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All living systems can grow, sustain and reproduces themselves. The different chemical changes occur in living body, fall in the domain of biochemistry. There are various molecules which take part in biochemical reactions of living body. These molecules are termed as Biomolecules.

These molecules interact with each other under optimum conditions and form different products. Hence, Biomolecules are complex chemical substances which form the basis of life and are responsible for their growth, maintenance and  ability to reproduce.

Biomolecules Definition:
Biomolecules is defined as "Biomolecules are those molecules that are involved in the maintenance and metabolic processes of all living organisms."


Types of Biomolecules

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The different bio molecules, which take part in maximum biochemical reactions are carbohydrates, proteins, enzyme, nucleic acid, lipids, hormones and compounds for energy storage like, adenosine diphospahte (ADP) and adenosine triphospahte (ATP). There are more than thousand bio molecules in different living systems.

Even though there are few basic classes of bio molecules. The major bio molecules included glycerine, polysaccharides, polypeptides and nucleic acids. These all bio molecules have giant molecular structures as they are polymers of certain small monomers.

For example:

Monomer Polymer
Fatty acid
Diglyceride, triglyceride
Amino acid
Nucleic acid(DNA, RNA)

Mass Spec Analysis

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Mass spectrometer analysis is used in detection and identification of chemical structure of given unknown mixture. It provides precise quantitative analysis and technique to detect the ultra-trace levels.

The spectrometer works in following steps

  1. Ionization: Ionization process takes place by knocking one or more electrons off to give a positive ion as mass spectrometers always work with cations.
  2. Acceleration: All cation gets accelerated to make them same as kinetic energy.
  3. Deflection: Cation gets deflected in magnetic field which depends upon their mass. Less mass correspond to less deflection. Deflection also depends on the positive ion given, as more charge is responsible for more deflection.
  4. Detection: The beam of ions detected by using electrical detector.

Mass spectrometer

Mass spectra contains several sets of peaks which corresponds to the molecular ion, isotope peak, fragmentation peaks and metastable peaks. Out of these peaks the molecular ion peak is mostly visible and can be weak or missing. The molecular ion is a cation (M+) formed by removing one electron from the molecule.

The relative intensity of the molecular ion peak decreases with branching and with molecular mass in a homologous series. For example in mass spectra of toluene the molecular ion peak is located at 92 m/z same as the molecular mass of toluene and preceded by a M-1 or M-2 peak.

The peak with the highest intensity is called the base peak which is not necessarily the molecular ion. Other peaks are larger than the molecular ion peak because of isotope distributions and known as isotope peaks.

Normal Phase Chromatography

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Normal-phase chromatography (NP-HPLC) is a different type of chromatography which involves the separation molecules. The separation of molecules is based on strength difference with a polar stationary phase during their interaction. In this chromatography method, the analyte mixture components are passed over the stationary-phase which contains particles with pores.

These pores are large enough in size for the molecules to enter, where a simple interaction with the polar surface helps remove them from the mobile-phase stream flow. Several types of Normal phase-HPLC columns with different pore sizes and coating are available. This could be utilized for the manipulation of the analyte stationary phase interaction strength. In this chromatography, the mobile phase is less polar than stationary phase. Many stationary phases are available for normal-phase chromatography. For example, silica which can provide very high selectivity for different applications.
There are two major types of silica in use:
  1. Newer type B silica - These are made of high silica purity with lesser acidic sites and are usually utilizede for separating out highly polar or basic compounds.
  2. Older less pure silica (type A) - These have some amount of trace metal content with highly acidic sites on the surface.
Another stationary phase is alumina (Al2O3) which has a unique selectivity of low theoretical plate number (N) and is considered as an disadvantage, the variable retention times, and low sample recovery.
Selecting solvents for normal-phase Chromatography, some factors like solvent strength, localization and basicity are taken into consideration.
Localization is a measure of the solvent and stationary phase interaction.

Non-polar solvents weakly interact with the stationary phase and the coverage of the surface is found to be random. The column of this chromatography has diameter of 4.6 mm and length of 150 to 250 mm.

HPLC Analysis

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High performance liquid chromatography is the best tool for the qualitative analysis. It's a highly improved column chromatography in which the solvent  is forced to drip through a column under high pressure of up to 400 atm. This external pressure makes the process faster and easier.

By using High performance liquid chromatography, very small particles can be used as packing material which provides a much greater surface area for interaction between both phases and allows much batter separation of components of given mixture. The other advantage of this chromatography is the detection method which is highly automated and extremely sensitive.

HPLC Analysis
There are mainly three steps in HPLC:
  • Injection of the sample: Sample injected to column under high pressure is very easy and automated.
  • Retention time: The time taken by a certain compound to travel through the column to detector is called as Retention time. Retention times depend on various factors like
    1. The pressure in column, the particles size and nature of the stationary phase.
    2. The composition of the solvent used in column.
    3. Temperature
  • The detector: Various detectors used for detecting the substance that has passed through the column. Generally ultra-violet absorption is used by using UV-detector and the output is recorded in the form of peaks.
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