1

Pharmaceutical Instrumental Analysis Laboratory Experiments:

Experiment # Title
1 Quantitative Analysis of Aspirin Tablets by Double-Beam Ultraviolet-
Visible Absorption Spectrophotometry
2 pKa determination of Acetaminophen drug by UV/Visible
spectroscopy
3 Determination of sodium ions concentration in Normal Saline Solution
using flame photometry
4 Determination of Ca and Mg ions in Zimacal Effervescent Tablets
using Atomic Absorption spectrophotometer (AA)
5 Polarimetry and Optical Activity of natural monosaccharides
6 Separation and quantitative determination of analgesic drug
combination (Aspirin, caffeine and acetaminophen) by HPLC
7 Gas chromatography-Mass Spectroscopy (GC-MS) of essential oils
from natural Palestinian thyme
8 Infrared spectroscopy (FT-IR) of solid and liquid selected Drugs
1
9 H-NMR of selected drugs
10 The simultaneous separation and determination of quinolone
antibiotics using reversed-phase HPLC

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 Faculty of Pharmacy (462 laboratory)

Exp. 1

UV/visible spectroscopy of Aspirin

2021

Prof. Saleh Abu-Lafi

3
Spectroscopy: interaction between matter (drugs) and electromagnetic radiation (EMR)

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 • Spectroscopy is the study of the interaction between matter (atom or
molecule) and electromagnetic radiation (EMR) as a function of
wavelength (l).
• Spectrometric methods are a large group of analytical methods that
are based on atomic and molecular spectroscopy

• Spectrum is a plot of response (absorption or emission of radiation)

vs wavelength (l)

• Spectroscopy provides information on chemical identity of a

compound, quantity present and structure based on the technique
selected and the wavelength of electromagnetic spectrum.

• Commonly used spectroscopic techniques are UV-VIS, FT-IR, NIR

(molecules), Atomic Absorption spectroscopy (AA) and atomic
emission such as flame photometry and ICP-MS spectroscopy (for
atoms).

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 Properties of Electromagnetic Radiation
• EMR can be described as a wave (wavelength,
frequency, velocity, and amplitude) and particles called
photons.
• EMR is sinusoidal wave which is composed of two
perpendicular fields.
• An electric field (explain absorption and emission of
radiation by analytes) and a
• Magnetic field at right angle to the electric field (used to
explain phenomena like nuclear magnetic resonance NMR).
7
 EMR

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9
Most drugs absorb UV radiation and some are
colored absorb visible radiation
Spectrum is a plot of
response (absorption) vs
wavelength (l)

Electronic:vibrational:rotational
100:1:0.01
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 The Absorption Process

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 The Absorption Process
A monochromatic radiation as it passes through a absorbing
solution cuvette of thickness b-cm and concentration C (moles
per liter). Because of interactions between the photons and
absorbing drug particles, the radiant power of the beam
decreases from P0 to P. The transmittance T of the solution is
the fraction of incident radiation transmitted by the solution.
Transmittance is often expressed as a percentage and called the
percent transmittance.
T = P / P0 %T = P / P0 *100
The absorbance A of a solution is related to the transmittance in
a logarithmic manner.
A= -logT = log (1/T) = log(P0/P)

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 Spectral nomenclature of shifts

Blue shift Red shift

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14
15
 Beer’s Law
According to Beer’s law, absorbance A is directly
proportional to the concentration of the absorbing species
c and the pathlength b of the absorbing medium
A = log P0 / P = abc
Here, a is a proportionality constant called the
absorptivity.
If, for example, c has the units of grams per liter (g L-1) and
b has the units of centimeters (cm), absorptivity has the
units of liters per gram centimeter (L g-1 cm-1).

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When we express the concentration in moles per liter and b in
cm, the proportionality constant, called the molar absorptivity,
is given the special symbol . Thus,
A = bc
where,
 has the units of liters per mole centimeter (L mol-1 cm-1).

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BP and USP used UV/visible to determine active pharmaceutical ingredients
(APIs).

