SCHOOL OF HEALTH SCIENCES

DISCIPLINE OF PHARMACEUTICAL SCIENCES

PHARMACEUTICAL ANALYSIS [PHRM 353]

PRACTICAL MANUAL

2017

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Please familiarize yourself with the following rules and how to write your
reports:

Practical Schedule and reporting

• The practical schedule will be e-mailed to students and posted on the notice
board.
• The class will be divided into about 32 groups, attending the morning session
(8.30 – 11.30 am) or the afternoon session ( 1- 4 pm) as per schedule. Group
lists may not be changed and there must be strict adherence to the schedule.
• Reports must be submitted at the next practical session following the practical.
Late submissions will incur a penalty.
• Compulsory lab attire: clean lab coat, closed flat shoes, long hair tied up and
away from the face, either long pants, long skirts or long dresses.
• Compulsory lab accessory: permanent marker
• Procedure for missed practicals: Medical certificates should be submitted to the
Academic Development Officer within 24 hours of returning to campus.
• DP requirements: Submit 100 % of all practical reports or 80 % with valid medical
certificate for the report not submitted
• Practical’s constitute 30 % of the DP mark (The DP mark for the practical’s is
comprised of an average of all the practical reports)

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GLP Good Laboratory Practice - is now a widely recognized term, covering standards of
working practice which are required by law in many laboratories and demanded by
clients in many others.

QA Quality Assurance - is a system of recording past history, sources and quality of all
materials, and calibration and performance of apparatus/equipment used for a process
or business, aimed at ensuring a consistently high quality of finished article. In our case
we aim to ensure the high quality of analytical results. Good documentation of methods
and standards is the key to good Quality Assurance.

QC Quality Control - is a system of controlling the quality of finished articles. In our case
the quality (accuracy) of analytical results can be controlled by regularly analysing check
control samples of known composition, and of course by regularly calibrating all
apparatus used in the analysis.

General aspects which should be considered in a GLP programme are:

1. Safety and cleanliness in the laboratory 


2. Keeping full and accurate records of work done 


3. Routine checking of performance of methods and equipment (normally done for you
at undergraduate level) 


4. Good housekeeping 


5. Cost and efficiency 


Safety and Cleanliness

1. Compulsory lab attire: lab coat, safety goggles, mask, gloves, closed flat shoes & hair
tied back. Report all accidents to staff.
2. Be aware of the location of the fire extinguishers and exits.
3. Use of the first aid box to be supervised by staff.
4. Do not eat drink or sniff any chemicals.
5. Do not run in lab.
6. Work in lab at the space allocated to you.

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7. Do not clutter your work area with glassware and equipment etc.
8. Connect electrical appliances as close as possible to the plug points and ensure that
the cords do not hang over the bench top. After use, switch of all appliances, allow
to cool and place in locker except for the water bath. When using the water bath
ensure that the level of the water is always above the element. Do not allow the
water bath to steam continuously – cover the spaces of the water bath that are not
in use.
9. Place all broken glassware in bins that are marked specifically for this purpose.
Mercury from thermometer breakages must be placed in appropriate bottles. Do not
touch the mercury.
10. Do not place sweet wrappings, tissue paper, broken glassware, etc. into the sink. and
ensure that all taps are tightly closed.
11. When working in the fume cupboard ensure that the extractor fan is switched on.
Close all lids /caps of reagent bottles and do not remove reagents from the fume
cupboard.
12. Do not weigh substances directly onto the balance. Place substances to be weighed
onto a weighing boat. Clean the balance after use and close all reagent bottles.
13. Dispose all used chemicals into waste chemical bottles.
14. Follow all additional safety instructions that are in the practical manuals and which
are given to you by the staff.
15. A laboratory notebook must be used at all times. No scribbling pads or loose pieces
of papers with instrument readings or weighing! Record all relevant data. 

16. Record the date and the experiment name on each page. Use the notebook also for
rough calculations, for example, at working out how much of chemical is needed for
preparing a solution. Should you have problems afterwards, such "thinking details"
can help indicate where you have gone wrong. 

17. It is expected that you will discuss the measurements and the results with other
students in the group - indeed, often you will have to pool your results for a fuller
picture, however, the report must be your own work. 
Good Housekeeping

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18. You will be working in a busy laboratory shared with many other people, and often
you will be using shared equipment. Co-operation is the only solution. Think about
the consequences for other people when you do something for yourself. 

19. Wash up regularly and tidy away clean apparatus. Clean the bench and the shelves
where you work. 

20. Put waste chemicals and solvents in clearly marked Waste bottles for safe disposal.
Label all bottles correctly and clearly. 

21. Keep instruction books beside instruments. Cost and efficiency 
Chemicals cost
money - look at the price of a bottle of solvent or acid. Scaling down will save on cost
and also on the disposal problem when you have finished. Consider the grade of the
chemical. Laboratory reagent grades are adequate for many purposes, other times
the higher purity of analytical grade reagents will be called for. Read the label! 


