Saturday, March 5, 2011
The most common questions about HPLC columns involve column care and storage. Good column care can extend the lifetime of HPLC columns, and in general requires little additional time. Column care involves operating and storing a column in a suitable mobile phase, following the manufacturers guidelines for column operation, and preventing and repairing column problems. The guidelines listed below will answer many of your column care and storage questions.
For further Queries about your problems feel free to mail to me -- kaushikzala@rediffmail.com
Choosing Mobile Phases for Column Operation and Storage
Mobile Phases for Column Operation
Mobile Phases for Column Storage
General Guidelines
For further Queries about your problems feel free to mail to me -- kaushikzala@rediffmail.com
Choosing Mobile Phases for Column Operation and Storage
Mobile Phases for Column Operation
- Choose appropriate organic mobile phase modifiers for reversed-phase or normal phase chromatography. Most columns are made for either reversed-phase or normal phase chromatography. For reversed-phase separations acetonitrile and methanol are the most common organic solvent choices. In addition, tetrahydrofuran (THF) and isopropanol are frequently used. For normal phase chromatography hexane and methylene chloride are popular choices. Acetonitrile, methanol and isopropanol are often used as modifiers with the appropriate primary solvents. Additional compatible solvents are often specified by the manufacturer in the column literature.
- Choose a mobile phase for your sample that falls within the recommended pH limits for that column. Most silica based columns can be operated over the pH range 2-8. Some columns, such as the StableBond (SB) columns, can be operated at lower pH (pH 1-2). Others, such as Eclipse XDB columns, can be operated up to pH 9. Remember to determine the mobile phase pH on the aqueous component alone, before mixing with organic modifiers.
- Initially, only pump solvents on to the column that are compatible with the shipping solvent. For reversed-phase separations, do not pump buffer on to a column shipped or stored in 100% organic. This will prevent buffer precipitation in the column. Equilibrate the column with the mobile phase without the buffer first, then equilibrate with buffered mobile phase. Some columns, such as CN or NH2, are used with normal phase and reversed-phase solvents. Check that your solvents are miscible with the shipping solvents before equilibration. To convert a normal phase column to a reversed-phase column may require flushing with a mutually miscible solvent, such as isopropanol, before equilibrating with mobile phase.
Mobile Phases for Column Storage
General Guidelines
- Do not store HPLC columns in buffers. A buffer may precipitate inside the column, resulting in plugged hits and packing material. A buffer may also encourage bacterial growth, which can plug both the column frit and packing material. This is more likely to occur with mobile phases which have close to or 100% buffer. Bacteria may also affect your analytes, and organic products from the dead bacteria may cause "ghost peaks" in chromatograms.
- Do not store HPLC columns in solvents that degrade easily -- tetrahydrofuran (THF), triethylamine (TEA), and trifluoroacetic acid (TFA). Unstabilized THF can form peroxides which may degrade the column. TEA and TFA are more likely to become contaminated from the lab environment and should be refrigerated during storage. Contamination may change the chromatography or change the column. Be aware of the quality issues with these mobile phase components when you use them for your chromatography
Short Term Storage
- Flush the column with your mobile phase minus the buffer or other additives for short term storage.
i.e. Mobile Phase: 65% Methanol : 35% Buffer
Flush: 65% Methanol : 35% Water
This will eliminate the risk of buffer salts precipitating in the column and minimize re-equilibration time when the column is next used. - For overnight storage, keep the mobile phase flowing at 0.1 - 0.2 mL/min. This is very helpful when using complex mobile phases (buffers and ion-pairing reagents), as it reduces re-equilibration time the following day to a few minutes rather than an hour or more. This slow purge will consume less than 100 mL of the mobile phase.
Short term storage solvent choices are often kept close to the original mobile phase. This is based on the desire for quick day-to-day re-equilibration. This is good unless your mobile phase is out of the normal operating range for a column.
Long Term Storage
- Return the column to the mobile phase it was shipped in. If you are using a buffered mobile phase, first wash the column with the same mobile phase composition without the buffer, then flush with the shipping solvent. It is not necessary (or desirable) to flush the column with 100% water just to wash out the buffer salts.
-OR- - Reversed-phase columns can also be stored in 100% acetonitrile. If you are using a buffered mobile phase, first flush the column with the mobile phase minus the buffer, then with 100% acetonitrile. In addition, a 100% ACN flush prior to long term storage can help remove contaminants from the column.
Other Operational Guidelines
- It is important to operate a column below the maximum temperature limits specified in order to obtain the longest possible lifetime. At high temperatures columns will degrade faster. This could involve loss of bonded phase or silica dissolution, depending on whether the mobile phase is at low (pH 1 - 3) or neutral pH (pH 6 - 8). It is important to control temperature for the most reproducible results, but not to exceed the maximum recommended operating temperature for any mobile phase condition.
- It is important to operate a column below the maximum pressure limits specified. Many silica based columns can be operated up to 5000 psi, but many other column materials have lower pressure specifications. You should note the column pressure when you start to use a column with your mobile phase. If you are running a gradient the column pressure will change during the gradient. In many cases, if the starting pressure increases during column operation, the column inlet frit may be plugged. Follow the procedures below for column repair in this situation.
- Choose a flow rate that will allow you to keep the column pressure below the limits specified. Typical flow rates are 1 - 2 mL/min for 4.6 mm i.d. columns, 0.4 - 0.6 mL/min for 3.0 mm i.d. columns and 0.2 -0.4 mL/min for 2.1 mm i.d. columns.
- An arrow on the column body is often used to indicate the recommended direction of flow through the column. Typically you should operate the column in the direction marked. It may not be harmful to the silica particles to operate the column in the reverse direction, but many columns only have a replaceable frit at the inlet of the column. This means you may not be able to replace the frit if the column is operated in the reverse direction and the frit becomes plugged.
Preventing Column Problems
- Column lifetime can be maximized by using a guard column. A guard column protects the analytical column from particulates which plug the column inlet frit and from strongly adsorbed material in the sample and matrix. Particulates will be present in every HPLC system, even when filtered samples are analyzed. Particulates can come from pump seals and the injector rotor seal when these components wear. An in-line frit/filter is also recommended as protection from particulates. these devices should also be used routinely.
- Use HPLC grade solvents whenever possible. You do not need to refilter HPLC grade solvents prior to use, but do filter buffers through 0.45 µL or 0.2 µm filters prior to use. Filter or centrifuge samples prior to use.
- Check that the filter type you are using is compatible with the sample solvent. Extractable components from the filter may show up as ghost peaks in a run. Incompatible filter types, which may partially dissolve in your solvent, introduce material into a column, which can cause plugging.
- Develop a column wash procedure to periodically remove contaminants from the column. Often times 100% strong solvent is a good choice for this.
