Part One

Determination of the Absorption

Coefficient of Chlorphenamine

1
Objectives
1. Learn the definition of absorption coefficient.
2. Master the method for determination of the absorption coefficients.

Equipment and chemicals

Equipment
752 spectrophotometer;
Volumetric flasks (50 ml, 100 ml);
Suction pipets (5 ml, 10 ml)
Chemicals
Chlorpheniramine stock solution ( 0.0150 g / 100 ml );
Sulfuric acid solution: 0.05 mol/L 2
三、Principles
3
The extinction coefficient of compounds, caused by absorption of
the UV-Vis light, can be measured by spectrophotometer.
A A A
I
A   lg T   lg  E  C  l E  E1cm 
1%

I0 C l C l C l
(C : mol / L) (C : g / 100ml )
For a drug, the absorptivity (a) is achieved by averaging
the results on five spectrophotometers of different types and the
relative standard deviation (RSD) must not be more than 1%. The
relative deviation between the two coefficients must be less than 1%.

3
Procedures
1.The preparation of solutions
Into two 50 ml volumetric flasks, pipet 5.00 and 10.00 ml
chlorpheniramine stock solution respectively, dilute to the
mark with 0.05 mol/L sulfuric solution, mix well.
2. Determination of absorption coefficient
Using the blank as the reference, measure the absorbance of
any one of the chlorpheniramine solutions at different
wavelength ranged from 250 nm to 270 nm. Take readings
about 2 nm apart. Select the wavelength of maximum
absorbance (λmax) for the determination. At the selected
wavelength, pair the Cuvette and measure the Abs of each
solutions.
Results
Calculate the absorption coefficient of each solution. The relative
deviation between the two results should not be more than 1%.
4
 The preparation of solutions (room 202)
Into two 50 ml volumetric flasks, pipet 5.00 ml (Ⅰ) and 10.00 ml
(Ⅱ) of Chlorphenamine stock solution, respectively, and then dilute
to the marks with 0.05 mol/L sulfuric acid solution, mix well.

Transfer 5 ml Dilute
stock
Ⅰ Ⅰ
solution

10 ml Dilute

Ⅱ Ⅱ
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 Part Two

Determination of Gatifloxacin by Fluorescence

Spectrometry

6
Objectives
1. Master the method of determining Gatifloxacin by fluorescence
spectrometry.
2. Learn the operation of SHIMADZU RF-6000 fluorometer.

Equipments and chemicals

Equipments
SHIMADZU RF-6000 fluorometer; quartz cell.
Chemicals
The prepared Gatifloxacin stock solution.

7
Principles
1. The fluorometer

Light
cuvette
source

detector
8
2. Excitation and emission spectra

F
Em
80 Ex
60
40
20250 300 350 400
0 λ(nm)

3. The relationship of fluorescence and concentration

F  KC
9
Procedures
1.Plot the fluorescence excitation and emission spectra of Gatifloxacin

Record the excitation and emission spectra of the working

solutions to determine the maximum wavelengths of excitation
(λex) and emission (λem).

Three characteristics of emission spectra: Stokes Shift,

mirror symmetry , the shape of emission spectra.

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