Spectrophotometric study of time stability and acid-base characteristics of chelerythrine and dihydrochelerythrine

Cen al Eu opean Jou nal o Chemis y 1. In oduc ion Qua e na y benzo[c]phenan h idine alkaloids (QBAs) a e p esen mainly in plan s o Fuma iaceae, Papa e aceae, Ramunculaceae and Runaceae amilies. Sanguina ine (SA) and chele y h ine (CHE) a e he only comme cially a ailable membe s o QBAs, in addi ion o mino membe s (i.e., sangui ubine, cheli ubine, sanguilu ine, chelilu ine and maca pine). QBAs, a g oup o low molecula compounds wi h N-he e oa om, belong o he highly bioac i e subs ances [1]. Some side e ec s we e epo ed ( oxic e ec on some cellula le el [2-5]) in addi ion o he bene icial pha macological ac ions (an imic obial, an iapop o ic, an iin lama o y e c. e ec s [6,7]). S anda dized plan ex ac s ha e been exploi ed as an i-plaque componen s o den al hygiene p epa a ions (mou h-wash, oo h pas e e c.), as ac i e componen s o myopa hy p epa a ions and in e e ina y p epa a ions. Many e y ex ao dina y p ope ies we e obse ed in he ame o de ail s udies o acidobasic and elec opho e ic beha io o sanguina ine (SA) and chele y h ine (CHE) [8-11] and mainly hei dihyd ode i a i es (dihyd ochele y h ine, dihyd osanguina ine, e c., he o me being ecognized as he i s me aboli e o de oxi ica ion o sanguina ine in a s [2]) in he p esence o ine elec oly es [3,4,10,11]. They exhibi e y cha ac e is ic spec al ea u es ha can be used o hei di ec de e mina ion, o s udies o hei in e ac ions wi h biomac omolecules using UV-VIS spec opho ome y, luo ime y, mass spec ome y e c. [12-15] The exac knowledge o physico-chemical cha ac e is ics is e y impo an o co ec in e p e a ion o expe imen s conce ning s udies o in e ac ions and beha io o he alkaloids a nea ly physiological condi ions. I is e iden ha he s udy o in e ac ions [3-6] wi h biologically impo an mac omolecules (i.e., ecep o s, anspo p o eins, nucleic acids e c.), as E-mail: [email p o ec ed]* Resea ch A icle Recei ed 14 Augus 2009; Accep ed 6 Ma ch 2010 1 Depa men o Chemis y and Biochemis y, Mendel Uni e si y, 613 00 B no, Czech Republic 2 Depa men o Chemis y, Masa yk Uni e si y, 611 37 B no, Czech Republic 3Depa men o Medical Chemis y and Biochemis y, Palacky Uni e si y, 775 15 Olomouc, Czech Republic 4 Depa men o Biochemis y and Food Analysis, Tomas Ba a Uni e si y, 762 72 Zlín, Czech Republic Helena Absolíno á1, Luděk Jančář2, I ena Jančářo á1, Ja osla Viča 3, Vlas imil Kubáň2,4* Spec opho ome ic s udy o ime s abili y and acid-base cha ac e is ics o chele y h ine and dihyd ochele y h ine Keywo ds: Chele y h ine•Dihyd ochele y h ine•Time s abili y•UV-VIS spec al cha ac e is ics• Equilib ium cons an s Abs ac : Time s abili y, acid-base and UV-VIS spec al p ope ies o dihyd ochele y h ine (DHCHE) we e s udied spec opho ome ically in wa e :me hanol and wa e :e hanol media. DHCHE is s able in s ongly acid milieu (pH < 3) and a he highe amoun s (60% / ) alcohol. Acid-base cha ac e is ics and UV-VIS spec al p ope ies o chele y h