Rapid Separation and Detection of Amino Acids by HPLC-HPIMS TM In addition to being the structural units that make up proteins, amino acids play important roles in gene expression, cell signaling, and immunity; also serving as metabolic intermediates in the synthesis of neurotransmitters and other biomolecules. 1,2 Due to their biological significance, efficient separation and detection of amino acids is essential for a variety of applications in the medical, pharmaceutical, and food industries. 3-5 Most High-Performance Liquid Chromatography (HPLC) methods for amino acid analysis (including commercial amino acid analyzers) require long run times and involve some form of derivatization to enhance separation or detection. 1-6 Like many compounds, underivatized amino acids lack a strong chromophore, fluorophore, or electroactive moiety for photometric, fluorometric, or amperometric detection, respectively. 1,3,4,7 In addition to being time consuming and inconvenient for the analyst, issues with derivative instability, side reactions, and reagent interferences can result in the presence of extra peaks in the chromatogram that can complicate interpretation and reduce analytical confidence. 1,3-6 For these reasons, direct detection is preferred whenever possible. Figure 1: Excellims IA 3100 HPLC-HPIMS TM displayed with a Shimadzu Prominence UFLC system. Excellims award-winning IA3100 (Fig. 1) provides a robust and efficient alternative for amino acid analysis that brings the rapid orthogonal separation and detection capabilities of High- Performance Ion Mobility Spectrometry (HPIMS TM ) to any existing HPLC system. This additional dimension of separation allows the analyst to condense long chromatographic methods by performing a partial separation with reverse phase (RP) chromatography and completing the separation in the gas-phase using ion mobility spectrometry. Using this technology, it is now possible to obtain complete separation of all 20 common amino acids in less than 15 minutes twice as fast as state-of-the-art amino acid analyzers. 9,10 With HPLC-HPIMS TM, as the sample elutes from the LC column, it flows through an adjustable flow splitter that diverts a small portion of the eluent (2 to 8 μl min -1 ) to the electrospray ionization (ESI) source, which converts the sample to gas-phase ions. The ionized analytes then enter the drift tube of the HPIMS TM and separate according to gas-phase mobility by the action of two opposing factors: 1) A strong electric field pulls the ions toward the detector with a force proportional to the charge on the ion; 2) The ions are slowed down to different extents by collisions with molecules of drift gas as they travel down the drift tube. The probability of these collisions is proportional to the cross-sectional area of the molecule reflecting its structural shape. Thus, to a first approximation, the mobility separation is dependent on the charge and crosssectional area (i.e., shape) of the ion. A Faraday plate detector at the end of the drift tube records the time at which each ion hits the detector and converts the current generated into an intensity value that is displayed on the IMS spectrum. The drift time of each ion can then be compared with standard or user-defined database values to determine the identity of the compound.
To demonstrate the utility of combining HPLC and HPIMS TM for orthogonal separation and detection, consider the separation that each individual method provides by itself (Fig. 2, 3). Figure 2: RP-HPLC separation of 20 common amino acids (100 µm each) using a Thermo Aquasil C18 column (100 x 4.6 mm, 5.0 µm). Method: H 2 O (A), ACN (B); Step Gradient: Hold 0% B for 3.5 min, 5% B for 2.5 min, 18% for 4 min, 35% for 4 min, 65% for 4 min, linear gradient to 100% B over 2 min and hold for 3 min; Injection: 5.0 µl). Table 1: Amino Acid Separation and Detection Methods Method Separation Detection - Requires derivatization IEC for UV or fluorometric + Standard column detection + Efficient - Requires desalting of + Reproducible eluent prior to ESI- HPIMS TM, ELSD, or CLND 12 IPC Specialty Column RP + Standard RP column 5,6,7 - Long reequilibration 2,6,7 - Poor reproducibility and intersystem transferability 6 - Ion-pairing agents toxic and expensive! + Efficient + Reproducible - Expensive - Single-purpose - Insufficient retention of polar amino acids 2,7,10 - Poor sensitivity direct UV detection 5 - Ion-pairing agents suppress ionization, which decreases sensitivity and complicates quantitation 12 - Poor sensitivity direct UV detection 5 - Often designed to be used with inorganic buffers that are incompatible with ESI-HPIMS TM, ELSD, and CLND - Eluent compatible with ESI-HPIMS TM, ELSD, and CLND Complete separation of 20 amino acids by HPLC takes approximately 30 minutes and can be accomplished using ion exchange chromatography (IEC), ion-pairing chromatography (IPC) on a RP-e C18 or porous graphitic column, or other methods that employ specialty amino acid columns. 