MODULE 4 CHROMATOGRAPHY THEORY & PRINCIPLES

Faculty of the School of Pharmacy CENTRO ESCOLAR UNIVERSITY Manila Malolos - Makati

CHROMATOGRAPHY: DEFINITION SCIENCE OF THE STUDY OF SEPARATION OF

MOLECULES BASED ON DIFFERENCES IN THEIR STRUCTURE AND / OR COMPOSITION.

CHROMATOGRAPHY: DEFINITION
It originated from the Greek words

chroma meaning color and graphein meaning to write It was coined by the Russian botanist Mikhail Semyonovich Tsvet [Tswett] during his research about chlorophyll where he separated the different plant pigments

CHROMATOGRAPHY:
PRINCIPLE

IT INVOLVES MOVING A
PREPARATION OF MATERIALS TO BE SEPARATED

(TEST PREPARATION ) OVER A STATIONARY SUPPORT. THE MOLECULES IN THE TEST

PREPARATIONS WILL HAVE DIFFERENT REACTIONS WITH THE STATIONARY SUPPORT.

Test molecules which display TIGHTER ( WEAK AFFINITY) interactions will tend to MOVE SLOWLY through the support, than those molecules with WEAKER (STRONG AFFINITY) interactions., which MOVE FASTER through the support.

CHROMATOGRAPHY:
PRINCIPLE

It is a collective term for a family of laboratory techniques for the separation of mixtures It involves passing a mixture dissolved in a mobile phase through a stationary phase which separates the analyte to be measured from other molecules in the mixture and allows to be isolated

CHROMATOGRAPHY:
PRINCIPLE

THIS WILL LEAD TO SEPARATION OF SIMILAR MOLECULES. DIFFERENT TYPES OF MOLECULES CAN BE SEPARATED FROM EACH OTHER, AS THEY MOVE OVER THE SUPPORT MATERIAL.

CHROMATOGRAPHY
It may be preparative or analytical Preparative chromatography It seeks to separate the components of a mixture for further use [and thus is a form of purification] Analytical chromatography It normally operates with smaller amounts of materials and seeks to measure the relative proportions of analytes in mixtures

Principal Objectives of Chromatography

Resolution of mixtures into constituent parts Determination of homogeneity Comparison of substances suspected of being identical Purification

Principal Objectives of Chromatography

Concentration of substances from dilute solutions Identification and control of technical products Quantitative separation from complex mixtures Indication of molecular structure

Terminologies
Analyte
The substance to be separated during chromatography

Chromatogram
The visual output/result in chromatography

Effluent
It is the mobile phase leaving the column

Stationary Phase
The stationary phase is the substance which is fixed in place for the chromatography procedure

Terminologies
Retention Time
Is the characteristic time it takes for a particular analyte to pass through the system [from the column inlet to the detector] under set conditions

Mobile Phase
It is the phase which moves in a definite direction It may be a liquid, a gas or a supercritical fluid The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column

SUPPORTS IN CHROMATOGRAPHIC PREPARATIONS

IMMOBILIZED SILICA ON GLASS THIN LAYER CHROMATOGRAPHY PLATES (TLC)
VOLATILE GASES GAS CHROMATOGRAPHY (GC)

PAPER

PAPER CHROMATOGRAPHY

LIQUIDS, CONTAINING HYDROPHILIC, INSOLUBLE MOLECULES

LIQUID CHROMATOGRAPHY

CHROMATOGRAPHY: THEORY
EXPLOITS THE DIFFERENCES IN PARTITIONING BEHAVIOR BETWEEN;

1. MOBILE PHASE II. STATIONARY PHASE

CHROMATOGRAPHY: THEORY
MOBILE PHASE
A GAS OR LIQUID THAT PASSES THROUGH THE COLUMN.

STATIONARY PHASE
A SOLID OR LIQUID THAT DOES NOT MOVE. IT REFERS TO THE CHROMATOGRAPHIC SUPPORT.

