LAB MANUAL

(SUBMISSION #01 SEMESTER FALL-2022)

BY
Group #4
Muhammad Aqib Shahzad 19014107-014
Hasanaat Ahmad 19014107-024
Muhammad Zulqernan 19014107-008
Ali Raza 19014107-034
Ayesha Ijaz 19014107-037
Hamna Masud 19014107-001

CHEM-456 (Analytical Chemistry-V-P)

BS-CHEM-VII (C)
Submitted to Ms. Shumaila Tufail
Department of Chemistry

UNIVERSITY OF GUJRAT
EXPERIMENT NO 1:
PREPARATION OF BUFFER SOLUTION OF ACETIC ACID AND SODIUM
ACETATE AND DETERMINATION OF pH

A buffer consists of a weak acid and its conjugate base or a weak base and its conjugate acid.
Buffer capacity is the amount of acid or base that can be added before the pH of a buffer
changes. An example of a buffer solution is bicarbonate in blood, which maintains the body's
internal pH.
Buffers can resist large change in solution pH upon the addition of small amounts of hydrogen
ions (𝐻 + ) or hydroxide ions (𝑂𝐻 − ).
Types of buffers
Acidic buffers: Acidic buffers are solutions that have a pH below 7 and contain a weak acid
and one of is salts. For example: boric acid + borax
Basic buffers: Basic buffer has a basic pH and is prepared by mixing a weak base and its salt
with strong acid. For example: ammonium hydroxide and ammonium chloride
pH is a logarithmic measure of hydrogen ion concentration, originally defined by Danish
biochemist Soren Peter Lauritz Sorensen in 1909. The p (small letter) of pH is taken from the
German word potenz used for power, so pH is an abbreviation for power of hydrogen.
pH= −log (𝐻 + )
Principle

According to Henderson Hasselbalch equation:

HA⇌ H + +A –
[H+ ][A–]
Ka= [HA]

K a [HA]
[H + ] =
A–
By taking negative log on both sides.
HA
– log[H + ]=–logK a –log
A–

– log[H + ]=pH
– logK a = pK a
[acid]
pH= pK a – log [salt]
[salt]
pH= pK a + log [acid]
Apparatus and Chemicals
 Beakers
 Weighing balance
 Stirrer
 Pipette
 Measuring flask
 pH paper
 Acetic acid
 Sodium acetate
Procedure
1. Firstly, we washed all the apparatus.
2. Then we pipetted out 0.17 ml of acetic acid in a beaker.
3. Then we weighted out 0.36 g of sodium acetate through weighing balance.
4. Then dissolve 0.36 g of sodium acetate in the tap water and mix it with a stirrer.
5. Then we mixed 0.17 ml of acetic acid in the solution we have made earlier.
6. Then we dipped pH paper in the solution to check the pH of the solution.
7. Let the pH paper to dry then we have matched it with the pH measuring scale.
8. pH was 4.9 so, it was acidic in nature.
Calculations
For acetic acid
𝒏𝒐 𝒐𝒇 𝒎𝒐𝒍𝒆𝒔
Molarity: 𝒗𝒐𝒍𝒖𝒎𝒆

Moles: molarity × volume

𝟏𝟎𝟎
moles: 0.17 × 𝟏𝟎𝟎𝟎

moles of acetic acid: 0.017 moles

𝒎𝒂𝒔𝒔
no. of moles:
𝒎𝒐𝒍𝒂𝒓 𝒎𝒂𝒔𝒔

no of moles of acetic acid × molar mass of acetic acid = mass of acetic acid
0.017 × 60 = 1.02g mass of acetic acid
To find the volume
Density is written on the bottle of acid
𝒎
d= 𝒗
𝒎
v= 𝒅
𝟏.𝟎𝟐
v=𝟏.𝟎𝟓
 v = 0.97 round of 1mL
 For sodium acetate
𝑛𝑜 𝑜𝑓 𝑚𝑜𝑙𝑒𝑠
Molarity: 𝑣𝑜𝑙𝑢𝑚𝑒

