Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Gas-Liquid Chromatography of Amino Acids
General Sample Preparation Procedure

A specified amount of internal standard, typically norleucine, is added to the protein hydrolysate because it resolves well from all Other Amino Acids, although pipecolic acid, tranexamic acid, and homoarginine have also been used. Some biological preparations require preliminary purification.

Purification of biological preparations. Plasma samples (100–500 µL) are deproteinized by Treatment with sulfosalicylic acid (50 mg/mL of plasma).

1) A Pasteur pipette (40×3 mm) is packed to a height of 10 mm with Dowex-50W ion-exchange resin (8% cross-linkage, 200–400 mesh, H⁺-form); small Glass wool plugs are placed at the top and bottom of the resin bed.

Add a known amount of internal standard to the sample, acidify to pH 1, and load onto the Column. Wash out non-cationic impurities with 2 mL of deionized Water. Amino acids are eluted with 2 mL of 4 M NH₄OH at a flow rate of ~1 drop per 5–10 s. The eluate is evaporated to dryness [96].

2) The amino acids are redissolved in 0.1 M HCl and applied to a column (150×15 mm) packed with 7 mL of Amberlite CG-120 cation-exchange resin (H⁺-form, 100–120 mesh). Wash twice with 10 mL of deionized water. Elute the amino acids with 2×10 mL of 7 M NH₄OH, followed by 5 mL of deionized water. Collect the eluate and evaporate to dryness in vacuo using a rotary evaporator at 60 °C [193].

8.17.1.1. Heptafluorobutyryl amino acid isobutyl esters [231–233]. In most cases, the sensitivity of a flame ionization detector is sufficient for detecting these derivatives. Electron-capture and thermionic detectors, as well as mass spectrometry, have also been employed for specialized Applications [96, 230].

Before and after Esterification, trace amounts of water can be removed from the sample via azeotropic drying by adding methylene chloride and evaporating. Ultrasonic treatment or vortex mixers are used to ensure efficient component mixing during esterification and acylation.

Preparation of alcoholic HCl solutions for esterification. The alcohol should be dried over granular calcium sulfate (~50 g/L) or by refluxing for 3–4 hours over magnesium turnings (with a crystal of iodine added to catalyze the reaction); after decantation, it is refluxed for another 3–4 hours over calcium hydride and distilled.

The required molar concentration of HCl is obtained by passing dry HCl gas into the alcohol until saturation or until the target mass is reached, followed by titration with alkali and dilution to the required volume. Store in small bottles in a cold place.

When a small quantity of reagent is needed for a single sample, it is prepared by adding the appropriate amount of acetyl chloride to the alcohol in the cold (this Procedure requires caution as the reaction is endothermic) and bringing it to the desired volume.

Esterification. The amino acid mixture sample (10–100 nmol of each amino acid) is dried at 50 °C in a Reacti-Vial (Pierce) under a stream of dry nitrogen. Add 3 M HCl in isobutanol (50–100 µL) and incubate at 120 °C for 30 min. After 5 min of heating, shake the reaction mixture on a vortex mixer for 20–30 s and resume heating. Cool to room Temperature. Evaporate the excess reagent in a stream of nitrogen; this step proceeds quickly if the vial is preheated to 50 °C. Traces of water can be removed by azeotropic drying with methylene chloride. Amino acid n-propyl esters were prepared in a similar manner [118].

Acylation. Add heptafluorobutyric anhydride (50 µL) and heat at 150 °C for 10 min. After cooling to room temperature, evaporate the sample to dryness under a stream of nitrogen. Excessive exposure to the nitrogen stream may lead to the loss of certain volatile derivatives.

Dissolve the residue in dry distilled acetonitrile or ethyl acetate (50 or 100 µL). Inject 1–2 µL onto the GLC column. If Histidine is present in the mixture, dissolve the sample in a 1:1 mixture of ethyl acetate and acetic anhydride and heat for 3 min at 150 °C; after cooling to room temperature, dilute with ethyl acetate. This reaction is best performed in a heating block with wells filled with silicone oil to a level matching the liquid height inside the reaction vial, with the upper part of the vial acting as a reflux condenser.

Because leaks in reaction vials can cause sample loss, it is preferable to use Pyrex glass tubes (50×3 mm), which are sealed and subsequently opened at each stage of the procedure.

It is recommended to perform the column analysis as soon as possible after the acylation reaction is completed (no later than 2–3 hours), which is particularly crucial for preserving derivatives of Cysteine, cystine, Methionine, Arginine, and histidine.

Columns. Good Separation and Quantitative determination of heptafluorobutyryl amino acid isobutyl esters have been achieved by several research groups. Although various column types (described below) were used, the elution order remained identical in all cases. Temperature programming modifications can be introduced to improve resolution. The carrier gas flow rate, injection port temperature, and detector temperature are optimized individually for each instrument and column under study.

1. Glass capillary column (25 m × 0.4 mm) coated with a 5% Chromosorb + 15% OV-101 phase mixture. Carrier gas: hydrogen (3 mL/min); makeup gases: nitrogen (30 mL/min), hydrogen (27 mL/min), air (350 mL/min). Column temperature: 90 °C, temperature-programmed increase (4 °C/min). Elution order over 35 min: Ala, Gly, Val, Thr, Ser, Leu, Ile, Nle, Pro, Cys, pipecolic acid, Hyp, Met, Asp, Phe, Glu, Lys, Tyr, Arg, CM-Cys, His, homo-Arg, Trp, Cys-Cys [57, 58].

2. Capillary column (25 m × 0.23 mm) coated with an OV-101 stationary phase (LKB) [96].

3. Glass SCOT capillary column (60 m × 0.5 mm) with an SL-30 stationary phase (SGE) [289].

4. Glass column (3.5 m × 2.5 mm) packed with Gas-Chrom Q (100–120 mesh) coated with 3% SE-30 phase (by weight) (Applied Science Laboratories) [231].

5. Glass column (3.1 m × 2 mm) packed with Chromosorb WHP (100–120 mesh) coated with 3% SE-30 phase [232, 233].

6. Glass column (3.5 m × 2 mm) packed with Gas-Chrom Q (80–100 mesh) coated with 3% SE-30 phase (Supelco) [289].

7. Glass column (6 m × 2 mm) packed with Gas-Chrom Q (80–100 mesh) coated with 3% OV-101 phase [339].