Instrumental Analysis Lab.

Course No.: 501418

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Faculty of Pharmacy and Medical Sciences

Instrumental Analysis laboratory

Course No : 501418

Prof. Eyad Mallah

Mr. Ammar Rasras

2024

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Contents
Introduction 5
General Laboratory Instructions 11

Experiment (1)
Calibration of volumetric glassware 15

Experiment (2)
Titration of Hydrochloric Acid with Sodium Hydroxide
(Direct titration) 22

Experiment (3)
Titration of Aspirin Tablets 30

Experiment (4)
Application of Qualitative Analysis by using
(Ultraviolet-Visible Spectroscopy) 36

Experiment (5)
Application of Quantitative Analysis by using
(Ultraviolet-Visible Spectroscopy) 47

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Experiment (6)
Qualitative Analysis of paracetamol in tablet by Using HPLC 53

Experiment (7)
Quantitative Analysis of paracetamol in tablet by Using HPLC 59

Experiment (8)
Determination of functional groups and identification of compounds by
using Infrared Spectroscopy (IR) 66

Experiment (9)
Determination of Ethanol in Commercial Products by using GC 77

Experiment (10)
Liquid Chromatography/ Mass Spectrometry 80

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Introduction

The Instrumental Analysis Laboratory is one of the most important

laboratories for pharmacy students, focusing on the use of modern instruments

and techniques to analyze the chemical composition, structure, and physical

properties of substances. In addition to, determine the levels of

pharmaceutical compounds.

Overview

Instrumental analysis involves the use of instruments to measure physical

quantities related to chemical species. These measurements help determine

qualitative and quantitative information about a substance or mixture.

Experiments focused on instrumental analysis use sophisticated tools to

detect, analyze, and quantify pharmaceutical substances.

Key Instruments

1. Spectroscopy:

- UV-Vis Spectroscopy: Measures the absorption of ultraviolet or visible

light by a substance to determine concentration.

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-IR Spectroscopy: Analyzes molecular vibrations to identify functional

groups.

-NMR Spectroscopy: Provides information about molecular structure by

studying the magnetic properties of nuclei.

-Atomic Absorption/Emission Spectroscopy: Used for detecting metals by

measuring the absorption or emission of light by atoms.

2. Chromatography:

-Gas Chromatography (GC): Separates volatile compounds in a mixture for

qualitative and quantitative analysis.

-High-Performance Liquid Chromatography (HPLC): Separates,

identifies, and quantifies components in a liquid sample.

-Ion Chromatography (IC): Separates ions and polar molecules for analysis.

3. Mass Spectrometry (MS):

-Used in combination with other techniques like GC or HPLC, it measures the

mass-to-charge ratio of ions to identify and quantify substances.

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4. Electrochemical Analysis:

-Techniques such as potentiometry, and voltammetry are used to measure

electrical properties that relate to chemical properties.

5. X-ray Diffraction (XRD):

- Provides information on the crystallographic structure of materials.

Applications

- Environmental Monitoring: Detection of pollutants and toxins in air, water,

and soil.

- Pharmaceuticals: Ensuring the purity and concentration of active ingredients

in drugs.

- Food and Beverage: Quality control, detecting contamination or

adulteration.

- Forensics: Identifying substances in criminal investigations.

- Importance in Research and Industry.

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The precise and accurate results produced by these instruments are essential

for understanding complex chemical systems, ensuring product quality,

complying with regulatory standards, and advancing scientific knowledge.

Instrumental analysis labs are crucial in areas like pharmaceuticals, materials

science, environmental analysis, and biochemistry.

In sum, an Instrumental Analysis Laboratory equips scientists and engineers

with the tools necessary to investigate chemical compositions and structures,

providing the backbone for modern analytical chemistry.

Common Laboratory Glassware

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10
General Laboratory Instructions

1. Laboratory Safety

- Each student must wear a clean, white laboratory coat at all times in the

laboratory. In addition, they should wear Safety Goggles, Closed-Toed

Shoes, Clothing that covers your legs, do not wear contact lenses.

- Eating or drinking in the laboratory is not permitted.

- Do not pipette by mouth or transport reagents across the laboratory.

- Return all equipment and bottles to their proper location after use.

- Assume all chemicals used in the experiment are hazardous.

- Wash your hands properly before leaving the lab.

2. Laboratory structure

- The students will work in groups.

- Students must read the theory and protocols of each experiment before

arriving to the lab.

- Each new experiment will be preceded by a brief lecture-discussion of the

theory and procedure.

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Instructions of Laboratory Report

-Laboratory Notebook

-The lab manual contains information about experimental procedure.

-The lab report with completed lab work should be delivered to the

instructor at the start of the following lab session.

-Abstract

The Abstract provides an insightful summary of everything you

accomplished and learned.

The abstract should contain the following:

- The objectives and scope of the experiment.

- A summary of the results and conclusions.

- Experiment's outcomes.

-Introduction of Report

This provides an overview of the analysis to be performed. The introduction's

objective gives students picture and contextualize about experiment.
The Introduction should not include any results or conclusions, and should

contain one or two paragraphs.

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- Experimental

Experimentation is a description of the materials and processes employed,

including what was done and how.

Describe the sample preparation process, including instrument specs. and

techniques.

While the Method does not need to include minute specifics (for example, if

you followed a set of printed instructions, you may not need to write out the

entire procedure - simply indicate what was done and credit the manual).

-Results

This part summarizes what you discovered; only mention your own

observed results in this section. Your Results will include the following:

- Pictures and spectra.

- Use tables and graphs whenever possible.

- Calculate samples for important lab aspects such as dilutions and the usage

of standard curves.

- Brief statements on the results in the text (without duplicating the data

from the graphs and tables). When writing about each picture, graph, or

table.
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- Discussion

Provide your interpretation of your findings, possibly comparing or

contrasting them with the literature. Reflect on your actual data and

observations.

