Phenylboronic Acid (PBA) Solid Phase Extraction Mechanisms and Applications Technical Overview Introduction Bond Elut PBA is a unique silica SPE sorbent containing a phenylboronic acid functionality that can retain analytes via a reversible covalent bond. This very strong covalent retention mechanism enables high specificity and cleanliness. The boronate group has a strong affinity for cis-diol containing compounds such as catechols, nucleic acids, some proteins, carbohydrates and PEG compounds. Aminoalcohols, alpha-hydroxy amides, keto compounds, and others can also be retained. PBA SPE Mechanism Interaction Example Energy [kcal/mole] Covalent H R 100-300 Most solid phase extraction 2 methodologies are based on non- H H polar, polar or ionic interactions. R1 H Phenylboronic acid (PBA) solid Ionic O 50-200 phase extraction media is unique in O that it employs a reversible covalent N interaction between the PBA and R sample molecule(s). Covalent Hydrogen Bonding H3C 5-10 chromatography involves an interaction O H H O of considerably greater energy than H other extraction mechanisms (Figure 1). Dipole-Dipole 3-10 This strong interaction allows for the Cl development of extraction methods of CH3 much greater specificity. C N The mechanism of boronate binding is illustrated in Figure 2. The immobilized Dipole-Induced Dipole 2-6 phenylboronic acid is first equilibrated H3C C N H with an alkaline solution to obtain the reactive boronate form RB(OH) -. 3 Dispersion CH3 1-5 The diol-containing compound is non-polar or Van der H next applied and is covalently bound Waals with the concomitant release of water. Once the compound of interest H C is retained, contaminants may be 3 selectively washed from the bonded Figure 1. The relative energetics of various retention mechanisms emplyed for the purpose of bonded phase provided that an alkaline pH is phase extraction maintained. Finally, the compound of interest is eluted by acidification of the boronate complex, which releases the HO- diol-containing compound and renders OH OH B B the immobilized phenylboronic acid H O+ OH OH 3 OH neutral (RB(OH)2). Equilibration HO R1 H2O OH O OH B R B HO R + 1 OH 2 H3O O OH R2 Retention + H3O HO R OH 1 O OH B R 1 H2O B HO R O OH 2 OH R2 Elution Figure 2. The three step process of borate binding including equilibration, retention and elution 2 Applicable Analytes Table 1. Molecules expected to bind to phenylboronic acid Compound Class Examples A variety of diol-containing and Polyhydroxy Glycoproteins, monosaccharides other compounds are retained by immobilized phenylboronic acid, Aromatic o-hydroxy Catecholamines, nucleic acids, ribavirin, glycoproteins, carbohydrates, SAMe, tannins including vicinal diols, 1,3-diols and 1,3,5-triols. Retention of vicinal diols α-hydroxy acids Lactic acid requires that the diol maintains a Aromatic ο-hydroxy acids and amides Salicylic acid, salicylamide coplanar or cis conformation. Intra- 1,3-Dihydroxy Tris, pyridoxine molecular hydrogen bonding often Diketo and triketo Dehydroascorbic acid, benzil, alloxan renders 1,3-diols coplanar and they Aminoalcohols β-Blockers are retained. Carbohydrate rings Other dihydroxys Steroids, lipids that exhibit pseudorotation may be Others Statins retained depending on other ring substitutions. Theoretically, any two or three hydroxyls spaced so as to fill the tetrahedral sites surrounding the Extraction Procedure trihydroxyboronyl functionality may be retained. Cartridge conditioning A variety of functional groups It is necessary to properly equilibrate Below pH 7 these secondary interaction other than diols will be retained the PBA bonded phase with an will generally predominate. Both by immobilized phenylboronic acid alkaline solution to obtain the active the neutral and anionic forms of the through the combination of charge- boronate form RB(OH)3-. The pKa of immobilized functionality contain transfer and covalent interactions. the immobilized phenylboronic acid is hydroxylated boron which functions Compounds not yet mentioned include approximately 9.2. Recall that at pH 9.2 as a hydrogen bond donor under aromatic o-hydroxy acids and amides, only half the immobilized groups will be appropriate conditions. Consequently, α-hydroxy acids, 1,3-dihydroxy-, diketo-, reactive (ionized). A sample preparation polar compounds including diols triketo- and aminoalcohol-containing column can be briefly equilibrated at pH may be retained by normal phase compounds. 