Quiz · laboratory methods
Lab methods quiz
Twelve questions on purity analysis, identification, units and handling. Each answer comes with an explanation and the guide that covers it.
Question 1 of 12
-
1. By which property does reversed-phase HPLC mainly separate peptides?
Show the answer
Answer Hydrophobicity
In reversed-phase HPLC the column packing is non-polar and the mobile phase starts out mostly water. Molecules with more hydrophobic surface cling to the packing for longer, so they leave the column later, as the proportion of organic solvent rises. Separating by mass is the job of a mass spectrometer, and separating by size that of size-exclusion chromatography.
-
2. A certificate states an HPLC purity of 99%. What does that figure measure?
Show the answer
Answer The main peak's share of all the peak area the detector recorded
HPLC purity is an area percentage: the area under the main peak divided by the area under every peak in the chromatogram, at the wavelength the detector used. It describes the peptide-related material that absorbs at that wavelength. Water and counter-ions do not appear in it at all, so a purity figure says nothing about how much of a powder's mass is peptide.
-
3. What does a mass spectrum confirm about a peptide?
Show the answer
Answer That its measured mass matches the mass expected for the sequence
A mass spectrometer measures the mass-to-charge ratio of ions. For a peptide the result is compared with the mass calculated from its sequence, and agreement is strong evidence of identity. It is not a purity measurement and it does not weigh the contents of a vial. Identity by mass spectrometry and purity by HPLC answer different questions, which is why a certificate reports both.
-
4. In an electrospray mass spectrum, why can one peptide give several peaks?
Show the answer
Answer The same molecule is detected carrying different numbers of charges
Electrospray ionisation usually puts more than one proton on a peptide. A spectrometer measures mass divided by charge, so the same molecule appears at one position with a single charge, at about half that value with two charges, at about a third with three, and so on. The peaks form a series, and each of them gives back the same molecular mass once its charge is accounted for.
-
5. What is net peptide content?
Show the answer
Answer The share of a powder's mass that is peptide; the rest is counter-ions and water
A lyophilized peptide is not pure peptide by weight. Basic groups on the chain are paired with counter-ions such as acetate or trifluoroacetate, and some water stays bound to the solid. Net peptide content is the percentage of the total mass that is the peptide itself, commonly determined by amino acid analysis or by nitrogen analysis. It is a different number from HPLC purity.
-
6. Why do many synthetic peptides contain trifluoroacetate or acetate?
Show the answer
Answer They are counter-ions paired with positively charged groups on the peptide
Trifluoroacetic acid is used to cleave a peptide from its resin and in the mobile phase during purification, so the positively charged groups on the finished peptide end up paired with trifluoroacetate. It is often exchanged afterwards for acetate or chloride. Either way the counter-ion is a normal part of a peptide salt, and it accounts for some of the mass of the powder.
-
7. A solution has a concentration of 1 mg/mL. What is that in micrograms per microlitre?
Show the answer
Answer 1 µg/µL
A milligram is 1,000 micrograms and a millilitre is 1,000 microlitres. Dividing both by a thousand leaves the ratio unchanged, so 1 mg/mL is exactly 1 µg/µL. The same reasoning shows that 1 mg/mL equals 1 g/L. Keeping these equivalences in mind prevents thousand-fold errors when a method and a pipette are marked in different units.
-
8. How is molarity defined?
Show the answer
Answer Moles of solute per litre of solution
Molarity is the amount of solute in moles divided by the volume of the solution in litres. Moles are found by dividing mass by molecular weight, so two peptides at the same concentration in mg/mL have different molarities when their weights differ. Moles per kilogram of solvent is molality, a separate unit, and grams per mole is the unit of molecular weight itself.
-
9. A solution is diluted. According to C1V1 = C2V2, what stays the same?
Show the answer
Answer The amount of solute
Concentration multiplied by volume is an amount of solute. Diluting adds solvent and nothing else, so the amount before, C1 times V1, equals the amount after, C2 times V2. Knowing any three of the four values gives the fourth. The equation only holds when both concentrations are written in the same unit and both volumes in the same unit.
-
10. What ties the results on a certificate of analysis to a particular vial?
Show the answer
Answer The batch or lot number
A certificate of analysis reports tests carried out on one production batch. The batch or lot number printed on the certificate is what connects those results to the material in hand: it should match the number on the vial label. A certificate with no batch number, or with a different one, describes some other material.
-
11. What does a bacterial endotoxin test detect?
Show the answer
Answer Lipopolysaccharide from the outer membrane of Gram-negative bacteria
Endotoxin is lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria. It remains after the bacteria themselves are dead and it passes through a sterilizing filter, so sterility and endotoxin are tested separately. The classical method uses a lysate of horseshoe-crab blood cells that clots in the presence of endotoxin, and results are reported in endotoxin units.
-
12. Why is a lyophilized peptide stored cold, dry and away from light?
Show the answer
Answer Because heat, moisture and light all speed up chemical degradation
The reactions that degrade a peptide, among them hydrolysis, deamidation and oxidation, run faster at a higher temperature, and most of them need water. Light can drive the oxidation of residues such as tryptophan. Keeping the dry solid cold, sealed against moisture and in the dark slows all of them at once, which is why those three conditions appear together in storage instructions.
How this works
A short check of what you know, built to teach as it goes.
-
One question at a time
Choose an answer and you see at once whether it was right, with a paragraph on why. There is no timer and no trick wording.
-
Settled science only
Every question has one textbook answer: bonds, units, methods. Nothing here is about what a compound does, and nothing is medical.
-
A reading list at the end
The result lists the guides behind the questions you missed first. No sign-up is asked for and your answers are not stored.
Related guides
The reading behind this tool, written as plain laboratory science.
- Analytical Methods for Peptides What HPLC, mass spectrometry, amino acid analysis, water, counter-ion, endotoxin and sterility tests each answer, and what they cannot.
- How to Verify a Peptide Reading an HPLC trace and a mass spectrum.
- What Does 99% Purity Mean? What a purity figure measures, and what it leaves out.
- Certificate of Analysis Guide Every field on a certificate of analysis, how to tell a batch document from a generic one, and what a certificate cannot prove.
- Peptide Units and Concentration Milligrams, moles, molarity and international units, and the arithmetic of making and diluting a peptide stock solution.
More research tools
This quiz covers research peptides as laboratory chemistry. It contains no dosing information and no guidance on use in people or animals.