Micellar liquid chromatography Original

Micellar Liquid Chromatography : 

Micellar Liquid Chromatography Franklin Ifeanyichukwu Uba CHEM 7750 (ORGANIZED MEDIA) LOUISIANA STATE UNIVERSITY OCTOBER 22ND, 2009

REFERENCES : 

REFERENCES Armstrong, Daniel W. ; Nome, Faruk. Partitioning behavior of solutes eluted with micellar mobile phases in liquid chromatography. Analytical Chemistry (1981), 53(11), 1662-6 5/3/2010 Amin Mohammad; Harrignton Katharine; Wandruszka Ray Von. Determination of Steroids in Urine by Micellar HPLC with Detection by Sensitized Terbium Fluorescence. Analytical Chemistry (1993), 65, 2346-2351 Kamande M.W. Analytical Separations using polymeric surfactants. Dissertation (2005), Louisiana State University, Baton Rouge. 181 pp

OUTLINE : 

OUTLINE Objective Liquid Chromatography Micellar Liquid chromatography Application Conclusion Critiques Acknowledgement

OBJECTIVE : 

OBJECTIVE To describe the partition behavior of solutes eluted with micellar phases in liquid chromatography. To show the mechanism for steroid analysis using micellar LC. 5/3/2010

LIQUID CHROMATOGRAPHY : 

LIQUID CHROMATOGRAPHY Liquid mobile phase Classifications Mode of operation Column and Planar Stationary phase Relative polarities Modern HPLC 5/3/2010 5 SEC IEC BPC LSC http://www.pltscientific.com/02int.jpg Chromatography; Concepts and contrasts, Wiley Int. Sci, 2nd edition, Pg 184

Stationary phase : 

Stationary phase 5/3/2010 6 Principles of Instrumental analysis, Thomas learning 5th edition, Table 26-1, Pg 677 Classification of Column Chromatographic methods.

Relative Polarities : 

Relative Polarities Normal Reverse 5/3/2010 7 Modern HPLC High sensitivity and resolution (tightly packed column) High Speed (minutes or seconds) Automation Ready adaptability Chromatography; Concepts and contrasts, Wiley Int. Sci, 2nd edition, Pg 184

MICELLAR LC : 

MICELLAR LC Micelles; MEKC and LSC. Micelle formation / types 5/3/2010 Normal micelle Reverse Micelles or Surfactants Conventional micelles CMC Prof. Isiah Warner, CHEM 7750, Lecture 9, Micellar HPLC

Micelles in MEKC : 

Micelles in MEKC Conventional micelles 5/3/2010 9 Dynamic equilibrium of normal Micelles and interaction with a Solute S, Solute; asterisk (*) denotes Chiral centre. CMC Prof. Isiah Warner,CHEM 7750, Lecture 9, Micellar HPLC

Micelles in MEKC : 

Micelles in MEKC Advantages; High Specificity Wide range of applications Disadvantages; Unstable dynamic Equilibrium Affected by factors; OC, [Surfactant], pH, etc. Chromatographic band Broadening Polymeric micelles. 5/3/2010 10 Kamande M.W. 2005. Analytical Separations using polymeric surfactants. LSU, Baton Rouge. 181 pp.

Polymeric Micelles : 

Polymeric Micelles Forms 5/3/2010 11 Schematic Representation of Type H and Type T micellar Polymerization. Prof. Isiah Warner, CHEM 7750, Lecture 9, Micellar HPLC

Polymeric micelles : 

Polymeric micelles 5/3/2010 12 Schematic diagram of the Principles of Polymerized T micelle and solute interactions; S, Solute; asterisk (*) denotes chiral centre Prof. Isiah Warner, CHEM 7750, Lecture 9, Micellar HPLC Solute partition

Studies on Sodium 10-Undecenoate : 

Studies on Sodium 10-Undecenoate Paleos, C.M., Stassinopoulou, C.I, Maillaris A. Comparative Studies between Monomeric and Polymeric Sodium 10-Undecenoate Micelles. J. Phys. Chem. 1983, 87, 251 – 254. 5/3/2010 13 Sodium 10-Undecanoate Paleos C.M, et al. J. Phys. Chem. 1983, 87, 251-254

Slide 14: 

5/3/2010 14 Monomer to polymer conversion vs γ-ray dose Paleos C.M, et al. J. Phys. Chem. 1983, 87, 252, fig. 1 Monomer to polymer conversion curve of L-SUV Prof. Isiah Warner, CHEM 7750, Lecture 9, Micellar HPLC

Slide 15: 

5/3/2010 15 Paleos C.M, et al. J. Phys. Chem. 1983, 87, 252, fig. 2 Electrical conductivity vs. surfactant concentration. The conductivity Is expressed in S cm -1 = Ω-1 cm-1

Micelles in Mobile Phases : 

Micelles in Mobile Phases Solute Elution efficiency Column Parameters Micelle Characteristics Three Partition coefficients 5/3/2010 16 h At As Am Vt Vs Vm Cm β ν KMW KSW KSM

Solute Partitioning : 

Solute Partitioning 5/3/2010 17 KSW KMW KSM Micellar Chromatography three ‘phase’ model. KMW = Partition coefficient between Micelle and water KSW = Partition coefficient between Stationary phase and water KSM = Partition coefficient between Stationary phase and Micelle Daniel Armstrong, et al, Anal. Chem., 1981, 53, 1663 ,figure 1. Shahab, Shamsi, et al, Anal. Chem., 2001, 148A ,figure 5. Proposed interaction of various chiral analytes with Polymeric dipeptide surfactants on the basis of electrostatic and hydrophobic interactions.

