Enzyme

Enzyme

1. INTRODUCTION: HPLC is a technique for separating components in a liquid mixture. Injection of a liquid sample into a stream of solvent (mobile phase) passing through a column filled with a separating medium (stationary phase).

2. components or Instrumentation of HPLC

   • Pressure gauge
   • Solvent storage bottle
   • De-gassing of solvents
   • Pumps
     • parameters for modern pumps or requirment for pumps
       • the generation of 6000 psi of pressure
       • Flow rate range: 0.01 to 10 ml/min
       • pumps must be able to operate with only one reservoir or many reservoirs, each with a different solvent.
       • Pressure pulsations : less than1 %
     • Types of Pumps
       • Displacement Pumps
         • It produces a flow that tends to be independent of viscosity & back pressure.
         • The contents of the reservoir are pumped into the syringe-like chambers through a plunger that is triggered by a screw.
         • a limited solvent capacity
       • PNEUMATIC PUMPS
         • Direct pressure pump
         • Amplifier pump
       • Reciprocating Piston Pump
         • 90% of HPLC’s, small internal volume, pulsed flow
         • Single piston reciprocati ng pump
         • Double piston reciprocating pump
         • Reciprocating diaphragm pump
   • Gradient controller and mixing unit
   • Column
     • Mobile Phases in LC
       • One of the factors that influences LC separations is the type and content of the mobile phase (eluent).
       • Desirable properties
         • Purity
         • Detector compatibility
         • Solubility of the sample
         • Low viscosity
       • LC modes have different mobile phases.
         • Most nonpolar phase solvents are in the normal phase
         • reversed-phase eluents often include water mixed with polar components a solvent composed of an organic base such as acetonitrile
     • Stationary Phase in LC
       • A crucial component of separation is the stationary phase, which has characteristics that affect the separation.
       • a distribution of particle sizes should be as narrow as feasible, and most commonly should be within 10% of the mean
       • particle size: 3 to 10 micrometers
       • Surface area: 50 to 250 square meters per gram
       • Most Popular Particle: Silica
       • PACKING Particle
         • Most Popular Particle: Silica
         • Larger particles 7 or 10 mm have been used with less efficient
         • Standard - 3 mm or 5 mm particles with good efficiency
       • Monolithic
         • Limitations of packed particle
           • Back pressure gets really high as the particle gets smaller – e.g. with UHPLC
         • A continuous porous stationary phase or SP support
         • Polymerization reactions that yield voids
         • Allow higher mobile phase flow rates with lower backpressure
         • Stable over a wide pH range
         • Particle Size Evolution
           • 1980’s to present day 3.5 - 5µm spherical micro-porous 1500-4000 psi (106.4-283.7 bar) 50,000 - 80,000 plates/meter 3.9 x 300mm
           • Early 1970’s 10µm Irregular micro-porous 1000-2500 psi (71-177 bar) 25,000 plates/meter 3.9 x 300mm
   • Detector
     • IDEAL DETECTOR PROPERTIES
       • High Sensitivity
       • Universality or predictable specificity
       • little distortion across a broad frequency range
       • known as reliable and easy to use
       • A single detector does not meet all of these requirements
     • UV-VISIBLE DETECTOR
       • UV visible detectors are extensively employed due to their ability to detect a large variety of chemicals.
       • classed as fixed or variable wavelength detectors
       • UV-visible spectrophotometers are used to offer dispersion of light
       • as the source for fixed wavelength detectors by allowing a certain wavelength to pass through.
       • For dispersion of wavelengths, diffraction gratings are often employed
     • FLUORESCENCE Detector
       • fluorescent as seen at a 90° angle from the excitation beam (add fluoro tag)
       • sample is stimulated by radiation emitted from the source (Hg, Xe, or laser)
       • Some substances give out fluorescence radiation.
       • More responsive and particular.
     • REFRACTIVE INDEX
       • changes in DENSITY when analytes comesthrough cell
       • sensitive to turbulence, temp, solvent changes
       • used w isocratic systems only
     • Diode Array Detectors
       • Diode array detectors can acquire all UV-Visible wavelengths at once.
       • Sensitivity (multiplex), Speed
       • High Resolution
     • EVAPORATIVE LIGHT SCATTERING DETECTORS (ELSD)
       • effluent nebulised into fine mist -- through laser & scattered radiation detected at right angle to laser
       • volatile mob. phase.
       • effluent nebulised into fine mist -- through laser & scattered radiation detected at right angle to laser
   • Recorder
   • Injector
     • Septum Injector
       • used for injecting the sample through a rubber septum.
       • injectors cannot be commonly used , since the septum has to withstand high pressures
     • Stop Flow Injector
       • In this type the flow of mobile phase is stopped for a while & the sample is injected through a valve
     • Rheodyne Injector
       • most popular injector and is widely used
       • a fixed volume of loop, for holding sample until its injected into the column
       • The injector is positioned just before the inlet of the column

