About author :
Jony Mallik*, Abdullah Al Faruq
Department of Pharmacy,
Southern University Bangladesh, Chittagong, Bangladesh
Protein is the collection of amino-acid moieties and essential part of human nutrition. Amino acids play central roles both as building blocks of proteins and as intermediates in metabolism. The 20 amino acids that are found within proteins convey a vast array of chemical versatility. The precise amino acid content, and the sequence of those amino acids, of a specific protein, is determined by the sequence of the bases in the gene that encodes that protein. The chemical properties of the amino acids of proteins determine the biological activity of the protein. Proteins not only catalyze all (or most) of the reactions in living cells, they control virtually all cellular process. In addition, proteins contain within their amino acid sequences the necessary information to determine how that protein will fold into a three dimensional structure, and the stability of the resulting structure. The field of protein folding and stability has been a critically important area of research for years, and remains today one of the great unsolved mysteries. It is, however, being actively investigated, and progress is being made every day. There are number of analytical technique that may perform analysis with a sample containing protein of particular amino acid sequences. Denaturation or folding occurs during analysis resulting fault in finale result. Modern Pharmaceutical analysis introduce a new technique to overcome these problems, the technique is “Fast Protein Liquid Chromatography” (FPLC)
Key words: Protein, Amino acids, Denaturation, Fast Protein Liquid Chromatography (FPLC).
Fast Protein Liquid Chromatography (FPLC) :
It is actually an automation of liquid chromatography and a reliable improvement of column chromatography. The FPLC instruments is used for separation of proteins (as amino acids) & their subsequent purification for future use. In other chromatographic analytical technique, the chance of denaturation is high because of their stainless steel made instruments which elevates the inner temperature and resulting denaturation of sample (protein) under investigation.
FPLC is said to be as “Protein loving chromatography”. It was introduced by PHARMACIA (Sweden) at 1982. (Pharmacia’s smart system).
FPLC vs. HPLC :
The instrumentation of FPLC is same as HPLC , hence said as Protein friendly HPLC. But the differences are
1. In HPLC, all parts of the whole instrument are made of high quality stainless steel where high quality plastics or glass used in FPLC. (For temperature control).
2. In HPLC, the pressurized pump generates pressure of about 0-550bar (14.6-8000psi), whereas in FPLC its about 0-40 bar ( For the safety of column).
3. In HPLC, standard analytical (4-5mm, 10-30 cm length) column is mostly used where in FPLC, microbore column (1-2mm * 10-25 cm length) is widely used.
4. In HPLC , available flow rate is in between 0.010 – 10 ml/min , but in FPLC its 1-499 ml/min.
5. HPLC technique is not suitable for protein separation, but FPLC is very reliable in separating & purifying proteins.
INSTRUMENTAL FEATURES :
The instrumental features are stated below
The stationary phase may be a liquid or a solid, in the form of a matrix.
The pumps control the constant flow rate of the solvents.
The solvents are accessed through tubing from an outside reservoir.
The flow rate of the solvent is set through computer input and controlled by pumps.
There are various columns used in liquid chromatography depending on the type of separation preferred. Each column contains a small diameter packing material. The column is a large (mm id) tube containing small (u) particles (gel beads) known as stationary phase.
The chromatographic bed is composed by the gel beads alone when they are inside the column.
The sample is introduced into the injector and then carried into the column by the flowing solvent.
Once in the column, the sample mixture separates as a result of different components adhering to or diffusing into the gel.
Biomolecules have various characteristics such as molecular weight, electric charge, and hydrophobicity.
As so, purification of the object is usually achieved by using a combination of chromatographic methods.
Solvent Degassing (To remove bubbles/ dissolved gases)
Subject the solvent to vacuum for 5-10 mins. to remove the gases.
Subject the solvent to ultrasonics for 10-15 mins. to remove the gases.
Mobile Phases mixing
Injection of sample
Flow rate maintenance by pump system
Separation take places
Elution of analyte & chromatograph generation
ANALYTICAL PRINCIPLE IN BRIEF :
FPLC system is a complete system for laboratory scale chromatographic separations of proteins and other biomolecules. This system consists of a programmable controller for developing and controlling automatic separation procedures, one or more pumps for liquid delivery, a mixer to ensure accurate and reproducible elution gradients, valves for sample injection and flow path control, one or more monitors for measuring chromatographic profile, a recorder for documenting chromatographic profile, a fraction collector and a chromatography rack for mounting the component in a compact laboratory bench top arrangement. Pharmacia’s SMART System is a micropurification chromatography system that recovers and purifies biologically active material present in a sample at very low concentrations (from micrograms to picograms). The system consists of specially designed instruments, software, and prepackaged columns to purify biomolecules for applications such as peptide sequencing and protein structure/function studies. When the micro-concentration of sample inject to column, due to solvent interaction the sample introduced to the stationary phase of the analytical column and separation take places. After elution of protein as amino acids, they are further use in analytical and biochemical purposes.
PHARMACEUTICAL IMPORTANCES OF FPLC :
1. Separation of biological compounds, and thermally labile compounds.
2. Qualitative and quantitative analysis of the biological compounds.
3. An importance for a pharmaceutical company in using fast protein liquid chromatography is the quality assurance this test provides.
4.The standard performance of FPLC testing enables contract manufacturers to transfer methods to their facilities from the company contracting production.
5. FPLC is used to analyze raw materials (Amino acids) and finished products (INFUSION) to assure that pre-established quality levels are being met.
6. With the use of FPLC, a company has the potential to discover faulty products which do not meet specification
7. This allows the manufacturer to avoid an expensive and reputation-damaging recall by not releasing the product in the first place.
In pharmaceutical analysis, FPLC occupies a vast area within which it play its role with biological substances (Protein, Nucleic acids etc.) in terms of their separation & purification. The pharmaceutical industry having LVPL (Large Volume Parenteral Liquids) area, must required FPLC setup to check the purity of the drug – products (Amino acid IVIF). Most of the industry do these job by utilizing HPLC. Its not unethical, but to perform an ethical & reliable separation & purification , there is no alternative except FPLC.