Jérémy Molineau

Maria Hideux

Caroline West
email: caroline.west@univ-orleans.fr

Chromatographic analysis of biomolecules with pressurized carbon dioxide mobile phases -A review

Keywords: amino acids, enhanced fluidity liquid chromatography, nucleosides, nucleotides, peptides, proteins, supercritical fluid chromatography

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Introduction

Biomolecules or biological molecules are molecular species present in living organisms of the fauna, the flora and microorganisms. Four principal categories of macromolecular biomolecules are usually distinguished: carbohydrates, lipids, proteins and nucleic acids.

Smaller molecules include primary and secondary metabolites. In this paper we will focus on amino acids, their oligomers and polymers (peptides and proteins), and on nucleic acid constituents like nucleobases, nucleosides and nucleotides (Figure 1). Peptides may be linear or cyclic, where the ring structure can be formed by an amide bond ("peptide bond") or other stable bonds like disulfide [START_REF] Joo | Cyclic Peptides as Therapeutic Agents and Biochemical Tools[END_REF]. Peptides and proteins differ in size and structure, in that peptides are usually smaller, with an undefined three-dimensional structure, while proteins are larger and with a defined, rather stable folding. However, there is no universally accepted limit value in the number of amino acid residues to distinguish between peptides and proteins. Nucleotides are the basic constituents of nucleic acids like DNA and RNA, and may be decomposed in (i) a nucleobase (purine or pyrimidine) and (ii) an osidic group, together forming a nucleoside, and (iii) a phosphate tail containing one, two or three phosphate groups linked to the 5' oxygen of the osidic group. The structures of the natural biomolecules may also be modified for specific purposes with other functional groups, as is the case with nucleoside antiviral drugs [START_REF] Mahmoud | Antiviral Nucleoside and Nucleotide Analogs: A Review[END_REF]. Indeed, in the recent years, peptide (linear or cyclic [START_REF] Joo | Cyclic Peptides as Therapeutic Agents and Biochemical Tools[END_REF][START_REF] Gang | Cyclic Peptides: Promising Scaffolds for Biopharmaceuticals[END_REF]), protein and nucleic acid biomolecules have largely emerged in the pharmaceutical arena, either as synthetic active pharmaceutical ingredients (API) [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF][START_REF] Sherman | Computational approaches in design of nucleic acid-based therapeutics[END_REF] or from natural ingredients [START_REF] Koehbach | The Vast Structural Diversity of Antimicrobial Peptides[END_REF][START_REF] Tang | Antimicrobial peptides from different plant sources: Isolation, characterisation, and purification[END_REF] as food products [START_REF] Lorenzo | Bioactive peptides as natural antioxidants in food products -A review[END_REF] or traditional Chinese medicine (TCM) [START_REF] Liu | Bioactive peptides derived from traditional Chinese medicine and traditional Chinese food: A review[END_REF]. The fast emergence of these types of bioactive molecules is setting new challenges to analytical chemists in general and chromatographers in particular.

Liquid chromatography is the most common method to analyze and purify such biomolecules [START_REF] Tang | Antimicrobial peptides from different plant sources: Isolation, characterisation, and purification[END_REF][START_REF] Sandra | Highly efficient peptide separations in proteomics[END_REF][START_REF] Temporini | Chromatographic tools for plant-derived recombinant antibodies purification and characterization[END_REF], preferably in the reversed-phase mode (RPLC) but also in the hydrophobic interaction (HIC) and hydrophilic interaction (HILIC) modes, ion-exchange chromatography (IEX) and size-exclusion chromatography (SEC). Beside liquid chromatography, capillary electrophoresis (CE), and microfluidics are other favorite analytical methods for biomolecules.. Supercritical fluid chromatography (SFC), employing pressurized carbon dioxide as a major constituent of the mobile phase, is another possible alternative that would deserve more attention in this field [START_REF] Lesellier | The many faces of packed column supercritical fluid chromatography -A critical review[END_REF]. In fact, the low viscosity and high analyte diffusivity allow for high flow rates together with high efficiency, thus the resolution per unit time is usually excellent with SFC. In addition, SFC is also advantageous at the preparative scale as it allows significant reductions in solvent consumption compared to liquid-phase preparative separations, and produces fractions that are more concentrated. As a result, the solvent cost, solvent evaporation time and waste disposal costs are all reduced compared to LC methods. For this reason, preparative SFC is considered a green purification method.

Interestingly, the benefits of pressurized carbon dioxide in the mobile phase are maintained whatever the proportions introduced in a liquid mixture. Traditionally, when the proportion of CO2 is the largest, the method is called subcritical or supercritical fluid chromatography (SFC). When the proportion of CO2 is the smallest, the method is rather called enhanced fluidity liquid chromatography (EFLC). Aside habits, there are no fundamental reasons to dissociate the two methods [START_REF] Olesik | Enhanced-Fluidity Liquid Chromatography: Connecting the Dots Between Supercritical Fluid Chromatography, Conventional Subcritical Fluid Chromatography, and HPLC[END_REF] and they will both be discussed in the following. While already an old technique, with the first experiments dating from 1962 [START_REF] Klesper | High-pressure gas chromatography above critical temperature[END_REF], SFC has faced a rapid surge of interest in the last decade, thanks to the introduction of improved instruments by several manufacturers. Before that time, the benefits of SFC were principally recognized at the preparative scale and for enantioresolution [START_REF] West | Recent trends in chiral supercritical fluid chromatography[END_REF][START_REF] West | Enantioselective Separations with Supercritical Fluids -Review[END_REF][START_REF] Speybrouck | Preparative supercritical fluid chromatography: A powerful tool for chiral separations[END_REF]. A variety of application fields that were previously little investigated with SFC are now being re-explored [START_REF] West | Current trends in supercritical fluid chromatography[END_REF]. Biomolecules analysis and purification to serve pharmaceutical and clinical research is one such field. As appears in Figure 2, the number of papers published on this topic may have been scarce during several decades but has been rising in the recent years. Although the total number of them remains modest, there is clearly an interest for alternative methods in this field. When analyzing biomolecules with SFC, difficulties are expected due to solubility issues of potentially large and polar species (see section 2 below). In addition, conformational changes may occur for proteins when exposed to carbon dioxide, as will be further detailed below.

The changes occurring nowadays in the way the technique is operated, typically with increasing portions of co-solvent and water in the pressurized carbon dioxide mobile phase are however raising new expectation.

The first example of peptide analysis in SFC was published in 1988 [START_REF] Kalinoski | Chemical ionization mass spectra of high molecular weight, biologically active compounds produced following supercritical fluid chromatography[END_REF], where a cyclic undecapeptide Cyclosporin A (1201.8 Da), was analyzed with SFC hyphenated to mass spectrometry (MS). Further attempts at analyzing such biomolecules initially concentrated on lipophilic peptides and proteins, while recent attempts also concern more polar species, as will be detailed in section 2. This paper reviews most significant articles related to SFC and EFLC analysis and purification of free (underivatized) amino acids, peptides, proteins, and building blocks of nucleic acids. It is not intended to be exhaustive but rather to spotlight trends and specific features. First, the polarity and size issues related to these biomolecules is discussed, to better relate them to the choice of stationary and mobile phases that were employed for their analysis. Detection issues are also discussed, with a particular focus on MS hyphenation.

