Introduction to Analytical Separations Introduction 1.)Sample Purity Many chemical analysis are not...

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

Electrophoresis (1997) 18:1259-1313

Introduction to Analytical Separations

2D Gel Electrophoresis of total protein extract from E. coli cells

Toxicological Sciences (2000) 57:326-337

NMR Spectra of Mouse Urine after treatment with a Drug

Introduction

3.) Illustration Biological Samples are Composed of Complex Mixtures

- Analysis of composition and changes help in understanding disease and the development of treatments

Analysis of Various Pesticides in Ground water using LC-MS

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

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


1

2

]S[

]S[K

K is called the partition coefficient

Immiscible liquids

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

21

1

KVV

Vq

where: q = fraction of moles of S remaining in phase 1V1= volume of phase 1V2 = volume of phase 2K = partition coefficient

n

n KVV

Vq

21

1 Assumes V2 is constant


2.) Extraction Efficiency Illustration

Ether layer

Water layer

1M UO2(NO3)2 (yellow)

After mixing, UO2(NO3)2

Is distributed in both layersAfter 8 extractions, UO2(NO3)2

has been removed from water


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.


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):

%..)mL()(mL

mL

KVV

Vq

n

n 2606205003100

1001

21

1

The fraction of S extracted (p) is simply:

%...qp 8939380062011


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):

%..)mL()(mL

mL

KVV

Vq

n

n 9800009801003100

1005

21

1

The fraction of S extracted (p) is:

%...qp 9029999902000098011

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


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)

1

2

PhaseinAofionConcentratTotal

PhaseinAofionConcentratTotalD


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:

1

2

PhaseinBaseofionConcentratTotal

PhaseinBaseofionConcentratTotalD

11

2

]BH[]B[

]B[D

00

1

]BH[

KBH


5.) pH Effects in Extractions The distribution of a weak base or weak acid is pH dependent

11

2

]BH[]B[

]B[D

1

2

]B[

]B[KB

]BH[

]B][H[KKK bwa

Substitute definition of KB and Ka into D:

]H[K

KKD

a

aB

(partition coefficient) (equilibrium constant)

D is directly related to [H+]


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)

]H[K

]H[KD

a

HA

1

2

]HA[

]HA[KHA where:


6.) ExampleButanoic 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


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 organic solvent

Soluble in organic solvent


7.) Extractions with a Metal Chelator Common complexing agents

Crown ethers

O N

N

-O

cupferron

N

OH

8-hydroxyquinolineS

HN

NH N N

dithizone


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

pH selectivity of dithizone metal ion extraction

Cu+2 is completely extracted at pH 5 while Zn2+ remains in aqueous phase

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)


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


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


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

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

Non-retained solute (void volume)

Retention time


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 (): ratio of adjusted retention time between two solutes

- Greater the relative retention the greater the separation between two components

mr'r ttt

where: tm = minimum possible time for a non-retained solute to pass through the column

'r

'r

t

t

1

2

where: tr2’ > tr1’ , so > 1


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:

m

mr

t

tt'k

phasemobileinspendssolutetime

phasestationaryinspendssolutetime'k


4.) Fundamental Measures of Solute Retention Capacity factor is equivalent to:

phasemobileinsoluteofmoles

phasestationaryinsoluteofmoles

phasemobileinspendssolutetime

phasestationaryinspendssolutetime'k

mm

ss

VC

VC'k

where: Cs = concentration of solute in the stationary phaseCm = concentration of solute in the mobile phaseVs = volume of the stationary phaseVm = volume of the mobile phase


4.) Fundamental Measures of Solute Retention Capacity factor is equivalent to:

Similar relationship for relative retention:

mm

ss

VC

VC'k

m

s

C

CK Under equilibrium

conditions(partition coefficient)

m

s

V

VK'k

Capacity factor is directly proportional to partition coefficient

1

2

1

2

1

2

K

K

k

k

t

t'

'

'r

'r


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.


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)


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)


5.) Efficiency of Separation Resolution (Rs) is defined as:

Or

Want Rs ≥ 1.5 for complete separation Rs ≥ 1.0 usually adequate for analysis

212

12

/)ww(

)tt(R

bb

rrs

where: tr2,tr1 = retention times of solutes 1 and 2 (tr2 > tr1)wb2,wb1 = baseline widths of solutes 1 and 2

14

NRs

where: N = number of theoretical plates = t2/t1 (>1)


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

22

21

55516

w

t.

w

tN r

b

r

where: wb = baseline width of peak (in time units)w1/2=half-height peak width


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)

A H decreases, more separation steps per column length are possible- Results in a narrower peak width and better separation between two

neighboring solutes

N/LH where: L = length of column

N = number of theoretical plates

H


6.) Measure of Column Efficiency H is affected by:

i. Flow-rate of mobile phaseii. Size of support: decrease size decrease Hiii. Diffusion of solute: increase diffusion decrease Hiv. Strength of retentionv. Others

Improved resolution by increasing column length


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


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 ()

Factors include:- Sample injection- Longitudinal diffusion- Finite equilibration between phases- Multiple flow paths- others


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


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


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 enter the column at the same time

Molecules exit the column at different times due to different path lengths


8.) Description of Band Spread Plate height (H) is proportional to band width

- The smaller the plate height, the narrower the band

xx

CB

AH

where: x = linear flow rateA,B,C = constants for a given column and stationary phase

Multiple paths Longitudinal diffusion

equilibrationtime

Van Deemter equation


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


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


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

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)


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



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

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Introduction to Analytical Separations Introduction 1.)Sample Purity Many chemical analysis are not...

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