Writing laboratory reports

The laboratory Notebook

The laboratory notebook is intended to be the complete data storage base for all the
work done in the laboratory. You must note everything in your laboratory notebook.
This will include measurement values, with sufficient notes to make clear what they are,
notes on planning the experiments and how to make the solutions, how to deal with the
sample, notes on information from the literature etc. It should be laid out in the
following manner.

The Laboratory report

The following paragraphs describe general outlines for how such a report may be
written. Do not forget that you are writing an accurate account of what happened for
anyone who wishes to read your report, not just the person who will mark it. You should
be able to give your report to a friend who has never seen the practical manual and he
or she should be able to follow what you did and what your results were, assuming they
have some basic knowledge in the subject.

The report should contain your results, how you achieved them, and an evaluation of
the results. You should demonstrate that you have understood the experiment as well

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as how to document and present it. Try to keep the text as short as possible without
leaving out important information or using a poor language. This is a difficult task but
practice makes perfect.

The layout is important for a good final result, hence the report should be computer
written with a word processing program. A recommended font is Times New Roman.
Figures should be constructed with the aid of a computer program or scanned. The
report is much easier to read if you don't have to flip back and forth between the main
text and an appendix. Remember that figures must be numbered, have a legend and be
referred to in the text.

Generally, lab reports should include the following sections, as in the usual outline of a
scientific paper.

Aim

This section states the aims of the experiment. In any experiment, you aim to do
something. For example, you aim to verify, to investigate, to measure, to determine, to
compare or to calculate. It should be short and concise. E.g. The purpose of this
experiment is to quantify the caffeine content of a diet soda sample using high
performance liquid chromatography (HPLC).

Introduction/Theory

In the introduction you supply the reader with some basic background which is
important to allow the work to be placed into perspective. You may use a textbook, the
laboratory manual and lectures as references. The references are included in a
reference list at the end of the report. Decide what the main purpose of the lab is. Does
it introduce a new instrumental method or does it demonstrate a new method of
standardisation?

Pictures may be included, e.g. a drawing of some equipment, an overview of the

chemical reactions etc. Mathematical and chemical formula should also be added to this
section. Do not forget figure legends and numbering as well as references if you take
pictures from the literature including the internet.

Common errors

Some parts are left out.
Numbering of figures and/or figure legends are missing.

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Methodology (Procedure)

In this section you describe equipment, chemicals and how you did the experiment.
Most people add too much, all you need are the main points. It is a good way for me to
see that you understand the key steps in the procedure. There is a complete list in the
manual it is senseless to just copy it down. There may be differences between what is
written down and what is given verbally i.e. slight difference in instructions, note these.

Common errors:

Wrong tense, the methods section should be written in the past tense. Text is copied
directly from the manual.

Results and Calculations

This is the most important part of the report where you should present the results you
obtained as clearly and concisely as possible, e.g. absorbance measurements and
observations.
This is the place where you can show one sample calculation and then
results go in table. The table should contain a heading, it should contain run number,
mean and standard deviation. If any runs left out, then these should be indicated. It is
best that separate tables are used for each unknown or are clearly separated from other
data. Make it as easy as possible for the reader to see the results
Extensive
presentations of raw data like long lists of absorbance measurements, chromatograms
and calculations should be included in an appendix. References to figures and tables
must be added both when they are present directly in the text as well in appendices.

Common errors:

Raw data sometimes missing and only the summary of the data presented.
Results not
described in the text, i.e. there is a table of results but no text refers to it. Legends,
headings and references left out.
Calculated values contain too many decimals, e.g. the
beer contained 5.457895% v/v ethanol. Samples not identified but simply presented as
"sample 1, sample 2. You need to specify what the sample or series is.
Labels missing
from plots.

Tables and figures

In manuscripts for submission to scientific journals, each table and figure is on a

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separate page at the end of the manuscript. In this type of lab report it is better to
include them at appropriate places.

Tables are numbered separately, Table 1, Table 2, etc. Each table should have a short
descriptive title placed above as a header. It should include necessary details to
distinguish from other tables. In general, you should be able to understand the table
without having to refer to the text.

Each column/row should have a descriptor and a UNIT (when applicable), comments can
be added as footnotes below the table.

Figures include drawings, diagrams, plots, chromatograms etc. They are also numbered,
Fig. 1, Fig. 2, etc. The title is usually put below the figure in what is called the "Figure
legend" which may also include a short description of what it is supposed to illustrate.

Common errors:

No numbering or reference inside the report.

UNITS and VALUES

Whenever you write a value you MUST also provide the unit. Otherwise the value is
completely meaningless. A good idea is to include the units in the calculations, thus you
will be able to make sure that your method of calculation is correct and yields the
correct units. Check also that you have the right order of magnitude.