Polarity and size features of the target biomolecules

To understand the chromatographic features of biomolecules analysis, it is useful to examine their structures first. In Figure 1, the generic structures of the target molecules related in this paper are presented. In addition, we have examined the structures of the molecules previously analyzed with SFC and EFLC in terms of size and polarity. For this purpose, structural descriptors were computed for the molecules mentioned in the literature reviewed with MolDesc, an open-source software (http://moldesc.icoa.fr/). Three descriptors were retained to discuss the relation to chromatographic analysis: molecular weight (MW), octanol-water partition coefficient (log P) and the topological polar surface area (TPSA). Note that, as any computed descriptors, they may not be entirely accurate, but they should be good enough to compare the features of the target molecules. They are summarized in Figure 3, where MW is plotted versus log P value, and bubble size is related to TPSA. Only the larger proteins and peptides are not represented in this figure, as it would have flattened all other molecules in an apparently small area at the bottom of the figure and because the computation of polarity features for such molecules has little meaning, as will be further discussed below. For instance, reports of large peptides and proteins with molecular weights as high as 3.7 kDa (issued from peptic digestion of myoglobin [START_REF] Emmett | Supercritical Fluid Chromatography Reduction of Hydrogen/Deuterium Back Exchange in Solution-Phase Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis[END_REF]), 5.8 kDa (biosynthesized human insulin [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF]), 13.7 kDa (ribonuclease A [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF]), 17.6 kDa (myoglobin [START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF]), 66.5 kDa and 80.3 kDa (bovine serum albumin and transferrin [START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF]) were excluded from this figure but are however discussed in the following sections.

Amino acids are all small molecules and are thus placed at the bottom of Figure 3 (small blue bubbles). Apart from the acid and amine functions, which may both be ionized depending on operating conditions, most amino acids possess a side chain (residue), which may be hydrophobic (hydrocarbon chain or aromatic group), hydrophilic bearing no ionizable function (like hydroxyl, thiol or amide functions), acidic thus possibly bearing a negative charge, or basic thus possibly bearing a positive charge. According to these varied residues, amino acids may be slightly hydrophobic (on the right of the figure) or hydrophilic (on the left of the figure). Note that several papers relating the analysis or purification of derivatized amino acids (for instance methyl esters, carboxybenzyl esters [START_REF] Nogle | Preparative separation and identification of derivatized β-methylphenylalanine enantiomers by chiral SFC, HPLC and NMR for development of new peptide ligand mimetics in drug discovery[END_REF][START_REF] Kraml | Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatography[END_REF] or dansyl esters [START_REF] Blackwell | Effect of Mobile Phase Additives in Packed-Column Subcritical and Supercritical Fluid Chromatography[END_REF])

will not be described in this review. Derivatization is usually facilitating the elution of amino acids in reducing their polarity, and often facilitating their chiral resolution.

When linear peptides are considered, some ionizable functions should remain at both ends, if the terminal amine and carboxylic functions are unmodified with protective groups.

When cyclic peptides are considered, there are not necessarily any pendant amine or carboxyl functions as they may have served to form the ring, unless some side chains are attached to the ring. Thus cyclic peptides are often observed to be less polar than linear peptide chains. This can be observed in Figure 3. Most cyclic peptides (light green bubbles) are situated in the right portion of the figure, with positive log P values, while most linear peptides (dark green bubbles) are situated in the left portion of the figure, with negative log P values. According to log P values, two polar cyclic peptides were reported, Linaclotide and Ocytocin (on the left of Figure 3), and two non-polar linear peptides or proteins were reported: the membrane proteins Gramicidin A and B.

However, as in any other molecule, the overall polarity of a peptide expressed by log P value is the result of the positive contributions of hydrophobic residues and the negative contributions of hydrophilic residues. When the two are counterbalanced, the overall log P value may be close to zero, although a significant number of polar groups could be present.

It is thus useful to observe a second polarity parameter, like the topological polar surface area (TPSA), which sums the individual contributions of all polar groups in a molecule, and is not counterbalanced by hydrophobic groups. In Figure 3, TPSA is represented by bubble size. Then it appears that all peptides reported, whatever their log P values, have significant TPSA values that should at least result from the peptide bonds (amide functions), whatever the residues. For instance, the membrane proteins Gramicidin A and B have some of the largest TPSA values (large bubbles), even though the overall polarity (expressed by log P) is low (around 4). In addition, in their stable conformation, the linear gramicidins are folded in helices with hydrophobic rings directed outside the helix, with little exposed polar surface.

The stable conformation of proteins may or may not be preserved during SFC and EFLC analysis [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF]. It is thus difficult to conclude on the exposed polarity of such molecules during the analysis. Actually, chromatographic retention on a polar stationary phase may be the best indicator of the exposed polarity, as was demonstrated by Goetz et al. [START_REF] Goetz | EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides[END_REF] for cyclic peptides: longest retention of peptides on a polar stationary phase in SFC conditions was indeed observed to be related to lower membrane permeability. Clearly, log P and TPSA are both insufficient to assess the polarity of large flexible molecules [START_REF] Caron | Molecular descriptors for polarity: the need for going beyond polar surface area[END_REF].

Nucleotides are also visible in Figure 1, with a representative structure based on a guanine nucleobase. The purine and pyrimidine structure of the five nucleobases (adenine, guanine, cytosine, thymidine and uracil) are all small and polar as they appear at the bottom of Figure 3, with negative log P values. Adding the pentose ring to form a nucleoside is slightly increasing molecular weight but also significantly increasing polarity, as the bubbles representing nucleosides are slightly higher and on the left. Finally, introducing a phosphate tail is also causing increased molecular weight, is not greatly affecting log P values but significantly increasing TPSA values (bigger red bubbles in Figure 3). Nucleic acid chains should then be most polar structures, in terms of log P or TPSA.

From Figure 3 and the above discussion, we can conclude that the target molecules reviewed in this paper constitute a very heterogeneous group, with a variety of polarities and sizes, mostly from 100 to 2000 Da, or even higher for some bigger peptides and proteins not represented in the figure. While all target molecules bear polar functional groups (expressed by TPSA), be they charge-neutral or ionizable, their overall polarity expressed by log P varies in a great range. As a consequence, it is unlikely that one generic chromatographic method would work for all of them. The different choices of stationary phases and mobile phases previously selected to achieve their analysis or purification, presented in Tables 1, 2 and 3, will be detailed in the following sections.

Mobile phase composition

CO2 has little polarity and is usually compared to hexane. This comparison is not entirely fair as CO2 has some polarity features (local dipole moments, polarizability) and reactivity.

However, a neat CO2 mobile phase in usual SFC operating conditions (temperature around 30°C, back-pressure of 11-18 MPa) is unlikely to solvate any of the target biomolecules.

When charge-neutral species are considered, mixing pressurized CO2 to a polar organic solvent, like the most frequently used short-chains alcohols [START_REF] West | Current trends in supercritical fluid chromatography[END_REF], is sufficient to achieve good solubility. Methanol is usually the preferred co-solvent observed in SFC literature, and the specific papers related to our target molecules are no exception, as methanol was observed in above 80% of the articles. A few papers mention other co-solvents, at least at the stage of method development: ethanol [START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF][START_REF] Blackwell | Effect of Mobile Phase Components on the Separation of Polypeptides Using Carbon Dioxide-Based Mobile Phases[END_REF][START_REF] Shao | Rapid Separation of Five Cyclosporin Analogs by Supercritical Fluid Chromatography[END_REF], isopropanol or acetonitrile [START_REF] Emmett | Supercritical Fluid Chromatography Reduction of Hydrogen/Deuterium Back Exchange in Solution-Phase Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF][START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Treadway | Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides[END_REF], the latter being often introduced in mixtures with methanol. The co-solvent increases the mobile phase polarity [START_REF] Deye | Nile Red as a solvatochromic dye for measuring solvent strength in normal liquids and mixtures of normal liquids with supercritical and near critical fluids[END_REF] and viscosity, modifies the mobile phase density [START_REF] Berger | Clarifying the relationship between density and viscosity of methanol/carbon dioxide mixtures used in supercritical fluid chromatography[END_REF], introduces other possible interactions between the mobile phase and the analytes and adsorbs onto the stationary phase [START_REF] Glenne | Systematic investigations of peak deformations due to co-solvent adsorption in preparative supercritical fluid chromatography[END_REF], thereby also changing the possible interactions between the analytes and stationary phase [START_REF] West | Effects of mobile phase composition on retention and selectivity in achiral supercritical fluid chromatography[END_REF].

