Importance of Controlling Mobile Phase ph in Reversed Phase HPLC

Transcription

1 TB Importnce of Controlling Mobile Phse ph in Reversed Phse HPLC By Aimee N. Heyrmn nd Richrd A. Henry INTRDUCTIN The importnce of controlling mobile phse ph when nlyzing ionizble compounds by reversed phse (RP) HPLC is often recognized nd esily understood, however it is often eqully importnt to control ph when working with field smples of non-ionizble compounds due to the presence of ionible impuritites. % Ionized Figure. Buffer Cpcity % Buffer Cpcity SELECTING THE RIGHT BUFFER A prtil list of common buffers nd their corresponding ph vlues is shown in Tble. Perhps the most common HPLC buffer is some form of phosphoric cid. A definition of buffer strength is given in Figure, where plot of how conjugte forms of phosphoric cid chnge with ph. Note tht buffer cpcity (the bility to resist ph chnge when smple is introduced t different ph) is only 00% t the pk vlue of the cid or bse. At ph 4, phosphte is poor buffer nd would chnge rpidly towrd one of its pk vlues if more cidic or bsic smple were introduced. As rule, one should work within ± ph unit of the buffer pk vlue for good ph control of the mobile phse. Adequte buffer concentrtions for HPLC tend to be in the 0-00 millimolr level depending on the size nd nture of the smple, s well s the column pcking mteril. Phses tht contin polr groups such s AQUASIL C8 nd PRISM RP, re often more comptible with dilute buffers thn trditionl lkyl pckings. When control t lower ph (-) is desired, phosphte, or stronger orgnic cids such s TFA or cetic cid when voltility is of concern, re commonly Tble. Properties of Common Buffers used. If control t ph 4-5 is desired, n orgnic cid buffer such s cette or citrte should be considered in plce of phosphte. Common Buffers pk Useful ph Rnge Phosphte pk..-. pk pk..-. Citrte pk..-4. pk pk Formte Acette Tris Ammoni Borte Diethylmine

2 WHEN IS ph CNTRL NECESSARY FR STRNGLY INIZABLE CMPUNDS? Smples contining ionizble compounds re strongly influenced by ph of the mobile phse s illustrted by the seprtions of sorbic nd benzoic cids shown in Figure. While orgnic cids re typiclly seprted under ion suppression conditions where the ph is djusted to or (Figure A), this smple required ph of 7 (Figure B) due to the tendency of the smple mtrix to precipitte t lower ph nd higher orgnic concentrtions. Low ph decreses the solubility of orgnic cids in wter nd requires the use of higher orgnic percentge in the mobile phse for prcticl elution under RP conditions. For cids, the retention time decreses s the ph of the mobile phse is incresed. Greter chrge cn be thought of s n extreme cse of polrity. At ph s bove the nlyte s pk, the cidic nlyte crries negtive chrge nd behves s n extremely polr molecule. In order to chieve dequte retention, the mobile phse should be highly queous. Below its pk, the cidic nlyte is neutrl nd much more hydrophobic. Under ion suppresion RP retention cn be more esily chieved nd nlysis times cn be longer. When the cid stndrds were introduced to unbuffered, neutrl mobile phse of 0% methnol (Figure C), poor pek shpe resulted. This result cn be trced to mismtch cused by the cidic nture of the smple nd zero buffer strength in the neutrl mobile phse. The smple therefore experiences ph grdient during the first prt of the seprtion, which usully cuses ionizble compounds to exhibit brod pek shpe nd poor retention reproducibility. As shown in Figure B, ddition of phosphte buffer t ph 7 eliminted the brod tiling peks nd creted rugged conditions suitble for successful ssy. Becuse the ssy of sorbic nd benzoic cids ws relted to food product contining protein, the wide pores nd neutrl ph prevented precipittion nd prolonged column life. Essentilly, the solutes re instntly ionized nd the wek cids re seprted in their nionic forms. This ph grdient effect is difficult to control nd cn lmost gurntee problems with reproducibility. CNCLUSIN: While it is not lwys strictly necessry to operte under buffered conditions, one should recognize tht poor pek shpe nd vrible retention cn result when the smple ph differs significntly from the ph of the nonbuffered mobile phse nd when ionizble compounds re present in the smple. Figure. Effect of ph Control on Seprtion of Ionizble Compounds A: ph.5, buffered B: ph 7.0, buffered C: ph 7.0, not buffered Smple:. Benzoic Acid. Sorbic Acid α,, =. α,, = MIN MIN MIN BioBsic 8, 5µm, 50x4.6mm Prt No: 55-7 Eluent: 0% MeH/80% 0.05M KH, ph.5 Flow:.0 ml/min Detector: 5 BioBsic 8, 5µm, 50x4.6mm Prt No: 55-7 Eluent: 0% MeH/90% KH, ph 7 Flow:.0 ml/min Detector: 5 BioBsic 8, 5µm, 50x4.6mm Prt No: 55-7 Eluent: 0% MeH/90% H Flow:.0 ml/min Detector: 5

