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Intro to HPLC

Intro to HPLC. Modes of HPLC. Reversed Phase Normal Phase Ion Pair Ion Exchange Size Exclusion. Modes of HPLC. Reversed Phase. Most commonly used HPLC mode. Excellent for water soluble compounds. Can analyze neutral cmpds, weak acids and bases. Good for MW less than 2000 amu

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Intro to HPLC

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  1. Intro to HPLC

  2. Modes of HPLC • Reversed Phase • Normal Phase • Ion Pair • Ion Exchange • Size Exclusion

  3. Modes of HPLC

  4. Reversed Phase • Most commonly used HPLC mode. • Excellent for water soluble compounds. • Can analyze neutral cmpds, weak acids and bases. • Good for MW less than 2000 amu • Proteins, peptides

  5. Reversed Phase • Typical runs are isocratic, same mobile phase composition throughout the run. • Gradient runs are also used, where the mobile phase composition varies (becomes stronger) through the run.

  6. Reversed Phase • Stationary phase is less polar than the mobile phase. • Polar solutes are eluted earlier than nonpolar solutes. • Typical columns have a silica support to which the stationary phase is covalently attached. • Polymer stationary phases may be used for extremely high or low pH values.

  7. Reversed Phase Columns • C-18 (octadecylsilyl) • C-8 (octasilyl) • C-4 (butyl) • Phenyl • Cyano • Amino

  8. Reversed Phase Columns • For neutral compounds, water and MeOH or acetonitrile are the most common mobile phases. • The exact %-composition would of course be optimized to get the best resolution in the shortest time. • MeOH and acetonitrile will not necessarily give the same elution order for solutes or of course the same retention times.

  9. Reversed Phase Columns • For weak acids, if just water and MeOH or acetonitrile are used, the peak shape will be extremely ugly, with a double peak. • The retention time may also be too short. • Therefore, a buffer to modify the pH is used: glacial acetic acid at a low pH (at least 2 pH units below the pKa) is commonly used. • At a low pH, the acid is in the free acid form (protonated form) instead of the deprotonated form.

  10. Reversed Phase Columns • For weak bases, a similar problem occurs. • The base will be attracted to the silanol groups on the stationary phase and thus give weird retention times and ugly peaks. • So a base modifier like TEA (triethylamine) is added. • TEA acts as a competitor for the silanol groups, lowering the probability that your base is attracted. • So better peaks.

  11. Reversed Phase Columns • For a mixture of solutes, can we predict the elution order? (at least roughly?) • Predict based on: • polarity or water solubility • degrees of unsaturation • branching • numbers of carbon atoms

  12. Elution Order: Polarity • Clearly the more polar the cmpd, the earlier it elutes. • General order from most polar to least: • carboxy acids • alcohols and phenols • amines, aldehydes, and ketones • hydrocarbons

  13. Elution Order: D. of Unsat. • Unsaturated cmpds containing pi bonds are more acidic than saturated cmpds. • So the more unsaturated the cmpd, the earlier it will elute. • It would still typically elute after amines.

  14. Elution Order: Branching • Hydrocarbon branching decreases retention time. • Due to steric hindrance, they would have less interactions with the stationary phase.

  15. Elution Order: Number of Carbons • The more carbon atoms, the more nonpolar a molecule becomes in general. • Thus the retention time increases for increasing C atoms.

  16. Elution Order for Reversed Phase • Here is the general guidelines for predicting elution order: • ions come out with void volume • strong or moderately strong acids • weak acids • weak bases • weakly polar (ketones, aldehydes, then unsaturated) • nonpolar

  17. Reversed Phase Mobile Phase • Although MeOH and acetonitrile are the most common, THF and IPA are also used. • What do you want in the organic portion of the mobile phase? • water soluble • low viscosity which means low pressure • low UV detection or interference • solutes are soluble in it • unreactive

