BIKAI PUBLIC APPLICATION NOTE

Automated Pre-Column
Derivatization for the Analysis
of 23 Amino Acids

BIKAI 1511 Pro HPLC • Dual-chemistry UV detection

Application overview

An automated pre-column derivatization workflow expands amino-acid coverage by combining OPA chemistry for primary amino acids with FMOC chemistry for secondary amino acids. The autosampler coordinates reagent handling and injection, supporting a consistent analytical sequence on the BIKAI 1511 Pro platform.

BIKAI 1511 Pro HPLC platform used for the reported application dataset.

01 • ANALYTICAL APPROACH

Extending UV Detection Across a Broad Amino-Acid Panel

Many amino acids have limited native UV absorbance. Pre-column derivatization converts them into UV-active products before reversed-phase separation, improving selectivity and enabling routine UV–VIS detection. Complementary chemistries are used because primary and secondary amino groups respond differently.

Amino acids are central to nutritional, biochemical and biopharmaceutical workflows.

BIKAI 1511 Pro System Configuration

02 • PRIMARY AMINO ACIDS

OPA Derivatization — 338 nm Channel

Twenty primary amino acids are evaluated as OPA derivatives. In the representative training dataset, the 338 nm trace displays a distributed series of well-defined responses across the separation window, supporting qualitative review and quantitative evaluation of a broad panel.

Figure 1. Representative OPA-channel chromatogram for the primary amino-acid panel at 338 nm.

Interpretation

The chromatographic profile demonstrates the capability of the system to resolve a multi-component amino-acid standard under the evaluated conditions. Complete peak-assignment and integration tables are deliberately not disclosed in this public version; they are supplied as part of the controlled application-transfer package.

03 • SECONDARY AMINO ACIDS

FMOC Derivatization — 262 nm Channel

The complementary FMOC channel extends the panel to hydroxyproline, sarcosine and proline. Monitoring at 262 nm provides a dedicated view of these secondary amino-acid derivatives and completes the reported 23-analyte scope.

Figure 2. Representative FMOC-channel chromatogram for secondary amino acids at 262 nm.

Calibration Performance

A four-level calibration evaluation spanning 25–1000 pmol/µL was reported for the complete amino-acid panel. Correlation coefficients exceeded 0.999 for all 23 analytes in the training dataset, indicating strong proportionality of detector response across the evaluated range.

Performance figures describe the supplied training dataset and should not be interpreted as a universal specification. Laboratories should establish acceptance criteria appropriate to their matrix, concentration range and intended use.