Charged Aerosol Detector (CAD) Application Brief--
Direct measurement without derivatization 17 amino acids

1, Preface

Amino acid analysis is one of the most important technologies in life science research. Amino acids are the basic components of human protein. Except for a few aromatic amino acids, their UV absorption is weak. Direct use of UV detectors has low detection sensitivity and high interference. In recent years of peptide drug research, amino acid composition analysis (Amino Acid Composition Analysis, AACA) is an indispensable core link. Such as amino acid sequence verification: the biological activity of peptide drugs is highly dependent on its amino acid sequence and composition. By analyzing the amino acid composition, it can be verified whether the synthesized peptide is consistent with the target sequence and avoid functional failure caused by synthesis errors (such as amino acid deletions, substitutions or unexpected modifications); amino acid structure characterization: combined with mass spectrometry (MS) and chromatographic techniques (e.g. HPLC-CAD), amino acid composition analysis can assist in analyzing the molecular structure of complex polypeptides (such as polypeptides containing unnatural amino acids or post-translational modifications) to ensure that they meet design requirements.

take advantage of Charged Aerosol Detector (CAD) combine high-performance liquid chromatography (HPLC)(HPLC) for direct detection without derivatization 17 amino acids, demonstrating significant technical advantages. Compared with traditional methods, this technology eliminates the need for cumbersome derivatization steps and avoids sensitivity fluctuations and quantitative deviations caused by instability of derivatization reagents, side reactions, or product diversity.CAD The universal principle of the detector enables it to respond with high sensitivity to non-volatile and weak UV-absorbing compounds (such as amino acids), enabling accurate detection without relying on specific functional group modifications. This method simplifies the pretreatment process, shortens the analysis time, and reduces the use of toxic reagents, which is in line with the concept of green chemistry. In addition, its wide dynamic range and good linear relationship support the simultaneous quantitative analysis of multiple amino acids in complex matrices (such as biological fluids and food samples), and is especially suitable for rapid screening and quality control in high-throughput laboratories. Research has confirmed that this method performs well in terms of retention time stability, repeatability and recovery, providing an efficient and reliable analytical solution for life sciences, clinical diagnostics and the food industry.

This article utilizes the use of reverse phase HPLC combine INSTRUMAX of Charged Aerosol Detector (CAD) direct measurement 17 This method is fast, simple, accurate, highly sensitive, reproducible and linear, providing strong technical support for amino acid detection and peptide drug research.

2, instruments and materials

Instruments:Agilent1200 liquid chromatograph

CAD detector(INSTRUMAX, Model: CADetector a1)

Reagents: pure water (Watson's distilled water),

Acetonitrile (chromatographically pure),

Trifluoroacetic acid (analytical grade)

Heptafluorobutyric acid (analytical grade)

Sample: in hydrochloric acid solution 17 Amino acid mixed solution standard material (Weiye measurement standard material)

3, methods and results

3.1 Chromatographic conditions

Column:ZORBAX SB-C18 3.5μm 4.6*250mm

Flow rate:0.8mL/min

Mobile phase A: water (0.7%Trifluoroacetic acid+5mm ol/L heptafluorobutyric acid) Mobile phase B: acetonitrile

gradient elution

Thinner: water

CAD Conditions:

Instrument model:INSTRUMAX CADetector a1

Drift tube temperature:35℃

Atomized air flow:3L/min

Corona airflow:1L/min

Corona current:1μA

Gain:0.05

RF：2.0

3.2 Experimental results

3.2.1 17 Separation of amino acids: under this method 17 The amino acids are well separated, and adjacent components can be effectively separated. The peak shapes of each amino acid are sharp and the column efficiency is high.

3.2.2 100pmol The sample signal-to-noise ratio is good (software P2P method automatically calculated).

3.2.3 Injection precision,1mm ol/L standard sample-injection 5μL, continuous injection 6 needle, each amino acid RSD% are better.

3.2.4 Linear

Inject 1μL,2μL,5μL,10μL and 20μL, use injection volume and peak area to make a linear equation.

The linearity is good, and the correlation coefficient R of each component is greater than 0.999.

The summary table is as follows:

4, conclusion

This study uses HPLC-CAD The method is used to determine amino acids. The sample can be directly injected and analyzed without derivatization. It does not require cumbersome pre-processing to avoid errors caused by derivatization. There is no need to screen derivatization conditions (derivatization reagents, reaction conditions, etc.), which reduces measurement errors. The operation is simpler, the results are accurate and reliable, and it provides strong instrument and method support for the determination of amino acids.