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1 w y wz 8«( 4y) 00~00, 2005 J. of the Korean Society for Environmental Analysis HPLC f y e t p»wá½»x w y w»y Detection Characteristics of a HPLC Method on Carbonyl-DNPH Derivatives in Relation with Column Flow and with Solvent Strength of Mobile Phase Ki-Han Lee and Ki-Hyun Kim Department of Earth & Environmental Sciences, Sejong University, Seoul , Korea A number of carbonyl compounds in the atmosphere such as ketones and aldehydes can harmfully affect the human health as primary and secondary pollutants, respectively. In order to analyze these compounds, one of the most reliable methods include the analysis of their 2, 4-dinitrophenylhydrazine (DNPH) derivatives through an application a reversed-phase High Performance Liquid Chromatographic (RP-HPLC) technique. The main purposes of this study are to examine the detection characteristics of the HPLC method against carbonyl-dnph derivatives in relation with column flow and solvent strength of mobile phase. The overall results of our study indicate that changes in column flow sensitively affect the detectability, while solvent strength influences more on their resolution. Key wordw : Carbonyl, Ketone, Aldehyde, HPLC, Column flow, Solvent strength, Mobile phase 1. w fm w e «Ÿyw mw ky y w w. e yw Ÿyw 1) O 3 w e» Ÿyw p ³ w w w w. 2) e w y» (Flame Ionization Detector: FID) w» w Gas Chromatography (GC) High-Performance Liquid Chromatography/ Ultraviolet detector (HPLC/UV) y w. GC w, HPLC/UV w ƒ ûš w ƒ. e yw w» 3), w yw yw w. p, 2, 4-DNPH (dinitrophenylhydrazine) e yw z w w x w. 4-6) 1984 y y (Environmental Protection Agency, EPA) w fmyw v w 2, 4-DNPH g w TO- 5 w. 7) 1987, 1999 DNPH ep w TO-11, 11A w z, w w ƒ ƒwš. e yw DNPH yw 8,9) HPLC/UV w Áü wš. w 10-12) w, e w» w, HPLC/UV w ƒ ù y g š. To whom correspondence should be addressed.
2 2»wÁ½»x HPLC» w w ww» w, DNPH,, (ph, q ) w w. 10,13) x HPLC»» w w wš,, w l w w w ƒ yw p w wƒ w. š j m v (Reversed-Phase (RP)-HPLC) w w w w s» w š w w, ƒ w p sƒwš w. RP-HPLC w Ì w š w ƒ. w p w» w, ƒ y(f ü y ) w. š w yƒ DNPH yw e yw p w w e ƒ š w. 2. HPLC sww j m vv» w, m y w e. w vj e x w. w, HPLC f w, ƒ y ƒw» w. y y ù e ƒ vj yw w w w w. HPLC» w UV w k wš,»» w ƒ w p ƒ š. 13) DNPH 349 nm Ÿ ùkü, s w - DNPH (364 nm), p w -DNPH (368 nm), p w -DNPH (369 nm) k ƒ ƒw λ max ƒ q w w. s w 10) C 1 ~C 6 w w w -DNPH sww 8 w (Table 1). w p š w, 360 nm q š w k w. 2.1»» w w HPLC/UV (Lab Alliance, Model 500) l» w x w. HPLC rv x rv (dual head pump) w, y (noise)ƒ y k w w (Table 2). DNPH yw xk e yw», f C18 (25 cm 4.6 mm, particle size 5 µm, Hichrom) w., w e 20 µl v w w t t w» w, Carbonyl-DNPH (Mix 1 Catalog NO U, Supelco) 15 ng/µl 15, 25, 50, 150, 300 mù p (Acetonitrile: w ACN) w. mw, ƒ (ƒƒ 0.05, 0.1, 0.3, 0.6, 1.0 ng/µl) t