1. LIQUID CHROMATOGRAPHY
CHAPTER 22

2. Liquid Chromatography
Chromatography with a liquid mobile phase, works very well with liquid samples (especially food testing, environmental samples, and biotechnology applications)
Can be both qualitative (retention time or volume) or quantitative (peak area or peak volume—standards)
LC chromatograms must show good resolution (difference in retention) and be based on a column set up with good efficiency (narrow peaks).

3. LC: Components

4. LC: Analyte Requirements
Must be able to place analyte into a liquid that can be injected into the column
Since it is usually possible to find some solvent for the analyte, LC has become very popular (especially for analytes that cause problems in GC, such as polymers and biological compounds).
Since volatilization is not required, LC can be run at lower temperatures (less likely to disrupt analyte).
Analyte interaction will be with both the stationary and mobile liquid phases (better resolution than GC with more flexibility in controlling retention times).

5. LC: Column Efficiency
In the 1960s, LC improved and became known as HPLC (High Performance Liquid Chromatography— narrow peaks and lower limits of detection).
The use of smaller support particles requires higher pressure to push the mobile phase (pumps exerting from a few hundred to a few thousand lb/in2).
HPLC tends to have a lower sample capacity and is more expensive than traditional LC.
Note the structure of the particles used in HPLC. Also note the trend toward smaller, more efficient particles.

6. LC: Particles in the Column
A smaller support size helps create faster mass transfer, smaller HETP, larger plate numbers, and narrower peaks.

7. LC: Particles in the Column
In a traditional porous particle the mobile phase flows around the particle while the analyte flows through it via slower diffusion, causing some band broadening.
Perfusion particles allow both to flow though, as do particles with a porous coating.

8. LC: Role of the Mobile Phase
Strong Mobile Phase: a solvent or solution that quickly elutes analyte (analyte is more soluble in the mobile phase)
Weak Mobile Phase: a liquid that slowly elutes analyte (analyte has better solubility in the stationary phase)
Isocratic Elution: using constant composition in the mobile phase (recall the “general elution problem”)
Solvent Programming: Begin with a weak mobile phase (A), then switch over time to stronger mobile phase (B).

9. Five Mechanical LC Techniques

10. Adsorption Chromatography
Separation based on adsorption on a
support (also called liquid-solid
chromatography, LSC)
Retention of an analyte depends on the binding strength of the analyte (A) to the support and on the surface area of the support.
The retention factor also depends on how much mobile phase is displaced by A and the relative amount of mobile phase that is displaced by A.
There is competition for binding sites on the support between (A) and the mobile phase (M).

11. Adsorption Chromatography
Elutropic Strength (eo): how strongly a solvent or mixture adsorbs to the surface of a support. A large value indicates strong absorbance to the support. This prevents the solute from binding to the support and, instead, encourages travel with
the liquid mobile phase.

12. Adsorption Chromatography: Stationary and Mobile Phases
Supports can be polar (SiO2; Al2O3), which strongly interact with polar compounds, or nonpolar organics (i.e., hexane). Mixtures can give non-linear changes— so use caution, and use pure solvents.

13. Adsorption Chromatography: Stationary and Mobile Phases
Isocratic elution of tocopherols in corn oil using 0.3% isopropanol in isooctane:

14. Liquid Partition Chromatography
Solutes are separated as they partition between a liquid mobile phase and a stationary phase that is coated (usually chemically bonded) on a support.
Normal Phase (NPLC): the stationary phase is polar, forms H-bonds and other dipole interactions with the solute, and retains polar compounds. The mobile phase most likely nonpolar.
Reverse Phase (RPLC): the stationary phase is nonpolar (i.e., alkanes); the mobile phase is polar. This is widely applied in common aqueous systems, drugs, and pharmaceuticals.

15. Liquid Partition Chromatography: Stationary Phases

16. Liquid Partition Chromatography: Mobile Phases
Solute distribution:
Related to retention factor:
Solvent Polarity (P) is rated based on polarity. Solvents can be mixed to achieve specific polarity (j = vol)

17. Liquid Partition Chromatography: Bonding Stationary Phase to Support
Bind organosilane (that contains desired side-group) with silica support.
If covering is not complete, use additional smaller organosilane to “cap” those still exposed silica areas to prevent mixed-mode interactions with solutes.

18. Liquid Partition Chromatography: Most Common Type of LC
AIDS drug study

19. Liquid Partition Chromatography
A low polarity solvent (weak mobile phase) will have a low value for (P(tot)) for NPLC.
A low polarity solvent (weak mobile phase for RPLC) will have a high value for P(tot).
For analytes that are weak acids (or bases), the pH of the mobile phase should be considered.
In RPLC, the neutral form of acid (HA) and base (B) will be more soluble in a low polarity solvent. The relative amounts of those forms are pH dependent.

