ANALYTICAL CHEMISTRY CHEM 3811

CHAPTER 21

DR. AUGUSTINE OFORI AGYEMANAssistant professor of chemistryDepartment of natural sciences

Clayton state university

CHAPTER 21

CHROMATOGRAPHYAND

MASS SPECTROMETRY

CHROMATOGRAPHY

- The most powerful tool for separating mixtures

- Used for both qualitative and quantitative analysis

CHROMATOGRAPHY

Comprises of Two Phases

Stationary Phase- A solid or liquid packed in a column (does not move)

Mobile Phase- A gas or liquid that passes through the column

CHROMATOGRAPHY

- A column is packed with the stationary phase

- Mobile phase passes through the stationary phase

- Separation process involves the interaction of themobile phase (a mixture) with the stationary phase

CHROMATOGRAPHY

A

B

eluent

eluate

CHROMATOGRAPHY

Adsorption- Occurs when a solute sticks to the surface of another species

- Consider a mixture containing solutes A and B

- A is more strongly adsorbed to the stationary phase than B

- A moves down the column more slowly than B

- B comes out of column before A

CHROMATOGRAPHY

Elution- The process of passing a liquid or gas through a column

Eluent- Fluid entering the column

Eluate- Fluid exiting the column

CHROMATOGRAPHY

Gas Chromatography (GC)

- Mobile phase is a gas

Liquid Chromatograpgy

- Mobile phase is a liquid

CHROMATOGRAPHY

Solutes may be retarded by the stationary phase based on various interactions

- Surface adsorption- Relative solubility

- Charge

Chromatography is classified based on the type of interactions

CHROMATOGRAPHY

Adsorption Chromatography

- Stationary phase is a solid

- Mobile phase is a liquid or a gas

- Solute adsorbs to the surface of the solid particles

CHROMATOGRAPHY

Partition Chromatography

- Stationary phase is a thin liquid coated on the surfaceof a solid support

- Mobile phase is a liquid or a gas

- Solute equilibrates between the stationary and mobile phases

CHROMATOGRAPHY

Ion-exchange Chromatography

- Allows separation of ions and polar molecules

- Ionic groups are covalently attached to a stationary solid phase

- Mobile phase is a liquid

- Ionic solutes are electrostatically attracted to the stationary phase

CHROMATOGRAPHY

Size Exclusion Chromatography(Gel Filtration, Gel Permeation)

- Solutes are separated based on size

- Stationary phase has small pores that exclude large molecules

- Small molecules enter the pores so spend more time in column

- Large molecules come out of column before small molecules

CHROMATOGRAPHY

Affinity Chromatography

- Very selective

- Based on specific interactions between a type of solute moleculeand another molecule covalently attached to the stationary phase

THE CHROMATOGRAM

- Detector response as a function of time or elution volume

- Different peaks correspond to different eluates

Retention Time (tr)- Time taken by a solute to reach detector after injection

THE CHROMATOGRAM

tr

tr

h

1/2h

w1/2 = 2.35σ

w = 4σ

Det

ecto

r re

spon

se

Time

THE CHROMATOGRAM

- An ideal chromatogram has a Gaussian shape

- h = height of peak

- σ = the standard deviation of the peak

- w = base width = 4σ

- w1/2 = width at half height (w at 1/2h) = 2.35σ

- tr and w can be measured in time or volume units

THEORETICAL PLATES

- Imaginary way to picture the separation process

- Imaginary discrete sections of the chromatography column

- Though the process is continuous

- Retention of solutes can be described by the number of equilibrium steps (theoretical plates)

21/2

2r

w

5.55tN

THEORETICAL PLATES

The number of theoretical plates on a column (N)

The Plate Height (H)

- The length of one plate

H = L/N

L = the length of column

THEORETICAL PLATES

- The higher the N the narrower the bandwidth

- The higher the N better the separation

- The smaller the H the narrower the peaks

- The smaller the H the better the separation

To Test a Column for Degradation

- Inject standards periodically

- Look forPeak asymmetry

Change in number of plates

THEORETICAL PLATES

- Peak separation (Δtr) divided by the average peak width (wav)

- Better resolution implies more complete separationbetween neighboring peaks

RESOLUTION

1/2(av)

r

av

r

w

t0.589

w

ΔtResolution

- Doubling the length of a column (2L) increases resolution by √2

QUALITATIVE ANALYSIS

- Identify peaks by comparing retention times to those of authentic samples

- Unknown sample is “spiked” (authentic sample is added)

- The relative size of a peak will increase if the authentic sample is identical to one of the components

- Different compounds may have the same retention time

- It is more likely for different compounds to have differentretention times on different stationary phases

