The Basics of Liquid Chromatography_1111

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1. Basics of Liquid Chromatography Week 2 1

2. 8 Types of Liquid Chromatography Normal phase liquid chromatography Reverse phase liquid chromatography High-performance liquid chromatography Flash chromatography : uses an inert gas to force the mobile phase through the stationary phase. Partition chromatography : MP and SP are liquid; partition coeffs . Size exclusion/Gel Permeation chromatography : it’s in the name Affinity chromatography : ligand to target/receptor Ion (Exchange ) chromatography : ionic compounds are separated depending on a positive or negative ionic exchange with a solid stationary phase. 2

3. Applications Che m ical Environmental polyaromatic hydrocarbons Inorganic ions herbicides Pharmaceuticals Consumer Products lipids an t i o x i d an t s sugars Clinical amino acids vitamins h o m o c y s t ei n e p o l y s t y re n es dyes phthalates tetracyclines corticosteroids an t i d e p r e s s a n t s barbiturates Bioscience proteins peptides nu cl e o t i d es 3

4. Flash Chromatography….Yawn…. 4 Stationary phases that are not resistant to air pressure include : Standard polysiloxanes : These have a repeating siloxane backbone and are characterized by the type and amount of functional groups on the silicon atoms. Dextran gels : These are used in exclusion chromatography and are produced by the action of certain bacteria on a sucrose substrate. Mobile phases for gas chromatography : Commonly used mobile phases include He, Ar , and N 2 , which are chemically inert toward both the sample and the stationary phase.

5. SEC vs GPC 5 SEC is the fractionation of proteins and other water-soluble polymers , while GPC is used to analyze the molecular weight distribution of organic-soluble polymers .

6. Affinity Chromatography 6 Basic Methodology : 1. A ligand is attached to a solid support to create a stationary phase . 2. The sample is incubated with the affinity support. 3. Non-bound components are washed away. 4. The target molecule is eluted from the immobilized ligand . Applications : Protein purification: Separates proteins from complex mixtures Clinical diagnostics: Used in the diagnosis of diseases Biopharmaceutical purification: Used to purify biopharmaceuticals Neuroscience: Used to capture specific proteins in complex mixtures Enzyme assays: Used to study enzymes and their substrates Nucleic acid analysis: Used to detect mutations and polymorphisms in nucleic acids

7. Ion Exchange Chromatography 7 Ion exchangers : Solid materials with positively or negatively charged functional groups Charged compounds : Adsorbed and retained by the ion exchanger with the opposite charge Neutral or similar charge compounds : Pass through the void volume and are eluted from the column Elution : Charged compounds are eluted with a salt or pH gradient

8. High-performance liquid chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). High performance liquid chromatography is now one of the most powerful tools in analytical chemistry. Has the ability to separate, identify, and quantitative the compounds that are present in any sample that can be dissolved in a liquid. Compounds in trace concentrations as low as parts per trillion (ppt) may easily be identified. Due to the high level of precision some HPLCs have, these instruments are also referred to as high precession liquid chromatography and Ultra-performance liquid chromatography (UPLC). What is HPLC ? 8

9. The sample is introduced through an injection system into the entrance to the column The stationary phase is porous silica. Mobile phase can either be aqueous. or organic solvent. HPLC Instrumentation/Mechanism Three recent developments have had an immense impact on the field of bioanalysis : The coupling (hyphenation) of liquid chromatography (LC) with mass spectrometry. the miniaturization of separation columns. the general availability of bioinformatics. Mobile Phase : carries the sample through the Stationary Phase as it moves through the column. Stationary Phase: remains fixed in the column. E.g. C18, Silica 9

10. Typical HPLC Set-Up Mobile Phases Flow Rate Composition Injection Volume Column Oven Temperature Wavelength Time Constant 10

11. Column Chromatography Chromatogram Separation Phase 11

12. Chromatograph t = column void time (injection to detection of unretained compound) t R = retention time (injection to detection of retained compound) t S = time spent in stationary phase (adjusted retention time) L = length of the column W = peak width W 1/2 = peak width at half height (column efficiency) The retention factor  k  is used to describe the retention independent of the column dimensions or flow rate. 12

13. Chromatograph: Retention Factor The retention factor  k  is used to describe the retention independent of the column dimensions or flow rate. The retention factor can have values between  k  = 0 (no retention) and  k  = ∞ (irreversible adsorption) with values of 1–20 most preferred for practical and economic reasons. 13

14. Chromatograph: Selectivity and Resolution To resolve two components, their retention factors must be different. The selectivity α of a chromatographic system describes the ability of a separation column to separate two compounds (1 and 2) based on their different retention factors k1 and k2. For a selectivity of α = 1 no separation is possible. To evaluate the quality of a separation not only the peak distance between the two components must be considered but also their respective peak width. The resolution,  R S , of two adjacent peaks in a chromatogram is defined by the ratio of peak distance and their peak widths. with the retention times  t R1  and  t R2  of two adjacent peaks and the respective peak widths  w 1  and  w 2 . Two peaks can be un-separated ( R S  < 1), partially overlapping ( R S  = 1), or baseline separated ( R S  > 1.5). 14

15. Theoretical Plates The higher the number of theoretical plates at a particular column length  L , the better the quality of column and the narrower the peaks. Does not account for the “mechanism” causing peak broadening No indication of other parameters’ effects No indication for adjusting experimental parameters 15

16. Zone Broadening: Kinetic Processes Van - Deemter Equation: relates the variance per unit length of a separation column to the linear mobile phase velocity by considering physical, kinetic, and thermodynamic properties of a separation. (M = 111) (M = 17 000)  The behavior of small molecules is determined by their diffusion; however, for large molecules the influence of the B term (longitudinal molecular diffusion) is negligible, particularly at higher flow velocities. The interaction with the stationary phase (C s -term) of large molecules results in band broadening. The optimal plate height for large molecules can be obtained at lower flow velocities. A =Eddy diffusion and mobile phase mass transfer effects and is a measure of the packing quality of the chromatographic bed (and is constant for a given column). B = longitudinal molecular diffusion effects. C = mass transfer resistances within the stationary phase microenvironment, which describes the interaction of the solutes with the stationary phase. 16

17. Optimization of your HPLC (or any LC system) Optimize Column Efficiency :Optimization of the peak efficiency, expressed as the theoretical plate number,  N , requires an independent optimization of each of the contributing factors that influence the band-broadening of the peak zones due to column and the extra-column effects. With a particular sorbent (ligand type, particle size, and pore size) and column configuration, this can be achieved through optimization of linear velocity (flow rate), the temperature, detector time constant, column packing characteristics, and by minimizing extra-column effects, by, for example, using zero-dead volume tubing and connectors.  Optimization of Selectivity : The most effective way to effect resolution. Mainly achieved by changing the chemical nature or concentration of the organic solvent modifier (acetonitrile, methanol, ethanol, isopropanol, etc.) in conjunction with the appropriate choice of mobile phase additive(s). Optimization of retention factors : Choose a different organic solvent modifier for the mobile phase. Alter flow rate. 17

18. Chromatographic Separation Modes for Peptides and Proteins Chemical and physical factors of the mobile and stationary phase that contribute to variation in the resolution, mass recovery, and bioactivity preservation of polypeptides, proteins, and other biomacromolecules in HPLC. 18