  18. Reversed Phase Mobile Phase • THF is unstable with time • IPA is very viscous • MeOH is more viscous and has a higher UV-cutoff than Acetonitrile • MeOH cutoff for UV interference is 210 nm • Acetonitrile is 190 nm • Acetonitrile is preferred choice (except right now due to global shortage)

  19. Reversed Phase Mobile Phase • What is the order of the “strength” of the typical solvents for the mobile phase? • Stronger solvents will give shorter retention times. • Strength from weakest: • water/buffers • water • MeOH • acetonitrile • IPA • THF

  20. Reversed Phase Column • You know that there are different stationary phases available for columns. • Columns also have different lengths, particle sizes of the stationary phase, pore sizes, and column diameter (and other differences such as endcapping to deactivate the silanol groups). • These differences lead to different retention times of solutes.

  21. Reversed Phase Column • The longer the column, the longer the retention time. But it can be good for method development to use a longer column until you know what is in your mixture. • Smaller diameter columns mean shorter retention times. • Smaller particle sizes mean shorter retention times. • But pressure can be an issue with the shorter and smaller columns: use lower flow rates.

  22. Ion Pair HPLC: Reversed Phase • Using Reverse phase HPLC, strong acids and bases can be separated. • This subset of reverse phase is called ion pair chromatography. • An ion-pair reagent is added to the mobile phase. • This reagent has a hydrophobic tail and a cation or anion group elsewhere.

  23. Ion Pair HPLC: Reversed Phase • To separate bases, an ion-pair reagent that has an anion group is used. • Common: • alkyl sulfonates (-SO3- ending) • TFA (trifluoroacetic acid) • other moderately strong or strong alkyl acids)

  24. Ion Pair HPLC: Reversed Phase • To separate acids, an ion-pair reagent that has an cation group is used. • Common: • tetramethylammonium phosphate (so quarternary amine) • quarternary alkyl amines (phosphate or acetate) • triethylammonium acetate • TEA

  25. Ion Pair HPLC: Reversed Phase • By adding an ion-pair reagent, the hydrophobic tail interacts with the stationary phase, leaving the ionic ending pointing out into the mobile phase. • Thus, very polar solutes can interact with the charged ion-pair and the retention time is increased.

  26. Ion Exchange HPLC and IC • Ion exchange is used to separate out organic and inorganic ions. • The stationary phase is a polymer or silica surface with charged groups bonded to the surface. • The charged groups may be cations or anions. • Typical charged groups include: • sulfonates (-) • carboxymethyl (-) • quarternary amines (+) • diethylaminoethyl (+)

  27. Ion Exchange HPLC and IC • The mobile phase contains counter ions and buffers and organic modifiers. • The counter ions in the mobile phase compete with the solute ions in the sample for interactions with the ionic endings on the stationary phase. • The more counter ions present, the less able the solute ions are able to interact with the stationary phase ions. • So retention time depends on the concentration of the counter ions.

  28. Ion Exchange HPLC and IC • Ion exchange is useful for the analysis of wines, fruit drinks, other foods, etc. • IC is used to analyze trace ions like halides, nitrates, phosphates, sulfates, and metal ions.

  29. Normal Phase HPLC • Normal phase HPLC was the first mode of HPLC developed. • It is adsorption chromatography. • Now, the mobile phase is organic. • The stationary phase is more polar than the mobile phase. • The more nonpolar the cmpd, the shorter the retention time.

  30. Normal Phase HPLC • It is useful to separate: • water insoluble cmpds • structural isomers • moderately polar to nonpolar cmpds

  31. Normal Phase HPLC • In the column, the polar end groups on the stationary phase interact with the solute molecules. • Again, the solvent can compete with solute molecules. • The solvent or solute particles are adsorbed on the surface via H-bonding, dipole-dipole forces, etc. • The stronger the solute interacts, the longer the retention time.

  32. Normal Phase HPLC Elution Order • It is basically backwards of reverse phase. • The more polar functional groups on the solute, the longer the retention time.

