UV-Vis Spectrophotometer — Standard Operating Procedure

1. Pre-Operation

• [ ] Warm up lamp for ≥15 min
• [ ] Verify wavelength accuracy with holmium filter
• [ ] Check photometric accuracy with neutral density filter
• [ ] Set baseline with blank solvent

2. Measurement

1. Dissolve peptide in appropriate solvent
2. Transfer to quartz cuvette (10 mm path)
3. Scan 240 – 400 nm
4. Read absorbance at 280 nm (and 220 nm if needed)
5. Calculate concentration using Beer-Lambert law

3. Calculation

$$A = \varepsilon \times c \times l$$

Where ε is calculated from Trp (5690), Tyr (1280), Cystine (120) contributions at 280 nm.

4. Shutdown

1. Clean cuvette with appropriate solvent
2. Rinse sample compartment
3. Turn off lamp