Description of chromatography paper and colomn

1. • HPLC it is a technique by which a mixture sample is
separated into components for identification, quantification
and purification of mixtures.
Liquid chromatography (LC)
It is an analytical chromatographic technique that is useful for
separating ions or molecules that are dissolved in a solvent.
Conventional LC is most commonly used in preparative scale
work to purify and isolate some components of a mixture.
Analytical separations of solutions for detection or quantification
typically use more sophisticated high-performance liquid
chromatography instruments.
HPLC is a popular method of analysis because it is easy to
learn and use and is not limited by the volatility or stability
of the sample compounds.
Modern HPLC has many applications including separation,
identification, purification, and quantification of various compounds.

2. Instrumentation:
HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.

3. The mobile phase is pumped through the column by a pump.
Solvents must be degassed to eliminate formation of bubbles .
The role of the pump is to force a liquid (mobile phase) through the
liquid chromatograph at a specific flow rate
The pump can deliver a constant mobile phase composition
(isocratic) or (gradient).
In isocratic elution compounds are eluted using constant
mobile phase composition.
In gradient elution different compounds are eluted by
increasing the strength of the organic solvent. The sample is
injected while a weaker mobile phase is being applied to the
system. The strength of the mobile phase is later increased in
increments by raising the organic solvent fraction, which
subsequently results in elution of retained components.
1- Mobile Phase

4. isocratic vs gradient elution hplc

5. 2- HPLC Pumps :
There are several types of pumps available for use with HPLC analysis,
3- Injection port
Samples are injected into the HPLC via an injection port.
The sample is typically dissolved in the mobile phase before injection.
Injection may be manual or auto-sampler.
4 - Stationary Phase:
There are a wide variety of stationary phases available for HPLC :
Normal Phase.
- Polar stationary phase and non-polar solvent. e.g. silica gel.
Reverse Phase.
- Non-polar stationary phase and a polar solvent. e.g. silica gel -C18
ion exchange.
Size Exclusion.
Affinity.

6. 5- Columns
• The heart of a HPLC system is the column.
• The column contains the particles that contains the stationary
phase.
• There are various columns that are secondary to the separating
column or stationary phase. They are: Guard, Derivatizing, and
Preparatory Columns.
• Guard Columns (precolumns): are placed before the separating
column. This serves as a protective factor that prolongs the life and
usefulness of the separation column. They are dependable columns
designed to filter or remove interferences.
Packings for columns :
Are diverse since there are many modes of HPLC. They are available
in different sizes, diameters, pore sizes, or they can have special
materials attached (such as an antigen or antibody for immunoaffinity
chromatography).
Packings available range from those needed for specific applications ,
to those for all-purpose applications.

7. 6- Detectors
The detector for an HPLC is the component that emits a response
due to the eluting sample compound and subsequently signals a
peak on the chromatogram.
There are many types of detectors that can be used with HPLC.
Some of the more common detectors include:
Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Near-Infra Red
(Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance
(NMR), and Light Scattering (LS).
Ultra-Violet (UV) detectors measure the ability of a sample to
absorb light. This can be accomplished at one or several
wavelengths:
A) Fixed Wavelength measures at one wavelength, usually 254 nm
B) Variable Wavelength measures at one wavelength at a time, but can
detect over a wide range of wavelengths
C) Diode Array measures a spectrum of wavelengths simultaneously.

8. Qualitative and Quantitative Analysis

9. Electrophoresis
Electrophoresis is a type of chromatography that relies upon somewhat
different principles than the others previously discussed chromatographic
methods.
The word electrophoresis connotes the movement of a charged particle
[Greek: electron + pherein (to carry)]. Electrophoretic methods of separation
thus rely on differences in the mobility of different charged substances in an
electric field.
Electrophoresis is a general term that describes the migration and
separation of charged particles (ions) under the influence of an electric field.
An electrophoretic system consists of two electrodes of opposite charge
(anode, cathode), connected by a conducting medium called an electrolyte.
Their rate of migration through the electrical field, depends on:
the strength of the field
the net charge
the size, and shape of the molecules
and also on the ionic strength, viscosity, and temperature of the medium
in which the molecules are moving.

10. Paper Electrophoresis
Paper electrophoresis makes the use of Chromatography paper
which has a different adsorption capacity of different molecules. To
perform this kind of electrophoresis, several steps involve:
First, the filter paper moistens with the buffer solution. After this,
immerse both the ends of filter paper in a buffer solution.
Then sample place at the centre, after which the electric field is
applied, that allows migration of molecules to their respective
poles depending upon their charge
Paper electrophoresis is
used for the analysis of
proteins like casein,
serum, albumin, myosin
etc
Electrophoresis
As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
It can be used analytically to study the properties of a single charged
species or mixtures of molecules. It can also be used preparatively as a
separating technique

11. Electrophoresis is usually done with gels
formed in tubes, or on a flat bed.
In many electrophoresis units, the gel is
mounted between two buffer chambers
containing separate electrodes, so that
the only electrical connection between
the two chambers is through the gel.
Gel Electrophoresis
In simple terms electrophoresis is a procedure which enables the sorting of
molecules based on size and charge.
When charged molecules are exposed to an electric field they will migrate
toward either the positive or negative pole according to their charge.
In contrast to proteins, which can have either a net positive or negative
charge, nucleic acids have a consistent negative charge imparted by their
phosphate backbone and will migrate toward the anode (positive pole).
The sample migrates through the gel in response to the electric current, the
small molecules move more easily and more quickly than the larger
molecules, which results in a distinct banded pattern forming in the gel.
The bands are visualized using stains. ***

14. In the separation of DNA fragments for DNA
fingerprinting to investigate crime scenes.
To analyze results of polymerase chain reaction.
To analyze genes associated with a particular illness.
In DNA profiling for taxonomy studies to distinguish
different species.
Applications of gel electrophoresis