CHE203L.

1 LAB REPORT EXPERIMENT 1A & 1B

Submitted By: Synthia Sadia Salim

ID: 2231669647
Submitted To: Dr. Mohammed Kabir Uddin (MKUn)
Date of Submission: 02/02/2023

North South University

Department of Biochemistry & Microbiology
Course name: Chemistry of Biomolecules Lab (CHE203L)
Section: 01
Semester: Spring 2025
 Experiment 1A: Separation of Mixtures by Using Simple
Distillation
 Purpose
This experiment aims to separate a mixture of water, acetone, and alcohol using simple
distillation and identify the components through chemical tests.

 Introduction
Distillation is a purification technique that exploits differences in boiling points of separate
volatile components in a mixture. When heated, the constituent with the lowest boiling
point vaporises first, condenses upon cooling through the condenser, and is collected as a
distillate in the receiving flask at the other end. In this experiment, acetone (boiling point:
56°C), tertiary butyl alcohol (boiling point: 56°C), and water (100°C) are separated.
Chemical tests such as the 2,4-DNPH test confirm the presence of acetone, while the Lucas
test differentiates primary, secondary, and tertiary alcohols. This experiment highlights the
practical application of distillation in isolating and identifying organic compounds.

 Material (Reagents and Apparatus)

The reagents or mixtures used in this experiment are a water-acetone-ethanol mixture,
which was used as a sample; 2,4-dinitrophenylhydrazine (2,4-DNPH); and Lucas reagent
(HCl/ZnCl₂), which was prepared by dissolving Zinc chloride in a concentrated
Hydrochloric acid solution.
The apparatus used in this experiment are a distillation kit, test tubes, a Bunsen burner,
and a pipette.
The distillation kit contains a 250ml round-bottom flask, a condenser connected to rubber
tubing containing a water inlet and water outlet opening, a thermometer, and a receiving
flask collecting the distillate at the end of the condenser.

 Methods
A 250 mL round-bottom flask was selected as the boiling flask. Two boiling chips were
added to ensure smooth boiling. The flask was clamped and connected to a distillation head
with a standard taper joint. A thermometer was positioned so its bulb rested just below the
sidearm of the distillation head/condenser to monitor the temperature of the vapour. A
water-cooled condenser was attached to the distillation head. Rubber tubing carrying
water was connected to the condenser, which had a lower inlet (water entry) and upper
outlet (water exit) to maintain continuous water flow. A receiving flask (250 mL) was
placed at the condenser’s end to collect distillates. All joints were lightly greased before
they were fixed together to prevent sticking. The boiling flask was filled with 100 mL of the
water-acetone-ethanol mixture.
The mixture was heated gently using a Bunsen burner. The heating was adjusted to
maintain a steady boiling rate, avoiding rapid vaporisation. As boiling commenced, vapours
rose into the condenser. The temperature was recorded when the first 1–3 mL of distillate
(acetone) condensed into the receiving flask (observed at 56–60°C). The first distillate was
collected until the temperature remained stable. Heating continued, and the temperature
rose to 82.2°C as tertiary butyl alcohol began distilling. The receiving flask was replaced
with a clean one to collect the second distillate, which was the tertiary butyl alcohol.
Distillation was halted once the temperature exceeded 82.2°C to avoid water
contamination.
In a test tube labelled 1, 4mL of the first distillate (acetone) was added using a pipette.
Using a dropper, 5–10 drops of 2,4-DNP reagent were introduced.
In test tube 1, 4 mL of the second distillate (tertiary butyl alcohol) was combined with 2 mL
of Lucas reagent (ZnCl₂ dissolved in concentrated HCl). The tube was shaken vigorously
and then observed

 Results
First Distillate (Test tube 1): Positive results with 2,4-dinitrophenyl hydrazine (2,4-DNPH)
where yellow or orange precipitate is seen due to the formation of hydrazone, confirming
the presence of acetone.
Second Distillate (Test tube 2): Positive results with Lucas reagent (ZnCl₂ dissolved in
concentrated HCl). It immediately forms two layers, the upper layer being a cloudy solution
due to the rapid formation of tert-butyl chloride, confirming the presence of tertiary butyl
alcohol, and the lower layer being a transparent/clear solution.

 Discussion
The boiling point order (acetone < tert-butyl alcohol < water) allowed sequential
separation of the components in the mixture used for this experiment. Acetone has a low
boiling point due to the absence of hydrogen bonding and the presence of weak Vander
Waals forces. The 2,4-DNPH tests specifically identified acetone’s carbonyl group. The
reaction of tert-butyl alcohol with Lucas reagent confirmed its classification as a tertiary
alcohol, which is highly reactive. It has relatively higher boiling point due to the presence of
OH groups hence strong hydrogen bonding occurs. The potential errors of this experiment
include incomplete separation due to rapid heating or contamination during testing. To
address incomplete separation, we maintain a controlled and steady heating rate to allow
for proper distillation. To prevent contamination, ensure all glassware is clean and dry, and
handle samples with care throughout the process.
 Experiment 1B: Preparation of Acid-Base Solutions, TE
Buffer, and PBS Buffer
 Purpose
The purpose of this experiment is to prepare 250 ml of 1.5 M NaOH, 100 ml of 1.5 % NaOH,
250 ml of 1M HNO3 solutions and 1000 ml of 10X TE buffer (10 mM Tris-HCl and 1 mM
EDTA) made from 1M Tris-HCl and 0.5M EDTA at pH 8.0 for laboratory uses.

