Sample preparation desalting

Dry the target under gentle vacuum and wash (also used for desalting if cmde hemolymph or digestion mixture) the sample preparation twice with 1 piL of 1 % TFA. Remove the washing solution after a few seconds using forced air, and dry the sample under vacuum. 

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. 

Sephadex G-25 is used at laboratory and production scale for sample preparation and clarification. Typically sample volumes of up to 30% of the total column volume are loaded. In a single step, the sample is desalted, exchanged into a new buffer, and low molecular weight materials are removed. The high volume capacity and speed of this step enable very large sample volumes to be processed rapidly and efficiently. The high sample volume load results in a separation with minimal sample dilution. A typical elution is shown in Figure 28. 

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. 

A number of microfluidic applications include the development of sample preparation tasks that are aimed at improving the detection limits. Most commonly, these tasks include processes such as cleanup, desalting, preconcentration, and enzymatic microdigestion. 

Figure 3.3 Sample preparation by miniaturized solid-phase extraction or affinity enrichment. The resin is used to trap the analyte and serves to concentrate and desalt...

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

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