For the most polar charge-neutral or ionized species, two options are usually considered.

First, (one or more) acidic, basic or salt additives are frequently employed. For instance, the second oldest example of SFC analysis of our target biomolecules (1992) reported the achiral analysis of free proteinogenic amino acids with SFC-UV [START_REF] Camel | Packed column subcritical fluid chromatography of underivatized amino acids[END_REF], and was mostly focused on adjusting the mobile phase composition with additives mixtures to elute the analytes with satisfying peak shapes. In general, additives nature and concentration is explored early on in method development, to determine adequate composition to achieve elution of the analytes with good peak shapes, taking account of detector issues. Unlike liquid-phase chromatography, the mobile phase in SFC and EFLC cannot be easily buffered because it is not possible to measure pH* precisely. As a result, the acid and base ionizable functions are not easily controlled. It was shown that the combined presence of CO2 and an alcohol cosolvent induces some acidic character in usual SFC [START_REF] West | Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase[END_REF][START_REF] Raimbault | Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography[END_REF] and EFLC [START_REF] Wen | Characterization of pH in Liquid Mixtures of Methanol/H2O/CO2[END_REF] conditions due to the formation of methoxylcarbonic acid, typically between pH* 4 and 6, depending on the proportion of water in the mobile phase, which is further acidifying the fluid through the formation of carbonic acid. It was also shown that the introduction of additives could modify the apparent pH*, with strong acids further reducing the apparent pH* down to 1-2, and bases only moderately increasing it to pH* 6-7 [START_REF] West | Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part I: Polarity and acidity of the mobile phase[END_REF][START_REF] Raimbault | Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography[END_REF]. Salts like ammonium acetate could act as stabilizers in gradient elution modes. However, we can only conjecture on the exact ionization state of the target molecules as their exact pKa values in such conditions are unknown. Whenever ionic groups are present, the use of additives serves not only in changing the apparent pH* and ionization state of the analytes, but may also contribute to the formation of ion pairs between the analyte and additive ions to favor their elution.

Furthermore, the additive can adsorb on the stationary phase to reduce strong interactions between an ionic analyte and polar groups of the stationary phase [START_REF] Raimbault | Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography[END_REF][START_REF] West | Unravelling the effects of mobile phase additives in supercritical fluid chromatography. Part II: Adsorption on the stationary phase[END_REF]. Other benefits of the co-solvent and additives may be found in detection issues, particularly when hyphenation to mass spectrometry is desired [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF]. This will be further detailed in the section related to detection issues. From a practical point of view, it is customary to use acidic additives for acidic analytes and basic additives for basic analytes, but the contrary is also observed when ion-pairing may help in favoring the elution [START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF]. When the analytes are large and complex, and possess several ionizable groups with acidic and basic functions, an acid and a base are sometimes used together, or salts like ammonium acetate. Examining the literature related to SFC/EFLC analysis of our target biomolecules, the most often cited additive to elute the target biomolecules was trifluoroacetic acid (TFA) [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF][START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF][START_REF] Camel | Packed column subcritical fluid chromatography of underivatized amino acids[END_REF][START_REF] Raimbault | Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Thurbide | Separation of linear gramicidins using carbon dioxide-containing mobile phases[END_REF][START_REF] Zheng | Feasibility of Supercritical Fluid Chromatography/Mass Spectrometry of Polypeptides with Up to 40-Mers[END_REF][START_REF] Zhang | Membrane Protein Separation and Analysis by Supercritical Fluid Chromatography-Mass Spectrometry[END_REF][START_REF] Tognarelli | Rapid peptide separation by supercritical fluid chromatography[END_REF][START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF][START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Miller | Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column[END_REF][START_REF] Ashraf-Khorassani | Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive[END_REF][START_REF] Ganipisetty | Supercritical fluid (CO 2 ) chromatography for quantitative determination of selected cancer therapeutic drugs in the presence of potential impurities[END_REF]. Other acids like formic acid [START_REF] Emmett | Supercritical Fluid Chromatography Reduction of Hydrogen/Deuterium Back Exchange in Solution-Phase Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis[END_REF][START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF][START_REF] Camel | Packed column subcritical fluid chromatography of underivatized amino acids[END_REF][START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Dos Santos Pereira | Green hydrophilic interaction chromatography using ethanol-water-carbon dioxide mixtures[END_REF] or methane-and ethane-sulfonic acids [START_REF] Blackwell | Effect of Mobile Phase Components on the Separation of Polypeptides Using Carbon Dioxide-Based Mobile Phases[END_REF][START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF][START_REF] Stringham | Chiral separation of amines in subcritical fluid chromatography using polysaccharide stationary phases and acidic additives[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF] were also mentioned in several occasions. Isopropylamine, a basic additive that is frequently used in SFC analysis of basic drugs, appeared only in a few papers [START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Ashraf-Khorassani | Analysis of the sulfomycin component of alexomycin in animal feed by enhanced solvent extraction and supercritical fluid chromatography[END_REF], suggesting that a basic additive may not be the best in this case. Ammonium acetate [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF][START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Zheng | Feasibility of Supercritical Fluid Chromatography/Mass Spectrometry of Polypeptides with Up to 40-Mers[END_REF][START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Ashraf-Khorassani | Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive[END_REF][START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Bolaños | SFC/MS in drug discovery at Pfizer, La Jolla[END_REF][START_REF] Hsieh | Supercritical Fluid Chromatography and High-Performance Liquid Chromatography/Tandem Mass Spectrometric Methods for the Determination of Cytarabine in Mouse Plasma[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF] and ammonium formate [START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF][START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF][START_REF] Dos Santos Pereira | Green hydrophilic interaction chromatography using ethanol-water-carbon dioxide mixtures[END_REF][START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF][START_REF] Sen | Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry[END_REF], which were both frequently employed in recent SFC methods [START_REF] West | Current trends in supercritical fluid chromatography[END_REF], were mentioned many times. More recently, ammonium hydroxide has become more and more popular in SFC analysis of basic drugs and was found to be equally useful for amino acids, peptides and proteins [START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF]. In older papers, several other exotic additives were employed, while in the past decade, the additives employed in SFC/EFLC analyses have mostly been standardized, largely favoring the additives that are compatible to mass spectrometric detection. Still a few original proposals can be found, like ammonium fluoride to achieve the elution of most polar amino acids in SFC [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF], or sodium phosphate for nucleotides in EFLC [START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF].