The method is based on the use of a std absorptivity A (1% w/v solution, 1 cm)
value (=E1%) for the active ingredient being assayed and this relies on the UV
spectrophotometer being accurately calibrated.
A = E1%.C
C(g/100ml) = A/E1%

Such method also presume that there is no interference from excipients

(preservatives, colorants, etc.) present in formulations and that the sample is
free of suspended matter, which would cause light scattering.
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19
 UV Vis Instrumental Components
• Source: D2/W
• l - Selector (monochromator)
• Sample holders: Cuvettes (typically b = 1 cm)
1. Glass or plastic (Vis)
2. Quartz or Fused silica (UV+Vis)
• Detectors
• Photodiodes
• PMTs

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 General UV-Vis Instrument Designs: Single beam

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 Double Beam: in space vs. in-time

T= P (sample)/P0 (reference)
P is less when absorption occurs
P is less when concentration increase

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 A multichannel diode-array spectrophotometer

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 Determination of Aspirin
drug by UV spectroscopy

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 SA and Aspirin lmax
is different

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How it works?
• Aspirin can be hydrolysed to salicylic acid by base in aqueous
solution.
• The calibration curve is constructed from standard solutions prepared
from pure salicylic acid.
• The acetylsalicylic acid in the unknown, a commercial aspirin tablet is
hydrolysed to salicylic acid in a 0.1 N NaOH solution, diluted, and the
absorbance of the solution measured at 297 nm.
• The amount of salicylic acid present in the solution is obtained from
the calibration curve, and the weight of acetylsalicylic acid in the
tablet is then calculated using the appropriate stoichiometric factor.
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27
 Faculty of Pharmacy (462 laboratory)
Prof. Saleh Abu-Lafi

Exp 2: Polarimetry and Optical Activity of drugs

Smell of (S)-limonene Smell of (R)-limonene

Limonene is a colorless liquid classified as a cyclic

monoterpene
It is the major component in the oil of citrus fruit peels.
The orientation around a chiral center of a drug can have a
dramatic impact on the pharmacological response of that
drug in the human body. A worst case scenario is one
which the non desired enantiomer causes serious toxicity
 Chiral drugs
• Approximately 25% of all drugs are marketed as either racemates
(mixtures of two enantiomers) or mixtures of diasteromers.
• Chiral purification is a crucial aspect of all successful drug
manufacture.
• FDA's Policy Statement for the Development of New
Stereoisomeric Drugs (1992), requires that the manufacture
should ensure its stereoisomeric composition with respect to
identity, toxicity, strength, quality, and purity.
 Polarimetry and Optical Activity

Spectroscopy: methods based on interaction of Electromagnetic radiation EMR with

matter (including drugs)
EMR = wave + particle
EMR is a self-propagating electromagnetic wave, with electric and magnetic
fields oscillating perpendicular to each other:
Polarized sunglasses
 Optical Rotation and Polarimetry
randomly oriented light

detector
chiral material

monochromatic light source

Na l=589nm
optical polarizer - only allows "vertical" light to pass through optical polarizer - only allows "horizontal" light to pass through

The maximum signal will be optained if the second polarizer is rotated to match the light rotation:

randomly oriented light

detector
chiral material

maximum signal

monochromatic light source

optical polarizer

optical polarizer - only allows "vertical" light to pass through

Schematic of a polarimeter
 Polax-2L polarimeter

Polarimetry is a sensitive, non-destructive technique for

measuring the optical activity exhibited by compounds
 Some definitions
Dextrorotary designated as (+), clockwise rotation (to the right)
Levorotary designated as (-), anti-clockwise rotation (to the left)

If only one enantiomer is present a sample is considered to be optically

pure.

Enantiomers will rotate the plane of polarization in exactly equal amounts

(same magnitude) but in opposite directions. When a sample consists of a
mixture of enantiomers, the effect of each enantiomer cancels out, molecule
for molecule.

A 50:50 mixture of two enantiomers or racemic mixture will not rotate plane
polarized light and is optically inactive.

