1. Introduction to Analytical Separations
1.) Sample Purity
Many chemical analysis are not specific for one compound
Actually respond to many potential interferences in the sample
Often it is necessary to first purify the compound of interest
Remove interfering substances before a selective analysis is possible
This requires a separation step.
2.) Techniques available for Chemical Separations:
Extraction
Distillation
Precipitation
Chromatography
Many others (centrifugation, filtration, etc)
Extractions and Chromatography are especially useful in analytical methods

2. Introduction to Analytical Separations
3.) Illustration
Biological Samples are Composed of Complex Mixtures
Analysis of composition and changes help in understanding disease and the development of treatments
NMR Spectra of Mouse Urine after treatment with a Drug
Analysis of Various Pesticides in Ground water using LC-MS
2D Gel Electrophoresis of total protein extract from E. coli cells
Journal of Chromatography A, 1109 (2006) 222–227
Toxicological Sciences (2000) 57:
Electrophoresis (1997) 18:

3. Introduction to Analytical Separations
Extractions
1.) Definition
The transfer of a compound from one chemical phase to another
The two phases used can be liquid-liquid, liquid-solid, gas-solid, etc
Liquid-liquid is the most common type of extraction
The partitioning of solute s between two chemical phases (1 and 2) is described by the equilibrium constant K
Immiscible liquids
K is called the partition coefficient

4. Introduction to Analytical Separations
Extractions
2.) Extraction Efficiency
The fraction of moles of S remaining in phase 1 after one extraction can be determined
The value of K and the volumes of phases 1 and 2 need to be known
The fraction of S remaining in phase 1 after n extractions is
where: q = fraction of moles of S remaining in phase 1
V1 = volume of phase 1
V2 = volume of phase 2
K = partition coefficient
Assumes V2 is constant

5. Is distributed in both layers
Introduction to Analytical Separations
Extractions
2.) Extraction Efficiency
Illustration
Ether layer
Water layer
1M UO2(NO3)2
(yellow)
After mixing, UO2(NO3)2
Is distributed in both layers
After 8 extractions, UO2(NO3)2 has been removed from water

6. Introduction to Analytical Separations
Extractions
3.) What happens as n approaches infinity?
Eventually the amount of S remaining in phase 1 becomes zero
Solution is infinitely diluted
This Situation Created a Strange Saga in Science – Water Memory
a founding principal of homeopathic medicine
the claim is that water remembers the activity of the drug after it has been removed
Nature (1988) 333:
Authors’ claim to still observe antibody activity even after a 1x10120 fold dilution.
Less than 1 molecule is present with a 1x1014 dilution
A number of subsequent studies have disputed the claim but the controversy is still popular in the press and as alternative medicine, even though the results are consistent with the placebo effect.

7. Introduction to Analytical Separations
Extractions
4.) Example #1:
Solute A has a K = 3 for an extraction between water (phase 1) and benzene (phase 2).
If 100 mL of a 0.01M solution of A in water is extracted one time with 500 mL benzene, what fraction will be extracted?
Solution:
First determine fraction not extracted (fraction still in phase 1, q):
The fraction of S extracted (p) is simply:

8. Introduction to Analytical Separations
Extractions
4.) Example #2:
For the same example, what fraction will be extracted if 5 extractions with 100 mL benzene each are used (instead of one 500 mL extraction)?
Solution:
Determine fraction not extracted (fraction still in phase 1, q):
The fraction of S extracted (p) is:
Note: For the same total volume of benzene (500 mL), more A is extracted if several small portions of benzene are used rather than one large portion

9. Introduction to Analytical Separations
Extractions
5.) pH Effects in Extractions
For weak acids (HA) and Bases (B)
Protonated and non-protonated forms usually have different partition coefficients (K)
Charged form (A- or BH+) will not be extracted
Neutral form (HA or B) will be extracted
Partitioning is Described in Terms of the Total Amount of a Substance
Individual concentrations of B & BH+ or HA & A- are more difficult to determine
Partitioning is regardless of the form in both phases
Described by the distribution coefficient (D)

10. Introduction to Analytical Separations
Extractions
5.) pH Effects in Extractions
The distribution of a weak base or weak acid is pH dependent
For a weak base (B) where BH+ only exists in phase 1:

11. Introduction to Analytical Separations
Extractions
5.) pH Effects in Extractions
The distribution of a weak base or weak acid is pH dependent
Substitute definition of KB and Ka into D:
(partition coefficient)
(equilibrium constant)
D is directly related to [H+]