Secondly, some portion of water can also be introduced in the mobile phase. Although already an old practice [START_REF] Pyo | Addition of water in carbon dioxide mobile phase for supercritical fluid chromatography[END_REF][START_REF] Geiser | Water as a stationary phase modifier in packed-column supercritical fluid chromatography : I. Separation of free fatty acids[END_REF], this was somewhat forgotten for several years, only to reappear most significantly in the last decade. Water may be present in small concentrations -typically 2 to 5% -and then acting like a polar additive [START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF], or in large concentrations (typically above 10%) and then acting as a modifier [START_REF] Wen | Chromatography of substituted benzoic acids with methanol-watercarbon dioxide mixtures[END_REF]. Because CO2 and water are not miscible in all proportions, large concentrations of water can only be introduced in ternary compositions of CO2-solvent-water. Such mobile phase compositions are most often encountered in EFLC experiments. Note that an EFLC method may be developed by introducing carbon dioxide in any sort of liquid mobile phase: either from organic solvents used in a normal-phase LC method [START_REF] Lee | Normal-phase high-performance liquid chromatography using enhancedfluidity liquid mobile phases[END_REF] or SEC [START_REF] Yuan | High-Performance Size Exclusion Chromatography Using Enhanced-Fluidity Liquid Mobile Phases[END_REF], or from buffer-organic mixtures used in RPLC [START_REF] Cui | Reversed-phase high-performance liquid chromatography using enhancedfluidity mobile phases[END_REF], HILIC [START_REF] Treadway | Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides[END_REF][START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF][START_REF] Philibert | Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides[END_REF], HIC [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF] and IEX [START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF] modes. The proportion of water possibly introduced in a CO2-solvent mixture depends on (i) the operating conditions of temperature and pressure, (ii) the proportions of CO2 and solvent and (iii) the nature of the solvent.

Ternary diagrams of such mixtures are scarcely available [START_REF] Lee | Phase diagram studies of methanol-H2O-CO2 and acetonitrile-H2O-CO2 mixtures[END_REF][START_REF] Olesik | Physicochemical properties of enhanced-fluidity liquid solvents[END_REF] but would be most useful to develop further analytical and preparative methods for the mostly polar target biomolecules.

For instance, Li and Thurbide [START_REF] Li | A comparison of methanol and isopropanol in alcohol/water/CO2 mobile phases for packed column supercritical fluid chromatography[END_REF] showed that using some isopropanol in conjunction with methanol as co-solvent was favorable to the introduction of larger proportions of water, thereby allowing the elution of most polar species like free amino acids without any other additive.

Additives and water may also have combined effects. For instance, Taylor had demonstrated that the combined use of water and ammonium acetate was more powerful in achieving the elution of nucleobases with good peak shapes than the single use of either one or the other [START_REF] Taylor | Packed column supercritical fluid chromatography of hydrophilic analytes via waterrich modifiers[END_REF]. Recently, Regalado and co-workers discussed the chaotropic effects of ammonium hydroxide combined with water in a CO2-methanol mobile phase when eluting peptides [START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF]. In fact, in many papers, the additives mentioned above were used in conjunction with water [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Miller | Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column[END_REF][START_REF] Ashraf-Khorassani | Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF][START_REF] Hsieh | Supercritical Fluid Chromatography and High-Performance Liquid Chromatography/Tandem Mass Spectrometric Methods for the Determination of Cytarabine in Mouse Plasma[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF][START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF][START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF][START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF][START_REF] Grand-Guillaume Perrenoud | Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-offlight mass spectrometry as a performing tool for bioactive analysis[END_REF]. Taking aside the synergistic effect that water may have with an additive, it may also favor the additive solubility, both in the pressurized CO2 mobile phase and after the decompression to avoid additive precipitation in the instrument.

Whenever a wide range of analyte polarities is encountered, the analyses are preferably conducted in gradient elution mode. With the evolutions in SFC practice, the proportion of cosolvent introduced in the carbon dioxide mobile phase has been increasing more and more.

In the early years, only neat fluids were employed (not only CO2 but also ammonia, fluorocarbons, etc.) Back in the years 1980s, when it was found that introducing a co-solvent brought more benefits in expanding the range of analytes amenable to the technique than the expected drawbacks of increased viscosity, only a few percents (1-5%) of solvent were employed. In the years 1990s, with the advent of packed column SFC over capillary SFC, the typical range of solvent proportion extended to 1-20%, then increased further in the years 2000s. In the years 2010s, the maximum amount of co-solvent encountered in most SFC papers was rather around 60%, which makes the fluid closer to a liquid than a supercritical fluid, although it does not change the way to operate the system.

As appears in Figure 4, SFC is usually operating in the low portion of solvent area, while EFLC usually works in the large portion of solvent area. While no fundamental nor practical limit exists between the two [START_REF] Olesik | Enhanced-Fluidity Liquid Chromatography: Connecting the Dots Between Supercritical Fluid Chromatography, Conventional Subcritical Fluid Chromatography, and HPLC[END_REF], the distinction is rather issuing from different approaches: SFC historically started with neat fluids, among which was neat CO2. A co-solvent was later introduced to expand the polarity limits of soluble analytes. At the other end of the solvent axis, EFLC started with LC conditions, where CO2 was introduced to reduce the viscosity of the mobile phase, thereby providing improved efficiencies and the possibility to use higher flow rates. In addition, the EFLC mobile phase should be a greener technique compared to a fully liquid mobile phase as the consumption of solvent should be reduced [START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF]. Olesik and co-workers are the main explorers of the EFLC concept, particularly at the analytical scale.

Specifically on biomolecules, they have successfully applied the method to nucleotides and nucleosides [START_REF] Treadway | Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides[END_REF][START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF][START_REF] Philibert | Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides[END_REF], amino acids [START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF] and proteins [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF][START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF]. In addition, Bennett and coworkers [START_REF] Bennett | Enhanced fluidity liquid chromatography: A guide to scaling up from analytical to preparative separations[END_REF] have brought EFLC to purification settings by introducing a guide to scaling up from analytical to preparative separations.

In 2014 [START_REF] Taguchi | Simultaneous analysis for water-and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography[END_REF], Bamba and co-workers proposed to use the full range of co-solvent proportion, starting with a mobile phase containing nearly 100% CO2 and ending the gradient elution at 100% hydro-organic composition. Because the first portion of the chromatogram was SFC, then crossing the EFLC area and ending in liquid chromatography conditions, the method was called "unified chromatography" (UC), a term previously used in similar experiments joining gas chromatography, SFC and LC [START_REF] Tong | Unified chromatograph for gas chromatography, supercritical fluid chromatography and micro-liquid chromatography[END_REF][START_REF] Ishii | Unified Fluid Chromatography[END_REF]. Most modern instruments are perfectly capable to operate these wide gradients, with some instruments being clearly designed for easy switching between SFC and LC operation. UC was found most useful to achieve the elution of all proteinogenic free amino acids, from the most hydrophobic to the most hydrophilic [START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF]. Guillarme and co-workers also used the same method to elute metabolites with a wide range of polarities, including free amino acids [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF] and nucleosides [START_REF] Losacco | Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices[END_REF]. EFLC and UC approaches might be a part of the answer to achieve the analysis of a wider range of biomolecules.