Explain or rationalize incorrect facts, or identify potential causes of mistake

and how they may have influenced the outcome.

The discussion must address the issue, "What do the results mean?" It's a

results-based argument.

-Conclusion

This is a summary of your argument, experiment, or research that should refer

back to the introduction. The Conclusion should be brief and reiterate the

results of your experiment/research. If relevant, offer ways to improve the

process and what more experiments or study may be beneficial.

-References

Cite any references you've used, making sure that each item in the reference

list has an in-text citation and a full reference at the end of your report.

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 Experiment (1)

Calibration of Volumetric Glassware

Aim:

Calibration of glassware for volumetric analysis.

Principle:

Calibration ensures that the marked volumes match the actual volumes

delivered by the equipment. It offers a remedy if agreement is missing.

One method used in calibration, to determine the mass of a liquid with a

known density delivered by the glassware, then compute its volume and

compare it to the market volume.

Theory:

Laboratory Technique for Burets

 Burets are used to transfer a pre-determined amount of liquid or solution

into another container. A burette is marked in milliliters, similar to a

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 graduated cylinder, except the numbers grow as you move down the

burette.

The stopcock regulates liquid flow. It is open while parallel to the burette’s

length, and closed when perpendicular to it.

 To clean a burette, wash the interior with soap and tap water. Next, rinse

the burette with (5-10) mL of DI water. Pour the water into the burette and

allow it to drain out the tip while holding it over the sink and opening the

stopcock. Pour solutions into the burette using a beaker; most breakage

occurs during the washing process, and burets do not fit under the faucet.

 Conditioning the burette: Once the burette has been thoroughly drained,

close the stopcock and pour approximately (5) mL of the titrant. To

thoroughly rinse the burette’s interior walls, tilt it sideways and roll the

barrel. Drain the solution through the burette tip to ensure it is well

conditioned. Repeat this step at least twice to ensure that all interior

surfaces are thoroughly rinsed with titrant.

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 To fill the burette, close the stopcock. Using a clean funnel, fill the burette

with titrant just over the "0" level. Place a container under the burette tip

and momentarily open the stopcock to fill the burette tip with solution,

leaving no air bubbles and bringing the meniscus level within the burette’s

markings. If the tip does not fill with solution while the stopcock is open,

the stopcock may contain an air bubble. Consult your instructor. Note: The

initial level of titrant does not have to be precisely at (0.00) ml. The

beginning level of liquid will be measured and subtracted from the final

volume to calculate the volume supplied.

 Before measuring the burette, remove the funnel that was used to fill it.

Titrant volume is recorded by noting the bottom of the meniscus. The

burette depicted below has numbers for every 1 mL, with ten lines between

each number for every (0.1) ml. Thus, the level of titrant in the burette can

be calculated to one decimal place higher than the markings, or to the

nearest (0.01) ml. As seen in the picture to the right, the meniscus is about

midway between (25.0) and (25.1) mL, therefore the amount of titrant can

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 be recorded as (25.04) ml, (25.05) ml, or (25.06) ml depending on whether

the bottom of the meniscus appears to be just above.

 Empty the burette and dispose of the titrant as per the waste disposal

instructions for the experiment. Wash the burette with soap and water, then

rinse with several batches of tap water, allowing some to run into the tip.

Finally, rinse the burette with small amounts of DI water, allowing it to

run through the tip before returning it to the stockroom.

Procedure:

I. Calibration of burette

1- Wash the burette with water and soap, then rinse it with purified water.

2- Fill the burette with (10) ml of distilled water.

3- Measure the temperature of the water.

4- Slowly open the stopcock and let the water drain until it reaches the

zero line.

5- Using a digital balance, weigh a conical flask and record its original

weight.

6- Drain the 10 ml of water from the weighted flask.

7. Weigh the conical flask and record the final mass.

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II. Calibration of a pipette

Follow the same technique using a (10 ml) bulb pipette instead of the

burette.

Data: (calibration of a burette)

Initial burette reading:

Final burette reading:

Initial mass of flask:

Temperature of water:

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Density of water at different temperatures:

Temperature Density Temperature Density

CO g/ml CO g/ml

10 0.9984 21 0.9970

11 0.9983 22 0.9968

12 0.9982 23 0.9966

13 0.9981 24 0.9964

14 0.9980 25 0.9962

15 0.9979 26 0.9959

16 0.9978 27 0.9957

17 0.9977 28 0.9954

18 0.9975 29 0.9952

19 0.9974 30 0.9968

20 0.9972 31 0.9947

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 Experiment (2)

Titration of Hydrochloric Acid with Sodium Hydroxide

(Direct titration)

1- Aim:

To determine the concentration of a hydrochloric acid solution by using acid‐

base titration.

2-Principle:

Titration is a technique used to determine an unknown concentration of a

known solution. Because we know what the chemical is, we can predict how

it will react with other chemicals and utilize that reaction to calculate the

concentration of the solution by detecting the creation of product (s). When

there is an unknown concentration of acid, we can utilize a known

concentration of hydroxide base. This is a neutralization process, and the

products are salt and water.

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A pH indicator, which changes color based on pH, then can indicate full

neutralization of a reaction. The equivalency point is the moment at which all

acid has been utilized and no excess base remains. We can use this

equivalency point to calculate the acid's initial concentration. The purpose of

the titration is to reach as close to the equivalence point as feasible by

gradually adding the base; this ensures that the computed acid concentration

is as close to the genuine value as possible. You'll run three titrations and

average the results.

2-Application: Titrations require computations to determine an unknown

acid concentration. The computations are presented below. For additional

examples, check to your lecture notes.

3-Theory:

• After each titration, determine the number of moles of sodium hydroxide

utilized. First, you need to know

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The standardized solution has a precise molarity determined by

experimentation.