  33. Normal Phase HPLC Mobile Phase • Fluoroalkanes are “weakest” solvent (long RT) • n-pentane (common) • n-hexane (common) • chlorobutane • benzene • xylene • chloroform • toluene (common) • methylene chloride • ethyl acetate • THF • Acetonitrile • MeOH

  34. Size Exclusion HPLC • Size exclusion is used for high MW cmpds • > 10000 amu • It is also called gel filtration (GFC) or gel permeation chromatography (GPC) depending on the mobile phase and stationary phase. • GFC uses silica columns and aqueous buffer mobile phases. It is used for biochemical applications. • GPC uses polymer stationary phases and organic mobile phases (like THF) to separate large MW organic cmpds, like polymers. • The separation is based on size of the solute.

  35. Size Exclusion HPLC • Whatever, the stationary phase, it contains pores of a very defined size. • Solutes which are too big to enter the pores are eluted first. • Solutes that kind of fit but get kind of stuck, elute next. • Solutes that are small enough to enter the pores and take a picnic break or take the scenic route down the column, elute last! • Different pore size columns are available for different MW separations.

  36. Back to Reverse Phase HPLC! • Let’s now go back and discuss the resolution between peaks. • Of course, this applies also to the other HPLC modes, with some exceptions and modifications. • Remember that we want a R of at least 1.5 to separate peaks sufficiently with no overlap, but we also want reasonable overall analysis times. • You learned how to calculate R between two peaks on a chromatogram. • But there is another equation.

  37. Resolution of Solutes in Reverse Phase • There is a “master” equation for resolution:

  38. Resolution of Solutes • What parameters are important in the resolution between peaks? • Based on the previous equation, there are 3 major parameters: • Efficiency (related to the theoretical plates) • Capacity (retention factor) • Selectivity

  39. Capacity, k’ • This is also called the retention factor. • It is both the interaction of the solute with the mobile and the stationary phases. • So it is about the equilibration of a solute particle partitioning between the two phases.

  40. Capacity, k’ • It is related to the retention time. • Mathematically: • The longer the RT, the higher the capacity factor. • You want it to between 1 to 5 (best for overall resolution) or between 2 and 10 for complicated mixtures.

  41. Capacity, k’ • The best way to change the capacity is to change the mobile phase and temperature. • The higher the %-water in the mobile phase, the “weaker” it is, and the longer the retention times. • Increasing the organic content of the mobile phase weakens the interactions of the solute with the stationary phase (nonpolar) and lowers the RT and k’. • Rule of thumb: a 10% increase in O-content decreases k’ by a factor of 2 to 3. • As increasing T tends to decrease RT, k’ would also decrease.

  42. Efficiency • Efficiency is related to N, the theoretical number of plates in the column. • It is related to the width of a peak. • Narrower peaks have a higher efficiency. • So for a higher efficiency, we need to lower the tendency of peaks to spread in a column. • We want to increase N, the number of plates.

  43. Efficiency • What is N mathematically? • N is ideally 5000 to 25000.

  44. Efficiency • Longer columns have more stationary phase and so have more theoretical plates, N. • They would give longer RT’s so would raise N in the equation. • Smaller particle sizes (3-5 microns) lower diffusion effects and so would increase N.

  45. Selectivity,  • It is how a stationary phase differentiates between two different solute particles and separates them. • So a higher selectivity means that the stationary phase can separate two solutes. • A selectivity of 1 is no separation. • It should be at least 1.2

  46. Selectivity,  • What is it mathematically?

  47. Selectivity,  • So what affects the selectivity? • Clearly the type of stationary phase is important. • So changing the type of column changes the selectivity.

  48. Selectivity,  • However, you can also change the selectivity by changes in the mobile phase and temperature. • A different mobile phase can change the strength of the solute’s interactions with the stationary phase.

  49. Selectivity,  • You can change: • the %-composition of the mobile phase • organic solvent in mobile phase • buffer pH • additives like TEA

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