For values it is important to estimate how many valid digits to provide. The precision of
a resulting value can never be higher than the least precise of the input values. More
difficult is to estimate the precision of the experimental methods. Until you have
statistical data (e.g. standard deviation), don't use more than 2-3 valid digits in lab
reports.

Common errors:

No units or incorrect units
Incorrect number of significant figures

Discussion

State again what the goal of the lab was. Discuss how these goals were accomplished
and how your results compare to previous knowledge. Did you expect these results?

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Why? It is not correct to simply state that a result is "wrong" or "right". Instead you
should make an effort to try to explain why you got a particular value or result.

Equipment that was used may be commented, maybe it is possible to develop or

improve the method. Discuss possible errors. Are your results unambiguous or would it
be possible to suggest additional experiments to exclude uncertainties?

Some of the experiments include questions and discussion points. These are meant to
point out things you should think about. Use them as support for your reports, in
particular in the results and discussion sections. Do not simply answer them one by one.

Common errors:
You write e.g. "The experiment went very well and we got the results
we had expected" i.e. the results are not compared to other facts.
You make no
attempt to evaluate your results.

The last two sections of the lab report may alternatively be combined into one section
titled Results and discussion. The general outline should still follow the above
mentioned recommendations.

References

Here you list the references you have given in your text. Each reference should list the
author(s), year, title and page of the reference. You may either number the references
as they follow in the text (1), (2) etc. Alternatively, you indicate the name of the first
author and publication year in the text e.g. (Smith et al., 1998). In the latter case the
references are listed alphabetically.

Internet sources can be difficult to reference; a typical example could be as

follows:
Steffen Thomas, Spectroscopic Tools, University of Potsdam, updated
30/8/2002, <http://www.chem.uni-potsdam.de/tools/>.

Examples of References

¨ United States Pharmacopoeia XXIII, Mack Publishing Company, Easton, PA,
1995, 456-458.

¨ Chowan, Z.T. Excipients and their functionality in drug product development.
Pharmaceutical Technology, 6:19 (1993) 72-82.

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 ¨ Parikh, N.H. Dibasic Calcium Phosphate In: Wade, A and Weller, P.J. (Eds.)
Handbook of Pharmaceutical Excipients, 2nd edition, American Pharmaceutical
Association, Washington, 1994, 56-60.

¨ Moffat, A.C., Jackson, J.V., Moss, M. S., and Widdop, B. Clarke’s Isolation and
Identification of Drugs, London, The Pharmaceutical Press, 1986, 533-534.
Common errors:

You forget to reference sources taken from the internet.

Appendix

The appendix may contain documentation that is too extensive to be inserted inside the
report, e.g. raw data, spectra, chromatograms and larger tables. Appendices should be
numbered and referred to in the text.

Common errors:

No numbering or reference inside the report.

Layout of Practical Report

Name:

Reg. No.:

Practical No.:

Aim: (1-2 lines) (0.5)

Theory: (half a page) (1.5)

Methodology: (1.5)

Results and Calculations: (2.5)

Discussion: (half a page) (2)

References: (1)

Presentation (1)

[10 marks]
Note: Parts of the above general instructions was adapted from the 2016 School of Chemistry and Physics CHEM 340 practical manual.

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PRACTICAL 1: MEASUREMENT OF THE REFRACTIVE INDEX OF
CHLORHEXIDINE GLUCONATE IN ALCOHOL SOLUTION USING A
REFRACTOMETER

CHLORHEXIDINE CLUCONATE 0.5% IN ALCOHOL 70%

FORMULA
CATAL. NO INGREDIENTS AMOUNT CHECK QC No. QC
1. 68 180 10 Chlorhexidine gluconate solution 5% 100.00L
2. 68 285 20 Alcohol (Ethanol) 96% (Batch no.) 729.20L
3. De-ionised water TO 1000.00L

TOTAL 1000.00L

PROCEDURE CHECK
1. Pour the alcohol 96% through a stainless steel sieve up to the 700L mark in the 1000L stainless steel
container.
2. Add the balance of the alcohol 96% (29.2L)

3. Add Chlorhexidine Gluconate 5% (100L)

4. Make up to volume (1000L) with de-ionised water

5. Mix with Silverson mixer No. 28 for 10 minutes.

6. Take 1x100ml sample for the QC lab.

7. Place lid on container and seal with clear tape.

8. When passed by the QC lab, mix well with Silverson mixer No.28 for 10 minutes.

9. Take 1x100ml sample for the QC lab.

10. Pack into 25L metal drums using Flux pump (No. 14), seal and label. Attach a security seal to each
drum.
Production
Pharmacis
t

QUALITY CONTROL REPORT

Limits Result Bulk Physical Checked

specifications characteristics
DD Appearance Transparent liquid
Assay 0.45 – 0.55% Colour Red
Refractive index 1.364 – 1.365 Clarity Clear
pH 7.1 – 7.5 Odour Spirituous
Maas/ml at 200C 0.877 – 0.887
QC No. :
Analyst`s signature :
Passed for packaging :