 Introduction
The preparation of an accurate biochemical solution is necessary for use in laboratory
experiments to yield accurate results. Both Sodium hydroxide (NaOH) and Nitric acid
(HNO3) solutions are used for pH adjustments. If the pH of a solution is too high (alkaline),
HNO3 is added for neutralisation or if the pH of a solution is too low (acidic), NaOH is used
for neutralisation. TE buffer is used for storing nucleic acids where EDTA, which is an ion-
chelating agent, is used to prevent the degradation of nucleic acid by nucleases and Tris,
which is an effective buffer, helps to maintain the pH of solution between 7 and 8.

 Material (Reagents and Apparatus)

To make 1.5 M NaOH in 250ml solution and 1% NaOH in 100ml solution we need: Sodium
hydroxide (NaOH) pellets, distilled water, a weighing balance, 250ml volumetric flask and a
stirrer
To make 1 M HNO3 in 250ml solution we need: Concentrated acid (conc. HNO3), distilled
water, a pipette, 250ml volumetric flask and a stirring rod.
To make 10X TE buffer we need: stock solution of Tris base
(Tris(hydroxymethyl)aminomethane) C₄H₁₁NO₃, stock solution of
Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na2·2H2O), a 1000ml
beaker or volumetric flask, distilled water, a pH meter, Hydrochloric acid (HCl) or sodium
hydroxide (NaOH) for pH adjustment and a magnetic stirrer.

 Methods
To make 1.5 M NaOH in 250ml solution, we measure exactly 15.0 g of NaOH pellets in an
analytical balance, transfer all of it to a volumetric flask and then start adding distilled
water. We need to stir the solution with a stirring rod at the same time to make sure the
pellets mix and dissolve in the solution. We stop pouring water when the volume of the
solution finally reaches 250ml.
To make 1.5% NaOH in 100ml solution, we measure exactly 1.5 g of NaOH pellets in an
analytical balance, transfer all of it to a volumetric flask and then start adding distilled
water. We need to stir the solution with a stirring rod at the same time to make sure the
pellets mix and dissolve in the solution. We stop pouring water when the volume of the
solution finally reaches 100ml.
To make 1 M HNO3 in 250ml solution, we take 16.04 ml of the given concentrated Nitric
acid in a 250ml volumetric flask, as per the calculations, and then add 100 ml of distilled
water into the acid. After that, we stir the solution using a stirring rod and then again add
distilled water until the volume of the entire solution reaches 250ml.
To make a 10X TE buffer, we must first prepare the stock solutionof Tris-base and EDTA
separately.
According to calculations, we prepare 10ml of 1M Tris-base using 1.21g of Tris-base, and
then adding 6-8ml of distilled or deionised water in a beaker. Then, we measure the pH of
this solution using a pH meter. If the pH of the solution is not close to 8, then we adjust it by
adding a base solution like NaOH (if the pH is low) or an acidic solution like HNO3 (if the
pH is high). After adjusting it, distilled water is added again until the volume reaches 10ml.
In the same manner, we prepare 10ml of 0.5M EDTA using 1.86g of EDTA, and then adding
6-8ml of distilled or deionised water in a beaker. Then, we measure the pH of this solution
using a pH meter. If the pH of the solution is not close to 8, then we adjust it by adding a
base solution like NaOH (if the pH is low) or an acidic solution like HNO3 (if the pH is high).
After adjusting it, distilled water is added again until the volume reaches 10ml.
Finally, we take 10ml of 1M Tris-base and 2ml of 0.5M EDTA from both the stock solution
and place them in 1L volumetric flask and add some distilled water and stir the solution
using a stirrer. The again distilled water until the volume reaches 1000ml.

 Results
**Calculations from performance paper**

 Discussion
Each of these solutions were prepared by carefully measuring the weights and volumes of
the compounds or solutions used. The accuracy of these measurements is vital, as
deviations can lead to significant changes in the solution's concentration, potentially
affecting subsequent experiments. The nitric acid solution required careful dilution, as
concentrated HNO₃ is highly corrosive and must be handled with caution. Potential sources
of error could have included inaccurate measurements, improper mixing, or failure to
achieve the desired pH. To minimise errors, we ensure precise measurements using
calibrated equipments and take multiple readings for accuracy. Additionally, thorough
mixing and regular pH monitoring during preparation can help achieve the desired solution
properties.