THE CHROMATOGRAPHIC SYSTEM

T STATIONARY PHASE (SILICA PLATE)

MOBILE PHASE (LIQUID)

BASIS OF INTERACTIONS OF AN ANALYTE TO THE

STATIONARY PHASE

CHARGE RELATIVE SOLUBILITY SURFACE ADSORPTION

CHROMATOGRAHY: THEORIES RATE THEORY

PLATE THEORY BOTH EXHIBITS THE CAPILLARYACTION, TO
MOVE THE SOLVENT THROUGH THE STATIONARY PHASE.

RATE THEORY AND PLATE THEORY HOW IT WORKS?

THE MOBILE PHASE MOVES
THROUGH THE STATIONARY PHASE, PICKING UP THE COMPOUNDS TO BE TESTED.

AT DIFFERENT POINTS
IN THE STATIONARY PHASE, THE DIFFERENT COMPONENTS OF THE COMPOUND ARE GOING TO BE ABSORBED; AND ARE GOING TO STOP (AT A SPECIFIC DISTANCE) MOVING THRU THE MOBILE PHASE.

AS THE MOBILE PHASE

TRAVELS THROUGH THE

STATIONARY PHASE, IT TAKES THE COMPOUNDS WITH IT.

RETENTION: DEFINITION
MEASURE OF SPEED AT WHICH A SUBSTANCE MOVES IN A CHROMATOGRAPHIC SYSTEM.

IN CONTINUOUS DEVELOPMENT SYSTEMS (HPLC & GC), WHERE THE COMPOUNDS ARE ELUTED WITH AN ELUENT, IT IS EXPRESSED AS RETENTION TIME (Rt).

RETENTION: DEFINITION
IN INTERRUPTED DEVELOPMENT SYSTEMS ( TLC & PC ), IT IS EXPRESSED AS RETENTION FACTOR (Rf). RETENTION FACTOR IS THE RUN LENGTH OF THE
COMPOUND DIVIDED BY THE RUN LENGTH OF THE ELUENT FRONT.

RETENTION FACTOR: FORMULA

Rf = distance travelled by the compound distance travelled by the solvent

D1 Rf = D2

RETENTION FACTOR: SIGNIFICANCE & IMPORTANCE

A QUANTITATIVE INDICATION OF HOW FAR A

PARTICULAR COMPOUND TRAVELS IN A PARTICULAR SOLVENT.

A GOOD INDICATOR OF WHETHER AN UNKNOWN & A KNOWN COMPOUND ARE IDENTICAL OR SIMILAR.

CHROMATOGRAPHY: APPLICATIONS

LIQUID CHROMATOGRAPHY TEST WATER SAMPLES FOR POLLUTION.

GAS CHROMATOGRAPHY 1. BOMB DETECTION IN AIRPORTS

2. IDENTIFY / QUANTIFY DRUGS & ALCOHOL. 3. IN FORENSICS TO COMPARE
FIBERS FOUND ON A VICTIM.

CHROMATOGRAPHY: APPLICATIONS

THIN LAYER
CHROMATOGRAPHY

PAPER
CHROMATOGRAPHY 1. SEPARATION OF AMINO ACIDS AND

1. DETECTION OF ANIONS. PESTICIDES & INSECTICIDES IN FOOD. 2. RNA FINGERPRINTING 2. IN FORENSICS TO 3. SEPARATION AND
ANALYZE DYE COMPOSITION OF FIBERS.
TESTING OF HISTAMINES & ANTIBIOTICS.

Principles of Separation

Partition
Stationary phase is liquid Mobile phase is liquid or gas Retention and separation occur due to the relative solubility of the analytes in the two fluids as determined by their partition coefficients

Principles of Separation
Ion-exchange
Stationary phase is a polymeric matrix bonded with ionic functional groups [usually styrenedivinylbenzene polymer] Mobile phase is always a liquid Retention and separation is mainly due to the electrostatic bonds with the functional groups

PRINCIPLES OF SEPARATION Adsorption

Stationary phase is solid Mobile phase is liquid or gas Retention and separation depends on the ability of the atoms on the surface to remove analytes from the mobile phase and adsorb them temporarily by means of electrostatic forces

PRINCIPLES OF SEPARATION
Molecular exclusion

Also known as size exclusion, gel permeation or gel filtration Stationary phase is a polymeric substance containing numerous pores of molecular dimensions Mobile phase is a liquid or gas Retention and separation depends on the differential migration of solute molecules based on molecular size