Moles: molarity × volume

100
Moles: 0.36 × 1000

Moles of sodium acetate: 0.036 moles

𝒎𝒂𝒔𝒔
No. of moles: 𝒎𝒐𝒍𝒂𝒓 𝒎𝒂𝒔𝒔

No of moles of sodium acetate × molar mass of sodium acetate = mass of sodium acetate
0.036 × 82 = 2.952g mass of sodium acetate
To find the volume
𝒎
d= 𝒗
𝒎
v= 𝒅
𝟐.𝟗𝟓𝟐
v= 𝟏.𝟓

v = 1.968 round of 1mL

Result
pH was 4.9 so the solution was acidic in nature.
EXPERIMENT NO. 2:
EXTRACTION OF ESSENTIAL OIL BY USING GC-MS
Different plant extracts is called essential oil. We can also define as essential oil is hydrophobic
liquid containing volatile compounds (easily evaporated at normal temperatures) from plants
it is also called volatile oil. It contains the essence of the plant’s. In this experiment we separate
oil from perfume sample by using the technique that called GC-MS Gas Chromatography Mass
Spectrometry.
Gas Chromatography Mass Spectrometry
The Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical
mixtures (the GC component) and identifies the components at a molecular level (the MS
component). It is one of the most accurate tools for analyzing environmental sample.
Principle
Gas Chromatography Mass Spectrometry is used for separation and detection of sample. The
GC works on the principle that a mixture will separate into individual substances when heated.
The heated gases are carried through a column with an inert gas (such as helium). As the
separated substances emerge from the column opening, they flow into the MS. Mass
spectrometry identifies compounds by the mass of the analyte molecule.
In GC-MS stationary phase is less volatile liquid and mobile phase is gas.

Instrumentation of GC-MS
Gas chromatography mass spectrometry (GC-MS) consists of two very different analytical
techniques:
 Gas chromatography (GC)
 Mass spectrometry (MS)
The sample is first introduced into the GC inlet. If Sample is in liquid form first it vaporated in
heated inlet and the sample vapor is transferred to the analytical column. The analytes
are separated by their differences in partitioning between the mobile phase (carrier gas) and the
liquid stationary phase (held within the column), or for more volatile gases their adsorption by
a solid stationary phase. In GC-MS analyses, a liquid stationary phase held within a narrow
(0.1-0.25 mm internal diameter) and short (10-30m length) column is most common. Lower
width separation column used because it causes fine separation. The neutral
molecules elute through a heated transfer line into the mass spectrometer. Within the mass
spectrometer the neutral molecules are first ionized most commonly by electron ionization. The
next step is to separate the ions of different masses which is achieved based on their m/z by
the mass analyzer After the ions have been separated by the mass analyzer based on their m/z
they reach the ion detector. Signals are recorded on the computer to produce a chromatogram
and a mass spectrum for each data point.

Apparatus
 Gas Chromatography Mass Spectrometry ( GC-MS )
 Essential oils / Perfume samples
Procedure
1. Took a 100µl of essential oil perfume sample.
2. For each GC-MS injection used 1µl sample.
3. Injector put into the gas chromatography mass spectrometry and then optimized the
conditions ( boiling point of solvent should known).
4. Set the temperature 220 °C it starts from 20 °C - 60 °C.
5. Heat the column using electricity.
6. Sample ionizes on a electron ionization check the readings after 10mints in analyzer.
7. Next step is to separate the ions of different masses on the charged to mass ration on
the mass analyzer.
8. The ions that have been separated by the mass analyzer reaches to the detector that
detect the signals and recorded on the computer.
9. A computer produce a chromatogram and mass spectrum of the extracted oil that we
extract from perfume sample solution by using GC-MS.
Graphical Interpretation

Fig: Total ion chromatogram (TIC) output from a GC-MS

This chromatogram shows Detector Responses on x-axises and Retention Time on y-axises.
Two peaks present in this chromatogram one is RT1 and secound is RT2. The RT1 is the
highest peak because its retention time is more and high boiling point. RT2 is the lowest peak
beacause its retention time is lower and lower boiling poit.
Base Peak: That shows more height and which compound is more abundent.
Molecular Peak: That shows which molecule is heavier.
If two compounds or components that shows similarity then we will see Retention Index Value
check.

