MIC-403

Practical 05: Use of SPECTROPHOTOMETER

A study of the interaction of light (or other electromagnetic radiation) with matter is an important and
versatile tool for the chemist. Indeed, much of our knowledge of chemical substances comes from their
specific absorption or emission of light. In this experiment, we are interested in analytical procedures
based on the amount of light absorbed (or transmitted) as it passes through a sample.

Suppose you look at two solutions of the same substance, one a deeper color than the other. Your
common sense tells you that the darker colored one is the more concentrated. In other words, as the
color of the solution deepens, you infer that its concentration also increases. This is an underlying
principle of spectrophotometry: the intensity of color is a measure of the amount of a material in
solution. A second principle of spectrophotometry is that every substance absorbs or transmits certain
wavelengths of radiant energy but not other wavelengths.

In recent years spectrophotometric methods have become the most frequently used and important
methods of quantitative analysis. They are applicable to many industrial and clinical problems involving
the quantitative determination of compounds that are colored or that react to form a colored Product.

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to

absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This
means it uses light in the visible and adjacent ranges. The absorption or reflectance in the visible range
directly affects the perceived color of the chemicals involved. In this region of the electromagnetic
spectrum, atoms and moleculesundergo electronic transitions. Absorption spectroscopy is
complementary to fluorescence spectroscopy, in that fluorescence deals with transitions from the excited
state to the ground state, while absorption measures transitions from the ground state to the excited state.
Ultraviolet and visible radiation interacts with matter which causes electronic transitions (promotion of electrons from
the ground state to a high energy state). The ultraviolet region falls in the range between190-380 nm, the visible
region fall between 380-750 nm.

OPERATING INSTRUCTIONS

1. Let the instrument “warm up” for at least 15 minutes.

2. Set the desired wavelength with the wavelength control.
3. Select the mode (absorbance or transmittance) by pressing mode button.
4. Add water (blank) in a cuvette and find the absorbance and press zero absorbance buttons.
5. Remove blank and add sample in cuvette and place in sample holder to find absorbance.
6. Before taking another reading on the same or different sample, you should again check that the
absorbance scale reads zero with the blank sample.
MIC-403

Nano drop quantification

 For measurement of 0.5 – 2 µL DNA, RNA, and protein samples

 Measure very low and high concentration samples (0.4 – 15,000 ng/µL dsDNA)

https://www.youtube.com/watch?v=vOtVI4ghWRs