Different Techniques of Chromatography

Techniques by Chromatographic Bed Shape Column Chromatography Planar Chromatography Paper chromatography Thin Layer chromatography Techniques by Physical State of Mobile Phase Gas chromatography Liquid chromatography HPLC

PARTITION CHROMATOGRAPHY:

COMMON TYPES

PAPER CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY GAS LIQUID CHROMATOGRAPHY GEL CHROMATOGRAPHY

PARTITION CHROMATOGRAPHY: SPECIAL TYPES

LIQUID CHROMATOGRAPHY (LC) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) SIZE EXCLUSION CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY

PARTITION CHROMATOGRAPHY:
TECHNIQUES

1.OPERATION OF A COLUMN (A TUBE FILLED WITH A SORBENT AND SOLVENT).

2. A SOLUTION CONTAINING THE SOLUTE IS
LAYERED ON TOP OF THE SORBENT AND IS

ALLOWED TO ENTER THE SORBENT.

PARTITION CHROMATOGRAPHY:
TECHNIQUES

PARTITION CHROMATOGRAPHY:
TECHNIQUES

3. THE SOLVENT IS ALLOWED

TO PASS CONTINUALLY THROUGH THE COLUMN.

PARTITION COEFFICIENT ( K ) IF TWO PHASES ARE IN CONTACT WITH ONE ANOTHER, AND IF ONE OR BOTH PHASES CONTAIN A SOLUTE, THE
SOLUTE WILL DISTRIBUTE ITSELF BETWEEN TWO PHASES.

IT IS THE RATIO OF THE CONCENTRATIONS OF THE SOLUTE IN THE TWO PHASES.

PARTITION COEFFICIENT ( K )

CONCENTRATION OF SOLUTE IN THE STATIONARY PHASE K = -----------------------------------------------------------CONCENTRATION OF THE SOLUTE IN THE MOBILE PHASE

PARTITION CHROMATOGRAPHY:
MATERIALS USED

COLUMNS CONTAINING A MATRIX THAT DOES NOT ADSORB SOLUTES. 1. DIATOMACEOUS EARTH (CELITE) 2 . SILICA GEL 3. CELLULOSE POWDER 4. CROSS-LINKED DEXTRANS (SEPHADEXLH 20)

PARTITION CHROMATOGRAPHY:
MATERIALS USED

STATIONARY PHASE CREATED BY SUSPENDING THE

SUPPORT OR WASHING THE COLUMN A PROPER SORBENT.

1. HYDROPHOBIC SOLVENT (BENZENE) 2. HYDROPHILIC SOLVENT (ALCOHOL)

PARTITION CHROMATOGRAPHY:
MATERIALS USED

MOBILE PHASE
1. ALCOHOLS & AMIDES FOR NONPOLAR MATERIALS. 2. PURIFIED WATER FOR POLAR
MATERIALS

TECHNIQUES IN PAPER CHROMATOGRAPHY

PAPER CHROMATOGRAPHY:
DESCRIPTION

THIS METHOD INVOLVES SPOTTING THE SAMPLE

SOLUTION ONTO A STRIP OF CHROMATOGRAPHIC

PAPER.

THE PAPER IS PLACED INTO A JAR, CONTAINING A SHALLOW LAYER OF SOLVENT AND THEN SEALED.

PAPER CHROMATOGRAPHY:
DESCRIPTION

AS THE SOLVENT RISES THRU THE PAPER, IT MEETS THE SAMPLE MIXTURE, WHICH STARTS TO TRAVEL UP THE PAPER WITH THE SOLVENT.
DIFFERENT COMPOUNDS IN THE SAMPLE MIXTURE TRAVEL DIFFERENT DISTANCES, ACCORDING TO HOW STRONGLY IT INTERACTS WITH THE PAPER.

THIS ALLOWS THE CALCULATION OF THE RF VALUES, AND COMPARE WITH A STANDARD.

