Separation of Amino Acids and Peptides with Supercritical Fluids Chromatography

ABSTRACT

This review article provides an overview of supercritical fluid chromatography (SFC) techniques for the separation of amino acids and peptides and modifications of mobile phases in SFC for the separation of highly polar and hydrophilic compounds. Furthermore, it covers SFC applications for the chiral and achiral separations of amino acids and peptides. In this review, detailed explanations of possible and already performed achiral and chiral separations by individual amino acids and peptides are presented. Different stationary phases, separation conditions such as backpressure and temperature, and compositions of the mobile phase with possible additives for each amino acid and peptide are summarized.

KEYWORDS:

• Supercritical fluid chromatography
• amino acid
• peptide
• separation

INTRODUCTION

Liquid chromatography (LC) is commonly used for the separation and analysis of hydrophilic and highly polar analytes. Supercritical fluid chromatography (SFC) began to develop in this direction too and pushed the limits of chiral and achiral separations. Due to the miscibility of CO₂ with organic solvents and changing the mobile phase with modifiers and additives, separations over a wide polarity range of both non-polar long-chain alkyl components and small polar components, such as amino acids, are possible. Because amino acids are the constituents of peptides and proteins, it is important to develop accurate and efficient techniques for their separation and purification. Many methods have been described for such separations – from electrophoresis, LC methods, thin-layer chromatography (TLC), gas chromatography (GC), to ion-exchange chromatography (IEC) and others, with IEC and high-performance liquid chromatography (HPLC) as most commonly used .^[1,2] Since the biological activity of amino acids also depends on their stereoisomeric configuration (D- and L-), the chiral analysis and separation of amino acids and peptides are very important. Very common techniques, in addition to SFC, for chiral separations are LC and GC at analytical scale, and LC at preparative scale .^[3]

SFC has emerged as a technique for separating components using supercritical fluids as mobile phases .^[4] This has many advantages, such as a short analysis time, high chromatographic efficiency, low cost, and, above all, the non-toxicity of the mobile phase with the use of CO₂, making SFC an increasingly popular technique for separation of different compounds. SFC uses the high permeability of gases and the property of liquids to dissolve materials into their components. It is considered a “hybrid” of GC and LC .^[5] Several reviews of separating amino acids and peptides have already been published, ^[2,6–8] including Molineau’s et al. ^[9] review of biomolecule separations with pressurized CO₂ mobile phases. The present work focused on the development of SFC, its modification to separate very polar and hydrophilic compounds and a detailed overview of recent analyses and chiral and achiral separations of amino acids and peptides with SFC.

PROPERTIES OF AMINO ACIDS AND PEPTIDES

Amino acids are involved in most metabolic pathways and processes that are crucial for the growth of organisms .^[10] Therefore, the synthesis, separation, and purification of amino acids are both important and interesting. Since pharmaceutical, peptide research, and other applications demand the production of enantiomerically pure compounds, chiral separations have become an important analytical task. There are different classes of amino acids (Figure 1) that need to be distinguished for further analysis. Nonpolar amino acids are those that have a hydrophobic R-group, with a small dipole moment and a tendency to be repelled from water. Next, polar and zwitterionic amino acids are those whose R-group has polar functional groups. The third class is acidic amino acids, which have carboxylic acid on their side chains that give them acidic (i.e., can donate a proton) properties. At neutral pH, all functional groups will be ionized, resulting in an overall charge of −1. The last group is basic amino acids, whose side groups are basic (i.e., can accept a proton) and exist in an overall charge of +1 at neutral pH.

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

When trying to separate amino acids, two characteristics can be exploited, namely the differences in charge and the differences in R-group polarity. Charge differences are affected by pH values. Increasing the pH deprotonates the acid functional group and makes the net charge on an amino acid more negative, while decreasing the pH protonates both functional groups and consequently makes the net charge more positive. On the other hand, the R-group polarity can also be exploited, as it determines whether a given amino acid will be soluble or insoluble in the solvent. Furthermore, the zwitterionic properties of amino acids also play an important role in their separation. With these characteristics, LC, as well as SFC, are more powerful and preferable separation techniques, since GC analyses are not possible unless analytes are subjected to some form of derivatization to increase their volatility.

Most studies of amino acid separation refer to chromatographies .^[2] Frank et al. ^[12] were the first group examining the separation of amino acids by GC using a chiral stationary phase. Later a lot of research on amino acid separation was done with GC .^[13–15] However, LC is today one of the most widely used methods, most notably HPLC, ^[16–18] which is the most popular among the various LC techniques .^[19] The separation of amino acids in the field of biochemical research is important because it is possible to further investigate the structure and composition of proteins, determine amino acids in biological tissues and fluids, and determine the presence of free amino acids in food products to estimate food values .^[20] Peptides usually contain both ionizable acidic and basic side chains, so they have a characteristic isoelectric point. The net charge of the peptides and the polarity in the aqueous solution varies with pH. Further, hydrophobicity/hydrophilicity and the number of charged groups present are essential factors in the separation of these compounds .^[21] The difference between some peptides may only be in a single amino acid. This is called a single amino acid polymorphism (SAAP), while a single amino acid chiral polymorphism (SAACP) is when peptides differ only by an inversion of chirality of a single stereogenic center .^[22,23] The separation of molecules like peptides or polypeptides often requires selective techniques, such as LC methods. More specifically reversed-phase liquid chromatography (RPLC) or LC combined with mass spectrometry (LC-MS), are the favored methods to achieve this, due to their robustness, applicability, and high speed of biomolecule analysis .^[24–26]

