SIC 1011

LAB REPORT C2- SEPARATION BY CHROMATOGRAPHIC

TECHNIQUES

PREPARED FOR: DR LIM SIEW HUAH

NAME SUMAYYAH BINTI LOKMAN

MATRIX NO. 22003285/1

SEMESTER 1

OCCURANCE 1

DATE OF 29th NOVEMBER 2022

EXPERIMENT
TITLE

Separation by chromatographic techniques

OBJECTIVES.

1. To separate the compound by column chromatography

2. To visualise the separation by TLC using UV-detection

RESULTS AND CALCULATION

Table 1: distance travelled by solute

Solute Distance travelled(cm) Retention factor (Rf)

Pure pyrene 4.1 0.58

Pure m-nitroaniline 1.1 0.15

Conical flask 1 4.3 0.61

Conical flask 2(a) 4.4 0.62

Conical flask 2(b) 1.4 0.20

Conical flask 3 1.4 0.20

Solvent front (hexane)= 7.1cm

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒

𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

4.1𝑐𝑚
𝑅𝑓 (𝑝) = = 0.58
7.1𝑐𝑚

1.1𝑐𝑚
𝑅𝑓 (𝑚) = 7.1𝑐𝑚
= 0.15

4.3𝑐𝑚
𝑅𝑓 (1) = 7.1𝑐𝑚
= 0.61

4.4𝑐𝑚
𝑅𝑓 (2𝑎) = 7.1𝑐𝑚
= 0.62
 1.4𝑐𝑚
𝑅𝑓 (2𝑏) = = 0.20
7.1𝑐𝑚
1.4𝑐𝑚
𝑅𝑓 (3) = = 0.20
7.1𝑐𝑚

ANALYSIS AND DISCUSSION

Chromatography is an essential biophysical method to separate, identify and purify the mixture
component for quantitative and qualitive study. Based on the traits including size and shape,
total charge, the presence of hydrophobic groups on the surface and binding capacity to the
stationary phase, proteins can be purified. One of the most used techniques for purification of
proteins is column chromatography (Coskun, 2016). The mixture is placed inside the top of
column to be analysed by column chromatography. By using air pressure or gravity, the liquid
solvent (eluent) is forced through the column. The solute adsorbed on the adsorbent and the
eluting solvent that passed through the column will reach to an equilibrium. Due to different
interactions that the mixture’s components have with the stationary and mobile phase, they
will be flow through the mobile phase to various degrees and the separation will be achieved.
As the solvent leaks from the bottom of the column, the eluents are collected.

In this experiment, attention must be given to the retardation or retention factor, Rf of the
component. It is the ratio of the analyte distance travelled to the solvent front on a
chromatogram. Analyte mobility with mobile solvents varies depending on the
chromatographic techniques added to stationary phases. The relative affinities of analytes with
stationary and mobile solvents account to this variation. Analytes will remain in place longer
when the relative affinity is higher and its Rf is lower (Team, 2021).

From this experiment, the Rf for pure pyrene is 0.58 while the Rf value for conical flask 1 and
2a is 0.61 and 0.62 respectively. Meanwhile, the Rf for pure m-nitroaniline is 0.15 while the Rf
for conical flask 2b and 3 is 0.20. Therefore, we can deduce that conical flask 1 contains pure
pyrene and conical flask 3 contains pure m-nitroaniline. Conical flask 2 however, is said to be
containing a mixture of pyrene and m-nitroaniline as it has 2 different Rf values.

Pyrene is a non-polar compound while m-nitroaniline is a polar compound that will react
strongly with the stationary phase (silica) which is also a polar compound. Therefore, m-
nitroaniline will move very slowly through the column and takes a longer time to be eluted. On
the other hand, pyrene will only make weak interaction with silica gel and move faster through
the column, closer to the solvent front. Hence, the Rf for pyrene is larger compared to m-
nitroaniline and the time taken for pyrene to be eluted from the column is shorter.
The precaution steps for this experiment includes making sure that the eluent never gets dry
by consistently adding the eluent (hexane) from time to time. Moreover, prevent physical
shock on the column by avoiding dropping the eluent from high position and drop the eluent
gently using a dropper. Furthermore, wear a mask and avoid inhaling silica dust as the
particle can become trapped in the lung and reduce the lung’s ability to take oxygen. Lastly,
mark the plate very lightly so that the silica gel on the surface will not be scratched.

QUESTION

1. i. Mobile phase: It is a phase that moves through the chromatographic medium.

Typically, the mobile phase dissolves the mixture that need to be separated.
Furthermore, it travels through the stationary phase which is the chromatography
structure that holds the material.
ii. stationary phase: It is the phase that is fixed to the column. Here, the forward
chromatography phase includes polar stationary phase while the reverse phase includes
non-polar stationary phase.
iii. Rf value: It is the ratio of the analyte distance travelled to the solvent front on a
chromatogram.
6.5𝑐𝑚
2. 𝑅𝑓 (𝑋) = 7𝑐𝑚
=0.93
2.5𝑐𝑚
𝑅𝑓 (𝑌) = = 0.34
7𝑐𝑚
3.5𝑐𝑚
𝑅𝑓 (𝑍) = = 0.50
7𝑐𝑚

3. Column chromatography advantage over TLC is that there is no limit for quantity of
mixture that need to be separated by using this method.
4. meta-nitroaniline, para-nitroaniline, ortho-nitroaniline.

REFERENCES
1. Coskun, O. (2016, November 11). Separation techniques: Chromatography. PubMed
Central (PMC). Retrieved December 4, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/

2. Column Chromatography. (n.d.). Column Chromatography. Retrieved December 5, 2022,

from
https://www.orgchemboulder.com/Technique/Procedures/Columnchrom/Columnchrom.shtml

3. Team, P. (2021, October 18). Rf values: Definition, Calculation and Explanation -

PSIBERG. PSIBERG. Retrieved December 4, 2022, from https://psiberg.com/rf-values/

4. What is the Difference Between Mobile Phase and Stationary Phase - Pediaa.Com.
(2019, October 13). Pediaa.Com. Retrieved December 5, 2022, from
https://pediaa.com/what-is-the-difference-between-mobile-phase-and-stationary-phase/