PAPER CHROMATOGRAPHY SET UP

TWO DIMENSIONAL
PAPER CHROMATOGRAPHY

PAPER CHROMATOGRAPHY:
DETECTION OF SPOTS

BY COLOR BY ITS FLUORESCENCE BY CHEMICAL REACTION, AFTER SPRAYING THE SPOTS

WITH A VISUALIZING REAGENT

BY RADIOACTIVITY

PAPER CHROMATOGRAPHY:
IDENTIFICATION OF SPOTS

COMPARISON WITH STANDARDS OF KNOWN RF VALUES.

ELUTION BY CUTTING OUT THE SPOT AND
SOAKING THE PAPER IN AN APPROPRIATE

SOLVENT.

TECHNIQUES THIN LAYER CHROMATOGRAPHY: THEORIES

1. WIDELY USED LABORATORY TECHNIQUE, SIMILAR TO

PAPER CHROMATOGRAPHY

2. STATIONARY PHASE USED IS A THIN LAYER OF ADSORBENT ON AN INERT, FLAT SUBSTRATE (GLASS PLATE); SILICA GEL
ALUMINA

CELLULOSE

THIN LAYER CHROMATOGRAPHY:

DETECTION OF SPOTS

BY ITS NATURAL COLOR

BY FLUORESCENCE BY SPRAYING WITH VISUALIZATION REAGENTS

SPECIFIC VISUALIZING AGENTS NINHYDRIN RHODAMINE B

SPECIFIC COMPOUNDS IDENTIFIED

AMINO ACIDS LIPIDS

ANTIMONY CHLORIDE
SULFURIC ACID + HEATING

STEROIDS TERPINOIDS
UNIVERSAL VISUALIZING AGENT FOR MOST ORGANIC SUBSTANCES

POTASSIUM PERMANGANATE IN SULFURIC ACID

ANISALDEHYDE IN SULFURIC ACID

HYDROCARBONS
CARBOHYDRATES

BROMINE VAPOR

OLEFINS

THIN LAYER CHROMATOGRAPHY:

ADVANTAGES

GREAT RESOLVING POWER BECAUSE SPOTS ARE SMALLER. GREATER SPEED OF SEPARATION; HIGHER RESOLUTION. WIDER CHOICE OF MATERIALS AS SORBENTS.

THIN LAYER CHROMATOGRAPHY:

ADVANTAGES

EASY DETECTION OF SPOTS. EASY ISOLATION OF

SUBSTANCES FROM THE CHROMATOGRAM.

DRUGS ANALYZED: ANALGESICS ANTIPYRETICS ASPIRIN, PHENACETIN, ACETAMINOPHEN

ANTI-INFLAMMATORY DRUGS URICOSURIC DRUGS CAFFEINE AND CAFFEINECONTAINING DRUGS

GAS LIQUID CHROMATOGRAPHY

IT IS BASED ON PARTITION
EQUILIBRIUM OF

STATIONARY PHASE IT IS ADHERED TO THE INSIDE OF A;

ANALYSES BETWEEN A SOLID STATIONARY PHASE AND A GAS MOBILE PHASE.

1. SMALL DIAMETER GLASS TUBE (CAPILLARY COLUMN) 2. SOLID MATRIX INSIDE A

LARGER METAL TUBE (PACKED COLUMN)

GAS LIQUID CHROMATOGRAPHY: APPLICATIONS

MEASUREMENT OF TOXIC SUBSTANCES IN WATER, SOIL OR AIR. IN DRUG QUALITY CONTROL, TO ASSURE THE QUANTITATIVE /
QUALITATIVE FEATURES OF PHARMACEUTICALS.

IN ANALYTICAL CHEMISTRY &

BIOCHEMISTRY

PETROCHEMICAL
ENVIRONMENTAL MONITORING

IN CHEMISTRY RESEARCH (EXTENSIVELY)

ASSAY OF VOLATILE OILS

CONTENT

GAS LIQUID CHROMATOGRAPHY:

INSTRUMENTATION

GAS LIQUID CHROMATOGRAPHY:

INSTRUMENTATION

Gc column ovens

GAS LIQUID CHROMATOGRAPHY:

PRINCIPLE AND THEORY

GLC USES A FLOW-THROUGH NARROW TUBE (COLUMN),

THRU WITH DIFFERENT CHEMICAL CONSTITUENTS OF THE SAMPLE PASS IN A GAS

THE FUNCTION OF THE

STATIONARY PHASE IN THE COLUMN IS TO SEPARATE THE DIFFERENT COMPONENTS.

