The MonoQ HR 5/5 is a high resolution, anion exchange column, prepacked for use with moderate pressure chromatography systems such as FPLC’s. The bed volume is 1 ml of resin and it can hold up to 20-50 mg of protein, although for optimal resolution only 5 mg should be loaded. It works at pressures less than 5 MPa. This column is top of the line in ion-exchange chromatography.
We have used this column repeatedly in our lab as well as in my previous lab, so in total I have about 6 years worth of experience with it. I have used it with both complex samples such as cell culture supernatants and crude cell extracts, as well as near homogenous protein preparations. In all instances, when used properly, this column has given me very high chromatographic resolution.
The most positive aspects of this prepacked column were its reproducibility, high resolution (again, when used properly), its relatively high capacity and the fact that it came prepacked. For complex samples where the protein of interest was less abundant, the capacity was limiting, but with more purified samples the capacity was rarely a limiting factor. As I can judge it, its reproducibility and high resolution make this column a required asset in any serious chromatography lab. The most serious limitation for this column is that to obtain its potential high resolving power, the conditions for its use must be carefully worked out. And that usually means very low flow rates (and long runs in the cold room), a lot of empirical work to determine the "ideal" running conditions with each system and a lot of cleaning. Indeed, the biggest working problem with this column is that it gets clogged surprisingly easily and requires constant cleaning and maintenance for optimal performance. As it gets dirtier (and this is almost unavoidable over time), the column will "eat" a larger proportion of your protein due to non-specific adsorption (up to 50% of protein at the end of its life). The other problem with this column is its price!
In summary, this column is best suited for serious chromatography of relatively pure samples where a very high level of resolution is required, typically at the polishing stages of purification protocols. The GE Healthcare MonoQ is thus an almost indispensable tool in any serious protein purification lab, yet requires a lot of pampering, attention and care for continued peak performance, like the high-priced jewel it is.
Pierre Beaurang, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco