ENGLISH FOR CHEMISTRY (3)

THIN LAYER CHROMATOGRAPHY (TLC)

Hong-Son NGUYEN 22147117

Thanh-Minh TRAN
Tai-Nguyen HUYNH-CONG 22147082
Hung-Anh NGUYEN-NGO

Lecturer (part-3): PhD. Thu-Phuong TRAN

Date

Template created by Matt Horwitz, Ph.D. Contact: [email protected]. 1

Content Thin Layer Chromatography – an overview.

What would we talk in this presentation

1) What is TLC and why we need it.

2) TLC mechanism and how it made?
3) How to prepare an TLC and analyze the result.
4) Some “rookie” mistake when running TLC.

Reaction tracking using TLC

2
 Scope of the presenation Thin Layer Chromatography – an overview.

Scope of the presentation:

Help you know about what is TLC

and how it work, know how to run a
TLC and reading result.

Not on scope:

This presentation not mention some

advanced technique like 2D-TLC,
HPTLC, electrophoresis
chromatography, enantiomer
seperation by modified TLC
(cyclodextrin, coated with chiral
compounds, poly(methacrylic) acid
amides,...). So any question not on
scope of the presentation would not
Relationship between TLC and HPLC
be answered.

[1] R. Ragesh Nath, C. Nethravathi, M. Rajamathi, ACS Omega 2021, 6, 12124–12132. 3

 What is TLC and why we need it. Thin Layer Chromatography – an overview.

• Scope of the presentation:

[1] L. Constantino, C. Dias, E. Valente, Comprehensive Organic Chemistry Experiments for the Laboratory Classroom 2016, 776–780. 4
 TLC mechanism and how it made? Thin Layer Chromatography – an overview.

[1] L. Constantino, C. Dias, E. Valente, Comprehensive Organic Chemistry Experiments for the Laboratory Classroom 2016, 776–780. 5
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Cutting TLC plate

The “a (cm)” value depends on the

purpose of the TLC, and how many
spots you need.

If you just want to know how many

compounds you have in a mixture
or the purify of a long time no-use
compound, one spot is enough.

Caution:
• Avoid touching the white
adsorbent layer (usually silica
or alumina) on the plate.
• Pen ink should not be used for
marking the line.

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 6

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Make a capillary TLC spotter

[1]D. Hickman, “Tips and tricks for the lab: How to make a capillary TLC spotter,” can be found under
https://www.chemistryviews.org/details/education/2102991/Tips_and_Tricks_for_the_Lab_How_to_Make_a_Capillary_TLC_Spotter/, 2022. 7
[2]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d.
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Spotting the plate

Caution:
• Try to avoid spotting too much
material, because this will deteriorate
the quality of the separation
considerably (‘tailing’).
• The spots should be far enough away
from the edges and from each other as
well.

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 8

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Developing a plate

Caution:
• The solvent level has to be below the starting line of the TLC, otherwise the spots
will dissolve away.
•The bottle and beaker must be closed. If not, the solvent will fly away and make the
solvent ratio change => solvent polarity change, the spot’s position will be changed,

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 9

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Another way to elution a plate

Solvent top-down elution

[1] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282. 10
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Visualization

Visualization
Visualization helps you
see what you don’t see.

Before visualize Visualized by UV

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 11

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Visualization

[1] “Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d.

[2] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282.
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 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Visualization

UV Light: For aromatics + Iodine: Visualizes ~half the p-Anisaldehyde: For Vanillin: For many
conjugated systems time. Strongly reacts with many aldehydes, ketones, aldehydes, ketones, and
aromatics and alcohols alcohols

Permanganate: For alkenes, Phosphomolybdic Acid (PMA): Iron(III) Chloride: For phenols Bromocresol Green: For
alkynes, or oxidizable groups For alcohols, phenols, alkenes, acidic compounds
(aldehydes, alcohols) and many carbonyl compounds.
[1]Libretexts, “2.3F: Visualizing TLC Plates,” can be found under
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Techniques_%28Nichols%29/02%3A_Chromatography/2.03%3A_Thin_Layer_Chromatography_%28T
LC%29/2.3F%3A_Visualizing_TLC_Plates, 2022. 13
[2]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d.
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Visualization
Reagent Works well for Colors Notes

Sulfuric acid General stain Brown or black spots

Chromic acid For difficult to stain Black spots

compounds
Cerium sulfate Good general stain, For more
very well for alkaloids information,
Ninhydrin Amino acids, amines Purple you can scan
2,4- Aldehydes, ketones Yellow/orange also called “DNP” this QR code.
Dinitrophenylhydrazine

Cerium molybdate (CAM, Good general stain, Blue or green spot Upon heating, very
‘Hanessian’s Stain’, very well with sensitive!
Ceric staining) polyhydroxylated and
carbonyl compounds
p-Anisaldehyde Good general stain, Varying colors on light Does not work with
particularly sensitive pink plate upon heating alkenes, alkynes or
towards nucleophiles aromatic system
unless functional
groups are present
Ehrlich’s Reagent Indoles, amines Pink or red-violet
(Dimethylaminobenzalde
hyde)
Dragendorff-Munier Amines even the ones Various colors
Stain that are low in
reactivity

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 14

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Analysing the data from TLC

Does my solvent
system separate
my mixture good?

