Please enable JavaScript.

Coggle requires JavaScript to display documents.

HPLC Method Development and Validation: Simultaneous Determination of…

- - - - Column (Stationary phase) - C18
        
        Octadecyl chains are hydrophobic and highly retains non-polar compounds (active ingredients)
        
        Denser packing increases surface area of compounds in mobile phase travels
    - - Diode-array UV detector
        
        to detect and identify analytes in the sample
        
        measuring the sample's absorption of light at different wavelengths, the analyte can be identified.
  - - - non-polar compounds in the sample will attach to the non-polar matric which then results in the slower movement of these compounds and polar sample in the compounds will move faster with the polar mobile phase
      - Increasing the non-polarity of organic solvents will produce the peaks faster
        
        Increasing the water content however slower down the peaks.
        
        Hence, the time retention increases and the peaks can be well separated.
        
        Like dissolves like
        
        When the analyzed compound is non-polar in nature, then in case of RP-HPLC, where the stationary phase is non-polar, strongly binds with that; but this should not be a irreversible bond. Therefore, when another stronger non-polar compound approaches to that compound (here, acetonitrile) then the reversible bond with stationary phase gets loosen and the compound elutes with the non-polar mobile phase.
    - - Excellent Elution Gradient & Good Separation
        The mobile phase was a mixture of
        38% methanol, 62% of 80 mM
        phosphate buffer : (38:62, v/v)
        
        Roles of Buffers in Reverse HPLC
        
        Precise control of pH using buffers is crucial for the reverse phase separations of polar and ionizable compounds which to guarantee the reproducible retention times and freedom from tailing
        
        pH value of 3.0 was selected because of optimum
        resolution
        
        Factors governing choice of buffers
        
        Compatibility with mobile phase
        
        Precipitate must be avoided to ensure none irreparable damage to the pump and the chromatography column.
        
        Buffer concentration
        
        Faster elution of polar molecules can be achieved by increasing the buffer concentration which can be applied to resolve co-eluting peaks. However, higher concentration tends to increase the viscosity in high column back pressure.
        
        UV cut off values
        
        Choice based on the transparency at the wavelength detection which commonly lie from about 230nm to below 200nm.
- - - - Preparation of Calibration curves
        
        Diluting stock solution in mobile phase with compounds at concentrations of:
        
        5-250 µg.mL-1 for pyrilamine maleate and diphenhydramine HCl
        
        10-250 µg.mL-1 for dextromethorphan HBr
        
        2.5-250 µg.mL-1 for chlorpheniramine maleate and triprolidine HCI
        
        0.5–250 µg.mL-1 for pseudoephdrine HCI and pheniramine maleate
        
        1–250 µg.mL-1 for guaifenesin
        
        0.2-250 µg.mL-1 for acetaminophen
  - - - Stock standard solutions of pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr and diphenhydramine HCI were prepared in ultrapure water
- - - - Significance
        
        Chromatographic selectivity being able to be manipulated by the changes in mobile phase characteristic can ease the experimental
        
        Stability of the sorbent materials under a wide range of mobile phase conditions will result to the excellent reproducibility of repetitive separations that can be carried over a long period of time
        
        Wide range of chromatographic conditions for closely related and distinct molecules can produce the excellent resolution
        
        High recoveries which enable the high productivity
      - Involves the separation of molecules on the basis of hydrophobicity
        
        Aqueous buffers are crucial during the application of solute mixture to the sorbent and adding the organic solvent to the mobile phase will elute.
        
        separation is depending on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands that attached to the sorbent (stationary phase)