Prepare Samples

TLA 5206
APPLY CHROMATOGRAPHIC TECHNIQUES
Element 2

TLA 5206 -2023-10 JM – Jiji Manu Varughese

TLA 5206 -2023-10 JM

PERRFOMANE CRITERIA

E2. Prepare samples and standard

• PC2.1 Obtain a representative analytical portion of the laboratory sample
• PC2.2 Prepare sample in accordance with testing requirements.
• PC2.3 Prepare validation checks for analytical portion
Sample preparation

• Sample preparation can be achieved by

using a wide range of techniques, with
the following purposes

• To remove any particle or impurity that

will affect the process.
• to increase the concentration of an
analyte
• if necessary, to convert the sample into a
more suitable form ( solid to liquid or
liquid to gas or liquid to solid)

TLA 5206 -2023-10 JM

 Drying
Powdering / Grinding
Crushing
Sample
Preparation Dissolving
methods Filtration
include Centrifugation
Evaporation
Distillation

TLA 5206 -2023-10 JM

CRUSHING Filtration
Filtration is done to remove large,
suspended and insoluble particles from
sample.

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CENTRIFUGATION
•Centrifugation is a technique of separating substances which involves the
application of centrifugal force.
•The particles are separated from a solution according to their size, shape,
density, the viscosity of the medium and rotor speed.
•The analyte may be the supernatant or pellet which can be separated

TLA 5206 -2023-10 JM

 Organic Solvents

DISSOLVING

• Acetone
In order to dissolve a sample completely, each • Acetic Acid
insoluble component must be converted into a
soluble form.
• Ether
• Alcohol
• Benzene
Samples are diluted in water if all components
are soluble in water
• Toluene
Samples are diluted in organic solvents if they are • Hexane
not soluble in water. Eg: oil • Chloroform
• Methanol
TLA 5206 -2023-10 JM
Commonly used solvents

Water ( Universal
solvent)
Ethanol Used for dissolving inks
Can be used with several other solvents such as water, acetone, ether, choloroform
Methanol processing of essential pharmaceutical ingredients and products such as cholesterol,
vitamins, streptomycin and hormones as a solvent.
Acetone Used as solvent for cosmetics, paints , pigments
Chloroform used as a solvent for adhesives, alkaloids, fats, oils and rubber
Toluene used as a solvent for paints, paint thinners, many chemical reactants, rubber, printing ink
etc
Hexane For extracting oil, dyes / pigments

TLA 5206 -2023-10 JM

Dissolution for sample preparation

TLA 5206 -2023-10 JM

Extraction
• Extraction is an important step in sample
preparation prior to chromatography.
• It involves the isolation of target analytes from a
complex sample, or much larger sample volume.
• The process removes interfering sample
components that may block HPLC and GC
columns. It also increases analyte concentration
• Example Liquid liquid extraction

TLA 5206 -2023-10 JM

Sample
Preparation

TLA 5206 -2023-10 JM

Extraction of
Chlorophyll
from leaves

TLA 5206 -2023-10 JM

https://www.youtube.com/watch?v=iPpy4khqtks

TLA 5206 -2023-10 JM

Cut out small pieces of spinach using scissors.

Add them to the mortar.

Accurately measure 5ml acetone using a measuring

cylinder and add it into the mortar.

With the help of mortar and pestle, grind the spinach

leaves into a smooth paste.

TLA 5206 -2023-10 JM

• Mobile phase used to run
chromatography for chlorophyll

• Petroleum ether : acetone – 9:1

• OR

• n- Hexane : Acetone – 7:3

TLA 5206 -2023-10 JM
TLA 5206 -2023-10 JM
Related video links
Chromatography of Chlorophyll

• https://www.youtube.com/watch?v=7q5HDMXSdtU

• https://www.youtube.com/watch?v=iPpy4khqtks

• https://www.youtube.com/watch?v=W56RHxu2Hpc

• https://www.youtube.com/watch?v=FQqVqVZgKjA - animation

TLA 5206 -2023-10 JM

Preparation of
food colour

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Food colour Chromatography

TLA 5206 -2023-10 JM

Water is used as Solvent
used for extracting
colour.

Place a drop of water in a

watch glass and place a
candy until the colour
seeps into the water drop

TLA 5206 -2023-10 JM

 Drop of
water in a • Place a drop of distilled
petridish water in a petri dish and
wait until the colour seeps
into the water drop.
• Load the sample onto
chromatography paper and
Place a candy in insert in chromatography
water drop chamber.

• Mobile phase used

0.1% Sodium chloride
soluiton

Prepare mobile
phase -0.1%NaCl

TLA 5206 -2023-10 JM

Related video links for food colours

• https://www.sciencebuddies.org/science-fair-projects/project-ideas/F
oodSci_p006/cooking-food-science/candy-chromatography

• https://www.youtube.com/watch?v=GDROkh1zlr8

TLA 5206 -2023-10 JM

Sample Preparation of medicines
Solvent used - Ethanol

Tablets are crushed using Mortar & pestle

Transfer it to a vial containing Ethanol and mix well.

Mobile phase used : ethyl acetate

https://youtu.be/zM3MrDkk5Nk

TLA 5206 -2023-10 JM

• https://www.youtube.com/watch?v=mgm8shbfmCc

• Sample prep for meat species determination & adulteration - Hi-

Res Mass Spec

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