SUPERCRITICAL FLUID CHROMATOGRAPHY

In recent years one of the separation processes which has been investigated by many researchers is SFC. This was originally developed as a form of gas chromatography almost 60 years ago by Klesper et al. .^[27] A few years later, Sie et al. ^[28,29] started using CO₂ as a mobile phase and studied its behavior from theoretical and experimental points of view. The physicochemical aspects of using supercritical CO₂ as a mobile phase were investigated by Bartmann and Schneider .^[30] It is the most commonly used fluid in pure form or combined with co-solvent .^[31] Moreover, the SFC process can also be performed when CO₂ is liquid, i.e. in the sub-critical range .^[32] Another advantage of this approach is the abundance of this material since it is a by-product in many industries .^[33,34] Because of all the features listed above and because of its mild critical temperature (31.1°C) and pressure (73.8 bar), it is ideal for pharmaceutical applications. On the other hand, it does have some disadvantages as it reacts with primary and secondary aliphatic amines. Nevertheless, due to the frequent reversible reaction in the case of primary amines, the original product can still be retrieved .^[35,36] Supercritical CO₂ is a non-polar solvent and has similar polarity to hexane. The general rule is that if a compound dissolves in hexane, it is also expected to dissolve in supercritical CO₂. This selectively dissolves water-insoluble substances such as fats, fatty acids, vegetable oils, and hydrophobic substances. The supercritical CO₂ itself does not dissolve hydrophilic components such as sugars, proteins, and minerals (e.g., salts and metals) .^[37,38] It is known that the solubility of an analyte can be significantly modulated by changing the pressure and temperature conditions.

The maximum achievable polarity index of pure CO₂ solvent is slightly over 2.0 .^[39] For comparison, at the bottom of the polarity index scale is pentane, with a value of 0.0, and at the top is water with a polarity index of 10.2 .^[40] The number for CO₂ is still very non-polar, making CO₂ unsuitable for working with polar analytes. The solution is to add a polar organic solvent as co-solvent. One of the first applications of the use of supercritical CO₂ and a co-solvent in the mobile phase was presented by Klesper and Hartmann .^[41] Methanol and n-pentane were used as co-solvents for the separation of the homologous species of styrene oligomers. The polarity range of solutes has expanded tremendously with the addition of organic solvents and additives, with strong acids, bases or salts often used. Many times, however, water is used as an additive, especially for the separation of highly polar compounds such as polypeptides .^[42] However, capillary SFC, where mostly neat CO₂ is used as the mobile phase, only allows the separation of less polar compounds (e.g., derivatized amino acids), so it is necessary to emphasize the importance of packed column SFC, as it allows the use of co-solvents, which enable separation of highly polar compounds (e.g., free amino acids) .^[43] Therefore, the SFC technique is useful for the separation of non-polar and polar components .^[44]

Considering the hydrophobicity and hydrophilicity of the R-groups of amino acids, it should be clarified that amino acids with hydrophobic R-groups are easier to separate with SFCs than those with hydrophilic R-groups. Above all, the most difficult to separate in SFCs are those amino acids that have basic hydrophilic residues (e.g., histidine) (see the Supplementary Material for classifications of amino acids based on side-chain properties, e.g., hydropathy, volume, chemical, charge, and polarity). Recently, however, the technique has evolved tremendously for the separation of chiral components ^[45] or enantiomers from racemates .^[46] Separation and determination of ethers, ^[47] lipids, ^[48] fat- and water-soluble vitamins, ^[49] ketones, ^[50] saponins, ^[51] steroids ^[52] and many other compounds is possible with SFC. Peptides up to 40 mers were also separated, but it is not a suitable technique for separating large biomolecules such as proteins .^[53] Furthermore, peptide isomers that differ in the position of one amino acid (pair of peptide isomers, where glycine is in the position of valine, and vice versa) were also well separated in a short time .^[54,55] Regarding the separation of peptides, the nature of these is also important, as it is easier to separate hydrophobic or lipophilic polymers with SFC than hydrophilic or lipophobic polar ones .^[56]

Initially, method development begins on a laboratory/analytical scale. The scheme of laboratory-scale SFC is shown in Figure 2. A basic laboratory-scale chromatograph consists of a tank for the mobile phase, unit for pressure control, tank for co-solvent/modifier, pumps, Rheodyne injection valve or automatic injector, separation column, detector, and Tescom valve backpressure regulator .^[58] From the analytical scale equipment, it is possible to obtain basic system parameters with a very small amount of material and to perform design and optimization to evaluate process performance and operating conditions .^[59]