A mixture that contains one enantiomer in excess will display a net plane of
polarization in the direction characteristic of the enantiomer that is in excess.
 specific rotation calculations

-The angle Φ = a = observed rotation

-It has been found that the magnitude of the observed angle of rotation depends upon the following
factors:
1-Nature of the substance and nature of the solvent used.
2-Length of the cell, usually 1.0 dm = 10 cm for turbid samples and 2.0 dm tubes for clear samples.
3-Concentration of the solution, usually expressed in gm solute/100ml
4-Temperature of the solution.
5-Wavelength of light used, usually sodium light (D-line) with wavelength 589 nm.
-The rotation power of a given solution is expressed as specific rotation [α]Dt
-The measurement is carried out at a temperature (t) and using the D-line of Na light.
-The specific rotation is a characteristic physical property associated with optically active molecules.
Example:
Calculate the observed rotation (a) of a solution of 0.5245g of (S)-1-amino-1-
phenylethane diluted to a volume of 10.0 mL with methanol at 23oC, using the
D-line of a Na lamp and a 1.00 dm tube. Specific rotation of this material is
[a]D23 = -30.0o.

Solution: Solving the specific rotation equation for observed rotation, we get

a = [a]lT L x C

The sample size is 0.5245 g, but this has been diluted to 10.0 mL, so the sample
concentration is 5.245 g in 100 ml.

Plugging in the numbers,

we get a = (-30.0o) (1.00 dm) (5.245 g in 100 ml).

Solving, we find a = -157o.

Enantiomeric access (ee)

ee % = ([R]-[S]) / ([R]+[S]) *100

where
[R] = concentration of the R-isomer
[S] = concentration of the S-isomer
Question: Determine the enantiomeric excess (ee) % of
the racemic mixture.

Answer: You would expect [R] = [S] = 50%

ee % = ([R]-[S]) / ([R]+[S]) *100

= (50-50) / (50+50) *100
=0%
Question: Consider that (S)-2-bromobutane has a specific rotation of +23.1o
and (R)-2-bromobutane has a specific rotation of -23.1o
Which isomer is dominant and what is the optical purity of a mixture, of
(R)- and (S)-2-bromobutane, whose specific rotation was found to be -9.2o?

Answer: The negative sign tells that the (R) enantiomer is the dominant one.

Optical purity % =[a]observed/[a]pure sample *100

= (-9.2)/-23.1o *100
= 40% this indicates 40% excess of R over S

Question: Then what is the percent composition of the mixture?

Answer: The 60% leftover, which is optically inactive, must be equal amounts of both
(R) & (S). The excess 40% is all (R), so there is a total of 70% (R) and 30% (S).

 Double check again using the ee%

ee% = 70-30/100 *100 = 40%
We will measure the Optical Rotation of a solution of Sucrose (table sugar), which will
allow us to determine Sucrose Specific Rotation [a]. We compare our measured value with
the accepted value as reported in the literature.

The molecule is a disaccharide combination of glucose and fructose with

the formula C12H22O11.
Ibuprofen

(S)-(+)-Ibuprofen
optical activity = [α]D26 equals +54.46°,
C = 0.6 in methanol (lit.)
Procedure:

Optical Activity of a Sucrose Solution

instrument must be calibrated before testing the samples
1. Each person will be assigned a Sucrose solution that is to be prepared between 5% and
20%. Prepare 100g of this solution by accurately weighing out both the Sucrose and the
Water. Using a magnetic stir bar, allow this solution to mix for at least 15 minutes.
2. Carefully load a polarimeter cell with this solution and measure the optical rotation of the
solution. Be sure to determine the length of the polarimeter cell. Be sure to thoroughly rinse
out the cell when you are done.
3. Determine the specific rotation of Sucrose.
4. [a]D20 = + 66.37o for Sucrose. Calculate the percentage error in your determination.

Optical Purity of S-(+)-Ibuprofen Sample

1. Dissolve your sample of resolve Ibuprofen in 2 mL of Ethanol. Transfer this to a clean 5
mL Volumetric Flask. Bring the solution up to the flask’s mark with additional Ethanol.
Mix the solution until it is homogeneous.
2. Load the sample into a polarimeter cell and measure the optical rotation of the solution. Be
sure to determine the length of the polarimeter cell.
3. Determine the “observed” specific rotation of the sample.
4. Determine the optical purity of the sample.
Report:
1. Determine if your sample is dextrorotatory or levorotatory
2. Determine the observed angle of rotation for all the
standard solutions of sucrose
3. Calculate the average specific angle of rotation
4. Is your result conceding with that recorded in literature?
5. Use excel program to plot the observed angle of rotation
versus the concentration, and draw a straight line
relationship. Find the equation of the straight line and R2
6. Take an unknown solution of sucrose from your instructor
and find its observed angle of rotation, then use the linear
relationship and calculate the concentration of this
unknown solution.
 Faculty of Pharmacy (462 laboratory)

