22. Chromatography

22. Chromatography

- Goal of the chromatography: - separation- identification (qualitative analysis)- quantitative determination

of components in various mixtures

Quantitative analysis: How much of a component is present?Qualitative analysis: What is the identity of the component?

22.1. Goals and fundamentals of chromatographic separations

Chromatography is a process in which we separate compounds from one another bypassing a mixture through a column that retains some compounds longer than others.

- Physico-chemical fundamentals of separation: different - adsorption

- solubility - exchange of ions between two phases - permeability

of the components of a mixture

- Concept of the technical realization: the components of a mixture are distributed differentlyin a two-phase system under dynamic conditions

stationaryphase

mobile phase mobile phase

mixtureseparated components

Methods of chromatography

according to the physico-chemical principles of the separation process:

- adsorption chromatography- partition chromatography- ion exchange chromatography - gel chromatography

Stationary phases:- adsorbents- liquid layers (wetted carrier substances)- ion exchangers- gels with a definite pore size

Mobile phases:- liquids ( liquid chromatography)- gases ( gas chromatography)

22.2. Adsorption chromatography

Principle of separation: different adsorption of the components on a solid phase (adsorbent)

Stationary phase: adsorbent filled in a tube (= column)Mobile phase: various solvents (eluents)

separated substances solventadsorbent

mixture of somecomponentssolvent

Column chromatography in practice

solvent

mixture

adsorbent

collectionflask strongly weakly

adsorbed component

22.2.1. Column chromatography

column

Elution:the components travel downward

with different speed

22.2.2. Thin layer chromatography (TLC)

- Principle of separation: different adsorption of the components on the surface of an adsorbent

- Stationary phase: adsorbent, mainly silica or aluminum oxide,forming a thin layer (0,1 – 0,3 mm) on a glass, plastic or aluminum plate

- Mobile phase: various organic solvents or mixture of solvents

- TLC in practice:

spotting developing visualization

developingchamber

solvent(eluent)

iodine crystals

iodinevapour

TLCplate

spots of mixture

- Evaluation

- number of components = number of spots - characteristic data for the components: retention factor, Rf

Rf =distance of the spot of a component

from the baseline 0 < Rf < 1

initialspot

solvent front

baseline

separatedcomponents

A, B, C

advantages: - rapid analyses- high sensitivity - high resolution

distance of the solvent frontfrom the baseline

Rf(A) = 14100

= 0.14

Rf(B) = 55100

= 0.55

Rf(C) = 86100

= 0.86

22.3. Partition paper chromatography

Principle of separation: differences in the partition coefficients of components of a mixture between two liquids

Stationary phase: thin layer of water adsorbed on the cellulose filaments of aprepared filter paper (10-20%)

Mobile phase: solvent, partly miscible or inmiscible with water

Advantages:- simple procedure,- sensitivity

Paper chromatography in practicesimilar as with TLC (sample, developing, evaluation)

separated components Disadvantages:

- long procedure,- limited resolving power

22.4. Gas chromatography

Principle of separation:different adsorption or solubilityof the components of the mixture

Stationary phase:

adsorbent or wetted carrier material

(adsorbent coated by a liquid layer)

Mobile phase: inert gas (nitrogen, argon, helium)

gas-solid-chromatographyGSC

gas-liquid-chromatographyGLC

Partition gas chromatography (GLC)

Principle of separation: different partition (solubility) of the components between the liquid layer and the carrier gas

Stationary phase: non-volatile liquid layer on the surface of the fine particles of the carrier substance

Mobile phase: gas

particle of the carriersubstance

liquid layer (film)

Liquid layer: non-volatile liquids, macromolecular products

Carrier substance: fine particles of silicate minerals (diatoma-earths, „Chromosorb”)

Cross-sectional view of wall-coated, support-coated, and porous-layer columns. Micrograph shows porous carbon stationary phase on inside wall of a fused-silicaopen tubular column.