12. Introduction to Analytical Separations
Extractions
5.) pH Effects in Extractions
A similar expression can be written for a weak acid (HA)
The ability to change the distribution ratio of a weak acid or weak base with pH is useful in selecting conditions that will extract some compounds but not others.
Use low pH to extract HA but not BH+ (weak acid extractions)
Use high pH to extract B but not A- (weak base extractions)
where:

13. Introduction to Analytical Separations
Extractions
6.) Example
Butanoic acid has a partition coefficient of 3.0 (favoring benzene) when distributed between water and benzene. Find the formal concentration of butanoic acid in each phase when 100 mL of 0.10 M aqueous butanoic acid is extracted with 25 mL of benzene at pH 4.00 and pH 10.00

14. Introduction to Analytical Separations
Chromatography
1.) Definition
A separation technique based on the different rates of travel of solutes through a system composed of two phases
A stationary phase
A mobile phase
Detect compounds emerging in column by changes in absorbance, voltage, current, etc
Chromatogram (not spectrum)

15. Introduction to Analytical Separations
Chromatography
2.) System Components and Process
Stationary Phase: the chemical phase which remains in the column (chromatographic system)
Mobile Phase (eluent): the chemical phase which travels through the column
Support: a solid onto which the stationary phase is chemically attached or coated
Solute are separated in chromatography by their different interactions with the stationary phase and mobile phase

16. Introduction to Analytical Separations
Chromatography
2.) System Components and Process
Solutes which interact more strongly with the stationary phase take longer to pass through the column
Strongly Retained
Weakly Retained
Solutes which only weakly interact with the stationary phase or have no interactions with it elute very quickly

17. Introduction to Analytical Separations
Chromatography
3.) Chromatogram
Chromatogram: graph showing the detector response as a function of elution time.
Retention time (tr): the time it takes a compound to pass through a column
Retention volume (Vr): volume of mobile phase needed to push solute through the column
Retention time
Non-retained solute (void volume)
The strength or degree with which a molecule is retained on the column can be measured using retention time or retention volume.

18. Introduction to Analytical Separations
Chromatography
4.) Fundamental Measures of Solute Retention
Adjusted retention time (tr’): the additional time required for a solute to travel through a column beyond the time required for non-retained solute
Relative Retention (a): ratio of adjusted retention time between two solutes
Greater the relative retention the greater the separation between two components
where: tm = minimum possible time for a non-retained solute to pass through the column
where: tr2’ > tr1’ , so a > 1

19. Introduction to Analytical Separations
Chromatography
4.) Fundamental Measures of Solute Retention
Capacity factor (k’):
The longer a component is retained by the column, the greater the capacity factor
Capacity factor of a standard can be used to monitor performance of a column
Capacity factor is equivalent to:
where: Vs = volume of the stationary phase
Vm = volume of the mobile phase
K = partition coefficient
Capacity factor is directly proportional to partition coefficient

20. Introduction to Analytical Separations
Chromatography
4.) Fundamental Measures of Solute Retention
Example:
The retention volume of a solute is 76.2 mL for a column with Vm = 16.6 mL and Vs = 12.7 mL. Calculate the capacity factor and the partition coefficient for this solute.

21. Introduction to Analytical Separations
Chromatography
5.) Efficiency of Separation
The width of a solute peak is important in determining how well one solute is separated from another
One measure of this is the width of the peak at half-height (w½ ) or at its baseline (wb)

22. Introduction to Analytical Separations
Chromatography
5.) Efficiency of Separation
The separation of two solutes in chromatography depends both on the width of the peaks and their degree of retention
The separation between the two solutes is given by their Resolution (Rs)

23. Introduction to Analytical Separations
Chromatography
5.) Efficiency of Separation
Resolution (Rs) is defined as: Or
Want Rs ≥ 1.5 for complete separation
Rs ≥ 1.0 usually adequate for analysis
where: tr2,tr1 = retention times of solutes 1 and 2 (tr2 > tr1)
wb2,wb1 = baseline widths of solutes 1 and 2
where: N = number of theoretical plates
g = t2/t1 (g>1)

