Separation of Amino Acids and Peptides with Supercritical Fluids Chromatography

Figures & data

Figure 1. Classification of 20 common amino acids considering their volume, hydropathy, and chemical classes as well as their charge and polarity ^[11].

Figure 2. Flow scheme of laboratory-scale SFC: V1-V3 – valves, AV – automated valve, PG – pressure gauge ^[57].

Figure 3. Flow scheme of pilot preparative to production scale SFC: 1 - CO₂ tank, 2 - CO₂ cooler, 3 - CO₂ pump, 4 - CO₂ heater, 5 - modifier tank, 6 - modifier pump, 7 - column with piston, 8 - sample tank, 9 - detector, 10 - flow meter, 11 – evaporator ^[76].

Figure 4. Specific mobile and stationary phases in SFC selected according to the polarity of soluble substances ^[54].

Figure 5. Flow chart of method development for SFC in general and for amino acids separations.

Table 1. Chiral separations of amino acids and amino acid derivatives with SFC using different stationary and mobile phases

Compounds	T(°C)	P(bar)	Chiral stationary phases (brand name)	Optimized mobile phase (% v/v)	Ref.
Leu, Val, Ala, Phe, Ile, Asp, Glu, Ser, Thr, Tyr, Cys, Met, Trp	40	200	Chiral diamide-bonded silica, remaining surface silanols were (CSP1) or were not (CSP 2) trimethylsilylated	CO2 with MeOH	^[Citation116]
Nα-FMOC proteinogenic amino acids	40	150	Chiralpak™ ZWIX (+) and Chiralpak™ ZWIX (-)	CO2 with 30% MeOH + 30 mM TEA + 60 mM FA	^[Citation117]
Leu, Met, Ala, Val, Nle, Nva	50–100	80–160	OV-225 L-Val-tert.-butylamide cross-linked with a glass capillary column	CO2	^[Citation118]
111 chiral compounds including heterocycles, analgesics, β-blockers, sulfoxides, DNPyr and CBZ N- protected amino acids and native amino acids	31	100	Chirobiotic™ T, Chirobiotic™ R, and Chirobiotic™ TAG	CO2 with MeOH + H2O + TEA + TFA + glycerol	^[Citation119]
Amino acid esters, amino acids (Pro, Pgl, Phe, Tyr), β-blockers, amines	Room	180	CHIRALPAK™ AD-H and CHIRALCEL™ OD-H	CO2 with 20% EtOH containing 0.1% ESA	^[Citation120]
6 amino acids derivatives (original phosphine-containing α-amino acid esters)	30	150	Lux™ Cellulose-1 and Lux™ Cellulose-2	CO2 with 3–5% EtOH	^[Citation121]
72 racemates including amines, amides, alcohols, amino acids (N-(3,5-dinitroben-zoyl)-DL-leucin) esters, imines, thiols, and sulfoxides.	Room	-	3 polymeric phases (3Me-P-CAP-DP, Naph-P-CAP-DP, and Cl-P-CAP-DP) and commercial polymeric phase (P-CAP-DP)	CO2 with 10% EtOH + 0.1% TFA	^[Citation122]
14 racemates including flavanones, thiazides, and amino acid derivatives (Dansyl-DL-α-amino-n-butyric acid, Dansyl-DL-norleucine, Dansyl- DL-α aminocaprylic acid, Dansyl- DL-phenylalanin)	40	150	Cationic β-cyclodextrin perphenylcarbamoylated derivatives (6^A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-cyclodextrinchloride and 6^A-(N,N-allylmethylammonium)-6-deoxyperphenylcarbamate-β-cyclodextrinchloride) were chemically bonded onto vinylized silica (with MPS)	CO2 with 40 % 2-propanol + 1% HAc	^[Citation123]
Aromatic amino acids (Phe, Tyr, Trp)	35	100	Chirobiotic™ T2	CO2 with 40 % MeOH + H2O (9:1)	^[Citation124]
β-blockers, amines, amino acids (Trp), andN-protected amino acids (N-benzoylAla, N-benzoylLeu, N-benzoylPhe, N-acetylTrp)	40	150	Chiralpak™ ZWIX (+)	CO2 with MeOH + 100 mM FA + 50 mM NH4FA	^[Citation125]
D- and L- FMOC amino acids(Ala, Asp, Glu, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val)	-	-	Lux™ Celulose-1	CO2 with 40% MeOH + 2% FA	^[Citation126]
PTH amino acids(Ala, Pro, Phe, AABA, Asn, Tyr, Val, Nva, Asp, Trp, Ile, Leu, Nle)	5–40	150	Chiralpak™ AD-H and Chiralcel™ OD-H	CO2 with 10% MeOH	^[Citation127]
Racemic mixture of 14 amino acids (His, Lys, Asp, Ser, Thr, p-Glu, Ala, Car, Val, Nva, Leu, Nle, Ile, Phe)	40	150	Chiralpak™ AS-H, Chiralcel™ OD-H, Chiralpak™ IB, Chiralpak™ AD-H, Chiralpak™ IA, Lux™ Cellulose-2, Lux™ Amylose-2, and Lux™ i-Cellulose-5	CO2 with 20% EtOH + 3% TEA	^[Citation128]
17 proteogenic amino acids (Gln, Asp, Phe, Met, Lys, Leu, Ile, His, Thr, Ala, Val, Ser, Pro, Tyr, Arg, Cys, Gly) and 2 non-proteinogenic amino acids (Taurine, Theanine)	25	150	Chiralpak™ ZWIX (+) and Chiralpak™ ZWIX (−)	CO2 with (10–100%) MeOH + 7% H2O + 70 mM NH4FA	^[Citation129]
18 underivatized amino acids (Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val)	35	100	CROWNPAK® CR-I (+)	CO2 with (25–55%) EtOH + 5% H2O + 0.8% TFA	^[Citation130]

