HPLC-UV

Non-interactive video download link (mp4).

The script that has been used to create the video shown above can be downloaded as excel script file.

• In liquid chromatography, the samples move through a column, this column is the [stationary phase].

Feedback if incorrect: You have not labelled the column correctly. In chromatography there is a phase that moves and a phase that is stationary. Does the column ‘move’?

• The liquid that passes through the column is the [mobile phase].

Feedback if incorrect: You have not labelled the liquid correctly. In chromatography there is a phase that moves and a phase that is stationary. Does the liquid ‘move’?

• In our case, the [answer_polar] is polar, and the [answer_apolar] is apolar.

Feedback if incorrect: You have not labelled the liquid correctly. In ‘normal’ liquid chromatography the column material is more polar than the mobile phase.

• In reverse phase chromatography it is the other way around. Check the column material and mobile phase in the list above. Analytes with a higher affinity with the [answer_higher] than the [answer_than_the] move through the column faster and arrive at the detector first.

Feedback if incorrect: Your answer regarding how affinity of the analytes with the phases determines what analyte runs off the column first is incorrect. Think of it like this: if the analyte rather ‘likes’ the stationary phase, it would ‘rather’ stay in the column then move along with the mobile phase.

Feedback if all answers Correct: The stationary phase, the column, should (slightly) repel the analytes to make them move through the column. Analytes that have higher affinity with the mobile phase than the stationary phase will run off the column and reach the detector first.

Each part of the HPLC-system serves its own purpose. Indicate what by selecting the right parts in the list below:

• This is what transports the sample through the column: [a] Mobile phase
  Feedback if Incorrect. The sample is brought through the system by the mobile phase.
• This is what releases your sample to be sent to the column. [b] Trigger
  Feedback if Incorrect. The sample is brought into the system by injection using a syringe.
• This ensures the right mobile phase flowrate. [c] Pump
  Feedback if Incorrect. What part of the system ensures the speed at which the sample flows through the system?
• This is where analyte separation takes place. [d] Column
  Feedback if Incorrect. The purpose of the liquid chromatography is to separate the analytes so that they can be detected separately. The separation takes place in the column (as long as the right conditions are set).
• This is the device that measures the analytes. [e] Detector
  Feedback if Incorrect. Your final answer is wrong.
  The sample is injected into the injection loop. Then the system switches and the mobile phase brings the sample through the column where separation of the analytes takes place.

Feedback if correct: The sample is injected into the injection loop.
Then the system switches and the mobile phase brings the sample through the column where separation of the analytes takes place.
At the end of the column, a detector registers the samples.

What mobile phase composition would you like to use? And what percentage of organic modifier would you like to add?

[a____] % [20 mM acetate buffer pH 4.5 / 0.1% formic acid pH 2.7 / 20 mM phosphate buffer pH 7.0/ ammonium buffer pH 9.0]: [b____] % methanol

• Feedback if a + b = NOT 100:
  These percentages don’t add up to 100%! That can’t be right.
• Feedback if b < 5:
  This percentage of methanol is very low! Are you certain this would lead to the desired separation based on your simulations using the HPLC simulator?

• Feedback if b > 75:
  This percentage of methanol is quite high! Are you certain this would lead to the desired separation based on your simulations using the HPLC simulator?

Feedback if correct:
Your desired mobile phase composition (based on the HPLC simulator) is [percentage_methanol]% methanol : [percentage_aqueous]% [chosen mobile phase]

In which order do you expect the parabens to elute? Place the first one on top.

Correct answer:

1. 1 Methyl paraben
2. 2 Ethyl paraben
3. 3 Propyl paraben
4. 4 Butyl paraben

• Feedback if correct
  Molecules with the highest affinity to the stationary phase will run off the column last. As you have determined earlier, methyl paraben has the highest water solubility, from which you can conclude it has the least affinity to the apolar stationary phase.
  This in contrast to the least water soluble paraben butyl paraben, that probably runs off the column last.

• Feedback if incorrect
  Incorrect. Consider the properties of the HPLC column and the properties of the analytes.

What requirements should the analytes have to make them suitable for analysis with liquid chromatography equipped with a UV-detector?

1. They should emit UV light
   Correct = no

You chose a wrong option. The LC system is equipped with a UV detector which is similar to a spectrophotometer. UV light is emitted through the sample and absorption at UV-wavelength is recorded.

1. They should absorb UV light
   Correct = Yes

1. They should be fluorescent
   Correct = no

You chose a wrong option. The LC system is equipped with a UV detector which is similar to a spectrophotometer. The analytes might be fluorscent but here the absorption is measured and not the emission of light.

1. They should have affinity with the solid phase of the column
   Correct = Yes

1. They should be able to dissolve
   Correct = Yes

1. They should be stable at solvent boiling point
   Correct = no

You chose a wrong option. In liquid chromatography, the samples are not evaporated, hence the term liquid. The analyzed sample will not reach boiling point.

1. They should be liquid at room temperature
   Correct = no

This is not necessarily the case. The liquid in liquid chromatography refers to the mobile phase. As long as the analytes can be dissolved, their phase in pure form at room temperature (solid, liquid or gas) is not really relevant.

Feedback if all correct options chosen:

You want separation of the analytes to take place, so that you can detect them separately. Here a UV-detector is suitable as all analytes absorb light in the UV-spectrum.

The needle used for injection of the sample into the chromatography system needs to be rinsed to remove any impurities or molecules that might interfere with the separation.

What are the properties that would make a solvent the most suitable for this?

1. Cheap
   Correct = yes

1. Miscible with water
   Correct = yes

1. Immiscible with water
   Correct = yes

1. Hydrophobic
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. What if there are water molecules present in the needle? Wouldn’t you want to remove those too? One or more of the properties you have selected now is in conflict with this.

1. Hydrophilic
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. What if there are water molecules present in the needle? Wouldn’t you want to remove those too? One or more of the properties you have selected now is in conflict with this.

1. A strong acid
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. Any acid or base properties are not relevant here, as long as you keep the purpose in mind.

1. A strong base
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. Any acid or base properties are not relevant here, as long as you keep the purpose in mind.

1. Low boiling point (<70°C)
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. The actual boiling point of the solvent used is not really relevant, as long as it is a liquid at room temperature, right?

1. High boiling point (>70°C)
   Correct = yes

1. Aqueous
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. The actual boiling point of the solvent used is not really relevant, as long as it is a liquid at room temperature, right?

1. Organic
   Correct = no

The purpose is to remove any impurities or molecules that might interfere with the separation. If your solvent is aqueous, you might not remove any hydrophobic impurities. Therefore an aqueous solution would not be suitable.

1. Safe to use
   Correct = yes

Feedback if all correct options:

The purpose is to wash any impurity away, so ideally both polar and non-polar compounds dissolve in the solvent used. Also you want it to be cheap and safe to not waste any money or take unnecessary risks.

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