Percentage composition of sample component

𝑨𝒓𝒆𝒂 𝒐𝒇 𝑷𝒆𝒂𝒌
٪ composition = 𝑻𝒐𝒕𝒂𝒍 𝑨𝒓𝒆𝒂

A = h ×𝒘𝟏𝟐
Graphs of GC-MS
EXPERIMENT NO. 3:
SEPARATION OF HYDROCARBONS BY USING GC-MS
Hydrocarbons are compounds comprised exclusively of carbon and hydrogen and they are by
far the dominant components of crude oil, processed petroleum hydrocarbons (gasoline, diesel,
kerosene, fuel oil, and lubricating oil), coal tar, creosote, dyestuff, and pyrolysis waste
products. The majority of hydrocarbons are colourless and hydrophobic, and their odours are
either weak or best characterized by those of gasoline and lighter fluid. They can be found in a
wide variety of molecular forms, including gases (like methane and propane), liquids (like
hexane and benzene), low-melting solids (like paraffin wax and naphthalene), and polymers
(such as polyethylene and polystyrene). Gas chromatography-mass spectrometry (GC-MS) is
a powerful technique for separating and detecting molecules. It is a widely-used method across
different industries like forensics, pharmaceuticals, environmental analysis, and
petrochemicals to detect for volatile organic molecules or gases.
Principle
GC-MS is use for separation and detection of samples in gas phase under vacuum. GC works
on the principle that the components to be separated will introduce into inlet system which
works under vacuum (10-4 – 10-7), and high temperature i.e. 300℃. This temperature is enough
to vaporize the volatile samples, non-volatile samples are vaouprize by spark. Heated gases are
carried through a column with an inert gas (such as helium, nitrogen or Argon). This will
vaporize our sample and then take the sample ionization source. The common method use for
ionization is bombarding of electrons on them. As the separated substances emerge from the
column opening, they flow into the Mass separator where these will detect on the base of charge
to mass ratio. They basically have unit charge on them so the separation will take place on the
basis of mass only.
Instrumentation
 autosampler
 inlet
 analytical column
 interface
 vacuum
 ion source
 mass analyzer
 ion detector and
 PC
A simplified diagram of a gas chromatograph–mass spectrometer showing carrier gas

Apparatus and Chemicals

 Gas Chromatography-Mass Spectrometry (GC-MS)
 N-heaxane • Diethyl ether
 Acetonitile
 Ethanol
 1-Propanol
 Chloroform
 Ethyl acetate
Procedure
1. A 100 µl perfume sample was taken from the essential oil.
2. A 1 µl sample was used for each GC-MS injection used.
3. Insert the injector into the gas chromatography-mass spectrometer and optimize the
conditions
4. Set the temperature to 220°C. Start at 20°C to 60°C.
5. Heat the column electrically.
6. The sample is ionized by electron ionization. Check the analyzer readings after 10
minutes.
7. The next step is to separate the ions of different masses by the charge-to-mass ratio of
the mass spectrometer.
8. Ions separated by the mass spectrometer reach the detector and pick up the signal. and
record it on computer.
9. A computer generates chromatograms and mass spectra of the extracted oil from which
it was extracted from Perfume sample solution using GC-MS
EXPERIMENT NO 04:
IDENTIFICATION OF UNKNOWN HYDROCARBON BY THIN LAYER
CHROMATOGRAPHY
Introduction
Thin layer chromatography (TLC) is a chromatographic technique used to separate the
components of a mixture using a thin stationary phase supported by an inert backing. It may be
performed on the analytical scale as a means of monitoring the progress of a reaction, or on the
preparative scale to purify small amounts of a compound. TLC is an analytical tool widely used
because of its simplicity, relative low cost, high sensitivity, and speed of separation.
Principle
The separation principle of the TLC procedure is based on the given compound’s relative
affinity towards the mobile and the stationary phase. The process begins here by moving the
mobile phase over the stationary phase’s surface. During this movement, the higher affinity
compounds gain less speed as compared to the lower affinity compounds. This results in their
separation.
Once the procedure gets completed, different spots can be found on the stationary surface at
distinct levels, reflecting various elements of the mixture. Basically, the compounds that are
more attracted towards the stationary phase secure their position at lower levels while others
move towards the higher levels of the surface. So their spots can be seen accordingly.
Equipment
 Beakers
 Pipettes
 Stirrer
 Weighting balance
 Led pencil
 TLC plate
 Capillary
 Bunsen Burner

