(±)-1-(9-Fluorenyl)ethyl Chloroformate Solution

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
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((+)-FLEC), C₁₆H₁₃ClO₂   272.73 CAS RN®: 107474-79-3. - Use a suitable grade with a concentration of NLT 18 mM in acetone.

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Enantiomeric Purity

Mobile phase: Acetonitrile and water (1:1)

Standard solution: 30 µg/mL of (±)-1-(9-uorenyl)ethanol in alcohol

Sample solution: 18 mM solution of (+)-1-(9-uorenyl)ethyl chloroformate in acetone

Carbonate buffer solution: Transfer 338 mg of sodium carbonate and 152 mg of sodium bicarbonate to a 100-mL volumetric ask. Dissolve in and dilute with water to volume.

Sodium hydroxide solution: Transfer 30 g of sodium hydroxide to a 100-mL volumetric ask, add 50 mL of water to dissolve, and cool and dilute with water to volume.

Acetate buffer solution: Transfer 0.3 mL of glacial acetic acid to a 100-mL volumetric ask, add 90 mL of water, adjust with Sodium hydroxide solution to a pH of 4.2, and dilute with water to volume.

Blank: Water

Procedure: Transfer 20.0 µL each of Sample solution and Blank to separate 10-mL test tubes. Add 80 µL of Carbonate buffer solution to each test tube and cap the tubes tightly. Let each solution react for 24 h at 45° over a water bath. Cool to room temperature, add 5.0 mL of Acetate buffer solution, mix by vortex, and transfer to separate vials.

Chromatographic System

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: Fluorescence

Excitation wavelength: 260 nm

Emission wavelength: 315 nm

Column: 4.6-mm × 15-cm; 3.0-µm packing L40

Column temperature: 25°

Flow rate: 0.5 mL/min Injection volume: 5 µL

Run time: 2 times the retention time of (±)-1-(9-uorenyl)ethanol

System Suitability

Samples: Standard solution, hydrolyzed Sample solution, and hydrolyzed Blank Suitability requirements

Relative retention times: About 0.95 for hydrolysate of (−)-1-(9-uorenyl)ethyl chloroformate and 1.0 for hydrolysate of (+)-1-(9- uorenyl)ethyl chloroformate

Resolution: NLT 1.5 between hydrolysate of (−)-1-(9-uorenyl)ethyl chloroformate and hydrolysate of (+)-1-(9-uorenyl)ethyl chloroformate. Absence of peaks at the retention times for hydrolysate of (−)-1-(9-uorenyl)ethyl chloroformate and hydrolysate of (+)-1-(9-uorenyl)ethyl chloroformate in the hydrolyzed Blank.

Analysis

Samples: Standard solution and hydrolyzed Sample solution Integrate only peaks at the retention times of (−)-1-(9-uorenyl)ethanol and (+)-1-(9-uorenyl)ethanol. Disregard any extraneous peaks in the chromatogram of hydrolyzed Sample solution.

Calculate the percentage of (+)-1-(9-uorenyl)ethyl chloroformate (P ) in the portion of (+)-1-(9-uorenyl)ethyl chloroformate taken:

Result = r_A/(r_A+ r_B) × 100

r_A = peak response of hydrolysate of (+)-1-(9-uorenyl)ethyl chloroformate from the Sample solution

r_B = peak response of hydrolysate of (−)-1-(9-uorenyl)ethyl chloroformate from the Sample solution

Calculate the percentage of (−)-1-(9-uorenyl)ethyl chloroformate (P ) in the portion of (+)-1-(9-uorenyl)ethyl chloroformate taken:

Result = 100 − P_A

Acceptance criteria: NLT 99% (RB 1-Jun-2021) of (+)-1-(9-uorenyl)ethyl chloroformate and NMT ▲1%▲ (RB 1-Jun-2021) of (−)-1-(9- uorenyl)ethyl chloroformate