FT-IR spectroscopy

X-RAY ULTRA-VIOLET VISIBLE MICROVAWE RADIO

0.2nm 190-380 nm 400-800 nm Infrared 3 mm-20 cm 10 m-30 Km

NEAR MID FAR

Principles:

EMR between 400-4000 cm-1 (2500-20,000 nm) is passed through a sample and is
absorbed by the bonds of the molecules in the sample causing them to stretch or
bend. The wavelength of the radiation absorbed is characteristic of the bond
absorbing it.

Applications:
• A qualitative fingerprint check for the identity of raw material used in
manufacture and for identifying drugs.
• Used in synthetic chemistry as a preliminary check for compound identity,
particularly for the presence or absence of a carbonyl group, which is difficult to
check by any other method.
• Can be used to characterize samples in the solid and semi-solid states such as
creams and tablets.
• Used as a fingerprint test for films, coatings and packaging plastics.
• Can be used to detect polymorphs of drugs (polymorphs are different crystal
forms of a molecule that have different physical properties such as solubility and
melting point, which may be important in the manufacturing process and
bioavailability).
Strengths:
• Provides a complex fingerprint which is unique to the
compound being examined.
• Matching of the spectrum with its standard fingerprint
(using authentic libraries) can now be readily carried out.

Limitations:
• Rarely used as a quantitative technique because of relative
difficulty in sample preparation and the complexity of
spectra.
• Usually can only detect gross impurities in samples.
• Sample preparation requires a degree of skill, particularly
when potassium bromide (KBr) discs are being prepared.
 Infrared region can be divided up into 3 regions:

• The middle-infrared region is commonly used for structural

confirmation
• Near-infrared (NIR) spectrophotometry, which has been used for
many years in quality control QC in the pharmaceutical industry.
Energy Levels in Molecules (UV-Vis between electronic levels,
while IR between vibrational levels)
• In IR, a molecule is viewed as being joined by bonds which
behave like springs.

• If the simple molecule HCl is examined in the gas phase, it can be

seen that it has an absorbance maximum at ca 2900 cm-1 which
results from the transition between the bottom vibrational state V0
and the first excited state V1
In order for the electrical component in EMR to interact with
a bond, the bond must have a dipole. Thus symmetrical
bonds, such as those in O2 or N2, do not absorb IR radiation.
Factors determining intensity of absorption in IR

Intensity of absorption

The intensity with which a bond absorbs radiation depends on its

dipole moment.

OH > NH > CH

The intensity depends on the relative electronegativity of the atoms

involved in the bond.
 Energy level of absorption
The equation which determines the energy level of vibration of a bond
is shown below:

• k is a constant related to the strength of the bond, e.g. double bonds

are stronger than single bonds and therefore absorb at a higher
energy than single bonds,
• m is related to the ratio of the masses of the atoms joined by the
bond. where m1 and m2 are the masses of the atoms involved in the
bond.
Instrumentation:
Two types of instrument are commonly used for obtaining
IR spectra:
1. Dispersive instruments, which use a monochromator
to select each wavenumber in turn in order to monitor its
intensity after the radiation has passed through the sample.
2. Fourier transform instruments, which use an
interferometer. FT-IR generates a radiation source in which
individual wavenumbers can be monitored within a one
second pulse of radiation without dispersion being
required.
 Dispersive IR instrument

The filament used is made of metal oxides, e.g. zirconium oxides, and is heated to
incandescence in air. The sample is contained in various ways within discs or cells
made of alkali metal halides. Once the light has passed through the sample, it is
dispersed so that an individual wavenumber or small number of wavenumbers can be
monitored in turn by the detector across the range of the spectrum.
In a Fourier transform IR, the monochromator is replaced by an
interferometer which uses a moving mirror to displace part of the
radiation produced by a source thus producing an interferogram,
which can be transformed using an equation called the ‘Fourier
transform’ in order to extract the spectrum from a series of overlapping
frequencies.
Full spectral scan can be acquired in about 1 s, compared with the 2–3
min required for a dispersive instrument.

