PepGenexShop our research products

Chromatogram

A chromatogram is the record of a chromatographic separation: a detector signal plotted against time as the separated components of a sample elute, with each detected component appearing as a peak at its retention time. ICH Q2(R2) names chromatograms among the representative data used to demonstrate the specificity of an analytical procedure.[1],[2]

What does a chromatogram record?

In HPLC of peptides, the signal is often ultraviolet absorbance. Mant and colleagues state that peptide bonds absorb light strongly in the far ultraviolet, below 220 nm, providing a convenient means of detection, generally at 210-220 nm.[1]

The same chapter notes that the aromatic side chains of tyrosine, phenylalanine and tryptophan absorb in the 250-290 nm range, but that these residues are not present in all peptides.[1]

Where the detector is a mass spectrometer, the trace can be a total ion chromatogram, as in the peptide map described by Mouchahoir and Schiel.[3]

What determines where a peak appears?

Mant and colleagues describe the three major HPLC modes for peptides as separating by differences in size (size-exclusion), net charge (ion-exchange) or hydrophobicity (reversed-phase). In reversed-phase HPLC, peptides elute with increasing overall hydrophobicity, least hydrophobic first.[1]

The same chapter reports that increasing temperature decreased peptide retention time and peak width in their reversed-phase examples, and that peaks co-eluted at some temperatures but not others.[1]

In size-exclusion chromatography, retention reflects size. Mant and colleagues show a size-exclusion separation of six peptides of 4 to 20 residues and relate each peptide's distribution coefficient to the logarithm of its number of residues.[1]

ICH Q6B states that aggregates, including dimers and higher multiples of the desired product, are generally resolved from the desired product and quantitated by procedures such as size exclusion chromatography or capillary electrophoresis.[4]

The number of peaks also depends on sample preparation: Mouchahoir and Schiel note that missed and non-specific cleavages in a digest can complicate a peptide map by increasing the number of peaks in the chromatogram.[3]

What is resolution, and why does it matter?

ICH Q2(R2) states that for critical separations in chromatography, specificity can be demonstrated by the resolution of the two components that elute closest to each other. Its example for separation techniques calls for absence of relevant interference between individual peaks of interest.[2]

Mant and colleagues note that peptide resolution in reversed-phase HPLC can be adjusted by varying the steepness of the acetonitrile gradient.[1]

What is system suitability?

ICH Q2(R2), citing ICH Q14, describes system suitability tests as tests developed and used to verify that the measurement system and the associated analytical operations are fit for the intended purpose.[2]

FDA guidance on analytical procedures describes system suitability acceptance criteria such as peak tailing, precision and resolution, and for chromatographic systems refers to USP General Chapter <621> Chromatography.[5],[6]

How does a peak become a number?

Quantitation relies on a relationship between analyte concentration and peak response. ICH Q2(R2) calls for experimental demonstration of a linear relationship between analyte concentrations and peak responses, or the ratio of peak responses where an internal standard is used, across the range of the procedure.[2]

ICH Q2(R2) also states that if an analyte has a different response from the reference material, for example a different specific ultraviolet absorbance, relative response factors should be calculated, and that a correction factor should be applied if a factor falls outside 0.8-1.2.[2]

ICH Q6B notes that the determination of absolute, as well as relative, purity presents considerable analytical challenges and that the results are highly method dependent.[4]

Limitations

A chromatogram shows only what the detector sees under the conditions used. Components that co-elute appear as one peak, and a component that responds weakly at the chosen wavelength is under-represented.[1],[2]

A peak at the expected retention time is not by itself an identity result; ICH Q6B states that more than one test may be necessary to establish identity.[4]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. Mant CT, Chen Y, Yan Z, et al. HPLC analysis and purification of peptides. Methods Mol Biol. 2007;386:3-55. PMID 18604941 · DOI 10.1007/978-1-59745-430-8_1 · PMC7119934
  2. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
  3. Mouchahoir T, Schiel JE. Development of an LC-MS/MS peptide mapping protocol for the NISTmAb. Anal Bioanal Chem. 2018;410(8):2111-2126. PMID 29411091 · DOI 10.1007/s00216-018-0848-6 · PMC5830484
  4. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  5. U.S. FDA. Analytical Procedures and Methods Validation for Drugs and Biologics. Guidance for industry, July 2015. FDA docket FDA-2015-N-0007
  6. United States Pharmacopeia, General Chapter <621> Chromatography (as named in FDA, Analytical Procedures and Methods Validation for Drugs and Biologics, 2015). USP <621>