Reversed-phase cartridges

Tannin crude extract partitioning Cl 8 reverse phase cartridge... 

Reverse phase Perkin Elmer Reverse Phase Cartridge/Varian PureDNA/Hamilton PRP-3... 

The encapsulated orange oil (0.1 g) was rehydrated in 7.5 ml distilled water via the use of a vortex mixer for 30 sec. Acetone (50 pi) containing 100 jug ethyl heptanoate (internal standard) was added and mixed for another 30 sec. This solution was forced through a preconditioned Sep-pak CIS reverse phase cartridge. 

Synthetic dyes may be isolated, purified, or concentrated from foods or from extracts by wool-dyeing procedures column chromatography with polyamide ion-pair or solvent extraction reverse-phase cartridges or ion-exchange resins (157,159,168). These techniques are discussed next. 

The SFE can also be used for extractions. Reverse-phase cartridges are available that, after activation with methanol, can be used to remove nonpolar materials from solution. This is true chromatography, not filtration. The adhering material can be eluted in step-gradient fashion with increasing nonpolar solvent fractions. Although they do not have the efficiency of an HPLC... 

Solvent exchange is very cmcial for HPLC analysis. The methylene chloride or acetonitrile extract is exchanged into methanol as described above, using ethylene glycol before passing through any C-18 reverse phase cartridge. 

Note If the experiments are highly sensitive to salt, then it is recommended to desalt the oligonucleotides by a reverse phase cartridge, such as a C-18 sep-Pak cartridge from Waters (Milford, MA), before speed-vac concentrating. Alternatively, the HPLC-purified oligonucleotides can be desalted and concentrated by ethanol precipitation. 

Silica gel-based reversed phase cartridge (SepPAK Waters Corp., Milford, MA, USA). 

The use of reversed-phase cartridges for serum sample preparation was also studied by Hartwick et al. (HIO) and excellent recoveries were reported for non-protein-bound compounds. 

Hom, T. and Urdea, M. S. 1988. Solid supported hydrolysis of apurinic sites in synthetic oligonucleotides for rapid and efficient purification on reverse-phase cartridges. Nucleic Acids Res., 16 11559-11571. 

Reverse phase cartridges should be washed with 6-10 holdup vol of acetonitrile or methanol. Ion-exchange cartridges should be washed with 6-10 vol of methanol (optional). Normal phase columns do not require a wash step. 

Capacity is a function of several factors total concentration of all components in the sample, sample-solvent polarity, and the affinity of the components for the adsorbents Overloading the adsorbent can result in poor or variable product recovery. If retention of the desired product is too low, the sample-solvent polarity may be too high or the adsorbent may be too weak. For example, if material in a 50% methanol extract of a fermentation broth does not bind sufficiently to a C8 reverse phase cartridge, the methanol concentration should be reduced (e g, by dilution with water or by evaporation of methanol), or the adsorbent should be changed to Cl8 (more hydrophobic). 

As a rule of thumb for particle-packed normal-phase and reversed-phase cartridges, the capacity is approximately 5% of the mass of the packed material. This means that not more than 5 mg of analytes should be applied on a 100 mg packed SPE cartridge. 

The reversed-phase cartridge Sep-Pak C18 (Waters) has been found applicable by Jacobson et al. (63) for the desalting of an extract from F1210 lymphoblasts. Elution of cobalamins from this type of column is brought about by 50% acetonitrile. 

Use a reversed-phase cartridge such as Macherey-Nagel C, 6 g in a 15-mL cartridge. Dissolve 800 mg product in 3 mL 2 1 Me0H/H20 and add to cartridge. Elute organic acid... 

Figure 5. Anlytical HPLC separation of 300 pg venom peptides (A) and 10 ml conditioned water (B) from Conus textile. Details on preparation of the substances are given in the legend to Fig. 4. Separations were performed on an analytical CIS reverse phase column (Vydac wide pore, 4.6 x 250 mm, 5 pm particle size) at a flow rate of 0.5 ml/min. Substances were loaded on the column in aqueous 0.1% tri-fluoroacetic acid (TFA) and eluted with a linear gradient of 0-60% acetonitrile in 0.1% aqueous TFA in 0-60 minutes. On-line detection and spectral analysis was performed with a Hewlett-Packard diode array detector. The spectrum of the main peak obtained from the CW (B) is not identical to those of any of the venom derived peptides (A) that are eluted at similar times from the column (not shown). Attempts to isolate the active component(s) of Conus textile CW on reverse phase cartridge columns and Amicon filters were not successful, due to loss of the biological activity.

---