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Method:

The above formula and procedure outlines the bulk preparation of chlorhexidine.
Using the formula provided in the production sheet, prepare 50ml of chlorhexidine
gluconate 0.5% in alcohol 70%. (Show all calculations).
After preparation of the solution, measure the refractive index , pH, and note the physical
characteristics of your sample

In your practical write-up include;

• A brief discussion on refraction

• Possible sources of errors if the refractive index is not within the specified limits

• Uses of chlorhexidine gluconate in alcohol solution

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PRACTICAL 2: THE ASSAY OF DEXTROSE INJECTION VIA
POLARIMETRY

THEORY
Optical rotation is defined as the ability of a substance to deflect a plane of polarized
light. If a molecule is asymmetric, ie. Possesses an asymmetric centre, it will exhibit
optical activity.
Optical rotation (α) is the angle through which the plane of polarization is rotated when
plane-polarised light passes through an optically active substance.
Substances which rotate plane-polarised light in a clockwise direction are said to be
dextrotatory, designated (+) and those which rotate the light in an anti-clockwise
direction are said to be laevorotatory, designated (-).
In order to measure the rotary power of a substance, a number of factors must be
considered;
a) The nature of the substance and its concentration (c)

b) The length of the light path through the solution (l)

c) The wavelength of the light (the shorter the wavelength, the greater the rotation)
(λ ) and

d) The temperature (t)

When these variables are controlled, one obtains the specific rotation [α] t λ, of the
particular substance. This is defined as the angle of rotation produced by a solution
containing 1g/ml of active substance when the length of the column through which the
light (usually the D line of sodium) passes is 1dm.
Optical rotation i.e. observed angle of rotation, is related to the specific rotation by the
formula;
[α] t λ = α
cl
t
where [α] λ = specific rotation (in degrees) at the specified temperature (usually 20-25
o
C) and wavelength (usually the D line of sodium),
α = observed angle of rotation, or optical rotation (in
degrees)
c = concentration of solute (in g/ml)
l = length of the column of liquid (in dm)

if the concentration is expressed as g/100ml, the equation becomes;

[α] t λ = 100α
cl

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The measurement of this property of optical rotation is called polarimetry and is carried
out with a polarimeter.
The main elements of a polarimeter are;
(i) A monochromatic light source, usually the D line of sodium,

(ii) A polarizer,

(iii)A sample tube,

(iv) An analyser,

(v) An optical system (eye piece, lenses, etc) and

(vi) A graduated rotary scale attached to the analyser

The polarizer and analyser are both Nicol prisms. Light from the source passes through
the polarizer and the emergent beam is plane-polarised. i.e. the electromagnetic vibrations
take place in one plane. Depending on the positions of the 2 Nicol prisms, the field of
view will appear either dark of light, and as the analyser is rotated, the field will change.
On complete rotation of the analyser prism through 180°, there will be 2 points of
maximum darkness and 2 points of maximum brightness.

Field of view as the analyser is rotated through 90°

The field of view is set with pure solvent in the sample tube to give a uniform
illumination. If a solution of an optically active substance is placed in the sample tube,
the filed will change. The analyser must be rotated through a certain angle (α) to restore
the original uniform illumination. Thus the optically active substance has rotated the
plane-polarised light through angle α. If the analyser is turned to the right (clockwise), the
substance is dextrotatory (+) and if it is turned to the left (anti-clockwise), the substance
is laevorotatory(-).
Since there are two points, 180° apart, to which the analyser can be turned, the sign of the
activity is determined by taking the direction required to give an angle of less than 90°.
HOW TO READ A VERNIER:
The “marker” that gives you the number before the decimal is the zero line on the bottom
scale, which slides past the fixed, upper scale. For example, in the example below, the
zero line of the sliding scale is between 12-13° on the main scale (first arrow). This

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means our reading must be 12.something. The number after the decimal is obtained by
looking along the sliding scale to see which of the 10 lines matches up perfectly with any
of the lines in the upper scale. This line on the sliding scale represents the digit after the
decimal. Here, the 6th line on the sliding scale (second arrow) matches with a line on the
upper scale, so our reading is 12.6°. Notice that we don’t care which line on the upper
scale matches; that is not relevant to our reading.

A laevorotatory sample would be read using the left-side of the scales. Be careful to
realize that the numbers are increasing to the left.

Method:
1. Determine the zero of the polarimeter by filling the 2-dm sample tube completely
with the pure solvent, ensuring that there are no air bubbles. Place the tube in the
polarimeter and set the field of view to give uniform illumination. Take the
reading on the rotatory scale.