AABA- α-Aminobutyric acid, Ala- Alanine, Arg- Arginine, Asn- Asparagine, Asp- Aspartic acid, Car- Carnitine, Cys- Cysteine, Eth- ethionine, Gln- Glutamine, Glu- Glutamic acid, Gly- Glycine, His- Histidine, Ile- Isoleucine, Leu- Leucine, Lys- Lysine, Met- Methionine, Nle- Norleucine, Nva- Norvaline, Pgl- 2-Phenylglycine, p-Glu– Pyroglutamic acid, Phe- Phenylalanine, Pro- Proline, Ser- Serine, Thr- Threonine, Trp- Tryptophan, Tyr- Tyrosine, Val- Valine.

ACHSA- Trans-2-aminocyclohexanesulfonic acid, Cl-P-CAP-DP - (1S,2S)-1,2-bis(2-chlorophenyl) ethylenediamine dihydrochloride, EtOH- Ethanol, ESA- Ethanesulfonic acid, FA- Formic acid, HAc- acetic acid, H₂O- Water, MeOH- Methanol, MPS - 3-methacryloyloxypropyl trimethoxysiliane, Naph-P-CAP-DP - (1S,2S)-1,2-di-1-naphthylethylenediamine dihydrochloride, NH₄FA- Ammonium formate, OV-225 - Polycyanopropylmethyl phenylmethyl silicone, PTH- Phenylthiohydantoin, TEA- Triethylamine, TFA- Trifluoroacetic acid, 3Me-P-CAP-DP - (1S,2S)-1,2-Bis(2,4,6-trimethylphenyl) ethylenediamine dihydrochloride.

*bold-labeled chiral stationary phases were most effective.

*see Appendix A for detailed properties of stationary phases.

Figure 6. Comparison of chromatograms of FMOC-DL-Alanine-OH in RP-HPLC and SFC. Experimental conditions: column: Lux™ (250 mm × 4.6 mm, 3 μm); mobile phase: 60:40 composition of supercritical CO₂ and MeOH with 0.1 or 2% formic acid; flow rate: 3 mL/min; detection mode: Agilent DAD UV-detector ^[126].

