To run a very basic test on the column and find out if the support may still be acceptable for use, you can purchase or mix up your own generic C18 type Test Mixture. Many test mixtures exist for this purpose and even the column supplier will have have some form of one for the initial QC testing of the column to evaluate the packing quality (but please do not use "their" test. Often a copy of the results are included with the column when new. While these are better-than-nothing, the test they use is often selected to allow the column to easily pass this check and may not actually evaluate it for real-world performance). Be sure to setup the HPLC system to rinse the column in pure water with some methanol added (e.g. 90/10, Water/DH20) before doing anything with the column to make sure any salts have been washed off it. After rinsing, gradually change the solvent composition from 90 % water, 10% MeOH (in example) to 5% Water and 95% MeOH, 5% water and run 20+ column volumes through it to flush off any bound material. If the column shows a clean baseline at low UV (~ 220nm) , then the column should be ready for testing after this.

• BTW: Could you please share with us which HPLC column you have? "GL" is usually an abbreviation for "GL Sciences", a business name only. What is the columns "brand name"? This will inform us of the column's properties. What are the column's dimensions (L x ID in mm) and also the particle size? As their are several thousand available "C18" columns, and most are different, these values are needed to describe the column you have. Depending on the exact column you have, the most suitable test mixture may vary.

TEST MIXTURES: As you are new to liquid chromatography, please spend a bit of time researching and learning about some of the "standardized" HPLC column test mixtures. One common example suitable for use with C18 columns is known as 'SRM 870'. This test mixtures includes five different organic compounds, in methanol. They are uracil, toluene, ethyl benzene, quinizarin and amitriptyline (amitriptyline hydrochloride); Normally sold in five separate ampoules with 1.1 mL of the mixture. *Each of these compounds tests for different properties of the support. You can mix up your own or purchase ready made test mixtures, as you prefer. In any case, please be sure to get the help of a local professional chromatographer as the HPLC system needs to be setup and running perfectly with a fully equilibrated HPLC column BEFORE injecting any samples (otherwise, any data obtained may be invalid).

• "HPLC COLUMN TEST MIXTURE SOLUTIONS; NP & RP EXAMPLES"; https://hplctips.blogspot.com/2017/07/hplc-column-test-mixture-solutions-np.html

Tường Vy,

You can give column flushing with the solvent in the ratio 90:10 (Water: Acetonitrile) for 30 minutes thereafter to re-generation procedure is to be done as follows,

1) Keep column oven temperature 35°C

2) Run Plain Water (H2O) for 30 minutes

3) 0.1% H3po4(Orthophosphoric Acid) for 30 min

4) Run Plain Water (H2O) for 30 min and check pH at outlet with pH paper to get the idea that orthophosphoric acid completely washed. pH of outlet should be toward alkaline more than 5.5pH.

5) Run Acetonitrile for 30 minutes

6)Run Methanol for 30 Minutes

Then you can use for further analysis.

Regards,

Vijay Bagul

William Letter thank you for your help!!!

Vijay Arjun Bagul thank you for your help!!!

Vijay Arjun Bagul

I would not agree with some parts of your recommendation. Not every C18 column can afford pure water. Some sorbents when exposed to water containing no organic solvent collapse their C18 tails because these are extremely hydrophobic. If this happens, it is nearly impossible to restore column's properties.

Tường Vy Please keep this in mind when trying to wash your column.