2. Rinse the sample tube twice with the test solution, fill it as before, place it in the
instrument and rotate the analyser till the original uniform illumination has been
restored (the angle of rotation must be less than 90°). Take the reading on the
rotary scale and subtract the zero reading from it to obtain the true angle of
rotation of the test substance. This angle (α) through which the analyser has been
rotated, is the optical rotation of the substance and from this, the specific rotation
( [α] t λ ) can be calculated.

Assay of dextrose injection:

To a volume of the dextrose injection equivalent to between 2g and 5g anhydrous
dextrose, add 0.2ml of dilute ammonia solution and sufficient water to produce 100ml.

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Mix well, allow to stand for 30 minutes to reach equilibrium and then measure the optical
rotation in a 2-dm tube.
At equilibrium, the specific rotation is +52.75°, thus 52.75° = 100α
cl
Therefore c (g/100ml) = 100 α
2 x 52.75
= 0.9477 α

Thus, observed rotation in degrees (α) multiplied by 0.9477 represents the mass in grams
of dextrose, C6H12O6, in the volume of the injection taken for assay.
Calculate the percentage purity of your sample and compare this with a literature
reference (BP, USP, etc). Please remember to quote your reference.

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PRACTICAL 3: ULTRAVIOLET SPECTROPHOTOMETRIC ASSAY OF
PARACETAMOL TABLETS

THEORY

The relationship between concentration and absorbance is given by the Beer-Lambert

Law.

The Beer-Lambert law states that the intensity of a beam of parallel monochromatic
radiation passing through a transparent solution of a particular absorbing species is
proportional to the concentration of the absorbing substance through which the light
passes and the length of the light path which the incident light must cross.

Absorbance (A) = log10 lo/lT = a b c

Where: lo= intensity of incident light

lT = intensity of transmitted light
a = absorptivity (a proportionality constant depending on wavelength, solvent
used and temperature)
b = path length (centimetres)
c = concentration (g/100ml)

Method:
The tablets you will be assaying claim to contain 500mg paracetamol per tablet. Your
task is to find out exactly how much paracetamol is in one tablet.

Standard preparation:
Stock solution:
Weigh out 100mg paracetamol powder into a beaker. Dissolve in hot distilled water.
Transfer to a 200ml volumetric flask. Add 10ml methanol and make up to volume with
distilled water.

Standard 1:
Pipette out 2ml of the stock solution into a 100ml volumetric flask and make up to
volume with distilled water.

Standard 2:
Pipette out 4ml of the stock solution into a 100ml volumetric flask and make up to
volume with distilled water.

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Standard 3:
Pipette out 6ml of the stock solution into a 100ml volumetric flask and make up to
volume with distilled water.

Blank preparation:
Pipette 10ml of methanol into a 200ml volumetric flask and make up to volume with
distilled water. Pipette 2ml of this solution into a 100ml volumetric flask and make to
volume with distilled water.

Sample preparation:
Crush and grind 1 paracetamol tablet.
Weigh out accurately an amount of tablet powder equivalent to 100mg paracetamol into a
50ml beaker. Dissolve in hot distilled water and filter into a 200ml volumetric flask.
Add 10ml methanol and make up to volume with distilled water.
Pipette 2ml of sample into a 100ml volumetric flask, make up to the mark with distilled
water.

Concomitantly measure the absorbance of the standard solutions and sample in 1cm cells
at 249nm, with a UV spectrometer, using the blank preparation as a reference.

Calculation
Using a calibration curve (absorbance vs concentration) calculate the amount of
paracetamol in the tablet.

Additional requirements for your write-up

• Look up the reference in any pharmacopoeia and indicate whether the product
meets the requirements stipulated. Record the Pharmacopoeia reference and
specification

• In preparing the sample for this assay, you were asked to accurately weigh out an
amount of tablet equivalent to 100mg paracetamol. Why were you asked to do
this and also explain how you obtained the calculated weight.

• Briefly explain the principles used in this assay method

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PRACTICAL 4: DETERMINATION OF MOISTURE CONTENT IN
PHARMACEUTICAL PRODUCTS

Introduction
Moisture content analysis is a critical component of material quality and essentially a
function of quality control in most production and laboratory facilities. From biological
research organisations, pharmaceutical manufacturers, to food producers and packers,
moisture content control greatly influences the physical properties and product quality of
nearly all substances and materials at all stages of processing and final product existence.
The primary methods of water content determination include spectroscopic, chemical,
conductivity and thermogravimetric analysis.
Moisture content determination accuracy for substance samples identifying a larger bulk
volume will be as precise as the representative sample quality. When choosing material
samples from a batch, the area of sample selection is crucial. For reproducible test results,
a test sample must be truly representative of the entire homogenous batch being tested.
For example, a bulk powder mixing process must be thoroughly blended before
extracting a representative sample. In the bulk mixing process a common condition
occurs where as a greater concentration of moisture exists away from the surface and
edges of the material. A test sample gathered from the top will not be a true
representation of the batch.
Optimum sample weight resolution is important for moisture content determination when
using the thermogravimetric drying process. A test sample's weight and placement can
influence the accuracy and test cycle time. To keep test cycle time to a minimum, a small
sample weight should be used. If a sample size is excessive, a larger volume of mass
must be vaporised decreasing the test performance and overall accuracy.

