Analytical chemistry: please anser part 1&2 as well as 3 Bile acids have been is
ID: 495095 • Letter: A
Question
Analytical chemistry:
please anser part 1&2 as well as 3
Bile acids have been isolated from plasma to assess the hepatotoxic effect of a drug on patients infected with Human Immunodeficiency Virus (again). They have been analyzed by HPLC with the conditions described below: Column with a silica-bonded C18 phase, 18 uM, 2.1 mm times 10 cm Solvents: Water (A) and acetonitrile (B) acidified with formic acid (0.1%) Mobile phase gradient: 10% B during 2 min, then 10% to 95% B from 2 to 12 min. 9S% B from 12 to 14 min and 10% B from 14 to 20 min. Explain each step of the gradient elution and their importance. Would have been a better idea to analy2e this extract in isocratic mode? Please explain why.Explanation / Answer
Steady changes of the mobile phase composition during the chromatographic run is called gradient elution. It may be considered as an analogy to the temperature programming in gas chromatography.
The main purpose of gradient elution is to move strongly retained components of the mixture faster, but having the least retained component well resolved.
Starting with the low content of the organic component in the eluent we allow the least retained components to be separated. Strongly retained components will sit on the adsorbent surface on the top of the column, or will move very slowly.
When we start to increase an amount of organic component in the eluent (acetonitrile) then strongly retained components will move faster and faster, because of the steady increase of the competition for the adsorption sites.
Gradient elution also increase quasi-efficiency of the column. In the isocratic elution, the longer a component is retained, the wider its peak. In gradient elution, especially with the smooth gradient shape without a flat regions, the tail of the peak is always under the influence of the stronger mobile phase when compared to the peak front. Thus, molecules on the tail of the chromatographic zone (peak) will move faster. This will tend to compress zone and narrow the resultant peak.
Performance of the gradient elution is strongly dependent on the instrumentation. Two main points the chromatographer needs to know about his instrument:
How large the volume between the component mixing point and column inlet is. For the low-pressure gradient systems this volume usually correspond to the pump volume, and about 2 - 3 ml.
How well does the system mix eluent components. If the system does not mix components well then it will supply for the certain time one component then another and so on. Chromatographic performance of such system will be very low especially for the least retained components.
2)
In column chromatography, usually the target molecule is bound while contaminants pass through the column, or alternatively, impurities can be bound to the column while the target passes through.
In gel filtration (GF), which separates sample components based on size, the components travel through the column at different speeds, giving different elution positions.
The composition of the eluent is unchanged during the entire purification (binding and elution). The target molecule passes through the column slower or fast than impurities. This type of elution is used in GF.
The eluent composition is changed stepwise, at one or several occasions. Several substances may be eluted in each step.
Gradient and stepwise elution can be combined by doing part of the elution in gradient mode and part of it in stepwise mode. This type of elution is not very common, but can be performed for purification methods that have been optimized.
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