Table 2. Achiral separations of amino acids and peptides with SFC using different stationary and mobile phases

Compounds	T(°C)	P(bar)	Stationary phases(brand name)	Optimized mobile phase (% v/v)	Ref.
Pro, Leu, Val, Ile, Nle, AIB, Nva, AABA, Ala, Met, Phe, Gly, Hyp, Ms, Thr, Ser, Gln, Tyr, Trp, Lys, Asn, His, Glu, Ca, Asp	40	241	Zorbax™ CN and Deltabond™ CN	CO2 with MeOH + TMAH	^[Citation132]
Val, Ala, Phe, Ser, Lys	42	270	Packed stainless-steel column with Nucleosil™ 100	CO2 with MeOH, H2O, and MMA(7–13% modifier)	^[Citation133]
Ala, Ser, Pro, Val, Thr, Cys, Ile, Leu, Asn, Asp, Gln, Lys, Glu, Met, His, Phe, Arg, Tyr, Trp, Taurine, Niacin, Serotonin, N-aceryl-serotonin, Melatonin, GSH, Kyneurine	40	150	Luna™ HILIC	CO2 with MeOH + NH4OAc/NH4FA + H2O	^[Citation134]
Leu, Val, GABA, Phe, Gly, The, Thr, Ser, Gln, Asn, His	25	100	Zorbax™ RX-SIL, Zorbax™ C18, Unitary™ Diol, Unitary™ NH2, Unitary™ XAmide, Venusil™ NP, Venusil™ PFP, Venusil™ Imidazolyl, Venusil™ HILIC, and Cosmosil™ 5X-SIL	CO2 with MeOH + 5% H2O + 0.4% TFA + 5 mM NH4OAc	^[Citation135]
Asp, His, Gln/Lys, Ile/Leu, Gly, Ala, Ser, Pro, Val, Thr, Cys, Asn, Glu, Met, Phe, Arg, Tyr, Trp	30	100	Viridis® BEH, Viridis® BEH 2-EP, Viridis® HSS C18 SB, Torus™ 2-PIC, Torus™ DEA, Torus™ Diol, Inertsil® SIL-100A HP, Inertsil® ODS-4 HP, Inertsil® ODS-4 NoEC HP, DCpak® P4VP, CROWNPAK® CR-I(+)	CO2 with 27% MeOH + 3% H2O + 0.15% TFA	^[Citation83]
Leu enkephalin, des-Tyr-Leu enkephalin, Met enkephalin, Oxt, and BK	10–60	125–250	Divinylbenzene polymeric column	CO2 with EtOH/2-methoxyethanol + 50 mM of MSA/TFMS/PFOS	^[Citation136]
Pro-Leu-Gly amide, Leupeptin hydrochloride, Met enkephalin, BK, BK fragment 1–8, BK fragment 2–9, Lys-BK, Ang I, II and III, Sauvagine, Urotensin II	40	120	Supelcosil™ SAX1, SFC Ethylpyridine-bonded silica, and SFC amino-bonded silica	CO2 with MeOH + TFA	^[Citation53]
Gly-Tyr, Val-Tyr-Val, Met enkephalin, Leu enkephalin, Ang II	-	-	2-ethylpyrindine column	CO2 with MeOH +0.2% TFA	^[Citation137]
2 peptide pairs that differ in amino acid sequences	40	100	2-ethylpyridine, Amino propyl, HILIC diol, 4-ethyl pyridine, HA-pyridine, Bare silica, and Luna™ Phenyl-Hexyl	CO2 with MeOH + 0.2% TFA	^[Citation138]
2 peptide pairs that differ in amino acid sequences	60	120	2-ethylpyridine, Silica, Diol, Luna™ C18	CO2 + 95% MeOH + 5% H2O + 0.2% TFA	^[Citation55]
Peptides among other hydrophilic compounds	35 and 40	100	GreenSep™ BASIC, GreenSep™ DEAP and GreenSep™ Ethyl Pyridine, Princeton™ SFC CN, Sepax™ SFC Diol, Chiralpak™ AD-H, Chiralpak™ AS-H, Chiralpak™ IA, Chiralpak™ IC and Chiralcel™ OD-H	CO2 + 95% MeOH + 5% H2O	^[Citation139]
Cyclosporin analogs	80	300	Bare silica and silica particles bonded or capped with pyridinyl, 2-ethylpyridinyl, diethylaminopropyl, glycopeptidyl, cyano and octadecyl groups	CO2 with EtOH	^[Citation140]
Peptide gramicidin isoforms	30–60	110–150	Kromasil™ SFC-2.5-XT and Waters™ Torus 2-PIC	CO2 with 95% MeOH + 5% NH4FA	^[Citation141]
57 metabolites including amino acids (Leu, Lys)	40	120	Poroshell™ HILIC, Nucleoshell™ HILIC, Sunshell™ Diol, Sunshell™ 2-EP, Cosmosil™ 3-HP, Kinetex™ C18 Polar, and Synergi™ Polar	CO2 with 95% MeOH + 5% H2O +50 mM NH4FA + 1 mM NH4F	^[Citation142]
BK, insulin	40	103	Waters™ Torus 2-PIC	CO2 with 3:1 MeOH:ACN + 5% H2O + 0.2% TFA	^[Citation143]

