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
Lecture 21 (4/12/2005)
Basic Principles of Chromatography (4)
Department of Food Science
Read Material: Chapter 27, page 437
Final Exam: April 29
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.
TLC- Factors of Consideration
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.
(b) The resolution of two bands
is a function of both their relative
Retentions and peak width.
Van Deemter Plot of Column
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