Estimation of Retention Factors of Nucleotides by Buffer Concentrations in RP-HPLC

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1 HWAHAK KONGHAK Vol. 38, No. 5, October, 2000, pp (Journal of the Korean Institute of Chemical Engineers) RP-HPLC Nucleotides ( , ) Estimation of Retention Factors of Nucleotides by Buffer Concentrations in RP-HPLC Ju Weon Lee and Kyung Ho Row Dept. of Chem. Eng., Inha Univ. (Received 1 February 2000; accepted 1 April 2000) buffer RP-HPLC!. "#!$ %& nucleotides(5'-cmp, 5'-UMP, 5'-GMP, 5'-IMP, 5'-AMP), buffer' sodium phosphate monobasic( acetic acid " ) mm*& +,-!. Buffer. / %& :; ) sodium phosphate monobasic( acetic acid 1<= A BC!. Abstract To estimate the retention behavior of ionized solute by the buffer concentration in mobile phase, the experiments were carried out by RP-HPLC(Reversed-Phase High Performance Liquid Chromatography). Five nucleotides(5'-cmp, 5'-UMP, 5'-GMP, 5'-IMP, 5'-AMP) were used as solutes. Buffers( mm) were sodium phosphate monobasic and acetic acid. We proposed the two nonlinear model equations in terms of buffer concentration to predict the retention factors. It showed the regression coefficients for the concentration of sodium phosphate monobasic and acetic acid were above 0.99, so the calculated by the model equations and experimental value were in good agreements. Key words: Nucleotides, Retention Factor, Buffer, emi-empirical Equation, RP-HPLC rowkho@inha.ac.kr 1. Nucleotides DNA, D-ribose, 3. Nucleotides nucleosides 3'! 5'" "#$% & ' ( )* +, -. Nucleotide./ 01adenosine-5'-triphosphate(ATP) 2345"6 /7 89. ATP ADP: ; & 9 A "B+* CDE F"6 "B+ GH I( J KL MN "B+* O7 F 45" PQ. R, adenosine 3'T 5'" UV phosphodiesterw& adenosine-3', 5'-monophosphate (cyclic AMP, camp) XY)Z" /7 89. [" /7 \)]^ NAD, NADP, FAD, Coenzyme A, vitamine B 12 _. nucleotide. [1]. Reversed-Phase High-Performance Liquid Chromatography(RP-HPLC) 9` Va b c Xde c MN f9g 'K, - [2-4]. RP-HPLC"6 hvn T \ in j + klm, nopqrs I Y9(. t hv" 9=K N I j uvq 9=Kw x& yz., {V& N }j u C 18 q C 8 Y9. 9= Kw x& yz N ~ j \? " }j,{v" +, ƒ 9DKw c w. h V" Q ˆ9` Šw c, x 2* C=Q }j,{v" &Œ c 9 [5]. hv ph" Ž vh" Ž ' MN ' " =6 dk [6-8]. Rtq hv ph* { ~ w, ~ h ph* w.! buffer " Ž vh š2* < œ. t w ž = Q '"6 hv ph š2* XŸQ hv F buffer. š2" Ž vh &Œ buffer. x " 'Q buffer." Ž vh [9, 10]. ' en + nucleotides, cytidine-5'-monophosphate disodium salt(5'-cmp), uridine-5'-monophosphate disodium salt(5'-ump), guanosine-5'-monophosphate disodium salt(5'-gmp), inosine-5'-monophosphate disodium salt(5'-imp), adenosine-5'-monophosphate disodium 626

