Title: Exercise K.1 Thin Layer Chromatography Thin Layer Chromatography of Analgesics and Natural Products
1Exercise K.1Thin Layer ChromatographyThin
Layer Chromatography of Analgesics and Natural
Products
2Chromatography
"Color Writing"
- A general term for a family of lab techniques
used for the separation of mixtures - Comes from two Greek words, chroma (color) and
grafein (to write) - Invented in 1900 by a Russian botanist ( Mikhail
Tsvet) for separating plant pigments - It involves passing a mixture dissolved in a
mobile phase through a stationary phase. - The analytes in the mixture are partitioned
differently between the stationary and mobile
phases which causes the separation. - Many types of chromatography - column, paper,
thin layer, gas, high performance liquid, ion
exchange
http//en.wikipedia.org/wiki/chromatography
3Thin Layer Chromatography
TLC plate
silica gel - silicon dioxide (SiO2)x (a common,
inexpensive stationary phase)
5 x 10 cm 250 µm silica gel layer impregnated
with a fluorescent indicator, on a foil backing
bulk (SiO2)x
4Effects of a Polar Stationary Phase
- Polar solutes are held more tightly than
non-polar solutes - So, will polar or non-polar solutes travel faster
up the TLC plate? - _______________________
- Always? Always.
5Effects of Solvent Polarity
- Polar solvents compete better with the polar TLC
plate so all solutes (even non-polar solutes)
elute faster with polar solvents - We say that polar solvents have greater eluting
power than non-polar solvents. - Always? Always.
6Thin Layer Chromatography
- Five Steps in a TLC Analysis
- Prepare Developing Chamber Saturate with solvent
vapor. - Apply Samples Capillary used to spot
solution of each sample. - Develop Plate This is when the separation
actually occurs. - Visualize Developed Plate View under UV
light. - Interpret Results Determine Rf values
identify components.
71. Prepare Developing Chamber
- 400mL beaker.
- Place a piece of filter paper against side of
beaker. (Cut one side so its flat on the
bottom.) - Pour 10mL of developing solvent (mobile phase)
into the beaker. - Cover beaker with a watch glass and allow to
stand undisturbed for about 15 minutes. - Allows development chamber to become saturated
with solvent.
82. Apply Samples (spot the plate)
TLC plate
Process
- Draw starting line lightly with pencil 1
cm from bottom - Make light x where each spot will be
- Use TLC capillary to transfer and spot dissolved
sample (keep spots very small 1-2 mm.)
93. Develop TLC Plate
- Place spotted TLC plate in developing chamber.
- The solvent is drawn up the plate by capillary
action. - Remove TLC plate when solvent front is 1 cm
from top. - Mark solvent front position with a pencil
immediately.
NOTE During this 20 min. developing stage,
compounds in the original spots are being pulled
through the silica gel.
Developing Chamber (400 mL beaker with 10mL
solvent)
104. Visualize Results
- Allow solvent to evaporate from surface of TLC
plate. - View results under UV light. Look for colored
spots on the fluorescent green background - Trace spots with a pencil while viewing under UV
light. - Mark the center of each spot.
UV
115. Interpret Results (Rf Values)
Solvent Front
Y
T
Z
X
Starting Line
125. Interpret Results (identify components)
- Compare Rf values of components in sample to Rf
values of standards.
13Purpose of Experiment
- To observe the effects of solvent polarity on
order and rate of elution - To identify components in an unknown using TLC
data
caffeine
acetaminophen
vanillin
14Pre-lab Preparation
- Read Technique K (pp. 92-97)
- Omit Resolution and Column Chromatography
- Prepare Notebook
- Header info
- Purpose statement
- Procedural reference
- Table of reagents
- Formula, structure, MW and hazards of ethyl
acetate, hexane, and the compounds to be
separated - Remember your data source(s)
15Pre-lab Preparation
- Notebooks Procedures
- Use the steps outlined earlier to write your
procedures (2-column format) - In addition, incorporate the following
- Check the newly spotted TLC plate under the UV
before you develop it. - Be sure you can see the spots before you start!!
- Two solvent systems will be used (run the known
compounds in both of these) - 100 ethyl acetate
- 50/50 mixture of ethyl acetate and hexanes
16Procedures continued
- Part One using Known Compounds
- Work in pairs. Each student will run one of the
two solvents. - Spot all three known compounds on each plate and
develop. - Compare and share Rf data for each solvent. Write
the data and a sketch of the TLC plate in your
notebook. - Part Two using Unknown Mixtures
- Work individually. Each student gets an unknown
containing at least one of the knowns. - Spot the unknown and all three known compounds.
You choose your development solvent system based
on your observations from part one.
17In Lab
- Follow your procedures to collect the Rf data for
Parts 1 and 2. - Recap
- Part One pairs, share data.
- Part Two individually, identify unknown.
18TLC Plates
Part One
Part Two
?
?
1 cm.
1 cm.
CAF ACE VAN
UNK CAF ACE VAN
19In/After Lab
- Calculations
- Calculate the Rf value for each known compound in
both solvents systems. (6 calcs) - Calculate the Rf value for each compound in the
unknown mixture. (4-6 calcs) - Results
- Discuss your observations concerning the known
compounds in the two solvent systems. - Identify the compounds in your unknown and the
rationale for your choice.
20Thin Layer Chromatography
- TLC lab technique hints
- Slide watch glass off beaker instead of lifting
it off to maintain solvent vapor saturation in
beaker. - Spot the solutes in the same order each time.
- Keep spots small (2 mm maximum).
- Never double dip spotting pipette.
- Dont get the solutes too close to the plate
edge. - Separate spotting points evenly.
- Do not disturb beaker during development.
- Ideal Rfs are between 0.25 and 0.75.
- Consider using mixed solvent to achieve this