A mixture of trans-resveratrol (left peak) and cis-resveratrol (right peak) were
ID: 1066598 • Letter: A
Question
A mixture of trans-resveratrol (left peak) and cis-resveratrol (right peak) were injected on an HPLC instrument with a C18 column in an 8-min run. The chromatogram is shown above. A 25:75 v/v mixture of water and methanol was the mobile phase. Which is more polar, trans-or cis-resveratrol? How can you tell? How will the retention times change if the mobile phase water is increased to 35%? How will the retention times change if the flow rate is increased from 1 mL/min to 1.5 mL/min? How will the resolution of the two peaks change if the flow rate is increased to 1.5 mL/min? Which term in the van Deemter equation describes this effect? The C18 column has bulky side groups in addition to the C18 groups. What is the benefit of these bulky side groups?Explanation / Answer
a) SInce C-18 is non-polar, more polar molecule (trans- resveratol) would elute first as it has low aafnity for C-18 and more affinity towars polar solvent (water, methanol mixture) while less polar molecule (cis- resveratol) would elute later as it has more affnity for non- polar C-18.
Trans molecule is more polar.
b) If mobile phase water is increased to 35 % that tends to make mobile phase more polar since OH bond is more polar than OC bond. Hence retention time would decrease/ sfift to left in chromatogram as resveratol has 3 OH bonds which have strong affinity for polar mobile phase.
c) If flow rate of mobile phase is increased, peaks are bound to get eluted faster (less retention time/ shift to left in chromatogram) as they would spend less time in column.
d) Vande meeter equation
HETP = A + B/u + C.u
where u is linear velocity
If linear velocity is increased from 1 to 1.5 ml/ min, then it will effect 2nd term (longitudnal diffusion) and HETP would be low giving good resolution.
Peaks will spend less time in column and give more sharper peaks which can be differentiated easily (higher resolution)
e) C18 molecule has bulky groups to provide shielding effect to water molecules and RP-HPLC (reversed phase high pressure liquid chromatophraphy) nature of column.
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