RCS1601 Chemistry 1A

EXPERIMENT 1

Separation of Mixtures by Chromatography

1. INTRODUCTION

Chromatography is a technique frequently used to separate mixtures. The basis of the method is the variation
in the ability of different compounds to be adsorbed onto solid surfaces. If a "solvent front" moves past a
compound that is adsorbed onto a solid surface, the compound may be moved along the surface by the solvent.
The greater the solubility of the compound in the solvent, then the faster the compound moves; the greater the
attraction of the compound to the surface, then the slower the compound moves. Due to the differences in
interactions between different compounds in a mixture with both the solvent and the surface, separation of the
compounds can be achieved. Many different substances can be used as solid adsorbents, e.g. alumina, silica
gel, filter paper, and resin beads, to name but a few.

In thin layer chromatography (TLC), the stationary phase is a layer of very finely divided solid, usually alumina or
silica, deposited on a solid support such as a glass plate or an aluminium sheet. A small drop of a solution of the
sample is placed near the bottom of the plate and the plate is placed in a glass jar or "developing tank"
containing a pool of suitable solvent at the bottom. This solvent then moves up the plate by capillary action
carrying the spot of sample with it. The compounds most strongly adsorbed tend to remain near the bottom of
the plate whilst those that are weakly adsorbed move further up the plate. At every point on the plate the
compounds pass from the solvent to the solid phase and vice-versa. The compound that is most strongly
adsorbed spends nearly all of its time bound to the stationary phase and will spend little of its time in the
solvent (moving) phase, hence it will move up the plate slowly. Compounds that spend little time bound to the
stationary phase (i.e. those that are weakly adsorbed) pass quickly up the plate.

A quantitative measure of the rate of movement of a given component is its !! value (i.e. the so-called
"relative-to-front" value) which is defined as:
!! = #"#$%#&'&( /#)#*+'&( (1)

where #"#$%#&'&( is the distance that the component moved and #)#*+'&( is the distance that the solvent
moved. These distances are measured from the point of application of the sample (see Figure 1).

Figure 1. Schematic representation of a developed chromatographic plate

In this experiment, the technique of chromatographic separation will be illustrated using TLC to investigate the
colourless compounds caffeine, aspirin and paracetamol which require either inspection under UV light or
 RCS1601 Chemistry 1A – Experiment 1, cont’d.

reaction with some colourizing reagent to make them visible on the chromatographic plate. The principles of
gas chromatography will also be considered and discussed.

2. EXPERIMENTAL SECTION

2.1 Thin Layer Chromatography

Solutions of caffeine, aspirin and paracetamol will be available. You will also be issued with an "unknown"
solution containing a mixture of two of these compounds.

(1) Prepare a chromatographic developing jar by removing its lid and adding butyl acetate-chloroform-formic
acid (6:4:1) solvent mixture to a depth of only 1 cm. Replace the lid on the jar and leave the jar to stand in
a fume hood, undisturbed.

(2) Obtain the separate solutions of caffeine, aspirin, paracetamol and your "unknown" mixture containing
two of these. Also obtain a thin-layer chromatography (TLC) plate.

(3) Draw a very feint pencil line (do not use a ball point or felt-tip pen) 2 cm from the bottom of the TLC plate.
This is the "baseline".

(4) Make four, very feint, evenly spaced, pencil marks (dots) that lie about 2 mm below the "baseline" to help
you to space your samples evenly across the plate. Ensure the outermost dots are at least 0.5 cm from the
edges of the plate.

(5) Draw a very feint line that is parallel to the first line and lies exactly 0.5 cm from the top of the plate. This
is the "top line". Place the prepared TLC plate aside.

(6) Very fine capillary tubes that have drawn tips will be made available to you for spotting your
chromatographic plate. Obtain four or five of these for use in the following steps.
[Note: To produce capillary tubes with fine tips an alternative procedure is to prepare these from melting
point tubes by holding the tube with one hand at each end and carefully heating it in a Bunsen flame at a
position near the centre of the tube. When the glass becomes soft, quickly pull the ends apart. Break the
tube in half, and trim the fine tips with a small spatula or file so that they are as square as possible. Your
demonstrator will assist you with this procedure.]

(7) Practise your technique of depositing a small, concentrated "spot" of sample onto the stationary phase, by
firstly taking a scrap piece of TLC plate. Dip the fine tip of the capillary tube into the test solution. Touch
the TLC plate lightly and quickly so that a small "spot" is deposited. Once the solvent evaporates to leave
the solid sample behind, deposit another "spot" on top of the first. Keep repeating this process until a
small, but very concentrated, "spot" of sample is deposited.
Important: The concentrated spot you produce should not be greater than ca. 1 mm in diameter.
Once you have perfected your technique using the scrap piece of TLC plate, you may then proceed to
deposit samples on your previously prepared TLC plate.

