Welcome to the Biotage Peptide Synthesis Blogs.

      How does media pore size impact peptide resolution?

      Sep 27, 2019 3:19:01 PM / by Elizabeth Denton posted in Developments, Peptides, reversed-phase, flash purification, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, stationary phase, particle pore size

      0 Comments

      Purification by reversed-phase chromatography relies primarily on a hydrophobic interaction of the molecule with the alkyl chains bonded to the stationary phase for column retention and elution through a partitioning mechanism.  While this is certainly true for purification of peptides, surface area accessibility and media particle size also play critical roles in the resolving power of a particular stationary phase.  The particle size influences the loading capacity, however pore size greatly influences molecular accessibility and therefore resolving power.

      In today’s post, I will demonstrate how pore size can impact your peptide purification using flash column chromatography.

      Read More

      How to use a scouting column for your peptide purification

      Sep 27, 2019 3:13:39 PM / by Elizabeth Denton posted in reversed-phase, flash purification, peptide, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, stationary phase, particle pore size, method development

      0 Comments

      In the past, when I have synthesized a new peptide, I always ran a “scout run” – a small scale injection, usually with an analytical HPLC column – to both get an idea of the crude purity and also to identify a shorter, more optimal gradient for the actual purification.  This strategy is still recommended when you want to use reversed phase flash chromatography for your purification strategy, but is there a better way?

      In today’s post, I’ll discuss using a scouting column to screen gradient conditions prior to peptide purification with reversed phase flash chromatography.

      Read More

      How to purify your peptide using mass directed flash chromatography

      Jul 31, 2019 4:02:19 PM / by Elizabeth Denton posted in Developments, Mass Detection, solid phase peptide synthesis, purify, loading capacity, peptide purification, peptides and flash chromatography, mass-directed purification

      0 Comments

      We've all used mass spectrometry to characterize our synthetic peptides.  It's often used to confirm that the peptide was in fact synthesized, then again as part of the purification process to make sure that we're collecting the correct peak.  But how many of you had the opportunity to use in-line mass spectrometry as an integral component during the purification itself?

      In today's post, I'll highlight some of the advantages to using in-line mass mass spectrometry for purification of peptides using reversed phase flash chromatography.

      Read More

      How much peptide is recovered from a reversed-phase C18 cartridge during flash purification?

      Jul 25, 2019 2:13:43 PM / by Elizabeth Denton posted in Developments, reversed-phase, flash chromatography, peptide, solid phase peptide synthesis, peptide purification, peptides and flash chromatography

      0 Comments

      Whether it’s the bonded stationary phase, particle size, or even particle pore size, scientists today are offered a plethora of choices when it comes to reversed phase HPLC columns.  An often acknowledged concern in the peptide community though is peptide recovery from reversed phase purification efforts, particularly for precious peptide mixtures.  But how is peptide recovery impacted when you use reversed phase flash chromatography for purification?

      In today’s post, I’ll compare recovery levels for two peptides that differ in length as well as crude purity using reversed phase flash chromatography.  In addition to comparing two peptides, I’ll also evaluate how recovery is impacted by altering the mobile phase pH.

      Read More

      How to choose an ion pairing agent to improve your peptide purification

      Jul 22, 2019 4:28:04 PM / by Elizabeth Denton posted in Developments, reversed-phase, flash chromatography, peptide, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, ion pairing agents

      0 Comments

      Ion pairing agents are used in a variety of strategies to improve overall purification efficiency. In a previous post, I utilized ion pairing agents to increase the peptide’s hydrophobicity, improving retention by the stationary phase and enabling purification.  But what other strategies can be improved by using ion pairing agents?

      In this post, I’ll utilize ion pairing agents to enable rapid peptide purification by flash chromatography.  The use of ion pairing agents can in fact alter the peptide’s apparent hydrophobicity sufficiently that the desired peptide and it’s closely eluting impurities can be resolved.  The question is, which one to choose?

      Read More

      Does loading method influence my peptide recovery after purification?

      Jul 17, 2019 6:25:29 PM / by Elizabeth Denton posted in Peptides, reversed-phase, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, loading method

      0 Comments

      In peptide purification, sample loading onto the column is rarely considered.  Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification.  If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column.  Alternatively, dry loading crude material is a strategy often used in small molecule purification, particularly when sample solubility concerns arise.

      The first question I asked myself when considering a new sample loading strategy is whether or not the purification efficiency will be maintained.  A close second though is whether or not the loading method will cause significant differences in peptide recovery.

      In today's post, I'll compare recovery efficiencies for peptides purified using reversed phase flash chromatography but loaded onto the cartridge using either direct liquid injection or dry loaded onto reversed phase material.

      Read More

      Can you use normal phase chromatography to purify protected peptides?

      Jun 11, 2019 8:29:38 PM / by Elizabeth Denton posted in normal phase, reversed-phase, flash purification, peptide synthesis, solid phase peptide synthesis, peptides and flash chromatography

      0 Comments

      Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature.  However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task.  Several ligation strategies have been developed that help to alleviate this challenge.  These strategies though, require a purified, yet fully protected peptide fragment.

      Purification of a fully protected peptide species can be challenging by standard reversed-phase techniques, primarily due to the limited solubility of protected peptides in aqueous solutions.  In today’s post, I will discuss using normal-phase chromatography for purification of protected peptides.

      Read More

      Can I improve my peptide purification by increasing the column length?

      May 23, 2019 4:41:39 PM / by Elizabeth Denton posted in reversed-phase, peptide workflow, v-10 touch, solid phase peptide synthesis, peptide purification, peptides and flash chromatography

      0 Comments

      There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC.  These strategies can include, changing column length, particle size, particle functionality (C4 vs C18).  I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.

      In today's post, I'll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.

      Read More