STREAM (MOBILE PHASE) AT

DIFFERENT RATES, WITH THE STATIONARY PHASE.

THIS CAUSES EACH OF THE

COMPONENT TO EXIT THE COLUMN AT A DIFFERENT TIME (RT).

AS THE CHEMICAL EXIT AT THE END OF THE COLUMN, THEY

ARE DETECTED AND ARE

IDENTIFIED ELECTRONICALLY.

GAS LIQUID CHROMATOGRAPHY:

PRINCIPLE AND THEORY

OTHER PARAMETERS THAT CAN BE USED TO ALTER THE RT ARE:

CARRIER GAS FLOW RATE
TEMPERATURE

1. A KNOWN VOLUME OF GAS OR LIQUID ANALYTE IS INJECTED INTO THE ENTRANCE / HEAD OF THE COLUMN, USING;
MICROSYRINGE
GAS SOURCE SWITCHING SYSTEM

2. THE CARRIER GAS SWEEPS THE ANALYTE MOLECULES THRU THE COLUMN. 3. THE SWEEPING MOTION IS INHIBITED BY THE
ADSORPTION OF THE ANALYTE MOLECULES ONTO THE; COLUMN WALLS PACKING MATERIALS IN THE COLUMN

4. SINCE EACH TYPE OF MOLECULE HAS DIFFERENT PROGRESSION, THE VARIOUS COMPONENTS OF THE
ANALYTE MIXTURE ARE SEPARATED AS THEY PROGRESS THRU THE COLUMN, AT DIFFERENT TIMES

(RT). 6. A DETECTOR IS USED TO

MONITOR THE OUTLET

STREAM FROM THE COLUMN.

Gas liquid chromatography: detectors / INJECTORS

FLAME IONIZATION DETECTOR

SPLIT / SPLITLESS INJECTOR

Gas liquid chromatography: advantages

EXCELLENT SEPARATION, USUALLY LESS THAN 60 SECONDS.

GC CAN BE RUN 1,000 TIMES FASTER THAN LC.

SENSITIVITY & SPEED ARE EXTRAORDINARY, WITH 10 TO 12 GRAM SAMPLE BEING

DETECTABLE FOR MANY SUBSTANCES.

LARGER PREPARATIVE GLCS CAN BE USED

FOR PURIFICATION OF

SAMPLES.

MOLECULAR SIEVE / GEL CHROMATOGRAPHY: DEFINITION

THEORY 1. A COLUMN IS PREPARED,

OF TINY PARTICLES OF AN INERT SUBSTANCE THAT CONTAIN SMALL PORES.

A COMMON TYPE OF
PARTITION

CHROMATOGRAPHY IN WHICH SEPARATION 2. IF A SAMPLE SOLUTION OF COMPONENTS IS (CONTNG. MOLECULES OF BASED ON VARIOUS DIMENSIONS) IS MOLECULAR SIZE. PASSED THROUGH THE COLUMN, THE FF. MOVEMENTS HAPPEN:

MOLECULAR SIEVE / GEL CHROMATOGRAPHY: THEORY

A. MOLECULES LARGER THAN THE PORES MOVE ONLY IN THE SPACE BETWEEN THE PARTICLES.

AND ARE NOT RETARDED MATERIAL.

BY THE COLUMN

MOLECULAR SIEVE / GEL CHROMATOGRAPHY: THEORY

3. MOLECULES ARE ELUTED FROM THE COLUMN IN THE ORDER OF;

A. DECREASING SIZE
B. DECREASING MOLECULAR WEIGHT

(FOR RELATIVELY CONSTANT SHAPES GLOBULAR OR ROD)

MOLECULAR SIEVE / GEL

GEL CHROMATOGRAPHY: DIAGRAM

CHROMATOGRAPHY: THEORY

B. MOLECULES SMALLER THAN THE PORES DIFFUSE IN AND OUT OF PARTICLES (WITH PROBABILITY THAT IT INCREASE WITH DECREASING MOLECULAR SIZE).

THIS SLOWS DOWN THEIR

MOVEMENT THRU THE COLUMN.