Partition equation : 

Partition equation Where; Vs, Ve, and Vm are the Stationary phase volume, Elution volume of the solute, and Mobile phase volume, respectively; ν is the partial specific volume of the surfactant in the micelle; Cm is the concentration of surfactant in the micellar form 5/3/2010 18 Daniel Armstrong, et al, Anal. Chem., 1981, 53, 1664.

Studies on SDS mobile phase : 

Studies on SDS mobile phase Stationary Phases Octadecylsilane reverse phase column Alkyl nitrile bonded-phase Mobile Phases Sodium dodecylsulfate (SDS) Solutes Hydroquinone Resorcinol p-nitrophenol p-nitroaniline Naphthalene 5/3/2010 19 Daniel Armstrong, et al, Anal. Chem., 1981, 53, 1664 ,figure 2.

: 

5/3/2010 Concentration of SDS in micelle, g/ml Chromatographic parameters vs Concentration of Surfactant in micelle. (C18 reversed Phase column) Hydroquinone, Resorcinol, p-nitrophenol, p-nitroaniline Daniel Armstrong, et al, Anal. Chem., 1981, 53, 1664 ,figure 2.

Slide 21: 

5/3/2010 21 Concentration of SDS in micelle, g/ml Chromatographic parameters vs Concentration of Surfactant in micelle. (alkyl nirile bonded Phase column); Resorcinol, p-nitrophenol, p-nitroaniline Naphthalene Daniel Armstrong, et al, Anal. Chem., 1981, 53, 1666 ,figure 3.

APPLICATION : 

APPLICATION Steroids in Urine Procedural requirement Sensitivity for free steroids down to 1-10ng Simultaneous determinations of different steroids. Why micellar HPLC? No sample preparation Direct urine injection 5/3/2010 22 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2346-2351.

Slide 23: 

Steroids Mohammad Amin, et al, Anal. Chem., 1993, 65, 2347 ,figure 1. Detection by sensitized Terbium Fluorescence. 5/3/2010 23

Why Terbium? : 

Why Terbium? 5/3/2010 24 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2348 ,figure 3. Energy level diagram of typical semi-quinoid steroid donor and lanthanide ion acceptor

Terbium Fluorescence : 

Terbium Fluorescence 5/3/2010 25 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2348 ,figure 2. Fluorescence emission spectrum of Tb (III)

Elution patterns : 

Elution patterns 5/3/2010 26 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2350 ,figure 5. HPLC separation of steroids with detection by post-column Terbium addition: Normal Phase Reverse Phase 0.03 M Tb (III) solution in 0.1 M SDS at 0.5 mL/min Testosterone acetate Progesterone Bolasterone (0.5 ng) Methyl testosterone Testosterone

Micellar LC spectra : 

Micellar LC spectra 5/3/2010 27 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2350 ,figure 6. Standard solution (20 ng of steroid) 200-µL urine sample Testosterone (300 ng/mL) Methyl testosterone (300 ng/ml) Bolasterone (100 ng/ml) Progesterone (100 ng/ml) Testosterone acetate (100 ng/ml)

Terbium with [SDS] : 

Terbium with [SDS] 5/3/2010 28 Mohammad Amin, et al, Anal. Chem., 1993, 65, 2349 ,figure 4. Variation of steroid-sensitized terbium fluorescence intensity with SDS concentration: 1.0 X 10-3 M Tb(III), 2.5 µg/ml testosterone; excitation 247 nm, emission 547 nm. Relative intensity Log [SDS]

Further studies : 

Further studies 5/3/2010 29 ZuLiang Chen, et al, Anal. lett., 1997, 30, 2319 ,figure 1. Mobile phase; (2.5 – 7.5) X 10-2 M and 9% 1-butanol. Flow rate: 1.0 ml/min, 20 µL 1. Hydroxycorticosterone 2. Corticosterone 3. Norethisterone 4. Testosterone 5. Mexdroprogesterone acetate 6. Progesterone Effect of the n-butanol in mobile phase on The retention time of the test solutes. 1. Hydroxycorticosterone 2. Corticosterone 3. Norethisterone 4. Testosterone 5. Mexdroprogesterone acetate 6. Progesterone Zuliang Chen, et al, Anal. lett., 1997, 30, 2320 ,figure 2.

CONCLUSION : 

CONCLUSION The behavior of solutes eluted by micellar liquid chromatography can be understood by studying the basic partitioning theory. Micellar LC is a simple, fast, sensitive and highly effective analytical method for steroid detection in biological samples. 5/3/2010 30

CRITIQUES : 

CRITIQUES ‘Sphere to Rod’ transitions of micelles can affect the elution The hydrophobic tail of an ionic surfactant can bind to the stationary phase, leaving the Polar head in contact with the bulk mobile phase. 5/3/2010 31

ACKNOWLEDGEMENTDr Isiah Warner : 

ACKNOWLEDGEMENTDr Isiah Warner 32 LSU chemistry faculty Fellow graduate students 5/3/2010

THANK YOU : 

THANK YOU 5/3/2010 LSU CHEMISTRY VILLAGES COLLEAGUES