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

Recently the physicochemical (e.g., high diffusivity, low viscosity, zero surface tension, and tunable solvent strength) properties of supercritical fluids, which are very relevant for the operation of SFC, were elucidated by Miller, Pinkston, and Taylor .^[60] The speed, sensitivity, and resolution of chromatographic methods are very important features. But on the other hand, ecological aspects such as lower consumption of toxic organic solvents, greater user safety, lower costs, faster analysis, and less sample preparation are also significant .^[61,62] In this context, modern SFC in the field of separation techniques has also evolved, ^[63] and due to its high efficiency, speed and robustness it has become a desirable meth .^[64] Furthermore, the miscibility of supercritical CO₂ with liquid organic solvents over a wide range of pressures and temperatures is also an important feature, as it provides wide polarity ranges. All these advantages, in addition to continuous improvements in the analytical SFC instrumentation (development of backpressure regulators, pumps, autosamplers, etc. ^[65]) and easy hyphenation with mass spectrometry (MS), ^[66] enable bioanalysis with good separation capacity and high throughput .^[67] Today SFC is used mainly on an analytical scale ^[68–70] for the separation and characterization of bioactive compounds ^[71] and has also been explored in preparative separations .^[72–75] The preparative SFC is shown in Figure 3.

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].

SFC is one of the fastest-growing analytical techniques for achiral and chiral small-molecule pharmaceutical separations .^[77–79] Because nowadays it is important to be environmentally friendly and provide the greenest technology possible, SFC has been developed in the context of green analytical chemistry .^[44] Due to its many good features, SFC is becoming an increasingly attractive and widely used method. Some of the advantages over other techniques are the short analysis time, the possibility of using a high flow rate, the low viscosity of the mobile phase and consequently high chromatographic efficiency, low costs, ease of recovery of the purified sample in preparative SFC, and, above all, lower toxicity of the mobile phase .^[80] Moreover, another advantage is the compatibility of almost all stationary phases with CO₂/co-solvent mixtures, resulting in a wider range of accessible analytes and incomparable separation options, about normal-phase LC (NPLC), reversed-phase LC (RPLC), hydrophilic interaction liquid chromatography (HILIC) and GC methods .^[81]

ADAPTING MOBILE AND STATIONARY PHASES FOR SEPARATIONS OF VERY POLAR AND HYDROPHILIC COMPOUNDS

The coupling of SFC with MS has greatly expanded the range of possible applications in recent years due to improvements in the sensitivity of MS, which in turn enables the analysis of compounds at very low concentrations on nano and even picomole scale .^[57,82–85] Analysis of compounds with a wide range of polarity is possible, ^[86] however, the analysis of polar and ionizable compounds (e.g., amino acids, peptides, polar drugs, metabolites) is much more complex. Typically, for the separation of such compounds, the mobile phase consists of 2 to 40% of co-solvents and polar additives .^[87,88] The most used co-solvent is methanol (MeOH), which represents about 75% of all used co-solvents, ^[89,90] followed by ethanol (EtOH), isopropanol (IPA), and acetonitrile (ACN) .^[91] Co-solvents are used to influence the polarity of neat CO₂, the polarity of the stationary phase (by adsorbing to its surface), the critical pressure and temperature, and the density of the mobile phase. These factors change analyte solubility, and more importantly, the elution strength of the mobile phase and separation selectivity .^[92] The choice of a good stationary phase with variable selectivity for resolving difficult peak pairs in the SFC is extensive, and it is quite simple to adjust the stationary phase to the solvent, as seen in Figure 4. Many times, the choice of different stationary phases is possible for specific separation. As the polarity of the analyte increases, a more polar phase must also be selected for separation.

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

In most cases, the addition of a co-solvent is not sufficient to obtain a chromatographic separation of components that are very polar. By using polar additives (in a concentration range of 0.1–2% ^[93]), improvements in component separation, peak shape, and selectivity are possible .^[94–96] The additives commonly used in SFC separations are acids (e.g., formic (FA), acetic (HAc), and trifluoroacetic (TFA) acid ^[96,97]), strong bases (e.g., amines and ammonium hydroxide ^[97,98]), and organic salts (e.g., ammonium formate and acetate ^{[94,97,99–101]}). Water also has a promising effect on the separation of highly polar components as an additive. A small amount of water (1–5%) alone or with other additives makes it possible to separate polar components and improve peak shapes .^[54] Moreover, as water is inorganic and moderately volatile, it can improve compatibility with MS .^[88] Water has very low solubility in supercritical CO₂, ^[102] but the addition of a co-solvent allows water to be mixed with supercritical CO₂. Water is more polar and becomes acidic in contact with CO₂ due to the formation and dissociation of carbonic acid .^[42,94,103] Based on such modified mobile phases, elution and separations of highly polar and hydrophilic compounds are possible .^[83] Figure 5 shows and explains the flow chart of method development for SFC in general and for amino acid separation. It can also be helpful in establishing the final method for the separation of very polar and hydrophilic compounds.

