EXPERIMENT 4 : CHROMATOGRAPHY Write in INDEX, leave

column of date blank

4a Separation of the constituents of a mixture of inorganic compounds containing two cations,

Pb2+ and Cd2+ , using chromatographic technique

4b To carry out the separation of photosynthetic pigments from the extract of spinach leaves by
paper chromatography.

EXPERIMENT 4(a)

On Ruled side
Aim

Separation of the constituents of a mixture of inorganic compounds containing two cations, Pb2+ and
Cd2+, using chromatographic technique

MATERIALS REQUIRED

Boiling tube, capillary, ruler, pencil, Whatman paper, mixture of inorganic compounds containing
two cations, Pb2+ and Cd2+and (NH4)2S solution as developer, A mixture of ethanol ,6.0 M HNO3 and
distilled water, in the ratio 8:1:1.

Theory

In paper chromatography, water molecules present in the pores of the filter paper act as the
stationary phase and the moving phase can be a solvent like hexane, toluene, acetone or a mixture
of solvents such as methanol-water mixture etc. As the moving phase passes through the spot-on
which sample has been adsorbed, it dissolves the components more or less readily; depending upon
the solubility and carries them along with it while moving on the support. At a given temperature
and for a given solvent, it is possible to determine the characteristic rate of movement of each
substance on the chromatographic paper, as the moving phase moves. This is represented by
relative front or retardation factor also called Rf value. Rf values of different compounds are
different even if the mobile phase (solvent) is same. Furthermore, Rf value of a compound may be
different in different solvents. Rf values can be calculated by using the following expression:

Since solvent front moves faster than the compounds, the Rf value of a substance will always be less
than one. Also note that Rf value has no unit. If the compound is coloured then its position on the
chromatographic paper may be easily located. However, if the substance is colourless, it may be
treated with a reagent, which imparts it a characteristic colour. This reagent is given the name
developer.
Procedure

(i) Take a strip of Whatman No. 1 filter paper.

(ii) With the help of a pencil, mark a line at a distance of 3 cm from one of the ends of this
paper.
(iii) Put a spot of the mixture on the marked line with the help of a fine capillary.
(iv) Hang the filter paper in a boiling tube containing a mixture of ethanol, 6.0 M HNO3 and
distilled water, in the ratio 8:1:1, such that spot lies above the level of the solvent.
(v) Allow the mobile phase (solvent) rises up to two third of the length of the paper.
(vi) Remove the filter paper from the jar, mark the solvent front.
(vii) Spray ammonium sulphide solution on the chromatography paper to obtain spots of
yellow and black colour.
(viii) Mark the position of spots with a pencil and allow the paper to dry.
(ix) Measure the distance moved by the solvent front and the different spots of the cations
with respect to the reference line.
(x) Record the observations in tabular form. Calculate the Rf value for each cation.

Result

(i) Rf values of Pb2+ ions is __________.

(ii) Rf values of Cd2+ ions is __________.

NOTE: PASTE THE CHROMATOGRAM ON THE BLANK SIDE AND MARK THE DISTANCE TRAVELLED BY
THE INDIVIDUAL COMPONENTS AND THE SOLVENT FRONT USING A PENCIL.

OBSERVATION TABLE TO DRAWN ON BLANK SIDE(LINES WITH PENCIL,DATA WITH PEN)

RESULT TO BE WRITTEN ON RULED SIDE

EXPERIMENT 4(b) On Ruled side

AIM: To carry out the separation of photosynthetic pigments from the extract of spinach leaves by
paper chromatography.

MATERIALS REQUIRED

Boiling tube, capillary, ruler, pencil, Whatman paper, pestle and mortar.

THEORY

The leaves of most plants contain many different pigments. Chlorophyll is the most abundant and
important plant pigment for absorbing sunlight during photosynthesis. It exists in two forms
chlorophyll a (bright green) and chlorophyll b (dull green). Other pigments such as Xanthophyll
(yellow) and carotene (orange red) may also be present. These pigments can be separated using
ascending paper chromatography in a suitable solvent.

PROCEDURE

1)Finally cut up some leaves and grind them with a pestle and mortar.
2)Add a pinch of sand and few drops of acetone while grinding.
3)Grind the mixture for at least three minutes.
4)On a strip of chromatography paper, draw a pencil line 3 cm from the bottom.
5)Use a capillary to put liquid from the leaf extract onto centre of line. Keep the spot small.
6)Allow the spot to dry, then add another spot on top before putting on the next.
7) Place the paper in a boiling tube containing small amount of solvent
(9 parts petroleum ether:1-part acetone), such that level of solvent is below the spot.

OBSERVATION TABLE:

S.No Colour of the Distance travelled by the Distance travelled by Rf value

spot components of the spots from the solvent from
the reference line in cm reference line in cm
1 Xanthophyll X Y X/Y=
(Yellow)
2 Chlorophyll a Z Y Z/Y=
(dark green)
3 Chlorophyll b W Y W/Y=
(light green)

Rf = Distance travelled by the substance from reference line (cm)

Distance travelled by the solvent front from reference line (cm)

NOTE: PASTE THE CHROMATOGRAM ON THE BLANK SIDE AND MARK THE DISTANCE TRAVELLED BY
THE INDIVIDUAL COMPONENTS AND THE SOLVENT FRONT USING A PENCIL.

OBSERVATION TABLE TO DRAWN ON BLANK SIDE (LINES WITH PENCIL,DATA WITH PEN)
RESULT TO BE WRITTEN ON RULED SIDE
PRECAUTIONS
(a) Use good quality pencil for drawing the reference line so that the mark does not dissolve in the
solvent .
(b) Dip the paper strip in the solvent in such a way that the spot of the mixture is above the solvent
level and the movement of the solvent front is not zig-zag.
(c) While spotting the test solution on the paper, do not allow the spots to spread. Use finely drawn
capillary to put the spot on the paper.
(d) Ensure that the filter paper strip hangs freely in the jar.
(e) Once the experiment is set, do not disturb the jar as long as the chromatogram is being
developed.
(f) Keep the jar covered with the lid when the chromatogram is being developed.
(g) Make the paper strip perfectly dry before developing the spots.