Finally, because many of the target molecules possess amine functions (amino acids, peptides, proteins), a few words of caution should be said about the possible reactions occurring between CO2 and amines. Indeed, CO2 is believed to react with some amines, possibly forming carbamic acids or ammonium carbamates [START_REF] Eckert | Sustainable Reactions in Tunable Solvents[END_REF]. However, this reaction is reversible so the original amine is usually found after CO2 depressurization [START_REF] Fischer | Reaction Monitoring of Aliphatic Amines in Supercritical Carbon Dioxide by Proton Nuclear Magnetic Resonance Spectroscopy and Implications for Supercritical Fluid Chromatography[END_REF]. This may not be an issue for smaller, unstructured molecules like amino acids and peptides, but may be a problem for 3D-stable structures like proteins, if the amine reaction causes permanent unfolding of the protein. In a paper from Regalado and co-workers [START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF], a co-solvent comprising methanol, 5% water and 0.2% ammonium hydroxide was employed, with gradient ranging from 5 to 50% co-solvent. The combined use of water and NH4OH were suggested to cause chaotropic effects favoring the elution of a variety of analytes including nucleobases, nucleosides, nucleotides and some peptides (linaclotide 1523 Da and gramicidin S 1882 Da). In a separate study from the same group [START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF], the authors found that some proteins like bradykinin and insulin could be recovered to their original higher-order structure after SFC purification, while others like ubiquitin, cytochrome C and apomyoglobin had permanently lost their higher-order structure. The co-solvent was a 3:1 mixture of methanol and acetonitrile, added with 5% water and 0.2% trifluoroacetic acid. The elution gradient ranged from 35 to 70% co-solvent in CO2, which is covering both the usual SFC and EFLC domains. Another recent paper from Govender and co-workers [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF] presented the purification of biosynthesized human insulin with SFC. The co-solvent was methanol comprising 5% water and 0.2% trifluoroacetic acid, thus identical to the Schiavone et al. [START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF] mobile phase. The purified insulins retained their bioactivity, suggesting that the higher-order structure was indeed retained.

On another hand, Wang and Olesik also relate the elution of intact proteins [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF] in EFLC mode starting from a hydrophobic interaction chromatography (HIC) method, and progressively introducing CO2 in the 70:30 methanol-ammonium acetate buffer mobile phase. In addition to improved chromatographic efficiency, the introduction of CO2 was shown to improve ESI-MS detection through facilitated desolvation. However, the claim was on intact molecular weight, suggesting that no permanent reaction had damaged the primary structure, but no information was given on preservation of higher-order structure.

Stationary phase nature, particle type and dimension

The range of stationary phases available for packed column SFC and EFLC was always large, as any stationary phase designed for liquid chromatography can also be used with CO2 in the mobile phase. The nature of stationary phases has however greatly expanded in the recent years as not only reversed-phase [START_REF] West | Characterization of stationary phases in subcritical fluid chromatography by the solvation parameter model: I. Alkylsiloxane-bonded stationary phases[END_REF][START_REF] West | Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model: IV. Aromatic stationary phases[END_REF] and normal-phase [START_REF] West | Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model: III. Polar stationary phases[END_REF] stationary phases are possible but also the most recent stationary phases designed for HILIC mode [START_REF] West | Characterization and use of hydrophilic interaction liquid chromatography type stationary phases in supercritical fluid chromatography[END_REF] and for mixed-mode liquid chromatography [START_REF] Lemasson | Mixed-Mode Chromatography-A Review[END_REF] and several novel stationary phases especially designed for SFC use [START_REF] West | An improved classification of stationary phases for ultra-high performance supercritical fluid chromatography[END_REF]. The diversity of available stationary phases is an interesting feature of SFC and EFLC because it means that different selectivities are available to achieve a given separation. The role of a stationary phase is two-fold: it must interact with the target analytes to (i) retain them and (ii) separate them. It is customary in SFC method development to start developing a method by screening several stationary phases with complementary selectivities, to identify a good starting point for optimization.

Judging from the above discussion on the biomolecules polarity, it appears that many different stationary phase chemistries could be useful as our target molecules comprise both polar and non-polar molecules. In addition, the molecules requiring some separation may have very different structural differences. For instance, when nucleobases must be resolved, polar interactions are necessary to distinguish between small polar analytes with subtle differences, thus a polar stationary phase is necessary. When large hydrophobic peptides must be separated, a non-polar stationary phase may be a best candidate.

As most of them possess a chiral center, underivatized amino acids were often analyzed on enantioselective stationary phases. The most cited phases were polysaccharide-based stationary phases [START_REF] Stringham | Chiral separation of amines in subcritical fluid chromatography using polysaccharide stationary phases and acidic additives[END_REF][START_REF] Lipka | Separation of enantiomers of native amino acids with polysaccharide-based chiral columns in supercritical fluid chromatography[END_REF], which are most popular in enantioselective SFC. Other possible enantioselective phases are Cinchona alkaloid-based zwitterionic stationary phases, which were specifically designed for amino acid enantioresolution in LC [START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF], macrocyclic glycopeptide stationary phase [START_REF] Raimbault | Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography[END_REF][START_REF] Sánchez-Hernández | Chiral analysis of aromatic amino acids in food supplements using subcritical fluid chromatography and Chirobiotic T2 column[END_REF] and crown-ether stationary phase [START_REF] Miller | Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column[END_REF], which are both also known to perform well for amino acids in liquid phase.

When no enantioresolution was desired and only intra-class separation of free amino acids was looked for, a variety of achiral stationary phases were used. Diol-type stationary phases are most often cited [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF][START_REF] Sen | Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry[END_REF][START_REF] Grand-Guillaume Perrenoud | Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-offlight mass spectrometry as a performing tool for bioactive analysis[END_REF], starting from the seminal paper from Camel and co-workers [START_REF] Camel | Packed column subcritical fluid chromatography of underivatized amino acids[END_REF]. Toribio and co-workers proposed to couple a chiral glycopeptide stationary phase to an achiral diol phase to resolve two pairs of enantiomers (phenylalanine and tyrosine) in a single analysis with SFC-UV [START_REF] Sánchez-Hernández | Chiral analysis of aromatic amino acids in food supplements using subcritical fluid chromatography and Chirobiotic T2 column[END_REF]. Cinchona alkaloid-based zwitterionic stationary phases also allowed a most complete separation of all proteinogenic amino acids from each other, together with enantioresolution for most of them [START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF]. Interestingly, the four isobaric leucine enantiomers and isoleucine enantiomers could all be resolved [START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF]. Finally, other achiral stationary phases cited for the SFC/EFLC analysis of free amino acids are phases normally used in HILIC mode, like the anionic polysulfoethyl stationary phase [START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF], the zwitterionic sulfobetaine-bonded stationary phase [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF], bare silica gel [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF] or amidebonded stationary phase [START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF].

For peptides and proteins, where chiral separation is rarely desired, achiral stationary phases with pyridine ligands were the most often cited stationary phases [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Zheng | Feasibility of Supercritical Fluid Chromatography/Mass Spectrometry of Polypeptides with Up to 40-Mers[END_REF][START_REF] Tognarelli | Rapid peptide separation by supercritical fluid chromatography[END_REF][START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF][START_REF] Jenny) Qian Cutrone | Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical consideration and method development[END_REF]. 2-Ethylpyridine is a stationary phase that was designed specifically for SFC at the beginning of the years 2000s by Princeton Chromatography, and found an immediate success for basic drugs because it provided nice peak shapes without the need of mobile phase additives, supposedly due to hydrogen bonding between residual silanol groups and the pyridine nitrogen atom [START_REF] Cazenave-Gassiot | Effect of increasing concentration of ammonium acetate as an additive in supercritical fluid chromatography using CO2-methanol mobile phase[END_REF]. Other variations include a 4-ethylpyridine ligand [START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF] or pyridyl-amide. A polymeric version of 4-ethylpyridine, poly(4-vinylpyridine) (P4VP) was also cited [START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF]. 2-Picolylamine, appearing in the latest publications [START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF], is a most recent version of 2-ethylpyridine, which was introduced to limit retention loss through silyl ether formation [START_REF] Fairchild | Chromatographic Evidence of Silyl Ether Formation (SEF) in Supercritical Fluid Chromatography[END_REF]. Other polar stationary phases used for peptides and proteins include again diol-type stationary phases [START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Patel | Packed column supercritical fluid chromatography of isomeric polypeptide pairs[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Ashraf-Khorassani | Analysis of the sulfomycin component of alexomycin in animal feed by enhanced solvent extraction and supercritical fluid chromatography[END_REF], bare silica gel [START_REF] Emmett | Supercritical Fluid Chromatography Reduction of Hydrogen/Deuterium Back Exchange in Solution-Phase Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis[END_REF][START_REF] Shao | Rapid Separation of Five Cyclosporin Analogs by Supercritical Fluid Chromatography[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF] and amide bonding [START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF], but also cyanopropyl-bonded silica [START_REF] Zhang | Membrane Protein Separation and Analysis by Supercritical Fluid Chromatography-Mass Spectrometry[END_REF][START_REF] Bolaños | SFC/MS in drug discovery at Pfizer, La Jolla[END_REF]. Non-polar and moderately polar phases, which are less frequently encountered in SFC applications, also appear in a few instances.