FT-IR instrument
Instrument calibration
In order to ensure that instruments conform with BP specifications, the
wavelength scale of the instrument is checked by obtaining an IR
spectrum of polystyrene film as shown below.
Sample preparation:
Three modes of sample preparation have been used prior to
IR analysis:
1. The sample is run as a film sandwiched between two
NaCl discs. For this method the sample must be a liquid, in
which it can be run without preparation.
2. The sample is ground to a powder with KBr. On a
weight-for-weight basis, sample weight is about 1% of the
weight of KBr used. About 200-300mg of the finely ground
powder are used and the sample is compressed into a disc
under vacuum by subjecting it to a pressure of 8 bar. This is
the procedure used in pharmacopoeial methods to prepare a
drug for analysis by IR.
3. IR spectra of liquids or solutions in an organic solvent,
commonly chloroform, may be obtained by putting the
liquid into a short pathlength cell with a width of ca 1 mm.
Types of samples:
• Gas in an evacuated cylinder
• Solutions in cuvette
• Neat Liquid between 2 NaCl plates
• Pellets: 2-3 mg sample is allowed to mix with
~0.2-0.3g of KBr which is transparent to IR
 An IR Spectrum

• A plot of % transmittance vs vibrational

frequency in wavenumbers  (cm-1)
How to convert cm-1 to microns or nanometers

Wavelength in µm = 10,000/cm-1

So, 300 cm-1 => 33 microns )10000/300)

Wavelength in nm = 10,000,000/cm-1

so, 20,000 cm-1 => 500 nm (10000000/20000)

How to convert microns or nm to cm-1 (inverse cm)

Wavenumbers in cm-1= 10,000/µm

So, 10.6 microns => 943 cm-1

Wavenumbers in cm-1= 10,000,000/nm

So, 632.8 nm => 15800 cm-1

IR source  sample (drug)  detector
Function of % transmission vs. wavenumber (cm-1)

IR spectrum
100

%T

0
4000 3000 2000 1500 1000 500

v (cm-1)
 IR spectrum
y axis is %T or A
x axis is wavenumber (or wavelength)
Io  sample  I
T = (I/Io) %T = 100 x (I/Io)

A = -log T
A absorbance (no units)

(Note: A (but not T)  concentration)

 Infrared region
Limit of red light: 800 nm = 0.8 mm = 12500 cm-1

Near IR: 0.8-2.5 mm, 12500-4000 cm-1

Mid IR: 2.5-50mm, 4000-200 cm-1 (mostly used)
Far IR: 50-1000 mm, 200-10 cm-1

The mid-infrared region is further broken down into:

The group frequency region (1280-4000 cm-1)
The fingerprint region (600-1280 cm-1).
Infrared Spectrum of Carbon Dioxide
 Fingerprint region
In the region from  1300-400 cm-1, vibrational frequencies are affected by the
entire molecule, as the broader ranges for group absorptions in the figure below
– fingerprint region.
Absorption in this fingerprint region is characteristic of the molecule as a whole.
This region finds widespread use for identification purpose by comparison with
library spectra.
Absorption Regions
Aldehydes
Carboxylic Acids
Ketones
 Esters
– C=O stretch at ~ 1730-1740 cm-1
strong
and
– C-O stretch at 1000-1300 cm-1 (broad)
(Note: other functional groups may have peaks in the 1000-1300 cm-1
region too!)
O

1743 1245
 Amides
• C=O stretch at 1640-1680 cm-1
• N-H stretch (if 1o or 2o) around 3300 cm-1
 Nitriles
• C  N absorbs just above 2200 cm-1 (med – strong)

• The alkyne C  C signal is much weaker and is just below

2200 cm-1
IR spectrum of Paracetamol drug (KBr disc)
The infrared spectrum of Aspirin as a KBr disc
The infrared spectrum of Dexamethasone (KBr disc)
 Faculty of Pharmacy (462 laboratory)
Experiment # 4
HPLC of Aspirin, Caffeine and Acetaminophen

