How high temperature eluents and column coupling can boost your resolution in peptide mapping
In UHPLC mode, the most effective tool to increase resolution is elevated eluent temperature. Using high temperatures allows separation of peptides and proteins with sharp peaks. Modern (U)HPLC columns, such as YMC-Triart C18, are designed to withstand up to 90°C. They are a perfect match for peptide mapping and large molecule analyses. Generally, larger molecules diffuse very slowly compared to small molecules.The resulting peak broadening causes peak overlapping, which is a difficulty in complex proteinous samples. About 25% more peaks can be resolved by increasing the column temperature to 70°C in the separation of tryptic digest of haemoglobin.
In order to further increase efficiency, column coupling can become an additional tool in your tool box. Conventional UHPLC columns are a maximum 150 mm long. The coupling of e.g. two 100 mm length YMC-Triart C18 UHPLC columns reduces the co-elution of peaks. It allows increased resolution and separation in the analysis of complicated samples as in peptide mapping.
Temperature is an easy and effective tool to increase resolution in the separation of proteins and peptides. The additional coupling of two columns is a further simple way to massively increase the resolution and efficiency.
Two application examples for peptide mapping can be downloaded here:
Peptide mapping of monoclonal antibodies
Peptide mapping of haemoglobin (2 columns coupled)
Check out YMC’s BioLC catalogue or visit our homepage to discover more about YMC-Triart C18 or to find further peptide applications.
Recommendations for the perfect (U)HPLC connection: MarvelXACT™
Are you looking for the perfect solution to connect your column?
Do you use columns with different port depths?
Are you uncertain which tubing diameter is most qualified?
With the help of the recommendations for the perfect (U)HPLC connection the choice of the right tubing internal diameter is simplified. Just connect your column with zero dead volume using the universal connector MarvelXACT™ and prevent any peak tailing and broadening. Easy handling: the finger-tight connection emits a haptic “click” when it reaches the optimum torque. Therefore, it is not possible to overtighten the connector and with various internal diameters available the ideal connection for your column is ensured.
Download YMC’s recommendations for the perfect (U)HPLC connection now.
Metal-free column hardware ideal for RP separation of phosphorothioate oligonucleotides
Oligonucleotides interact with metals. This effect can also be found in LC. When using conventional (U)HPLC column hardware peak tailing is commonly observed. The solution is to use metal-free, bioinert column hardware from YMC.
This application note shows optimum peak shapes for phosphorothioate oligonucleotides using a metal-free YMC column hardware. Also the analyses times can be reduced by the use of a YMC-Triart C8 phase with reduced hydrophobicity.
Download the application note for more details.
