The supernatant after the separation of the sub cellular components contain soluble proteins, amino acids, different pigments like chlorophyll, xanthophylls etc. These soluble components can be separated by different biochemical methods.
The most common method followed for the separation of proteins, amino acids and pigments is chromatography (Greek chrome-a color; graphing- to write). This procedure was originally applied by a Russian chemist Michael Semonovich Tswett in 1906 for the separation of different pigments.
The principle underlying chromatography is first to absorb the components of a mixture on an insoluble material (known as Stationary phase), then to differentially remove (or elute) individual components one by one with a suitable solvent (known as Mobile phase).
There are various kind of chromatography like Thin layer chromatography(TLC), Column Chromatography, paper chromatography, Ion- exchange chromatography etc. The most common one is the paper chromatography.
Two Russian scientists, Ismailia and Schraiber in 1938 for the first time developed the procedure of paper chromatography. In this method the soluble components present in a mixture are separated into different colored spots on a piece of special type of filter paper. The filter paper containing the colored spots is called as chromatogram.
Whatman No.1filter paper is the most commonly used for this. A narrow strip of the filter paper is taken and to its one end a drop of the solution containing the mixture of the compounds to be separated is applied as a spot and dried.
The paper strip is then kept in a cylindrical glass jar containing a mixture of solvents for the compound to be separated in such a way that its end nearer to the spot is inserted in the solvent mixture but the spot remains above. For the separation of different compounds different solvent mixtures are used. For the separation of amino acids the solvent mixture used is n- butanol: acetic acid: water (BAW) in the ratio of 4:1:5 (v\v). For pigments the solvent mixture is petroleum ether: acetone in the ratio of 9:1 (v\v).
The filter paper is kept hanging inside the glass jar by a glass rod tightly fitted to a rubber bung. The solvent mixture then rises (ascends) on the filter paper and different compounds depending on their differential solubility ascend to different heights on the filter paper.
This process is, therefore, known as ascending paper chromatography. In another method the solution mixture contained in a narrow glass trough is mounted near the top of the container.
The end of the filter paper strip nearer to the application spot is put in the solvent mixture and the mixture is allowed to move down (descend) the filter paper.
This is known as descending paper chromatography. After some time the paper strip is removed, dried and the separated compounds are located. For amino acids the paper strip is sprayed with 0.5% ninhydrin (in acetone) to develop the colored spots of the separated amino acids. The separated pigments themselves develop the colored spots and no further treatment is necessary.
RF = The separated compounds are identified by computing their RF value (Resolution front) from the chromatogram. / Distance moved by the compound (distance of the colored spot) from the point of application
Distance moved by the solvent mixture from the point of application.
For any compound separated in a particular solvent mixture the RF value is fixed. This value of a particular compound varies with different solvent mixtures. Paper chromatography can also be used for quantitative estimation of a compound after it is separated as a colored spot on a chromatogram and identified by its RF value.
The identified spot is cut out from the chromatogram and the compound present in the spot is dissolved in suitable solvents for quantitative estimation. Then other biochemical analysis procedures like colorimetric, spectrophotometry are followed for quantification.
Ultra Structure of Prokaryotic and Eukaryotic Cells
On the basis of cellular organization in living organisms two types of cells are recognized: Prokaryotic cell with no organized nucleus and Eukaryotic cell with organized nucleus bound by nuclear envelope. Prokaryotes (Gk. Pro-primitive; Karyon- nucleus) include primitive organisms like bacteria, cyan bacteria and mycoplasma.Eukaryotes (Gk.Eu-true) includes higher organisms.
Prokaryotic cells are the simplest kind of cells. The cells are smaller in size, consisting of cytoplasm surrounded by a cell membrane and encased within a rigid cell wall (except in Mycoplasma).
These cells do not have interior compartments. These cells vary in size from o.ipm-o.25 m m (as in Mycoplasma), a few micrometers in length in bacteria and a little larger in Cyan bacteria.
The cellular contents constitute a dense cytoplasm and a less electron dense nuclear area but no organized nucleus. The nuclear material or prokaryotic chromosome is a variously coiled naked DNA molecule without any histone protein and not enclosed within an envelope.
It is known as genophore or nucleoid or incipient nucleus.’ Some prokaryotes like bacteria also contain extra chromosomal, additional small DNA entities called plasmids. Cyan bacteria and photosynthetic bacteria posses layered membranes involved in photosynthesis. These membranes appear to be derived from the infoldings of the cell membrane. Membrane bound cell organelle like mitochondria, endoplasmic reticulum, Golgi comlex, lysosomes etc. are absent. Micro tubules are also absent. The prokaryotic cell contain 70s ribosome for protein synthesis as compared to 80s ribosome in eukaryotes.
The cytoplasm does not show streaming movement or cyclosis and spindle fibers are never formed during cell division (refer Cell division).True vacuoles are absent.
Some prokaryotes contain flagella but the internal organization of prokaryotic flagellum is different from that of eukaryotes. The bacterial cell divides by simple fission and both the bacteria and cyan bacteria form resting spores to pass off adverse conditions.
The eukaryotic cells found in higher forms of plants and animals are far more complex structures.
The characteristic features of eukaryotic cell is the presence of a membrane bound organized nucleus enclosing the genetic materials and also the compartmentalization of the interior of the cell by extensive end membrane system. The exterior of the cell is bound by a plasma membrane in animal cell.
The plant cells have rigid cell walls external to plasma membranes. The cytoplasm contains a number of membrane bound organelles like mitochondria, endoplasmic reticulum, Golgi complex; lysosomes. Cytoskeleton, micro fibrils and microtubules are found in eukaryotic cells. The eukaryotic ribosome is 80s type.
Features -Cell size
Prokaryote – Generally 1-10 mm in linear dimension.
Eukaryote- Generally 1-100 mm in linear dimension.
Prokaryote- Relatively rigid, no vacuolated, resistant to desiccation, osmotic shock and thermal denaturation.
Eukaryote- More fluid, vacuolated, more sensitive to dessication, osmotic shock and thermal denaturation.
Prokaryote – Membrane bound organelles absent.
Eukaryote- Membrane bound organelles like mitochondria, endoplasmic reticulum etc. present
Prokaryote- True nucleus absent; no nuclear membrane, no nucleolus, no nu – cleoplasm
Eukaryote- True nucleus present with nuclear membrane, nucleolus and nucleoplasm
Prokaryote- Small, 70S type.
Eukaryote- Large, 80S type. (Organelles like Mitochondria and chloroplasts have 70S ribosome)