This experiment describes how much time it takes to remove sodium N- dodecyl sarcosinate by dialysis. This experiment is from the Laboratory Guide for Protein Purification and Identification, by Zhu Houzhu.

Operation method

Dialysis for the removal of sodium N-dodecylsarcosinate

Materials and Instruments

Glacial acetic acid Trifluoroacetic acid (TFA) Acetonitrile Solvent A (0.1% TFA) Solvent B (80% acetonitrile + 0.086% TFA)
Reversed-phase high performance liquid chromatography (HPLC) instrument C18 reversed-phase HPLC columns

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Materials and equipment

Reversed Phase High Performance Liquid Chromatography (HPLC) Instruments

C18 reversed-phase HPLC column (VYDAC?, 4.6x250 mm)

Glacial acetic acid

Trifluoroacetic acid (TFA) (2% aqueous solution reservoir)

Acetonitrile

Solvent A (0.1% TFA) (filtered through 0.2-um nylon membrane)

Solvent B (80% acetonitrile + 0.086% TFA) (filtered through 0.2-um nylon membrane)

Operating Procedures

1) Analyze samples taken during and after dialysis by reverse-phase HPLC (see P.149).

2) Take 100ul of each sample, add 5ul of glacial acetic acid and mix well.
Note: If the sample is extremely turbid or precipitated, it may be centrifuged in a microcentrifuge prior to injection.

3) Equilibrate the C18 column with 0.1% TFA/water/acetonitrile system and feed 50ul of the sample adjusted with acetic acid for detection at 215nm. After injection, wash the column with solvent A for 5 min at a flow rate of 1 ml/min. Linear gradient elution: from 100% solvent A to 100% solvent B in 10 min, then solvent B for 3 min and solvent A for 7 min.

4) A series of diluted (0.005~0.075%) sodium N-dodecyl sarcosinate (SKL) standards were added to the sample, and the SKL peak height was plotted as a function of concentration. The concentration of SKL in the sample was calculated from the peak height of the sample and plotted as the concentration of SKL in the sample as a function of time.

5) After use, rinse the column with 20 ml of Milli-Q water, followed by 20 ml of 20% methanol, and store at room temperature.
Note: Prepare a blank sample with 100 ml of Buffer A and 5 glacial acetic acid and analyze as described above. This can be used to correct for any solvent peaks eluting from the column.

Results

A typical reversed-phase HPLC chromatogram is shown in Figure 3-6.

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