ine (CHE) we e s udied in aqueous solu ions in he p esence o di e en concen a ions o HCl, HNO3, H2SO4, H3PO4 and hei mix u es. Rema kable shi s o o ma ion pa s o abso bance-pH (A-pH) cu es o he alkaline medium we e obse ed depending on he ype and concen a ion o ine elec oly e (mos ema kable o HNO3 and HCl). The co esponding equilib ium cons an s pKR+ o he ansi ion eac ion be ween cha ged iminium Q+ and uncha ged QOH (pseudo-base, 6-hyd oxy-dihyd o de i a i e) o ms o chele y hine we e calcula ed using a nume ical in e p e a ion o A-pH cu es by a SQUAD-G compu e p og am which anged om 8.51-9.31. The highes changes o ∆pKR+ (0.75 and 0.53) we e obse ed o H3PO4 and H2SO4, espec i ely. The p io i y e ec o ionic species and ionic s eng h was con i med in he p esence o addi ions o NaCl and KCl. The s eng h o in e ac ion o CHE wi h biomac omolecula compounds (i.e., pep ides, p o eins, nucleic acids e c.) may be a ec ed because o he obse ed in luence o bo h ca ion and anion o he ine elec oly e on acid-base beha io . © Ve si a Sp. z o.o. Cen . Eu . J. Chem. • 8(3) • 2010 • 626–632 DOI: 10.2478/s11532-010-0038-7 626 Am esco®, Solon, Ohio, USA) and sodium hyd oxide ( eagen g ade pu i y, Pen a, Ch udim, Czech Republic) solu ions we e used o he pH adjus men in a pH in e al pH = 2 – 11 wi h s eps ∆pH = 0.2 – 0.5. All o he expe imen al condi ions we e in acco dance o p e ious pape [17]. All spec opho ome ic measu emen s we e pe o med using a UV-VIS Lambda 25 double beam spec opho ome e con olled by UV WinLab so wa e ( ange 190 -1100 nm, sli 1 nm, ixed, qua z cu e e 10 mm, Pe kin Elme , Shel on, USA) o a Helios Be a UV-VIS single beam spec opho ome e ( ange 190 – 1100 nm, sli 1 nm, ixed, qua z cu e e 10 mm, Unicam, Camb idge, UK) con olled by a Vision so wa e. The inal pH o he solu ions was con olled using a pH-me e model WTW pH 527 wi h a WTW SenTix 21 combined elec ode. The elec ode was egula ly calib a ed (se e al imes pe day, a leas a he beginning and a he end o A-pH cu e measu emen ) using a se o s anda d bu e solu ions o pH = 4.01, 7.00 and 9.01 (all WTW GmbH, Wilheim, Ge many). The pKR+ cons an s we e calcula ed om he abso bance alues a six selec ed wa eleng hs be ween 268 and 405 nm (268, 272, 280, 316, 339 and 405 nm) using he SQUAD-G compu e p og am [18]. The abso bance alues o bo h alkaloids we e measu ed h ee imes a each pH and he mean alues om hese measu emen s we e used o calcula e o pKR+. Fo mo e de ails see ou p e ious pape [17]. 3. Resul s and Discussion 3.1. UV-VIS spec al and acid-base beha io o dihyd ochele y h ine (DHCHE) Ve y complex abso p ion spec a o DHCHE we e egis e ed in he wa eleng h in e al 220 – 380 nm in p esence o 60% ( olume pe olume / pe cen age is well as he o en men ioned o ma ion o in e cala ion complexes wi h cyclodex ins and biopolyme s, belongs o his class o expe imen s. The main goals o ou e o we e (i) s udy o he acid- base beha io and ime s abili y o CHE and DHCHE as he unc ion o expe imen al condi ions (pH, ionic s eng h, concen a ion o elec oly e and i s composi ion, e c.), (ii) de e mina ion o UV-VIS spec al cha ac e is ics and hei “ ue” pKR+ cons an s, (iii) iden i ica ion o expe imen al condi ions and equi emen s ha quali y he possibili y and co ec ness o in e ac ion s udies wi h hese compounds in almos neu al and weakly basic solu ions (close o physiological condi ions). 