Prof. Saleh Abu-Lafi

1
Separation and quantitative determination of analgesic drug
combination (Aspirin, caffeine and acetaminophen)
by HPLC/PDA using external calibration standards

Figure 1: Illustration of the basic chromatographic process

Figure 2: Schematic of an HPLC system
Expected elution order in reversed-phase HPLC
3 Analgesic APIs: Extra strength headache powder combination

1. Aspirin (2-acetoxybenzoic acid),

2. Caffeine (1,3,7-trimethylxanthine) and
3. Acetaminophen (4-acetamidophenol)

O CH3 OH
COOH H3C
N N
OCOCH3
O N N
CH3
NHCOCH3

Figure 6: Structure of Aspirin, caffeine and acetaminophen

Objectives:
1. To learn the basics of liquid chromatography
2. To learn how to operate the HPLC/PDA system
3. To identify the 3 compounds using reversed phase HPLC/PDA
4. To calculate the chromatographic parameters: tr, tr’, k’, N, a, Rs
5. To calculate the concentration of unknown

Chromatographic operating conditions:

Flow rate: 2 mL/min.
Injection volume: 10-mL.
Column dimensions: 25 cm x 4.6 mm i.d. (5-mm particle size)
Column type: C18, ODS-Xterra or X-bridge analytical column with guard column.
A photodiode array UV–Vis detector is set at 275 nm to monitor absorbance.
Column Temperature: 45Co
Preparation of Mobile Phase:

1. Prepare a suitable mixture of water, methanol

and glacial acetic acid (69:28:3; v/v/v).
2. Mixed thoroughly, and degassed using sonicator
bath (make adjustments if necessary). Or use in-
line degasser

Solvent Mixture: prepare a mixture of methanol and

glacial acetic acid (95:5).
Preparation of Analgesic standard solutions:

Standard stock solution:

Dissolve accurately weighed quantities of acetaminophen RS,
Aspirin RS, and caffeine RS in solvent mixture to obtain a
solution having a known concentration of about 0.25mg/ml
Sample preparation

1. Weigh and finely powder not less than 20 tablets.

2. Transfer an accurately weighed portion of the powder
(equivalent to about 250 mg of acetaminophen) to a 100 ml
volumetric flask.
3. Add about 75 ml of solvent mixture and shake by
magnetic stirrer for 15 min.
4. Dilute with solvent mixture to volume and mix.
5. Transfer 2.0 ml of this solution to a 50.0 ml volumetric
flask, dilute with solvent mixture to volume and mix.
5. Filtrate the solution with 0.45μm nylon membrane
disposable filter
6. Inject 10 μL of the filtered solution and standard solution
to the HPLC as recommended by the analytical method
Report:

1. Record the retention times RT of the three compounds,

calculate RRT of each compound, elaborate on the order of
elution.
2. Calculate the adjusted retention time tR’, retention factors
(k’), selectivity (a) between each adjacent compounds.
3. Calculate resolution (Rs) between each adjacent compound.
4. Calculate efficiency (N) for each peak based on its peak
width. Discuss any significant differences.
5. Plot a calibration curve of relative peak area against
concentration (g/mL) of the 3 components.
6. Estimate the concentration of the 3 compounds in the
unknown.
 Faculty of Pharmacy (462 laboratory)
Experiment # 5
Atomic Emission Spectroscopy (AES)
(Flame photometry)

Prof. Saleh Abu-Lafi

1
Spectroscopy is the interaction between matter and EMR
Mater is atom or molecule

If atom  atomic spectroscopy/line spectrum

(AAS or AES such as flame photometry and ICP)

If molecule  Molecular spectroscopy/continuous spectrum

(if absorption: UV-VIS
if emission: Florescence)
 Lithium Flame Test

LiCl

3 Li atoms in the flame produce carmine red color

Spectral line 610.4 nm.
 Sodium (Na) Flame Test

Na atoms in the flame produce yellow-orange color.

Spectral lines 589.0 and 589.6 nm
Emission spectra of some common elements

Characteristic ‘spectral fingerprint’.