AABA- α-Aminobutyric acid, AIB- α-Aminoisobutyric acid, Ala- Alanine, Ang- Angiotensin, Arg- Arginine, Asn- Asparagine, Asp – Aspartic acid, BK- Bradykinin, Ca- Cysteic acid, GABA- γ-Aminobutyric acid, Gln- Glutamine, Glu- Glutamic acid, Gly- Glycine, GSH- Glutathione, His- Histidine, Hyp- Hydroxyproline, Ile- Isoleucine, Leu – Leucine, Lys – Lysine, Met- Methionine, Ms- Methionine Sulfone, Nle- Norleucine,, Nva- Norvaline, Orn- Ornithine, Oxt- Oxytocin, Phe- Phenylalanine, Pro – Proline, Ser- Serine, The- Theanine, Thr – Threonine, Trp- Tryptophan, Tyr- Tyrosine, Val- Valine.

ACN- Acetonitrile, EtOH-Ethanol, H₂O- Water, MeOH- Methanol, MMA- Methylamine, MSA- Methanesulfonic acid, NH₄F- Ammonium fluoride, NH₄FA- Ammonium formate, NH₄OAc- Ammonium acetate, PFOS- Perfluorooctanesulfonic acid, TEA- Triethylamine, TFA- Trifluoroacetic acid, TFMS- Trifluoromethanesulfonic acid, TMAH- Tetramethyl-ammonium hydroxide.

*bold-labeled chiral stationary phases were most effective.

*see Appendix A for detailed properties of stationary phases.

Figure 7. Effects of ammonium acetate, trifluoroacetic acid, and water in the mobile phase on the separation of amino acids by SFC: 1. Leucine, 2. Valine, 3. Gamma Aminobutyric Acid, 4. Phenylalanine, 5. Glycine, 6. Theanine, 7. Threonine, 8. Serine, 9. Glutamine, 10. Asparagine, 11. Histidine. Column: Unitary™ XAmide (250 mm × 4.6 mm, 5 μm); mobile phase: supercritical CO₂ with indicated additives: 3 mL/min; 25°C; 100 bar; detection mode: selected ion monitoring mode using the precursor ions. Adapted from ^[135].

Table 3. Advantages and disadvantages of SFC over other separation techniques in the separation of amino acids and peptides .^{[80,81,146,147].}

	General Property
Advantages	Low viscosity and high diffusivity of SF fluid make SFC 3–5 times faster than HPLC. Lower operating temperature due to high-pressure conditions. Wide polarity range of analytes; lipophilic and hydrophilic analytes can be separated in a single run. Wider molecular weight range of analytes compared to GC. No derivatization needed, compared to GC. Analysis of non-volatile and thermally labile components. Due to higher diffusivity of supercritical fluids from liquids (HPLC), distribution into the mobile phase and separation is better. Wide range of stationary phases and operating parameters. A wider range of selectivity compared to HPLC. High throughput; Fast calibration; Direct coupling with MS. CO2 is non-flammable; CO2 much less toxic than most organic solvents. Reduced solvent costs and waste disposal costs. Less pressure drop (longer column and/or small particle size used). Reduced organic solvent emission; Less waste disposal. High resolution; Excellent interfacing.
Disadvantages	Limited choice of mobile phases. Limited analyte solubility in the mobile phase. Expensive high-pressure vessels.

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