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Module 1 Lesson 2 Spectrometric Calculations

This document provides information on spectrometric calculations and analysis. It discusses photometry and how light can undergo absorption, scatter, reflection, emission, or fluorescence. It describes Beer's law and how absorbance increases linearly with concentration. Beer's equation relates absorbance, absorptivity, path length, and concentration. Two sample problems are provided to demonstrate calculating percentage purity based on absorbance measurements and reference standards.

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Veronica Deldio
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Download as PDF, TXT or read online on Scribd
484 views

Module 1 Lesson 2 Spectrometric Calculations

This document provides information on spectrometric calculations and analysis. It discusses photometry and how light can undergo absorption, scatter, reflection, emission, or fluorescence. It describes Beer's law and how absorbance increases linearly with concentration. Beer's equation relates absorbance, absorptivity, path length, and concentration. Two sample problems are provided to demonstrate calculating percentage purity based on absorbance measurements and reference standards.

Uploaded by

Veronica Deldio
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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SPECTROMETRY

MODULE 1
Lesson 2
SPECTROMETRIC
CALCULATIONS
Photometry
 Is the measurement of light.
 It is the most commonly used
measurement in the laboratory because
light can undergo
 Absorption – spectrophotometry
 Scatter – nephelometry
 Reflection – turbidimetry
 Emission – flame spectroscopy
 Fluorescence – fluorometry
Light, Wavelength & Energy

 Light
is a form of electromagnetic energy
that appears to travel in waves.
 The wavelength or distance between peaks
of light waves is a function of its energy.
 As
the wavelength is increased, emitted energy is
decreased.
 Asthe wavelength is decreased, emitted energy is
increased.
Light, Wavelength & Color

The color of light is a function of


its wavelength.
  in nm Color Absorbed Color Reflected

 400-435 Violet Yellow-green


 435-500 Blue Yellow
 500-570 Green Red
 570-600 Yellow Blue
 600-630 Orange Blue-green
 630-700 Red Green
Beer’s Law
 A law stating that the power of a
transmitted radiant beam decreases
exponentially as the concentration of the
solution containing the absorbing
chemical species increases arithmetically.

Transmittance Concentration
Transmittance vs. Concentration
Beer’s law
 But if the logarithm of the
%T is plotted against the
concentration, a straight
line is obtained.
Absorbance vs. Concentration
Beer’s law
Absorbance
 Theterm “absorbance” is used to
represent the logarithm of
transmittance.

 Absorbance increases linearly with


concentration.

 Absorbance is also known as “optical


activity”, “absorbancy” or “extinction
coefficient”.
Beer’s Equation

 The
relationship of absorbance to
concentration is expressed in the
equation known as Beer’s equation.
A = abC
 Where:
A = absorbance
 a = absorptivity coefficient (constant)
 b = length of light path (constant)
 C = concentration
 Inspectrophotometric analysis, the
absorbance of an unknown
concentration (u) of a particular
constituent is compared with the
absorbance of a known
concentration (s).

Au / As = Cu / Cs
Lambert’s or Bouguer’s Law

 It states that the power of a


transmitted radiant beam
decreases exponentially as the
thickness of the solution containing
the absorbing chemical species
increases arithmetically.
 Thickness is inversely proportional
to transmittance and directly
proportional to absorbance.
Beer-Lambert’s or
Beer-Bouguer’s Law

A combination of the above


laws and relates the power
of the incident beam and
transmitted beam to the
thickness and concentration
of the solution containing the
absorbing chemical species.
Spectrometric Analysis
SAMPLE PROBLEMS
Sample Problem 1

Assay of Sulfisoxazole Tablets


Materials

10 tablets of sulfisoxazole 0.5g


100 mg USP sulfisoxazole reference
standard
1 L of 1N HCl
200 mL of 3% USP vanillin in alcohol
 Experiment Procedure

 Preparation of Calibration Curve


 Dissolve 100.6 mg sulfisoxazole reference standard in 100 mL of 1N
HCl.
 Transfer 25 mL sulfisoxazole solution to another container and add
75 mL of 1N HCl. This serves as the “stock solution”.
 In four separate volumetric flasks, prepare the following solutions.
 VF1 – 10mL stock solution + 20mL 3% vanillin + qs ad 100mL 1N
HCl
 VF2 – 14mL stock solution + 20mL 3% vanillin + qs ad 100mL 1N
HCl
 VF3 – 18mL stock solution + 20mL 3% vanillin + qs ad 100mL 1N
HCl
 VF4 – 22mL stock solution + 20mL 3% vanillin + qs ad 100mL 1N
HCl
 Get the absorbance at 390 nm.
 Procedure