For example, aromatic phases like pentafluorophenyl (PFP) [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF] and polystyrenedivinylbenzene (PS-DVB) [START_REF] Blackwell | Effect of Mobile Phase Components on the Separation of Polypeptides Using Carbon Dioxide-Based Mobile Phases[END_REF][START_REF] Thurbide | Separation of linear gramicidins using carbon dioxide-containing mobile phases[END_REF][START_REF] Li | A comparison of methanol and isopropanol in alcohol/water/CO2 mobile phases for packed column supercritical fluid chromatography[END_REF][START_REF] Zhang | Direct analysis of gramicidin double helices using packed column supercritical fluid chromatography[END_REF] were mentioned in a few occasions, starting with the first paper reporting SFC analysis of both cyclic and linear peptides [START_REF] Blackwell | Effect of Mobile Phase Components on the Separation of Polypeptides Using Carbon Dioxide-Based Mobile Phases[END_REF]. Finally, alkyltype stationary phases were also mentioned: octadecylbonded-silica (ODS) was used in the first report of cyclic peptide analysis (Cyclosporin A) [START_REF] Kalinoski | Chemical ionization mass spectra of high molecular weight, biologically active compounds produced following supercritical fluid chromatography[END_REF] and later to resolve isomeric 12-mer peptides [START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF]. Polybutyl and polypentyl phases normally used in HIC mode (hydrophobic interaction chromatography) were recently employed to analyze intact proteins in EFLC [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF].

To those less familiar with the technique, it may be surprising to observe that there seems to be little rationale behind the use of one column or another as very different stationary phases were found to be useful for the same purpose by different users. For instance, the cyclic peptides of cyclosporins were analyzed on an ODS phase [START_REF] Kalinoski | Chemical ionization mass spectra of high molecular weight, biologically active compounds produced following supercritical fluid chromatography[END_REF] or on bare silica gel [START_REF] Shao | Rapid Separation of Five Cyclosporin Analogs by Supercritical Fluid Chromatography[END_REF]. Gramicidin, a membrane protein comprising several members, was also analyzed by several different authors on different stationary phases: cyanopropyl-bonded silica [START_REF] Zhang | Membrane Protein Separation and Analysis by Supercritical Fluid Chromatography-Mass Spectrometry[END_REF][START_REF] Bolaños | SFC/MS in drug discovery at Pfizer, La Jolla[END_REF], PS-DVB [START_REF] Thurbide | Separation of linear gramicidins using carbon dioxide-containing mobile phases[END_REF][START_REF] Zhang | Direct analysis of gramicidin double helices using packed column supercritical fluid chromatography[END_REF], bare silica gel [START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF] or pyridine phases [START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF][START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF]. Different pairs of 12-mer isomeric peptides (about 1100 Da) were successfully resolved on a variety of stationary phases including ODS, bare silica gel or 2-ethylpyridine [START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF]. Actually, it is often observed in SFC that several very different stationary phases can be successful for the same separation problems, although different elution orders would be observed [START_REF] West | A classification of columns for SFC use[END_REF].

The case of nucleic acid bricks is different because, as appeared in Figure 3, nucleobases, nucleosides and nucleotides are all polar, with negative log P values. Thus retention and selectivity in that case is favored by polar stationary phases with bare silica [START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Dos Santos Pereira | Green hydrophilic interaction chromatography using ethanol-water-carbon dioxide mixtures[END_REF][START_REF] Hsieh | Supercritical Fluid Chromatography and High-Performance Liquid Chromatography/Tandem Mass Spectrometric Methods for the Determination of Cytarabine in Mouse Plasma[END_REF], diol [START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF][START_REF] Ashraf-Khorassani | Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive[END_REF][START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Sen | Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry[END_REF], amide [START_REF] Treadway | Enhanced fluidity liquid chromatography for hydrophilic interaction separation of nucleosides[END_REF][START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF][START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF][START_REF] Philibert | Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides[END_REF] and pyridine-type [START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF][START_REF] Ashraf-Khorassani | Subcritical fluid chromatography of water soluble nucleobases on various polar stationary phases facilitated with alcohol-modified CO2 and water as the polar additive[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF][START_REF] Ganipisetty | Development of supercritical fluid (carbon dioxide) based ultra performance convergence chromatographic stability indicating assay method for the determination of clofarabine in injection[END_REF] being equally cited.

The improved instruments for analytical SFC introduced at the beginning of the years 2010 tended towards higher efficiency through reduced dead volumes and detector cells.

Concomitantly, there has been a trend to high efficiency packed columns, either through small fully porous particles (FPP) with sub-2 µm diameter, or through superficially porous particles (SPP) with sub-3 µm diameter. In the recent papers, sub-2 µm FPP stationary phases appear [START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF][START_REF] Ganipisetty | Development of supercritical fluid (carbon dioxide) based ultra performance convergence chromatographic stability indicating assay method for the determination of clofarabine in injection[END_REF]. However, when a large elution gradient is desired, the viscosity of the mobile phase at the end of the gradient is high. Because small particles also generate high pressure drops, not all SFC instruments can sustain the high pressure. Indeed, some instruments are still limited to 400 bars inlet pressure (Waters, Jasco), while others can reach 600 bars (Agilent) or 660 bars (Shimadzu). In that case, the flow rate and/or back-pressure must be reduced to avoid reaching the upper pressure limit. Another option is to favor columns packed with SPP stationary phases [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF].

Detection issues and hyphenation to mass spectrometry

Packed-column SFC and EFLC make use of the same detectors as LC, namely UVvisible or diode-array detection (DAD), evaporative light scattering detection (ELSD), Corona charged-aerosol detection (CAD) and mass spectrometry (MS). The latter is preferably equipped with LC-type ionization source, namely electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Among all these detectors, not all are equally useful for the target biomolecules. Amino acids, their oligomers and polymers mostly lack of chromophores, thus UV and DAD detection are of little use, unless the desired detection limit is not too low [START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF]. Bigger peptides and proteins and those comprising aromatic residues may however be easier to detect with UV at 210 or 280 nm. Because the nucleobases are good chromophores, UV and DAD detection is appropriate for the nucleobases, nucleosides, nucleotides and nucleic acids.

While UV detection is placed before the back-pressure regulator, ELSD and CAD are placed after the BPR. Only a few studies explored ELSD possibilities in SFC [START_REF] Camel | Packed column subcritical fluid chromatography of underivatized amino acids[END_REF][START_REF] Patel | Supercritical fluid chromatographic resolution of water soluble isomeric carboxyl/amine terminated peptides facilitated via mobile phase water and ion pair formation[END_REF][START_REF] Lipka | Separation of enantiomers of native amino acids with polysaccharide-based chiral columns in supercritical fluid chromatography[END_REF] or in EFLC [START_REF] Beres | Enhanced-fluidity liquid chromatography using mixed-mode hydrophilic interaction liquid chromatography/strong cation-exchange retention mechanisms[END_REF] for underivatized amino acids and small peptides.