2. Expe imen al P ocedu e 2.1. Ma e ials and me hods S ock solu ion o chele y h ine (c = 130 µmol L-1) was p epa ed by dissolu ion o chele y h ine chlo ide (Sigma Ald ich) in dis illed wa e . Wo king solu ions o CHE (c = 6.5 o 9.4 µmol L-1) we e p epa ed by dilu ion o he s ock solu ion wi h cold eshly boiled dis illed wa e . Dihyd ochele y h ine (DHCHE, MP 189-191°C, 99% pu i y – checked by HPLC and con olled by calcula ion using mola abso p i i y) was p epa ed om CHE by educ ion wi h NaBH4 in me hanol [16]. S ock solu ions o dihyd ochele y h ine (cm = 60 μg mL-1) we e p epa ed by dissolu ion o DHCHE in me hanol o e hanol. Wo king solu ions (cm = 2.4 µg mL-1) we e p epa ed eshly by dilu ion o he s ock solu ion wi h me hanol, e hanol and/ o eshly boiled dis illed wa e . All he solu ions we e s o ed in he e ige a o and we e p o ec ed om ligh . S ock solu ions o elec oly es we e p epa ed om HCl, HNO3, H2SO4, NaCl, KCl, (all Pen a, Ch udim, Czech Republic), H3PO4, e hanol and me hanol (Lach- Ne , Ne a o ice, Czech Republic), all o p.a. pu i y. T is- (hyd oxyme hyl)aminome hane (TRIS – ul a pu e g ade, Figu e 1. Abso p ion spec a o dihyd ochele y h ine (cDHCHE = 2.4 µg mL-1, cHCl = 0.01 mol L-1, he pH was adjus ed by NaOH) in 60% me hanol (A) and in 60% e hanol (B): A: 1 – pH = 2,10; 2 – pH = 3,11; 3 – pH = 4,05; 4 – pH = 8,97; B: 1 – pH = 2,08; 2 – pH = 2,95; 3 – pH = 3,64; 4 – pH = 9,58. 2 H. Absolíno á, L. Jančář, I. Jančářo á, J. Viča , V. Kubáň 627 12% a e 30, 60 and 120 min, espec i ely, in all cases. The same end (bu much slowe ) was obse ed o highe concen a ions o me hanol and e hanol (10 – 30%). The solu ions in 60% me hanol and 60% e hanol we e mos ly s able in he ime in e al (≤ 120 min) and insigni ican inc ease o abso bance a 317 and 353 nm was obse ed (app ox. up o 104 % o hei ini ial alues) in me hanolic solu ions. The g aphs o ime s abili y o DHCHE in 4, 10, 30 and 60% me hanol (A) and me hanol (B) a λ = 274 nm a e depic ed in Fig. 2. Highe ime s abili y o DHCHE in e hanol is e iden om he Fig. 2B. 3.1.2. In luence o acidi y on ime s abili y. The solu ions o DHCHE a e ela i ely s able only in s ongly acidic milieu a all con en s o me hanol o e hanol. A pH 2 in he p esence o 0.01 mol L-1 HCl abso bance dec eased be ween 95-92% o hei ini ial alues a e 30 min in 4% me hanol and e hanol, espec i ely. In he p esence o o mia e, ace a e and phospha e bu e s (pH 2.8, 4.2 and 6.7, espec i ely) he dec eases we e mo e e iden (see Fig. 3 o 4% wa e /me hanol – A - and wa e /e hanol mix u es - B) due o lowe s abili y a highe pH alues and p obably due o p esence o componen s o bu e s. used h ough he ex i no s a ed di e en ly) me hanol o e hanol (Fig. 1) and 0.01 mol L-1 HCl in he pH ange 2 – 10 wi h ∆pH = 0.3 – 0.5 s eps (pH was adjus ed by a s epwise addi ion o a NaOH solu ion). Dis inc abso p ion maxima a 230, 274, 317 and 353 nm a e p esen in acidic media pH < 3. Only wo dis inc abso p ion maxima appea a 227 and 280 nm in pH egion 4 – 10. The abso p ion maximum a 230 nm is shi ed o sho e wa eleng hs (λmax = 227 nm) and he abso p ion maximum a 274 nm is shi ed o longe wa eleng hs (λmax = 280 nm) when pH was inc eased (pH > 3). P esence o isosbes ic poin s a 232, 268, and 333 nm con i med e e sible acid- basic equilib ium be ween he cha ged iminium Q+ and uncha ged QOH o ms o DHCHE a pH 4 - 10. 3.1.1. In luence o alcohol con en on ime s abili y Time s abili y o DHCHE solu ions was egis e ed in 4, 10, 30 and 60% me hanol o e hanol a pH = 2 (0.01 mol L-1 HCl), in he p esence o o mia e (pH = 2.8), ace a e (pH = 4.2) and phospha e bu e s (pH = 6.7) a ou selec ed wa eleng hs 274, 278, 317 and 353 nm. The mean alues ( h ee eplica es) o abso bance apidly dec eased in 4% aqueous me hanol o abou 38, 14 and Figu e 3. Time s abili y o DHCHE solu ions con aining 2.4 µg mL-1 DHCHE in 4% aqueous me hanol (A) and in 4% aqueous e hanol (B) measu ed a 274 nm. Cu e 1 – 0.01 mol L-1 HCl (pH = 2); 2 – o mia e bu e (pH = 2.8), 3 – ace a e bu e (pH = 4.2); 4 – phospha e bu e (pH = 6.7) Figu e 2. Time s abili y o solu ions con aining 2.4 µg mL-1 DHCHE in aqueous me hanol (A) and e hanol (B) media and measu ed a 274 nm. Alcohol con en (in % / ): 1 – 4%; 2 – 10%; 3 – 30%; 4 – 60% 3 Spec opho ome ic s udy o ime s abili y and acid-base cha ac e is ics o chele y h ine and dihyd ochele y h ine 628 eac ion be ween a cha ged iminium Q+ and an uncha ged QOH (pseudo-base, 6-hyd oxy-dihyd ode i a i e) o ms o chele y h ine is con i med by he p esence o h ee sha p isosbes ic poin s a 239, 275 nm and 302 nm. The mola abso p ion coe icien s ε1 a ε2 o bo h acidobasic o ms o CHE in 10 and 100 mmol L-1 HCl (calcula ed using SQUAD-G) a e gi en in Table 1. 3.2.2. Abso bance-pH cu es Abso bance-pH cu es (A-pH cu es) we e measu ed a cons an concen a ion o chele y h ine c = 6.5 µmol L-1 and 10-100 mmol L-1 HCl (see Fig. 5), HNO3, H2SO4 and H3PO4 (no g aphically p esen ed). The da a we e collec ed in pH in e al pH = 3 – 11 wi h s eps ∆pH = 0.3 – 0.5 o six wa eleng hs (268, 272, 280, 316, 339 and F om he esul s i is clea ly e iden ha he solu ions o DHCHE a e s able only in mo e hen 60% me hanol/ e hanol and wi h a dec easing con en o alcohols he s abili y dec eases. Time s abili y is also se iously inc eased by acidi y. The highes ime s abili y was obse ed in s ongly acidic media (0.01 mol L-1 HCl o me hanol and e hanol and in o mia e bu e o pH 2.8 o e hanol). In sligh ly acidic, neu al and alkaline media he solu ions a e uns able and s abili y dec eases wi h dec easing con en o alcohols and inc easing pH. 3.2. UV-VIS-spec al and acid-base p ope ies o chele y h ine 3.2.1. Abso p ion spec a Abso p ion spec a o CHE (c = 6.5 µmol L-1) we e egis e ed be ween 