ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 21 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of...

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