Solid-phase peptide synthesis (SPPS) was invented by Bruce Merrifield1, and he was rewarded with the 1984 Nobel Prize in chemistry for this major advance. The advantage of this technique is that peptides can be built efficiently with a standard reaction procedure that is well-suited for automation. After attachment of the first protected amino acid onto the resin with its carboxyl function, the excess reagents are washed from the resin, the protecting group on the amino function is removed, and the resin is washed again. The next protected amino acid can then be coupled, and these steps are repeated until the required peptide sequence has been built up. Cleavage of the peptide from the resin produces the crude peptide, which is then purified by preparative chromatography to achieve high final purity.

Merrifield developed SPPS using tert-butyloxycarbonyl (BOC)-protected amino acids, but more recently the Fluorenylmethoxycarbonyl (FMOC)-protecting group was introduced2,3, and these have become the raw materials of choice for the preparation of synthetic peptides. The main advantage of the FMOC strategy is that the removal of the protecting groups, as well as the cleavage from the resin, are safer and scalable to manufacturing scale. Using this methodology, peptides can be prepared in a few days with high yield from lab scale to manufacturing scale (kg).

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The final purity of the peptide produced is directly affected by the chiral purity of each amino acid. For example, if every coupling is made with FMOC amino acids pure at 98%, a 30-amino-acid peptide would have a final purity of approximately 55%. But if those chiral amino acids are pure at 99.5%, we would obtain final purity of approximately 86%. Currently, for the 19 most common commercially available natural FMOC-protected amino acids, the expected chiral purity is 99.5% with less than 0.5% of the other enantiomer. This level of precision can be achieved with very few analytical techniques, chiral HPLC being one of them. The main advantages of chiral HPLC over other analytical techniques are speed, detection level and ease of use. There have been few publications describing the chiral separation of protected amino acids using various chiral stationary phases (CSPs) and separation modes4.

In this article, we will describe the chiral separation of the 19 most common, commercially available, FMOC-protected amino acids for SPPS, using polysaccharide-based CSPs5 under reversed-phase (RP) mode, which is amenable to LC/MS analysis and routinely used by peptide chemists to analyze fractions and peptide purity.


Click to Enlarge - Table 1

Five polysaccharide-based CSPs, depicted in Figure 1, were explored in the RP HPLC enantioseparation of the 19 most common FMOC-protected α-amino acids. Due to the acidic nature of FMOC amino acid derivatives and based on our previous extensive screening work in RP mode6, we decided to use trifluoroacetic acid (TFA) or formic acid (FA) as additives with acetonitrile (ACN) or methanol (MeOH) organic modifiers. These mobile phases are arguably the most widely used in RP mode.

All analyses were performed using an Agilent 1100 series LC system (Agilent Technologies Inc., Palo Alto, Calif.) equipped with quaternary pump, inline degasser, multi-wavelength UV detector and autosampler. Phenomenex Lux analytical columns were used for analysis. The HPLC column dimensions were 250 x 4.6 mm ID and all columns were packed with 5 μm particles. FMOC-protected L and D amino acids were obtained from Bachem (Bubendorf, Switzerland) and all solvents were purchased from EMD (San Diego, Calif.). For the screening, the HPLC flow rate was set to 1 mL/minute and the temperature was ambient with UV detection set at 220 nm. The injection volume was 5 μL and the sample concentration was 2 mg/mL in methanol or acetonitrile (pure FMOC amino acids enantiomer L and D were mixed in a ratio of 2:1).

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All the analyses were performed in isocratic mode with run times less than 25 minutes. Initial screening was performed with 0.1% TFA/ACN in a volume ratio of 40:60. For retention time (Rt) less than six minutes and resolution (Rs) less than 1.5 (no baseline resolution), the amount of ACN was decreased in order to improve retention and chiral recognition. If no chiral separation was obtained with ACN as modifier, columns were screened with 0.1% FA/MeOH in a volume ratio of 20:80. In general, we observed more retention with TFA as an additive than with FA when using ACN as modifier and, as expected, ACN elution power is stronger than MeOH. Quite a few FMOC amino acids can be separated with either ACN or MeOH as modifier.

Click to Enlarge - Table 2

Table 1 summarizes all the separations and chiral recognition observed after performing RP screening using the protocol described above. As shown in Table 1, all the amino acids tested were successfully resolved on at least one of the five polysaccharide-based CSPs screened. In the case of Ile, Leu, Met, Phe and Val FMOC derivatives, baseline resolution was achieved on the five CSPs. Under our RP screening protocol, Cellulose-2 was the most successful phase, with 18 chiral recognitions, followed by Cellulose-3, as represented in Figure 2.

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Table 2 describes some of the optimal separations observed for each FMOC amino acid. For both enantiomers, retention time, alpha value, resolution achieved and order of elution are shown. All the separations reported are baseline-resolved and the run time is less than 25 minutes.

Interestingly, Trityl (Trt) side-chain-protected FMOC amino acids such as His, Asn and Cys derivatives are more challenging to separate and baseline resolution is only achieved using Cellulose-2, Cellulose-3 and Cellulose-1, respectively. Representative chiral separation of FMOC-Asp(OtBu)-OH and FMOC-Tyr(tBu)-OH are shown in Figure 3.


Five different polysaccharide-based chiral stationary phases were explored in reversed phase HPLC for the separation of the 19 most common FMOC-protected amino acids. Under our RP screening protocol, Cellulose-2 was the most successful chiral stationary phase with 18 chiral recognitions (15 baselines resolved), followed by Cellulose-3.

All FMOC amino acids evaluated were fully resolved (Rs greater than 1.5) in less than 25 minutes by RP HPLC. TFA acidic additive and Acetonitrile as organic modifier are the best combination for successful chiral separation of FMOC α-amino acids derivatives.

This study demonstrates that with a proper screening protocol, most of the FMOC-protected amino acids can be resolved with five polysaccharide-based chiral stationary phases.



  1. Merrifield R. B. J. Am. Chem. Soc., 1963, 85, 2149.
  2. Carpino L.A. and Han G.Y. J. Org. Chem., 1972, 37, 3404.
  3. Bruckdorfer T., Marder O. and Albericio F. Current Pharmaceutical Biotechnology 2004, 5, 29.
  4. Li Y. H., Baek C-S., Jo B. W., Lee W. Bull. Korean Chem. Soc. 2005, 26, 998.
  5. Chankvetadze B. J. Chromatogr. A, 2012, 1269, 26.
  6. Peng L., Jayapalan S., Chankvetadze B. and Farkas T. J. Chromatogr. A 2010, 1217, 6942.