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BIOLOGY LABORATORY
PRELIMINARY PERIOD
EXPERIMENT 1
Use of Micropipettor and
Spectrophotometer
Micropipettor
Micropipettor is used to
transfer volumes that are less
than 1mL
Measures as little as one
microliter (uL), one millionth
(10^-6) of a litter
Most useful units of liquid
measurement in molecular
biology are milliliter (mL) and
microliter (uL)
Micropipettor is a precision
pump with a disposable tip
Spectrophotometer
1. Simple spectrophotometer
- Visible light
- 380 to 750 nm
- Light is produced by a
tungsten lamp
2. UV-vis spectrophotometer
- Second lamp is used:
DEUTERIUM turns a
visible
light
spectrophotometer into a
UV-visible unit that can
measure from: 190-380
nm
- Available with a variety of
features:
scanning,
multiple cells, intergral
printers,
and
user
interfaces
Types of Spectrophotometers
ABSORBANCE AND
CONCENTRATION OF
ABSORBING MOLECULE
Absorbance vs Transmittance
Absorbance: The amount of
light that can be absorbed
Transmittance: The amount
of light that passes through
the solution as it is not
absorbed
Absorbance rather than the
transmittance is most useful
in spectrophotometry
If no light is absorbed
Absorbance = 0
Transmittance = 100%
Each unit in absorbance
corresponds with an order of
magnitude in the fraction of
light transmitted
A=1, 10% of the light is
transmitted (0.10) 90% is
absorbed
A=2, 1% of the light is
transmitted and 99% is
absorbed
is
is
r= Pearsons correlation
coefficient/correlation coefficient
r= value should be near 1 or 1 for
the graph to be a linear graph
Formula:
Transmittance, T = P / P0
% Transmittance, %T = 100 T
Absorbance,
A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T
What is the relation of
Concentration
and
Absorbance?
Explained by the Beers Law
Beers Law
A=ebc
Where A is absorbance (no units,
since A
=
log10 P0 /
P)
e is the molar absorbtivity with units
of
L
mol-1 cm-1
b is the path length of the sample - that is, the path length of the
cuvette in which the sample is contained. We will express this
measurement
in
centimetres.
An
increase
in
the
absorbance will also lead to
an
increase
in
the
concentration
(direct
relationship)
Linear Regression
y=mx + b
ACCURACY VS PRECISION
Accuracy
How close the measured
value is to the actual value or
true value
Precision
How close the value of the
measurement is close to the
other measured values by the
group
Analytical Balance
Great precision in quantitative
chemical analysis
Can give up to 4 decimal
places (0.0000g)
Water: 1:1 ratio with mL to g
DENSITY of water:1
Formulas used:
Computing for Concentration (c2)
C1V1=C2V2
Bromophenol blue: 1.25g/mL