Spektrofluorometri

Summary

This video demonstrates a step-by-step laboratory procedure for analyzing salicylic acid using spectrofluorometry. It begins with preparing stock and intermediate solutions, carefully measuring and diluting samples, and correctly handling cuvettes. The tutorial then explains how to operate the spectrofluorometer software, including connecting the instrument, setting emission and excitation wavelengths, performing auto-zero with ethanol as a blank, and scanning to determine optimal wavelengths. After identifying emission and excitation peaks, a calibration series of standard solutions is prepared and measured to build a quantification curve. Finally, unknown samples are analyzed, and the resulting data and spectra are exported and saved as PDF reports.

Takeaways

• 😀 The spectrofluorometer must be preheated before use, indicated by a green light on the device.
• 😀 Cuvettes for fluorometry should be handled only by the top and do not have opaque sides, unlike UV-Vis cuvettes.
• 😀 A stock solution of salicylic acid is prepared by weighing 100 mg and dissolving it in 10 mL of ethanol, then transferred to a 50 mL volumetric flask.
• 😀 Intermediate stock solutions are prepared by sequential dilutions from the original stock solution, allowing accurate concentration series for analysis.
• 😀 The L Solution RF software is used for spectrofluorometry, requiring connection, auto zeroing, and selection of emission mode at specific wavelengths (e.g., 297 nm for excitation).
• 😀 Blank samples (ethanol) must be used for auto zeroing, and cuvettes need to be rinsed and cleaned before insertion to avoid contamination.
• 😀 The emission spectrum is measured first to determine the appropriate emission wavelength, followed by the determination of the excitation wavelength.
• 😀 A concentration series is prepared from 100 µL to 700 µL for calibration and quantification purposes.
• 😀 Quantification involves entering excitation and emission wavelengths into the software, naming samples, inputting concentrations, and running each sample sequentially.
• 😀 After completing measurements, data is saved and exported as PDFs for reporting and analysis, ensuring proper documentation of standards and samples.
• 😀 Proper technique includes wiping cuvettes in a single direction and always rinsing with either blank or sample solution to maintain accuracy.

Q & A

What is the first step before using the spectrofluorometer?

The first step is to heat the instrument until it is ready, indicated by a green light on the device.

Where should the sample be placed in the spectrofluorometer?

The sample should be placed in the central cuvette holder of the spectrofluorometer, where the light will pass through and be detected.

What is the proper way to handle cuvettes for fluorometry?

Cuvettes for fluorometry should be handled by the top only and should not have any opaque sides.

How is the stock solution of salicylic acid prepared?

Weigh 100 mg of salicylic acid, dissolve it in 10 mL of ethanol, and transfer it to a 50 mL volumetric flask, then fill up to the mark with ethanol.

What are intermediate stock solutions and how are they prepared?

Intermediate stock solutions are diluted forms of the main stock solution. Intermediate 1 is made by taking 100 µL of the stock solution, and Intermediate 2 is prepared by taking 1000 µL of Intermediate 1.

How is the spectrofluorometer software set up for emission measurements?

Connect the instrument, delete previous messages, select spectrum mode, set spectrum type to emission, choose the emission wavelength (297 nm), and set the range (320–500 nm).

What is the purpose of the 'auto zero' step?

Auto zero calibrates the spectrofluorometer using a blank (ethanol) to eliminate background signal before measuring actual samples.

How are the excitation and emission wavelengths determined for a sample?

First, measure the emission spectrum to find the emission peak, then set the excitation wavelength accordingly (in this case, 403 nm was used for emission).

How are the serial dilutions of the sample prepared?

Take varying volumes of the intermediate stock solution, ranging from 100 µL to 700 µL, to prepare a series of concentrations for analysis.

What steps are necessary before running a new sample in the spectrofluorometer?

Rinse the cuvette with the sample or blank, fill it to 3/4 height, wipe it with tissue in a single direction, and insert it properly into the instrument.

How are the data recorded and saved from the spectrofluorometer?

After measurements, view the data in the table, execute the data display, and save or print it as a PDF with appropriate file naming.

What is the procedure for quantifying samples after obtaining emission and excitation data?

Enter the obtained wavelengths into the quantification software, add sample concentrations, connect to the instrument, measure each sample, and save or print the results as PDF.