Precipitation

Precipitation is also used to remove proteins from biological samples (e.g. blood serum, urine samples etc.) and involves precipitating interfering materials by appropriate regents. The materials of interest, however, must not be co-precipitated, or the analysis will be unreliable. Proteins can damage LC columns as they may be precipitated by the mobile phase, block the interstitial column voids, increase the flow impedance of the column and actually arrest the flow. Protein can be removed by reducing the pH by adding trifluoroacetic acid or perchloric acid causing denaturation and almost instant precipitation. The protein is separated by filtration, or by centrifugation and the upper layer used for further examination. Proteins can also be precipitated from aqueous media with polar solvents such as methanol or acetonitrile.

The efficiency, however, varies considerably with the sample/solvent ratio and should be avoided if acid precipitation is possible. When employing precipitation, co-precipitation and/or adsorption of the solutes must be carefully avoided. It is important to understand that 100% recovery is not essential as long as the actual recovery efficiency is known. However, trace quantities of heavy metals from aqueous media can often be co-precipitated as an isolation technique and the heavy metals removed in a concentrated form from the precipitate.

Dialysis

Dialysis extraction depends on the selectivity of semi permeable membranes. The membranes are fabricated to have the desired pore size. During dialysis the material is transported across the membrane by a concentration gradient. Dialysis can either concentrate a sample or selectively extract and dilute it, depending on whether the material is smaller than the pores or larger. For example consider a very dilute aqueous solution of fairly large molecules on one side of the membrane and a strong sugar solution on the other. Water would pass through the membrane to dilute the sugar solution (as a result osmotic pressure) and, the material would be concentrated. If the solution contained salt and pure water was on the other side of the membrane then salt would pass though the membrane and the sample desalted. Dialysis is often used as a desalting procedure after protein precipitation with salt. Membranes with a wide range of pores are readily available.

Ultra Filtration

Ultra filtration also utilizes a semie permeable membrane that actually filters the sample. It differs from dialysis, in that the material is transferred across the membrane by hydraulic pressure, as opposed to a concentration gradient. Ultra filtration results in sample concentration and can be carried out on a continuous basis. Due to the relatively high pressures involved, and the relative fragile nature of the membranes, they are usually supported on a web structure to provide adequate mechanical strength. Dialysis and ultra filtration are gentle processes and can be used for thermally labile materials.