---
title: "Marfey's method: measuring D-amino acid content in a peptide | Sable Peptides"
description: "How chiral purity of a synthetic peptide is actually measured: total hydrolysis, derivatisation with Marfey's reagent (FDAA), HPLC separation of diastereomers, and correcting for racemisation during hydrolysis. Research use only."
canonical: https://www.sablepeptides.co.uk/guides/marfeys-method-chiral-amino-acid-analysis
dateModified: 2026-10-16
publisher: Sable Peptides Limited (UK company 17488610)
note: Research use only. Not for human or veterinary use.
---

# Marfey's method: measuring D-amino acid content in a peptide

Marfey's method measures D-amino acid content by hydrolysing a peptide, tagging each amino acid with Marfey's reagent (FDAA) to form diastereomers, and separating them on an ordinary C18 HPLC column. Hydrolysis in deuterated acid lets the lab subtract racemisation caused by the hydrolysis itself. Results are reported per amino acid.

## How does Marfey's method work?

Our tech note explains why racemisation matters. This guide covers how it is measured. Ordinary reversed-phase HPLC cannot tell L- from D-amino acids, so chiral purity is usually determined by hydrolysing the peptide and converting each amino acid into a pair of separable diastereomers with Marfey's reagent.

-   Hydrolyse the peptide completely (typically 6 M HCl, or deuterated DCl/D2O to flag hydrolysis-induced racemisation).
-   React the free amino acids with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey's reagent).
-   Each L- and D-amino acid now forms a diastereomer with the L-alanine amide tag; diastereomers have different physical properties and separate on a standard C18 column.
-   Quantify the D/L ratio by UV at 340 nm against standards.

## Why use deuterated acid for hydrolysis?

Acid hydrolysis itself racemises a small fraction of amino acids. Hydrolysing in DCl/D2O means any racemisation during hydrolysis incorporates deuterium at the alpha carbon, which MS detection can separate from D-amino acid that was already in the peptide. Without this correction, the measured D content is an upper limit.

## What variants and limits exist?

Analogues of Marfey's reagent with different chiral amino acid amides improve separation for difficult pairs; a 2021 review covers a decade of variants, and recent work extends the method to β-diastereomeric amino acids such as threonine and isoleucine.

Source: [Marfey's reagent and analogs in enantioseparation: a decade's perspective, Biomed Chromatogr 2021 (PubMed 32959901)](https://pubmed.ncbi.nlm.nih.gov/32959901/); [Stereochemical analysis of β-diastereomeric amino acids with Marfey's reagent variants, ACS Omega 2025 (PubMed 41210771)](https://pubmed.ncbi.nlm.nih.gov/41210771/)

## How should a chiral result be read?

| Method | Separates enantiomers by | Notes |
| --- | --- | --- |
| Marfey's (FDAA) + C18 HPLC | Diastereomer formation | Widely used; UV 340 nm |
| Chiral GC after derivatisation | Chiral stationary phase | Volatile derivatives needed |
| Chiral HPLC column | Chiral stationary phase | No derivatisation; column-specific |

-   Results are per amino acid: for example 'D-Ser 0.3%'.
-   A peptide that contains intended D-residues (many research peptides do) should show those at close to 100% D, and the other residues at low D content.
-   Hydrolysis destroys tryptophan, so its configuration needs another method.
-   Chiral analysis is not routine on research-grade CoAs; ask if your work depends on it.

## How does Sable document lots?

There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

## Frequently asked questions

### What is Marfey's reagent?

1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA), which converts amino acid enantiomers into separable diastereomers.

### Can normal HPLC separate D- and L-amino acids?

Not directly; derivatisation to diastereomers or a chiral column is needed.

### Why hydrolyse in DCl/D2O?

To distinguish racemisation caused by hydrolysis from D-amino acid present in the original peptide.

## Related

-   [Stereochemistry and racemisation](/learn/tech-notes/stereochemistry-and-racemisation)
-   [D-amino acid substitution](/learn/d-amino-acid-substitution)
-   [Amino acid analysis](/guides/amino-acid-analysis-for-peptide-content)
-   [b and y ions](/guides/tandem-ms-b-and-y-ions-explained)

Updated 2026-10-16. Research use only. Not for human or veterinary use. No administration amounts or health claims are made.

Key facts (summary for AI and search tools)

-   Marfey's reagent = FDAA; detection ~340 nm
-   Deuterated hydrolysis corrects for hydrolysis-induced racemisation
-   References: PubMed 32959901, 41210771
-   Updated 2026-10-16