3. Type of HPLC

   • Based on the saparetion
     • Normal Phase
       • utilizes a non-polar mobile phase and a polar stationary phase
       • The most frequent mobile phases are ethyl acetate + n-hexane, dichloromethane + n-hexane, isopropanol + n-hexane, and so on.
     • Reverse Phase
       • It employs a polar mobile phase and a non-polar stationary phase.
       • example: C18 column , and polar solvents such as water, methanol or acetonitrile
       • Retention Mechanisms in LC
         • Ionic interactions in ion-exchange
         • Hydrophobic in Reverse Phase
         • Dipole-dipole (polar) in normal phase
   • Based on the principle
     • Absorbation
       • Adsorption Chromatography is the process of utilizing analytical techniques to separate a chemical mixture based on the interaction of the adsorbate with the adsorbent.
       • When a gas or liquid mixture passes over an adsorbent bed, different components adsorb at different rates, separating the mixture.
       • Adsorbent - An adsorbent is a porous substance with a high surface area that has the ability to adsorb chemicals on its surface via intermolecular forces.
     • Ion-exchange
       • Sulfonate (strong acid) and carboxylic acid gp are active sites for cation exchange (weak)
       • injection of amines and alcohol into a sulfonate-based cationic resin that has been equilibrated with NaOAc
       • silica/zirconium/organic polymer as a foundation material
       • Strongly basic tertiary amine gps / weakly basic primary amine groups are anionic exchangers.
     • Ion pair
       • org salt w large org counter ion
       • added in small conc
       • counter ion pairs w analyte ion & this pair partition into stat. phase.
     • Gel permeation
       • packings (~10 ångströms) polymer particles with uniform holes is called a silica-polymer network
       • Molecular mass sp.
       • Polymers can expand or shrink depending on the solvent.
       • See also weight average molecular weight (Mw) (molar mass distribution), size average molecular weight (Mz), and viscosity molecular weight (Mv).
       • GPC can tell you both the Ð and Mv, and with further data, the Mn, Mw, and Mz may be calculated.
   • Based on the Affinity
     • HIC
     • Affinity
     • Chrial
       • Ceramic Mg silicate cyclodextrin Some H bonding works on π acceptor/ π donor interactions.
       • A chiral molecule will need to be in either the mob or stat phase for enantiomers to be made.
       • While a chiral resolving agent is employed with one of the isomers, complexation occurs, leading to separation.
   • Based on the Elution
     • Isocratic
       • The eluent composition remains consistent throughout the study, which is why the eluent composition doesn't change.
     • Gradient
       • As the eluent composition (and strength) changes over the run, so does the eluent's emulsifying and emulsification strength.

4. Disadvantages

   • An expensive, purity-free solvent is used.
   • moveable parts, connections
   • one must be a skilled individual
   • A SUSTAINABLE LEVEL OF PERFORMANCE IS Achieved by making a regular, ongoing effort.
   • The cost of chromatography apparatus is very high

5. Advantages

   • requires a small sample size
   • has the capability to do varying analyses
   • the data is trustworthy
   • OPTIMIZED DETECTION
   • Separate based on size, charge, polarity, and solubility
   • a large range of substances: inorganic, metallic, ionic, polymeric, organic, and so forth.
   • detectors in many different ranges and attempt to retrieve samples

6. HPLC vs UPLC

   • Particle size
     • UPLC 1.7µm particles
     • HPLC 4µm particles
   • Column
     • AQUITY UPLC BEH C18,50 ×2.1mm
     • XTerra,C18,50 × 4.6mm (HPLC)
   • Flow rate
     • UPLC 0.6 ml per min
     • HPLC 3.0 ml per min
   • Injection volume
     • UPLC 3 µl partial loop fill or 5 µl full loop fill
     • HPLC 20 µl
   • Total run time
     • HPLC 10 min
     • UPLC 1.5 min
   • Theoretical Plate count
     • UPLC 7500
     • HPLC 2000