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KROMATOGRAF İ

KROMATOGRAF İ. Sedat Türe. HPLC. Liquid Chromatography. ÖZET. HPLC. The original development of HPLC used higher pressures than previously used ----High Pressure Liquid Chromatography However, over the years the preferred term has become: High Performance Liquid Chromatography.

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KROMATOGRAF İ

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  1. KROMATOGRAFİ Sedat Türe

  2. HPLC Liquid Chromatography

  3. ÖZET

  4. HPLC The original development of HPLC used higher pressures than previously used ----High Pressure Liquid Chromatography However, over the years the preferred term has become: High Performance Liquid Chromatography

  5. Advantages of HPLC High resolution Speed Re-usable columns Great reproducibility Control of physical parameters flow rate, polarity, packing efficiency, and particle size. Easy automation of instrument and data analysis. HPLC

  6. HPLC Chromatograph of Muscadine Grape Juice

  7. HPLC SOLVENTS Includes both liquid phase and solid materials (Buffers) dissolved in the liquid.

  8. HPLC • Solvent properties affecting detection • Solvent properties affecting separation • Solvent properties affecting flow • Viscosity • Miscibility

  9. HPLC Solvent Properties Affecting Detection UV Cutoff -Solvent may interfere with detection For peptide analysis UV = 215 nm. Solvents that absorb UV at this wavelength would not be good candidates for the mobile phase. Refractive Index of Solvent vs Sample for Refractive Index detection (Carbohydrates) Volatility needed for HPLC Mass Spectrometry (trifluoroacetic acid is a typical volatile buffer)

  10. HPLC BUFFERS 1)Buffers are needed to control the pH differences caused by the sample matrix. 2)Buffers are used to control the ionization of compounds and therefore their retention by the column.

  11. HPLC Retention Time and pH in Reversed Phase When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases. not charged partially charged Basic Compound Relative Retention Time fully charged pK a 3 4 5 6 7 8 9 pH

  12. HPLC SOLVENT SELECTIVITY The less time a compound spends in the stationary phase, the faster it will move through the column (less retention time). If two compounds are added to the column, the ratio of their retention times is called the selectivity. The higher the selectivity, the better the separation. Selectivity can be increased by adjustment of the mobile and stationary phases.

  13. HPLC Solvent Selectivity Triangle Representing 3 “Polarity” factors 1) Each dot in the triangle represent a different solvent 2) Solvents can be grouped based on their type of polarity 3) Solvents and solvent mixtures are available for just about any separation you may desire.

  14. HPLC Viscosity - resistance to flow Difficult to force high viscosity solvents through the column. Mixing solvents can drastically change viscosity

  15. HPLC Viscosity of Water-Organic Solvent Mixtures

  16. HPLC Viscosity vs. Pressure

  17. HPLC EXAMPLE P = 250 L  F / Dp2 Dc2 column length = 15 cm, column diameter =.5 cm, particle diameter = 5 mm, flowrate = 2.0 mL/min For water n = 1.0 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psi For methanol n = 0.54 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psi For 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% methanol

  18. Miscible with Water? SOLVENTS UV Cutoff Viscosity Polarity HPLC

  19. HPLC Peripheral Properties • Toxicity • Flammability • Reactivity solvent should not react with sample • Cost • Disposal can be more than purchase cost

  20. HPLC Geometry of HPLC Columns Diameter Length Particle Size What is the effect on pressure?

  21. HPLC P = 250 L  F / Dp2 Dc2 Where P = pressure drop in psi. F = flow rate (mL/min) L = column length (cm) Dp = particle diameter (mm) = solvent viscosity (cP) Dc = column diameter (cm)

  22. HPLC Geometry of HPLC Columns Diameter Length Particle Size What is the effect on Theoretical Plates?

  23. HPLC What is the effect of column geometry on Theoretical Plates? Remember that separation is best on columns with high number of theoretical plates. N=L/H where N is number of plates, H is plate height and L is Column Length Therefore, doubling the column length will double N but this will double analysis time and pressure!

  24. HPLC What is the effect of column geometry on Theoretical Plates? Decreasing column diameter by half For comparison purposes, let’s keep the mobile phase velocity constant. Therefore, flow would be reduced 4X and analysis won’t take any longer! halving the column diameter can also increase N slightly This reduces the amount of solvent used by 4X but also reduces the amount of sample that can be injected by 4X. http://www.catatankimia.com

  25. HPLC What is the effect of column geometry on Theoretical Plates? Decreasing particle size by half Will increase pressure by 4X However, halving the particle size can double N Decreasing particle size and making the column half as long will keep N the same but cut sample time in half and solvent use in half.

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