Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Introduction Fast Protein and Peptide Cleanup with C4, C8 or C18 SpinColumns When working with protein and peptide samples some purification is usually required, therefore, to remove the salts, solvents, and protecting groups that can interfere with mass spectrometric or spectrophotometric analysis of the peptides, or to trace enrich small quantities of organism water or to isolate products in serum. The C4, C8 & C18 SpinColumns from Harvard Apparatus offer a rapid method for sample cleanup by isolating and/or purifying components of your sample. The C4, C8 & C18 SpinColumns use reversed-phase...

Introduction (cont.) There is an easy acronym for determining polarity of your sample. It relies on samples dissolving in solvents they are like so polar compounds dissolve in polar solutions and non-polar compounds dissolve in non-polar solvents. So an easy way to determine the polarity of the sample is to dissolve it in different solvents. Wet Match is an easy acroymn to assist you, and an easy way to remember is, you cannot start a fire with a "wetmatch" or Polar protic Polar aprotic Nonpolar Guide to Reverse Phase SpinColumns Chromatography for Sample Prep 3

Modes of Separation Step Gradient Prepare sample. Add sample. Sample remains in column. Impurities pass through the column. Elute sample from column with buffer. Low ionic strength or solvent and increase the concentration by steps. Elution Volume

Modes of Separation (cont.) Direct Elution Prepare sample. Elute sample with single volume. separate By – Retaining/Retarding unwanted materials on the column and have the molecule of interest pass through the column with the void volume and collect. Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

Modes of Separation (cont.) Trace Enrichment Elution Buffer Prepare sample. Impurities remain in column, sample is eluted with buffer. separate By – Retaining the molecule of interest to the column, then remove them with elution buffer.

SpinColumn Selectivity Harvard Apparatus SpinColumns good selectivity, and feature: • Faster protein, peptide separations than traditional column formats • Macro SpinColumns in 200 µl packing • Fast protein and peptide separations without conventional HPLC systems The 150 µl, 300Å packing’s are ideal for fast bioseparations in life science applications. The C18 packing’s and short 5 mm column coupled with centrifugation give faster sample throughput. Guide to Reverse Phase SpinColumns Chromatography for Sample Prep

SpinColumn Selectivity (cont.) Sample Volume Sample Concentration Elution Ultra Volume

Fast Protein Analysis Optimization of Step Gradient Parameters for Fast Protein Analysis SpinColumns are the perfect tool for fast reversed-phase protein separations. These are not columns so their plate count is low. In gradient selectivity come from exchange of polarities of solvent in and the volume. So selectivity in sample prep with prep columns is dependent on getting the sample to stick and use different polarities to elute off the sample or to have the molecule of interest to come off in a particular volume. Proteins adsorb at the head of the column and then desorbs and elute once the...

Specifications C18 Specifications: Particle Size Pore Size Pore Volume Surface Area %Carbon (w/w) Silica Class Acid and alkali stable C8 Specifications: Particle Size Pore Size Pore Volume Surface Area %Carbon (w/w) Silica Class Acid and alkali stable C4 Specifications: Particle Size Pore Size Pore Volume Surface Area %Carbon (w/w) Silica Class Acid and alkali stable

Chemical Stability The C18 polymer has a 300 Å pore size to provide access to all adsorbent surfaces for large peptides and proteins. The chemistry of the 20 µm particles allows easy scale-up from Ultra-micro SpinColumns to Macro SpinColumns with minimal modifications to conditions. The polymer-based reversed-phase SpinColumn is chemically stable and, more importantly, provides good performance for the separation of peptides and proteins. The packing material is based on porous (300 À pore diameter), highly crosslinked polystyrene-divinylbenzene spheres. Because of the high crosslinkage, the...

Protocol Peptides in aqueous buffers adsorb to C18 SpinColumns and can be subsequently eluted with an organic based buffer. As with reverse phase chromatography, the addition of trifluoroacetic acid as an ion-pairing agent to both the binding and elution buffers can greatly enhance adsorption and elution, respectively. Depending upon the nature of the peptide (i.e., composition and length), the elution buffer can be a combination of aqueous and organic solvents. This allows for washing of bound peptide at lower concentrations of organic solvent, and elution at a higher concentration. Macro SpinColumn...

Protocol (cont.) Solid Phase Extraction SpinColumns Apply the sample to the top of the SpinColumn Centrifuge for 1 minute at low speed. Impurities pass through column and collect in receiver tube #1. Place column into receiver tube #2. Add elution buffer to the column to remove bound components. Centrifuge briefly to collect purified sample. Guide to Reverse Phase SpinColumns Chromatography for Sample Prep 13

Protocol (cont.) For example: Reverse Phase C-18, Somatostatin-14 binds in aqueous phase and after washing the SpinColumn. The peptide was eluted with 70% isopropanol (after) and injected to HPLC. The peptide is desalted as well concentrated by a factor of 2. The analysis was performed on HPLC Column (PolyHydroxyethyl A) manufactured by PolyLC, Inc

Discussion Analysis of both the crude peptide solution and the cleaned peptide demonstrates that the C18 SpinColumn effectively and rapidly separates synthetic peptides from contaminating solutes. This ability to capture a desired peptide without purifying an entire sample and the compatibility of the SpinColumn with standard centrifuges can greatly speed the analysis of libraries stored in microwell plates. Choice of Buffer pH Optimum buffering capacity occurs at a pH equal to the pKa of the buffer. In general, the effective pH range for a buffer is within ± 1 pH unit of the pKa. The buffer...