3 WHEN IS ph CNTRL NECESSARY FR NN-INIZABLE SAMPLES? DELTABND AK ws engineered for the ssy of ldehydes nd ketones present in uto emissions. Since then, other groups hve employed DELTABND AK for the ssy of ldehydes nd ketones in mbient ir smples nd smples from other sites where ldehyde nd ketone pollution is present. Trditionlly, the smples re first derivtized with DNPH for esy detection by UV-Vis, then ssyed with mobile phse consisting of H /ACN. The DNPH-derivtized ldehydes nd ketones re not ionizble nd therefore do not require buffered mobile phse. Auto emissions generlly re free of ionizble compounds; however, recently the usul method ws used with DELTABND AK for the nlysis of ldehydes nd ketones in mbient ir smples surrounding fcility which mnufctures nylon (Figure A). Although the sme method hd been used successfully for this nlysis t other mnufcturing fcilities, suddenly lrge ghost pek ws observed tht interfered with the quntittion of formldehyde. The ghost pek hd poor pek shpe nd ws not present in blnk runs or stndrds. Although the pek shpe ws lwys brod, the retention time vried column-to- column nd lot-to-lot, indicting tht the ghost pek ws intercting strongly with the residul silnol groups on the surfce of the silic. The H component of the mobile phse ws replced with buffer (KH ph.5 with H ) (Figure B). The lrge spurious pek shifted to the solvent front, mking ccurte quntittion of the formldehyde pek possible. The retention times of the ldehydes nd ketones were unffected since they re not ionizble nd therefore re unffected by chnges in ph. The ghost pek is most likely mine compound tht hs been prtilly derivtized with DNPH. A dimine is used in the mnufcturing process of nylon, which could be the source of the spurious pek. The DNPH derivtiztion of one of the mino groups renders the other mino group inctive, resulting in prtilly derivtized compound tht is still ionizble nd consequently does not behve well under the non-buffered chromtogrphic conditions. Although unbuffered, the current mobile phse is pproximtely ph 7, resulting in prtil ioniztion. Mobile phse ph will chnge slightly s the percent orgnic in the mobile phse chnges, which in turn ffects the percent ioniztion of the ghost pek. The retention mechnism of this pek is probbly due in prt to ion exchnge effects with the residul silnol groups, which cn vry from lot-tolot nd even column-to-column. This effect cn be eliminted by buffering the mobile phse t bout ph.5. Under these conditions the mino compound will be extremely ionized, move to the solvent front, nd behve consistently nd the silnols will not dissocite nd imprt negtive chrge to the pcking. FIGURE. Effect of ph Control on Seprtion of Non-Ionizble Compounds CNCLUSIN: Mobile phse ph should be controlled when ssying non-ionizble or neutrl nlytes in the presence of ionizble contminnts or impurities. Ionizble compounds re esily recognized by their inconsistent run-run nd smple-smple behvior under non buffer conditions. A: H /ACN 4 Smple-DNPH derivtized mbient ir smples :. Formldehyde. Acetldehyde. Acetone 4. Acrolein Impurity B: 0.05M KH, ph.5/acn Impurity Grdient: T %A %B T=Time (min.) MIN DELTABND AK, 5µm, 50x4.6mm Prt No: Eluent: A H 0 B: ACN Flow:.5 ml/min Detector: MIN DELTABND AK, 5µm, 50x4.6mm Prt No: Eluent: A: 0.05M KHP4 in H, ph.5 B: ACN Flow:.5 ml/min Detector: 65

4 IMPRTANCE F CAREFUL ph CNTRL? Smll chnges in the mobile phse ph cn lso hve drmtic effect on the selectivity of wekly ionizble compounds. A smple of 7 common ntiinflmmtory drugs ws seprted t ph. (Figure 4A) nd ph.5 (Figure 4B). Although 6 of the 7 nlytes behved very similrly under both conditions, Diflunisl eluted pproximtely minute erlier t ph.5 thn t ph., indicting tht it is more ionized t the higher ph. This behvior indictes the presence of crboxylic cid group in the molecule tht ws sensitivie to ph in this rnge CNCLUSIN: Adequte ph control should lwys be employed when working with mildly ionizble compounds to ensure mximum run-run reproducibility. C H F H F Diflunisl FIGURE 4. Effect of Smll Chnges in ph on the Seprtion of Mildly Ionizble Compounds A: ph. Smple:. Urcil. Tolmetin. Nproxin 4. Fenoprofen 5. Diflunisl 6. Indometcin 7. Ibuprofen B: ph MIN 0 6 MIN BETASIL C8, 5µm, 50x4.6mm Prt No.: Eluent: 50% ACN/ 50% 5mM H, ph=. Flow: 0.8 ml/min Detector: 0 nm BETASIL C8, 5µm, 50x4.6mm Prt No.: Eluent: 50% ACN/ 50% 5mM KH, ph=.5 Flow: 0.8 ml/min Detector: 0 nm 4

