High performance reversed-phase liquid chromatography
High performance reversed-phase liquid chromatography
High performance reversed-phase liquid chromatography can be applied to: Separate non-polar, polar or ionic compounds.
Operation method
High performance reversed-phase liquid chromatography
Principle
Reversed phase chromatography (RPC) is a chromatographic mode based on the hydrophobic effect between the solute, polar mobile phase and the surface of the non-polar stationary phase. There is a non-polar hydrophobic portion in the structure of any organic molecule, and the larger the portion is, the higher the retention value is, which is the most widely used separation mode in high-performance liquid chromatography. In high performance liquid chromatography (HPLC), this is the most widely used mode of separation. Under the conditions of reversed-phase liquid chromatography of biological macromolecules, the mobile phase mostly adopts acidic, low ionic strength aqueous solution with a certain proportion of organic modifiers such as isopropanol, acetonitrile, or methanol, etc., which is miscible with water, and a large number of fillers are silica gel alkyl-bonded phases with pore sizes of more than 30 nanometers, except for a small number of polymer microspheres. Experiments have shown that alkyl chain length has no significant effect on the reversed-phase retention of proteins, but there is a difference between short-chain hydrocarbon groups (e.g., C4, C8, phenyl) and long-chain alkyl (e.g., C18, C22) reversed-phase packings in the active recovery of proteins. The longer the alkyl chain, the more hydrophobic the stationary phase is, thus increasing the organic component of the mobile phase is required for faster elution of proteins. Excessive hydrophobicity and too much organic solvent can lead to irreversible adsorption of proteins and loss of biological activity. In conclusion, reversed-phase chromatography on alkyl-bonded silica gel is a widely used method for the separation, analysis, purification and structure elucidation of proteins due to its high column efficiency, good separation and clear retention mechanism.
Materials and Instruments
huBPP Move I. Sample Preparation 1. Extraction At room temperature, weigh (2 ± 0.05) g of tissue samples in a 50 mL centrifuge tube, add ethyl acetate 15 mL, vortex mixing extraction for 3 min, centrifugation for 10 min (4000 rpm), transfer the ethyl acetate layer to a 50 ml pear-shaped bottle. To the residue, add 10 mL of 0.1 mol/L NaOH solution, mix well, add 15 mL of ethyl acetate to extract the ethyl acetate layer. Combine the two extracts, concentrated under reduced pressure at 40 ℃ nearly dry, add ethyl acetate 1.0 mL and hexane 5 mL, so that it is fully dissolved, to be purified. 2. Purification SPE was carried out using a Silica solid phase extraction column. After activating the SPE column with 5 mL of hexane, the sample was eluted with 5 mL of hexane, dried, and then eluted with 5 mL of hexane-acetone (6:4, v/v). The eluate was blown dry at 40 ℃ under N2, and the residue was dissolved in 20% aqueous acetonitrile 1.0 mL, and then passed through a 0.45 μm filter membrane for the determination of liquid chromatography and mass spectrometry conditions by LC/MS/MS method. Chromatographic conditions 1. Chromatographic column: Thermo Hypersil Gold (150 × 2.1 mm, 5 μm); see Table 1 for gradient elution, in which A: water (containing 0.01% formic acid); B: acetonitrile; flow rate: 250 μL/min; injection volume: 10 μL. 2. Mass spectrometry conditions Electrospray ionization source (ESI), negative ion mode; selective reaction monitoring (SRM) scanning mode, spray voltage: 3500 V; ion transfer tube temperature: 350 ℃; nitrogen was used as the sheath gas and auxiliary gas, of which 40 arb for the sheath gas and 5 arb for the auxiliary gas, and argon was used as the collision gas, with a collision pressure of 1.5 mTorr; selective reaction monitoring (SRM). Caveat Chromatographic column operation instructions, (Diamonsil(TM) column as an example)1. General parameters of chromatographic columnCatalog.No. Product Patent No. Serial No.Date DatePacking Column Paking such as, Diamonsil（TM） Diamond C18 5цmColumn Dimension 250×4.6mmMaterial Lot No. 0206Guarantee Plate Number N>70,000/m (usp 1/2 Peak height)Asmmetry Factor As=0.8-1.4 (5% Peak height)Test report (with chromatogram) omitted2. Acceptance of the column(1) Check whether the appearance of the column is intact.(2) Check whether the column parameters, such as specifications, are consistent with your needs, item by item. 3. Use of mobile phase(1) Check the permitted mobile phase and pH of the column, for example, buffer solution is routinely used for gel column, organic solvent is used for reversed-phase column, and the permitted pH of diamond C18 column is 2-7. 5.(2) Pay attention to the viscosity of the mobile phase on the post-column, such as methanol/water system is higher than acetonitrile/water system.(3) Filtration of the mobile phase, sample use, commonly used 0.45μm membrane, distinguish between water-soluble and fat-soluble membrane. Generally in the mouth of the column has 0.2μm membrane protection, so as not to clog the column.(4) Flow corresponding degassing. 4. Chromatographic column installation(1) Confirm that the column and instrument connectors, piping match.(2) Before installing the column, check whether the solvent in the flow path system is normal.(3) In order to reduce the dead volume so that the injection valve and the connection between the column and the detector piping inside diameter as thin as possible, such as 0.01 inches. 5. Requirements for the sample(1) The sample should be soluble in the mobile phase.(2) The sample concentration should be within a certain range, different column requirements.(3) The sample should not have insoluble matter, and should be filtered with a needle or filter before use.(4) The sample should not react with the mobile phase. 6. Use of the chromatographic column(1) The solvent of the last package of the chromatographic column is generally the same as that of the test report or specially indicated. If the mobile phase is changed, the flow rate should be slow, generally not more than 0.5ml/mim.(2) Do not change the mobile phase frequently, otherwise, it will accelerate the reduction of column efficiency.(3) Note that different columns have different degrees of pressure resistance, the pressure should be within the range of column pressure resistance, in order to obtain the best separation effect, diamond C18 columns do not use more than 200bar (300psi).(4) Pay attention to the influence of temperature when using the heat preservation column. 7. Storage of the column(1) If salt or acid reagents are used in the mobile phase, rinse with water (about 20 times the column volume) before storing.(2) Pump in preservation solution, use different preservation solution for different columns, diamond C18 column should be preserved with 100% acetonitrile or methanol as far as possible.(3) After removing the column, immediately seal it with a connector and store it in a stable environment. 8. Column regenerationDifferent columns can be regenerated in different ways, please refer to the description of each column.For diamond reversed-phase bonded-phase C18, C8, pH, and CN columns, use the following procedure:Flush the column with 20 times the column volume of solvent according to the following procedure. (Flush in the direction of the arrow)Water → methanol → chloroform → methanolFor water rinsing, use hot distilled water (55°C) or add a small amount (0.8%) of DMSO.Reverse rinsing of the column is generally not advocated, but can be done if necessary. Common Problems The use of the method is limited to some extent by the use of expensive and toxic reagents such as acetonitrile and methanol in reversed-phase chromatography. For example, it can inactivate some of the proteins. Therefore, the method is used a little more for analytical identification, especially for samples that are tolerant to organic solvents. Peptide samples, for example, can be analyzed by HPRPC for the synthesized peptide huBPP. For more product details, please visit Aladdin Scientific website.
Methanol (HPLC grade) Trifluorohexanoic acid (TFA) Acetonitrile (HPLC grade) Biodistilled water
High-performance liquid chromatograph Beckman gold system Sampling needle Tubes Centrifuge Refrigerator Test tubes Chromatographic column Diamonsil TM (Diamond)