Make a note of this when you begin the titration. Next, calculate the volume

of the given solution to reach the equivalency point. Next, determine the moles

of base utilized in the titration:

• Determine the moles of HCl in the flask:

The balanced reaction between sodium hydroxide and hydrochloric acid

follows a 1:1 ratio. Each hydroxide (-OH) ion neutralizes one hydronium

(H+) ion. Neutralization reactions and acid-base titrations do not always

follow the same pattern. To calculate the general formula, multiply the

determined moles of base from the previous equation by the stoichiometric

ratio found in the balanced equation.

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•Determining the acid concentration:

The number of moles present at the start of the titration can be used to

calculate the initial acid concentration in the flask. In the end, only water and

salt remain. To compute the acid concentration, use the following formula

given the starting volume:

4-Requirement:

The following terms will help you comprehend the terminology used in the

experiment:

• The solution with a known concentration is known as the titrant or

standardized solution. This lab uses sodium hydroxide solution as a titrant.

• A burette is a long, cylindrical piece of glass used to accurately measure

minute amounts of solution. A burette is regulated by a stopcock, which is a

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white Teflon piece that may be rotated to release the solution. The markings

on the burette require you to deduct the beginning reading (where the titrant

level is initially) from the end reading to get the volume of base supplied. The

burette precisely measures the second digit following the decimal point.

• A volumetric pipette or pipette bulb is a narrow glass tube with a single

marking used to measure volume.

•Phenolphthalein, a pH indicator. In acidic and neutral solutions, the indicator

is colorless, whereas in basic solutions, it is a vivid pink.

Pink color intensity increases with higher pH levels. The equivalent point

occurs when the color is extremely faint pink. To observe the color shift, place

the acid and indicator flask on a white piece of paper.

 50‐mL Burette with clamp

 Phenolphthalein indicator

 125 mL or 250‐mL Erlenmeyer flasks

 Burette funnel

 250‐mL beaker

 25‐mL volumetric pipette

 Pipette bulb

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5-Procedure:

You'll do at least three titrations. If you add too much base and the solution

becomes too bright pink, you must delete the results and repeat the

experiment. In addition, if your titrations deviate by more than 1%, you will

need to repeat them. Four columns of data are provided for this purpose.

Patience in this lab will save you from having to perform further

experiments!!!

1. Record the molarity of the sodium hydroxide

solution on the note paper.

2. In a clean beaker, prepare roughly 100 mL of sodium

hydroxide solution. This should be sufficient for

cleaning your burette and doing your first three trials.

3. To fill the burette, pour approximately 5 mL of the

base solution from the beaker using a funnel. Move the

funnel around to coat the burette’s sides with base. To

coat all inner surfaces, carefully tilt and spin the burette

with 5 mL of titrant after removing it from the stand.

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Using the stopcock, drain the solution into a waste beaker. Rinse with a second

5 mL of base.

4. Add more sodium hydroxide solution to the burette until it reaches around 0.00

ml. Open the stopcock and let a few droplets rinse through the burette’s tip.

This should clear out any air bubbles in the burette tip. Fill out the data sheet

with your initial burette reading for trial 1. The volume does have to be accurate

(0.00 mL).

5. Take 25.00 mL of the acid solution into the volumetric pipette and transfer

this solution into an Erlenmeyer flask. Add 2‐3 drops of phenolphthalein to the

acid solution in the flask.

6. Begin by adding the base solution to the Erlenmeyer flask while it is under

the burette. Assign one lab partner to spin the flask while the other controls the

stopcock.

When pink begins to appear, add the solution more gradually. To achieve a

light pink tint, add one drop at a time and swirl for 30 seconds. Remember:

the lighter the pink, the better!!

7. Record the final reading of the burette. Wash the contents of the flask down

the drain with water.

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8. If necessary, fill the burette with extra sodium hydroxide solution. Fill in
the revised volume for trial 2 on the data sheet. Pipette another acid sample,
add phenolphthalein, and repeat the titration process.

9. Repeat the titrations until three of them deviate by no more than 1.0%.

10. Fill out the data sheet and post-lab questions. Present your work for full
credit!!!

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 Experiment (3)

Titration of Aspirin Tablets

(Back Titration)

1-Aim:

To determine the purity of commercially available aspiring tablets using an

acid-base titration. In general, acid and base react to produce salt and water

by exchanging a proton (H+):

2.Principle:
Acetylsalicylic acid is the active ingredient of aspirin, as well

as the chemical known by the same name. To quantify the

amount of aspirin (acetylsalicylic acid) in a sample, the exact

volume and concentration of NaOH, as well as the total

reaction, must be determined. NaOH is used as a

supplementary standard since its concentration varies over time.

To determine the precise concentration of NaOH, it must be

titrated against a primary standard, which is an acid that completely dissolves

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in water, has a high molar mass, remains pure when standing, and is not

hygroscopic (attracts water from the air). Sodium hydroxide is hygroscopic,

therefore it pulls water from its surroundings.

This means that you cannot simply weigh a sample of sodium hydroxide,

dissolve it in water, and then use the mass to calculate the number of moles of

sodium hydroxide present, because any sample of sodium hydroxide is likely

to be a mixture of sodium hydroxide and water. Thus, the most frequent

method for determining the concentration of any sodium hydroxide solution

is titration. Standardization is the process of precisely determining the

concentration of NaOH using a primary standard. You will first standardize

your NaOH solution before using it to test aspirin tablets for aspirin content

and purity.

3-Theory:

- A titration is a method for estimating the concentration of a solution (the

analyte) by allowing a precisely determined volume of this solution to react

with another solution with a known concentration (the titrant). The

equivalency point is the point in the titration at which enough titrant has been

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added to react perfectly with the analyte, as determined by the balanced

equation.

- An indicator, which changes color at the equivalence point, is commonly

used to mark it. 1 There are various sorts of titrations. In this lab, you will do

an acid-base titration.

