The Modern Student lclborotory:
HPLC Identification of Amino Acids in Unknown Dipeptides A Derivatization with 9-Fluorenylmethyl Chloroformate and HPLC Charles H. Clapp and James S. Swan Bucknell University, Lewisburg, PA 17637 James L. Poechmann Williamsport High Schwl, Williamsport, PA 17701 High performance liquid chromatography (HPLC) has bemme one of the most powerful and versatile tools of biochemical research Incorporating this technique into biochemistry laboratory courses is an important challenge. In this paper we will describe an experiment in which students use HPLC to identify the amino acids in unknown dipeptides. Since most amino acids do not absorb UV light, detecting these compounds requires derivatization with a chromophoric group. A large number of derivatives have been investigated, and their relative advantages have been discussed in recent reviews (1,2).We chose the method of Einarsson et al. (3)in which amino acids are treated with 9-fluorenylmethyl chloroformate (FMOC) to provide the corresponding FMOC derivatives.
Preparation of FMOC Derivatives Standard amino acid solutions of 0.5 mM are prepared in 0.2 M borate buffer, pH 7.7. The derivatization reaction is started bv addition of 0.5 mL of 15 mM 9-fluorenvlmethvl chlomfo&ate in reagent-grade acetone to 0.5 m ~ ' bamiAo f acid solution in a 15-mLcentrifuee tube. ARer about 40 s. 2 mL of hexanes is added. Then the tube is stoppered with a plastic cap and shaken. The tube is briefly centrifuged to speed the separation of the layers, and the hexanes layer is removed by pipet. The aqueous layer is extracted with two additional 2-mL portions of hexanes. ARer the fmal extraction, the lower layer is carefully transferred by pipet to a vial and stored in a refrigerator for HPLC. Storage for several weeks leads to no detectable decomposition, except in the case of histidine.
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Peptide Hydrolysis and Derivatization of the Products
FMOC *H* *Ct
This reaction is attractive for a laboratory murse because it is wmplete in less than 1min. FMOC derivatives are usually detected by fluorescence. However, in this experiment we use the less sensitive but more commonly available UV detector, taking advantage of the stmng absorption of FMOC derivatives at 254 nm. Experimental Procedures Materials FMOC derivatives of alanine, arginine, glycine, isoleucine, leucine, methionine, pmline, phenylalanine, and valine were obtained from Sigma Chemical Company. The other FMOC derivatives referred to in the paper were prepared from the corresponding amino acids (Sigma) and 9-fluorenylmethylchlomformate (Sigma) by the procedure described below. The unknown dipeptides were also obtained from Sigma. HPLC-grade acetonitrile and methanol were obtained from Burdick and Jackson. A122
Journal of Chemical Education
Approximately 2 mg of unknown dipeptide is dissolved in 0.2 mL of 6 M HCl. The resulting solution is sealed in 6-mm-0.d. pyrex tubing and heated for 18-24 h in an oven at 110-120 '6.After cooling, a 50yL aliquot of the hydmlysate is transferred to a 1.5-mL Epindorftube and mncentrated to dryness in a Speed-Vaccentrifuge (Savant Instruments). The residue is dissolved in 0.2 mL of 50 mM borate buffer, pH 9.0. Then it is diluted with 2.0 mL of 0.2 M borate buffer, pH 7.7. The pH of the resulting solution should be checked and adjusted if necessary to between 7.5 and 8.0 by addition of HC1 or NaOH. Then a 0.5-mL aliquot of this solution is transferred to a 15-mL centrifuge tube and treated with 0.5 mL of 15 mM 9-fluorenylmethylchloroformate in acetone. After 40 s the reaction mixture is extracted with three 2-mL portions of hexanes as described above, and the aqueous layer is analyzed by HPLC. High Performance Liquid Chromatography
The minimum requirements are a binary HPLC system with a UV detector operating at 254 nm. We used a Beckman system with two 114 M pumps, a 421 controller, a 210A injector with a 20pL sample loop, and a 1064 variable wavelen&h detector. FMOC derivatives were s~paratedon a 250 x 4.6 mm Alltech Adsorbosohcrc 5-u C18 column (cataloe no. 287062). Mobile has;^ was 10:40:50 mixture o? acetonitrile, methanol, and pH 4.2 acetate buffer. Mobile Phase B was a 50:50 mixture of acetonitrile and pH 4.2 acetate buffer.
The Modern Student laboratory: HPLC some variability due to residual HC1 in the hydrolysate. Consequently, the pH should always be checked and adjusted if necessary to 7.58.0. Einarsson et al. (3) reported that the use of phosphate buffer in the derivatization caused a large impurity peak in the chromatogram when fluorescence detection was used. When we substituted phosphate buffer for borate buffer, no impurities were detected a t 254 nm. However, the yields of the FMOC derivatives were decreased, and the yield of the product of FMOC hydrolysis (see below) was increased relative to the results obtained with borate buffer. The Extraction Procedure
10
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20
time (mi")
Chmmatoaram o-f a- mixture acids. The.detector sen.- - o -f FMOC ..amino .-... ....... s t v l y was 0.3 AUFS. ana tne chromatogram was ponea with a Spectra Pnysln SP4290 Integrator. wnich was set at an anemat on of 8. Other chromatographic parameters and the preparallon of the standard mixture are described in the Experimental Procedures sec~
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~
The acetate buffer was prepared by adding 3.00 mL of glacial acetic acid and 1.00 mL of triethylamine to 900 mL of deionized water. The pH was adjusted to 4.2 with sodium hydroxide solution. Then this was diluted to l L in a volumetric flask. The elution program used is described below. A 3-min isacratic elution with 100%A A 9-min linear gradient fmm 100%A to 100%B An 11-minisaeratic elution with 100%B A flow rate of 1.3mumin
The final isocratic elution must be extended to 20 min if histidine and lysine are present. A standard mixture of the FMOC derivatives that are not commercially available was prepared by combining 50-uL aliauots of the solutions obtained from each individuai derivative preparation. A 150-pL aliquot of this mixture was combined with 50 pL of a solution that contained 0.22 mg/mL of each commercially available FMOC derivative. A 20+L injection of the resulting solution gave the chromatogram in the figure. Results and Discussion Monitoring the pH
Conversion of amino acids to their FMOC derivatives proceeds rapidly in borate buffer a t pH 7.7 (3).As discussed by previous workers, controlling the pH is critical for this reaction (3, 4). Most importantly, the rate of the reaction decreases sharply below pH 7 (4). The buffering capacity of borate a t pH 7.7 is poor. If the residue from the mixture of amino acid hydrochlorides obtained from the peptide hydrolysis is dissolved directly in 0.2 M borate buffer, pH 7.7, the pH ofthe resulting solution is below the acceptable range for the derivatization. The procedure described in the Experimental Procedures section usually yields a solution of pH 7.5-8.0, but there is
After the derivatization reaction, the excess FMOC i~ removed bv extraction with hexanes. Betner and Foldi have developid a n alternative procedure in which excess FMOC is consumed by reaction with 1-adamantylamine (3.We decided to use the extraction procedure because smallscale extraction in a centrifuge tube is a useful technique in biochemical research that is usually not encountered by students in other chemistry laboratory courses. The Separation
Achromatogram of a mixture of FMOC amino a