220 – 430 nm and pH 2.5 - 11 in he p esence o 10 mmol L-1 concen a ions o HCl, HNO3, H2SO4 and H3PO4. Two dis inc abso p ion maxima a 268 and 316 nm and less p omo ed maxima a 339 and 405 nm a e p esen in he pH in e al pH = 2.5 – 8. Abso p ion maxima shi ed o longe wa eleng hs in alkaline media. The wo dis inguished abso p ion bands a 280 nm and a less dis inguished one a 316 nm cha ac e ize he spec um o he alkaline solu ions (pH 8 – 11). Abso p ion maxima a 339 and 405 nm disappea wi h inc easing pH (see Fig. 4). The e e sible ansi ion Figu e 4. Abso p ion spec a o chele y h ine (cCHE = 6.5 µmol L-1, cHCl = 0.01 mol L-1, pH adjus ed by NaOH). Cu e - pH: 1 – 3.16; 2 – 5.16; 3 – 7.07; 4 – 8.03; 5 – 9.05; 6 – 10.03 Table 1. Values o mola abso p i i y coe icien s ε1 and ε2 and hei a e age s anda d de ia ion o e he whole da a se o he cha ged iminium Q+ (ε1) and he uncha ged QOH (pseudo-base, 6-hyd oxy-dihyd ode i a i e, ε2) o ms o chele y h ine calcula ed using a nume ical in e p e a ion o he A-pH cu es by a SQUAD-G p og am. (cCHE = 6.5 µmol L-1, cHCl = 0.01 o 0.1 mol L-1, pH adjus ed by NaOH). Medium ε1(268 nm) [L mol–1 cm–1] ε2(268 nm) [L mol–1 cm–1] ε1(316 nm) [L mol–1 cm–1] ε2(316 nm) [L mol–1 cm–1] 0.01 mol L-1 HCl 59 660 ± 220 33 520 ± 370 37 060 ± 120 16 790 ± 260 0.1 mol L-1 HCl 59 970 ± 200 34 350 ± 270 38 230 ± 150 17 990 ± 200 Figu e 5. Abso bance-pH cu es o chele y h ine (cCHE = 6.5 µmol L-1) in he p esence o mine al acids a c = 0.01 mol L-1 measu ed a λ = 268 nm, neu aliza ion wi h NaOH (a) o TRIS (b). Cu e – acid: 1 – HCl; 2 – HNO3; 3 – H2SO4; 4 – H3PO4 H. Absolíno á, L. Jančář, I. Jančářo á, J. Viča , V. Kubáň 629 4 405 nm) co esponding o he abso p ion maxima o bo h acidobasic o ms. The o ma ion pa s o he A-pH cu es (and o cou se he co esponding pKR+ alues) we e shi ed o he mo e alkaline medium wi h inc easing ini ial concen a ion o acids (ionic s eng h) and depended on he cha ac e o he co esponding anion o he acid. The co esponding pKR+ alues calcula ed using a nume ical in e p e a ion o he A-pH cu es by he SQUAD-G p og am (see Table 2) a ied om 8.51 – 9.31. The mos se ious shi o he pKR+ was ound in he p esence o H3PO4 (∆pKR+ = 0.75) and H2SO4 (∆pKR+ = 0.53), while he less dis inc shi was obse ed o HCl (∆pKR+ = 0.50), HNO3 (∆pKR+ = 0.46) and he smalles one o CH3COOH (∆pKR+ = 0.37). F om he esul s i can be concluded ha he co esponding pKR+ alues a e in luenced by he cha ac e ( ype) o anions and also by o al mass concen a ion o anions in he o de PO4 3– > SO4 2– > Cl– > NO3 – > CH3COO–. To e i y he in luence o ca ionic species on pKR+ alues, he pH o he CHE solu ions in HCl, HNO3, H2SO4 and H3PO4 a he concen a ions c = 10 mmol L-1 was adjus ed by s epwise addi ion o NaOH o TRIS solu ions in he in e al 2 - 10. The co esponding pKR+ alues (see Table 3 and Fig. 5) we e again shi ed o he mo e alkaline medium wi h inc easing concen a ion o acids and hey we e signi ican ly highe o TRIS (mainly in he p esence o H2SO4 and H3PO4) compa ed o he alues ob ained o he A-pH cu