4-Application:

Determination of the purity of aspirin tablets

You will titrate a sample of your aspirin (acetylsalicylic acid) with the

standardized NaOH to determine the moles of acid in a given weight of your

product. This will allow you to assess its purity.

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Assuming the aspirin is not contaminated with other acids, the titration

allows you to quantitatively determine its purity. (The reactive hydrogen is

circled in the following equation.)

The net ionic equation for the titration in this experiment is:

5-Procedure:

A) assay of aspirin

Saponification of aspirin does not give a sharp end point with an indicator by

direct titration because the reaction is slow and needs heat so back titration is

the solution.

Aspirin is to be hydrolyzed with an accurately measured volume of (0.5) M

NaOH in excess; the excess is then titrated with (0.5) M HCl.

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1- of a known amount of powdered aspirin tablets, take a weight that is

equivalent to (100) mg aspirin

2- add (20) ml 0.5 M NaOH

3- heat for 10 minutes

4-titrate the excess of alkali with standardized 0.5 M HCl using phenol red

solution as indicator

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5- calculate the percentage claim and see whether it follows the range allowed

in the British pharmacopoeia.

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 Experiment (4)

Application of Qualitative Analysis

by Ultraviolet-Visible Spectroscopy

1-Aim:

Determination of maximum absorption and effect of solvents for organic

compounds.

2-Instrumentation Ultraviolet-visible spectroscopy

The instrument used in ultraviolet–visible spectroscopy is called a UV/Vis

spectrophotometer. It measures the intensity of light after passing through a

sample and compares it to the intensity of light before it passes through the

sample.

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2.1 Design and Principles of Single and Double-beam UV-Visible

Spectrophotometers

Single beam spectrophotometer

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Feature Single Beam Double Beam

Single beam splits into reference and

sample beams; each beam passes

Single beam passes through
through monochromator, cuvette
Light Path monochromator, sample cuvette,
(reference: blank/solvent, sample:
and detector.
sample solution), and recombines

before reaching dual detector.

Absorbance Single detector directly measures Dual detector measures intensities of

Measurement transmitted light both reference

Baseline drift affects both beams

Subject to drift due to environment
Baseline proportionately, minimizing influence
and instrument aging, affecting
Stability on absorbance ratio; superior
accuracy.
precision compared to single beam.

Higher accuracy and precision, wider

Simpler design, lower cost, suitable dynamic range, ideal for demanding

Advantages for routine analyses or educational applications requiring reliable data

settings. (research, quality control, high-

throughput analysis).

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 Limited accuracy and precision due
Typically, more expensive and
Disadvantages to source fluctuations and baseline
complex design.
instability.

Monochromator, beam splitter,

Monochromator, sample cuvette,
Components reference cuvette, sample cuvette,
detector, source.
dual detector, source.

Routine analyses, teaching Research, quality control, high-

Suitable
laboratories, applications where cost throughput analysis, applications
Applications
and simplicity are prioritized. requiring high accuracy and precision.

2.2 Components of spectrophotometer:

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2.2.1 Light Source:

a- Deuterium Arc Lamp

UV Region

Wavelength Range: 190~420nm

b- Tungsten Lamp:

Wavelength Range:

Part of the UV and the whole of the Visible range (350 ~ 2,500)
nm

c- Xenon Lamp:

Wavelength Range: 190~800nm

2.2.2 Monochromator

The monochromator itself houses the mirrors, slits, and grating. panchromatic

light from a light source is introduced into the monochromator through the

entrance slit and collimated onto a diffraction grating which is rotated to select

discrete wavelengths.

The light is then refocused by another mirror

onto the exit slit so that can be adjusted to

control the spectral bandwidth (SBW).

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The light is then refocused by another series of mirrors and directed to the

sample where it is either transmitted, absorbed, or reflected.

2.2.3 Cuvette:

The cuvette is rectangular test tubes. They are used to hold aqueous solutions

like normal test tubes. Normal test tubes are useful in chemical reactions. In

contrast, cuvettes are used in

UV-Vis spectrophotometer

or fluorometer for the

measurement of

transmittance or absorbance of radiation at a particular wavelength.

2.2.4 Detectors

Detectors are used to measure the transmitted or reflected light from a sample

and convert it into a signal. The type and material of the detector will

determine the sensitivity and wavelength range of the data that can be

acquired. While photomultiplier tubes and silicon photodiodes are sensitive in

the ultraviolet and visible wavelength ranges, Lead sulfide (PbS)

photoconductive cells and indium gallium arsenide (InGaAs) photodiodes are

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used to measure the near-infrared region of the spectrum. However, all the

detectors mentioned below exploit the photoelectric effect where light or

photons that are incident on a material result in the emission of electrons.

3. Principle
The solvent dissolves the drug substance and exerts an intense influence on

the quality and shape of the UV-visible spectrum. Hence the absorption

spectrum of drug substance changes mostly as per the change of solvent that

has been used to dissolve the drug substance. Here, the change in either

wavelength (absorption maxima) or absorption intensity is monitored by

changing the different solvents.

4.Theory
Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry

(UV–Vis or UV/Vis) refers to absorption spectroscopy or reflectance

spectroscopy in part of the ultraviolet and the full, adjacent visible regions of

the electromagnetic spectrum.

Absorption spectroscopy deals with the spectroscopic techniques that measure

the absorption of radiation, as a function of frequency or wavelength, due to

its interaction with a sample. The sample absorbs energy, i.e., photons, from

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the radiating field. The intensity of the absorption varies as a function of

frequency, and this variation is the absorption spectrum. Absorption

spectroscopy is employed as an analytical chemistry tool to determine the

presence of a particular substance in a sample and, in many cases, to quantify

the amount of the material present. Infrared and ultraviolet–visible

spectroscopies are particularly common in analytical applications.

Lambda max or absorption maxima (λ max)

Lambda max refers to the wavelength along the absorption spectrum where

a substance has its strongest photon absorption. Simply, the wavelength at

which a substance displays maximum absorption is called as lambda max .