w. t j vr w ( w š), t Table 1. A list of target carbonyl compounds for the analysis Full name Short name Chemical Formula Mol. Weight g/mol Formaldehyde Form-A HCHO 30 Acetaldehyde Acet-A CH 3 CHO 44 Propionaldehyde Propion-A CH 3 CH 2 CHO 58 Crotonaldehyde Croton-A CH 3 CH=CHCHO 70 Butyraldehyde Butyr-A CH 3 CH 2 CH 2 CHO 72 Benzaldehyde Benz-A C 6 H 5 CHO 1061 Isovalerldehyde Isovaler-A (CH 3 ) 2 CHCH 2 CHO 86 Valealdehyde Valer-A CH 3 (CH 2 ) 3 CHO 86
3 HPLC f y e t p 3 Table 2. An operating condition of HPLC/UV system for the analysis of carbonyl compounds Order HPLC/UV Lab Alliance, Model Pump: Dual head pump system 2 Mobile phase ratio (ACN : Water): [1] 6 : 4, [2] 6.5 : 3.5, and [3] 7:3 3 Column: C18 (Hichrom: particle size 5 ) 25 cm x 4.6 mm ¾ 4 Column flow rate (ml/min): [1] 1.5, [2] 2.5, and [3] Loop size (µl): 20 ACN yww w. e ({ )» w, ACN q v w w x HPLC p w» w, ƒ (f ü, )» w. w y w HPLC»» p q wš w., ƒ x w ù x y w, w» w y x HPLC f ü 3 (1.5, 2.5, 3.5 ml/min) y g. š w t ƒ 1z w w. y w x r x 1 w., x 7:3 (Acetonitrile: Water) š w w. w x y w 0.1 ng/µl t 3z w ( ) y x x w Acetonitrile:Water» 3 ( 7 : 3, 6.5 : 3.5, 6 : 4) w. š y g, w t ƒ 1z w. w x r x 2 w. w w ml/min š w w., w x y w, 0.1 ng/µl 3z w. 3. š y x( x 1) y x( x 2) y w l š»» (r 2 ) w. Table 3 ƒ x l w w w. ƒ x 0.31~0.93 µg/m 3 w, 2.35% w t w x w» w (Table 4A, B) y x, w, vj j ùkû (Fig. 1). s w, 3.5 ml/min 143,290, 2.5 ml/min 201,790 vj» w., 3.5 ml/min w 2.5 ml/ min 1.4 ƒw. š 1.5 ml/min, vj 343, ml/min 2.4 ƒw. s w ƒ 3.5 ml/min 1 ml/min w, vj ƒƒ 1.4, 1.7 ƒw., 3.5 ml/min 2.4~2.5 vj ƒw ùkû., vj w ü yw ùkû.»» w r ùkû y x»zw x 2 y
4 4»wÁ½»x Table 3. Results of calibration analysis of carbonyl compounds under varying conditions Mobile Phase (ACN : H 2 O) (ml/min) Compounds Form-A Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A n-valer-a 7 : Slope r Slope r Slope r : : Slope r Slope r Mean SD RSE Table 4. Comparison of two fundamental analytical parameters: Detection Limit & Reproducibility A. Detection Limit Mobile Phase (ACN : H 2 O) (ml/min) Compounds ¾ ¾ ¾ ¾ ¾ ¾ Form-A Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A n-valer-a 7 : µg/m ppb* µg/m ppb* µg/m ppb* : µg/m ppb* µg/m : ppb* * A total sample volume of 15 L was assumed B. Reproducibility (RSE %) Mobile Phase (ACN : H 2 O) (ml/min) Compounds Form-A Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A n-valer-a 7 : : :
5 HPLC 분석에서 컬럼유속과 이동상 용매강도의 변화에 따른 카보닐 표준시료의 검출특성 5. Comparison of calibration curve under the conditions of varying column flow rates. Fig. 1 율하기 위해, 이동상의 비율을 변화시켜 주면서 진행하 할 수 있다. 그러나 검량선의 기울기에 대한 r2 값은 였다. 그 결과, 용매강도의 변화가 검량선의 기울기 값 이상으로 상당히 높은 수준을 유지하였다. 에 별다른 변화를 주지 않는 것을 확인할 수 있다(Fig. 2). 이 결과는 Table 3에 제시한 1.5 ml/min에서의 상 고찰 대표준오차 값이, 5% 이하로 나타난 것으로부터 확인 본 연구에서 두 가지 분석인자의 변화에 따른 3.3.
