GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS AS ETHYL CHLOROFORMATE DERIVATIVES.
MICHAEL R. SCHILLING, HERANT P. KHANJIAN, & LUIZ A. C. SOUZA
1 APPENDIX 1: AMINO ACIDS THAT MAY BE FOUND IN ACID HYDROLYSATES OF PROTEINS
2 APPENDIX 2: EXPERIMENTAL PROCEDURE
2.1 VAPOR-PHASE ACID HYDROLYSIS
As a precaution, gloves should be worn during all sample handling steps. Samples were weighed onto small, V-shaped aluminum pans to the nearest 0.1 μg using a microbalance. Prior to analysis, the samples of saliva, guano, and blood were dried at room temperature for 48 hours. Samples ranged from 50 to 200 μg. After being weighed, samples were transferred to 1 ml glass sample tubes by gentle tapping. Sample tubes (a maximum of 10) were placed inside a 25 ml glass hydrolysis chamber. Accompanying the sample tubes were an empty tube that served as a blank, a tube containing rabbit skin glue that served to monitor the yield of the hydrolysis reaction, and a tube containing 10 μl of a calibration standard. To each tube was added 2 μl of L-norleucine internal standard solution (1,500 ppm in 0.1M HCl).
A 200 μl aliquot of 6M HCl was introduced to the bottom of the chamber, which was subsequently capped tightly with a Mininert valve. To minimize losses of amino acids during hydrolysis due to oxidation, the concentration of oxygen inside the chamber was reduced by thrice alternating between vacuum for 30 seconds (supplied by a mechanical pump) and nitrogen purge for 30 seconds. Access to the atmosphere inside the chamber was provided by penetrating the septum of the Mininert valve with a 20 gauge needle attached to a vacuum hose and a gas-switching valve. The chamber was evacuated for a fourth and final time prior to hydrolysis, then kept at 105°C in an oven for 24–36 hours to fully hydrolyze the samples (Halpine 1992).
After the chamber was cooled, the Mininert valve was unscrewed slowly to release the vacuum, and the outsides of the tubes were wiped dry. To eliminate the last traces of liquid, the tubes were reinserted into a clean, dry chamber, and vacuum was applied for 30 minutes while the chamber was maintained at 50°C. Remaining traces of acid were removed by washing each tube with 15 μl of water and drying them at 50°C in a gentle stream of nitrogen. The hydrolysate was dissolved in 120 μl of 25mM HCl.
An aliquot of the hydrolysate was transferred to a 1.2 ml flat-bottomed vial. It is advantageous to retain a portion of the hydrolysate in the event that reanalysis is required. Experience has shown that 60 μl of hydrolysate gives adequate results for all but submicrogram paint samples. The aliquot must contain less than 100 μg of amino acids (Hušek 1991). The solution was adjusted with 25mM HCl to give a final volume of 60 μl.
After mixing the 60 μl hydrolysate aliquot with 32 μl of ethanol and 8 μl of pyridine, 5 μl of ethyl chloroformate was added, and the vial was shaken gently for 5 seconds until evolution of carbon dioxide subsided. A 100 μl aliquot of 1% ECF in chloroform was added to extract the amino acid derivatives. After shaking, the vial was gently tapped to facilitate separation of the two layers (Hušek 1991).
The chloroform layer (usually, but not always, the lower of the two) was transferred to a flat-bottomed vial containing 100 μl of chloroform and 10–20 mg of anhydrous sodium sulfate (to remove traces of water) and then transferred to a third clean, flat-bottomed vial. The solution of ethanol:pyridine:acid hydrolysate was extracted a second time with 100 μl of chloroform. The chloroform layer was transferred to the vial containing sodium sulfate, and the chloroform solutions were combined into the third vial.
The derivatives were concentrated by evaporating the chloroform to dryness under a slow stream of nitrogen. This step must be performed with great care to minimize loss of volatile derivatives. Evaporation of the chloroform is nearly complete when condensation forms on the vial; at this instant the solvent remaining inside the vial is greatly enriched in pyridine, as evidenced by the odor. Complete evaporation may be facilitated by warming the bottom of the vial between the fingertips. A small volume of benzene is added to the vial to dissolve the derivatives prior to injection (5–10 μl was the typical amount for microsamples from objects, whereas 100 μl may be used for larger paint samples and for pure proteinaceous materials). Approximately 20 minutes were required for derivatization and sample preparation. The benzene solutions of the derivatives are stable for at least 2 days when stored at 4°C.
2.3 GAS CHROMATOGRAPH INSTRUMENTAL PARAMETERS
A Hewlett Packard 5890A gas chromatograph equipped with a flame ionization detector, split/splitless inlet, and Pascal ChemStation was used for this study. Amino acid derivatives were separated on a 15 M × 0.25 mm fused silica capillary column coated with a 0.25 μm film of HP-INNOWAX. Helium was used as carrier gas at a nominal linear velocity of 95 cm/second (measured at 70°C and an inlet pressure of 17 psig); the split vent flow rate was 50 ml/minute and the septum purge flow rate was 0.4 ml/minute. Samples were injected into the inlet held at 240°C, with a purge delay of 30 seconds. The oven, maintained at 70°C for 1 minute, was heated at a rate of 27°C/minute to 250°C and held at that temperature for 9.33 minutes. The detector, set to 240°C, was adjusted to detect peaks with widths greater than 0.01 minute. The total analysis time was 17 minutes.
