Separations - see text for chapters on each topic

1. Solvent Extraction

2. What is Chromatography

3. Efficiency of Separation

4. Why Bands Spread

5. Electrophoretic Separations (gel and capillary)

6. Electrochromatography

Separations – two-phase separation: partitioning

Liquid Solid Gas GLC GSC distillation sublimation vapor phase chrom. (VPC) Liquid L-L extraction liquid chrom. (e.g., HPLC) Hg electrochem deposition Solid precipitation adsorption chrom. electrodeposition zone refining (melting)

Single phase separation: electrophoresis; ultracentrifugation ; diffusion; mass spectrometry; excited state reactions

Solvent extraction

21

1

1

1

2

11

22

1

2

:dunextractefraction where

/

/)1(

/

/

KVV

Vq

m

mq

Vqm

Vmq

Vm

Vm

S

SK

Partition coefficients and undissociated speciesPartition coefficient:

i phase of volume:

totalmoles :

i phasein moles :

i

i

V

m

m

Multiple extractions

n

n

KVV

Vq

21

1

For multiple extraction with each extraction using same volume of V, : the fraction unextracted after n extractions

For “infinite number of extractions (i.e., the limit):

2

1

)( V

VK

eq

Dependence of multiple extractions on Particitition Coefficient

(V1=V2)

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100 120

Number of extractionsFr

actio

n un

extra

cted

K=1

K=0.2

K=0.1

K=0.05

K=0.01

n=99%95%90%82%37%

e.g. Consider Kp=2 and single vs 5

extractions (V1 = 100 mL; V2(total) = 1 L)

Single: q = 100/[100+(K*1,000) = 0.048 (4.8% unextracted or 95.2% extracted)Multiple: q=[100/(100+(K*200)]5 = 0.00032 ( 0.032% unextr. or 99.998% ext.)

][

1 phasein ion concentrat total

2 phasein ion concentrat totalt coefficienon Distributi

HK

KKD

KHBBH

D

a

a

a

Extraction of acid/base species

B +H+

B

BH+

Extraction of metal chelates

Counter current extraction (equilibrium-based partitioning)

1 2 3

1 2 3 1 2 3

1 2 31 2 3

1 2 3

Calculations after many extractions

1 2 3

r: tube #

fraction present in the r th tube after n extractions

nr

qqrnr

nf rnr

rn

where

)1()!(!

!,

For large values of n:

stationary

mobile

Counter current vs. “chromatography”

Chromatography

Separating Molecules

Science of Chromatography

Separation (relative speeds of molecules) depends on Polarity of solvent Polarity of substrate Other molecular properties (b.p., chirality, etc.)

Like moves fastest with like Polar molecules move fastest with polar solvents

Chromatography

Two phases• Stationary phase

Solid or liquid Spatially fixed throughout experiment

• Mobile phase Liquid or gas In motion relative to the stationary phase

Chromatography is classified as to type of mobile phase and stationary phase • LC (liquid mobile) . . . . liquid or solid stationary• GC (gas mobile) . . . . usually liquid stationary

Types of chromatography

adsorption partition ion-exchange molecular exclusion (size exclusion) affinity chiral separations

Subtypes of Chromatography

Paper chromatography Thin layer chromatography (TLC) Gas chromatography (GC) High-performance liquid chromatography

(HPLC) And several others

Liquid Chromatography

Paper Chromatography

Spottingsample(pencil marknear spot)

Developingchamber

Solventfront

Original spot(pencil line)

Final position ofsolvent front (pencil line)

Visualizing Spots

May be visible If not, try

• UV light• I2

• Mark spots with pencil when visualized

Lichen extract

Retardation Factor, Rf

R Ad

dfA

S

( )

Rf is relatively imprecise Cut out spots, extract solute and run further tests

to complete identification In paper chromatography,

• The mobile phase is a• The stationary phase is a• The chromatographic classification is LSC

liquid

solid?

?

?

Cellulose

Absorbed water in thepolar regions is actually the stationary phase

Thus, paper chromatography’s actual classificationis LLC

Thin Layer Chromatography (TLC)

Stationary layer is a thin layer of a solid bonded to an inert plastic or glass binder• Silica (SiO2)

• Alumina (Al2O3)

• Binder is often plaster of Paris

Silica

Surface O’s converted into O-H’sSmall particles mean very enormous surface areaAlumina works similarly

TLC is an example of LSC TLC is exactly like paper chromatography in terms

of the actual procedure Rf values are more stable than on paper In forensics, TLC is routinely used for identifying

and/or individualizing• Inks• Dyes• Drugs

Thin Layer Chromatography

What components

are in the unknown

from the case?

Thin Layer Chromatography

Identifying

gasoline

by separation

of dye

additives in

the fuel

High Performance Liquid Chromatography (HPLC)

Directly analogous to GC• Column is relatively short (10-30 cm), with inside

diameter of 4-10 mm• Column is very tightly packed• LSC

Packed with microparticles (3-10 m) of silica or alumina

• LLC Packed with microparticles coated with a liquid

Liquid solvents (up to four) replacecarrier gas in GC experiment

Column is at roomtemperature

Liquid pressure isenormous (in excessof 6000 psi = 45 bar)

Even at enormous pressurethe flow rate is 0.1-10 mL/min

Detector is double-beam UV-Visspectrometer, constantlyscanning a selected range of ’s

Can also hook-up the columndirectly to a MS to give LC-MS

HPLC Data

HPLC – use of solvent gradients

Ion Chromatography

Ion Chromatography

Size Exclusion Chromatography

Gas Chromatography

Gas Chromatography (GC)

