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Food Analysis Lecture 21 (4/12/2005). Basic Principles of Chromatography (4). Qingrong Huang Department of Food Science Read Material: Chapter 27, page 437 Final Exam: April 29. Affinity Chromatography. Affinity Chromatography : separation is based on the specific, reversible

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Food Analysis

Lecture 21 (4/12/2005)

Basic Principles of Chromatography (4)

Qingrong Huang

Department of Food Science

Read Material: Chapter 27, page 437

Final Exam: April 29


Affinity Chromatography

  • Affinity Chromatography: separation is based on the specific, reversible
  • interaction between a solute molecule and a ligand immobilized on the
  • chromatographic stationary phase.
  • The ultimate extension of adsorption chromatography
  • Involved biological materials as the stationary phase, including antibodies,
  • enzyme inhibitors, lectins, or other molecules that selectively and reversibly
  • bind to complementary analyte molecules in the sample;
  • Separation exploits the lock and key binding of biological systems;
  • The ideal support for affinity chromatography should be a porous, stable,
  • high-surface-area material that does not adsorb anything itself;
  • Elution methods: nonspecific and biospecific.
  • Nonspecific method: disrupt ligand analyte binding by changing the mobile
  • phase pH, ionic strength, dielectric constant, or temperature;
  • Biospecific method: free ligand, either identical to or different from the
  • matrix-bound ligand, is added to the mobile phase.

A: The support presents the

immobilized ligand to the analyte

to be isolated;

B: The analyte makes contact

with the ligand and attaches


C: The analyte is recovered by

the introduction of an eluent,

which dissociates the complex

Holding the analyte to the ligand;

D: The support is regenerated,

ready for the next isolation.


Chromatographic Techniques

  • Paper chromatography: introduced in 1944
  • (1) The dissolved sample is applied as a small spot one-half inch
  • from the edge of a strip or square of filter paper;
  • (2) The strip is then suspended in a closed container, the atmosphere
  • of which is saturated with the developing solvent (mobile phase);
  • (3) The end closer to the sample is placed in contact with solvent,
  • which travels up or down the paper by capillary action, separating sample
  • components in the process.
  • In paper or thin-layer chromatography (TLC), components of a mixture
  • are characterized by their relative mobility (Rf) value:
  • Rf = Distance moved by component/Distance moved by solvent

Thin-Layer Chromatography


  • First introduced in 1938;
  • Better than Paper Chromatography because it is faster, more sensitive,
  • better resolution, and more reproducible;
  • The particles on the plate are smaller and more regular than paper
  • fiber  better resolution;
  • Advantages:
  • TLC has high sample throughput and low cost;
  • The possibility to analyze several samples and standards
  • simultaneously;
  • Minimal sample preparation (since the stationary phase is disposable);
  • A plate may be stored for later identification and quantitation.

TLC Procedures

  • TLC utilizes a thin (ca. 250 m thick) layer of stationary phase bound
  • to an inert support in a planar configuration;
  • The support is typically a glass plate (20 cm by 20 cm);
  • Typically used TLC stationary phases: silica gel; alumina, cellulose,
  • and diatomaceous earth;
  • Quantitative evaluation of TLC:
  • (1) in situ (directly on the layer by using a densitometer);
  • (2) after scraping a zone off the plate, eluting compound from the
  • stationary, and analyzing the resultant solution.
  • The selection of mobile phase for TLC:
  • (1) selected based on their chemical characteristics and solvent
  • strength;
  • (2) In simple adsorption TLC, the higher the solvent strength, the
  • greater the Rf value (range 0.3-0.7) .

TLC- Factors of Consideration

  • Stationary phase and mobile phase;
  • Type of developing chamber used;
  • Vapor phase conditions (saturated vs. unsaturated);
  • Development mode (ascending, descending, horizontal, radials);
  • Development distance.

Column Chromatography

  • The most useful method of separating compounds in a mixture;
  • Elution: the process of passing the mobile phase through the column;
  • Eluate (or effluent): The portion that emerges from the outlet end of
  • the column;
  • Isocratic: the elution of constant mobile-phase composition;
  • Gradient elution: refers to changing the mobile phase (e.g. increasing
  • solvent strength or pH) during elution in order to enhance resolution
  • and decrease analysis time;
  • Retention Volume (VR): The volume of liquid required to elute a
  • compound from an LC column; the associated time is the retention
  • time tR
  • Adjusted Retention Time (t’R): by subtracting the time required for
  • the mobile phase or a nonretained solute to travel through the column
  • to the detector.

Column Chromatography

  • Advantages Limitations
  • Inexpensive • Slow
  • Simple • Low resolution
  • Good Prep Method • Quantitation Difficult

Separation and Resolution


Where Rs = resolution;

t = Difference between retention times of peak 1 and 2;

W2 = width of peak 2 at baseline;

W1 = width of peak 1 at baseline.

Chromatographic resolution is a function of column efficiency, selectivity, and capacity factor.



  • Idealized Gaussian
  • chromatogram;

(b) The resolution of two bands

is a function of both their relative

Retentions and peak width.




  • Where
  • N = number of theoretical plates;
  • tR = retention time;
  •  = standard deviation for a Gaussian peak
  • w = peak width at baseline (4)
  • W1/2 = peak width at high height
  • The number of theoretical plates (N) is generally proportional to column length (L). HETP=L/N
  • HETP: height equivalent to a theoretical plate.

Van Deemter Plot of Column

Efficiency (HETP)

HETP: height equivalent

to a theoretical plate;

A, B, C: constants;

u: mobile phase velocity


Diffusion in Column


A: Eddy diffusion or multiple flowpaths, refers to the different micro-

scopic flowstream that the mobile phase can take between particles

in the column. As a result, solute molecules spread from an initially

narrow band to a broader area within the column. Can be minimized

by good column packing and the use of small diameter particles of

narrow particle size distribution.

B: Longitudinal diffusion, exists because all solutes diffuse from an

area of high concentration to one of low concentration.

C: The mass transfer term arises from the finite time required for solute

to equilibrate between the mobile and stationary phases. Can be

minimized by using porous particles of small diameter or pellicular

packing materials.