Introduction to Analytical Separations

Introduction

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

 Introduction to Analytical Separations
Introduction

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 Analysis of Various Pesticides
after treatment with a Drug 2D Gel Electrophoresis of total
in Ground water using LC-MS
protein extract from E. coli cells

Journal of Chromatography A, 1109 (2006) 222–227

Toxicological Sciences (2000) 57:326-337 Electrophoresis (1997) 18:1259-1313
 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

Immiscible
[ S ]2
K
liquids

[ S ]1

- The partitioning of solute s between two chemical phases (1 and 2) is

described by the equilibrium constant K

K is called the partition coefficient

 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

V1
q
V1  KV2 
where: q = fraction of moles of S remaining in phase 1
V1 = volume of phase 1
V2 = volume of phase 2
K = partition coefficient

 The fraction of S remaining in phase 1 after n extractions is

n
 V1 
qn   
 V1  KV2  
Assumes V2 is constant
 Introduction to Analytical Separations
Extractions

2.) Extraction Efficiency

 Illustration

Ether layer

Water layer

1M UO2(NO3)2
(yellow)

After mixing, UO2(NO3)2 After 8 extractions, UO2(NO3)2

Is distributed in both layers has been removed from water
 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) 333:816-818

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.
 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):

n 1
 V1   100 mL 
qn       0.062  6.2%

 1V  KV 
2   100 mL  ( 3 )  ( 500 mL ) 

The fraction of S extracted (p) is simply:

p  1  q  1  0.062  0.938  93.8%

 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):
n 5
 V1   100 mL 
qn        0.00098  0.98%
 V1  KV2   100 mL  ( 3 )  ( 100 mL ) 

The fraction of S extracted (p) is:

p  1  q  1  0.00098  0.99902  99.902%

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
 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)

Total Concentration of A in Phase 2

D
Total Concentration of A in Phase 1
 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:

Total Concentration of Base in Phase 2

D
Total Concentration of Base in Phase 1
0
K BH   0

[ BH ]1

[ B ]2
D
[ B ]1  [ BH  ]1
 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:

[ B ]2 [ B ]2 [ H  ][ B ]
D KB  Ka  K w Kb

[ B ]1  [ BH ]1 [ B ]1 [ BH  ]
(partition coefficient) (equilibrium constant)

K B Ka
D D is directly related to [H+]

Ka  [ H ]
 Introduction to Analytical Separations
Extractions

5.) pH Effects in Extractions

 A similar expression can be written for a weak acid (HA)

K HA [ H  ] [ HA ]2
D where: K HA 
Ka  [ H  ] [ HA ]1

 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)

 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
 Introduction to Analytical Separations
Extractions

7.) Extractions with a Metal Chelator

 Metal ions may be separated from one another by using various organic
complexing agents.
- Soluble in organic solvent

Insoluble in Soluble in
organic solvent organic solvent
 Introduction to Analytical Separations
Extractions

7.) Extractions with a Metal Chelator

 Common complexing agents

OH

- N
O NH N N

N HN

O N S
cupferron 8-hydroxyquinoline dithizone

Crown ethers
 Introduction to Analytical Separations
Extractions

7.) Extractions with a Metal Chelator

 Many of the complexing agents bind to a variety of metals
- Different strengths or equilibrium constants

 A metal ion extraction may be made selective for a particular metal by:
- Choosing a complexing agent a high affinity to the metal (small K)
- Adjusting the pH of the extraction
Cu+2 is completely
extracted at pH 5 while Zn2+
remains in aqueous phase

pH selectivity of dithizone
metal ion extraction
 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)

 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
 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
 Introduction to Analytical Separations
Chromatography

3.) Chromatogram
 Chromatogram: graph showing the detector response as a function of elution
time.

Retention time
Non-retained solute
(void volume)

 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

The strength or degree with which a molecule is retained on the column can
be measured using retention time or retention volume.
 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

t'r  t r  t m
where: tm = minimum possible time for a non-retained
solute to pass through the column

 Relative Retention (a): ratio of adjusted retention time between two solutes

t'r 2
a
t'r1
where: tr2’ > tr1’ , so a > 1

- Greater the relative retention the greater the separation between two
components
 Introduction to Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor (k’):

t t
k'  r m
tm

 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:

time solute spends in stationary phase

k' 
time solute spends in mobile phase
 Introduction to Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor is equivalent to:

time solute spends in stationary phase moles of solute in stationary phase

k'  
time solute spends in mobile phase moles of solute in mobile phase

C sVs
k' 
C mVm

where: Cs = concentration of solute in the stationary phase

Cm = concentration of solute in the mobile phase
Vs = volume of the stationary phase
Vm = volume of the mobile phase
 Introduction to Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor is equivalent to:

C sVs
k' 
C mVm

Cs
Under equilibrium K (partition coefficient)
conditions Cm

Vs
k'  K
Vm

Capacity factor is directly proportional to partition coefficient

 Similar relationship for relative retention:

t'r 2 k'2 K2
a  
t'r1 k1' K1
 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.
 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)
 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)
 Introduction to Analytical Separations
Chromatography

5.) Efficiency of Separation

 Resolution (Rs) is defined as:

( t r2  t r1 )
Rs 
( wb2  wb1 ) / 2

where: tr2,tr1 = retention times of solutes 1 and 2 (tr2 > tr1)

wb2,wb1 = baseline widths of solutes 1 and 2
Or

Rs 
N
  1
4
where: N = number of theoretical plates
 = t2/t1 (>1)

 Want Rs ≥ 1.5 for complete separation

 Rs ≥ 1.0 usually adequate for analysis
 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

2
 t 
2  t 
N  16  r   5.55  r 
 w1 
 wb   2 
where: wb = baseline width of peak (in time units)
w1/2=half-height peak width
 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)

H  L/ N
where: L = length of column
N = number of theoretical plates

 A H decreases, more separation steps per column length are possible

- Results in a narrower peak width and better separation between two
neighboring solutes
 Introduction to Analytical Separations
Chromatography

6.) Measure of Column Efficiency

 H is affected by:
i. Flow-rate of mobile phase
ii. Size of support: decrease size decrease H
iii. Diffusion of solute: increase diffusion  decrease H
iv. Strength of retention
v. Others

Improved resolution by
increasing column length
 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
 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
 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
 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
 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

Molecules enter the
different times due to different
column at the same time
path lengths
 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

B
Van Deemter H  A  C x
equation x

Multiple paths Longitudinal equilibration

diffusion time

where: x = linear flow rate

A,B,C = constants for a given column and
stationary phase
 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
 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
 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
 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)
 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
 Introduction to Analytical Separations
Chromatography

9.) Types of Liquid Chromatography

 Packed and Open Tubular Columns

 Open tubular columns:

- higher resolution, increased sensitivity, but small sample capacity
- higher flow rates, longer columns more theoretical plates and resolution
- No band spreading from multiple pahts