Required chemicals
 Diphenyl imine
 Aldehyde
 n-hexane
  Methanol

Procedure
1. The process starts by making a thin mark on the TLC plate’s bottom with a pencil.
The line which are drawn on the TLC plate is called base line.
2. It helps in the application of sample spots. These spots are kept at equal distances.
3. The sample is then applied to these spots made on the line.
4. Sample spots means mixture of diphenyl imine and aldehyde and also spot these two
chemicals separately.
5. Then the TLC chamber is filled with the mobile phase up to a few centimeters of its
bottom.
6. Mobile phase which was used here n-hexane.
7. Finally, the prepared stationary phase plate is put inside the chamber. At this point,
the sample spots are kept on the mobile phase’s side.
8. Remember to keep the sample spots well above the level of the mobile phase. Do not
immerse it in the solvent.
9. The chamber is then closed after placing the plate into it.
10. Once the solvent front neared the top of the plate, I removed the plate from the TLC
chamber, the solvent front was marked with a pencil, and allowed it to dry.
11. At last, the sample spots get analyzed through a suitable method for the sample, such
as UV light, KMnO4 stain, and iodine staining.
Retention Factor
After a separation is complete, individual compounds appear as spots separated vertically. Each
spot has a retention factor (Rf) which is equal to the distance migrated over the total distance
covered by the solvent.
The Rf formula is
Rf=distance traveled by sample/distance traveled by solvent
Result
Rf value of unknown compound is 0.65.
EXPERIMENT NO 05:
GRAVIMETRIC ANALYSIS
Introduction
Gravimetric analysis describes a set of methods in analytical chemistry for the quantitative
determination of an analyte based on the mass of a solid.
In most cases, the analyte in solution is first converted to a solid by precipitation with an
appropriate reagent. The precipitate can then be collected by filtration, washed to remove
impurities, dried to remove traces of moisture from the solution, and weighed. The amount of
analyte in the original sample can then be calculated from the mass of the precipitate and its
chemical composition. This approach has been used to determine the atomic weights of many
chemical elements.
In other cases, it may be easier to remove the analyte by vaporization. The analyte may be
collected perhaps in a cryogenic trap or on some absorbent material such as activated carbon
and measured directly. Alternatively, the sample may be weighed before and after it is dried;
the difference between the two masses gives the mass of analyte lost. This approach has been
especially useful in determining the water content of complex materials such as foodstuffs.
General procedure:
A general procedure for gravimetric analysis is outlined below.

The sample is dissolved, if it is not already in solution.

The solution may be treated to adjust the pH (so that the proper precipitate is formed, or to
suppress the formation of other precipitates). If it is known that species are present which
interfere (by also forming precipitates under the same conditions as the analyte), the sample
might require treatment with a different reagent to remove these interferents.
The precipitating reagent is added at a concentration that favors the formation of a "good"
precipitate. This may require low concentration, extensive heating (often described as
"digestion"), or careful control of the pH. Digestion can help reduce the amount of
coprecipitation.
After the precipitate has formed and been allowed to "digest," the solution is carefully filtered.
The filter needs to be appropriately chosen to trap the precipitate; smaller particles are more
difficult to filter.
Depending on the procedure followed, the filter might be a piece of ashless filter paper in a
fluted funnel, or a filter crucible. Filter paper is convenient because it does not typically require
cleaning before use; however, filter paper can be chemically attacked by some solutions (such
as concentrated acid or base), and may tear during the filtration of large volumes of solution.
The alternative is a crucible that has a bottom made of some porous material, such as sintered
glass, porcelain, or sometimes a metal. These materials are chemically inert and mechanically
stable, even at elevated temperatures. However, they must be carefully cleaned to minimize
contamination or carryover (cross-contamination). Crucibles are often used with a mat of glass
or asbestos fibers to trap small particles.
After the solution has been filtered, it should be tested to make sure that the analyte has been
completely precipitated. This is easily done by adding a few drops of the precipitating reagent;
if a precipitate is observed, the precipitation is incomplete.
After filtration, the precipitate, along with the filter paper or crucible, is heated. This achieves
three purposes:
The remaining moisture is removed (drying).
Secondly, the precipitate is converted to a more chemically stable form. For instance, calcium
ion might be precipitated using oxalate ion, to produce calcium oxalate (CaC2O4); it might
then be heated to convert it into the oxide (CaO). It is vital that the empirical formula of the
weighed precipitate be known, and that the precipitate be pure; if two forms are present, the
results will be inaccurate.
The precipitate cannot be weighed with the necessary accuracy in place on the filter paper; nor
can the precipitate be completely removed from the filter paper in order to weigh it. The
precipitate can be carefully heated in a crucible until the filter paper has burned away; this
leaves only the precipitate. (As the name suggests, "ashless" paper is used so that the precipitate
is not contaminated with ash.)
After the precipitate is allowed to cool (preferably in a desiccator to keep it from absorbing
moisture), it is weighed (in the crucible). The mass of the crucible is subtracted from the
combined mass, giving the mass of the precipitated analyte. Since the composition of the
precipitate is known, it is simple to calculate the mass of analyte in the original sample.