CHEM2310 Fundamentals of Analytical Chemistry

Assignment 1 Model Answer

Please submit hand-written, scanned copy to Canvas by 23:59 of 8th October 2024

(Q1 = 5, Q2 = 1, Q3 = 2, Q4 = 2; total = 10)

1. A). (0.5 x 2 = 1) What is reversed-phase liquid chromatography (RP-LC)? Name two of the
commonly used stationary phases and mobile phases for RP-LC.
Reversed-phase liquid chromatography using polar starting mobile phase and non-polar
stationary phase, mainly use to separate low to moderate polarity compounds.
Stationary phase: Octadecyl (C18); Octyl (C8); Phenyl
Mobile phase: Water; MeOH; ACN

B). (0.5) Explain why caffeine has a stronger retention than theobromine in RP-LC.
Caffeine has an extra non-polar methyl group than theobromine, which allows strong
non-polar interactions with the non-polar stationary phase in RP-LC.

C). (0.5) Ceteris paribus, how will the chromatogram change if a larger proportion of polar
solvent is used as the mobile phase?
The retention time of both compounds will be longer as the increased polarity of mobile
phase strengthen the interactions between analytes and non-polar stationary phase, also
weaken the elution strength of the mobile phase.

D). (0.5 x 2 = 1) Ceteris paribus, how will the analyte (detector) response (i. area under
the curve; ii. peak height) change if the internal diameter (i.d.) of HPLC column used was
changed from 4 mm to 2 mm?
The area under curve remains similar while peak height may increase for the 2 mm i.d.
column.
E). (0.5) The optimal flow rate used in the 4 mm internal diameter (i.d.) column was 1
mL/min, to maintain the same height equivalent to a theoretical plate (HETP), what
should be the flow rate used in the analysis for a 2 mm i.d. column?
The flow rate is inversely proportional to the square of the diameter ratio. So, to maintain
the same height equivalent to a theoretical plate (HETP), the flow rate should change
from 1 mL/min to 0.25 mL/min.

F). (0.5) Ceteris paribus, how will the chromatogram change if the packing material used
was changed from 5 µm to 1.7 µm particle size?
Smaller particle sizes (decrease H) result in larger number of theoretical plates (increase
N) provide higher column efficiency, resulting in sharper, narrower and taller peaks and
better resolution. Also, the retention time remains relatively similar.

G). (0.5) In additional to changing from 5 µm to 1.7 µm particle size, how will the
chromatogram change (compared to that when the column was packed with 5 µm
particle size material) if the flow rate used was doubled?
The retention time of all compounds will be shorter and the peaks will be sharper and
narrower.

H). (0.5) Predict, with reason, the migration order of benzene, naphthalene, toluene, and
phenol in RP-LC.
Elution Order: Phenol à Benzene à Toluene à Naphthalene
In reversed-phase liquid chromatography (RP-LC), the migration order is mainly
determinted by the polarity of compounds. In general, more non-polar substances elute
slower due to more non-polar interaction to the stationary phase. Phenol carries polar
hydroxyl group, which will be eluted first. For the other three non-polar substances, the
number of carbon increase from benzene to naphthalene, so naphthalene will be eluted
the last.
2. (0.5 x 2 = 1) Explain, with the aid of a van Deemter plot, why HPLC columns with i).
different internal diameter; ii). different particle size has different optimal flow rates.
The van Deemter plot illustrates the relationship between the height equivalent to a
theoretical plate (HETP) and the linear flow rate of the mobile phase. The plot typically
has three main components: A-term (Eddy diffusion), B-term (longitudinal diffusion), and
C-term (mass transfer).

i) Internal diameter
According to Van Deemter plot, to maintain an optimal linear velocity, the flow rate
should be adjust accordingly. For example, an increased column i.d. requires a
proportionally larger flow rate (i.d.2).

ii) Particle size

Different particle size have different optimal flow rate as smaller particles have a larger
surface area-to-volume ratio, which facilitates faster mass transfer (C-term) between the
stationary phase and the mobile phase. The flow rate should be smaller to allow enough
time for mass transfer.

3. A). (0.5 x 2 = 1) What’s the advantage of using: i). peak area; ii). peak area ratio (vs an
internal standard); over peak height for quantitative analysis?
i) For peak area, it is more accurate as it will less likely to be affected by column
performance.
ii) For peak area ratio of analyte against internal standard, it can normalize the
variations between samples as it compared to constant amount of IS. Besides, it
can improve the precision by correcting errors arise from instrumental
fluctuation.
 B). (0.5 x 2 = 1) Retention time is the most used parameter for qualitative analysis in
analytical chromatography. Name two techniques that can be used to enhance the
confidence of compound identification using chromatography.
For chromatography coupled with UV detector, we can compare the UV absorption
spectrums of sample and standard. For those coupled with MS detector, we can check
the m/z value and fragmentation pattern of target analyte and compare the sample to
the standard. Moreover, co-chromatography can be used by adding standard solution of
the analyte to see if there is an increase signal.

4. A). (0.5) Predict, with reason, the migration order of benzene, naphthalene, toluene, and
phenol in capillary GC using a 100% dimethylpolysiloxane (HP-1/DB-1) column.
Benzene → Toluene → Phenol → Naphthalene
(Benzene → Toluene → Naphthalene → Phenol)

The analytes are separated based on boiling points solely as HP-1 is a non-polar column.
The molecular size and boiling points are increased from benzene, toluene and phenol
to naphthalene.
(For phenol, it carries polar hydroxyl group which can have dipole- induced dipole
interaction and elutes later.)

B). (0.5) Ceteris paribus, how will the chromatogram change if the column used was
changed to a 5% phenyl-95% dimethylpolysiloxane (HP-5/DB-5) column?
The HP-5 column has higher polarity than HP-1 which allows weak dipole or hydrogen
bonding in analyte interactions. Therefore, polar phenol elutes slower due to stronger
polar interaction and retention while the other three compounds will also elute slower
due to more dipole- induce dipole interaction.
C). (0.5) Why only “ultra-high purity” and inert gas should be used as mobile phase/carrier
gas in GC?
The ‘ultra-high purity’ gases can reduce the interference of impurities from the mobile
phase which improve the precision. Beside, it can minimize any side reactions occurs
between the carrier gas and analytes, such as oxidation by oxygen or hydrolysis by water.
Also, inert gases, such as helium or nitrogen, do not react with the sample compounds.
This is crucial for maintaining the integrity of the sample.

D). (0.5) In general, capillary GC has a smaller loading capacity than that in HPLC. Briefly
explain this phenomenon.
For the size of column, capillary GC columns are usually narrower than HPLC columns,
which have a much smaller cross-sectional area and smaller amount of statioanary
phase. This smaller diameter limits the loading capacity.

For the volatility of analytes, GC is mainly used for volatile compounds. The sample size
must be small enough to ensure that the analytes can vaporize effectively without
causing overload, which can lead to peak distortion and poor separation.