Different compounds may have very different absorption maxima and

absorbances. Intensely absorbing compounds must be examined in dilute

solution (absorbance value less than 1), so that significant light energy is

received by the detector, and this requires the use of completely transparent

(non-absorbing) solvents.

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The most commonly used solvents are water, ethanol, hexane and

cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S,

Br & I) are generally avoided.

Figure: Typical example of unknown sample depicting absorption

maxima at

279.50 nm

-Choice of solvents

Every solvent is supposed to exhibit UV-vis absorbance cut-off wavelength.

The solvent cutoff is the wavelength below which the solvent itself absorbs

all of the light. So, when choosing a solvent student has to be careful of its

absorbance cut-off. If the solvent is showing cut-off near the absorption

maxima of the substance under examination, another solvent is to be chosen.

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5.Requirements
Apparatus: Glass beakers, measuring flasks, filter paper, Measuring cylinder,

etc.

Chemicals: Paracetamol, Distilled water, Ethanol, 0.1 N NaOH, 0.1N HCl etc.

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6. Procedure
1- Record Lambda Max:

Sample preparation: Use the following solvents:

water, ethanol, 0.1 M HCl, 0.1 M NaOH.

Step 1: Transfer an amount of Phenolphthalein equivalent 10 mg to a 100 ml

volumetric flask, add 80 ml Solvent and sonicate for 10 minutes. Complete up

to 100 ml with diluent. (Stock solution) (Note: Repeat the process for each

solvent).

Step 2: Transfer 1 ml from (Stock solution) to 25 ml volumetric flask and

dilute to volume with ethanol. And then read the absorbance by using UV-Vis

spectroscopy at different wavelength: (200 nm, 250 nm, 300 nm, 350 nm, 400

nm, 450 nm, 500 nm, 550 and 600 nm).

Report: Draw the standard curve between Absorbance vs. λ (Determine the

lambda max)

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 Experiment (5)

Application of Quantitative Analysis

by Ultraviolet-Visible Spectroscopy

1- Aim:

Determine the stability of Phenolphthalein by UV spectroscopy.

2- Principle:

Quantitative analysis is all about measuring the amount or concentration of a

specific substance within a sample. This precision is essential for determining
drug dosage, ensuring efficacy, and verifying compliance with regulatory
standards.

3- THEORY

create linear equation (calibration curve) specific to Phenolphthalein, so we

need some information before we start like:

Calibration of Instrumental Methods

All types of analytical methods require calibration for quantitation.

Calibration is a process that relates the measured analytical signal to the

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concentration of analyte. We can’t just run a sample and know the relationship

between signal and concentration without calibrating the response.

The three most common calibration methods are:

• Calibration curve

• Standard addition method

• Internal standard method

Linearity and range:

The linearity of an analytical procedure is its ability (within a given range) to

obtain test results, which are directly proportional to the concentration of

analyte in the sample.

Linearity should be evaluated by visual inspection of a plot of signals as a

function of analyte concentration or content. If there is a linear relationship,

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test results should be evaluated by appropriate statistical methods, for

example, by calculation of a regression line by the method of least squares.

4-Application:

• There are different ways to perform a calibration curve including serial

dilution by (V1M1=V2M2).

• Preparation Standard calibration level:

• Step 1: Transfer an amount of Phenolphthalein Standard equivalent 10

mg to a 100 ml volumetric flask, add 80 ml methanol and sonicate for

10 minutes, dilute to volume with diluent. (Stock solution)

• Step 2: prepare calibration points (use diluent based on solvent as

diluent).

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ID Sample Concentration Volume of Volume taken Absorbance
(mg/ml) volumetric flask from stock
used to prepare solution
the calibration
points

Stock solution 0.1 - -

Level (1) 0.05

Level (2) 0.01

Level (3) 0.005

Level (4) 0.001

Level (5) 0.0005

Notes: the stock solution point should not be included in the calibration curve

calculation.

You should know how to calculate the volume taken from stock solution using

the following law: (V1M1=V2M2) regardless the volume of volumetric flask

used to prepare the calibration points.

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5-Stability study of Phenolphthalein in (HCl, NaOH, Water, ethanol)

Procedure:

Sample preparation:

Step 1: Transfer (10) mg of Phenolphthalein equivalent to (100 ml) volumetric

flask, add 80 ml diluent and sonicate for 10 minutes, complete up to 100ml
using the diluent. (Stock solution).
Step 2: prepare (25 ml volumetric flask) and then transfer (1 ml) of (Stock

solution) to 25 ml volumetric flask and dilute to volume with (HCl, NaOH,

Water, ethanol), respectively.

And then read a sample by using UV-Vis spectroscopy at lambda max at

different time points: (0, 5,10 and 15) minutes.
Draw the standard curve between Absorbance vs time:

Time (min) Absorbance

10

15

51
To draw the standard curve between concentration vs time rather than between

absorbance vs time, we need to perform a calibration curve and find out the

linear line equation.

Report: Draw the standard curve between conc (from Phenolphthalein

stability study) and observe the change in conc vs time.

Time (min) concentration

10

15

52
 Experiment (6)

Qualitative Analysis of Paracetamol Tablet Using HPLC

Aim
Determine presence of Paracetamol in tablet using HPLC.

Theory
Paracetamol (acetaminophen) is a common over-the-counter analgesic and

antipyretic medication. Paracetamol is available in several dose forms,

including tablets, capsules, drops, elixirs, suspensions, and suppositories.

Various pharmacopoeias list paracetamol dosage forms as well as

combinations with other medications. There have been numerous methods

published for analyzing paracetamol and its mixtures in pharmaceuticals or

biological fluids. In pharmaceutical preparations, paracetamol has been

determined in combination with other medications using titration,

voltammetry, fluorimetry, colorimetry, UV-spectrophotometry, quantitative

thin-layer chromatography (TLC), high-performance liquid chromatography

(HPLC), and gas chromatography (GC).

