principles of chromatography. chromatography is the most powerful tool for separating &...

Principles of Chromatography

Chromatography is the most powerful tool for separating & measuring the components of a complex mixture.

Quantitative & qualitative analysis

What is Chromatography?

1) Solvent Extraction :

transfer of a solute from phase 1 phase 2

S (in phase1) S (in phase 2)

partition coefficient

1

2

s

sK

2) Chromatography : same as extractiona) One phase: held in place stationary phase. solid material (packing material)

Another phase : fluid phase mobile phase. sample gas (GC) liquid (LC)

What is Chromatography?

What is Chromatography?

b) A solute equilibrates between a mobile and a stationary phase.The more it interacts with the stationary phase, the slower it is moved along a column.

Xm Xs

Ks = [X]s / [X]mSolutes with a large Ks value will be retained more strongly by the stationary phase.

What is Chromatography?

c) The science & art of separation

d) Originator : adsorption chromatography by M.Tswett in 1903

e) Eluent, eluate, elution.

What is Chromatography?

elution : always (100%) dilution

What is Chromatography?

sam plein

eluentin

CaCO 3

(adsorption)

colum n

eluantout

detector

chrom atogram(m ass spect. IR

spect. etc)

3) Types of Chromatography

Is divided into categories on the basis of the mechanism of interaction of the solute v.s. the stationary phase.

What is Chromatography?

polar s.p.

What is Chromatography?

for GC & LC for GC

21.1 What is Chromatography?

resin-SO3- gel filtration

resin-N(CH3)3+ by size

What is Chromatography?

Ask Yourself 20-A p.461pH, and ionic strength

the most selective one

How do we describe a chromatogram

1) Chromatogram :

A graph showing the detectors

response as a function of elution

time : band’s shapes, position,

resolution.

2) For individual band :

a) Retention time (tr) :the time needed after injection for an individual solute to reach detector.

b) An ideal chromatographic peak Gaussian shape. w½ = 2.35σ, w = 4σ

How do we describe a chromatogram

How do we describe a chromatogram

How do we describe a chromatogram

3) For pairs of bands

a) Efficiency : two factors contribute to how well components are separated :

the widths of the peaks :

the wider the peak, the poorer separation.

the spacing in time :

the further apart, the better separation.

How do we describe a chromatogram

b) Theoretical plates (N): (from distillation)the more plates on a column, the more

equilibration steps, and the better the

separation.

Number of plates on column :

N = 5.55(tr/w½)2

Plate height : H = L/N

The smaller plate height

narrower peaks better separation

How do we describe a chromatogram

c) Resolution (Rs)

How do we describe a chromatogram

Rs2 s.p. theoflength 2

1.5Rs analysis, vequantitatiFor

Lw

0.589t

ww21

tt

w

ΔtRs

1/2av

r

21

rr

av

r 12

Qualitative: • Co-chromatography• Detector:

– Mass spectrometer– IR, UV-VIS spectrophotometer

d) Qualitative & Quantitative analysis

How do we describe a chromatogram

• Figure illustrates the point that computers and humans may not choose the same baseline for measuring area.

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Qualitative and Quantitative Qualitative and Quantitative AnalysisAnalysis

Internal Standards

• An internal standard is known amount of a compound, different from analyte, that is added to an unknown.

• To use an internal standard, we prepare a known mixture of standard and analyte and measure the relative response of the detector to the two species. In Figure 5-6, the area under each peak is proportional to the concentration of each compound injected into the column.

P.119

• [X] and [S] are the concentrations of analyte and standard after they have been mixed together.

P.119

ExampleExample ： ： Using an Internal StandardUsing an Internal Standard• In a chromatography experiment, a solution containing

0.083 7 M X and 0.066 6 M S gave peak areas of Ax=423 and AS=347.

• To analyze the unknown, 10.0 mL of 0.146 M S were added to 10.0 mL of unknown, and the mixture was diluted to 25.0 mL in a volumetric flask.