Method
Determine the moisture content of the given samples using the Moisture Analyser as
demonstrated.
Record the results. Comment on the results in terms of USP/BP limits and answer the
following questions:
1. What is moisture?
2. What is LOD?
3. What are "moisture analyzers”
4. How does a moisture analyzer work?
5. Briefly discuss other methods/applications for moisture analysis / loss on drying
of pharmaceutical products

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PRACTICAL 5: ATTENUATED TOTAL REFLECTION (ATR)
SPECTROSCOPY - Identification of the active ingredient in a tablet /capsule

THEORY
This is a versatile and powerful technique for infrared sampling

How an ATR accessory works

• Internal reflection spectroscopy passes infrared radiation through an infrared-

transmitting crystal of high refractive index, allowing the radiation to reflect in the
crystal one or more times

• An attenuated total reflection accessory measures the totally reflected infrared

beam when the beam comes in contact with a sample

• In this way, an evanescent wave penetrates into the sample in contact with the
crystal, producing a spectrum of the sample

Evanescent Wave

• The infrared radiation interacts with the sample through a series of standing
waves, called evanescent waves

• An evanescent wave is a penetrating electromagnetic field whose intensity

quickly decays as it moves away from its source

This diagram depicts the path of the infrared beam from the IR source, through the
crystal and to the detector

• When the beam is directed from the IR onto an optically dense crystal at a certain
angle, the beam is reflected through the crystal. When a sample is placed on the
crystal, the infrared radiation interacts with the sample, producing a
“transmission-like” spectrum

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 • The evanescent waves penetrate 1 to 4 micrometers into the sample at each
reflection point. A portion of the wave is absorbed by the sample. The altered
(attenuated) beam from each wave exits at the opposite end of the crystal and is
directed to the detector. The detector records the attenuated beam as an
interferogram signal, which generates an infrared spectrum

• Because the evanescent waves decay rapidly as they move away from the surface
of the ATR crystal, the sample must contact the crystal surface to produce an
accurate spectrum

• When collecting ATR data, samples are run as % Reflectance or log (1/R)

• % Reflectance is equivalent to % Transmittance

• Log (1/R) is equivalent to Absorbance

http://www.brukeroptics.com/applications/microscopy.html; Thermo Electron

Corporation

Method:

1. You will be given 2 reference powders and 2 unknown tablets/capsules. Compare

the spectra of the reference powders and the samples by looking at the principal
peaks and identify the active ingredient in the unknown tablet/capsule. Also verify
the spectra by comparing it to a standard spectrum on BP, USP etc.

2. Using a mortar and pestle crush the tablet/ contents of the capsule into a fine
powder. Follow the ATR protocol for further analysis.
ATR protocol

1. Create a folder with the name at C:\ or C:\UKZN.

2. Clean the crystal of the ATR instrument using Methanol and cotton
3. Wait for the crystal to Dry completely.
4. Start the application OPUS in the computer.
5. Wait for the application to initialize.
6. Once the accessory is test done and ready, close the window
7. Go to MEASEURE and select Measurement
8. Enter the Sample name.
9. Leave the file name empty. (If something is written, delete it)
10. Select the path (Personal folder in C drive or in respective place).
11. Select Start Background Measurement
12. Once done, place your sample on the crystal and close the sample compartment

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13. Press Start Sample measurement on the application.
14. Wait for the spectra to appear.
15. Go to Manipulate, select Baseline correction.
16. Go to Smooth, select smooth (optimum smooth value is 17).
17. Place the cursor on the spectra and right click. Then choose single peak pick.
18. Pick all the representative/principal peaks
19. Go to File – Select Save as (A window will appear)
20. Go to Mode – Select OPUS format (To access the file in OPUS again) and save
21. Go to file – Select save as (again)
22. Go to Mode – Select Data point table (.dpt) (To access the file in software like
Origin)
23. Once you save both the formats, Go to the spectral window
24. Right click – Copy all
25. Open Paint from the computer – paste the spectra (make sure it gets pasted with
the frequency values)
26. Save it as .jpg file in the same personal folder of yours.
27. Finally, you should have three formats of a spectra (OPUS, .dpt, .jpg)
28. When one spectrum is done, close the window completely (Spectral window not
the OPUS window)
29. Open a new fresh file from File option (File – default) and repeat the process from
step 7.

