HPLC analysis applications

Reverse-phase high-performance liquid chromatography (RP-HPLC), also known as high-pressure liquid chromatography, is a powerful and key technique used to separate, quantify and identify individual compounds within a mixture. It is suitable for a wide array of compounds and is routinely used to partition or purify peptides or proteins due to its high resolving power, robustness and extreme reproducibility.

With over 50 years of experience in peptide chemistry, AltaBioscience provides expert analytical and preparative HPLC services for peptide and protein chemistry. Our services include:

• Determination of peptide purity and impurity content
• Purification of intact peptides following solid-phase peptide synthesis
• Separation and identification of peptide fragments resulting from protein enzymatic digestion.

Our HPLC analysis service is often offered as part of the following related services:

• Custom peptide synthesis
• Molecular weight analysis by mass spectroscopy
• Peptide mapping or fingerprinting
• Protein identification using Edman N-terminal sequencing service
• Net weight determination by amino acid analysis

Reverse-phase HPLC for peptides and proteins

HPLC Basic Principle

High-performance liquid chromatography (HPLC) is a general term that encompasses several techniques such as Reverse-phase chromatography (RP-HPLC), ion-exchange chromatography (IEX), size exclusion chromatography (SEC) or hydrophilic interaction chromatography (HILIC). HPLC works by passing a mobile phase under high pressure through a column in order to separate a mixture of compounds. These techniques, developed over the last 40 years and often used hand in hand together, have played a key role in the development of peptide and peptide-based therapeutics.

Reverse-Phase HPLC

In the case of RP- HPLC analysis, a polar mobile phase – usually a blend of water with a volatile and miscible organic solvent, such as acetonitrile – is commonly used in combination with a non-polar surface-modified silica column. A small amount of an ion-pairing agent, i.e., trifluoracetic acid (TFA), is added to the eluent to maintain a low pH and to ensure a good peak shape.

Contrary to ion exchange chromatography, where compounds are separated based on their net charge, in RP-HPLC analysis, compounds are partitioned based on their difference in hydrophobicity. Polar (hydrophilic) or ionic compounds will stick more to the highly polar aqueous mobile phase and elute first, whereas less polar compounds will interact more strongly with the non-polar stationary phase and elute at a later stage. Elution can be performed either via an isocratic or a gradient separation mode. Eluted peptides are detected by UV at 214-215 nm.

HPLC analysis services with AltaBioscience

Poor quality column or condition choice can lead to multiple issues, including low resolution and peak tailing. Our highly-experienced technical team only uses the best analytical and preparative monolithic columns available to tailor our RP-HPLC analysis services to your project’s needs and ensure the best possible outcome during the separation and purification of your peptide mixture.

We use a range of C18, C8 and C4 columns. These columns have free silanol covalently bound to linear alkyl silica chains. C18 columns with octadecyl silica chains are the least polar of all and are commonly used for the purifications of small peptides less than 300 Daltons. On the other hand, C4 columns lend themselves better to the separation of larger peptides and proteins.

If you have any questions about HPLC analysis or would like to discuss your project with us, please get in touch.

Reverse-phase HPLC applications

Peptides have countless applications. Whether produced as biotherapeutics, used as antigens to generate antibodies, antibody epitope mapping or protein function studies, their applications within the biotechnology, pharmaceutical and agrochemical industries have been booming over the last decades. This would certainly not have been possible without cutting-edge advancements in HPLC techniques, especially in RP-HPLC, combined with the development of high-resolution and accurate mass spectrometers, recombinant technologies and improved peptide synthesis methodologies.

RP-HPLC in peptide drug discovery

The increase in chronic diseases (cancer, cardiovascular, diabetes) as top causes of death in the world as well as the COVID-19 pandemic, have spurred the discovery, development and repurposing of novel therapeutic peptides.

• Isolation of peptides from natural products using RP-HPLC
  Natural peptides are not only present in the human body as hormones, neurotransmitters or growth factors but can also be found in natural products such as microorganisms, plants and animals. These bioactive peptides have interesting therapeutic properties, and for instance, peptides isolated from snake venoms have shown bioactivity on some cancer cell lines. Over the last 20 years, there has been a renewed interest in isolating peptides from natural sources to harness their therapeutic properties. Elucidating their unknown structures is therefore crucial in order to design new leads for drug discovery with improved biological properties. To achieve this, peptides are often isolated by RP-HPLC and subsequently characterised by mass spectroscopy analysis (using, for instance, MALDI–TOF/MS or LTQ Orbitrap Mass Spectrometry) and N-terminal sequencing to determine the amino acid sequence.
• Isolation of therapeutic peptides with diverse structures
  Although natural peptides are highly specific and well tolerated compared to small molecules, they suffer from low metabolic stability, poor cell membrane penetration and rapid clearance. Because of this, they were once thought to have limited applications. However, the development of new routes of administration (e.g., intradermal, nasal), as well as the emergence of multiple synthetic strategies to overcome these issues, have led to the design and synthesis of a wide range of peptide structures. These therapeutic peptides may, for instance, incorporate non-natural amino acids, be cyclic or contain cross-links through side chains to enhance their stability. The extreme variety of structures has generated the need for robust technologies suitable for their purification. For that purpose, RP-HPLC has proven to be an ideal technique, allowing the efficient and reliable purification of structurally diverse peptides on many different scales.

RP-HPLC analysis and monitoring of biopharmaceuticals

The manufacturing of biopharmaceuticals is complex and challenging. This is due because of their relatively larger size compared to small molecule drugs, but also because they are subject to a high degree of variations as they are produced from living cells. They must consequently meet strict criteria to ensure product quality and consistency in line with the ICH Q6B specifications.

The use of RP-HPLC analysis is particularly useful in this area, not only to determine the purity of the biopharmaceuticals produced and their impurity content during manufacturing but also as a tool to monitor any changes in their primary structures during cell culture or storage. Alterations can result, for instance, from post-translational modifications (methylation, phosphorylation, glycosylation), deamidation, oxidation or from the proteolytic cleavage of C-terminal to arginine or lysine residues. Because of its high resolving power and sensitivity, RP-HPLC can detect those minimal changes and is, therefore, an ideal tool for quality compliance testing.

If you wish to discuss your project with us or have any further technical questions, please do not hesitate to contact our technical team or download our technical documents.