Structure of paracetamol

53
Instrumentation

High Performance Liquid Chromatography (HPLC) is a type of column

chromatography in which a solvent (known as the mobile phase) is

pumped at high pressure through a column containing chromatographic

packing material (the stationary phase). HPLC can separate and identify

chemicals in any material that can be dissolved in a liquid, even at trace

quantities as low as parts per trillion. Because of its versatility, HPLC

is utilized in a wide range of commercial and scientific applications,

including pharmaceuticals, environmental, forensics, and chemicals.

The sample retention period will vary based on the interaction between

the stationary phase, the molecules being studied, and the solvent or

solvents utilized. As the sample moves across the column, it interacts

with the

54
Glossary of HPLC Terms
• High-performance liquid chromatography (HPLC) is a separation

technique for organic mixtures that involves retaining components on

a stationary phase inside a column based on physicochemical

interactions and sequential elution.

• The stationary phase is a solid bed inside the column with particles

coated in the retention phase.

• The mobile phase is a liquid carrier media used to convey samples

through the HPLC equipment.

• Normal phase separation refers to polar retention materials and

55
 nonpolar mobile phases. The retained sample components are eluted

in ascending order of polarity.

• Reverse phase separation occurs when the stationary phase is

nonpolar and the mobile phase is polar. Elution of components occurs

in a decreasing polarity order. It is the most often utilized method of

HPLC separation.

• Isocratic elution using mobile phase.

Qualitative analysis
In most cases, identification of a sample component is performed by

comparing its retention time with that in a standard sample. If a

complex chromatogram with many peaks is obtained or if the retention

time of the target component differs between the standard and the actual

sample, the target component is identified by adding the standard

sample to the unknown sample. Analyzing the HPLC-collected

components by IR or mass spectroscopy enables reliable qualitative

analysis.

56
Requirements
Paracetamol

Acetonitrile

Methanol

Paracetamol

Procedure:

1. Prepare diluent and mobile phase: (Acetonitrile – pH= 3, (40:60))

Mix 400 ml Acetonitrile with 600 ml Distilled water.

2. Wight 50 mg of paracetamol standard in 100 ml volumetric Flask, dissolve

it with the (Acetonitrile - pH3 (40:60)) →stock solution then sonicates for 5

minutes.
57
3. Prepare the following trial sample by using diluent (mobile phase) in 25 ml

Volumetric flask from the stock solution.

5. Chromatographic system consisted of Thermo HPLC model LC-20AT with

UV detector model LW0-20A and Hypersil C18 (25 cm×4.6 mm), 5 µm

particle size.

HPLC condition were given in the Table:

Column Hypersil C18 (25 cm×4.6 mm), 5 µm

Wavelength 243 nm

Mobile phase Acetonitrile - pH3 (40:60)

Retention time 3.2 minutes

Flow rate 1.0 mL/min

Temperature Ambient

Injection volume 10 µL

58
 Experiment (7)

Quantitative Analysis of Paracetamol in Tablet Using HPLC

Aim
To determine the concentration of Paracetamol in a tablet using HPLC.

Theory
There are two methods for quantification: the external and internal standard

methods, both of which are performed using a calibration curve. The

external standard method creates a calibration curve for a standard sample

and unknown samples are quantified using the calibration. In the internal

standard method, a fixed amount of an internal standard substance is added

to an unknown sample when creating a calibration curve using a standard

sample, and a calibration curve is created with the concentration ratio vs.

peak area ratio for quantification.

The internal standard substance is required to be a component not included

in the actual sample, to produce peaks that are completely separable from

those for any contamination components, to elute at a retention time close to

the quantitative target component, to be chemically and physically stable,

and to be highly pure.

59
The advantage of the internal standard method to prevents errors in the

injection volume or those caused by evaporation of the solvent.

Quantitative Analysis Overview

After integrating and identifying the peaks, the next step in the study is
quantification.
Quantification employs peak areas or heights to calculate the concentration of
the component in the sample.

A quantitative analysis consists of several processes, which are simply

explained below:
• Identify the compound to be analyzed.

60
 • Create a method for evaluating samples containing the compound.
• Analyze a sample or samples (the Standard) with a known
concentration of the compound to determine the response (known as
'Calibration').
• You may examine a number of these samples with varying
concentrations of the chemicals of interest if your detector has a non-
linear response or if a large concentration range is to be evaluated in the
samples.
• examine the sample having an unknown quantity of the substance to
obtain the reaction caused by the unknown concentration.
• Determine the amount of component present by comparing the
response of the unknown concentration to that of the known (standard)
concentration.

To establish a reliable comparison between the unknown sample

response and the known standard, the data must be collected and
processed under the same conditions.

Requirements
Paracetamol, Acetonitrile – pH=3, (40:60), Paracetamol

61
Procedure

A. Preparation of calibration curve level:

1. Prepare diluent and mobile phase: (Acetonitrile – pH=3, (40:60))

Mix 400 ml Acetonitrile with 600 ml Distilled water.

2. Wight 50 mg of paracetamol standard in 100 ml volumetric Flask dissolve

it with the (Acetonitrile – pH=3, (40:60)) →stock solution then sonicates for

5 minutes.

62
3. Prepare the following standards by using diluent (mobile phase) in (25) ml

Volumetric flask from the stock solution using the following table.

ID Sample Volume taken from stock solution (mL) Area

(AUC)

Level (1) 2.5

Level (2) 5

Level (3) 7.5

Level (4) 10

Level (5) 15

63
B. Sample preparation

1. Wight Paracetamol tablet then crush it, take 25 mg from the tablet and

dissolve it with the mobile phase in (100) ml volumetric Flask then sonicate

for 5 minutes.

