Question

As with extraction, there are different types of chromatographic separations…one type that we haven’t discussed yet is HPLC. Let’s say that you’re trying to perform an HPLC analysis of a mixture of sweeteners found in soft drinks; the compounds present in this mixture are listed below. What order would these compounds elute (come off of the HPLC column)? Please explain your choice. Note: I’ll encourage you to first do a little background reading on HPLC (and reverse-phase HPLC in particular) before you answer this question. The mixture of sweeteners contained: saccharin sodium, aspartame, sucralose

Answer 1

Hplc is used to identify these analytes or sugars in this case and also you can know their individual concentrations as well. Reverse hplc is a mode of separating these sugars. Now there are two phases in the column called stationary and mobile phases and compounds are separated based on their polarity. Solvents are injected into the column that the beverage sample is injected into the column. They travel through the high pressure column where all the non polar molecules are dissolved in stationary phase and the polar molecules in mobile phase. So the molecules in the polar phase elute first and the non polar later, to make it better molecules are coated with non polar substances to make them soluble in stationary phase. Sometimes analytes can be ionic and so the mobile phase is usually water buffered with some weak acid in order to retain them in a better way. Therefore this whole described process above sums up to reverse hplc where we separate compounds based on their polarity. However to analyse and quantify the compounds we have a detector ( most of the times it is UV-Vis) and a computer of course. The detector is set with some benchmark to analyse them as they pass through the exit.
The elution order could be (1)Sodium saccharin (2)Sucralose (3)Aspartame

Reason for this elution order : So after reading above info it should be clear that we use an
aqueous (water & buffered with a weak acid) mobile phase which is polar. Also you should
know by now that stationary phase is hydrophobic (water-fearing) and mobile phase is hydrophilic.
So if there are hydrophobic molecules in mobile phase, they tend to get adsorbed into hydrophobic
stationary phase. Hydrophilic analytes are more soluble in hydrophilic phase (because like attracts like)
and the most hydrophilic molecule (high solubility value) in the mobile phase will pass through
the column and is eluted first.
Aspartame is least soluble in water among the three analytes and saccharin sodium is the most soluble
in water. This is how I predicted the order