2 salt(5'-amp) vh -klm hv" sodium phosphate monobasic! acetic acid* Q buffer." Ž vh ª«. 2.,{V C 18 }j +, -&E x& 9=Kw - N,{V" v +. 9=Kw x& y z }j,{v"6 (k) T x& yz? ( ): }x& yz? ( ) & q ± < -. k= (1 F )+ F (1) Q6 F hv F"6 x& yz ²³. ª hv" 9=Kw +1 µx F\F, 1 x& yz, { F T d < -. K [ ][ C + ] = [ C] 1 F = [ C + ] K Q6 K x2 V<, C x2k ) q F, /"6 x ), C + µx ) Xd. (1)" (3) X n¹: N < - [10]. k [ C + ] K + k = [ C + ] * I º»" hv" - µx C + buffer" =6 ¼½(, { < µx ¼½ buffer x2 V< K B T. [ C + ][ B ] K B = [ BC] Q6 BC buffer ) XdL B buffer"6 q¾ 1 x Xd. C +.* hv F buffer." X < T WT ¼ÀÁ } W& {Â. [ C + ] = K 1 C B [ C + ] = K 2 C B (6)T (7)"6 C B hv F"6 buffer., K 1,K 2 V<. (6)T (7) (4)" X buffer. " Ž < < -&L T. K C 1 K B + k = K C K B K C K B + k = K C K B (8)T (9) hv F"6 buffer." Ž š2* q F 4 (semi-empirical equation) ¼ÃÂ. RP-HPLC Nucleotides 627 (2) (3) (4) (5) (6) (7) (8) (9) Ä "6 Å ( Æ + ~Çš<,,K 1,K 2 } W ÈÉ cê /"6 Levenberg-Marquardt Ëe2" Q Â&L Ÿ Ì.: Í9¾Î ÏÏ 0.95, 0.05Â. 3. "6 Y9( nucleotide cytidine-5'-monophosphate disodium salt(5'-cmp), uridine-5'-monophosphate disodium salt(5'-ump), guanosine-5'-monophosphate disodium salt(5'-gmp), inosine-5'-monophosphate disodium salt(5'-imp), adenosine-5'-monophosphate disodium salt(5'-amp) Fluka"6 Â&L hv& N ÐÑ ÐQ ULTRA-PURE WATER YTEM Milli-Q50(Millipore)* 9Q 18 MΩcm ÒÓ<* Šw Y9Â&L klmn HPLCgrade* J. T. Baker"6 Q Y9Â. hv ph* )Z =6 Y9(Ô + buffer acetic acid, sodium phosphate monobasic. HPLC columnn 0.39Õ30 cm empty column" Lichrospher 100 RP-18(15 µm, MERCK) packing ˆÖQ Y9Â. HPLC WatersY 616 pump: 600 controller, 2487 detector* Y 9Â. N 1 ml/min&,{â,, hvf klm ) N 5, 10, 15, 20 vol% š2 Ø.hV F" buffer. acetic acid» " mm š2q Â,, sodium phosphate monobasicn " mm š2l Â. amplen 150 ppm <9` Šw 20 µl* I Â. ª N Vx"6 ÙÂ. 4. (8)T (9) hv F buffer." Ž * { model & + 5'-nucleotides" X=6 acetic acid* buffer Y9Â?" Ô + } W ª«, (8)T (9) parameter* Table 1" q F.Table 2 buffer sodium phosphate monobasic Y9Â? (8)T (9) parameter* \, -. Buffer acetic acid* Y9»" (8)" X V P <(regression coefficient) , (9)" X VP < (9)\ (8) Ú, -. : X )e& buffer sodium phosphate monobasic Y9» VP < (8)» , (9)»" (8)\ (9) Á^+ }Ûe Ú, - \, -. (8)T (9) ÎÜN µx.* { CÊ" -. (8)N hv F µxn hv F buffer.(c B ) 1Î" }Ý (6) Y9Â, (9) hv F µxn hv F buffer.(c B ) ¼ÀÁ" }Ý (7) Y9Â. : N ÎÜ, = Þ?" acetic acid"6 q¾ µx H +. acetic acid. 1Î" }Ý (6)" =6 ß < -&L sodium phosphate monobasic"6 q¾ µx Na +. sodium phosphate monobasic. ¼ÀÁ" }Ý (7)" =6 ß < -. à6 Y9( buffer " à 6 buffer." }Ý {. áàâ < -. Fig. 1T 2 buffer acetic acid* Y9Â? ãt (8)T (9)" = ( ã q ä & data point ã, & q ä N ã q ä. Fig. 1T 2"6 Þ < -å acetic acid. 4mMV»" Ï " X { ã \, - ã& < -. t 0 (8)N Fig. 1"6: acetic acid 4mM V æ "6 <ç ã Ú q F, -+ (9)" =6 ( ãn HWAHAK KONGHAK Vol. 38, No. 5, October, 2000

3 628 Table 1. Parameters of Eqs. (8) and (9) by acetic acid Material Methanol (vol%) Eq. (8) Eq. (9) K 1 R 2 K 2 R 2 5'-CMP '-UMP '-GMP '-IMP '-AMP Table 2. Parameters of Eqs. (8) and (9) by sodium phosphate monobasic Material Methanol (vol%) Eq. (8) Eq. (9) K 1 R 2 K 2 R 2 5'-CMP '-UMP '-GMP '-IMP '-AMP Fig. 2"6: acetic acid. 4mMV æ"6 { ã& <ç» nè 661 л q F, -. Fig. 1T 2"6 \ ƒ" =6. acetic acid» (8) ã (9) ã \ Ú, acetic acid. š2" Ž š2. Ú d, - < -. Fig. 3T 4 hv F" sodium phosphate monobasic buffer Šw -»" sodium phosphate monobasic š2" Ž * ãt ã" XQ d & data point ã N ã q FL Fig. 3N (8)" X ã Fig. 4 (9)" X ã q F. Acetic acid»: X)e&»" sodium phosphate monobasic. Ð Q. { ã& <ç» nè л \, -. (8)T (9) VP < ni Á^ ã& (9) én ã \, -.! Fig. 3"6: (8)N sodium phosphate monobasic. Ð <ê л Ú q F+ ë, sodium phosphate monobasic. Ð <ê ã\ in ã \, -&q, Fig. 4" 6 (9) monobasic. Ð <ê Ð ã

