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parfocality. This can result in damage to the
objective lens or slide.
The microscope could be accidentally bumped
against objects (or your lab mates) and damaged by
The whole eyepiece tube could fall off the
microscope and be badly damaged.
This can result in damage to the mechanical stage
This results in a loss of the friction that holds the
stage at a selected focus. In this condition the
microscope will not stay focused.
Time will be wasted in finding the plane of the focus
of the next specimen.
This will make finding a desired object much more
difficult and time consuming.
Less light will be available for viewing the specimen.
The usefulness condenser diaphragm for adjusting
specimen contrast will be lowered. 13. Attempting to view low contrast specimens with
the light too bright and the condenser diaphragm The specimen will probably not be visible.
too widely open.
It will be difficult if not impossible to gain a three
14. Improper adjustment of the inter-ocular distance.
dimensional image of the specimen.
15. Attempting to view specimen with eyes too close The specimen will be difficult if not impossible to
to the oculars.
see. Biology 05LA – Fall Quarter 2012 Lab 2 – page 1 LAB 2: SPECTROPHOTOMETRY AND QUANTITATIVE DATA ANALYSIS
This exercise will introduce the use of a spectrophotometer (or colorimeter) to measure a
particular chemical property of many biological (and non-biological) materials. We will also learn to
quantify spectrophotometric data, learn several "wet lab" techniques, and learn how to properly prepare
scientific graphs. Be advised that you will be using all of these newly acquired skills in the labs of the
weeks to come, so come to lab prepared to master these skills – your potential success in these future
labs will be greatly enhanced if you do so.
All solutions that are colored to the human eye absorb electromagnetic radiation in the visible
portion of the electromagnetic spectrum (Fig. 1). The color that we perceive represents those
wavelengths of light that are not absorbed by the substance, or, conversely, represents those
wavelengths that are transmitted. For example, if a solution is green, it absorbs in the blue and red
portions of the spectrum. If it is red, we would expect it to absorb in the green and blue portions of the
spectrum. Another way of saying this is that a solution transmits light of a color complementary to
that which it absorbs (Fig. 1, color wheel).
The absorption of certain wavelengths of
electromagnetic radiation is as characteristic of a
compound as is its molecular weight, solubility
properties, melting point or any other intrinsic
property. Thus, the absorption spectrum of a
compound may be used to identify it.
The fact that a compound, solution or
substance is not colored does not mean that it does
not absorb electromagnetic radiation. It merely
means that it does not absorb visible light.
However, it may absorb strongly in other regions
of the spectrum. Water, for example, is colorless,
but it absorbs far infrared light, a band of wavelengths to which the human eye is insensitive.
The radiation that is absorbed by water is
converted to heat, and the water becomes warm.
Many other compounds of biological importance
absorb in non-visible regions of the electromagnetic spectrum.
Absorbance and transmittance are measured
with either a colorimeter or a spectrophotometer.
The instruments used in Biology 05LA are
technically called colorimeters. A colorimeter is
an instrument that selectively projects narrow
bands of visible light upon a solution (see Fig. 2).
A spectrophotometer does the same thing but also
can utilize wavelengths in non-visible regions of
the near ultraviolet and infrared.
Both colorimeters and spectrophotometers
function in the same manner. Light from a source Comparable Wavelength
to Rubidoux 106 Size of
blue whale. 104 Common
Name McDonald ’s
hamburger. 102 Hertzian waves;
radio, TV, and
microwaves. 100 Size of
an amoeba 10-2 Infra red. Red blood
cell. 10-4 Visible
Ultra violet. AIDS
virus 10-6 H atom. 10-8 Violet
(400nm) Gamma ray. 10-12
(450nm) (500nm) X-ray. 10-10
(650nm) Cosmic ray. Figure 1: The Electro magnetic Spectrum. The range of
wavelengths of the EMS is shown. The visible spectrum
represents a narrow band of the EMS. The approximate
wavelengths of the colors in the visible spectrum are
given in the color wheel. Colors that are complementary
fall on opposite sides of the wheel. Biology 05LA – Fall Quarter 2012 Lab 2 – page 2 that emits light at the required wavelength is focused upon a monochromator by a lens-like collimator.
The monochromator, either a prism or diffraction grating, separates the light into its component
wavelengths. Once the light has been separated, the wavelength required for the analysis is selected
from all the available wavelengths. This selection occurs when the operator adjusts a control on the
front of the instrument which moves a slit ( selector) that allows onl...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.
- Fall '12