Chapter 25. HPLC High-Performance Liquid Chromatography. Typical HPLC. Application Microdialysis. Monitor Aspirin in Blood. Increased Efficiency. In any method the object is to increase mobile phase / stationary phase interaction. Decrease particle size. Better packing – slower flow.
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Chapter 25 HPLC High-Performance Liquid Chromatography
Increased Efficiency • In any method the object is to increase mobile phase / stationary phase interaction. • Decrease particle size. • Better packing – slower flow.
Smaller is Better • N ~ 3500 L (cm) / dp (mm) • Smaller particle size leads to • Higher plate number • Higher pressure • Shorter optimum run time
What is a bar? • A measure of pressure. • One bar = 1.01325 Atm • One bar is 100 000 Newtons/m2 • Under water you gain about one atm for each ten meters in depth.
Stationary Phase / Support • Support is the scaffolding that the stationary phase sits on. • Support - Microporous Silica • Solvent can get inside • Surface area of 100’s m2 / gram • Silica degrades above pH 8 so keep pH below. • Special supports have been developed for higher pH
Bonded Phases • SiOH + ClSi(CH3)2 - R = Si-O-(CH3)2 - R • Change surface polarity • Amino (CH2)3NH2 • Cyano (CH2)3C#N • Diol (CH2)2OCH2CH(OH)CH2OH • Octadecyl (CH2)17CH3 • Octyl (CH2) 7CH3 • Phenyl (CH2)3C6H5
Elution • Competition for the surface. • Which substance has the greater surface affinity – a solvent with a higher affinity will push the analytes along.
Isocratic and Gradient Elution • Isocratic - same composition • Gradient - composition changes over time • Method Development. • A mixture of compounds. Separated with acetonitrile (B) and aqueous buffer (A) • 1) benzyl alcohol 2) phenol 3) 3’,4’-dimethyloxyacetophenone 4) benzoin 5) ethyl benzoate 6) toluene 7) 2,6-dimethoxytoluene 8) o-methyoxybiphenyl
Too Long - Over Two Hours! • We can do a gradient. Examine when we get the best separations and then change composition over time.
Practical Issues • Solvents • Must be very pure, lack UV absorbing species • Should be filtered • Guard columns should be used to protect column from strong absorbing compounds • Solvents should be degassed – bubbles and oxygen (sparging) • Normal phase solvents should be 50% saturated with water • Gradients in reversed phase can require 10 – 20 column volumes to return to starting conditions • Add 3% 1-propanol to each solvent and you will cut this to 1.5 empty volumes
Reducing Waste Solvent(Save some money – be a hero) • Shorter columns with smaller particles • Switch from 4.6 mm to 3.0/2.0 mm id columns • In isocratic systems – use an electronic recycler.
Quality Assurance • Inject a QC sample each day to insure that you have consistent peak shapes and retention times. • Keep a log of your column performance
Symmetric Band-shape • Asymmetry Factor A/B should rarely be worse than 0.9 to 1.5. • Tailing a bigger issue and fronting generally. • Amines interacting with support active sites. • Add 30 mM TEA • Acidic compounds • Add 30 mM Ammonium Acetate • Or for mixes or unknowns – 30 mM triethylammonium acetate. • Persistent problem – add dimethyloctylamine or dimethyloctylammonium acetate These take a long time to wash on on changing mobile phases.
Other issues • Voids can develop at the column inlet. • Repack with fresh stationary phase to get rid of this problem. (Might want to get new column) • Columns should be washed to get rid of salts and strongly adsorbed compounds • Frits should be cleaned. Back wash or replace • Samples should be dissolved in mobile phase or a weaker solvent.
Overloading • Care should be taken not to overload the injection on the column. • Inject 10x less and see if peaks look any better. • Reversed Phase can deal with 1 to 10 mg sample per gram of silica. (About 10 cm on a 0.46 mm column) • Injection volume • < 15% of the peak volume at baseline
Minimize Dead Volume • Minimize connection tubing • Ensure that fittings are proper matches.
Injection and Detection • Pumps • Piston Type under program control. Up to 400 bar (40 MPa or 6000 psi). Gradients made by proportioning valves.
Injection • Sample loop filling either done manually or by and autoinjector
Evaporative Light Scattering • Solutes less volatile than mobile phase • Light scattered from mass of analyte. • Poor linearity – polynomial calibration • Good with gradients. No solvent front. • Same buffers as used with mass spec. Acetic acid, formic acid and TFA, ammonium acetate, diammonium phosphate, ammonia or TEA.
Electrochemical • Analytes that can be oxidized or reduced • Phenols, aromatic amines, peroxides, mercaptans, ketones, aldehydes etc. • Can be very sensitive but are difficult to work with.