This document discusses chromatographic techniques for detecting antimicrobials in biological fluids. It provides a brief history of chromatography, explaining that Mikhail Tswett first developed the technique in 1906 to separate plant pigments. The key principles of chromatography are explained, including that it works by allowing molecules in a mixture to distribute between a stationary and mobile phase. Various types of chromatography are classified based on the interaction with the stationary phase and physical state of the mobile phase. Sample preparation techniques for biological fluids like blood, urine and milk are also outlined, including removal of debris, cleanup, and concentration.

1. CHROMATOGRAPHIC
TECHNIQUES FOR
DETECTION OF
ANTIMICROBIALS IN
BIOLOGICAL FLUIDS
SUBMITTED TO
DR. SOUMEN CHOUDHARY
ASSISTANT PROFESSOR
DEPT OF PHARMACOLOGY AND
TOXICOLOGY,DUVASU,MATHURA
1 SUBMITTED BY
RAKTIM SAIKIA
MVSC 1ST YEAR
V-1818/17

2. HISTORY
o Tswett used chromatography to separate
plant pigments (1906)
o colorful separation of plant pigments was
done using a column of calcium
carbonate(chalk)
o the new technique was called
chromatography because the result of the
analysis was written in color.(Chroma
means color and graphein means to write)
Mikhail Tswett,
Russian Botanist,
1872-1919
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3. CHROMATOGRAPHY
Technique used to separate and identify the
components of a mixture
PRINCIPLE: Works by allowing the molecules
present in the mixture to distribute themselves
between a stationary and a mobile medium.
Molecules that spend most of their time in the
mobile phase are carried along faster.
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10. 10

11. Components:
mobile phase: a solvent that flows through the supporting
medium
stationary phase: a layer or coating on the supporting
medium that interacts with the analytes
supporting medium: a solid surface on which the stationary
phase is bound or coated
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12. BASIC TERMS….
 Adsorbtion: Interaction of solute molecules with the
surface of the stationary phase
 Eluent: The mobile phase
 Elution: Motion of the mobile phase through the
stationary phase
 Elution time: The time taken for a solute to pass
through the system. A solute with a short elution time
travels through the stationary phase rapidly, i.e. it elutes
fast
 Stationary phase: The part of the chromatography
system that is fixed in place
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13.  Resolution: Degree of separation of different solutes.
In principle, resolution can be improved by using a
longer stationary phase, finer stationary phase or
slower elution.
 Rf value: distance travelled by solute
distance travelled by solvent
 Rf = retardation factor. The Rf value has to be
between 0 and 1, and it is typically reported to two
decimal places.
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14. CLASSIFICATION
On the basis of
interaction of
solute to the
stationary phase
On the basis of
chromatographic
bed shape
Techniques by
physical state of
mobile phase 14

15. On the basis of
interaction of
solute to the
stationary phase
Adsorption
chromatography
Partition
chromatography
Ion exchange
chromatography
Molecular
exclusion
chromatography
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16. On the basis of
chromatographic bed shape
Column
chromatography
TLC
Paper
chromatography
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17. Techniques by
physical state of
mobile phase
Gas
chromatography
Liquid
chromatography
Affinity
chromatography
Supercritical
fluid
chromatography 17

18. • In chromatography, two immiscible phases are brought into
contact; one is the stationary (nonmoving) phase and the
other is the mobile (moving) phase.
• A sample mixture is introduced into the mobile phase and
interacts (partitions) with both mobile and stationary phase
many times as it moves through the system carried by the
mobile phase.
• Differences in chemical and physical properties in the
sample components influence the rate of migration of each
individual component to different degrees, i.e., some
components move faster than others. The result is a
separation of the components.
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19. • A column is an integral part of contemporary
chromatography, and this may be packed or
coated with stationary phases from a wide
variety of different materials, solids or liquids.
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21. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
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24. Normal phase: “Unmodified”
stationary phase where POLAR
solutes interact strongly and run
slowly
Reverse phase: “Modified”
stationary phase where POLAR
solutes run fast i.e. reverse order
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26. GAS CHROMATOGRAPHY
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31.  UPLC
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33. SAMPLES
 WHOLE BLOOD
 PLASMA
 URINE
 FAECES
 SEMEN
 MILK
 MEAT
 EGGS……
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34. Although urine and blood
specimens are both commonly
tested for antibiotic activity, urine
is generally accepted as being
more sensitive than serum or
plasma because chemical
compounds are often
concentrated in urine. 34

35. SAMPLE PREPARATION
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36. 1.REMOVAL OF DEBRIS
 A large component of crude cell or tissue lysate will
consist of debris from insoluble biological material
and particulates. Centrifugation at 45, 000 rpm for
30 minute will isolate the bulk of this.
 Further clarification— even the smallest of
particulates can clog pores in any chromatography
medium; they can also affect hardware such as
HPLC flow lines and rotary injection valves---
achieved by passing the sample through a 0.45- or
0.2-µm filter to remove fine particulates.
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37. 2.CLEANING UP THE SAMPLE…
 The removal of contaminants may be achieved in a
single step via size-exclusion gel filtration, in which
proteins are eluted before salts and other small
contaminants because they cannot access the
interior of the gel matrix.
 This technique is also known as buffer exchange,
and there are several commercially available kits
for researchers to use. They provide the advantage
of sample elution into a buffer that is appropriate for
the next steps in your chromatography workflow.
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38. 3.CONCENTRATION OF SAMPLE
 Most chromatography experiments require only a
small volume of sample; therefore, the samples
must have a high protein concentration.
 For larger volumes, spin columns with appropriate
spin concentrator can be used to concentrate the
sample.
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