Amino acid calibration standards were prepared by serial dilution of a standard mixture obtained from Sigma Chemical Co. (no. A2161); nominal concentrations ranged from 100 ppm to 0.1 ppm. L-norleucine was added as an internal standard at a concentration of 150 ppm. Aliquots of the calibration standards (10 μl) were derivatized in the same manner as the samples. The derivatives were dissolved in 100 μl of benzene prior to injection. Calibration curves for all analytes exhibited correlation coefficients of 0.999 or better. Reproducibility of the method was established for nine replicate analyses of a solution of Knox gelatin. Relative standard deviations were 1–7% for all amino acids except methionine, which was 24%. Methionine is unstable under conditions of acid hydrolysis unless a preservative is present.
3 APPENDIX 3: TROUBLESHOOTING
Although the ECF method has proven to be extremely reliable, a certain amount of routine troubleshooting was needed to obtain consistent results on a daily basis. It is our experience that, when properly maintained, a column should last for more than 300 injections.
The primary concern was the effect of capillary column and liner activity on peak areas and detector response factors. Reaction of the fused silica and quartz surfaces with water and oxygen produces silanol groups that can undergo hydrogen-bonding with certain analytes. Hydroxy-substituted amino acid derivatives (serine, threonine, and hydroxyproline) were most severely affected by activity because ECF does not derivatize every active hydrogen. Activity was minimized by: (1) installation of oxygen and moisture traps in the carrier gas line; (2) carefully separating the chloroform layer from the aqueous layer and drying the chloroform layer over sodium sulfate to remove water from the injection volume; (3) changing the inlet liner every 30–50 injections to keep semivolatile active contaminants out of the column; (4) baking the column for 4 hours at 250°C if peak tailing is observed.
Peak resolution is another consideration in maintaining optimum system performance. In developing a temperature program for a particular column the problem of “tunable selectivity” is encountered (the phenomenon in which elution order changes with heating rate). This effect is evident for the triplet of methionine, serine, and glutamic acid in figure 2. As a column ages, the resolution between serine and glutamic acid decreases. Improved resolution may be obtained by increasing the heating rate in the temperature program by 2°C or more. Of course, this effect cannot be maintained indefinitely; column replacement will be necessary when improvement in resolution between serine and glutamic acid is obtained at the expense of a decrease in resolution between methionine and serine.
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SOURCES OF MATERIALSEthyl chloroformate 18,589–2:
Aldrich Chemical Co. Inc., 1001 W. St. Paul Ave., Milwaukee, Wisc. 53201C32 Microbalance with remote weighing unit
Cahn Instruments, Inc., 16207 S. Carmenita Rd., Cerritos, Calif. 90701HP 5890 Gas Chromatography with HP 5895 GC ChemStation; HP-INNOWAX capillary column, 0.25 mm ID, 0.25 μm film thickness, 15M length, 19091N–131
Hewlett Packard Co., 2850 Centerville Rd., Wilmington, Del. 19808DB-WAX capillary column, 0.25 mm ID, 0.25 μm film thickness, 15M length, 122–7012; DB–1701 capillary column, 0.25 mm ID, 0.25 μm film thickness, 15M length, 122–1312
J and W Scientific, 91 Blue Ravine Rd., Folsom, Calif. 95630–4714OV–1701 capillary column, 0.25 mm ID, 0.25, μm film thickness, 15 M length, 415–2502
Ohio Valley Specialty Chemical, 115 Industry Rd., Marietta, Ohio 457506N hydrochloric acid–sequanal-grade 24309; HPLC-grade water 51140 pyridine 27530; 25 ml screw-top septum vials 13074; Mininert valves for 25 ml screw-top vials 10130
Pierce Chemical Co., 3747 N. Meridian Rd., Rockford, Ill. 61105–0117,2, 20 and 100 μl Pipetman micropipettors
Rainin Instrument Co., Inc., P. O. Box 4026, Woburn, Mass. 01888–4026AA calibration standard A2161
Sigma Chemical Co., 3050 Spruce St., St. Louis, Mo. 63178anhydrous 200 proof ethanol E1028
Spectrum Chemical Mfg. Corp., 14422 S. San Pedro St., Gardena, Calif. 90248
MICHAEL R. SCHILLING earned his B. S. (1983) and M.S. (1990) in chemistry from California State Polytechnic University, Pomona. He has worked at the Getty Conservation Institute since 1983 and presently holds the position of associate scientist. He has been active in the examination of painted museum objects, pigment identification, binding medium analysis, and analysis of volatile organic compounds in the museum environment. He has also been involved in a number of special GCI collaborative projects in Egypt, China, and Israel. Address: Getty Conservation Institute, Scientific Program, 4503 Glencoe Ave., Marina del Rey, Calif. 90292.
HERANT P. KHANJIAN received his B.A. in chemistry from California State University, Northridge. His research interests involve the detection and identification of binding media found in art objects using gas chromatography and infrared spectroscopy. Address as for Schilling.
LUIZ A. C. SOUZA received his B.S. (1986) and M.Sc. (1991) in chemistry from the Federal University of Minas Gerais, Brazil. In 1987–88 he held an internship at the Scientific Department of the Royal Institute of Cultural Properties (IRPA) in Brussels, Belgium. Since 1989 he has been teaching and researching at the Center for Conservation and Restoration of Cultural Movable Properties, Federal University of Minas Gerais, Brazil. His research interests are preventive conservation in the tropics and the history of techniques and technology of paintings and polychromed sculptures. During 1992 and 1993 he had a research fellowship in the Scientific Program at the Getty Conservation Institute, working with scientific analysis and identification of materials of baroque and rococo polychromed sculptures from Minas Gerais, Brazil. Address: Luiz A. C. Souza, CECOR, EBA, Universidade Federal de Minas Gerais, 31270–901, Belo Horizonte, MG, Brasil.