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

SEPARATION OF AMINO ACIDS AND PEPTIDES WITH SFC

Chiral Separations

Enantiomer analysis is especially important for the characterization of chiral drugs in the pharmaceutical industry and in food science, as chiral analysis can provide valuable information on the safety, quality, traceability, and bioactivity of the analyzed samples .^[104,105] The biological activity of molecules depends on chirality since enantiomers can trigger different reactions in a living organism because different enantiomers bind to different receptors .^[106] High diffusivity and low viscosity using a mobile phase with supercritical CO₂ make SFC a widely used technique for separating chiral compounds .^[107] Several general SFC strategies for fast chiral separations, including screening and fine-tuning of the method, have already been described .^[108] In the chiral separation of enantiomers, a particular component must interact enantioselectively with the enantiomers to be separated .^[109] For that, chiral selectors are required. Among the various chiral selectors, the most commonly used are cation and anion exchangers (e.g., aminosulfonic acid-based chiral strong cation exchangers, Chinchona alkaloid-based chiral weak anion exchangers), ^[110] zwitterionic chiral selectors (e.g., quinine and quinidine-based zwitterionic CSPs), ^[111] and macrocyclic antibiotic-based materials (e.g., teicoplanin aglycone) .^[112] While the most used and universal chiral stationary phases are made with derivatized macrocrystalline cellulose or amylose coated on silica .^[54] Given the fact that the polarity of free and derivatized amino acids is very different, the choice of the composition of the stationary and mobile phases is also distinctive. Ordinarily, less polar derivatized amino acids are analyzed on polysaccharide chiral selectors, while very polar free amino acids are best analyzed on ionic chiral selectors .^[113]

SFC applications concerning amino acid derivatives analysis have mostly been focused on chiral separations. Extremely important are the facts that, for example, some D-amino acids have long been considered non-functional, and with the introduction of enantiomeric separations have gained an important role in many physiological processes in living organisms, including the human body, and have also been associated with many diseases (e.g., schizophrenia, age-related disorders, and amyotrophic lateral sclerosis) .^[114] Early research related to chiral/enantiomeric separation of derivatized amino acids appeared in the late 1980s .^[115] Chiral separations of amino acids and amino acid derivatives with SFC using different stationary and mobile phases are depicted in Table 1. Dobashi et al. ^[116] studied the enantiomer resolution of D and L-alpha-amino acid derivatives on chiral valine-diamide phases with supercritical CO₂ and methanol as a co-solvent. The column temperature was 40°C and the pressure used was 200 bar. Enantiomer separation was carried out in less than five minutes, except for the tryptophan derivative, which showed the highest retention. D-enantiomers were always eluted before L-enantiomers. Lajkó et al. ^[117] carried out separations of the enantiomers of N_α-proteinogenic amino acids on alkaloid-based zwitterionic CSPs. Amino acids were protected with N_α-Fmoc and with additional protecting groups (t-butyloxycarbonyl, t-butyl, O-t-butyl, triphenylmethyl, and N_ω-2,2,4,6,7-pentamethyl-dihydro benzofuran-5-sulfonyl) to make them more appropriate for peptide separation protocols. The study was performed at 40°C and 150 bar. On Chiralpak™ ZWIX (+), all D-enantiomers eluted before L-enantiomers, while on Chiralpak™ ZWIX (-) the elution sequence was opposite.

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.

Additionally, a variable-temperature study was performed to investigate its effect on chromatographic parameters. Retention time slightly increased, while the selectivity decreased with increasing temperature. Lajkó et al.’s findings are in line with those of Lou et al. .^[118] Changing the temperature can improve the enantioselectivity and capacity factor. Enantioselectivity decreases with increasing temperature, while the column pressure has no connection with enantioselectivity. However, it should be considered that the mobile and stationary phases and the nature of the analyte (e.g., free amino acids or derivatized amino acids) strongly influence the effect of the temperature on enantioresolution, and such studies are difficult to compare. Furthermore, in the conditions applied by Lou et al., ^[118] the enantioselectivity was not affected by the mobile phase density. However, it should be noted that the researchers used an uncommon column, with OV-225-L-valine-tert.-butylamide chiral stationary phase, as otherwise in most cases mobile phase density does affect enantioselectivity in SFC .^[131]

Liu et al. ^[119] investigated chiral separations of 111 chiral compounds, including N-protected and native amino acids, with macrocyclic glycopeptide CSPs at 31°C and 100 bar. Because of the decrease in enantioselectivity with increasing temperature, a low and constant temperature was selected. Furthermore, constant outlet pressure was used due to the effect of decreasing enantioresolution factors with rising pressure. Seventy percent of the compounds were separated in less than 4 min, and a maximum of 15 min was required to separate the remaining compounds. Regarding the separations of N-protected amino acids, most baseline separations could be achieved with 40–60% MeOH in the mobile phase, but the addition of 0.1% TEA was also required, meaning that the stationary phase and the solutes were in negatively charged forms. On the other hand, due to the nature of the polarity of native, underivatized amino acids, 2–2.8% of water and/or 0.3% of glycerol with 0.1–0.15% TEA and TFA needed to be added to the mobile phase to increase the solubility of polar analytes and to improve peak shapes.