2. Chromatographic system consisted of Thermo HPLC model LC-20AT with

UV detector model LW0-20A .

64
HPLC condition were given in Table.

Column Hypersil C18 (25 cm×4.6 mm), 5

µm

Wavelength 243 nm

Mobile phase Acetonitrile - pH3 (40:60)

Retention time 3.2 minutes

Flow rate 1.0 mL/min

Temperature Ambient

Injection volume 10 µL

3. Insert all samples and then Apply system.

4.Create calibration curve.

5.Determine the amount of sample.

6.Calculate assay of tablet depend on equation:

65
 Experiment (8)

Determination of Functional Groups and Identification of

Compounds by Using Infrared Spectroscopy (IR)

Aims:

1- To investigate the relationship between the molecular structure of

(Amoxicillin) and its absorption in IR spectrum.

2-To find functional groups of (Amoxicillin) by interpreting its IR spectrum.

Principle:

IR spectroscopy is a simple technique that can identify the presence or absence

of a functional group in about 10 minutes. The infrared spectra of pure

compounds are similar to fingerprints.

66
Instrumentation

An IR instrument consists of an IR light source, a sample holder, a means of

selecting individual wavelengths or frequencies of the light, some means of

detecting the amount of incident light that the sample absorbs, and a device

for plotting the amount of light absorbed or transmitted as a function of

wavelength or frequency or wavenumber.

Simplified diagram of IR spectrometer

67
IR Spectrophotometer, 7800 to 350 cm-1
The plot of absorbance or transmittance on Y-axis vs. wavelength or

frequency or wavenumber (cm-1) on X-axis is referred to as the IR Spectrum.

The Reference IR spectrum of Amoxicillin is shown below together with the

Amoxicillin structure

FTIR Spectrum of Amoxicillin Trihydrate, Diluted Potassium

Clavulanate and Optimized formulation

68
Since IR light is absorbed by most materials, the optics of an IR Spectrometer

requires special materials. The sample to be investigated must be prepared in

an inert matrix that does not absorb IR light. Most frequently, such matrix is

made of NaCl or KBr water soluble salts.

Requirement:

IR Spectrometer, manual hydraulic pressure unit, metal unit that can

withstand pressure to prepare the disk, metal spatula, mortar, pestle, KBr solid

pure material, Amoxicillin pure standard material, balance.

Sample preparation:

IR spectra can be determined for solids, liquids, or gases. Spectra of solid

samples may be obtained by mixing the sample (1-2 mg) with dry KBr (300

mg), grinding to a fine well mixed powder, and then forming a disk of the

mixture by applying high pressure in a specially designed unit. The resulting

KBr disk is used for IR analysis, which will eventually produce an IR

spectrum.

69
Interpretation of IR Spectra:

The IR spectra should be interpreted by using the below correlation table,

which shows wavenumbers (cm-1) of different chemical bonds that would

appear in an IR spectrum:

Application:

Obtain an IR spectrum of Amoxicillin, and then interpret it by determining

the relationship between molecular structural features (functional groups) and

absorptions in the IR spectrum.

70
Procedure

1- Sample holder

• The sample to be analyzed is held in front of an infrared laser beam, in

order to do this, the sample must be contained in something,

consequently this means that the very container the sample is in will

absorb some of the infrared beams.

Type of container the sample:

Transparent Ranges
Material Solubility Notes
-1
(cm )

NaCl 40,000 – 625 H2O Easy to polish, hygroscopic

Silica glass 55,000-3,000 HF Attacked by HF

Quartz 40,000-2,500 HF Attacked by HF

Sapphire 20,000-1,780 - Strong

40,000-2,500 and Very strong, expensive, hard,

Diamond -
1,800-200 useless for pellets

71
 Attacked by acids, avoid
CaF2 70,000-1,110 Acids
ammonium salts

BaF2 65,000-700 - Avoid ammonium salts

ZnSe 10,000 – 550 Acids Brittle, attacked by acids

AgCl 25,000-400 - Soft, sensitive to light.

Hygroscopic, soft, easily

H2O, Et2O,
KCl 40,000-500 polished, commonly used in
acetone
making pellets.

Hygroscopic, soft, easily

KBr 40,000-400 H2O, EtOH polished, commonly used in

making pellets.

H2O, EtOH,
CsBr 10,000-250 Hygroscopic soft
acetone

H2O, EtOH,
CsI 10,000-200 Hygroscopic, soft.
MeOH, acetone

72
-The fact that all materials have vibrations makes this a bit more

complicated. Thus, if the sample holder has an optical window made of

something that absorbs near where your sample does, the sample might not

be distinguishable from the optical window of the sample holder.

-Proper plate treatment ensures a long and useful life. Here are a few simple

tips on how to manage plates:

1. Avoid contact with solutions where the plates are soluble.

2. Place the plates in a desiccator to remove excess moisture, even if they are

water-insoluble.

3. Use clean gloves.

4. Avoid wiping plates to prevent scratches.

Collecting spectra through using IR goes about one of two general ways.

73
1. Nujol mulls Liquid sample

-A common method of preparing solid samples for IR analysis is mulling.

-The principle here is by grinding the particles to below the wavelength of

incident radiation that will be passing through there should be limited

scattering.

-To suspend those tiny particles, an oil, often referred to as Nujol is used.

-IR-transparent salt plates are used to hold the sample in front of the beam in

order to acquire data.

74
2-Pressed pellets Solids sample

-Spectra of gases can also be obtained but will not be discussed in this guide.

-The alternate method is similar to the nujol mull, but instead of mineral oil

as the suspending medium, it uses salt.

-Use an agate mortar and pestle to grind the solid into a fine powder, then add

suspending salt.

-This approach uses KBr or CsI, both soft salts.

75
76
 Experiment (9)

Determination of Ethanol in Commercial Products Using GC

Aim:

Determination of Ethanol in Bronchicum using GC.

