solid phase
Examples for Chromatographic methods
• Thin Layer Chromatography (TLC)
• High performance liquid chromatography (HPLC)
• Gas chromatography (GC)
• Ion-Exchange chromatography (IEC)
• Affinity chromatography (AC)

• Each chromatographic metod differs in the kind of stationary and mobile phase they use.
General Information about TLC/FID
TLC can be used for:
- Determining the number of components in a mixture
- Determining the identity of compounds
- Determining the purity of a compound
- Monitoring the progress of a reaction

Figure 1. khanacemy.org/18.10.2020
General Information for TLC/FID
• Stationary phase: Chromarods are thin quartz rods coated with an adsorbent such as silica gel or
aluminum oxide embedded in porous sintered glass can be prepared by coating the rods with a
suspension of the adsorbent

• Mobile phase: The solvent in which the rod is dipped and that runs up the plate by capillary
action is the mobile phase.

• The stationary phase (i.e. Silica) is very polar in nature, while the solvent is less polar compared to
silica.
General Information about TLC/FID
• TLC/FID analysis consists of three steps:
• spotting,
• development
• monitorizing and quantification
1- Spotting
Spotting of the sample on chromarod:
• Sample is dissolved in a solvent to produce a dilute (1%) solution.
• Spotting is done using a micro pipet to transfer a small amount of this dilute solution to
chromarod (stationary phase)
2. Development
• Step 1: Chromarods are placed in a development tank containing solvent
(mobile phase).
• Step 2: The mobile phase is adsorbed by silica of the chromarods and travels up
the rod by capillary action.
• Step 3: When the mobile phase is traveling up the chromarod, it moves over
the original spot sample.
• A competition starts between the silica gel rod and the development solvent
for the spotted material. (silica gel: polar, development solvent: non-polar)
• Different components in the sample, having different polarities, will move
different distances from the original spot location and show up as separate
spots.

• When the solvent goes up to nearly top of the silica, the rod is removed the
solvent is allowed to evaporate.
Separation Principle of TLC/FID
• The polar components of the sample has high affinity to silica gel and adhere to the silica
strongly and travel up the rod slowly.
• The less polar or non-polar components has low affinity to the silica and do not adhere
that strongly to the silica and travel up the rod relatively fast with the mobile phase.
 Separation Principle of TLC/FID (Continued)
• What is adsorption and solubility?
• Adsorption shows how well a component of the mixture sticks to the stationary phase,
while solubility is the property of how well a component of the mixture dissolves in the
mobile phase.
• Higher the adsorption to the stationary phase, the slower the molecule will move through
the chromarod.
• Higher the solubility in the mobile phase, the faster the molecule will move through the
chromarod.
Separation Principle of TLC/FID (Continued)
• Question: Why do different compounds have different affinities on the stationary and
mobile phases?
• Answer: “Polarity” of the compounds dictates their affinities towards the stationary and
mobile phases.
3. Monitorizing and Quantification
• After development the rods are burned and ionized in a hydrogen flame. An electrode in the detector is
disposed above the hydrogen flame and the ionized gas. Between this electrode and the gas burner there is a
high voltage and thus negative and positive ions migrates respectively to the burner and collector electrode.
An electric current, proportional to the amount of each separated substance, is detected quantitatively and
amplified by a detector electrode surrounding the negative electrode.
• A data processing unit converts the amplified signal and calculates the percentage area of each peak in the run
as a percentage of total area of all peaks, which in turn corresponds to the relative amount of each
components.
• The peaks are identified as the different fractions based on a retention time specified for each reference peak
in the reference method
Chromatographic separation

The different compounds in the sample mixture move through the stationary phase at different rates, due to
different attractions for the mobile and stationary phases. Separation occurs because each component of a
mixture has a different affinity for the stationary phase, and thus will be adsorbed to a greater or lesser extent
than the other components. A component which is quite soluble in the stationary phase will take longer to
travel through it than a component which is not very soluble in the stationary phase but very soluble in the
mobile phase. As a result of these differences in mobilities, sample components will become separated from
each other as they travel through the stationary phase
Flame-Ionization Detector Working Principle
• After chromatographic separation the rods are burned and ionized in a hydrogen flame. An electrode in the
detector is disposed above the hydrogen flame and the ionized gas. Between this electrode and the gas
burner there is a high voltage, which generates a positive polarity for the burner, and a negative polarity for
the collector electrode. This makes negative and positive ions migrates respectively to the burner and
collector electrode. An electric current, proportional to the amount of each separated substance, is detected
quantitatively and amplified by a detector electrode surrounding the negative electrode. A data processing
unit converts the amplified signal and calculates the percentage area of each peak in the run as a percentage
of total area of all peaks, which in turn corresponds to the relative amount of each components. The peaks
are identified as the different fractions based on a retention time specified for each reference peak in the
reference method (Figure 6).
Example for a chromatogram
3. Monitorizing and Quantification
(Continued)
• Identification of the peaks
• The compounds (peaks) of chromatograms are identified by retention time. Since the retention
time is a specific property of a component, it may be used to identify the component. The
retention time of the unknown component (peak) is compared to the retention time of a so-
called standard.
3. Monitorizing and Quantification
(Continued)
𝑔 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑝𝑒𝑎𝑘 1
Peak 1 %( ) = ×100
𝑔 𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑒𝑎𝑘𝑠 (1+2+3)

𝑔 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑝𝑒𝑎𝑘 2
Peak 2 %( ) = ×100
𝑔 𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑒𝑎𝑘𝑠 (1+2+3)

𝑔 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑝𝑒𝑎𝑘 1
Peak 3 %( ) = ×100
𝑔 𝑡𝑜𝑡𝑎𝑙 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑒𝑎𝑘𝑠 (1+2+3)
REPORT
REPORT
• 1. Purpose (10 p.)
• 2. Theory (10 p.)
• 3. Material/Metod (10 p.)
• 4. Procedure (5 p.)
• 5. Calculation (20 p.)
• 6. Discussion (45 p.)
CALCULATION PART
• Calculate the percentages of each peak (each component).
Discussion Part
• Identify the ethanolysis reaction products (2-monoacylglycerol,
diacylglycerol, and fatty acid ethyl ester) and unreacted triacylglycerol
on the chromatogram considering their polarities. Explain the reason.
• Explain why activation is done before the analysis.