pharmceutical analysis m.pharm modern bio analytical technique

1. KARNATAKA COLLEGE OF PHARMACY
SUBJECT : MODERN BIOANALYTICAL TECHNIQUES
SEMINAR ON TOPIC: Analysis of biological samples using HPLC
PRESENTED TO: Dr. Harsha K Tripathy
Department of pharmaceutical analysis
KCP
PRESENTED BY: KAVANA BB ( M pharma II SEM )
Department of pharmaceutical analysis
KCP
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2. Contents:
• Introduction
• Principle
• Types
• Advantages
• Applications
• Limitations
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3. Introduction
• High-performance liquid chromatography (HPLC) is a broad analytical chemistry
technique used to separate compounds in a chemical mixture.
• These separations utilize the pressure-driven flow of a mobile phase through a
column packed with a stationary phase.
• In the 1960s, the column chromatography LC with its low-pressure suitable glass
columns was further developed to the HPLC with its high-pressure adapted metal
columns.
• HPLC is thus basically a highly improved form of column liquid chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is
forced through under high pressures of up to 400 atmospheres.
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4. Principle:
• The purification takes place in a separation column between a stationary and a
mobile phase.
• The stationary phase is a granular material with very small porous particles in a
separation column.
• The mobile phase, on the other hand, is a solvent or solvent mixture which is forced
at high pressure through the separation column.
• Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to
the separation column using a syringe.
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5. • Subsequently, the individual components of the sample migrate through the
column at different rates because they are retained to a varying degree by
interactions with the stationary phase.
• After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
• At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
• The chromatogram allows the identification and quantification of the different
substances.
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7. Types of HPLC
1.Normal phase:
Column packing is polar (e.g silica) and the mobile phase is non-polar. It is used for water-sensitive
compounds, geometric isomers, cis-trans isomers, and chiral compounds.
2.Reverse phase:
The column packing is non-polar (e.g. C18), the mobile phase is water+ miscible solvent (e.g.
methanol). It can be used for polar, non-polar, ionizable, and ionic samples.
3.Ion exchange:
Column packing contains ionic groups and the mobile phase is buffer. It is used to separate anions
and cations.
4.Size exclusion:
Molecules diffuse into pores of a porous medium and are separated according to their relative size
to the pore size. Large molecules elute first and smaller molecules elute later.
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8. Application
The HPLC has developed into a universally applicable method so that it finds its use
in almost all areas of chemistry, biochemistry, and pharmacy.
• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides
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9. Limitations of HPLC
1.Cost: Despite its advantages, HPLC can be costly, requiring large quantities of
expensive organics.
2.Complexity
3.HPLC does have low sensitivity for certain compounds, and some cannot be
detected as they are irreversibly adsorbed.
4.Volatile substances are better separated by gas chromatography.
Advantages:
• Speed
• Efficiency
• Accuracy
• Versatile and extremely precise when it comes to identifying and
quantifying chemical components.
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10. HPLC is historically divided into two different sub classes based on the
polarity of the mobile and stationary phases.
• 1. Normal phase high performance liquid chromatography
• 2. Reverse phase high performance liquid chromatography
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11. Normal phase high performance liquid chromatography:
Techniques in which the stationary phase is more polar than the mobile phase is
call normal phase high performance liquid chromatography.
Stationary Phase –Polar nature e.g.: SiO2, Al2O3.
Mobile Phase – Non-polar nature e.g.: Heptane, hexane, cyclohexane, CHCl3
Mechanism:
• Polar compounds travels slower and eluted slowly due to higher affinity b/w
solute and stationary phase.
• No polar compound travels faster and eluted first due to lower affinity b/w solute
and stationary phase.
• This technique is not widely used in pharmaceutical separation.
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12. Reverse phase high performance liquid chromatography:
• Techniques in which the mobile phase is more polar than the stationary phase is
called reverse phase high performance liquid chromatography. ¸
• Stationary phase – Non-polar nature. e.g.: n-octadecyl, n-octyl, ethyl, phenyl diol
• Mobile Phase – polar nature. e.g.: Methanol or Acetonitrile/water or buffers
Mechanism:
• A polar compound travels faster and eluted first due to lesser affinity b/w solute
and stationary phase.
• Non polar compounds travel slower and eluted slowly due to higher affinity b/w
solute and stationary phase.
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13. Example of biomolecule separation by HPLC
• To determine if the two genes of interest differ, they are first amplified by the
polymerase chain reaction and then injected together into a so-called reversed-
phase column.
• In this type of column, the stationary phase is less polar than the mobile phase,
which is the opposite of the arrangement found in standard columns.
• Once the genes are injected, their DNAs bind to the stationary phase. Increasing
the temperature causes each gene to separate into its two strands (a phenomenon
called denaturing).
• Once denatured, the strands can enter the mobile phase and move through the
column.
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14. • Cooling the column causes the strands of the genes to rejoin, and the
DNAs reattach to the column.
• The temperature is manipulated to make the strands constantly
separate and rejoin, with the balance determined by the strength of
the attraction that exists between the strands.
• If the two genes are exactly identical, they will spend more time in
the stationary phase, and elute from the column more slowly.
• If the genes differ even by a single nucleotide, however, they will
spend more time in the mobile phase, and leave the column more
quickly.
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15. Questions
• Write a brief biomolecule separation by HPLC.
• Give the general principle and method of separation of biomolecule
by HPLC.
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