es in p esence o NaOH. The lowes di e ences ∆pKR+ be ween alues o A-pH cu es neu alized wi h TRIS o NaOH we e obse ed in he p esence o HCl and, on he o he hand, he di e ences we e compa able in he p esence o o he acids. To con i m he in luence o he ype o anions on co esponding pKR+ alues, he A-pH cu es we e measu ed in he mix u es o acids a cons an o al concen a ion 100 mmol L-1 and he co esponding pKR+ alues we e again calcula ed using a nume ical in e p e a ion o he A-pH cu es by he SQUAD-G p og am (see Table 4). Mix u es o HCl + HNO3, HCl + H2SO4, HCl + H3PO4, HNO3 + H2SO4, HNO3 + H3PO4 and H2SO4 + H 3PO4 o mass concen a ion a ios 0.07 mol L-1 + 0.03 mol L-1, 0.05 mol L-1 + 0.05 mol L-1 and 0.03 mol L-1 + 0.07 mol L-1, espec i ely, we e es ed. The co esponding pKR+ alues con i m ha he p io i y e ec o dominan anion o mine al acid is highly ques ionable since he pKR+ chele y h ine a e p ac ically he same. 3.3. In luence o ionic s eng h and cha ac e o he elec oly e The changes in acid-base beha io o CHE depending on ionic s eng h and cha ac e o ine elec oly e we e e alua ed in he p esence o NaCl and KCl a I = 0.01, 0.10 and 1.0 and a he cons an concen a ion o HCl, HNO3, H2SO4, H3PO4 (c = 0.01 mol L-1). The co esponding pKR+ alues a e p esen ed in Table 5 and in he g aphical o m in Fig. 6. The o ma ion pa s o A-pH cu es and also he co esponding pKR+ alues we e shi ed o he alkaline egion wi h inc easing ionic s eng h in he ange ∆pKR+ = 0.17 – 0.61. The highes e ec was obse ed in he p esence o H2SO4 while he lowes in he p esence o HCl. A no able in luence was obse ed in he p esence o KCl (∆pKR+ = 0.69 – 1.01) wi h he ex eme e ec s in he p esence o H3PO4 (highes ) and HNO3 (lowes ), espec i ely. Medium pKR+ as(A)bUcMedium pKR+ as(A)bUc 0.01 mol L-1 0.1 mol L-1 HCl 8.80 ± 0.072 0.0100 0.0030 HCl 9.30 ± 0.037 0.0039 0.0005 HNO38.81 ± 0.046 0.0180 0.0183 HNO39.26 ± 0.017 0.0023 0.0002 H2SO48.77 ± 0.038 0.0158 0.0177 H2SO49.31 ± 0.021 0.0033 0.0004 H3PO48.51 ± 0.053 0.0240 0.0450 H3PO49.29 ± 0.031 0.0046 0.0011 a (pKR+ = 8.9 – 9.0 [6], pKR+ = 9.0 [9]; pKR+ = 8.77 ± 0.028 and 9.14 ± 0.040 we e ob ained o 0.01 and 0.1 mol L-1 ace ic acid ( his wo k); b he a e age s anda d de ia ion o abso bance (s(A)) o e he whole da a se ; c he sum o squa es o abso bance esiduals U= Σ ( Aexp,i – Acalc,i ) 2 o i = 1 – n, whe e n is he o al numbe o abso bance da a o all solu ions and wa eleng hs used Table 2. Values o pKR+ (and hei a e age s anda d de ia ion o e he whole da a se ) o chele y h ine in dependence on he ype and concen a ion o ino ganic acids calcula ed using a nume ical in e p e a ion o he A-pH cu es by a SQUAD-G p og am. (cCHE = 6.5 µmol L-1, cHA = 0.01 o 0.1 mol L-1 mine al acid, pH adjus ed by NaOH). Table 3. Values o pKR+ o chele y h ine in dependence on he ype and concen a ion o ino ganic acids (cCHE = 6.5 µmol L-1, cHA = 0.01 mol L-1 mine al acid, pH adjus ed by NaOH o TRIS). medium pKR+pKR+ TRIS NaOH HCl 8.85 ± 0.064 8.80 ± 0.072 HNO38.89 ± 0.063 8.81 ± 0.046 H2SO49.33 ± 0.067 