6 6»wÁ½»x Fig. 2. Comparison of calibration curve under the conditions of varying solvent strengths. HPLC p y w. š» w ù ƒ (N) ù (Rs) w š w (Table 5, 6). y x, t ƒ ƒ w ü f ü w vj ƒw. w vj y w w, vj s ƒw w., vj z (broadening effect)ƒ w š w (Table 5). ù vj w s ƒw
7 HPLC f y e t p 7 Table 5. The number of theoretical plates (N) and Resolution (Rs) with carbonyl compounds at varying column flow rates (7:3, 2 ng injection) ml/min Compounds PH T R min W b N Rs Form-A Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A Form-A ¾ Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A Form-A ¾ Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A Table 6. The number of theoretical plates (N) and resolution (Rs) in the analysis of carbonyl compounds with respect to solvent strength (1.5 ml/min, 2 ng injection) ml/min 7 : : : 4 Compounds¾ PH T R min W b N Rs Form-A Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A Form-A ¾ Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A Form-A ¾ Acet-A Propion-A Croton-A Butyr-A Benz-A Isovaler-A Valer-A
8 8»wÁ½»x w j ùkû., ƒ ƒw š w. y x vj wš s ùkû., vjƒ w w (Table 6). ù w yƒ slope w e y w., y w,, vjs, vj w, vj slope w w. x (Rs) w, x 1 w vj x ƒw w w y w., x 2 ƒ w ƒw, ƒ ƒw (Table 5, 6). f ü w, ƒ ƒw., w yƒ w e. wr, ƒ w ƒw, w e ùkû. 4. DNPH x w w yw HPLC/UV w» wš w. w w w, ùkù e yw» p» wš w. j m v w w yw w, f ü w w. š y y x š w. f ü y ƒ w ü y w e, w e y w. y ( z ) y w e y w. j m v w»», w» w x w wƒ v w. w n» w, wùwù y wš» w û w. mw y w HPLC», j m v» p q w» y w» w. š x 1. Müller, K., Chemosphere, 1997, 35(9), x, ³,,»y œwz, 2002, 24(10), , x, Ÿ,, w wz, 2000, 10(1), Kuwata, K. Uebori, M. and Yamazaki, Y., Journal of Chromatography A, 1979, 211(3), Andersson, G., Andersson, K., Nilsson, C. A. and Levin, J.-O., Chemosphere, 1979, 8(10), Beasley, R. K., Hoffmann, C. E., Rueppel, M. L. and Worley, J. W., Anal. Chem., 1980, 52(7), Environmental Protection Agency (EPA), 1984, Method for the determination of aldehyde and ketone in ambient air using high performance liquid chromatography (HPLC), method TO Environmental Protection Agency (EPA), 1987, Method for the determination of formaldehyde in ambient air using adsorbent cartridge followed by high performance liquid chromatography (HPLC), method TO Environmental Protection Agency (EPA), 1999, Method for the determination of formaldehyde in ambient air using adsorbent cartridge followed by high performance liquid chromatography (HPLC) method TO-11A (Active Sampling Methodology). 10.,, wywz, 1999, 43(4), y, z,,, w, ½ y, w y wz, 2006, 32(1), Grandy K., carsten S. Christensen, Atmo. Environ., 1997, 31(10), y, ½»x, w wz, 2005, 18(1),
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