Because it provides additional information on analyte structure, mass spectrometry is certainly the most desirable detection mode hyphenated to SFC/EFLC instruments, especially now that it is easier to access in most laboratories, with simpler instruments available at a moderate cost. Hyphenation of SFC/EFLC to MS may be done several ways [START_REF] Guillarme | What are the current solutions for interfacing supercritical fluid chromatography and mass spectrometry?[END_REF][START_REF] Tarafder | Designs and methods for interfacing SFC with MS[END_REF]. First, flow splitting may be done -or not -prior to entering the MS, with one portion of the column effluent entering the MS and the other being directed towards the backpressure regulator (BPR) and waste or fraction collection [START_REF] Grand-Guillaume Perrenoud | Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds[END_REF]. When the chromatographic flow rate is high, the flow is preferentially divided because the ionization sources cannot all cope with the large volume expansion caused by CO2 depressurization. However, this can affect sensitivity for mass-sensitive detectors. As the largest portion of SFC instruments sold are based on flow-splitting configuration, this is quite logically the most often cited in the literature related to biomolecules. However, when the chromatographic flow rate is low, the whole effluent can be directed to the MS, which is then placed right after the BPR. Second, a make-up fluid may be introduced prior to entering the MS, and prior to the flow splitting, when flow splitting is desired. The interest of a make-up fluid is to avoid analyte precipitation upon the cold CO2 depressurization and to introduce ionization enhancers prior to entering the MS source. However, the make-up fluid may cause further dilution of the column effluent, thereby may be reducing sensitivity for concentration-sensitive detectors. Deconvoluting the positive and negative effects of the make-up fluid is difficult, so the variations observed are not always in accordance with intuition [START_REF] Akbal | Effects of liquid post-column addition in electrospray ionization performance in supercritical fluid chromatography-mass spectrometry[END_REF].

The composition of make-up fluid is most often methanol, comprising a small proportion of water (1 to 10%) and one acidic or salt additive. This additive is sometimes the same as the one employed in the mobile phase [START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF][START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF], sometimes different [START_REF] Miller | Chiral separation of underivatized amino acids in supercritical fluid chromatography with chiral crown ether derived column[END_REF][START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF] because the composition favoring good chromatography is not necessarily the same as the composition favoring good ionization. A supposedly generic composition and flow rate are often adopted and their impact was rarely studied [START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF]. When large elution gradients are employed, especially in UC experiments, a constant flow rate of make-up fluid could be inadequate.

The choice of ionization source, which depends on analyte size and polarity, has a significant impact on the intensity of molecular and fragment ions detected. ESI and APCI were employed in almost all studies. Wolrab et al. [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF] compared ESI and APCI sources specifically for SFC-MS/MS analysis of amino acids and related compounds. They concluded that ESI seemed to be the best choice for the least polar amino acids. However, for polar amino acids and non-polar amino acids with an additional heteroatom as well as acidic amino acids, APCI seemed the best choice. In other words, the preferred ionization source will be greatly dependent on analyte, even within one molecular family.

In terms of analyzers, some recent investigations were done with triple-quadrupole [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF] or quadrupole-time-of-flight [START_REF] Wang | Enhanced-Fluidity Liquid Chromatography-Mass Spectrometry for Intact Protein Separation and Characterization[END_REF][START_REF] Bennett | Protein separations using enhanced-fluidity liquid chromatography[END_REF][START_REF] Laboureur | Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry[END_REF][START_REF] Grand-Guillaume Perrenoud | Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-offlight mass spectrometry as a performing tool for bioactive analysis[END_REF] analyzers, which can provide precise structural information the target analytes. When complex matrices are present, or high molecular weight biomolecules are considered, high resolution is necessary.

Applications areas

Due to the limited number of papers published to date, applications only articulated around three themes. First, food products were the topic of a few papers. Sánchez-Hernández et al. [START_REF] Sánchez-Hernández | Chiral analysis of aromatic amino acids in food supplements using subcritical fluid chromatography and Chirobiotic T2 column[END_REF] coupled chiral and achiral stationary phases to control the quality of food supplements and confirm the absence of enantiomeric impurities of free amino acids with UV detection. Raimbault et al. [START_REF] Raimbault | Analysis of free amino acids with unified chromatographymass spectrometry-application to food supplements[END_REF] used a single chiral stationary phase for quantitative assessment of free branched amino acids in food supplements with single-quadrupole MS detection. Huang and co-workers [START_REF] Huang | Simultaneous determination of amino acids in different teas using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF] determined 11 free amino acids in several varieties of tea with an achiral stationary phase and single-quadrupole MS detection. The method was fully validated. Ashraf-Khorassani et al. [START_REF] Ashraf-Khorassani | Analysis of the sulfomycin component of alexomycin in animal feed by enhanced solvent extraction and supercritical fluid chromatography[END_REF] focused on depsipeptides, which are peptides with some amides functions replaced by esters. The authors developed a quantitative extraction method for some growth-promoting animal feed additives, and analyzed them with SFC-UV. The target molecules were the major components of alexomycin like sulfomycin (see structure in Figure 5) and promothiocin.

Secondly, human/animal [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF][START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF][START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF][START_REF] Sen | Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry[END_REF][START_REF] Losacco | Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices[END_REF] or plant [START_REF] Grand-Guillaume Perrenoud | Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-offlight mass spectrometry as a performing tool for bioactive analysis[END_REF] metabolomics studies are also concerned with biomolecules separations in SFC/EFLC or UC. Liquid-phase chromatography hyphenated to mass spectrometry is currently dominating the metabolomics scene. However, because the samples are complex and the metabolites are extremely varied in terms of structures and polarities, the potential of other methods to offer complementary information to other existing techniques is of great interest, as exposed by Shulaev and Isaac [START_REF] Shulaev | Supercritical fluid chromatography coupled to mass spectrometry -A metabolomics perspective[END_REF].

Wolrab et al. [START_REF] Wolrab | Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization[END_REF] focused on MS detection of free amino acids and related compounds in human serum. Amino acids, peptides, and nucleosides were all included in SFC-MS methods from Sen and co-workers [START_REF] Sen | Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry[END_REF], and from Akbal and Hopfgartner [START_REF] Akbal | Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine[END_REF]. The latter compared the results to those of RPLC and HILIC. Many metabolites previously included in the method development were identified in human urine within fast SFC-MS runs. In a different study, Desfontaine et al. [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF] focused on a most generic method to analyze a wide range of metabolite polarities, including most polar amino acids as well as non-polar lipids.

For this purpose, a wide UC elution gradient was favored, and was later applied to real samples of plasma and urine [START_REF] Losacco | Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices[END_REF]. Similarly, Grand-Guillaume and co-workers [START_REF] Grand-Guillaume Perrenoud | Ultra-high performance supercritical fluid chromatography coupled with quadrupole-time-offlight mass spectrometry as a performing tool for bioactive analysis[END_REF] developed a widely applicable SFC-MS/MS method including a great variety of plant metabolites, from the most polar free amino acids to the least polar lipids and terpenes. They later demonstrated the interest of combining LC-MS/MS and SFC-MS/MS to acquire a most complete knowledge on plant extracts [START_REF] Grand-Guillaume Perrenoud | Combining the full potential of UHPSFC-QToF/MS and UHPLC-QToF/MS to improve the workflow efficiency of both plant metabolic profiling and natural bioactive discovery[END_REF].