Flame Photometry Principle:

This is simply measures the atoms excited by a flame

(temperature range: 2000–31000K).

After excitation, atoms will readily lose the gained energy

and revert back to the ground state and the emission
occurs.

It is that emission that actually being measured.

The wavelengths of the emitted light will almost be similar

as those that were absorbed in the atomic absorption,
since exactly the same energy transitions occurs
 Evaporation
+ - + -
M X M X MX
Solution Mist Solid

Vapourisation

Thermal M (gas) Dissociation

*
M (g) MX
X (gas)
excitation Gas
Flame
emission Absorption of radiant
Measure for atomic
energy h
h absorption spectroscopy
Measure for
flame emission *
spectroscopy M (g)
o
Re-emit radiation at 90 Measure for atomic
(Atomic fluorescence) fluorescence spectroscopy

Process by which gaseous atoms are produced in flames

 EXAMPLES OF FLAME TEMPERATURES

Fuel Oxidant Temperature

(K)
Acetylene Air 2400-2700

Butane Air 1700-1950

Acetylene Oxygen 3300-3400

Hydrogen Air 2300-2400

8
 Solution Samples introduction
Nebulizers
Samples in solution are usually easily
introduced into the atomizer by a simple
nebulization, aspiration, process.
Nebulization converts the solution into an
aerosol of very fine droplets using a jet of
compressed gas. The flow of gas carries the
aerosol droplets to the atomization chamber.

9
 Pneumatic Concentric Tube Nebulizer

10
Emission of excited atoms is proportional
to concentration of analyte.
Flame emission is good for such atoms that
do not require high temperatures for
atomization and excitation, like Na, K, Li,
Ca, and Mg.
The instrument is very simple and excludes
the need for a source lamp. The filter is
exchangeable in order to determine the
analyte of interest and, in most cases, a
photomultiplier tube is used as the
detector.
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 Flame photometer

consists of burner, nebulizer, monochromator (grating or filter) and detector

Flame photometer used in the experiment
 Atomic Emission Spectroscopy
Absorbance = -log(It/I0)
I0 =intensity of radiation reaching detector
in the absence of sample.
It = intensity of radiation reaching detector
when sample is being aspirated.

14
• Determination of Na ion in an i.v. infusion
using flame photometry

• To determine the concentration of 0.9% NaCl

normal saline solution after dilution.
• Based on a standard curve. (standard
concentration range: 5-80 mg/L or ppm).

15
I.V. infusion solution

Normal Saline: 0.9% Sodium Chloride Solution

Student determination of Na

1 mL 2 mL 3 mL 4 mL 5 mL

Na:
stock
mg mL-1
50.00 mL volumetric flasks
 Determination of Na
Calibration curve for absorbance of Na

1.5
Absorbance

1 y = 0 093x + 0 001

0.5

0
0 1 2 3 4 5 6

Na concentration / ppm
Analytical procedure

Preparation of Stock solutions (500 mg Na+/L)

Dissolve an accurate weight of 0.636 g NaCl in 500ml volumetric flask with distilled
water.
Preparation of standard solutions
Standard solutions are prepared by dilution of stock solutions. Use different glass
pipettes and numbered 50 ml (or 25 ml) volumetric flasks and prepare the solutions
using serial dilution (concentration range 5-80 mg/L). Fill it up to the mark with
distilled water and mix.

Preparation of Normal Saline solution.

Dilution if necessary the normal saline solution to range of standard solutions (5-80
mg/L)
Determination of Na by flame photometry:

1. Let the instrument warm up for 5-10 minutes.

2. Feed distilled water to the instrument.
3. Select the element Na by turning the selector.
4. Pull the knob slightly out and adjust readout to 0.
5. Aspirate the most concentrated standard solution and
adjust readout to approximately 250-300.
6. Aspirate distilled water–the instrument should read 0.
7. Aspirate standard solutions no. 1, 2, 3, test solution, and
then standards 4, 5. Record the results by triplicate
measurement.
8. Aspirate distilled water for at least 5 minutes to clean the
system.
 Calculations
• Find average emission intensity for each
concentration, n=3
• Calculate RSD
• Construct a calibration curve
• State the best fit equation and R2 value
• Calculate the unknown Na concentration