5 USE F ACID MDIFIERS T ADJUST ph It is lso common to employ strong or wek cids lone to control ph t low vlues (Figure 6), s shown in Tble, for commonly used trifluorocetic (TFA) nd cetic (HAC) cids. For more thorough tretment of this topic plese see reference. Equtions used to clculte pproximte ph vlues re shown in Figure 5 for strong (nerly dissocited) nd wek cids, where C is the concentrtion of the cid in mol/l nd K is the cid-dissocition constnt. As shown in the cse of TFA, clculted vlues cn differ significntly from mesured vlues when the cid hs properties between tht of wek nd strong cid. Eqution is more rigorous estimtion of ph thn eqution 4 nd offers better pproximtion of ph for moderte cids such s TFA. When TFA nd HAC re used, this method of ph control does not provide buffered mobile phse nd my not be s effective for ll types of smples, especilly bsic ones. However, it hs become populr for djusting the ph of mildly ionizble compounds such s peptides nd proteins. As Figure 6 illustrtes, TFA cn be used to not only control mobile phse ph but lso the selectivity s well. An order of mgnitude chnge in concentrtion of TFA results in significnt chnge in ph nd drmticlly different chromtogrm. At 0.% TFA (ph.0) (Figure 6A), Angiotensins II nd III coelute nd t 0.0% TFA (ph.4) (Figure 6B), they re bseline resolved. While some of the chnge cn be ttributed to ioniztion differences t the two ph vlues, TFA lso hs unique properties which my result from its reported bility to form strong ionpirs with positively chrged species. Tble. Properties of Acid Modifiers Modifier Concentrtion Mesured Clculted Clculted Constnt Buffer (v/v) (Moles/L) ph ph () ph () K pk 0.% TFA.5x % TFA 6.75x % TFA.5x % HAC.75x x ph clculted using Eqution 4. ph clculted using Eqution Figure 5 Wek Acids Figure 6. Effect TFA Concentrtion on Seprtion of Mildly Ionizble Compounds () + [ H ] K = ± K + 4K C A: 0.% TFA (v/v) ph.0 B: 0.0% TFA (v/v) ph.4 () () ~ K ± K + 4K C ph = log [ H ] + = K C, Smple:. Angiotensin III. Angiotensin II. Angiotensin I (4) log ph = ( K C ) ~ Strong Acids (5) + [ H ] = C A (6) ph = log C MIN MIN PRISM RP, 5µm, 50x4.6mm Prt No.: 55- Eluent: A: 0.0% TFA in H B: 0.0% TFA in ACN 0% 50% B in 0 min. Flow:.0 ml/min Detector: PRISM RP, 5µm, 50x4.6mm Prt No.: 55- Eluent: A: 0.0% TFA in H B: 0.0% TFA in ACN 0% 50% B in 0 min. Flow:.0 ml/min Detector: 5

6 Figure 7 is nother exmple of the importnce of the cid concentrtion. Figure 7A shows seprtion of tryptic digest of β-lctoglobulin with 0.0% TFA while Figure 7B shows the sme seprtion without TFA present in the mobile phse. In this cse there ws drmtic loss of retention nd selectivity for ll of the peptide frgments. A trce mount of cid is usully required to mintin dequte ph control nd improve the seprtion. Generlly the lowest concentrtion possible should be employed s long s results show ruggedness nd reproducibility. Lower concentrtions of buffers nd dditives cn reduce mintennce requirements, be more comptible with detectors, nd improve the lifetime of columns nd other system components. Figure 7. Tryptic Digest of β Lctoglobulin on BetBsic 8 A: 0.0% TFA ph.4 Smple: Tryptic Digest of β Lctoglobulin 75-0 B: 0.00% TFA ph MIN 0 0 MIN 75-9 BetBsic 8, 5µm, 50x4.6mm Prt No.: Eluent: A: TFA in H B: TFA in ACN 0% 50% B in 0 min. Flow:.0 ml/min Detector: 0 Figure 8. Effect of Acid Modifier on Selectivity A: 0.0% TFA ph.4 B:.0% Acetic Acid Smple: ph.0. Angiotensin III. Angiotensin II. Angiotensin I Figure 8 illustrtes the effect of different cid modifiers on selectivity. Although the ph of the two mobile phses vries by less thn ph unit, the cetic cid mobile phse offers much greter selectivity for the sme pir of components tht re only prtilly resolved with TFA. This is nother indiction tht the mechnism of seprtion, especilly with orgnic cids, cn involve specific interctions between the solute nd cid, such s ion-piring mentioned bove or simply ph effects. The incresed noise observed in Figure 8B is cused by higher bckground UV bsorbnce of.0% cetic cid compred to 0.0% TFA MIN BetBsic 8, 5µm, 50x4.6mm Prt No.: Eluent: A: 0.0% TFA in H B: 0.0% TFA in ACN 0% 50% B in 0 min. Flow:.0 ml/min Detector: 0 nm MIN BetBsic 8, 5µm, 50x4.6mm Prt No.: Eluent: A:.0% HAC in H B:.0% HAC in ACN 0% 50% B in 0 min. Flow:.0 ml/min Detector: 0 nm 6