Finally, analysis and purification of biomolecules for pharmaceutical and clinical applications were the most abundant among applied studies. Therapeutic peptides may contain a large number of impurities requiring identification and elimination, where chromatographic and electrophoretic methods are most useful [START_REF] Hondt | Related impurities in peptide medicines[END_REF]. Method development for peptides and proteins were well exposed in several papers [START_REF] Govender | The development of a supercritical fluid chromatography (SFC) based method for the purification of biosynthesised human insulin[END_REF][START_REF] Schiavone | Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography[END_REF][START_REF] Goetz | EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides[END_REF][START_REF] Ventura | Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries[END_REF][START_REF] Bolaños | SFC/MS in drug discovery at Pfizer, La Jolla[END_REF][START_REF] Liu | Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale[END_REF][START_REF] Enmark | Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode[END_REF][START_REF] Jenny) Qian Cutrone | Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical consideration and method development[END_REF]. Typical examples of therapeutic peptides are presented in Figure 5, like gramicidin, cyclosporin, angiotensin and ocytocin. As mentioned above, proteins set particular problems in that the ternary structure should be preserved in purification methods. Free amino acids were analyzed in a traditional medicinal plant with SFC-MS [START_REF] Raimbault | A chiral unified chromatography-mass spectrometry method to analyze free amino acids[END_REF]. Nucleobases and nucleosides were also searched in ginseng, a traditional Chinese medicine [START_REF] Huang | Simultaneous analysis of nucleobases, nucleosides and ginsenosides in ginseng extracts using supercritical fluid chromatography coupled with single quadrupole mass spectrometry[END_REF]. Other synthetic nucleosides were the topic of clinical studies: azacitidine, clofarabine and cytarabine (see structures in Figure 6) were analyzed with SFC along with their impurities following ICH guidelines [START_REF] Ganipisetty | Supercritical fluid (CO 2 ) chromatography for quantitative determination of selected cancer therapeutic drugs in the presence of potential impurities[END_REF][START_REF] Hsieh | Supercritical Fluid Chromatography and High-Performance Liquid Chromatography/Tandem Mass Spectrometric Methods for the Determination of Cytarabine in Mouse Plasma[END_REF][START_REF] Ganipisetty | Development of supercritical fluid (carbon dioxide) based ultra performance convergence chromatographic stability indicating assay method for the determination of clofarabine in injection[END_REF]. For instance, cytarabine was quantified in mouse plasma and the SFC-MS/MS method was compared to LC-MS/MS [START_REF] Hsieh | Supercritical Fluid Chromatography and High-Performance Liquid Chromatography/Tandem Mass Spectrometric Methods for the Determination of Cytarabine in Mouse Plasma[END_REF]. Finally, Pirrone et al. showed an interesting application of SFC-MS for impurity fate mapping of a nucleoside drug substance, where reversed-phase liquid chromatography failed to deliver the desired answers due to keto-enol tautomerization in aqueous conditions [START_REF] Pirrone | Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances[END_REF].

Conclusion and perspectives

As appeared in this review, amino acids, peptides, proteins and nucleic acid bricks are widely diverse molecules in terms of sizes and polarities. It comes as no surprise that no consensus stationary phase and mobile phase compositions have emerged for their SFC/EFLC analysis. Significant diversity in stationary phase chemistry and mobile phase composition was observed, although recent papers mostly cite polar stationary phases and wide elution gradients comprising some portion of water in the co-solvent, along with additives. Rather than a source of confusion, this diversity should be perceived as a great potential for successful method development and optimization. MS proves to be an essential tool for structure identification but UV detection still comes in handy for quantitative analysis of samples with low complexity, or when sensitivity is not an issue. This is typically the case in quality control of pharmaceuticals, food products or extracts of natural products.

Applications of biomolecules with SFC/EFLC clearly deserve more attention than is currently the case. At the analytical scale, the complementary information that can be obtained when comparing to most often used RPLC methods should justify the investment.

At the preparative scale, the time and solvent economy are well worth the effort. Finally, unified chromatography (UC) slowly emerges with very promising capacities shared from both LC and SFC/EFLC technologies. Inosin triphosphate [START_REF] Desfontaine | Applicability of supercritical fluid chromatography -mass spectrometry to metabolomics. I -Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances[END_REF] Uridin monosphate [START_REF] Philibert | Characterization of enhanced-fluidity liquid hydrophilic interaction chromatography for the separation of nucleosides and nucleotides[END_REF] 7-phenyl-7-deazaadenosine [START_REF] Kozlov | Evaluation of separation properties of stationary phases in supercritical fluid chromatography; deazapurine nucleosides case study[END_REF] Adenosin diphosphate [START_REF] Beilke | Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique[END_REF]

Figure 1 .Figure 2 .

 12 Figure 1. Generic and representative structures of the target molecules reported in the literature reviewed in this paper.

Figure 3 .

 3 Figure 3. Molecular weight and polarity features related to biomolecules previously analyzed with SFC or EFLC. The bubble size reflects the topological polar surface area (TPSA). The three descriptors were computed with MolDesc freeware (http://moldesc.icoa.fr/).

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Usual operating domains of SFC, EFLC and LC. Unified chromatography (UC) operates with large elution gradients covering a wide range of mobile phase compositions.

Table 1 .

 1 Published SFC/EFLC methods to analyze free amino acids

	Reference	Year	Samples	Stationary phase nature	Mobile phase co-solvent	Detection
	[40]		Standards	Diol	Methanol with water , triethylamine and pyridine	UV (225 nm), ELSD
	[60]		Standards	Chiral polysaccharides	Ethanol with 0.1% ethanesulfonic acid	UV
	[80]		Standards	Polystyrene-divinylbenzene	Methanol-isopropanol-water	UV
					Acetonitrile or methanol, with 20%
	[33]		Standards	Polysulfoethyl	buffer (15 mM ammonium formate /	ELSD
					formic acid, pH 3)
	[67]		Standards and human urine	Diol	Methanol with 20 mM ammonium formate	DAD and ESI(+/-)-MS/MS
	[82]		Standards & plant extracts	Diol, C18, 2-ethylpyridine	Methanol with 2% water and 10 mM ammonium formate	ESI(+/-)-MS/MS
	[97]		Standards and food supplements	Diol and chiral macrocyclic glycopeptide	Methanol with 10% water	UV (200 & 220 nm)
	[34]		Standards and human serum	Diol	Methanol with 1% water and ammonium formate	ESI(+)-MS/MS & APCI(+)-MS/MS
					Methanol with 5% water, 50 mM
	[66]		Standards	Silica and sulfobetaine	ammonium formate and 1 mM	ESI(+/-)-MS/MS
					ammonium fluoride
	[54]		Standard and tea leaves	Amide	Methanol with 5% water, 5 mM trifluoroacetic acid ammonium acetate and 0.4%	ESI(+)-MS
	[53]		Standards and plant extract	Chiral zwitterionic Cinchona alkaloid	Methanol with 5% water and 50 mM ammonium acetate	ESI(+)-MS
	[61]		Standards and food supplements	Chiral zwitterionic Cinchona alkaloid	Methanol with 2% water and 20 mM methanesulfonic acid	ESI(+)-MS
	[55]		Standards	Chiral crown-ether	Ethanol with 5% water and 0.8% trifluoroacetic acid	ESI-MS
	[45]		Standards and human urine	Diol	Methanol with 10 mM ammonium hydroxide	ESI(+/-)-MS/MS