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Day 1. The two most widely used organic modifiers are acetonitrile and methanol, although acetonitrile is the more popular choice. Isopropanol 2-propanol can be employed because of its strong eluting properties, but is limited by its high viscosity which results in lower column efficiencies and higher backpressures. Both acetonitrile and methanol are less viscous than isopropanol. All three solvents are essentially UV transparent.
This is a crucial property for reversed phase chromatography since column elution is typically monitored using UV detectors. Acetonitrile is used almost exclusively when separating peptides. Most peptides only absorb at low wavelengths in the ultra-violet spectrum typically less than nm and acetonitrile provides much lower background absorbance than other common solvents at low wavelengths. Ion suppression: The retention of peptides and proteins in reversed phase chromatography can be modified by mobile phase pH since these particular solutes contain ionisable groups.
The degree of ionisation will depend on the pH of the mobile phase. The stability of silica-based reversed phase media dictates that the operating pH of the mobile phase should be below pH 7. The amino groups contained in peptides and proteins are charged below pH 7. The carboxylic acid groups, however, are neutralised as the pH is decreased. The mobile phase used in reversed phase chromatography is generally prepared with strong acids such as trifluoroacetic acid TFA or ortho-phosphoric acid.
These acids maintain a low pH environment and suppress the ionisation of the acidic groups in the solute molecules. Varying the concentration of strong acid components in the mobile phase can change the ionisation of the solutes and, therefore, their retention behaviour. The major benefit of ion suppression in reversed phase chromatography is the elimination of mixed mode retention effects due to ionisable silanol groups remaining on the silica gel surface.
The effect of mixed mode retention is increased retention times with significant peak broadening Figure 4. Reversed phase separations are most often performed at low pH values, generally between pH The low pH results in good solubility of the sample components and ion suppression, not only of acidic groups on the sample molecules, but also of residual silanol groups on the silica matrix. Acids such as trifluoroacetic acid, heptafluorobutyric acid and ortho-phosphoric acid in the concentration range of 0. Note that phosphate buffers are not volatile.
It is important to maintain the pH of the mobile phase in the range of 2.
Handbook of Pharmaceutical Analysis by HPLC, Volume 6 - 1st Edition
This is due to the fact that the siloxane linkage area cleaved below pH 2. Absorbance: An UV-visible detector is based on the principle of absorption of UV visible light from the effluent emerging out of the column and passed through a photocell placed in the radiation beam. UV detector is generally suitable for gradient elution work. The mobile phase used should not interfere in the peak pattern of the desired compound hence it should not absorb at the detection wavelength employed Selectivity is affected by the surface chemistry of the reversed phase medium, the nature and composition of the mobile phase, and the gradient shape Figure 5.
Both high column efficiency and good selectivity are important to overall resolution. However, changing the selectivity in a chromatographic experiment is easier than changing the efficiency. Selectivity can be changed by changing easily modified conditions like mobile phase composition or gradient shape.
Viscosity: Solvent of lowest possible viscosity should be used to minimize separation time. An added advantage of low viscosity is that high efficiency theoretical plate HETP values are usually lower than with solvents of higher viscosity, because mass transfer is faster. Viscosity should be less than 0. Temperature: Temperature can have a profound effect on reversed phase chromatography, especially for low molecular weight solutes such as short peptides and oligonucleotides.
The viscosity of the mobile phase used in reversed phase chromatography decreases with increasing column temperature. Since mass transport of solute between the mobile phase and the stationary phase is a diffusion-controlled process, decreasing solvent viscosity generally leads to more efficient mass transfer and, therefore, higher resolution. Increasing the temperature of a reversed phase column is particularly effective for low molecular weight solutes since they are suitably stable at the elevated temperatures 8.
However, among these the five dominant detectors used in LC analysis are the electrical conductivity detector, the fluorescence detector, the refractive index detector, mass spectrometry detector and the UV detector fixed and variable wavelength. The detector selected should be chosen depending upon some characteristic property of the analyte like UV absorbance, fluorescence, conductance, oxidation, reduction, etc.
Characteristics that are to be fulfilled by a detector to be used in HPLC determination are:. RP-HPLC is probably the most universal, most sensitive analytical procedure and is unique in that it easily copes with multi-component mixtures. While developing the analytical methods for pharmaceuticals by RP-HPLC, must have good practical understanding of chromatographic separation to know how it varies with the sample and with varying experimental conditions in order to achieve optimum separation.
To develop a HPLC method effectively, most of the effort should be spent in method development and optimization as this will improve the final method performance. Int J Pharm Sci Res. Article Information Sr No: 9. Download: Cited By: 1.
Authors: Sanjay Kumar D and D. Published: 01 December, This choice should be based on the following criteria: The unique requirements of the application, including scale and mobile phase conditions. The molecular weight, or size of the sample components. The hydrophobicities of the sample components. The class of sample components. The aim of sample preparation is a sample aliquot that, Is relatively free of interferences, Will not damage the column, and Is compatible with the intended HPLC method that is, the sample solvent will dissolve in the mobile phase without affecting sample retention or resolution 12 Sample preparation begins at the point of collection, extends to sample injection onto the HPLC column and encompasses the various operations summarized in table 1.
Option Comment 1. Sample collection Obtain representative sample using statistically valid processes 2. Sample storage and preservation Use appropriate inert, tightly sealed containers; be especially careful with volatile, unstable, or reactive materials; biological samples may require freezing.
Preeliminary sample processing Sample must be in a form for more efficient sample pretreatment e. Weighing or volumetric dilution Take necessary precautions for reactive, unstable, or biological materials; for dilution, use calibrated volumetric glasswares. Alternative sample processing methods Solvent replacement, desalting, evaporation, freeze drying, etc. Removal of particulates Filtration, solid-phase extraction, centrifugation. Sample extraction Different methods used for liquid samples and solid samples 8. Derivatization Used mainly to enhance analyte detection; sometimes used to improve separation.
Critical Parameters in Reversed Phase Chromatography: Classifying the sample: The first step in method development is to characterize the sample as regular or spherical. Chrom-Ed Book Series, ; High performance liquid chromatography [Internet].
HPLC Method Development for Pharmaceuticals, Volume 8
Available from: en. CBS Publishers and distributors, 7 th edition ; , Connors AK. A Text Book of Pharmaceutical Analysis. Comprehensive Analytical Chemistry. Elsevier; Amesham Biosciences: Reversed Phase Chromatography.
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