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
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 Chromatograph of Muscadine Grape Juice
SOLVENTS Includes both liquid phase and solid materials (Buffers) dissolved in the liquid.
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) Solvent Properties Affecting Detection
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.
Retention Time and pH in Reversed Phase pH Relative Retention Time pK a partially charged fully charged not charged 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. Basic Compound
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.
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.
Viscosity - resistance to flow Difficult to force high viscosity solvents through the column. Mixing solvents can drastically change viscosity
Viscosity of Water-Organic Solvent Mixtures
Viscosity vs. Pressure
P = 250 L F / D p 2 D c 2 EXAMPLE column length = 15 cm, column diameter =.5 cm, particle diameter = 5 m, flowrate = 2.0 mL/min For water n = x 15 x 1.0 x 2 / 5 2 x.5 2 = 7125/6.25 = 1200 psi For methanol n = x 15 x.54 x 2 / 5 2 x.5 2 = 2025/6.25 = 648 psi For 60% water n = x 15 x 1.9 x 2 / 5 2 x.5 2 = 7125/6.25 = 2280 psi 40% methanol
SOLVENTS UV Cutoff ViscosityPolarity Miscible with Water?
–Toxicity –Flammability –Reactivity solvent should not react with sample –Cost –Disposal can be more than purchase cost Peripheral Properties
Geometry of HPLC Columns Diameter Length Particle Size What is the effect on pressure?
P = 250 L F / D p 2 D c 2 Where P = pressure drop in psi.F = flow rate (mL/min) L = column length (cm)D p = particle diameter ( m) = solvent viscosity (cP)D c = column diameter (cm)
Geometry of HPLC Columns Diameter Length Particle Size What is the effect on Theoretical Plates?
What is the effect of column geometry on Theoretical Plates? N=L/H where N is number of plates, H is plate height and L is Column Length Remember that separation is best on columns with high number of theoretical plates. Therefore, doubling the column length will double N but this will double analysis time and pressure!
What is the effect of column geometry on Theoretical Plates? Decreasing column diameter by half halving the column diameter can also increase N slightly For comparison purposes, let’s keep the mobile phase velocity constant. Therefore, flow would be reduced 4X and analysis won’t take any longer! This reduces the amount of solvent used by 4X but also reduces the amount of sample that can be injected by 4X.
What is the effect of column geometry on Theoretical Plates? Decreasing particle size by half However, halving the particle size can double N Will increase pressure by 4X 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.
In general small diameter columns with small particles are best for rapid separation, ….but require higher pressures, smaller samples, and can plug easier. The problem with plugging should not be underestimated and care should be exercised in keeping the sample, mobile phases, and columns CLEAN!