24. Introduction to Analytical Separations
Chromatography
6.) Measure of Column Efficiency
Number of Theoretical Plates (N)
Similar to number of extractions performed in an extraction separation
As N increase (number of separating steps)  greater the separation between two compounds
where: wb = baseline width of peak (in time units)
w1/2=half-height peak width

25. Introduction to Analytical Separations
Chromatography
6.) Measure of Column Efficiency
Height Equivalent of a Theoretical Plate (H or HETP)
The distance along the column that corresponds to one “theoretical” separation step or plate (N)
As H decreases, more separation steps per column length are possible
Results in a narrower peak width and better separation between two neighboring solutes
where: L = length of column
N = number of theoretical plates H

26. Introduction to Analytical Separations
Chromatography
6.) Measure of Column Efficiency
H is affected by:
Flow-rate of mobile phase
Size of support: decrease size decrease H
Diffusion of solute: increase diffusion  decrease H
Strength of retention
Others
Improved resolution by increasing column length

27. Introduction to Analytical Separations
Chromatography
6.) Measure of Column Efficiency
Example:
Two compounds with partition coefficients of 15 and 18 are to be separated on a column with Vm/Vs = 3.0 and tm = 1.0 min. Calculate the number of theoretical plates needed to produce a resolution of 1.5

28. Introduction to Analytical Separations
Chromatography
7.) Why Bands Spread?
Remember: Efficiency is dependent on peak width
A band of solute spreads as it travels through the column
described by a standard deviation (s)
Factors include:
Sample injection
Longitudinal diffusion
Finite equilibration between phases
Multiple flow paths
others

29. Introduction to Analytical Separations
Chromatography
7.) Why Bands Spread?
Sample injection – sample is injected on the column width a finite width, which contributes to the overall broadening
Similar broadening may occur in the detector
Longitudinal diffusion – band slowly broadens
as molecules diffuse from high concentration
in band to regions of lower concentration

30. Introduction to Analytical Separations
Chromatography
7.) Why Bands Spread?
Finite Equilibration Time Between Phases – a finite time is required to equilibrate between stationary and mobile phase at each plate
Some solute is “stuck” in stationary phase as remainder moves forward in mobile phase
Results in band broadening
Distribution of solute between mobile and stationary phase
Solute in mobile phase moves down column  broader peaks

31. Introduction to Analytical Separations
Chromatography
7.) Why Bands Spread?
Multiple Flow Paths – As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances.
Molecules exit the column at different times due to different path lengths
Molecules enter the column at the same time

32. Introduction to Analytical Separations
Chromatography
8.) Description of Band Spread
Plate height (H) is proportional to band width
The smaller the plate height, the narrower the band
Van Deemter equation
Multiple paths
equilibration
time
Longitudinal
diffusion
where: mx = linear flow rate
A,B,C = constants for a given column and stationary phase

33. Introduction to Analytical Separations
Chromatography
9.) Types of Liquid Chromatography
Adsorption Chromatography
Solutes are separated based on their different abilities to adsorb to the support’s surface
Uses an underivatized solid support (stationary phase = solid support)
Oldest type of chromatography, but not commonly used

34. Introduction to Analytical Separations
Chromatography
9.) Types of Liquid Chromatography
Partition Chromatography
Solutes are separated based on their different abilities to partition between the stationary phase and mobile phase.
Uses a solid support coated or chemically derivatized with a polar or non-polar layer
Most common type of liquid chromatography at present. Good for most organic compounds
Reversed Phase: stationary phase is non-polar
Normal Phase: stationary phase is polar

35. Introduction to Analytical Separations
Chromatography
9.) Types of Liquid Chromatography
Ion-Exchange Chromatography
Used to separate ions based on their different abilities to interact with the fixed exchange sites.
Uses a solid support containing fixed charges (exchange sites) on its surface
Cation-Exchange: support with negative groups
Anion-Exchange: support with positive groups

36. Introduction to Analytical Separations
Chromatography
9.) Types of Liquid Chromatography
Size Exclusion Chromatography
Separates large and small solute based on their different abilities to enter the pores of the support
Uses a porous support that does not adsorb solutes
Commonly used to separate biological molecules or polymers which differ by size (MW)

37. Introduction to Analytical Separations
Chromatography
9.) Types of Liquid Chromatography
Affinity Chromatography
Separates molecules based on their different abilities to bind to the affinity ligand
Uses a support that contains an immobilized biological molecule (affinity ligand)
Commonly used to purify and analyze biological molecules
Most Selective type of Chromatography