Later, Stringham ^[120] separated 36 of 45 basic compounds, including four underivatized amino acids, with amylose tris-(3,dimethyl phenyl carbamate) chiral stationary column 20% of EtOH containing 0.1ethane sulfonic acid (ESA) in the mobile phase. It was found that the addition of acid was already required during the preparation of the analytes for successful separation, and the acid-free mobile phase did not result in elution of the peaks. The cause is probably in the formation of salts from the basic compound and ESA, which are then separated as intact salts throughout SFCs. Further West et al. ^[121] studied packed-column SFC with two polysaccharide-based chiral stationary phases to separate six amino acid derivatives (PP, PFP, PBzt, oMPP, oMPFP, PPY). Lux™ Cellulose-1 enabled enantiorecognition for five out of six racemates, and Lux™ Cellulose-2 complemented Lux™ Cellulose-1, as it enabled enantiorecognition of two out of six racemates, but one of the two was the one (PPY) that Lux™ Cellulose-1 did not recognize. Therefore, the separation of five racemates was studied on Lux™ Cellulose-1, while Lux™ Cellulose-2 was studied with only two racemates. SFC was performed at 30°C, 150 bar, and with a modifier proportion of 10%. For better resolution, the percentage of modifier was lowered from 10 to 3%.

Payagala et al. ^[122] investigated the applicability of three polymeric CSPs in SFC for the application of amino acids and other chiral compounds. With simple free-radical-initiated polymerization in solution, CSPs were synthesized based on three (1S,2S)-(−)-1,2-diphenylethylenediamine derivatives and were evaluated and compared with commercial polymeric CSP in HPLC and SFC. In SFC, 65 of 100 racemates were separated with 24 baseline separations. Among all of these, a derivatized amino acid N-(3,5-dinitrobenzoyl)-DL-leucine was separated only with commercial P-CAP-DP CSP, with 10% EtOH and 0.1% TFA in the supercritical CO₂ mobile phase. Wang et al. ^[123] studied enantioseparation on 14 racemates, including amino acid derivatives. They used chemically bonded cationic β-cyclodextrin perphenylcarbamoylated derivatives as CSPs. Dansyl amino acids were separated using 2-propanol as a modifier, as it has a lower polarity compared to MeOH and does not react as much with the substituents on the cyclodextrin rim, allowing better enantioseparation. On the other hand, the alkyl chain length of the substituent at a position to the carboxylic acid group affects retention and selectivity. Shorter alkyl chains form looser inclusion complexes with a cyclodextrin cavity, so retention and selectivity are lower for dansyl amino acids with a shorter alkyl chain.

Another study by Sánchez-Hernández et al. ^[124] reported the enantioseparation of aromatic amino acids with SFC, carried out on glycopeptide-based CSP. As analytes had no acidic or basic group in the residue, just MeOH and H₂O were necessary as additives to the mobile phase. The effects of pressure and temperature were also evaluated. The optimized parameters were 100 bar and 35°C, as lower pressure (90 bar) can cause slightly higher retention and broader peaks, while higher pressure (150 bar) causes lower retention and higher pressure drops. As the temperature increased (from 20 to 40°C), the resolution and retention increased, as well as the analysis time. It took less than 7 min for individual separation and identification of amino acids. Wohlrab et al. ^[125] studied the enantioseparation of N-protected amino acids and other basic analytes with HPLC and SFC. SFC was carried out at 40°C and 150 bar. Because the HPLC separations were performed at 20°C, the researchers also performed a separation with subcritical CO₂ at 20°C to compare the measurements. Enantioselectivity increased at 20°C, but resolution decreased. In addition, chromatographic conditions were investigated for increasing the ratio of a polar co-solvent (alcohol) with subcritical CO₂, and with that retention times decreased. Interestingly, under certain conditions, no enantiomers were separated by HPLC, while the same analytes were separated by SFC. The reason could be in the physical-chemical properties of CO₂, which is a non-polar solvent but has the property of accepting protons which makes it completely miscible with protic organic solvents.

Vera et al. ^[126] compared the chiral separation of D- and L-fluorenylmethyl oxycarbonyl (FMOC) chloride amino acids with HPLC and SFC conditions. RPLC gave the best resolution between enantiomers in the range of amino acids, but with the addition of higher concentrations of formic acid (2%) in the mobile phase at SFC allowed similar resolution and a shorter run time. SFC exhibited the best resolution per minute overall separations. A comparison of chromatograms is shown in Figure 6. Vera et al. ^[126] also noted the advantage of SFC over HPLC due to the lower consumption of organic solvents, which reduces environmental as well as economic costs.

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].