Column • Contains stationary phase• Long coil (2-3 m) of tubing• Relatively narrow internal diameter (2-4 mm) • Glass or metal

Stationary phase• May be solid (GSC)

Silica, alumina• May be liquid at operating temperatures (GLC)

Squalane, C30H62

Dimethyl silicone oil, (CH3)3-Si-O-[Si(CH3)2-O]n-Si(CH3)3

Coated onto inert beads or granules

Equilibration between Phases

Molecules divide up between those in mobile phase and those in the stationary phase

Rapid equilibrium established Avoid column overloading

The ratio in each phase depends on Temperature Polarities of column material, mobile phase, and

molecules “other” factors

Gas Chromatograms

Complex mixtures can have many peaks

Broad peaks (later) High backgrounds can

come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)

Identification of species: retention times

Capacity factor: k’= (tr

- tm) / tm

t solute is in stationary phase = ---------------------------------------- t solute is in mobile phase

Qualitative Analysis

Retention times, tR

• Difficult to develop data base because of the variety of factors which alter tR

Nature of stationary and mobile phases Length and diameter of column Flow rate of carrier gas Temperature, etc.

Compare original chromatogram to a known chromatogram Use GC to separate components, then identify them in some

other fashion (IR, MS, etc.)• Detector must be nondestructive

Band broadening and resolution

van Deempter equation

'

'2

2

2

1Resolution

16

avg

r

k

kN

H

L

w

tN

• plates ( ‘theoretical plates’)• plate height, H• Height equivalent of a theoretical plate, HETP• number of plates• resolution

Improved resolution with larger N, longer retention on column (i.e., larger k’)

Band Broadening Description –assumes Gaussian peaks

Pressure reducers

Flow rate valve

Mobile phase iscalled “carrier gas”(He, Ar, N2)

Column housed in oven

User selectstemperature

Must be sufficient to volatilize allcomponents

Lower temperatures lead to better resolution but longer retention times

GC Improvements

Capillary Columns• Fused silica• 10-60 m long• 0.1-0.3 mm internal

diameter• Coated with a liquid

(GLC) Temperature

programming

Types of columns

Packed high capacity common in many types

of chromatography adsorption, LC (e.g., HPLC), affinity, frontal

Open tubular less capacity better resolution (less

band broadening) preferred approach for

most GC applications

Flame Ionization Detector (FID)

2 H2 + O2 → 2 H2O

Proceeds withoutionic intermediates

Cathode-

Amp

Flame Ionization Detector (FID)

CH4 + 2 O2 → CO2 + 2 H2O

Proceeds by forming a variety of ionic intermediates

Cathode-

Amp

Gas Chromatograms

Complex mixtures can have many peaks

Broad peaks (later) High backgrounds can

come from molecules that decompose as they move through the column or from column degradation etc. (column bleed)

Temperature Isothermal runs can suffer from:

Very broad peaks after long tR at low temps.

Poorly Resolved peaks at short retention times at high temps.

Use programmed temperature ramp Start at low temp

Allows resolution of early eluting cmpds.

Ramp to high temp Prevents band broadening of

late eluting cmpds.

Quantitative Analysis

The area under each peak is proportional to the percent (by mass) of the analyte in the original sample

A1 : A2 : A3 : … = %1 : %2 : %3 : …• Get areas by

Numerical or electronic integrator Does not account for different detector response to different

molecules

Alternatively, prepare a calibration curve for each analyte • Area versus concentration

GC-MS

GC is excellent for separating mixtures MS is excellent for qualitative analysis Make the MS the detector for the GC

• Always use a capillary column• No “regular” detector at the end• The column is plumbed directly into the MS ion source

subsystem

Experiment starts whensample is injected into GC

At that instant, start the MSrecording spectra sequentially

Experiment starts whensample is injected into GC

It takes about 0.6 s to record a MS.Thus, about 100 spectra are recorded (and stored) every minute

It may take 15-45 min forall the components to elutefrom the GC column

Thus the MS may record andstore 1500 – 4500 spectra duringthe lifetime of the experiment

Most of these are blanks (only carrier gas coming through)

It may take 15-45 min forall the components to elutefrom the GC column

When an analyte componentappears, its band may take 10 sto completely elute

During that time, perhaps 17 orso spectra are recorded. The peakswill get more and more intense,and then will wane

Quantitative Analysis For each chromatographic peak

• Calculate the Total Ion Count (TIC)• Plot TIC versus time

Has the appearance

of a regular GC Perform the same

sorts of quantitative

analysis as you

would do on any

other chromatogram

Qualitative Analysis w/ MS

Sort through the 1500 – 4500 spectra and pick out the ones that aren’t blank

Of the sets of non-blanks, pick out the most intense from each set

Identify the component based on the MS fragmentation patterns (MS is covered in a separate section of the course)

Problem Samples

Substances with enormous molecular masses (e.g.: polymers)• Not volatile• Not soluble

Much forensic evidence falls in this category• Paint• Grease• Tars• Fibers• Plastics

Pyrolysis GC (PGC)

The analyte is pyrolyzed (heated to 500 – 1,000 oC in the absence of oxygen)• Giant molecules fragment (often into radicals)

Take GC of the fragments (pyrogram) Will not identify the analyte, but can be used to

individualize it• Compare complex pyrograms of known and questioned

samples

Pyrolysis GC Data (with FIDetector)