Why do we use internal standards?

Answer the following questions in 1-3 sentences maximum.

1. A standard plant metabolite extraction protocol uses methanol and water. Why do
we use both of these solvents and not just Methanol?

2. Why do we use internal standards?

3. Why do we derivatise compounds for GC-MS?

4. What is the difference between isocratic and gradient elution in HPLC? Why is this

5. You would like to extract the beta-carotene from plant tissue.

a) Is this compound polar or apolar? Provide a brief explanation.
b) What solvent types would you use to extract (provide 2 examples) and explain your

Answer the following questions in 1-3 sentences (4-5 lines) maximum.
Using the dataset given in the attachment, follow the steps in the bioinformatics
practical session using MetaboAnalyst and use it to answer the following questions.

1. Under �Data transformation� explain (with 2 reasons) why a log transformation is
done before statistical analysis of metabolomics data.

2. In the �t-test� see what happens when you change the P-value threshold to 0.10.
Explain with reasons your observations.

3. In the �PCA 3D Scores� plot, HFD4 groups with the ND samples. Explain why this
might be the case and what you would recommend be done for further statistical
analysis of the dataset.

4. In your words explain why it is important to correct for false positives when
performing multiple significance tests.

5. Run a pathway enrichment analysis using a significant metabolite list after
performing a t-test with a P-value threshold of 0.05 and 0.10. What are the top 5
enriched pathways for each threshold? What has contributed to the change in
top 5 order?

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