Recently, Khater et al. ^[127] separated 13 pairs of enantiomers belonging to the group of phenylthiohydantoin (PTH)-amino acids. To examine the thermodynamic behaviors of enantioseparations, temperatures from 5 to 40°C were applied on cellulose and amylose-based CSPs. The thermodynamic relationship between temperature (T) and chromatographic retention factor (k) was expressed using the Van’t Hoff equation and the separation factor (α) was defined from it. An equilibrium between enthalpy and entropy, where enantiomers coelute (α = 1), exists at the isoelution temperature (T_iso), while below this temperature the separation is enthalpically-driven. Here, enantioselectivity decreases with increasing temperature. In contrast, over the T_iso, the separation is entropically-driven, and with temperature the enantioselectivity increases. It was observed, that when the temperature rises above 20–30°C, both CSPs underwent structural changes. The trend of comparing retention and enantioselectivity values at different CSPs is important. The values of ln(α) can be related to the energy of interactions in the chromatographic system, so the diversity of these values is reflected in the diversity of the chiral cavities in the CSP. In the cellulose-based phase, where all cavities are similar, all chiral cavities were affected by temperature to the same extent. On the contrary, in the amylose-based phase, cavities may be more diverse, the chiral cavities were affected differently, which enables more diverse separation factors and exhibit a diverse level of sensitivity to temperature changes. This means that the stationary phase with amylose may allow more options and variety of chiral binding, and thus the analytes can access different cavities.

The separation of the 13 native amino acid enantiomers was studied with different polysaccharide-based chiral selectors by Lipka et al. .^[128] The separation with eight different columns with coated or covalently immobilized chiral selectors and with the addition of 20–40% ethanol or methanol as a co-solvent at 40°C and 150 bar was compared. Five out of 8 CSPs (Chiracel™ OD, Chiralpak™ AS-H, Chiralpak™ AD, Chiralpak™ IB, and Chiralpak™ IA) did not provide separation of any pair of enantiomers. Lux™ Amylose-2 resolved only enantiomers of Phe, while Lux™ Cellulose-2 separated enantiomers of Phe, Val, Ile, and Leu. The cellulose-based column (3,5-dichlorophenylcarbamate), Lux™-I-Cellulose-5, showed the best ability to separate the enantiomers, as it exhibited recognition ability for the enantiomers of 10 amino acids. The composition of the mobile phase and the nature of the chiral selector play a key role in the differentiation of the enantiomers. Raimbault et al. ^[129] described the enantioresolution of 19 amino acids from two food supplements, tablets containing a mixture of proteinogenic amino acids and capsules containing taurine and theanine. To achieve elution of all amino acids, a wide elution gradient ranging from 90% CO₂ to 100% co-solvent was employed at 25°C and 150 bar. For elution of the most basic amino acids, it was necessary to add water and ammonium formate.

Recently, Miller and Yue ^[130] performed chiral separations of 18 underivatized amino acids in SFC with a chiral crown ether derived column CROWNPAK® CR-I (+). Detection was performed with MS, and the type of modifier and acidic additives were optimized, while the role of water in the mobile phase was determined. The use of IPA as a modifier resulted in the separation of nine amino acids, while the use of EtOH or MeOH alone resulted in the separation of 17 of the 18 amino acids, with only histidine, the most basic amino acid, not resolved. This is in line with previous observations, as amino acids with a basic side group are more difficult for SFC. Furthermore, all amino acids were separated by gradient elution, with all three modifiers: MeOH, EtOH, or IPA with the addition of 5% H₂O and 0.5% TFA. Because EtOH provided exceptional resolution, the influence of water and the type and concentration of acid additives on the separations were checked only with EtOH as the modifier. The addition of 5% water achieved sharper peaks and better resolution for all amino acids except histidine, while the addition of 10% resulted in decreased resolution (compared to 5% H₂O). Among HAc, FA, and TFA, TFA, the strongest acid, resulted in the separation of 17 of the 18 amino acids, and with its addition, the resolution was also improved. Stronger acids provide full protonation, thus enabling the interaction of crown ether and ammonium ion (-NH₃⁺) and the consequent enantioselectivity and chiral recognition. Moreover, a higher concentration of TFA increased the resolution. It is important to emphasize the effect of the acid additive on histidine in particular. Near baseline resolution was achieved with 0.8% TFA, while 1.5 and 2.0% TFA enabled baseline separation of DL-histidine. For all amino acids, the D-enantiomer eluted first and was separated using gradient methods, and then transferred to optimized isocratic conditions. Detailed methodologies of possible and already performed chiral separations by individual amino acids, which include CSPs, separation conditions such as temperature and backpressure, and the composition of the (modified) mobile phase with possible other additives are listed in the Supplementary Material.

Achiral Amino Acid Separations

So far, only a few studies have been devoted to the achiral analysis of amino acids by SFC. Different such achiral separations using different stationary and mobile phases are depicted in Table 2. Most amino acids lack suitable chromophores, so MS is usually used to detect them .^[83,144] Previously, UV and FID detection was used .^[132,145] In 1989 Berger et al. ^[132] separated 24 PTH-amino acids using a packed cyanopropyl Zorbax™ column. Separation was achieved in less than 20 min at 40°C and 240 bar. Veuthey et al. ^[133] separated amino acids with SFC after a pre-derivatization step with FMOC and (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) reagents in 40 min at 42°C and 270 bar. Further, they separated three FLEC-amino acid diastereomers on bare silica with low polar modifier concentration on supercritical CO₂. L-enantiomer eluted before D-enantiomer, and a further advantage of SFC over HPLC is that the excess of FLEC reagents is eluted with the acetone solvent and does not interfere with the amino acid peaks.

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.