10 or 12 without damage to the silica mechanisms, if extracted from organic substrate. For example, equilibrate PBA solvents. The propyl linkage and PBA SPE has been used extensively with 50-100 mM phosphate buffer, pH phenyl ring through which the boron for the extraction of catecholamines, 10. If samples are base-sensitive, it can functionality is immobilized may provide nucleic acids, proteins and be effective to condition the extraction sites of nonspecific retention owing to carbohydrates from a variety of column with 0.05-1.5 M alkaline buffer, Van der Waals and pi-pi interactions if matrices, as well as for the successful pH 10-12, followed by 0.01-0.50 M compounds of hydrophobic character extraction of brassinosteroids, lipids buffer at pH 8-8.5. are present. Ionic repulsion may occur and polyhydroxyflavones, and the between the boronate anion and successful separation of gycosylated Secondary interactions anions on the compound of interest. proteins from non-gycosylated For example, because of the ionic proteins and ribonucleotides from The secondary interactions anticipated repulsion between the boronate and deoxyribonucleotides1-16. The cis-diol for an immobilized phenylboronic acid phosphate groups of nucleic acids, it is found in many biomolecules lends itself are illustrated in Figure 3. necessary to add a divalent cation to to PBA extraction. Unpublished work the mobile phase in order to get good has shown that PEG (polyethylene hydrophobic Ionic TT-TT binding. Finally, cations may be retained glycol) metabolites can also be cleared by the anionic boronate functionality. from biological samples using PBA If not removed in the wash step, these SPE through covalent bonding between N OH ions will coelute with the compound PEG metabolites and PBA. The scope H B OH of interest when the bonded phase of molecular species that can interact hydrogen OH is acidified, and may subsequently with the di- or trihydroxyboronyl interfere with detection, although functionality is summarized in Table 1. charge-transfer most ionic species elute in the void Figure 3. The anticipated secondary interactions volume of the analytical column. Ionic exhibited by immobilized PBA secondary interactions may also be 3 avoided by using buffers of higher ionic The effective pKa of an amine- the aqueous component of the wash strength, between 50 and 500 mM, in organoboron charge-transfer complex solvent remains alkaline, the covalent the equilibration, sample application is lower than that of the isolated complex will remain intact. and/or wash steps. organoboron. Consequently, complex formation can reduce the pKa of Elution Buffer selection the immobilized functionality thus A variety of acids can be employed An additional secondary interaction increasing the capacity of the bonded as elution solvents. In most cases, of considerable importance phase. Therefore, zwitterionic primary reduction to pH 5 is sufficient to in understanding immobilized and secondary amine buffers such effect elution and significant buffer phenylboronic acid is the potential as HEPES can enhance binding. capacity is not required. Acetic, of boron to form a charge-transfer The zwitterionic character of these formic, hydrochloric, phosphoric complex with 1’, 2’, 3’ amines. compounds has the added advantage of and trifluoroacetic acids (TFA) have Unprotonated amines readily form functioning as a local acid scavenger, been used. In many cases, HPLC such complexes. Although complex removing protons associated with the mobile phase for reverse phase formation is believed to involve only sample matrix and thus ensuring that chromatography is an effective elution the neutral phenylboronic acid form, the organoboron retains its reactive solvent. equilibrium considerations allow for ionic form. Buffers employed for this The addition of up to 90% organic complex formation under alkaline purpose include HEPES, glycine, modifier is permissible and ensures conditions. In the charge-transfer diglycine and morpholine17-18. that the compound of interest is not complex the amine obtains a positive In selecting an appropriate amine for retained by secondary interactions. charge and the boron a negative charge the purpose of charge-transfer complex Charge transfer complexes involving rendering the complex neutral (Figure formation, ß-hydroxyl amines should amines require reduction to pH 3 to 4). Charge-transfer complex formation be avoided, as they form highly stable ensure complete elution. Competitive with an immobilized phenylboronic acid complexes in which the amine and elution with a borate ion, competing can have both favorable and deleterious ß-hydroxyl groups interact with the diol such as sorbitol or mannitol, or impact on a bonded-phase extraction. dihydroxyboronyl functionality to form an α-hydroxyl such as tris provide an adduct with both charge-transfer an alternative to acidification when and covalent components (Figure extraction of proteins or acid labile R1 4). Therefore, bicine, tricine, tris and compounds is required. R2 N 1’,2’,3’-ethanolamine buffers should not B R3 be used. HO OH Removing interferences 100 mg 1 mL Bond Elut PBA (p/n 12102019) A -Charge-Transfer complex with amine It should be assumed that once the Condition: sample has been applied to a properly 1. 1 mL 70:30 H2O:ACN with 1% formic acid R1 conditioned column, compounds 2. 1 mL 50 mM phosphate buffer, pH 10 R2 N are retained by both covalent and B nonspecific mechanisms. It is at this HO O Apply Sample: Sample should be buffered to stage of the extraction process that the pH 8.5 with 50 mM phosphate buffer, pH 8.5 advantages of covalent chromatography B - Charge-Transfer complex further stabilized by become apparent.