20. Ion-Exchange Chromatography
Solutes are separated by adsorption onto a support containing fixed charges on its surface.
Home water softener is an example.
Other applications include separations of inorganic ions, amino acids, nucleic acids, organic ions

21. Ion-Exchange Chromatography
A charged analyte competes with a bound ion on the support for the ion-exchange site. This is described by the selectivity coefficient, KA,C
Larger KA,C describes higher retention.
Affected by:
The nature and accessibility of the ion-exchange groups
The type and concentration of analytes
The nature and concentration of competing ions in the mobile phase

22. Ion-Exchange Chromatography: Stationary and Mobile phases
Cation Exchanger: the stationary phase has a negatively charged group used to separate cations in mixture.
Anion Exchanger: the stationary phase has a positively charged group used to separate anions in mixture.
Support for Cation or Anion Exchange: Chromatography is often modified silica (where surface now has charged groups).
Properly treated Polystyrenes may also be a support for IC. These are referred to as resins.
Naturally occurring carbohydrates may be modified (functional groups added These supports are “gels.”

23. Ion-Exchange Chromatography – Stationary and mobile phases
Polystyrene modified with divinylbenzene affects pore size and rigidity:
Agarose support systems have -OH groups, creating hydrophillic forces. Also, the large pore size works when separating proteins

24. Ion-Exchange Chromatography – Stationary and Mobile Phases

25. Ion-Exchange Chromatography: Stationary and mobile phases
A strong mobile phase usually has a high concentration of competing ions. This means the analyte will not have as many binding sites, so its not retained as much.
Changing the competing ion concentration will then change anion retention times.
If analytes are weak acids (or weak bases), the pH of the mobile phase can be used to change the HA/A– ratio.
Adding a complexing agent to the mobile phase can also change the charge on analytes. (Fe3+ analyte can be changed to FeCl4–)

26. Ion-Exchange Chromatography: Applications
Applications include de-ionizing H2O by replacing cations in water by H+ and replacing anions with OH–, purifying proteins, nucleotides, and inorganics in food and the environment.
Ionic chromatography (IC) uses a low number of charged sites for the stationary phase—this requires a lower concentration of competing ions.
This method (IC) often uses a second column, which replaces ions of high conductivity with ones with low conductivity (suppressor ion chromatography.)

27. Ion-Exchange Chromatography: Suppresser IC
F–, Cl–, and Br– example: A mixture of these anions is applied to a low-capacity anion exchange column; this is eluted with solvent containing OH–.
As analytes elute from the first column they enter the suppresser column. All cations are replaced by H+, which reacts with competing OH–  H2O.
The F–, Cl–, and Br– retain high conductivity (as competing ion concentration decreases).
See Figure for a visual explanation.

28. Ion-Exchange Chromatography: Suppresser IC

29. Size-Exclusion Chromatography
SEC is a form of liquid chromatography where separation is based on analyte particle size.
No true stationary phase is present. Rather, solutes travel (in the mobile phase) through various sized pores within the support.
The retention volume (VR) will be between the excluded volume (the mobile phase volume outside the pores, VE) and the true void volume (VM).
Solutes will, ideally, elute at or before void volume, so the retention factor (k) is not used in this method.

30. Size-Exclusion Chromatography
Use retention time or retention volume to calculate Ko.
This shows the fraction between VM and VE in which solute elutes. Smaller molecules will have Ko values approaching (or equaling) 1. Larger molecules will have Ko values approaching or equaling) 0.

31. Size-Exclusion Chromatography
Ideally, the porous support does not interact with the analytes. (polyacrylamide gel, others)
The range of pore size determines separation.
Polar or nonpolar mobile phases may be selected depending on analyte solubility.
Aqueous mobile phase is gel filtration chromatography.
Organic mobile phase is gel permeation chromatography.

32. Size-Exclusion Chromatography
SEC can be a preparative technique for biological samples when it is necessary to remove smaller molecules from larger proteins.
It can be used to remove salts from a sample.
It can separate biomolecules and polymers.
When it’s used to determine molecular weights, a series of known standards are employed (standards cover the range from totally included from pores [eluting at VM] to totally excluded [VE]).
Exercise 22.3 shows an example for molar mass

33. Affinity Chromatography
The AC method is based on biological interactions. These are selective, reversible interactions such as substrate–enzyme, antibody–antigen, and hormone–receptor.
One of the partners is immobilized on the support and placed into a column (affinity ligand) used in on/off format.
The sample is applied to the column (with a buffer), and the affinity ligand binds analyte selectively. After non-retained analytes wash through, a separate buffer is applied that releases analyte.
The column and affinity ligand are regenerated.

