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• The earliest colorimeters relied on the human • The depth of the color
eye to match the color of a solution with that is proportional to the
concentration of the
of one of a series of colored discs. The results compound being
obtained were too subjective and not measured, while the
particularly accurate. amount of light is
proportional to the
intensity of the color
and hence the
concentration.
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Light source slit condenser cuvette filter photocell galvanometer Light source slit condenser cuvette filter photocell galvanometer
lens lens
Light source slit condenser cuvette filter photocell galvanometer Light source slit condenser cuvette filter photocell galvanometer
lens lens
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The Spectrophotometer:
• All types require a Blank:
which is a solution that contains the entire
reagents except the substance to be measured.
• It is used to adjust the device to zero.
Here is a summary of the steps of operation of Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer: a Spec 20 & spectrophotometer:
5- Blank 1- Power
Fill tube half full with water. Turn on power.
Place in sample holder and close cover. 2-Warm up
Adjust meter needle to 100%T (or 0 A) using light Allow about 5 minutes when first turned on.
control knob. 3-Wavelength
Select appropriate wavelength.
4- Zero
With sample holder empty and closed, adjust meter
needle to 0%T (or infinite A) using zero control knob.
Here is a summary of the steps of operation of Here is a summary of the steps of operation of
a Spec 20 & spectrophotometer: a Spec 20 & spectrophotometer:
6- Standard (Measure absorbance (or %T) of known solution). 6- Standard (Measure absorbance (or %T) of known solution).
Fill tube half full with sample of known Fill tube half full with sample of known
concentration. concentration.
Place in sample holder and close cover. Place in sample holder and close cover.
Read absorbance value (or %T) from meter. Read absorbance value (or %T) from meter.
Repeat this step if making a calibration curve or Repeat this step if making a calibration curve or
verifying proportionality (Beer's Law). verifying proportionality (Beer's Law).
7- Sample
Measure absorbance (or %T) of solution with
unknown concentration as in previous step.
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Types of spectrophotometer:
1- Visible spectrophotometer.
COLORIMETRIC
DETERMINATION OF 2- Ultraviolet (UV)
VITAMIN B-12 spectrophotometer.
Vitamin B12
- Vitamin B12 is water soluble vitamin.
3 4 5 Unk. - RDA= 2.4 µg /day for adult.
B 1 2
- It is found only in animal sources.
Functions:
1- Aids folic acid in synthesis of heme.
St. Viatamin (ml) 0 1 2 3 4 5 0 2- It prevents anemia.
Shake Well with vortex
Unk. Vitamin (ml) 0 0 0 0 0 0 5 3- Required for protein digestion and absorption.
Read
H O (ml)the Optical
2 5 4 Density
3 2 1at 555
0 nm
0
* Calculations Results
Absorbance of Unknown Absorbance of Standard 0.6
= 0.5
Concentration of Unknown Concentration of Standard 0.4
Abs.
0.3
0.1
A St. 0
0 1 2 3 4 5 6
Conc.
Samar A. Damiati