8.77 ± 0.038 H3PO49.27 ± 0.068 8.51 ± 0.053 Spec opho ome ic s udy o ime s abili y and acid-base cha ac e is ics o chele y h ine and dihyd ochele y h ine 630 5 Table 4. Values o pKR+ o chele y h ine in dependence on he ype and concen a ion o ino ganic acids mix u es calcula ed using a nume ical in e - p e a ion o he A-pH cu es by a SQUAD-G p og am (cCHE = 6.5 µmol L-1, cHA = 0.1 mol L-1 mine al acid, pH adjus ed by NaOH). Medium pKR+ s(A)aUb 0.03 mol L-1 HCl + 0.07 mol L-1 HNO39.02 ± 0.028 0.0100 0.0076 0.05 mol L-1 HCl + 0.05 mol L-1 HNO39.03 ± 0.019 0.0081 0.0051 0.07 mol L-1 HCl + 0.03 mol L-1 HNO38.99 ± 0.021 0.0081 0.0051 0.03 mol L-1 HCl + 0.07 mol L-1 H2SO48.99 ± 0.031 0.0122 0.0115 0.05 mol L-1 HCl + 0.05 mol L-1 H2SO48.99 ± 0.030 0.0130 0.0134 0.07 mol L-1 HCl + 0.03 mol L-1 H2SO48.90 ± 0.033 0.0148 0.0180 0.03 mol L-1 HCl + 0.07 mol L-1 H3PO48.85 ± 0.041 0.0190 0.0290 0.05 mol L-1 HCl + 0.05 mol L-1 H3PO48.91 ± 0.034 0.0120 0.0190 0.07 mol L-1 HCl + 0.03 mol L-1 H3PO48.97 ± 0.032 0.0149 0.0172 0.03 mol L-1 HNO3 + 0.07 mol L-1 H2SO48.84 ± 0.027 0.0063 0.0031 0.05 mol L-1 HNO3 + 0.05 mol L-1 H2SO48.96 ± 0.032 0.0070 0.0034 0.07 mol L-1 HNO3 + 0.03 mol L-1 H2SO49.11 ± 0.037 0.0078 0.0047 0.03 mol L-1 HNO3 + 0.07 mol L-1 H3PO48.91 ± 0.031 0.0158 0.0193 0.05 mol L-1 HNO3 + 0.05 mol L-1 H3PO48.99 ± 0.021 0.0102 0.0080 0.07 mol L-1 HNO3 + 0.03 mol L-1 H3PO49.06 ± 0.014 0.0072 0.0040 0.03 mol L-1 H2SO4 + 0.07 mol L-1 H3PO48.87 ± 0.028 0.0060 0.0025 0.05 mol L-1 H2SO4 + 0.05 mol L-1 H3PO48.89 ± 0.023 0.0053 0.0022 0.07 mol L-1 H2SO4 + 0.03 mol L-1 H3PO48.91 ± 0.024 0.0058 0.0026 a he a e age s anda d de ia ion o abso bance (s(A)) o e he whole da a se ; b he sum o squa es o abso bance esiduals U = Σ ( Aexp,i – Acalc,i ) 2 o i = 1 – n, whe e n is he o al numbe o abso bance da a o all solu ions and wa eleng hs used Medium pKR+ HCl HNO3H2SO4H3PO4 no addi ion o Cl-8.80 ± 0.072 8.81 ± 0.046 8.77 ± 0.038 8.51 ± 0.053 0.01 mol L-1 NaCl 8.84 ± 0.021 8.82 ± 0.039 8.83 ± 0.035 8.80 ± 0.035 0.10 mol L-1 NaCl 8.97 ± 0.026 9.11 ± 0.028 9.04 ± 0.013 8.97 ± 0.021 1.00 mol L-1 NaCl 9.13 ± 0.031 9.33 ± 0.018 9.38 ± 0.014 9.00 ± 0.055 0.01 mol L-1 KCl 8.81 ± 0.088 8.88 ± 0.034 8.89 ± 0.038 8.65 ± 0.043 0.10 mol L-1 KCl 9.08 ± 0.017 9.15 ± 0.024 9.21 ± 0.019 9.09 ± 0.020 1.00 mol L-1 KCl 9.53 ± 0.055 9.48 ± 0.024 9.49 ± 0.022 9.52 ± 0.012 Table 5. Values o pKR+ o chele y h ine in dependence on he ype and concen a ion o ine elec oly e (ionic s eng h - as NaCl o KCl addi ions) calcula ed using a nume ical in e p e a ion o he A-pH cu es by a SQUAD-G p og am (cCHE = 6.5 µmol L-1, cHA = 0.01 mol L-1 mine al acid, pH adjus ed by NaOH). Figu e 6. Dependence o pKR+ alues o chele y h ine on he cha ac e o ine elec oly e and ionic s eng h (NaCl – A, KCl – B), in he p esence o 10 mM HCl (cu e 1), HNO3 (cu e 2), H2SO4 (cu e 3) and H3PO4 (cu e 4) H. Absolíno á, L. Jančář, I. Jančářo á, J. Viča , V. Kubáň 631 6 4. Conclusions Changes in acid-base cha ac e is ics o chele y h ine we e s udied by measu ing