Passage
The N-terminus of an amino acid must be protected with 9-fluorenylmethoxycarbonyl (Fmoc) during solid state peptide synthesis to avoid side reactions (Scheme I) .
Scheme IThe Fmoc protecting group is typically removed with piperidine (Scheme II) , a heterocyclic secondary amine.
Scheme IIBecause the use of piperidine is restricted, structurally similar amines were examined as possible replacements. To determine whether piperidine analogues were suitable alternatives for Fmoc removal, deprotection of Compound 2 was carried out with several amine bases (Scheme III) .
Scheme IIICompound 2 was prepared from L-tryptophan and Fmoc-Cl, and the reaction was monitored by thin-layer chromatography (TLC) and visualized by ultraviolet light. The deprotections were done with piperidine and each analogue using a microwave set to 155 watts at 75 °C for 15 seconds. The products of the deprotections were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry; the retention times of the products were compared to tryptophan and Compound 2 standards.
Adapted from O.P. Luna et al, "Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?." Molecules. ©2016 MDPI.
-What can be inferred about the HPLC column used in the passage if tryptophan has a shorter retention time than Compound 2?

Question

Passage
The N-terminus of an amino acid must be protected with 9-fluorenylmethoxycarbonyl (Fmoc) during solid state peptide synthesis to avoid side reactions (Scheme I) .

Scheme IThe Fmoc protecting group is typically removed with piperidine (Scheme II) , a heterocyclic secondary amine.

Scheme IIBecause the use of piperidine is restricted, structurally similar amines were examined as possible replacements. To determine whether piperidine analogues were suitable alternatives for Fmoc removal, deprotection of Compound 2 was carried out with several amine bases (Scheme III) .

Scheme IIICompound 2 was prepared from L-tryptophan and Fmoc-Cl, and the reaction was monitored by thin-layer chromatography (TLC) and visualized by ultraviolet light. The deprotections were done with piperidine and each analogue using a microwave set to 155 watts at 75 °C for 15 seconds. The products of the deprotections were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry; the retention times of the products were compared to tryptophan and Compound 2 standards.
Adapted from O.P. Luna et al, "Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine?." Molecules. ©2016 MDPI.
-What can be inferred about the HPLC column used in the passage if tryptophan has a shorter retention time than Compound 2?

Quizplus · Accepted Answer

11ecba2d_12a9_b89f_a3bf_9b01c1dd594d_MD0008_00 High-performance liquid chromatography (HPLC) is a purification technique used for small sample sizes. The instrumentation consists of a sample injector, a column (stationary phase), solvent under pressure (mobile phase), a detector, and a computer for data acquisition. Two types of columns-normal-phase (NP) or reverse-phase (RP)-can be used, depending on the polarity of the compounds being separated. NP-HPLC is used to separate relatively nonpolar compounds and consists of a polar stationary phase (typically silica) and a nonpolar mobile phase. RP-HPLC is used to separate polar compounds and has a nonpolar stationary phase (typically C-18 alkyl hydrocarbon) and a polar mobile phase. Nonpolar compounds in a mixture will interact more with the stationary phase than polar compounds, causing nonpolar compounds to have a longer retention time. The only structural difference between Compound 2 and tryptophan is the Fmoc protecting group on Compound 2, which contains a polycyclic aromatic hydrocarbon and an ester. The large nonpolar hydrocarbon group decreases the polarity of Compound 2 relative to tryptophan. Because tryptophan has a shorter retention time and is more polar than Compound 2, tryptophan interacted less with the stationary phase than Compound 2. Therefore, the mobile phase is polar and the stationary phase is nonpolar. These conditions indicate that an RP-HPLC column was used. (Choice B) An RP-HPLC column was used but RP columns have a nonpolar stationary phase and a polar mobile phase. (Choices C and D) In NP-HPLC, nonpolar compounds have a greater affinity for the mobile phase (nonpolar), and therefore travel through the column faster than polar compounds. Because tryptophan is more polar than Compound 2, it would have had a longer retention time than Compound 2 on a NP-HPLC column. Educational objective: In high-performance liquid chromatography (HPLC), two types of columns-normal-phase (NP) or reverse-phase (RP)-can be used, depending on the polarity of the compounds being separated. NP-HPLC is used to separate nonpolar compounds and consists of a polar stationary phase and a nonpolar mobile phase. RP-HPLC is used to separate polar compounds and consists of a nonpolar stationary phase and a polar mobile phase.

Passage the N-Terminus of an Amino Acid Must Be Protected with with 9-Fluorenylmethoxycarbonyl