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PRACTICAL 6: ASSAY OF A RIBOFLAVIN SOLUTION USING
FLUORESENCE SPECTROSCOPY

INTRODUCTION

This assay is very similar to the preceding spectrophotometric assay in that a series of
standard riboflavin are made, and the fluorescence of these standards is measured. Using
the data, a standard curve of fluorescence versus concentration of riboflavin is plotted. In
a manner analogous to the spectrophotometric assays, the fluorescence of the unknowns
is measured and the potency of the solutions is ascertained by relating the fluorescence to
the standard curve.

THEORY

Many organic and some inorganic compounds will emit radiant energy after absorbing
radiant energy of some particular frequency. This is known as photoluminescence. If
there is a time delay no longer than 10-9 second between absorption and the re-emission
of the energy, the effect is known as fluorescence. Usually the emitted radiation is of the
longer wavelength than the absorbed radiation.
Adsorption of the energy raises the electrons in a molecule from their normal energy
level known as ground state to one of any vibration levels of the first excited electronic
state of the molecule. Any excess energy above the lowest vibrational level in the first
excited state is quickly lost in collisions and by partition of this energy to other modes of
rotation and vibration in the molecule. The molecule then returns to an excited vibrational
level of the ground electronic state by the loss of energy through the emission of radiation
of a longer wavelength than that originally absorbed. Therefore, it can be seen that
fluorescent transition involves less energy than the absorption transition.

Since the fluorescent radiation is at a different wavelength than the radiation used to
excite the molecules, an Emission Filter which passes fluorescent radiation but not the
exciting radiation is placed between the fluorescent sample and the photosensitive tube.
To ensure that the radiation used to stimulate the sample is essentially monochromatic, a
Primary Filter is used between the light source and the sample.

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PROCEDURE

Determine the concentration of Riboflavin in the Vitamin B complex tablet

Standard Solution preparation

1. Prepare a standard stock solution of 10 mg riboflavin in 100 ml of 1% acetic acid.

2. Make a series of dilutions of this standard, in 1% acetic acid such that each
standard contains 0.2 mg through to 0.8 mg of riboflavin. (i.e. you are preparing
four solutions containing 0.0002%, 0.0004%, 0.0006% and 0.0008% of riboflavin
in 1% acetic acid. (Pipette 1, 2, 3 & 4 ml of standard stock solution into 50 ml
flasks respectively and make up to volume with 1 % acetic acid ).

Sample preparation

1. Crush 1 tablet, using the mortar and pestle, and transfer into a 100 ml flask, and
make up to volume with 1% acetic acid. Pipette 5ml of this solution into a 50 ml
flask and make up to volume with 1 % acetic acid.

3. Read the intensity of fluorescence obtained through each of the four solutions,
starting with a blank solution containing only 1% acetic acid, followed by the
0.0002% and then the progressively stronger solutions.

4. Finally read the intensity of fluorescence of the sample (tablet) solution.

Calculations

1. Plot a graph of fluorescence intensity versus concentration and calculate the

concentration of riboflavin in the tablet by extrapolation from the graph.

2. Does the concentration of riboflavin comply with the label claim of the tablet.

3. Remember to include the following in your write-up:

• All your calculations in preparing the stock solution and the serial
dilutions, submit your graph with your write-up
• End your write-up with a satisfactory discussion and conclusion on your
method and results achieved.

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PRACTICAL 7: GAS CHROMATOGRAPHY (GC)
DETERMINATION OF ALCOHOL CONTENT IN PARACETAMOL SYRUP

THEORY

Gas chromatography is the most suitable technique for the separation of liquid stable and
volatile organic and inorganic compounds. Gas-liquid chromatography (GLC)
accomplishes the separation by partitioning the components of a chemical between a
moving (mobile) gas phase and a stationary liquid phase held on a solid support. What
are the other types of gas chromatography?

The standard gas chromatograph consists of several basic modules joined together;

• Provide a constant flow of carrier (mobile) gas

• Permit the introduction of sample vapours into the flowing gas stream
• Contain the appropriate length of stationary phase
• Maintain the column at the desired temperature
• Detect the sample components as they elute from the column
• Provide a readable signal proportional in magnitude to the amount of each
component.

METHOD

GC parameters
Injector temperature : 200 0C
Detector temperature : 250 0C
Column : Restek K,(800-356-1688) 30 m; 0.32mm ID; 0.25um
Column temperature : 700C
Detector type : Flame Ionization Detector (FID)
Flow rate : Nitrogen (carrier gas) - 30ml/min
: Hydrogen - 10ml/min
: Air - 10ml/min

Preparation of standard solutions:

1. Pipette 8ml of absolute ethanol into a 100 ml volumetric flask. Make up to the
mark with deionised water and mix well.

2. Pipette individual amounts of 10ml, 15ml, and 20ml into each of the 50ml
volumetric flasks. Dilute to volume with deionised water and mix standard
solutions well.

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Preparation of the sample solution:
1. Pipette 10ml of paracetamol syrup into a 50ml volumetric flask. Dilute to volume
with deionised water and mix well.

