S Al-Shokair

1
College of Veterinary Medicine Animal
Resources Department of Clinical
Studies Toxicology Forensic Medicine
Chromatography

• By
• S Al-Shokair

2
Chromatography (from Greek chroma, colour and
graphein to write).

• http//en.wikipedia.org/wiki/Chromatography

3
They all have a stationary phase (a solid, or
a liquid supported on a solid) and a mobile phase
(a liquid or a gas).
4
Chromatography is used to separate mixtures of
substances into their components. All forms of
chromatography work on the same principle.
5
The mobile phase flows through the stationary
phase and carries the components of the mixture
with it. Different components travel at different
rates.
6
Classfication of Chromatography
7
Types of chromatography Paper
chromatography Thin layer chromatography Gas
chromatography Liquid chromatography
8
High Performance Liquid chromatogrphy
(HPLC)Affinity ChromatographyColum
Chromatography
9
Paper Chromatography
10
In paper chromatography, the stationary phase
is a very uniform absorbent paper. The mobile
phase is a suitable liquid solvent or mixture of
solvents.
11
Amino acid Rf value
alanine 0.38
arginine 0.20
asparagine 0.5
aspartic acid 0.24
cysteine 0.4
glutamine 0.13
glutamic acid 0.30
glycine 0.26
histidine 0.11
isoleucine 0.72
leucine 0.73
lysine 0.14
methionine 0.55
phenylalanine 0.68
prolinenot a true amino acid - shows up as yellow 0.43
serine 0.27
threonine 0.35
tryptophan 0.66
tyrosine 0.45
valine 0.61
12
AimsTo demonstrate separation of amino acid
by paper chromatography To use chromatography
to identify amino acids
13
Uses Clinical research hospitals
Manufacturing industries Forensic science
studies.
14
AdvantagesThe advantages of paper
chromatography are easy and economy.
15
DisadvantagesThe disadvantage of paper
chromatography is that many a times complex
mixtures cant be separated by using this method.

16
Materials
17

• Chromatography tank and lid
• Chromatography paper
• Capillary tubes
• Amino acid samples
• Pencil metric ruler

18
Unknown samples Solvent (BAW)
Butan-1-ol Acetic acid Water 60 15 25
(FLAMMABLE, TOXIC) Hairdryer or heating tray
19
Spray can of Indanetrione (Ninhydrin) 1 in
acetone butan-1-ol (FLAMMABLE, TOXIC) Latex
gloves Access to fume cupboard Drying oven
at 100C
20
Methods
21
Touch the tip of the capillary to the first
mark and pull it away Let this dry and apply
the sample again Repeat for each sample or
mixture Let the paper dry.
22
Developing the chromatogram
23
A. The eluting solution for this experiment is
1-butanol H2O Acetic Acid 60 ml 15
ml 25 ml.
B. Develop using Ninhydrin (triketohydrindene
hydrate) acetone (0.2 mg ninhydrin dissolves
in 100 ml).
24
Draw a line 1.5 2cm above the bottom of the
chromatography paper

• Make small marks at 0.5cm intervals along the
  line.
• Fill a capillary tube by capillary action with
  your first sample

25

• Stand the chromatography paper in the tank so
  that the bottom edge is in the solvent but the
  remaining paper does not touch the tank.
• Place the lid on the tank and leave for about 1
  hour.
• Wearing gloves, remove the damp paper and mark
  where the solvent has reached

26
Dry the paper in the fume cupboard. Still
in the fume cupboard, spray the whole of the
paper with ninhydrine in butanol. Dry the paper
and transfer it to the drying oven for 1-2min.

27
B. Develop using Ninhydrin (triketohydrindene
hydrate) acetone (1g ninhydrin dissolves in
100 ml).
28
Mark the position of each spot that develops.
Note the colour and Rf (or travel distance) of
each standard and hence find the amino acid
composition of the unknown samples.
29
Resu ts
30

31

Quantitative analysis of chromatographic data
To calculate the Rf (retention factor or
retardation factors ), we use the equation Note
that an Rf value has no units because the units
of distance cancel.

• Rf distance traveled by component from
  application point distance traveled by solvent
  from application point

32
Retention Factor (Rƒ) Calculation
33
In our example, this would be
Rf distance traveled by component
from application point
distance traveled by solvent from
application point
Rf 2.5 cm5.0 cm 0.5
Note that an Rf value has no units because the
units of distance cancel.
34
determination of the components a plant
contains analyzing ceramides and fatty
acids detection of pesticides or insecticides in
food and water analyzing the dye composition of
fibers in forensics, or assaying the
radiochemical purity of radiopharmaceutica
35
(No Transcript)
36
Thank yous2000ss_at_yahoo.com
37
Analytical Conditions Column Zorbax SIL Mobile
phase Toluene-ethylacetate-formic
acid-methanol (897.52.01.5) Flow rate
1.0ml/min Column temp. 40C Detection
Shimadzu spectrofluorophotometer (Em365nm,
Ex425nm)