Wolrab et al. ^[134] directly coupled SFC to MS for the analysis of amino acids and related compounds. They described a method for separating metabolites from the tryptophan metabolic pathway and other amino acids present. Supercritical CO₂ and co-solvent (MeOH, EtOH, or 1:1 mixture of MeOH: ACN) were used for the mobile phase. For better ionization and separation, they included NH₄FA or NH₄OAc as additive. They were added to the modifiers to support the ionization of the analytes and improve peak shapes. They successfully separated all the components of the standard mix at 40°C and 150 bar. The efficiency of ionization using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and positive and negative ion modes was also compared. APCI was found to be more suitable for amino acids with polar side chains, while ESI provided better ionization efficiency of amino acids having hydrophobic residues.

Huang et al. ^[135] studied the presence of amino acids in various teas (green tea, Oolong tea, black tea, and Pu-erh tea). Ten different columns were used for separation with SFC. Due to the polarity of the analytes, the analytes have no retention on the Zorbax™ C18 column. More polar stationary phases (Venusil™ PFP, Venusil™ NP, and Venusil™ Imidazolyl) resulted in increased analyte retention on the column and co-elution of some peaks. The other six hydrophilic stationary phases (Zorbax™ RX-SIL, Cosmosil™ 5X-SIL, Unitary™ Diol, Venusil™ HILIC, Unitary™ XAmide, and Unitary™ NH₂) resulted in successful separation, shorter retention, and better resolution. All eleven amino acids could be baseline separated with a Unitary XAmide column. The addition of TFA and water in the supercritical CO₂ mobile phase significantly improved the separation of amino acids, as shown in Figure 7. The study was carried out at 25°C and 100 bar. In all tea samples, L-theanine was the most abundant amino acid. Green tea had the highest total content of amino acids, followed by Oolong tea, while black and Pu-erh teas contained a much lower total amino acid content.

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].

Konya and colleagues ^[83] developed a new method using SFC and tandem MS for the simultaneous analysis of polar metabolites. Using 11 columns, the separation of 20 proteogenic amino acids was optimized. The retention time, peak shape, and chromatographic separation of two pairs of amino acids with the same multiple reaction monitoring (MRM) transition (e.g., Gln/Lys and Ile/Leu) were important factors in selecting the optimal column and mobile phase. They found that replacing FA with TFA shortens the retention time of almost all amino acids and dramatically improves peak shapes. However, only five (Viridis® BEH, Viridis® HSS C18SB, Torus™ Diol, DCpack® P4VP, and CROWNPAK® CR-I (+)) out of 11 columns were able to separate the Gln/Lys pair, with the CROWNPAK® CR-I (+) column being the only one separating the Ile/Leu pair. Furthermore, it was found that amino acid separations with the same MRM transitions are improved by increasing the ratio of CO₂ in the mobile phase to 70%. Therefore, a CROWNPAK® CR-I (+) column with a mobile phase composition of 70% CO₂ and 30% modifier under isocratic elution was selected as the most optimal for the separation of polar metabolites. It would also be important to point out that there is a trend of using smaller particles and even superficially porous particles in chiral separations using SFC, and in the case of chiral amino acid separations we observe that particles of size 3 and 5 μm are still used.

Achiral Separations of Peptides

Blackwell and Stringham ^[136] were already studying the separation of a group of small polypeptides (leucine enkephalin, des-Tyr-leucine enkephalin, methionine enkephalin, oxytocin, and bradykinin) in 1999. Bradykinin, which contained the largest number of amino acid residues, was the last of these to be eluted. A study of the effect of temperature (10–60°C) on the gradient elution of polypeptides was also carried out. As the temperature increased the retention also rise, but a greater selectivity between the peaks of the polypeptides and impurities was detected at the expense of lower efficiency. Achiral separations of peptides with SFC using different stationary and mobile phases are depicted in Table 2. Zheng et al. ^[53] showed that 40-mer peptides, including a variety of basic and acidic residues, can be separated with SFC. This is of great importance to rapidly perform determinations and separations of bioactive peptides in complex biological matrixes, which is useful for pharmaceutical and other branches of bioindustries. TFA was used as additive in the supercritical CO₂/MeOH mobile phase to suppress ionization of the carboxylic acid groups and to protonate amino groups of peptides. A higher concentration of additive allowed less retention and fronting peak shapes, indicating electrostatic reflectance between peptides and some part of the functional groups in the stationary phase. The ethylpyridine bonded silica was the only stationary phase to allow successful elution of polypeptides, which is attributed to the deactivation of silanol groups due to the hydrogen bond between silanols and pyridine functional groups on the stationary phase. Also, the pyridine aromatic ring causes a steric barrier and consequently limited access to the silica surface.

