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Automated amino acid analysis with choices of LCs and high-resolution columns

By John Henderson
Agilent Chemist

Many LC methods for amino acid analysis (AAA) require an offline derivatization step that introduces variability. This article describes a simplified, highly reproducible AAA method that uses online derivatization and lets you choose among Agilent ZORBAX Eclipse Plus C18 columns and Agilent LCs.

Reduce variability

Although quite popular, amino acid analysis by reversed phase HPLC can be complicated. AAA usually uses a mobile phase gradient to separate twenty or more amino acids from each other and components in the sample matrix. Mobile phase gradients are often difficult to reproduce because flow-path volume can vary from instrument to instrument. AAA also typically involves chemical derivatization of the amino acids so you can detect them using UV or fluorescence detectors. Manual or offline derivatization presents several challenges, such as reaction variability, introduction of contamination, and stability of amino acid derivatives until analysis.

Here, we present an easier, online, automated derivatization with OPA-FMOC (o-phthalaldehyde and 9-fluorenylmethyl chloroformate). We combined it with a reversed phase method that includes a linear mobile phase gradient, Agilent ZORBAX Eclipse Plus C18 columns, and the most popular LC systems – the Agilent 1200 Series LC system and Agilent 1200 Series Rapid Resolution LC (RRLC) system. This method makes complicated amino acid analyses straightforward and reproducible.

Figure 1. You can achieve high-resolution amino acid analyses with a choice of Agilent ZORBAX Eclipse Plus C18 columns and Agilent LCs. (Click here to see this image larger.)

Three high-resolution options

We developed amino acid methods for three different Agilent LC systems: a 1200 Series with a quaternary pump, a 1200 Series with a binary pump, and a 1200 Series RRLC system. In fact, we developed only one method and then modified it by proportionally scaling between the analytical columns used with each of the instruments. Each column configuration provided similar high-resolution analyses for all three HPLC systems (Figure 1).

Each of the methods shown in Figure 1 has its advantages. The 4.6 x 250 mm, 5 µm Agilent ZORBAX Eclipse Plus C18 column has excellent resolution, with the closest pair of peaks having a 2.4 resolution factor (Rs). The long column provides the high resolution, but requires a 40-minute runtime, including re-equilibration.

The 2.1 x 150 mm, 3.5 µm Agilent ZORBAX Rapid Resolution column reduces analysis time by three-fifths, and maintains a 2.0 resolution factor by using smaller, more efficient particles in a shorter column length. It has an added benefit because its narrow bore reduces solvent consumption. It uses only 12 mL of mobile phase per analysis, compared with 64 mL for the standard-bore 4.6 x 250 mm column.

The third option, a 4.6 x 100 mm, 1.8 µm Agilent ZORBAX Rapid Resolution High Throughput (RRHT) column, provides an even shorter analysis time of only 16 minutes, and gives excellent resolution (Rs for all peaks at least 2.6). Mobile phase consumption is 28 mL per analysis – less than half that of the 4.6 x 250 mm column. The sub-two-micron particle size provides superior performance, but requires a 1200 Series RRLC system.

Figure 2. Outstanding method reproducibility is shown by an overlay of nine back-to-back injections and percent relative standard deviations (% RSDs) of peak areas. (Click here to see this image larger.)

Outstanding reproducibility

Automated precolumn derivatization is another key benefit of the Agilent amino acid protocol because it reduces or eliminates the variability and error sources of offline derivatization, and it is easier. All the reagents are readily available from Agilent, and you simply position them in the autosampler tray. Each sample is derivatized using the programmable robotic injector arm and metering device of the Agilent 1200 Series Standard Autosampler or 1200 Series High Performance Autosampler SL Plus, and is immediately injected. The injection-to-injection reproducibility is excellent, as is demonstrated in Figure 2.

Method adapts to your existing Agilent LC

The scalability of the Agilent ZORBAX Eclipse Plus C18 stationary phase between three particle sizes (5, 3.5, and 1.8 µm) and three column dimensions, offers a high-resolution amino acid protocol that you can adapt to Agilent 1200 Series LC (or 1100 Series LC) and 1200 Series RRLC systems. You can choose the appropriate combination based on resolution, speed of analysis, LC model, or even mobile phase consumption.

The ZORBAX Eclipse Plus C18 columns coupled with reliable Agilent LC instrumentation and proven automated online derivatization makes high-resolution analysis of free amino acids far easier and more reproducible. For details, please see a related poster.

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