Inject 4 ul of each of the standards until a consistent representative area value is obtained.
Inject 4ul of sample solution.

Calculations

1. Plot a calibration graph of Standard Area vs Concentration of alcohol. Extrapolate

the alcohol content of the paracetamol syrup from the graph.

2. Does the alcohol content of the paracetamol syrup comply with the label claim.

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PRACTICAL 8: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC) ASSAY OF PARACETAMOL SYRUP

THEORY
Chromatography is a physical method in which the components to be separated are
distributed between two phases. If this division is based on the nature of the stationary
phase, and the separation process, four modes can be specified;

1. Absorption chromatography - the stationary phase is an absorbent and the

separation is based on repeated absorption - desorption steps.

2. Partition chromatography - the separation is not based on absorption but rather

partition between the mobile and stationary phase.

3. Ion exchange chromatography

4. Size chromatography

Concerning the first two modes, it is sometimes not clear whether the dominant process is
absorption or partition or both. For this reason, in practice, two or more modes are
defined depending on the relative polarity of the two phases: normal and reversed
chromatography.

In the normal phase chromatography, the stationary phase is strongly polar in nature (e.g.
Silica) and the mobile phase non-polar (e.g. n-hexane or tetrahydrofuran). Polar samples
are thus retained on the column longer than the less (or non) polar materials.

Reversed phase chromatography is the exact inverse of this. The stationary phase is non-
polar (Hydrocarbon) in nature, while the mobile phase is a polar liquid such as water or
an alcohol, here the less polar the material is, the longer it will be retained. Sometimes the
mobile phase can be modified to adjust its polarity. In the normal mode, this may be done
by the addition of a more polar substance, while the reversed phase chromatography, the
additive is a less polar substance.

The mobile phase usually plays an important role in liquid chromatography systems. This
is one of the more important differences between liquid and gas chromatography. In gas
chromatography, the mobile phase is inert.

In liquid chromatography the mobile phase may be a single substance or two or more
substances, to properly adjust the characteristics of the phase. Also, one may maintain a
constant mobile phase composition during analysis or change it. The first mode is called
isocratic operation, while the second is called gradient elution.

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The stationary phase is often chemically bonded to the support particles in the packings
of liquid chromatography. Bonded phases are prepared by chemical reaction between the
surface hydroxyl groups of silica particles and a linear organic molecule or organosilane.
These columns are used in the reversed mode and are useful in the separation of a wide
range of substances. Polar bonded phases, e.g. having an amino or cyano group at the end
of the hydrocarbon chain, also exist and are used in the normal mode.

The support material used in the preparation of bonded phases may be one of two types.
The first type is porous through its whole body while in the second type, there is a solid
core within a thin porous skin. The latter is called a pellicular support.

The chromatograms obtained will be measured in terms of the area of each peak or the
height of each peak. Either of these can be used as a measure of the response to the
sample and standard.

METHOD

HPLC parameters
HPLC column : C18 column
Mobile phase : Acetonitrile : Methanol : Water
100 150 250
Wavelength : 254nm
Flow rate : 2ml/min
Injection volume : 4µl
Elution time : 2-6 minutes

Optimisation of HPLC
Ensure that the correct column is attached.
Run the mobile phase at 2ml/min.
Purge the system and ensure that the pressure is stable.

Preparation of Standard solutions

Stock solution
1. Dissolve 0.3g of paracetamol powder in 50 ml hot deionised water in a beaker.

2. Transfer to a 100ml volumetric flask and make up to the mark with cool deionised
water.

Standard solution

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 3. Pipette 2ml of the stock solution into a 25ml volumetric flask, followed by 5ml
acetonitrile and 7ml methanol and make up to the mark with deionised water.
(Repeat using 4ml and 6ml of the stock solution).

4. Filter the standards via a syringe through a 0.45µm microfilter.

Preparation of sample

1. Pipette 0.5ml of paracetamol syrup into a 25ml volumetric flask.

2. Pipette 5ml acetonitrile and 7ml methanol into the flask and make up to the mark
with deionised water.

3. Filter the sample via a syringe through a 0.45µm microfilter into the sample vials.

4. Place in order, standards, and then sample vial into the sample tray and slot the
tray into the sample holder of the HPLC. (Multiple sampling can be done with the
use of the autosampler ie: 3 or more groups can read samples in 1 run.

5. Open the analysis file and edit the standard and sample details.

6. Start the analysis – The auto-injector will perform each sample analysis and the
results will be stored in the data file. (calculate the run time – based on the
retention time per sample.

7. When the analysis is complete rinse the column with water and then with
acetonitrile.

8. Download the results from the data file and print.

Calculation
Calculate the amount of paracetamol in 5ml of syrup: Using a calibration curve (standard
area vs concentration) extrapolate the paracetamol concentration
Does this comply with the label claim.

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