HPLC

Question 1: What should we do first before using the HPLC instrument?

✓ Prepare the sample by crushing the tablet and dissolving it in solvent
– Mix the mobile phase with the stationary phase
– Insert the tablet into the HPLC instrument
– Perform UV detector analysis

Question 2: Great job! You have crushed the tablet using the mortar and dissolved it. Now that you
have created a sample solution, what should we do next?
✓ Filter it through a 0.5 µm filter
– Measure using a spectrophotometer
– Boil the solution
– Inject it into the HPLC instrument

Question 3: Before we start measuring our sample, we need to ... so that we will be able to
determine our sample's concentration.
✓ Create a standard curve
– Perform a plate count
– Perform a system suitability test
– Adjust the flow rate

Question 4: Let's have a look at these chromatograms. The number written next to the peak is the
area under the curve. It is proportional to the sample's ...?
✓ Concentration
– Ionic charge
– Polarity
– Retention time

Question 5: Which analysis can we use to identify a sample concentration based on the area under
the curve of a specific peak?
✓ Use the linear regression analysis
– Use the differential analysis
– Use the multiple regression analysis
– Use the logarithmic analysis

Question 6: Using the regression analysis formula y = mx + y0, find out the value of m and y0. Scroll
down to see the options

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 ✓ m = 21.5, y0 = 2.2
– m = 18.4, y0 = 3.2
– m = 24.5, y0 = 1.5
– m = 30.1, y0 = 5.4

Question 7: What is the retention time for the metformin hydrochloride?

Click the "View Image" button to see the chromatogram.

✓ 5 minutes
– 16 minutes
– 10 minutes
– 8 minutes

Question 8: Which molecule emerge last from the column? Scroll down to see the options

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 ✓ Magnesium stearate
– All of them together
– Povidone
– Metformin hydrochloride

Question 9: Which molecule has the highest lipophilicity? Scroll down to see the options

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 ✓ Magnesium stearate
– All of them together
– Povidone
– Metformin hydrochloride

Question 10: The measurement of the time the sample component resides in the stationary phase
relative to the time it resides in the mobile phase is called?
✓ Retention factor
– Plate number
– Resolution
– Selectivity

Question 11: If you would like to change the mobile phase or stationary phase, what do you need to
do first before you can start measuring?
✓ Wait until the pressure is constant again
– Re-start only the UV detector
– Do several injections using water
– Re-start the whole machine

Question 12: The key of a successful HPLC procedure is good sample separation which is
represented by a clean peak for each individual compound. What should we do to ensure a good
sample separation?
✓ Use a column and a mobile phase with different polarity
– All of those
– Use a column and a mobile phase with the same polarity
– Use a lower temperature higher than the mobile phase boiling point

Question 13: What can we do to separate metformin hydrochloride (non-polar) and magnesium
stearate (polar)?

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 ✓ either of these
– Perform HPLC using C-18 and acetonitrile
– Perform normal phase HPLC
– Perform reverse phase HPLC

Question 14: Which of the following is considered a good peak?

✓A
–D
–C
–B

Question 15: Running the separation at higher temperatures decreases the ... resulting in lower
backpressure.
✓ Mobile phase viscosity
– Mobile phase retention
– Mobile phase selectivity
– Mobile phase sensitivity

Question 16: We want to elute metformin hydrochloride first before eluting the other drug
components. Can you suggest the correct HPLC parameters (column, mobile phase) to achieve
this goal?
✓ C-18, methanol or acetonitrile
– Silica, methanol or acetonitrile
– Silica, hexane
– C-18, hexane

Question 17: Which is the best mobile phase?

✓ C-18, acetonitrile, higher temperature
– C-18, acetonitrile, lower temperature
– C-18, methanol, lower temperature
– C-18, methanol, higher temperature

Question 18: What should we do if we want to have one solid peak instead of three peaks?
✓ We can use silica as stationary phase and methanol as mobile phase
– We can use C-18 as stationary phase and acetonitrile as mobile phase
– We can use C-18 as stationary phase and methanol as mobile phase
– We can use silica as stationary phase and hexane as mobile phase

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Question 19: Based on your investigation, how can we elute magnesium stearate first, instead of
metformin hydrochloride?
✓ We can use silica as stationary phase and hexane as mobile phase
– We can use C-18 as stationary phase and hexane as mobile phase
– We can use C-18 as stationary phase and methanol as mobile phase
– We can use silica as stationary phase and acetonitrile as mobile phase

Question 20: What will happen if we use a nonpolar substance for both the stationary and the
mobile phase?
✓ There is no distinct difference in the retention time between the three substances
– Povidone will elute first
– Metformin hydrochloride (non-polar) will elute first
– Magnesium stearate (polar) will elute first

Question 21: What is the difference between the 2 chromatograms? Scroll down to see the options

✓ The first peak is lower in the second chromatogram

– There are more peaks in the second chromatogram
– The peaks are delayed in the second chromatogram
– The first peak is higher in the second chromatogram

Question 22: What would be the explanation of the lower peak in the sample that has been stored
under extreme condition?
✓ It has a lower concentration
– It has higher lipophilicity
– It has higher polarity
– It has lower lipophilicity

Question 23: To calculate the concentration you will need the standard curve you made earlier and
the AU values.
In order to get the AU value, a computer software is used to get the area under the curve, or the
integral. Often one must select the boundary of the peak manually.

