HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY - PowerPoint PPT Presentation

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HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY

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Title: HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY


1
HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY
2
HPLC
  • Presented By -
  • Mr. Shaise Jacob
  • Faculty
  • Nirmala College of Pharmacy
  • Muvattupuzha, Kerala
  • India
  • Email jacobshaise_at_gmail.com

3
  • Liquid chromatography is a separation technique
    that involves
  • the placement (injection) of a small volume of
    liquid sample into a tube packed with porous
    particles (stationary phase)
  • where individual components of the sample are
    transported along the packed tube (column) by a
    liquid moved by gravity.

4
  • The components of the sample are separated from
    one another by the column packing that involves
    various chemical and/or physical interactions
    between their molecules and the packing
    particles.
  • The separated components are collected at the
    exit of this column and identified by an external
    measurement technique, such as a
    spectrophotometer that measures the intensity of
    the color, or by another device that can measure
    their amount
  • ?NoteThe modern form of liquid chromatography is
    now referred to as flash chromatography

5
Four types of high performance liquid
chromatography (HPLC)
  • partition
  • adsorption (liquid-solid)
  • ion exchange
  • size exclusion or gel

6
? improved performance
  • ? high pressure
  • HPLC- Separation is accomplished by
    partitioning b/w a M.P Stationary column
    material.
  • Packing material
  • small, uniform particle
  • gives high column efficiencies
  • High pressure
  • to achieved desired flow rates

7
Types of HPLC techniques
  • Based on Modes of chromatography
  • 1. Normal phase mode
  • S.P is polar
  • M.P is non polar
  • 2. Reverse phase mode
  • S.P is non polar
  • M.P is polar
  • Different columns used ODS,C18,C8,C4

8
Based on principle of separation
  • 1. Adsorption chromatography
  • 2. Ion exchange
  • 3. Ion pair
  • 4. Gel permeation / Size exclusion
  • 5. Affinity
  • 6. Chiral phase
  • Based on elution technique
  • Isocratic separation
  • Gradient separation

9
Based on scale of operation
  • Analytical HPLC
  • Preparative HPLC
  • Based on the type of Analysis
  • Qualitative analysis
  • Quantitative analysis
  • HPLC offers numerous advantages
  • ? Capable of handling macromolecules
  • ? Suitable for pharmaceutical compounds
  • ? Efficient analysis of liable natural
    products
  • ? Reliable handling of inorganic ionic
    species

10
? Dependable analysis of biochemical's
  • PRINCIPLE
  • Adsorption
  • Particle size of the S.P material plays a
    crucial role in HPLC
  • Micro particulate column packing
  • Silica particles ? uniform, porous, with
    spherical or irregular shape
  • Diameter ? 3.5 to 10 µm

11
HPLC instrumentation comprises
  1. M.P reservoirs
  2. Eluent degas module
  3. Solvent delivery pumps
  4. Manual / Auto injector
  5. Analytical column
  6. Detector
  7. Data processor

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Mobile phase reservoir
  • stores M.P (HPLC grade solvents)
  • ? Resolution Speed of analysis
  • Flow rate, polarity pH
    of M.P
  • Can't use metallic reservoir
  • Eluent degas module
  • Dissolved gases in M.P pose a number of problems
  • ? flow ? excessive
    detector noise
  • ? Rt fluctuation
  • ? Bubbling the pump detector,

17
  • Degas module with reservoir of inert gases
  • He or N2
  • Vacuum filtration
  • Helium purging
  • Ultrasonication (converts ultra high frequency to
    mechanical vibrations.)
  • SOLVENT DELIVERY PUMPS
  • Reciprocating pumps
  • widely used
  • maintain accurate flow rate

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Cross-sectional diagram of a simple single
piston reciprocating pump
21
Solvent delivery systems
  • two types 1. Isocratic system
  • 2. Low pressure gradient
  • 3. High pressure gradient
  • Injection system
  • a. Syringe system- results best
  • b. Injection valve - Rheodyne injector
  • Loading through the sample loop
    (20-50µl)

22
u
  • c. Automated injection device -
  • commercially available, automatically
    inject 100samples

23
Guard column
  • Pre-filter - useful for industry

24
Analytical column
  • Heart of any chromatographic system
  • Actual separation of components takes place
  • Several S.P available
  • depending upon tech. or mode of separation
  • Column material
  • S.S, glass, polyethylene, PEEK
  • Column length Column diameter Particle size
  • 5-30 cm 2mm-50mm 1µ-20µ

25
Particle nature
  • Spherical, uniform sized porous material
  • Surface area
  • 1g S.P provides surface area 100-860 sq.m
  • Functional group
  • Depends on the type of chromatographic
    separations.
  • Normal phase mode hydroxy group
  • Reverse phase mode C18 (octa decyl silane)
  • Bondapak
    ( waters)
  • C8 octyl column, C4 butyl column, CN Nitrile
    column
  • NH2 Amino column

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b
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Column packing
  • three forms
  • 1.Microporous support
  • 5-10 µm in d.m
  • 2. Pellicular
  • Porous 40 µm in d.m
  • 3.Bonded phase
  • S.P bonded onto an inert support

29
DETECTORS
  • 1. UV DETECTOR Based on UV light ab.
  • gt fixed WL detector (254nm)
  • gt variable WL detector (190-600nm)
  • 2. REFRACTIVE INDEX DETECTOR
  • Non specific / Universal detector
  • ? sensitivity specificity
  • 3. PHOTODIODE ARRAY DETECTOR (PDA)
  • similar to UV detector, non destructive
  • 190-600 nm for quantization identification
  • Spectra is 3D, Response vs time vs WL

30
Photodiode Array Detector
31
Flourimetric detector
  • Excitation emission WL can be selected
  • ? sensitive than UV
  • Disadvantage Some comp. are not fluorescent
  • Conductivity detector
  • based on electrical conductivity
  • Amperometric detector
  • Reduction / oxidation

32
RECORDERS INTEGRATORS
  • Recorders to record the responses
  • Integrators - data processing capabilities
  • ? record individual peaks with Rt, height,
    width
  • of peaks, peak area, of area..
  • Selection of solvent systems
  • Solvent compatibility with the detector
  • e.g.. Hexane, chloroform, ACN , Methanol
  • Most widely used solvent in HPLC is water
  • Millipore Milli-Q apparatus produce water

33
Selection of Column
  • Non polar moderately polar comp. ?
  • ADSORPTION CHRO.
  • Highly polar molecules by ? R.P Chro.
  • Acids Bases by ? Ion exchange Chro.

34
APPICATIONS OF HPLC
  • ? Pharmaceutical field
  • ? Chemical Petrochemical industry
  • ? Forensic
  • ? Biochemical separations
  • ? Food analysis
  • Qualitative analysis
  • Checking the purity of a compound

35
Quantitative Analysis
  • Direct comparison method
  • injecting the sample std. separately
    comparing their peak areas.
  • Area of the peak peak height x width of peak at
    half height
  • Calibration curve method
  • Multi component analysis
  • Isolation identification of drugs
  • Stability studies

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