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The importance of a chemically stable HPLC packing material

In preparative chromatography it is of uttermost importance that all parameters are optimized to give the lowest possible cost for the target substance purified. We have in previous articles described the importance of optimization of the HPLC separation. However, another factor that could greatly influence your separation cost is the lifetime of the packing material. To run the separation using a stable packing material will not only result in a lower cost due to a longer lifetime of the packing material, but the separation can also be run with less disturbances, with more stable conditions for a long time.

Design of a silica-based HPLC packing material

A spherical, totally porous silica-based packing material for HPLC is manufactured from colloidal silica, consisting of small, solid primary particles that are agglomerated into the final particle. The space in between the primary particles will make up the porous network. The resulting particles are subsequently derivatized using organosilanes, if the silica is to be used for reversed phase HPLC.

The chemical stability of the final packing material is a result both of the manufacturing process of the silica particle, and of the bonding process.

The chemical attack on a silica-based RP packing material

Click on the attack arrows for illustrated info

Stability at low pH

A well-covered RP silica-based packing material should be possible to use down to at least pH 1.5 continuously. The acidic breakdown consists of an attack on the siloxane bond between the silane and the silica matrix. This bond will break, thus producing a free silanol, and a free silane (see figure). The free silanes could combine to form dimers during this process.

silica acidic attack silica acidic leackage

As more and more free silanols are generated on the silica surface, retention times will change — hydrophobic substances will show decreased retention times, and compounds interacting with the silanols can instead show increased retention times.

An effect of the release of silanes from the packing material is that these can contaminate the collected product fractions. One should be very observant for this phenomenon!

Stability at high pH

The recommended upper pH limit for many silica-based packing materials is 7-8, but a good material with a high silane density should be possible to use up to around pH 9.5-10 at least. In some cases we have seen that our classic packing material can be used even up to above pH 11, also for extended times.

silica alkalic attack silica alkalic leackage

At high pH the breakdown process is different from at low pH — it is now the silica matrix itself that will dissolve. In the alkaline attack fragments with silica and silane will be released from the packing, resulting in a deterioration of the packed bed, and formation of a void at the column inlet, provided the process continues for some time (see figure). The result will be broader peaks, loss of performance, and usually increased back pressure.

Also during this process there is a risk for contamination of the collected product fractions, in this latter case both from Si(OH)4 and silane.

How to extend the lifetime of the packing

By taking some precautions when running the chromatography the lifetime of the packing can be significantly extended. Below some recommendations are listed:


  • Check the recommended pH range for the packing material that will be used, and go outside of this range only after careful checking of the effect.
  • Be careful when using certain buffers, e.g. carbonate or phosphate buffers, that can produce a significantly higher pH after mixing with organic modifiers (see table below).
  • Be sure to filter the crude before injection in case the feed contains any particulates.
  • Use regular CIP (cleaning in place) to avoid accumulation of contaminants on the column bed. A CIP could consist of a wash, going to much higher organic modifier content for a certain time, to wash out substances that stick more strongly to the packed bed. To avoid precipitation of certain buffers, e.g. phosphate buffer, one should preferably start with a high water content to wash out the buffer, and then switch to a high concentration of the organic modifier.

pH shift upon addition of organic modifier
– in 0.1 M buffer/methanol (50/50)
buffer (0.1 M)pH beforepH afterdifference
Glycine9.949.83-0.11
Sodium borate9.9310.17+0.24
Sodium phosphate10.0010.65+0.65
Sodium bicarbonate10.0011.40+1.40

G. W. Tindall et al; J. Chromatogr. A 988 (2003) 309

Conclusion

Preparative HPLC in large scale can be a rather costly process. However, if some of the ideas above are applied, one might at least keep the cost for packing material down to a minimum, and maintaining a cost-effective process!

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