• This mixture gave the chromatogram in Figure 5-6, with peak areas Ax=533 and AS=582. Find the concentration of X in the unknown.

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SOLUTIONSOLUTION ：：

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Because X was diluted from 10.0 to 25.0 mL when the mixture with S was prepared, the original concentration of X in the unknown was (25.0/10.0)(0.057 21 M)=0.143 M.

Why do bands spread ?

1) Why broadening?a) diffusionb) slow equilibration of solute between the m.p and s.p.c) irregular flow paths.

Why do bands spread ?

2) Longitudinal diffusion :

the faster the flow

the less a band spends in column.

the less time for diffusion.

broadeningu

1

Why do bands spread ?

3) solute requires time to equilibrate between phases.

(s.p.m.p.) with temp. broadening u

Can’t equilibrate rapidly enough.

m.p.

s.p.

Why do bands spread ?

Solute requires a finite time to equilibrate between the mobile and stationary phases.

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4) A Separation Has an Optimum Flow Rate4) A Separation Has an Optimum Flow Rate

• The rate of mass transfer between phases increases with temperature.

Optimum resolution (minimum plate height) occurs at an intermediate flow rate. Curves show measured plate height in gaschromatography of n-C17H36 at 175°C, using N2, He, or H2 mobile phase.

Why do bands spread ?

Why do bands spread ?

5) Multiple paths

Band spreading from multiple flow paths. The smaller the stationary-phase particles, the less serious is this problem. This process is absent in an open tubular column.

Why do bands spread ?

6) Plate height equation

Plate height equation

Why do bands spread ?

Why do bands spread ?

7) open tubular columns

Packed column (A, B, C 0 in van Deemter’s eqn.)

Open tubular column (A = 0 in van Deemter’s eqn.)

resolution (∵ H & column length) sample capacity (∵ less s.p.)

Why do bands spread ? 8) Funny shapes

polarsolute

OH OH

SiSi S i S iOO

OSi(CH 3)3(CH3)3SiO

s.p. silanization

Mass Spectrometry

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Mass Spectrometry• Mass spectrometry measures the masses and

abundances of ions in the gas phase.

A Mass SpectrometerA Mass Spectrometer

• Figure next page shows a transmission quadrupole mass spectrometer, which is the most common mass separator in use today.

• The mass separator consists of four parallel metal rods to which a constant voltage and a radio-frequency oscillating voltage are applied.

Transmission quadrupole mass spectrometer.

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Figure 21-13

• Ionization: 1) Electron ionization

2) Chemical ionization

Mass Spectrometry

1) Electron ionization

M + e- M+ + e- + e-

70 eV -55 eV 0.1eV

Molecular ion break into fragments.

Base peak: most intense peak.

2) Chemical ionization

CH4 + e- CH4+ + 2e-

CH4+ + CH4 CH5

+ + CH3

CH5+ + M CH4 + MH+

CH4+ CH3

+ + H

CH3+ + CH4 C2H5

+ + H2

• Total ion Chromatograms is a reconstructed total ion chromatogram showing all ions from seven opium alkaloids found in street heroin.

• Selected ion Chromatograms:– Simplify analysis – improve S/N

P.473

Information in a Mass Spectrum

Nominal Mass : CNominal Mass : C44HH99Br is 136 Br is 136

Information in a mass spectrum

Rxn : CH3(CH2)2CH2–OH + Br- CH3(CH2)2CH2–Br

1–Butanol 1–Bromobutane

CH3 15

CH2 14

Br 79

C4H979Br+ 50.0%

C4H981Br+

Information in a mass spectrumFragmentation Patterns

Information in a mass spectrumIsotope PatternsCnHxOyNz

12C/13C

Intensity = n x 1.1%

Ex: C6H6

(M+1)/M+ = 6 x 1.1 %

Nitrogen Rule: A compound: odd nominal mass / odd number of N atoms;

even nominal mass/ even number of N atoms