7,10,11 Traditional reverse phase chromatography is ineffective as it provides insufficient retention of the most polar amino acids. 2,7,10 In the chromatogram in Figure 2, the first ten amino acids elute in under two minutes and seven of the ten co-elute as a single LC peak. (See Table 1 for a comparison of advantages and disadvantages of the separation and detection options for each type of chromatography). Although this separation is not trivial, it is generally recognized that the challenge of amino acid analysis lies not in their separation, but in their detection. 1,3 The detector that is most commonly used for amino acid analysis is the electrochemical detector; however, this method of detection requires an experienced operator. In addition, the number of amino acids that can be detected at carbon-based electrodes is limited and the electrodes are prone to fouling. 1,3 UV and refractive index detectors are also commonly used, but they respond to amino acids with poor sensitivity and changes in solvent composition elicit a strong response which further clouds the weak analyte signal. 1,3,4,7 These strong solvent responses can be seen in Figure 2 at 8, 12, 16, and 21 minutes. For this reason, these detectors are generally considered to have poor compatibility with step-gradient elution and are incompatible with all other gradient elution profiles. 3,5,7 In contrast, direct detection by ESI-HPIMS TM is highly sensitive, reproducible, and has the added benefit of providing a rapid orthogonal separation based on size-to-charge ratio (Fig. 3). While many of the amino acids can be rapidly separated and detected using HPIMS alone, some amino acids exhibit ion mobility coincidences, necessitating a complementary method to resolve them. Thus, the addition of the gas-phase separation capabilities of HPIMS TM to HPLC allows for greater flexibility in the chromatographic separation as compounds that coelute in the HPLC can be resolved by the HPIMS TM detector. One capability that distinguishes ion mobility from other orthogonal separation techniques is its ability to rapidly separate isomers and other molecules with slight structural differences that do not separate by HPLC. For example, the
HPIMS TM separates leucine and isoleucine as shown below (Fig. 4) with just a few second acquisition. Figure 3: HPIMS TM separation of 20 common amino acids on Excellims GA2100 stand-alone ion mobility spectrometer equipped with a Directspray TM ionization source. Sample: 100 µm each in 50:50 MeOH/H 2 O, Flow Rate: 1.0 µl min -1 ; Source: 3.0 kv; Drift Tube: 8.0 kv; Desolvation/Drift Region Temp: 180 o C; Drift Gas: 2.0 L min -1, air; Exhaust Flow: 1.8 L min -1. Therefore, this isomer capability enables complete two-dimensional separation of amino acids using a standard reverse phase column without the need for ion-pairing agents. Ion-pairing agents are generally added to increase retention of polar compounds that are poorly retained in reverse phase columns, thus enabling their separation. 2,4,5 Longchain perfluorinated carboxylic acids are often used as ion-pairing agents as they are volatile enough to be used with ELSD, CLND, and instruments that rely on electrospray ionization for sample introduction, such as MS and HPIMS TM. 2,4 However, ion-pairing chromatography has a number of major drawbacks with respect to efficiency, reproducibility, and detection sensitivity. 2,6,7 Depending on the chain length of the ion pairing agent used, it can take up to 3 hours for the ion-pairing agent to re-equilibrate with the stationary phase after each run, which severely limits throughput. 2,7 Furthermore, variation in the extent of re-equilibration results in poorly reproducible retention times, complicating data analysis. These ion-pairing agents have also been shown to reduce ionization efficiency by up to 80%, which negatively impacts detection sensitivity. 