Sample preparation desalting procedures

Because of the favorable sorptive properties of the reversed-phase supports, batch adsorption and desorption can be a very effective way to desalt a chromatographed sample or to partially fractionate a peptide mixture during a purification procedure. For example, 1-2 gm of an oc-tadecyl silica packed into a silanized glass or plastic pipette can be used for the batch fractionation of small amounts of a crude peptide extract from tissues, such as the pancreas or pituitary, or from a synthetic experiment. A number of commercial products, such as the Waters Sep-Pak, have found use in this manner 10) as a purification or sample preparation aid. Protocols for batch extraction procedures on alkyl silicas have been discussed 17a,b) and applied to neuropeptides 10, 158, 166) and other hormonal peptides 88, 162, 167, 168). With these methods recoveries of peptides present in a tissue extract are generally higher than those found with classical fractionation techniques due in part to the fact that proteolytic degradation is minimized. 

Sometimes the sample preparation is a difficult problem, especially in clinical and environmental chemistry. General procedures are filtration (perhaps by means of a dedicated membrane which retains compounds selectively), solid phase extraction with disposable cartridges (also with dedicated selectivity), protein precipitation and desalting. A special case is sample preparation for biopolymer analysis. 

The ideal sample preparation scheme will minimize the final HPLC separation problem, reduce the risk of HPLC column contamination, and provide good analyte recovery. At the same time, the procedure should be as simple and rapid as possible. The number of steps can be minimized by coupling steps with complementary rather than similar selectivity. The product of each step should be directly compatible with the successive step to minimize interstep manipulations such as desalting or concentration. Many devices are commercially available to streamline, automate, and miniaturize many steps in sample preparation. 

DESALTING. A sample preparation procedure involving removal of salts by some procedure such as ion exchange or dialysis. 

Depurination, alteration of nucleic acids by, 945 Desalting procedures in sample preparation, 14-15 Detection, 205-218 measurement of radioactivity, 345-355 mictdbioiogical methods, 218 microchemical methods, 211-218... 

Sample application, 20-23 application of spots, 20-21 choice of sample solvent, 20 formation of bands, 21-22 for preparative layer chromatography, 22-23 Sample preparation, 9-16 cleanup of extracts by column chromatognqrhy, 11 cleanup of extracts by solvent partitioning, 10-11 deproteinization, 18 derivatization, 18 desalting procedures, 14-15 direct application of sample solutions or extracts, 9-10... 

Fig. 3 Schematic of a nano-scale capillary ESI interface. This specialized LC/MS interface, operating at flow rates from 20-500 nL/min and using 50-100 pm ID columns, typically provides low femtomole sensitivity. Fully automated sample handling and preparation procedures (i.e., desalting and preconcentration) combined with specialized devices for high separation and variable nL gradient flow rates provide unique capabilities for high-throughput analysis of proteins. (Courtesy of New Objective, Cambridge, MA.)...

---