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A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic(meaning constant composition). The word was coined by Csaba Horvath who was one of the pioneers of HPLC.[citation needed],
The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution.[3] One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column. In reverse-phase chromatography, solvent Ais often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.
In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.
Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times - all according to the desire for optimum separation in minimum time.
In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change - that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.[citation needed]
The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.
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ProfessorIn chromatography, isocratic elution involves using a constant mobile phase composition throughout the separation process, while gradient elution involves changing the mobile phase composition over time. Isocratic elution is simpler but may have limitations in resolving complex mixtures, whereas gradient elution offers more flexibility and improved separation of components with different polarities.
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Why retention time vary during isocratic analysis?
Retention time can vary during isocratic analysis due to changes in experimental conditions such as flow rate, temperature, or column stability. Retention time is influenced by the interactions between the analyte, stationary phase, and mobile phase, which can fluctuate during the analysis leading to variations in retention time.
What is the difference in concentrations of a substance between two areas called?
This is called a concentration gradient. It represents the difference in the concentrations of a substance between two regions, with molecules naturally moving from high to low concentration areas to reach equilibrium.
What is the difference between concentration gradient of molecule and ions?
A concentration gradient of molecules refers to the difference in the concentration of a specific molecule across a distance or region, which drives passive diffusion. In contrast, a concentration gradient of ions specifically refers to the variance in the concentration of charged particles (ions) across a space, influencing cellular processes like ion channel transport.
How does the concentration gradient affect osmosis?
The concentration gradient in osmosis refers to the difference in solute concentration between two solutions separated by a semi-permeable membrane. Water will move from an area of low solute concentration to an area of high solute concentration in an attempt to equalize the concentration on both sides of the membrane. The steeper the concentration gradient, the faster the rate of osmosis.
Does the steepness of con centration gradient affect the rate of difussion?
Yes, the steeper the concentration gradient, the faster the rate of diffusion. This is because there is a greater difference in concentration between two regions, driving molecules to move from areas of high concentration to areas of low concentration more rapidly.