4 RP-HPLC Nucleotides 629 Fig. 1. Comparison of calculated and experimental retention factor[eq. (8), acetic acid]. Fig. 2. Comparison of calculated and experimental retention factor[eq. (9), acetic acid]. (8)\ Ú d, -. Fig. 5: 6N acetic acid» (8) sodium phosphate monobasic» (9)* e9â?" (8)T (9)"6 }x" X * q F parameter ã hv F" klm µ š2" XQ.. Fig. 5: 6 }Û» N " X acetic acid: sodium phosphate monobasic ÏÏ»" X=6 ~ Î* \, -. R acetic acid» H + µx Na + x\ hv F" ~ M yz? " Na H UKw disodium salt Wa nè 5'-nucleotide Wa }x yz? " 5'-nucleotide * q FL sodium phosphate monobasic»" Na + x M yz? " Na H UK+ N disodium HWAHAK KONGHAK Vol. 38, No. 5, October, 2000

5 630 Fig. 3. Comparison of calculated and experimental retention factor[eq. (8), sodium phosphate monobasic]. Fig. 4. Comparison of calculated and experimental retention factor[eq. (9), sodium phosphate monobasic]. salt Wa 5'-nucleotides disodium salt * q ä. : }x Wa ÏÏ 5'-nucleotide: 5'-nucleotide disodium salt % q q? " buffer " à N ì Îq & \. R, klm µ Ð <ê í^ < -. t» N hv F" modifier µ Ð <ê í^ 4e» T, - [2, 4]. Fig. 7T 8N (8)T (9)"6 x& yz? * q F parameter ã acetic acid: sodium phosphate monobasic" XQ klm Ð" Ž ã». & klm µ ÐQ. v š2+ &L ~ in ã& { +K Þ < -. t x

6 RP-HPLC Nucleotides 631 Fig. 5. Changes of with methanol contents in mobile phase[eq. (8), acetic acid]. Fig. 8. Changes of k 1 with methanol contents in mobile phase[eq. (9), sodium phosphate monobasic]. Fig. 6. Changes of with methanol contents in mobile phase[eq. (9), sodium phosphate monobasic]. µ ÐQ. j x " +. RtE x }j,{v" v +, 9 Dï < RP-HPLC"6 hv" buffer Kw ph š ' ŠN hv ph" Ž š2* Â. Rtq µ buffer " X=6 : N ' ÖÙK+ ð. '"6 buffer " à6 Ž ª«e9 &Œ ph"6, K+ N buffer " Ž 0, Â. T" acetic acid»" (6) P e9( (8), sodium phosphate monobasic»" (7) P e9 ( (9) Ú, -. Buffer 0" ñ " P ª«=6 buffer: µx." P Pò & {=ó.! model "6 }xt x *q F : ã {» q F, -? " buffer.: modifier.* h ", model A (. '"6 ¼Ã ª«[ (8), (9)] 9Q ô buffer." Ž æ < -. t ª«9Q buffer* 9 RP-HPLC c õ )ö < -, Ù¾Îe CÊ\ Ž æ F" Ëe c õ )ö < -. ' X3Û Û<'Öø}" Q <ÙK &L " íyùúû. Fig. 7. Changes of k 1 with methanol contents in mobile phase[eq. (8), acetic acid]. Wa -»" ~ j + KL ~ j N }j,{v" î;%+, 9D.! klm k F K K B : retention factor : retention factor of non-ionized solute : retention factor of ionized solute : mole fraction of ionized solute : equilibrium constant of solute : equilibrium constant of buffer HWAHAK KONGHAK Vol. 38, No. 5, October, 2000

7 632 K 1,K 2 : empirical constants : concentration of buffer in mobile phase [mm] C B 1. huler, M. L. and Kargi, F.: Bioprocess Engineering, Prentice Hall, London(1992). 2. Lee, Y. W., o, M.., Lee, J. W., Chung,. T. and Row, K. H.: Korean J. Chem. Eng., 13, 578(1996). 3. Row, K. H. and Lee, J. W.: Korean J. Chem. Eng., 14, 412(1997). 4. Row, K. H. and Lee, J. W.: eparation cience and Technology, 35, 271(2000). 5. Row, K. H.: Principles and Applications of Liquid Chromatography Inha Univ.(1999). 6. Bosch, E., Bou, P., Allemann, H. and Roses, M.: Anal. Chem., 68, 3651(1996). 7. Roses, M., Canals, I., Allemann, H., ilgur, K. and Bosch, E.: Anal Chem., 68, 4094(1996). 8. Kaltenbrunner, O. and Jungbauer, A.: J. Chromatogr. A, 769, 37(1997). 9. Kaliszan, R.: J. Chromatogr. B, 717, 125(1998). 10. Hajnos, M. W.: J. Chromatogr. B, 717, 93(1998)

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