Chromatography is a general analytical technique used to separate a mixture into its individual components. High performance liquid chromatography, commonly known as HPLC, has a variety of applications in the chemical biology research laboratory.
In chemical biology, individual analytes, such as peptides, are often chromatographically purified for use as a functional tool. High performance liquid chromatography (HPLC) is a method used to analyze and separate liquid samples. In chemical biology laboratories, HPLC is considered indispensable for the purification of peptides (synthesized manually or automated with a synthesizer) and other small to medium-sized organic molecules. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.
There are many types of HPLC columns developed for specific applications such as normal-phase HPLC (NP-HPLC) and reversed phase HPLC (RP-HPLC). The right choice of column is critical for obtaining good HPLC results. Column choice is governed by characteristics of components in the mixture we wish to separate.
Also known as normal-phase HPLC (NP-HPLC), or adsorption chromatography, this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers.
Everything that goes into the HPLC must be filtered first, through a 0.45 mm or 0.2 mm filter and special glassware to remove particles that can get caught up on the column and interfere with absorption and separation.
The crude peptide, prepared by manual or automated synthesis, will be supplied as a lyophilized (dried by freezing in a high vacuum) substance.
Filter your buffers, using the designated glassware and following the specific instructions provided by your TA. This can be done prior to use and buffers stored at room temperature until you are ready to use the HPLC.
Your TA will provide specific instructions pertaining to the use of the HPLC. Typically, a run starts by attaching your buffers and washing the column. Next allow the column to re-equilibrate to conditions that will start your run.
Once a specific separation method is specified, you may review the parameters such as pump flow gradient, run time, and the acquisition. On some instruments, you will need to specify the lamp used for detection.
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|Mouse IgG2a Protein (HPLC-51094-MNAH), > 90 % as determined by SDS-PAGE, >95% as determined by SEC-HPLC|
Gundinger T, et al. (2018) A sensitive and robust hplc method to quantify recombinant antibody fragments in e. Coli crude cell lysate. J Chromatogr B Analyt Technol Biomed Life Sci 1083: 242-248.
Mant CT, et al. (2007) Hplc analysis and purification of peptides. Methods in molecular biology (Clifton, N.J.) 386: 3-55.