7 GUIDELINES FR PREPARING MBILE PHASES Becuse slight vritions in ph nd cid concentrtion cn hve drmtic impct on seprtion, consistent certin techniques should be employed when prepring mobile phses to ensure good reproducibility. As described in the literture, it is generlly good ide to mesure n pproprite mount of pure wter into volumetric flsk with n ccurte mount of slt or cid. The ph of the mobile phse should be djusted, if required, by dding regent before diluting to finl volume nd prior to blending of ny orgnic solvents. For exmple, blending 5% methnol will rise the pprent, mesure ph of the combined mobile phse by bout 0.5 ph units. Alterntively, equimolr solutions of different ionic forms of the sme buffer (i.e. mono nd dibsic phosphte) cn be blended to rech the desired ph. When developing rugged method, it is desirble to select mobile phse with finl ph t lest one ph unit wy from ny nlyte s pk vlue to cuse ioniztion or suppression of the nlytes. There is often some guesswork in this becuse the effect of type nd concentrtion of orgnic solvent on either mobile phse ph of solute pk vlues is not ccurtely known. During method development, it is importnt to monitor chromtogrphic (k, α) reproducibility with severl btches of mobile phse, s it cn be difficult to consistently reproduce ph precisely. The equtions in Figure 5 re good strting point for clculting the concentrtion of cid required to chieve desired ph for seprtion. However, s Tble shows, ph cn vry significntly from those clcultions. It is therefore very importnt to experimentlly determine nd report the vlue of the mobile phse ph with clibrted ph meter to ensure reproducible results. The use of pre-mixed mobile phse (pumping from single reservoir) is essentil to ensure ccurte nd reproducible mobile phse composition. However, it hs become populr to prepre n queous buffer nd progrm the instrument to blend orgnic solvent with queous buffer for grdient elution or fst isocrtic method development. This prctice cn result in poor ccurcy nd incomplete mixing, depending on system mintennce nd clibrtion, mgnitude of dwell volume, flow rte nd other fctors. Isocrtic methods tht hve been developed using instrument blending should be confirmed by premixed mobile phses, nd grdient methods should be compred between more thn one instrument when possible. SUMMARY AND RECMENDATINS Controlling the seprtion of ionizble compounds cn be difficult, nd creful ttention must be pid to ll experimentl detils in order to ccomplish rugged method. Slight vritions in mobile phse preprtion cn result in ph chnges tht cn hve drmtic effects on selectivity, cpcity fctor (retention fctor), pek shpe, resolution, nd reproducibility. ptimum ph control will usully result in mobile phse contining buffer nd cid compositions tht will resist chnge when the smple is introduced nd force ionizble nlytes into predominntly one form (ionized or neutrl) s they enter the column. Good lbortory prctice in prepring mobile phses should be followed to ensure tht results cn be reproduced within nd between lbortories. While instrument solvent blending hs become very convenient for fst method development, it is best to evlute pre-mixed solvent whenever possible to ensure ccurcy nd equilibrtion before completing nd publishing n HPLC method. This extr step cn eliminte the possibility tht instrument fctors could mke seprtion results difficult for others to reproduce. Mobile phse ph should be selected so tht it is t lest ±.5 ph units from the nlyte s pk. This ssures tht the nlytes re either 00% ionized or 00% non-ionized nd should help control run-run reproducibility. At high ph, cidic compounds re ionized nd re much more hydrophilic thn under ion suppression conditions. These conditions should be selected when fst nlysis nd low retention re desired. BioBsic 8 is good choice under high ph conditions. REFERENCES: ) R.C. West, ed., Hndbook of Chemistry nd Physics, (CRC Press: Clevelnd, H), 974. D-D4. ) R.A. Henry, D Ghgn, Design of Voltile Buffer Systems for LC Applictions, (Keystone Scientific, Inc. Bellefonte, PA) ) Seprtions Solutions: Mobile Phse ph, U.D. Neue, Americn Lbortory, Mrch 999, p