12 However, if ESI-HPIMS TM is used for detection, these polar amino acids can be separated by gas-phase ion mobility, eliminating the need for ion-pairing agents Figure 4: HPIMS TM separation of leucine and isoleucine with Excellims GA 2100 stand-alone ion mobility spectrometer equipped with a Directspray TM electrospray ionization source. Sample: 100 µm each in 80:20 MeOH/H 2 O, Flow Rate: 1.0 µl min -1 ; Source: 2.4 kv; Drift Tube: 10.0 kv; Desolvation/Drift Region Temp: 180 o C; Drift Gas: 1.4 L min -1, air; Exhaust Flow: 1.2 L min. -1 Figure 5: A three-dimensional HPLC-HPIMS TM spectrum showing the separation of 20 amino acids. HPLC retention time is on the y- axis, ion drift time is on the x-axis, and signal intensity is on the z- axis. The HPLC separation conditions are identical to those listed in Figure 2. The UV absorbance detector trace at 190 nm (blue) and 210 nm (red) is displayed on the left. The HPIMS TM separation was conducted on Excellims IA3100 equipped with an adjustable flow splitter and an Infusion TM electrospray ionization source. Sample Flow: 4.0 µl min -1, Sheath Flow: 2.0 µl min -1 ACN; Source: 2.4 kv; Drift Tube: 10.0 kv; Desolvation/Drift Region Temp: 180 o C; Drift Gas: 2.0 L min -1, air; Exhaust Flow: 1.2 L min -1. and the problems that come with using them. This simplification of the chromatographic separation conditions results in a more robust and efficient amino acid separation. In addition, the mobility data from the HPIMS TM provides an additional metric for compound identification that enhances detection confidence. Since HPLC separations are carried out on a timescale of minutes, whereas IMS separations are completed in milliseconds, dozens of IMS spectra can
be acquired across each chromatographic peak. These IMS slices can be stacked in-parallel to create a three-dimensional contour plot (Fig. 5). Each peak in the contour plot has a unique drift time and retention time that can be used to identify each amino acid in the spectrum. This identification is readily accomplished using the user-defined compound library feature in Excellims VisION Control software. To populate this library, run standards of each amino acid and enter a compound identifier with the calibrated drift time. The software will automatically assign the peaks in the spectrum based on this library data and will even perform quantification if calibration data is supplied. Most of the samples for which amino acid analysis is of interest are present in complex biological or other matrices. Thus, the practical utility of HPLC-HPIMS TM as a method for amino acid analysis is dependent on its ability to handle samples in complex matrices, such as fermentation and cell culture media. The efficiency of these bioprocesses is currently limited by the availability of on-line monitoring data that can guide decision making in real time. Lysogeny broth (often referred to as Luria Broth or simply LB broth) is a common nutrient mixture used in E. coli cell culture work. Although a variety of formulations exist, a typical recipe contains 10 g L -1 NaCl (~170 mm), 10 g L -1 tryptone, and 5 g L -1 yeast extract. Tryptone is produced by the tryptic digestion of casein protein and serves as an amino acid fuel source for growing bacteria. The digestion products are predominantly free amino acids and some small peptides. Yeast extract is produced by the autolysis of yeast and provides an assortment of free amino acids, peptides, carbohydrates, vitamins, and other organic compounds necessary for growth. The depletion of these nutrients and accumulation of waste products are essential parameters influencing the success of these processes and must be monitored and managed to optimize process efficiency. As Figure 6 shows, the integrity of the HPLC-HPIMS TM method for amino acid analysis is preserved despite the complex biological matrix of the cell culture medium. A variety of small molecules and tryptic peptides from the yeast extract and the tryptone can be seen in the inset, but they do not Intensity Scaling: 5.8x Figure 6: A three-dimensional HPLC-HPIMS TM spectrum of Lysogeny broth obtained on an Excellims IA3100 interfaced with a Shimadzu Prominence UFLC. The instrument parameters are the same as those used in Figure 5. No modifications to the HPLC or HPIMS TM separation conditions were necessary to accommodate for the matrix. appear to overlap with the amino acid signals. The Excellims IA3100 is a promising alternative to traditional HPLC methods for amino acid analysis that involve derivatization. Not only does it deliver results in half the time of industry-leading amino acid analyzers, the orthogonal separation provided by HPIMS TM enhances detection confidence by providing an additional metric for compound identification. This orthogonal separation also enables greater matrix compatibility without tedious and time-consuming adjustments to HPLC methods. Authors: William J. Warren, Anthony J. Midey, Adam M. Graichen, and Ching Wu*, Excellims Corp., Acton, MA
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