STANDARD OPERATING PROCEDURE (SOP)

FOR PERKIN ELMER LAMBDA XLS UV-VIS SPECTROPHOTOMETER

(Quantification Method)

Location: 3121 Biltmore Hall/Robertson Labs

Contact: Barbara White, Research Operations Manager, or Ying Xue, Graduate Student

Telephone: 919-513-0298 or 919-753-8523

CAUTION

UV LIGHT EXPOSURE

Do not look directly into the light path that is inside the UV-VIS instrument. This can cause damage to eyesight.

STANDARD OPERATING PROCEDURE (QUANTIFICATION):

To set up method:

Select Standard Methods.

Select Choice 5 (Standard Curve)

Hit à

Choose wavelength.

Mode (absorbance for most people)

Curve fit (regression or zero regression are the two most common)

Units (g/L is commonly used)

Enter standards (Gang’s protein method: 0, 0.2, 0.4, 0.6, 0.8)

Calibration—standards

Replicates select à (3 typically)

Concentration à

Next

To measure samples:

Insert cuvette with a blank solution

Hit OA/100%T.

Then measure standards (hit -à button for each replicate)

Then measure unknowns.

To transfer data to a text file on flash drive:

Data

Methods

Select button that looks like (located in the middle of the four arrows):

Show graph

Select middle button

9 à Autoprint

Goes to software and can be transferred to a flash drive.

Hit “Stop”

Go back to main menu.

Go to computer and select Lambda XLS program.

Find the data file that has the latest date/time. This will be your data file.

Export to .txt file and save on flash drive.

Notes:


Cuvette should be stored in 100% ethanol.

Use quartz cuvettes for wavelengths below 400 nm.

Wear gloves when handling cuvettes.

Make sure the clear side, not the frosted one, is in the light path. The frosted side should be facing you as you stand in front of the spectrophotometer.

When using the same cuvette for multiple samples, rinse the cuvette between samples with the buffer solution, discarding the solution each time.

Make sure the cuvette is filled.

If solution is spilled on the cuvette, wipe clean with a Kimwipe.

Make sure the cuvette is placed on the right hand side, in the spring-loaded holder, against the source.