Tognarelli et al. ^[137] explored the SFC separation of five peptides ranging in molecular weight from 238.2 to 1046.2 Da. The separation lasted less than 12 min, compared to the HPLC method which lasted nearly 1 hour. Patel et al. ^[138] demonstrated that a packed-column SFC is suitable for separating water-soluble peptides of the same mass, composition, and charge that differ only in the sequence of amino acids. Furthermore, it was also shown ^[55] that polar, high molecular mass polypeptides could be separated using ion-pairing SFC. Liu et al. ^[139] studied the separation of peptides among other hydrophilic compounds by SFC using water-containing modifiers at 35 and 40°C and 100 bar. Shao et al. ^[140] used SFC to develop a method for the separation of five cyclosporin analogs. These are cyclic peptides and contain eleven amino acids but differ at the side chain of their amino acid residues. With an increase in temperature (0–80°C) the selectivity remained almost the same, but the efficiency of the column was increased as the widths of the peaks were significantly reduced. Baseline resolution for all five peaks was achieved at 50°C and resolution continued to improve up to 80°C. With increasing temperature, the retention times were shifted for approx. 3 min longer at 80°C compared to 0°C. Increasing back pressure (100–300 bar), the retention times decreased, and the selectivity was significantly improved. In summary, the best conditions for separating cyclosporin analogs with SFC are at 80°C and 300 bar back pressure using ethanol modified supercritical CO₂ mobile phase as a mobile phase on a bare silica column due to the directly exposed hydroxyl group on the silica surface.

Enmark et al. ^[141] compared the separation of the peptide gramicidin, using either isocratic or gradient elution, and found that gradient elution is about three times more robust than isocratic elution. Desfontaine et al. ^[142] studied the suitability of SFC-MS for the analysis of lipophilic and highly hydrophilic substances. To cover the widest possible range of analytes, a gradient from 2 to 100% co-solvent in CO₂ was systematically used. The best conditions for the separation of a wide range of chemical compounds have proved to be bare silica or silica bonded with a zwitterionic ligand for the stationary phase at 40°C and 120 bar. A considerable amount of salt in the mobile phase makes it possible to reduce the retention, to increase the solubility, and to obtain the corresponding peak shapes for ionizable species. Schiavone et al. ^[143] reported the potential of SFC for the purification of peptides and proteins. An organic-aqueous modifier-based preparative SFC method was developed for the purification of peptides. Purification of bradykinin and insulin were demonstrated using preparative SFC for peptide purification. Peptides were dissolved in water or TFA solution. The mobile phase modifier was 3:1 MeOH:ACN with 5%/0.2% H₂O/TFA additive. Purification was carried out at 25°C and 100 bar. However, the analyzed proteins (ubiquitin, cytochrome C, and apomyoglobin) changed during the SFC process and were unable to return to their original higher-order structure.

Detailed methodologies of possible and already performed achiral separations by individual amino acids and peptides are listed in the Supplementary Material. Different stationary phases, separation conditions such as temperature and backpressure, and the composition of the (modified) mobile phase with possible other additives are also included.

CONCLUSIONS

While SFC is not a new concept, it has experienced incredible commercial success in the last few years, driven by the introduction of new instruments with improved performance and robustness, and hyphenation to MS. SFC also has major advantages in the separation of amino acids and peptides (Table 3) over the well-known HPLC and GC methods, which are still considered to be standard approaches for the analysis of complex samples. Analysis of a wider molecular weight range of compounds is possible with SFC compared to GC, along with good detection of chromophore-free compounds, separation of thermally labile compounds at low temperatures, and low viscosity and high diffusivity, allowing the SFC method to be three to five times faster than HPLC, among other advantages .^[148,149] Mixing polar organic solvents with non-polar CO₂ allows the elution of polar compounds, while more polar additives such as TFA, TEA, and water further extend the polarity range of solutes suitable for SFC .^[150] Direct coupling of SFC with MS makes the technique even easier to operate. Although the number of published studies on separating amino acids and peptides in recent years is relatively low, the interest in the achiral and chiral separation of these compounds continues to increase. The applicability of SFC in chiral separations of amino acids has been demonstrated to be successful in numerous reports. Most applications have been developed on polysaccharide-based CSPs while applying a chiral selector was also described as an effective method for separating enantiomers of amino acids. Regarding the achiral separations of amino acids and peptides, SFC provides an extremely wide choice of stationary phases and a wide range of operating parameters. Compared to HPLC, these properties show the strength of the SFC technique, which allows a wider range of selectivity.

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.

Finally, the increasing number of published articles focused on the development of the SFC method for the analysis of highly polar and hydrophilic compounds confirms the important role of SFC in the family of separation techniques. Due to its excellent properties and obvious benefits, it will probably become a method of choice for many other applications in the near future.

Author Contributions

M.L. and M.P. conceived and designed the study. K.K. studied the literature and wrote the manuscript. M.L., M.P., and Z.K. reviewed the manuscript. K.K. edited the manuscript. Z.K. and M.L. were responsible for the financial part of the project. All authors accepted the final version of the review.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This research was supported by the Slovenian Research Agency 2187/FS-2019,J2-1725,J2-3037,P2-0046 (ARRS) within the frame of program P2-0046 (Separation Processes and Production Design), project No. J2-1725 (Smart materials for bioapplications), project No. J2-3037 (Bionanotechnology as a tool for stabilization and applications of bioactive substances from natural sources), program P2-0118 (Textile Chemistry), young researcher ARRS fellowship contract No. 2187/FS-2019.