Process of the separation

Scheme of a gas chromatograph

control unit(flow of carrier gas)

column(stat. phase)

carrier gas

thermostat

detector amplifier

recorder

sample injection

gas chromatogram

thermometer

Detectors:- flame ionization detector (FID)- thermal conductivity detector (TCD)

- flame ionization detector (FID) - thermal conductivity detector (TCD)

Evaluation of the gas chromatogram

- number of peaks = number of components - retention time, tR (min): characteristic data (qualitative analysis)

(tR = time elapsed between the sample injection and the maximum of the peak). Each analyte in a sample will have a different retention time.

- peak area: proportional to the relative amount of the separated components (quantitative analysis)

GC analysis of fuels/oils(„Gasoline Range Organics”)

Column: 30 m x 0.25 mm I.D.Stat. phase: SLBOven: 50 - 200 oCDetector: FIDCarrier gas: heliumInjection: 0.5 L

1. methanol (solvent) 7. toluene2. methylene chloride (imp.) 8. ethylbenzene3. 2-methylpentane 9. m-xylene4. benzene 10. o-xylene5. 2,2,4-trimethylpentane 11. 1,2,4-trimethylbenzene6. heptane

Column: SLB, 30 m x 0.25 mm I.D.Oven: 180 oCDetector: FIDCarr. gas: helium, 25 cm3/minInjection: 1 l

GC analysis of edible oils(analysis of fatty acid methyl esters, FAMEs)

1. Lauric acid methyl ester (C12:0)2. Myristic acid methyl ester (C14:0)3. Palmitic acid methyl ester (C16:0)4. Palmitoleic acid methyl ester (C16:1)5. Stearic acid methyl ester (C18:0)6. Oleic acid methyl ester (C18:1)7. Linoleic acid methyl ester (C18:2)8. Linolenic acid methyl ester (C18:3)

GLC analysis of melamine and some closely related derivatives in food

Melamine:

- contaminant in pet and baby food- added to increase the N-content- causing illnesses, fatalities

Column: 30 m x 0.25 mm I.D.Stat. phase: SLBOven: 115 - 325 oCDetector: FIDCarrier gas: heliumInjection: 1.0 L

SLB: silphenylene polymer

22.5. High Performance Liquid Chromatography (HPLC)

Schematic representation of an HPLC unit.(1) Solvent reservoirs, (7) Sample injection loop, (2) Solvent degasser, (8) Pre-column (guard column), (3) Gradient valve, (9) Analytical column, (4) Mixing vessel for delivery of the mobile phase, (10) Detector (i.e. IR, UV), (5) High-pressure pump, (11) Data acquisition, (6) Switching valve in "inject position", (12) Waste or fraction collector. (6') Switching valve in "load position",

- separation in a densely packed column- high pressure is required to move the solution through the column

Solvent molecules compete with solute molecules for binding sites on the stationary phase.The more strongly the solvent bindsto the stationary phase, the greaterthe eluent strength of the solvent.

Detector (UV),

Comparison of HPLC and GC Sample Volatility Sample Polarity

HPLC• No volatility requirement

• Sample must be solublein mobile phase

GC• Sample must be volatile

HPLC

GC

• Separates both polar andnon polar compounds

• PAH - inorganic ions

• Samples are nonpolarand polar

Comparison of HPLC and GC

Comparison of HPLC and GC Sample Thermal Lability Sample Molecular Weight

HPLC• Analysis can take place

at or below roomtemperature

GC

• Sample must be ableto survive high temperature injectionport and column

HPLC

GC

• No theoretical upper limit

• In practicality, solubility islimit.

• Typically < 500 amu

Comparison of HPLC and GC Sample Preparation Sample Size

HPLC• Sample must be filtered

• Sample should be insame solvent as mobilephase

GC

• Solvent must be volatileand generally lower boiling than analytes

HPLC

GC

• Sample size based uponcolumn i.d.

• Typically 1 - 5 L

Comparison of HPLC and GC Separation Mechanism Detectors

HPLC• Both stationary phase

and mobile phase takepart

GC

•Mobile phase is a sample carrier only

HPLC

GC

• Most common UV-Vis• Wide range of non-

destructive detectors• 3-dimensional detectors• Sensitivity to fg (detector

dependent)

• Most common FID,universal to organiccompounds

How can We Analyze the Sample?

Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose

Zorbax NH2 (4.6 x 250 mm)

70/30 Acetonitrile/Water

1 mL/min Detect=Refractive Index

1

23

4

5

mAU

time

6

The Chromatogram

Injection

to

tRmAU

time

tR

to - elution time of unretained peaktR- retention time - determines sample identity

Area or height is proportionalto the quantity of analyte.

HPLC Analysis Parameters

Mobile Phases

Flow RateComposition

Injection Volume

Column Oven Temperature

WavelengthTime Constant

HPLC Applications

Chemical

Environmental

Pharmaceuticals

Consumer Products

Clinical

polystyrenesdyesphthalates

tetracyclinescorticosteroidsantidepressantsbarbiturates

amino acidsvitaminshomocysteine

Bioscience

proteinspeptidesnucleotides

lipidsantioxidantssugars

polyaromatic hydrocarbonsInorganic ionsherbicides

HPLC analysis of some corticoids

Prednisone(internal standard)

Some steroids (e.g. synthetic corticosteroids) are highly efficient, widely used drugs(in treatment of inflammatory, rheumatic, allergic, etc. disorders)

Column: 10 cm x 4.6 mm I.D.Stat. phase:- fluorinated non-polarMobile phase:- water-methanol, 50:50Temp.: 35 oCDetector: UV at 240 nmFlow rate: 0.8 cm3/minInjection: 5 L

- Physico-chemical fundamentals of separation: different - adsorption

- solubility - exchange of ions between two phases - permeability

of the components of a mixture

- Concept of the technical realization: the components of a mixture are distributed differentlyin a two-phase system under dynamic conditions

stationaryphase

mobile phase mobile phase

mixtureseparated components

22.6. Ion-exchange chromatography

Ion-exchange: RH + K+A- RK + H+A- :ions of an electrolyte solution will be replaced with an equivalent amount of ions of the same sign from an ion-exchanger

Principle of separation:different affinity of the ions of the mixture to the ion-exchanger

Stationary phase: cation- or anion exchangers- zeolithes (sodium-aluminum-silicate) (cation exchanger)- cation exchanger resins, - anion exchanger resins

Mobile phase: aqueous solution of ionic compounds

Structures of polystyrene ion-exchange resins. Cross-links are covalent bridges between polymer chains.

Anion exchangers contain bound positive groups.

Cation exchangers contain bound negative groups.

Ion-exchange chromatography in practice

Use:- e.g. in analysis of mixtures of amino acids

(amino acid analyzers)

solution of an ionic mixture

ion-exchanger

The selectivity depends on the geometrical size and electrical charge

of the ions.

R CH COOH

NH2

H+ R CH COOH

NH3+

amino acid

cations of amino acids (in acidic solution):

Deionized water is prepared by passing water through an anion-exchange resin loaded with OH and a cation-exchange resin loaded with H.

Measuring extremely low levels of analyte is called trace analysis. Trace analysis is especially important for environmental problems in which low concentrations ofsubstances, such as mercury in fish, can become concentrated over many years inpeople who eat large quantities of fish.

Preconcentration

22.7. Gel permeation chromatography (gel filtration)(also known as size-exclusion chromatography)

Principle of separation: the small particles of a mixture can penetrate easier into the pores of the packing material than the larger molecules

Stationary phase: grains of a swelling three-dimensional polymer with definite pore size (modified polysaccharides, Sephadex gels)

Mobile phase: solution of components to be separated

The larger particles will pass the column faster,than the smaller (inverse filtration)Use:

- separation of peptides, proteins- semi-quantitative determinationof the molecular mass

(a) A mixture of large and small molecules is applied to the top of a molecular exclusion chromatography column.

(b) Large molecules cannot penetrate thepores of the stationary phase, but small

molecules can. Therefore less of the volume is available to large molecules and they move down the column faster.

Separation of proteins by molecular exclusionchromatography, using a TSK 3000SW HPLC column. The highest molecular masses are eluted first.

Molecular Mass Determination

An electric arc struck between graphite rods creates nanometer-size carbon products, including tubes with extraordinary strength and possible use in electronic devices.

Molecular exclusion chromatography separates nanotubes (fraction 1) from other forms of carbon in fractions 2 and 3.