Introduction:

Gas chromatography is a technique for separating and analyzing volatile

substances in the gas phase.

All chromatography uses a stationary and a mobile phase. In this

chromatography, the mobile phase is invariably gas. However, the stationary

phase might be either liquid or solid. If the stationary phase is solid, the

process is known as gas-solid chromatography (GSC). In addition, if the

stationary phase is liquid, the process is known as gas-liquid

chromatography, or GLC. In GLC, the mobile gas phase resembles helium,

while the stationary phase is a high boiling point liquid deposited onto a

solid. As with other types of chromatography, the mobile phase in this case

is a chemically inert gas that transports the analyte through the heated

column to separate it into discrete molecules.

77
 This chromatography

consists of an injection port,

a column, an oven, a heater

to control the temperature, a

carrier gas, flow control

equipment and a detector.

Procedure
Purification and Extraction: Both methanol and ethanol standards must

be treated by C18-SPE. Collect 30 ml of methanol and 6 ml of ethanol.

Bronchium syrup must be centrifuged for heating purposes in 2x1.5

Eppendorf tubes. Then it is C18-SPE treated. Collect 2 ml of the

Bronchium filtrate and keep it for the analysis. GC Conditions: Using

TR-WAX column and nitrogen as the carrier and backup gases start

with 3min@45deg. Then raise the temperature to 60 deg. at 5 deg./min.

Hold at 60 for 3 min. Use 1.00 micro as injection volume, 20 as splitting

ratio."

78
Standards
Prepare the following standards

- Measure the concentration of the standard and the sample using GC.

79
 Experiment (10)

Liquid Chromatography/ Mass Spectrometry

Liquid chromatography is an important separation technique in the life

sciences and other branches of chemistry. Unlike gas chromatography, which

is ineffective for nonvolatile and thermally sensitive molecules, liquid

chromatography can safely separate a wide range of organic substances,

including small-molecule drug metabolites, peptides, and proteins.

Traditional liquid chromatography detectors include refractive index,

electrochemical, fluorescence, and ultraviolet-visible (UV-Vis). Some of

devices produce two-dimensional data, which represents signal strength as a

function of time. Others, such as fluorescence and diode array UV-Vis

detectors, produce three-dimensional data. Three-dimensional data includes

both signal intensity and spectral data at each point in time.

Mass spectrometers also produce three-dimensional data. In addition to signal

strength, they produce mass spectral data, which can reveal important

information about a sample's molecular weight, structure, identification,

quantity, and purity. Mass spectrum data adds specificity to qualitative and

quantitative investigations, increasing trust in the conclusions.

80
For most substances, a mass spectrometer is significantly more sensitive and

selective than any other LC detector. It can evaluate chemicals without a

sufficient chromophore. It may also detect components in unresolved

chromatographic peaks, which eliminates the necessity for flawless

chromatography.

Mass spectral data supplements information from other LC detectors. Two

compounds may have comparable UV or mass spectra, but they are unlikely

to have both. The two orthogonal data sets can be utilized to accurately

identify, confirm, and measure substances.

81
Instrumentation:

Mass spectrometers work by ionizing molecules, sorting and identifying the

ions based on their mass-to-charge (m/z) ratios. This procedure requires two

important components: the ion source, which generates the ions, and the mass

analyzer, which sorts the ions. LC/MS typically use a variety of ion sources.

Each is appropriate for various types of chemicals. Various types of mass

analyzers are also used. Each has advantages and cons based on the type of

information required.

Electrospray ionization Electrospray uses chemistry to produce analyte ions

in solution before they reach the mass spectrometer. The LC eluent is sprayed

(nebulized) into an atmospheric-pressure chamber containing a strong

electrostatic field and a hot drying gas.

82
 Mass analyzers

Although any mass analyzer could be used for LC/MS, the four most

commonly utilized types are quadrupole, time-of-flight, ion trap, and Fourier

transform-ion cyclotron resonance (FT-ICR or FT-MS).

Each has advantages and downsides, depending on the requirements of the

analysis.

1- Quadrupole A quadrupole mass analyzer has four parallel rods placed

in a square. The analyte ions travel down the center of the square.

Electromagnetic fields are generated by applying voltages to the rods. These

fields control which mass-to-charge ratio of ions can flow through the filter at

any particular time. Quadrupole mass analyzers are typically the most simple

and inexpensive. Quadrupole mass analyzers operate in two modes:

• Scanning mode.

• Selected ion monitoring mode (SIM)

83
2-Time-of-flight

In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is

applied to all ions at the same time, causing them to accelerate down a flight

tube.

- Ion trap mass analyzer consists of a circular ring electrode plus two end caps

that together form a chamber. Ions entering the chamber are “trapped” there

by electromagnetic fields. Another field can be applied to selectively eject

ions from the trap.

Fourier transform-ion cyclotron resonance (FT-ICR)

84
An FT-ICR mass analyzer (also called FT-MS) is another type of trapping

analyzer. Ions entering a chamber are trapped in circular orbits by powerful

electrical and magnetic fields. When excited by a radio-frequency (RF)

electrical field, the ions generate a time dependent current. This current is

converted by Fourier transform into orbital frequencies of the ions which

correspond to their mass-to charge ratios.

85
 Instrumental Lab Report

Name

Group

Lab Experiment

Lab Date

Introduction: (What do you expect to learn? What is the purpose of this lab?)

Hypothesis: (Predict the outcome(s) of the experiment.

86
Materials: (What equipment and materials used).

Procedures:

87
Data Recording: (Record the data that is required at each step of the lab:

tables, charts, graphs, sketches, etc.)

88
Analysis: (Explain your data in words.)

Discussion: (Discuss what happened in the lab., mention details.

89
Conclusion: (What conclusions can you draw form the results of this lab

assignment? Compare the results of the experiment with your hypothesis.)

90
91
Note paper

92
Note paper

93
Note paper

94