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What is the boundary for the first peak (metformin)?
✓ Between 2 and 6 minutes
– Between 7 and 15 minutes
– Between 0 and 6 minutes
– Between 3 and 5 minutes

Question 24: The area under the curve for the normal conditions sample is 1513.56.
The area under the curve for the extreme conditions sample is 1465.55.

Using the standard curve regression data, the concentration of metformin hydrochloride in normal
conditions is ..... grams per liter and in extreme conditions is ..... grams per liter
✓ 70.3 and 68.1
– 57.3 and 54.2
– 89.6 and 87.3
– 45.6 and 56.3

Question 25: You have just calculated the concentration of the two samples. Now calculate the
original amount. The original amount of metformin hydrochloride in normal condition tablet is ..... mg
and in extreme conditions tablet is ..... mg. Remember that the samples came from the tablets
diluted in 10 mL of liquid.
✓ 703 and 681
– 5.73 and 5.42
– 896 and 873
– 45.6 and 56.3

Question 26: What is the decline in concentration compared to the normal conditions sample?
✓ 3.1%
– 7.2%
– 5.6%
– 2.4%

Question 27: In an HPLC system, the beads inside the column are also called the ... phase?
✓ Stationary
– Mobile
– Column
– Solid

Question 28: What is making the mobile phase pass through the column?
✓ Pump
– Electricity
– Gravity
– Capillary force

Question 29: What are the most common mobile phases in reversed phase HPLC?
✓ Methanol and acetonitrile
– Ether and acetone
– Methylenechloride and chloroform
– Isopropanol and toluene

Question 30: How should you prepare a calibration curve?

✓ By injecting a known amount of analyte and determining the peak area
– By setting the detector response to zero

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 – By accurately measuring the flow rate
– By using a RI-detector and Sucrose as analyte

Question 31: In reversed-phase HPLC ...

✓ A hydrophobic stationary phase is combined with a polar mobile phase
– A hydrophilic stationary phase is combined with a polar mobile phase
– A hydrophilic stationary phase is combined with a non-polar mobile phase
– A hydrophobic stationary phase is combined with a non-polar mobile phase

Question 32: In the reversed-phase HPLC, we use nonpolar stationary phase. Which of the
following is NOT commonly used as a stationary phase?
✓ Silica
– Polar end-capped
– Phenyl
– Octadecyl C-18

Question 33: Which of the following mobile phase is not commonly used in reversed-phase HPLC?
✓ Hexane
– Water
– Methanol
– Acetonitrile

Question 34: The ratio of how long a sample takes to pass the stationary phase compared to how
long it would take to pass through the column with the velocity of the mobile phase, is called?
✓ Retention factor (k)
– Selectivity (a)
– Retention time (tR)
– Resolution (Rs)

Question 35: In reversed-phase HPLC, you injected a sample containing 2 compounds. Compound
A is more lipophilic than compound B. Which compound will elute first from the column?
✓ Compound B
– You cannot answer this without first conducting the experiment
– Both of them at the same time
– Compound A

Question 36: Depending on the interaction, different compounds will travel through the column at
different speeds and elute at different times. This specific time is called?
✓ Retention time
– Peak time
– Elution time
– Base time

Question 37: The ratio of the retention factors of both peaks is called?
✓ Selectivity
– Efficiency
– Resolution
– Sensitivity

Question 38: The column efficiency can be measured by calculating the theoretical plate number by
comparing ... to ...
✓ Retention time (tR); plate width at baseline (W)
– Concentration in stationary phase (CS); concentration in mobile phase (CM)
– Signal strength; time

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 – Retention factor 1 (k1); retention factor 2 (k2)

Question 39: The theoretical plate number can be increased by ...?

✓ Decreasing the beads size
– Decreasing the flow rate
– Increasing the flow rate
– Increasing the beads size

Question 40: When using a smaller beads size, the resolution is improved, however ...?
✓ High pressure is reached
– It can only be used with lipophilic compounds
– Higher sample concentration is needed
– Longer run time is needed

Question 41: The column is also called the stationary phase and it is where the ... takes place.
✓ Sample separation
– Sample excretion
– Sample injection
– Sample preparation

Question 42: The oven regulator is used to set the temperature for the separation. One of the
advantages of using a higher temperature is?
✓ Enabling a faster run time
– Enabling stability of biological compounds, such as enzymes or proteins
– Increases the chiral recognition
– Able to analyze more sample at the same time

Question 43: What are the two ways of injecting the sample into an HPLC instrument?
✓ Manual or automated
– Manual or continuous
– Mixed or not mixed
– Single or multiple

Question 44: In reversed-phase HPLC, the mobile phase is ...

✓ Polar
– Large
– Ionic
– Non-polar

Question 45: What is the function of the pump?

✓ To create a high pressure in the mobile phase and sample
– To create low pressure in the mobile phase and sample
– To pump the waste out
– To pump the solid phase into the column

Question 46: Compounds that are eluted from the column will pass through the detector recording
the ... and the ..., which corresponds to the x and y axis in a chromatogram.
✓ Retention time; signal strength
– Sensitivity; gradient
– Flow rate; resolution
– Plate number; efficiency

Question 47: Lipophilic molecules are attracted to other lipophilic molecules. The weak interaction
between two non-polar (lipophilic) molecules, as seen in the figure is called?

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✓ London dispersion force
– Keesom force
– Ionic bonding
– Hydrogen bonding

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