The stationary phase is PLgel MIXED-A, a polystyrenedivinylbenzene resin with pore sizes corresponding to a molecular mass range of 2 000–40 000 000 Da.

Images of carbon in each fraction were made by atomic force microscopy.

Purification of carbon nanotubes by molecular exclusion chromatography.

What is AC?

• Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure.

• AC is designed to purify a particular molecule from a mixed sample.

Matrix

Affinity Ligand

The resin

Step 1. Loading affinity column.

Step 2. Proteins sieve through matrix of affinity beads.

Step 3. Proteins interact with affinity ligand with some binding loosely and others tightly.

Step 4. Wash off proteins that do not bind.

Step 5. Wash off proteins that bind loosely.

Step 6. Elute proteins that bind tightly to ligand and collect purified protein of interest.

http://www.bio.davidson.edu/Courses/genomics/method/Affinity.html

Chocolate is great to eat but not soeasy to analyze.

(Real samples rarely cooperate withyou!)

Animals that metabolize theobromine, such as dogs, can succumb to theobromine poisoning from as little as 50 grams of general chocolate for a smaller dog and 400 grams for an average-sized dog. It should be observed the concentration of theobromine in dark chocolates (approximately 10 g/kg) is up to 10 times that of milk chocolate (1-5 g/kg) - meaning dark chocolate is far more toxic to dogs per unit weight or volume than milk chocolate.

Sample Preparation

Extracting fat from chocolate to leave defatted solid residue for analysis. (Fat needs to be removed because it would interfere with chromatography later in the analysis.)

The next step in the sample preparation procedure is to make a quantitativetransfer (a complete transfer) of the fat-free chocolate residue to an Erlenmeyerflask and to dissolve the analytes in water for the chemical analysis.

Principle of liquid chromatography. (a) Chromatography apparatus with anultraviolet absorbance monitor to detect analytes at the column outlet. (b) Separation of caffeine and theobromine by chromatography. Caffeine is more soluble than theobromine in the hydrocarbon layer on the particles in the column. Therefore caffeine is retained more strongly and moves through the column more slowly than theobromine.

The Chemical Analysis

Chromatogram of 20.0 microliters of dark chocolate extract.A 4.6-mm-diameter 150-mm-long column, packed with 5-micrometer particles of Hypersil ODS, was eluted (washed) with water:methanol:acetic acid (79:20:1 by volume)at a rate of 1.0 mL per minute.

Only substances that absorb ultravioletradiation at a wavelength of 254 nanometers are observed inBy far, the major components in the aqueous extract are sugars, but they are not detected in this experiment.

Chromatogram of 20.0 microliters of a standard solution containing 50.0 micrograms of theobromine and 50.0 micrograms of caffeine per gram of solution.

Calibration curves, showing observed peak heights for known concentrations of pure compounds. One part per million is 1 microgram of analyte per gram of solution. Equations of the straight lines drawn through the experimental data points were determined by the method of least squares

Interpreting the Results

Mass Spectrometry

Background

• Mass spectrometry (Mass Spec or MS) uses high energy electrons to break a molecule into fragments.

• Separation and analysis of the fragments provides information about:– Molecular weight– Structure

Background

• The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation.– A species with a positive charge and one

unpaired electron

+ e-C HH

HH H

HH

HC + 2 e-

Molecular ion (M+) m/z = 16

Background• The impact of the stream of high energy

electrons can also break the molecule or the radical cation into fragments.

(not detected by MS)

m/z = 29

molecular ion (M+) m/z = 30

+ CH

HH

+ H

HH CH

HCH

H

H CH

HCH

H

H CH

H

+ e-H C

H

HCH

HH

m/z = 15

Background• Molecular ion (parent ion):

– The radical cation corresponding to the mass of the original molecule

• The molecular ion is usually the highest mass in the spectrum– Some exceptions w/specific isotopes– Some molecular ion peaks are absent.

HH

HHC H C

H

HCH

HH

Background

• The cations that are formed are separated by magnetic deflection.

Background

• Mass spectrum of ethanol (MW = 46)

SDBSWeb : http://riodb01.ibase.aist.go.jp/sdbs/ (National Institute of Advanced Industrial Science and Technology, 11/1/09)

M+