34. Affinity Chromatography
Complexation reaction:
KA association constant:
mL refers to moles of active ligand sites VM is void volume.
Note Exercise 22.4.

35. Affinity Chromatography
Many of the affinity ligands have large KA due to forces (dipoles, H-bonding, etc.).
Specificity ligands bind specifically to one type of analyte.
General ligands bond to a family of ligands.
Supports can be carbo gels or silica (when converted into diol-bonded phase with low specificity).

36. Affinity Chromatography
The affinity ligand can be attached to the support via “immobilization” that uses carboxyl, amine, or sulfhydryl groups on the ligand.
A “spacer arm” between the ligand and the support may be needed to avoid steric hindrance possibilities.
An application buffer is a weak mobile phase (that allows strong binding between analyte and ligand) used in application, washing, and regeneration.
A strong mobile phase is a solvent that can readily remove analyte from ligand, and is called an elution buffer.

37. Affinity Chromatography: Applications
Large scale purification for proteins and enzymes
Preparation of sample by isolation of cellular proteins
Direct analysis of complex biological systems (boronate affinity column to measure glycated hemoglobin)
Use with HPLC to measure hormones, proteins, drugs, herbicides
Difficult separations in chiral mixtures can be done with affinity chromatography with a stationary chiral phase—See Box 22.2.

38. Liquid Chromatography Detectors
Detection methods such as Refractive Index, Absorbance, Fluorescence, Conductivity, Electrochemical methods, and Mass Spec. are available

39. Liquid Chromatography Detectors
General Detector: Absorbance uses UV or Vis in a flow-through cell that allows mobile and analytes to flow through the detector continuously.
An example of this is the simple, fixed-wavelength absorbance detector. (Often 254 nm is set because mercury vapor lamps have intense emission at that l. Many aromatics or unsaturated compounds absorb light in this range.)
A variable wavelength detector monitors l in the nm range using an advanced monchromator.
A photodiode array simultaneously monitors many wavelengths.

40. Liquid Chromatography Detectors
The Refractive Index changes in the mobile phase—analyte mixture can be used in another general detector.
Note that there are
two flow cells
One with reference
Difference in RI of materials in the two cells causes the light beam to bend
RI works well for compounds without chromophores and analytes that may be unknown.
Disadvantage: not having low limits of detection

41. Liquid Chromatography Detectors
Evaporative Light-scattering Detector (ELSD): a general type of detector used with solutes that are less volatile than mobile phase.
The mobile phase leaving the column is converted to a spray of small droplets. As they evaporate, solute is left behind.
Solutes then pass through a beam of light; scattering relates to measurement. There is low background signal and a better limit of detection—good for lipids and carbohydrates.

42. Liquid Chromatography Detectors
Fluorescence measurements can be very specific for analytes eluting from the column. Specific excitation and emission is selective but can be limited.
Conductivity measurements work for ionic compounds in the mobile phase.
A flow cell with electrodes measures current This works well with ion chromatography (and gradient elutions).

43. Liquid Chromatography Detectors
Electrochemical methods are used to measure the oxidation or reduction of analytes by measuring change in current at a constant voltage, or by measuring voltage at constant current. Since electrical measurements can be very accurate, the analyte measurements can also be accurate.

44. Liquid Chromatography Detectors
Liquid chromatography/mass spectrometry (LCMS) can be both qualitative and quantitative.
Examining all ion fragments (full scan mode) allows general analyte identification.
Examining a few ion fragments of a known pattern (LCMS) is used as a selective detector.
LCMS works well for proteins producing low-mass to-charge ratios.

45. Liquid Chromatography Detectors
In electrospray ionization (ESI), droplets form (with excess + or – charge) from a spray containing analyte. Then when coulombic forces exceed the cohesive force, the droplets divide (coulombic explosion), releasing ions into the gas phase. See Figure 22.22: two AIDS drugs.

46. LC Equipment and Sample Pretreatment
Samples are typically introduced via injection valve.
The sample is applied into a loop; excess sample passes through the loop and then into waste.
A switch places the sample into the mobile phase and into the column.

47. LC Equipment and Sample Pretreatment
HPLC requires high pressure for the mobile phase.
Pressure can be generated by a reciprocating pump (rotating cam/piston mL/min range). Or, for lower pressure, a motorized syringe pump is used (mL/min).

48. LC Equipment and Sample Pretreatment
HPLC columns typically are 10 or 25 cm in length with an inner diameter of 4.1 to 4.6 mm.
Using longer, narrower columns provides a larger number of plates with more efficient separations but can handle only small samples.
Some samples are derivatized to enhance detection.