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Chemistry
Posted 3 months ago
Figure 1 shows the chromatogram of an HPLC analysis of an environment water sample containing eight (8) compounds of interest possessing non-polar and mid-polar properties. Answer following questions related to the chromatogram.

Figure 1: A chromatogram of a mixture of different polarity in a packed HPLC column. The flow rate of mobile phase was 0.40 mL/min0.40 \mathrm{~mL} / \mathrm{min}. The column labelling denoted as 2.1 mm×100 mm×4.8μm2.1 \mathrm{~mm} \times 100 \mathrm{~mm} \times 4.8 \mu \mathrm{m}.
i. What is the meaning of ' 2.1 mm×100 mm×4.8μm2.1 \mathrm{~mm} \times 100 \mathrm{~mm} \times 4.8 \mu \mathrm{m} '? Recognize each of them.
ii. Suggest the most suitable type of HPLC stationary phase column for successful separation of these analytes.
iii. Explain the interaction between analyte and the suggested stationary phase column mentioned in d (ii).
iv. Comment on the overall separation criteria for the 8 analytes.
(3+2+2+6(3+2+2+6 marks ))
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Answer from Sia
Posted 3 months ago
Solution
1
Identify the column dimensions: The notation 2.1 mm×100 mm×4.8μm2.1 \mathrm{~mm} \times 100 \mathrm{~mm} \times 4.8 \mu \mathrm{m} refers to the dimensions and particle size of the HPLC column
2
Recognize each dimension: 2.1 mm2.1 \mathrm{~mm} is the internal diameter of the column, 100 mm100 \mathrm{~mm} is the length of the column, and 4.8μm4.8 \mu \mathrm{m} is the particle size of the stationary phase
Answer
The column has an internal diameter of 2.1 mm, a length of 100 mm, and a particle size of 4.8 micrometers.
Key Concept
HPLC column dimensions
Explanation
The dimensions and particle size of an HPLC column are crucial for its separation capabilities and are denoted by the notation 2.1 mm×100 mm×4.8μm2.1 \mathrm{~mm} \times 100 \mathrm{~mm} \times 4.8 \mu \mathrm{m}.
Solution
1
Suggest stationary phase: For compounds with non-polar and mid-polar properties, a C18 column (octadecylsilane bonded phase) is most suitable
Answer
A C18 stationary phase column is most suitable for the separation of non-polar and mid-polar compounds.
Key Concept
Stationary phase selection
Explanation
A C18 column is recommended for non-polar and mid-polar compounds due to its non-polar nature, which interacts well with similar analytes.
Solution
1
Explain interaction: The interaction between analytes and the C18 stationary phase is primarily hydrophobic, where non-polar compounds are retained longer due to stronger interactions with the non-polar stationary phase
Answer
Non-polar and mid-polar analytes interact with the C18 stationary phase through hydrophobic interactions, affecting their retention time.
Key Concept
Hydrophobic interactions
Explanation
Non-polar analytes are retained longer on a C18 column due to hydrophobic interactions with the non-polar stationary phase.
Solution
1
Comment on separation criteria: The separation of the 8 analytes will depend on their polarity, molecular weight, and the strength of their interaction with the stationary phase
2
Optimization: To achieve optimal separation, factors such as column temperature, mobile phase composition, and flow rate must be optimized
3
Resolution: The goal is to achieve sufficient resolution between the peaks of the analytes to allow for accurate identification and quantification
Answer
The separation of the 8 analytes will be based on their individual interactions with the stationary phase, and optimization of the HPLC conditions is necessary to achieve clear resolution between the analytes.
Key Concept
Separation criteria in HPLC
Explanation
Successful separation of analytes in HPLC is achieved by optimizing conditions to exploit differences in analyte interactions with the stationary phase, leading to sufficient resolution

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