Spectrophotometer
An instrument used for measuring
the transmission or reflection of light by comparing various wavelengths of the
light. It also makes use of the Beer-Lambert’s
principle which is “Light transmitted is inversely proportional to the
concentration, while the absorbance is directly proportional to it.”
Some of
the precautions in this test:
1.
Never brush cuvettes because it will lead to
scratches and give inaccurate results
2.
Use rather a clean soft cloth to wipe the
cells before reading it
3.
Ideal amount of the sample is ¾ full so that
the light will strike the given sample and not the empty space.
4.
Let the machine be warmed up first for about
10-15 min before use, so that fluctuation is avoided.
There are four
essential parts of a spectrophotometer
1.
Light source
2.
Monochromator or filter
3.
Sample cell with holder
4.
Detector
Turbidimetry
-
The determination of the quantity of
insoluble suspended matter in a liquid by measuring the loss of intensity of
light in the direction of propagation of the incident beam, with reference to a
standard solution
Gravimetry
-
Gravimetric
methods of analysis are used where weights of reactants and products of
chemical reactions are reproducible, stable and reflect the presence of
constituents which are important in the establishment of identity.
-
Two
important methods deal with the trapping and weighing of products in the solid
and gaseous phases.
Osmometry
-
Measurement of the amount of osmotic pressure
that a particular solution exerts
-
Can be vapor pressure osmometry, freezing
point depression osmometry or membrane osmometry
Emission
Flame Photometry
A type of flame
photometry in which molecules in a flame are volatilised to generate free atoms
that are excited to higher energy levels. When these atoms return to the ground
state, they produce a characteristic emission spectrum. These typically use a low-temperature, air-gas, laminar flame burner which
is inherently subject to drift and therefore lithium (Li), which is not a
normal serum constituent, is excited as a known added constituent to
"standardize" the results despite the drifting of the flame output.
This approach does not permit addition of Ca as a third determined constituent
as the emission of the Ca at serum levels in the low-temperature flame is below
measurable intensity; furthermore the Li would interfere optically with the Ca
determination.
Atomic Absorption Spectrophotometry
Atomic Absorption Spectrophotometry is the
measurement of the concentration of an element in a given sample. This
technique can be done in three ways: Flame type, Electrothermal type, and Color
Vapor type. In the flame type, gas and combustible substance are used to ignite
the mixture of gases.
The second type which is
electrothermal makes use of electric current and slit width. On the other hand
is the cold vapor method wherein a reducing agent is added and vaporized.
Volumetry
Volumetry is t he measurement
of volumes of liquids, solids, and gas.
-the quantitative analysis of an unknown chemical solution by
determining the amount of reagent of known concentration necessary to effect a
reaction in a known volume of the solution
Chromatography
It is a broad range
of physical methods used to separate and or to analyze complex mixtures. The
components to be separated are distributed between two phases: a stationary
phase bed and a mobile phase which percolates through the stationary
bed.
- Thin
Layer Chromatography
TLC
is a simple, quick, and inexpensive procedure that gives the chemist a quick
answer as to how many components are in a mixture. TLC is also used to support
the identity of a compound in a mixture when the Rf of a compound is
compared with the Rf of a known compound (preferably both run on the
same TLC plate).
A
TLC plate is a sheet of glass, metal, or plastic which is coated with a thin
layer of a solid adsorbent (usually silica or alumina). A small amount of the
mixture to be analyzed is spotted near the bottom of this plate. The TLC plate
is then placed in a shallow pool of a solvent in a developing chamber so that
only the very bottom of the plate is in the liquid. This liquid, or the eluent,
is the mobile phase, and it slowly rises up the TLC plate by capillary action.
B.
Gas
chromatography
specifically gas-liquid chromatography - involves a sample
being vaporized and injected onto the head of the chromatographic column. The
sample is transported through the column by the flow of inert, gaseous mobile
phase. The column itself contains a liquid stationary phase which is adsorbed
onto the surface of an inert solid.
C.
‘High
Performance Chromatography
High-performance
liquid chromatography (HPLC) is a form of liquid chromatography to separate
compounds that are dissolved in solution. HPLC instruments consist of a
reservoir of mobile phase, a pump, an injector, a separation column, and a
detector. Compounds are separated by injecting a plug of the sample mixture
onto the column. The different components in the mixture pass through the
column at different rates due to differences in their partitioning behavior
between the mobile liquid phase and the stationary phase.
Electrophoresis
Electrophoresis is a separations technique
that is based on the mobility of ions in an electric field. Positively charged
ions migrate towards a negative electrode and negatively-charged ions migrate
toward a positive electrode. For safety reasons one electrode is usually at ground
and the other is biased positively or negatively. Ions have different migration
rates depending on their total charge, size, and shape, and can therefore be
separated.
Instrumentation
An
electrode apparatus consists of a high-voltage supply, electrodes, buffer, and
a support for the buffer such as filter paper, cellulose acetate strips,
polyacrylamide gel, or a capillary tube. Open capillary tubes are used for many
types of samples and the other supports are usually used for biological samples
such as protein mixtures or DNA fragments. After a separation is completed the
support is stained to visualize the separated components.
Resolution can
be greatly improved using isoelectric focusing. In this technique the support
gel maintains a pH gradient. As a protein migrates down the gel, it reaches a
pH that is equal to its isoelectric point. At this pH the protein is neutral
and no longer migrates, i.e., it is focused into a sharp band on the gel.
