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Chapter 25

HPLC

High-Performance Liquid Chromatography

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Typical HPLC

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ApplicationMicrodialysis

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Monitor Aspirin in Blood

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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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How Does the Smaller Size Help

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Smaller is Better

• N ~ 3500 L (cm) / dp (m)

• Smaller particle size leads to – Higher plate number– Higher pressure– Shorter optimum run time

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Size Considerations

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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.

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HPLC Columns

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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

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Active SitesGenerally Bad

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Active Sites Can Lead to Tailing

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We Can Modify the Surface

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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

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New TechnologyMonolithic Silica Columns

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Micrographs

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Elution

• Competition for the surface.

• Which substance has the greater surface affinity – a solvent with a higher affinity will push the analytes along.

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The Eluotropic Series

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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

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Too Long - Over Two Hours!

• We can do a gradient. Examine when we get the best separations and then change composition over time.

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Separation Design

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Separation Design

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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

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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.

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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

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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 acetateThese take a long time to wash on on changing mobile phases.

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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.

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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

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Minimize Dead Volume

• Minimize connection tubing

• Ensure that fittings are proper matches.

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Injection and Detection

• Pumps – Piston Type under program control. Up to 400

bar (40 MPa or 6000 psi). Gradients made by proportioning valves.

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Injection

• Sample loop filling either done manually or by and autoinjector

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DetectorsMass or Concentration Types

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UV- Vis (Photometric)Variable Wavelength Detector

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Photodiode ArrayAll wavelengths at once

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Refractive IndexUniversal Detection with major issues.

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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.

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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.

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Method Development

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Eluent Strength Nomogram

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Gradient Separations

• Same purpose as temperature or pressure programming in GC. Speed up analyses.

• Increase the eluent strength as the run progresses. That is increase the amount of organic phase as the run progresses in reversed phase methods.

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Dwell Time

• Time it takes for the composition change at the pump to reach the head of the column.

• Important on method transfer

• Can be determined by running a gradient of 0.1% acetone (detect at 260 nm) without a column. Start gradient at time 0 and see when the acetone starts to be absorbed.

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Since gradients require a post equilibrium time then perhaps isocratic will work better.

• What is the span of all the peaks. That time span is t

– If t/tg > 0.25 then use a gradient. If less you can make an isocratic system. (See next slide)

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Optimize Gradient

• Run a broad gradient. (5% to 100% over 40 to 60 min)

• Eliminate gradient before first peak and after last peak. Run over same time.

• Cut time if above works well to save time.

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