[1] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282. 15
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Analysing the data from TLC

Is my compound
pure?
One dot, huh! It’s not simple
like what you see.

[1]Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282. 16
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Analysing the data from TLC

How is my
reaction going?

Relationship
between TLC and
HPLC and the use
of HPLC to
monitoring reaction

[1] R. Ragesh Nath, C. Nethravathi, M. Rajamathi, ACS Omega 2021, 6, 12124–12132.

[2] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282.
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 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Analysing the data from TLC

The Retention Factor a (cm) d: the distance of the start

(Rf) – What’s that? Rf = line to the solvent front.
d (cm) a: the distance of center of
Just a way to know how far a the spot to the start line,
compound traveled

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d. 18

 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Rf affected by:

plate’s activity, grain size,

plate properties
thickness,...
solvent system’s ratio,
solvent properties
solvent polarity.
(Rf)
amount of spot’s size, concentration =>
material spotted semi-quantitative property of TLC

temperature

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d.

[2] ] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282
19
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

What do we know from Rf?

(Rf)
0< Rf <1

The Rf can be used to identify compounds due to their uniqueness

to each compound
By comparing with standard compound in similar condition (same plate,
same plate’s size, same solvent system,...)

Rf value help you choose suitable solvent for flash column

Optimized Rf value is from 0.3 - 0.8 (optimized for separartion of which
compound you care about should be around 0.2 – 0.3). As a result, you
can optimize your solvent system for better separation.

[1]“Thin Layer Chromatography,” can be found under https://www.chem.ucla.edu/%7Ebacher/General/30BL/tips/TLC1.html, n.d.

[2] ] Nguyễn Kim Phi Phụng, Các phương pháp cô lập hợp chất hữu cơ, Nhà xuất bản đại học quốc gia TP Hồ Chí Minh, 2007, P. 213 – 282
20
 How to prepare an TLC and analyze the result. Thin Layer Chromatography – an overview.

Software approach

[1] N. Mac Fhionnlaoich, S. Ibsen, L. A. Serrano, A. Taylor, R. Qi, S. Guldin, J. Chem. Educ. 2018, 95, 2191–2196. 21
 “Rookie” mistake Thin Layer Chromatography – an overview.

Over-large spots

Increasing the spot’s size would

overlap other component spots with
similar Rf values on your TLC plate =>
loss of the separation

On the other hand, spotting too small

can make you difficult to see.

Increasing spot’s size

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
22
 “Rookie” mistake Thin Layer Chromatography – an overview.

Over-large spots

Solving: Spotting sizes of your

sample should not be larger than 1-2
mm in diameter.

Increasing spot’s size

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
23
 “Rookie” mistake Thin Layer Chromatography – an overview.

Two high concentration

the solvent can not handle the

concentrated sample and in result,
moves as much of the substance as it
can up the stationary phase
=> now you got a lovely streak rather
than a single separate spot.

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
24
 “Rookie” mistake Thin Layer Chromatography – an overview.

Two high concentration

Solving: if you see a streak, then you

should dilute your sample and take
another plate to do again.

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
25
 “Rookie” mistake Thin Layer Chromatography – an overview.

Spotting under the solvent line

Get ready to see your spot washed

away and you are full of wondering
where your compound is?

Solvent line

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
26
 “Rookie” mistake Thin Layer Chromatography – an overview.

Spotting under the solvent line

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
27
 “Rookie” mistake Thin Layer Chromatography – an overview.

Spotting under the solvent line

Solving: please sketch the solvent

line and spot over there.

Solvent line

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
28
 “Rookie” mistake Thin Layer Chromatography – an overview.

Un even elution - No flat bottom:

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
29
 “Rookie” mistake Thin Layer Chromatography – an overview.

Un even elution - Plate is not cut evenly:

[1]Libretexts, “Thin Layer Chromatography,” can be found under

https://chem.libretexts.org/Ancillary_Materials/Demos_Techniques_and_Experiments/General_Lab_Techniques/Thin_Layer_Chromatography, 2022.
30
“Rookie” mistake Thin Layer Chromatography – an overview.

Another mistake

Find out more mistakes at Not Voodoo X.4

by University of Rochester

Put a TLC in the jar and

walked away to to
something else...
remembered the TLC half
an hour later.
Forgot to
make a co-
spot on the
TLC plate.
Ran TLC
Applied spots without
too close on spotting plate.
TLC, so lanes Didn't realize
Tried to run
merged in until
TLC in two
subsequent visualization
solvents
running. stage.
that were
immiscible.

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Thank you for
listening us!

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Any question?

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 Our question after the presentation

Why don’t we run TLC for metal containing

compounds even if it’s air stable?

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