A practical ways to return a contaminated column to — or close to — its original state.What Causes Contaminant Buildup in Reversed-Phase Columns?
Usually, sample matrices contain compounds that are of no interest to analysts. Salts,lipids, fatty compounds, humic acids,hydrophobic proteins and other biological compounds are a few of the possible substances that can come in contact with an HPLC column during its use. These
materials can have lesser or greater retention than the analytes of interest.
Those compounds that have lesser retention, such as salts, will usually be eluted from the column at the void volume. These undesired interferences might be observed by a detector and appear as chromatographic peaks, blobs, baseline upsets or even negative peaks.
Washing Bonded-Silica Columns: The keys to rejuvenating a contaminated HPLC column are knowing the nature of the contaminants and finding an appropriate solvent that will remove them.
A recommended column washing system for a typical bonded-silica column and a mobile phase without buffer salts is to use:
• 100% methanol, • 100% acetonitrile, • 75% acetonitrile–25%isopropanol, • 100% isopropanol, • 100% methylene chloride and • 100% hexane.
When using methylene chloride or hexane, the column must be flushed with isopropanol before returning to an aqueous mobile phase because of solvent immiscibility. A minimum of 10 column volumes of each wash solvent should be passed through a column. For 250 mm x 4.6 mm analytical columns, analysts can use a typical 1–2 mL/min HPLC flow-rate. To return to the original mobile phase, chromatographers can usually skip going through the entire series in reverse order. Using isopropanol as an intermediate solvent is recommended, followed by mobile phase without buffer, then finally with the starting mobile-phase composition. Tetrahydrofuran is another popular solvent that can be used for cleaning contaminated columns. If users suspect severe fouling, they can mix dimethyl sulphoxide (DMSO) or dimethylformamide mixed 50:50 with water and pass them at flow-rates less than 0.5 mL/min. The successful regeneration of a reversed-phase column can be a time-consuming process, and solvent washings can be programmed into a gradient system for overnight operation.
Bonus Question: A question arises as to whether to reverse the HPLC column during the washing procedure? --- tips: This situation is particularly true if metallic ions are sorbed to the silica
or bonded phase. A chelating reagent such as 0.05 M ethylenediaminetetraacetic acid
(EDTA) can be flushed through a column. The EDTA complexes with many metallic
species and solubilizes them. After treatment with an EDTA solution, analysts
can wash the column thoroughly with water.
To control bacterial growth that could be present in a buffer system or in columns
left unattended in aqueous buffer, chromatographers can use common household bleach diluted 1:10 or 1:20 or 35% Nitric Acid . Run at least 50 column volumes followed by another 50 column volumes of HPLC-grade water. Do not run the bleach through the detector, because it could attack the flowcell.