QUANTATIVE ANALYSIS

- Chromatographic peak area is proportional to quantity of solute

- A good measure of solute concentration is obtained byusing internal standards

- Internal standards eliminate the effect of variable conditions

- Conditions mostly vary from run to run

QUANTATIVE ANALYSIS

Conditions Include

- Sample injection errors or changes

- Column changes

- Detector variations

QUANTATIVE ANALYSIS

Internal Standard Method

- Concentration of analyte (canalyte) can be determined using the concentration of internal standard (cIS) and both peak areas

analyte

IS

analyte

IS

Area

Area

c

c

SCALING UP

Analytical Chromatography

- For small-scale analysis

Preparative Chromatography

- For large-scale analysis

SCALING UP

- A developed procedure for analytical chromatography can be scaled up and used for preparative chromatography

- Maintain column length and increase cross-sectional area

- Volume flow rate should also be increased by the same factor

2

radiuscolumnsmall

radiuscolumnlarge

(g)loadsmall

(g)loadlarge

BAND BROADENING

BAND BROADENING

May be due to

Diffusion- Diffusion of solute molecules away from the center

of the band in both directions

- Longitudinal diffusion

- The faster the flow rate the sharper the peaks

- Broadening is inversely proportion to flow rate

BAND BROADENING

May be due to

Solute Equilibration

- If solute equilibrates slowly between mobile and stationary phases

- Solute in stationary phase tends to lag behind solute in mobile phase

- Broadening is directly proportional to flow rate

BAND BROADENING

May be due to

Irregular Flow Paths

- Occurs since column is packed with solid particles

- There are random multiple paths for solute particles

- These multiple paths are unequal

- Independent of flow rate

BAND BROADENING

van Deemter Equation

- The plate height equation as a result of the three band broadening mechanisms

Cuu

BAH

Multiplepaths

Longitudinaldiffusion

Equilibrationtime

BAND BROADENING

van Deemter Equation

u = flow rate

A, B and C are constants dependent on - Column

- Stationary phase- Mobile phase- Temperature

OPEN TUBULAR COLUMN

- Hollow capillary column

- Inner wall is coated with thin layer of stationary phase

- Gives better separation than packed columnNo multiple paths (A = 0)

Can be much longer (gives less resistance to gas flow)Smaller plate height

- Only useful for analytical chromatography(can only handle small samples due to less stationary

phase)

ASSYMETRIC PEAKS

- When a band is overloaded by too much solute

- Band emerges gradually in front

- An abrupt cut off is observed behind the concentration region

- Overloading leaves very little trails of solute behindthe concentrated region

ASSYMETRIC PEAKS

- Tailing is when the trailing part is elongated

- Occurs when the stationary phaseis strongly polar

has highly adsorptive sites (-OH groups)

Salinization- Chemical treatment to reduce tailing

- Converts -OH groups to nonpolar -OSi(CH3)3 groups

- Column should be replaced when tailing increases

MASS SPECTROMETRY

- Measures the masses and abundances of ions in the gas phase

- Detector is sensitive to low analyte concentrations

- Distinguishes different substances with the same retention time

- Used for both qualitative and quantitative analysis

MASS SPECTROMETRY

- Molecules are converted to ions prior to separation

- Molecules entering the ionization chamber of a mass spectrometer are converted into ions

- Ions are separated based on mass-to-charge ratio (m/z)

MASS SPECTROMETRY

Two Common Methods of Ionization

Electron Ionization (EI)- Electrons emitted from a hot filament are accelerated by 70 V

- Molecules are ionized by striking electrons as they absorb energy

M + e- → M+ + e- + e-

M+ is called the molecular ion

M+ breaks into fragments after ionization

MASS SPECTROMETRY

Two Common Methods of Ionization

Electron Ionization (EI)

- The most intense peak from fragments is called the base peak

- Other peaks are expressed as percentages of the base peak intensity

MASS SPECTROMETRY

Two Common Methods of Ionization

Chemical Ionization (CI)

- Ionization chamber contains a reagent gas (CH4)

- Pressure is maintained at about 1 mbar

- Energetic electrons convert gas into a variety of products

MASS SPECTROMETRY

Two Common Methods of Ionization

Chemical Ionization (CI)

CH4 + e- → CH4+ + 2e-

CH4+ + CH4 → CH5

+ + CH3

CH5+ then protonates the analyte

CH5+ + M → CH4 + MH+

- Fragmentation is less than EI

MASS SPECTROMETRY

Types of Mass Spectrometers (Analyzers)

Electrostatic

Magnetic

Time of flight

Ion trap (quadrupole ion storage)

Quadrupole mass spectrometer

THE MASS SPECTRUM

- Fragmentation patterns from the mass spectrum provideinformation about the structure of analyte molecule

Nominal Mass- Integer mass of the species with the most abundant isotope

of each element

For benzene (C6H6)- The most abundant isotopes are 12C and 1H

Norminal mass = (6 x 12) + ( 6 x 1) = 78

THE MASS SPECTRUM

Isotope Pattern- Information is obtained from relative intensities at M+1 and M+

- M+1 is one mass unit above the molecular ion

Nitrogen Rule- Used to propose composition of molecular ions

- Odd nominal mass implies compound has odd number of N atoms

- Even nominal mass implies compound has even number of N atoms