 Assay of Sulfisoxazole Tablets


 Weigh 10 tablets of sulfisoxazole (500mg) =
6,490mg.
 Powderize and get a sample powder (100mg) =
98.5mg.
 Dissolve sulfisoxazole in 100 mL 1N HCl.
 Transfer 25 mL sulfisoxazole solution to another
container and add 75 mL of 1N HCl. This serves
as the “tablet stock solution”.
 In a volumetric flask (VF5), transfer 15mL of the
tablet stock solution, add 20mL of 3% vanillin and
sufficient amount of 1N HCl to make 100 mL.
 Get the absorbance at 390 nm.
Experimental Data

Weight of USP Reference Standard


Sulfisoxazole = 100.6 mg

Weight of 10 Sulfisoxazole tablets = 6,490 mg

Weight of sample tablet = 98.5 mg

Soln # Conc (ug/mL) Abs @ 390nm


1 25.2 0.210
2 35.3 0.295
3 45.4 0.375
4 55.4 0.465
5 ? 0.235
Calculation Step 1

 Determine the concentration of active constituent


(Cu) in the final dilution.
 Cu = ?

 Using the formula


Au/As = Cu/Cs

 Then Cu = Au/As x Cs
1 and 5 Cu = 0.235/0.210 x 25.2 ug/mL = 28.2 ug/mL

2 and 5 Cu = 0.235/0.295 x 35.3 ug/mL = 28.1 ug/mL

3 and 5 Cu = 0.235/0.375 x 45.4 ug/mL = 28.5 ug/mL

4 and 5 Cu = 0.235/0.465 x 55.4 ug/mL = 28.0 ug/mL

Average Cu
Cu = Cu/n = 112.8 ug/mL / 4 = 28.2 ug/mL
Calculation Step 2

 Determine the concentration of the


sample (U) taken in the final dilution.

U = wt/mL x # of dilutions
U = wt/mL x # of dilutions

Then,
98.5 mg x 25mL x 15mL x 1000ug
100 mL 100mL 100mL 1 mg

= 36.9 ug/mL
Calculation Step 3

 Determine the quantity of the active


constituent in a given quantity of sample.

Amount/tab
= Cu/U x Average weight of tablet
Amount/tab = Cu/U x Average weight of
tablet

Amount/tab = 28.2 ug/mL x 649mg


36.9 ug/mL

= 496 mg/tab
Calculation Step 4

 Compute for the % labeled amount.

%LA
= Amount per tablet x 100%
Labeled claim
%LA
= Amt per tab/Labeled claim x 100%

%LA
= 496mg/500mg x 100%
= 99.2%
 TheUSP Specification is
that…
 Sulfisoxazole tablets contain
NLT 95% and NMT 105% of the
labeled amount of C11H23N3O3S
 What will be your disposition
based on the given
requirement?
Sample Problem 2

Assay of Trioxalen USP XIX


 Exactly51.0 mg of trioxalen USP was
placed in a 100-mL volumetric flask
and chloroform was added to volume.
Exactly 1 mL of the solution was
transferred to a 100-mL volumetric flask
and chloroform was added to volume.
The absorbance of the final dilution
was found to equal to 0.530.
 Exactly
49.0 mg of USP trioxalen
reference standard was treated in the
same manner as the sample. The
absorbance of the final dilution was
found to equal to 0.520.

 Calculate the % purity of trioxalen USP.


Calculation Step 1

 Determine the concentration of


reference standard (Cs) in the final
dilution.

Cs = wt/mL x # dilutions
Cs = wt/mL x # dilutions

Cs = 49.0 mg x 1 mL x 1000 ug
100mL 100mL 1mg

= 4.9 ug/mL
Calculation Step 2

 Determine the concentration of


trioxalen (Cu) in the final dilution
using the spectrometric equation.

Cu = Au/As x Cs
Cu = Au/As x Cs

Cu = 0.530 x 4.9 ug/mL


0.520

= 5.0 ug/mL
Calculation Step 3

 Determine the concentration of


sample present in the final dilution.

U = wt/mL x # dilutions
U = wt/mL x # dilutions

U = 51.0 mg x 1 mL x 1000 ug
100mL 100mL 1mg

= 5.1 ug/mL
Calculation Step 4

◦ Determine the % purity.

% purity = Cu/U x 100%

% purity = 5.0 ug/mL x 100%


5.1 ug/mL

= 98%
Calculation Shortcut

 Short-Method

% Purity = Au/As x S/U x 100%

= 0.530 x 49mg x 100%


0.520 51mg

= 98%
 TheUSP/NF Requirement is
that…
 TRIOXSALEN contains NLT 93%
and NMT 103% of the labeled
amount of C14H12O3, calculated
on a dried basis.
 What will be your disposition?
The end!!!

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