(8) Using a clean capillary tube for each different sample, deposit small "spots" of: (i) caffeine, (ii) aspirin, (iii)
paracetamol and (iv) your "unknown" sample mixture onto the TLC plate. Ensure the "spots" are
positioned evenly across the plate, in line with the four feint pencil marks that were previously made.
Ensure that each "spot" is deposited on the very feint "baseline" so that it will lie a short distance above
the top of the solvent when the plate is eventually placed in the developing jar. The spots should be no
more than ca. 1 mm in diameter.

(9) Allow all of the spots to dry and, if necessary, carefully label the samples (C, A, P, U) in the space available
above the "top line".

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

(10) Remove the lid of the appropriate developing jar and carefully place the TLC plate upright (vertically) in
the jar. Ensure that the test "spots" are at the bottom and that these lie just above the solvent level in the
jar. Immediately replace the lid on the jar and allow the system to stand undisturbed until the solvent
front reaches the "top line".

(11) When the solvent front reaches the "top line", remove the TLC plate from the jar and stand it upright to
dry on a piece of paper towel in the fume hood. Pour the solvent that remains in the tank into the solvent
residue bottle located in the fume hood.

(12) Dry the TLC plate in warm air and examine it under UV light (254 nm). Your demonstrator will assist you
with this step.

(13) Whilst the TLC plate is under the UV light, mark the positions of all of the spots with a pencil.

(14) Remove the TLC plate from the UV source and, using a ruler, make the necessary measurements to enable
you to calculate the !! values of the three known compounds and those !! values of the compounds in
the unknown mixture. Record these measurements in Table 1 of the laboratory report sheet (LRS). [See
Equation (1) and Question (1) in Section 3.]

2.2 Gas Chromatography

The principles of chromatography illustrated in the previous exercise form the basis of a very powerful technique
that is used extensively in modern analytical chemistry. Modern chromatography takes many different forms
such as gas chromatography, high performance liquid chromatography, ion chromatography, size exclusion
chromatography, etc., but each of these techniques is capable of delivering information on the two key
objectives of analytical chemistry: the identification of the analyte and the quantitation of the analyte.

In this part of the session your demonstrator will discuss with you the technique of gas chromatography and, if
the opportunity arises, may show you a modern gas chromatograph instrument in one of our laboratories. There
are some questions on gas chromatography that can be answered from the discussion and the introductory
notes that follow.

Gas chromatography (GC) is a chromatographic technique that is used to separate complex mixtures of
compounds that can be readily volatilized. It is much more sensitive and sophisticated than thin-layer or paper
chromatography. It not only allows the identity of chemicals in a mixture to be identified but also enables the
amount of each chemical present to be determined. The basic elements (functional elements) of a gas
chromatograph are represented in Figure 2.

Figure 2. Functional elements of a gas chromatograph.

In GC the "carrier" or "mobile" phase is an inert gas such as helium, nitrogen or argon (the "mobile" phase in
chromatography is also known as an "eluant"). The sample is injected and vapourized into a column where it is
fractionated by being swept over an adsorbent (stationary phase) that is on the surface of the column. The

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

stationary phase is often a very thin layer of an inert, non-volatile liquid on an inert solid support – such as
beads of silica packed into a long, thin, flexible tube. The tube is coiled many times and is situated inside a
thermostatically-controlled oven. The fractionation process that takes place within the column consists of
multiple partitions of the analyte between the liquid and gas phases.

Substances that have a greater affinity (attraction) for the mobile phase reach the detector at the end of the
column more quickly than substances with a greater affinity for the stationary phase. Hence, different
substances remain in the column for different periods of time and hence "elute" from the column at different
times. The time that a given substance remains in the column under a specified set of experimental conditions
(type of column, flow rate of carrier gas, temperature, etc.) is very much a characteristic of that compound and
is known as the "retention time". The retention time,%, , of an unknown substance can be compared with
standard reference data to aid in the identification of the unknown compound.

The separate species emerging from the column are commonly detected by measuring a change in thermal
conductivity of the exit gas stream or by using a flame ionization detector (FID). A typical gas chromatogram
(i.e. the output from a GC instrument) is a plot that shows time along the &-axis and the intensity of the
detector response along the '-axis. There will often be a series of peaks with each peak corresponding to a
different compound. One can thus determine the number of different compounds in the mixture. The position
of the peak along the &-axis (retention time) provides information about the identity of the compound and the
intensity of the peak ('-axis), as determined by the area under the peak, provides information about the
amount of the compound that is present.

Gas chromatography is commonly used for samples containing 10 mg or less of material. The technique can
separate closely related polar compounds, hydrocarbons with similar boiling points, and high-temperature
boiling point compounds. The technique is used extensively for drug analyses (including the detection of
banned substances in urine samples from athletes) and pesticide analyses.

