This document discusses different methods for detecting molecules in solutions, including indicator solutions and spectrophotometers. It describes how spectrophotometers work by shining light on a sample and measuring how it absorbs, reflects, or transmits the light. Spectrophotometers can use ultraviolet or visible light and measure transmittance, converting it to absorbance using Beer's Law to determine concentrations of unknown samples. The document provides details on the inner parts of spectrophotometers including lamps, prisms or gratings, and outer parts like sample holders and displays.

2. Mujahid ali
Roll No: 44
B.s applied geology
Institute of geology, University of the Punjab Lahore

3. 1. Indicator – Quickest method.
 Indicator solutions change colors
when a molecule of interest is
present.
 Allows scientist to detect colorless
molecules in a solution.
 Examples: Bradford protein reagent
Diphenylamine (DPA) or Biuret
(NaOH+CuSO4).

4. 2. Spectrophotometer (Spec)
 An instrument that measures the
amount of light that passes
through (is transmitted through) a
sample.

5. Uses a type of light to detect
molecules in a solution
Light is a type of energy, and
the energy is reported as
wavelengths, in nanometers
(nm).

6. 1. Ultraviolet (UV)
Spectrophotometers.
 Uses ultraviolet light of wave lengths
from 200 nm to 350 nm.
2. Visible (VIS) Light Spectrum
Spectrophotometers.
 Uses visible light (white light) of wave
lengths from 350 nm to 700 nm.

7. R O Y G B I V

8.  Shines a beam of light on a sample.
 The molecules in the sample interact
with the light waves in of 3 ways:
 Absorb the energy
 Reflect the energy
 Transmit the energy between and
through the atoms and molecules of
the sample.

9. Consider blue molecules, all the
wavelengths of light are absorbed,
except for the blue ones.
The blue wavelengths are transmitted
or reflected off the molecules. If these
blue wavelengths hit a detector (such
as in the spectrophotometer or the
nerve cells in your eye), they appear
blue.

10.  Molecules are whatever color of
light that they do not absorb.
 Green molecules appear green
because they absorb most
wavelengths of visible light,
except the green wavelengths.

11. The spectrophotometer measures
the amount of light transmitted
through the sample
(Transmittance).
By using an equation (Beers law),
it converts the transmittance data
to an absorbance value.
It is quantitative analysis

12. The concentration of an unknown
sample can be determined by
comparing the absorbance data to
standards of known concentration.
The data generated with the set of
known standards is called a
standard curve.

13.  Inner parts
● Lamp
● Prism or grating that direct light of
a specific wavelength.

14.  Outer parts:
Sample Holder
Display
Knobs or buttons used to calibrate the
spec to measure the designated molecule.
Wavelength
Selection

16. Visible Spectrophotometer
White light hits the prism or grating,
it is split into the colors of the rainbow
(Visible Spectrum).
The wavelength knob rotates the
prism/grating, directing different
color of light toward the sample.

17.  The wavelength of light produced
by the tungsten lamp range from
about 350 nm (Violet light) to 700
nm (red light).
 The molecules in the sample either
absorb or Transmit the light energy
of one wavelength or another.

18.  The detector measures the amount
of light being transmitted by the
sample and reports that value
directly (% transmittance) or
converts it to the amount of light
absorbed in absorbance units (au)
using Beers Law.

19. 1. Determines the presence and
concentrations of samples.
2. Determines the purity of a
sample.
3. Look at the change of samples
over time.