he abso bance-pH cu es a cons an concen a ion o chele y h ine wi h a ying cha ac e ( ype) and concen a ion o s ong ino ganic acids (HCl, HNO3, H2SO4, H3PO4). The o ma ion pa s o he abso bance-pH cu es shi ed o a mo e alkaline egion depending on he cha ac e and ini ial concen a ion o acids and his esul ed in a co esponding inc ease in equilib ium cons an s pKR+.The mos ema kable shi s we e obse ed in he p esence o phospho ic acid (H3PO4). The abo emen ioned esul s indica e ha i is p ac ically impossible o iden i y he p io i y e ec o anions since he pKR+ alues o chele y h ine ( o concen a ion o indi idual acids 0.1 mol L-1) di e ed e y li le om each o he . The cha ac e and o al concen a ion (ionic s eng h) o ine elec oly es (NaCl and KCl) also signi ican ly in luence he pKR+ alues o chele y h ine. The pKR+ alues inc eased wi h inc easing ionic s eng h o solu ions (NaCl and KCl addi ions I = 0.01 – 1.0) in HCl, HNO3, H2SO4 and H3PO4. DHCHE solu ions in 4% me hanol and e hanol a e ime s able only a pH = 2 (0.01 mol L-1 HCl). Howe e in he p esence o o mia e (pH = 2.8), ace a e (pH = 4.2) and phospha e bu e s (pH = 6.7) hei abso bance apidly dec eases om 92% – 17% in 4% me hanol and 95-25% in 4% e hanol a e 120 minu es (see Fig. 3). DHCHE solu ions a e s able a ela i ely high con en s o me hanol o e hanol (60%, no p esen ed g aphically). The esul s con i med ha he expe imen al condi ions (pH, ionic s eng h, ine elec oly e concen a ion, ype and concen a ion o bu e s and o ganic sol en s, e c.) se iously in luence he acid-base and UV-VIS spec al p ope ies o chele y h ine and mainly i s dihyd o- de i a i e. None o he in es iga ed componen s in solu ions (anions, ca ions e c.) is indi e en o he alkaloids, hus, exac e alua ion o he side e ec s as well as he de e mina ion o pH-dependencies o spec al and/o acidobasic p ope ies and ime s abili y o he alkaloids in di e en media is highly impo an be o e s a ing any in e ac ion s udies wi h biomac omolecules. Applica ion o di e en ypes and concen a ions o bu e s, elec oly es and componen s o solu ion, acid- base p ope ies and in e ac ions could be e y ca e ully s udied. This s udy poin s ou ha he beha io o chele y h ine and especially i s dihyd o-de i a i e in aqueous solu ions is mo e complex han be expec ed [7,8,10,11]. Acknowledgmen s Financial suppo om he G an Agency o he Czech Republic (GA ČR), g an No. 525/07/0871 is g a e ully Re e ences [1] V. Šimánek, in: A. B ossi (Ed.), The Alkaloids (Academic P ess, O lando, 1985) 26, 185 [2] J. Pso o á e al., J. Ch oma og . B., Analy . Technol. Biomed. Li e Sci. 830, 427 (2006) [3] M. Mai i, G.S. Kuma , Med. Res. Re . 27, 649 (2007) [4] R. Sinha, M. Hossain, G.S. Kuma , DNA Cell Biol. 28, 209 (2009) [5] J. U bano á, P. Lubal, I. Slanino á, E. Tábo ská, P. Tábo ský, Anal. Bioanal. Chem. 394, 997 (2009) [6] I. Slanino á, J. Slanina, E. Tábo ská, Chem. Lis y 102, 427 (2008) (in Czech) [7] Z. D ořák e al., He e ocycles 68, 2403 (2006) [8] M. 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