3. QUESTIONS AND CALCULATIONS

Instructions
• Answer ALL questions using the space provided in the LRS. Attach a separate sheet, clearly labelled with
the question number/s, if you have insufficient space in the LRS.
• Reports that are not submitted using the LRS will NOT be accepted.
• Answer Question (1) in Box (1), Question (2) in Box (2) and so on.
• Marks awarded for each question are shown at the bottom of each box along with the assessment
criteria.
• Show all working and/or reasoning for every question. Answers that are given as a single word, number,
letter, etc., will score zero.
• Final numerical answers should be quoted to the correct number of significant figures.
• Ensure that all developed chromatographic plates obtained in the laboratory are included in either your
own or your partner’s report.

Questions
(1) Use the data obtained in Step (14) in Section 2.1 calculate the !! values of the three "known" compounds
and each of the compounds in the "unknown" mixture. Ensure these values are all entered in Table 1 of
the LRS. On the basis of these data determine the identity of each compound in the "unknown" mixture.

(2) What are the two key objectives with which analytical chemistry is concerned and that can be achieved
using gas chromatography?

(3) What is gas chromatography (GC) used for?

(4) In GC, what is commonly used as the: (i) mobile phase and (ii) stationary phase?

(5) What determines the order in which each compound is eluted from a GC column?

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

(6) What can be said about the retention time for a given compound run under a given set of experimental
conditions in a GC instrument?

(7) Shown in Figure 3 is a schematic chromatogram.

With reference to Figure 3 answer the questions below and give reasons for your answers.
(i) How many different compounds are present in the chromatographed mixture?
(ii) Which compound is present in the smallest amount?
(iii) Which compound has the shortest retention time
(iv) Which compounds are present in equal amounts?
(v) Which compound has the longest retention time?
(vi) Which compound is present in the greatest amount?
(vii) Which compound has the greatest affinity for the stationary phase?
(viii) Which compound has the greatest affinity for the mobile phase?

Figure 3. A schematic chromatogram.

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

Detailed Marking Scheme for LRS

Assessable Detail Allocation

Aim Conduct investigations into Chromatography (up to three topics @ 2 ea) 6
Total 6
Q1 dcomp correct (five values for a total of 3 marks) 3
dsolvent correct 1
Correct Rf values (five values for a total of 3 marks) 3
Correct sig. figs. 1
Correct identification of unknowns (two compounds @ 1 ea) 2
Total 10
Q2 Mention of ‘analyte’ characteristics (two @ 1 ea) 2
Total 2
Q3 Mention of purposes/abilities of GC (up to three @ ⅔ ea) 2
Total 2
Q4 Identification of mobile phases (up to three @ ⅔ ea) 2
identification of stationary phase components (up to three @ ⅔ ea) 2
Total 4
Q5 Identification of characteristic controlling elution order 1
Mention of correct link between elution order and retention time 1
Total 2
Q6 Mention of nature of retention time under a given set of conditions 1
Mention of the relationship of retention time to the analyte 1
Total 2
Q7 Correct answer and explanation (eight pair @ ½ + ½ per pair) 8
Total 8
Conclusion Mention of each of the aims (three @ 1 ea) 3
Reasonable spelling & sentence construction 1
Total 4
Grand Total 40

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 Name and Student ID
Student: ____________________________________
RCS1601 Chemistry 1A Partner: ____________________________________

EXPERIMENT 1
Separation of Mixtures by Chromatography
LABORATORY REPORT

Aim

Aim/s clearly identified and stated. ___/6

Method
As per laboratory manual
Changes:

Results and Calculations

Q1 Table 1

System dcomponent/mm dsolvent/mm Rf

1. caffeine __________ __________ __________
2. aspirin __________ __________ __________
Mixture
3. contains: (a) ________________
paracetamol __________ and (b) ________________.
__________ __________
4. unknown (a) __________ __________ __________
(b) __________ __________ __________

Mixture contains: (a) ________________ and (b) ________________.

Correct calculation of Rf values, sig. figs. and correct identification of compounds in mixture. ___ /10

Q2

Correct identification of key objectives of analytical chemistry. ___ /2

Q3

Correct answer, succinctly stated. ___ /2

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

Q4

Correct identification of mobile and stationary phases, succinctly stated. ___ /4

Q5

Correct answer, succinctly stated. ___ /2

Q6

Correct answer, succinctly stated. ___ /2

Q7

Correct answers, succinctly stated. ___ /8

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 RCS1601 Chemistry 1A – Experiment 1, cont’d.

Conclusions

Succinct and accurate conclusions ___ /4

Attach developed TLC plate HERE