WHAT IS CHROMATOGRAPHY ? PRINCIPLE AND OPERATION CLASSIFICATION OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY PAPER CHROMATOGRAPHY APPLICATION OF CHROMATOGRAPHY

1. CHROMATOGRAPHY
NAME : SAKHARELIYA SHUBHAM A.
ENROLLMENT NO. : 180470105047
BRANCH : CHEMICAL ENGINEERING
SUBJECT : ADVANCE SEPERATION PROCESS

2. CONTENTS
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✗ WHAT IS CHROMATOGRAPHY ?
✗ PRINCIPLE AND OPERATION
✗ CLASSIFICATION OF CHROMATOGRAPHY
✗ COLUMN CHROMATOGRAPHY
✗ PAPER CHROMATOGRAPHY
✗ APPLICATION OF CHROMATOGRAPHY

3. A TECHNIQUE FOR THE SEPARATION
OF A MIXTURE BY PASSING IT IN
SOLUTION OR SUSPENSION THROUGH
A MEDIUM IN WHICH THE
COMPONENTS MOVE AT DIFFERENT
RATES.
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WHAT IS CHROMATOGRPHY ?

4. PRINCIPLE AND OPERATION
✗ CHROMATOGRAPHY IS BASED ON THE PRINCIPLE WHERE
MOLECULES IN MIXTURE APPLIED ONTO THE SURFACE OR INTO THE
SOLID, AND FLUID STATIONARY PHASE (STABLE PHASE) IS
SEPARATING FROM EACH OTHER WHILE MOVING WITH THE AID OF A
MOBILE PHASE.
✗ THE FACTORS EFFECTIVE ON THIS SEPARATION PROCESS INCLUDE
MOLECULAR CHARACTERISTICS RELATED TO ADSORPTION (LIQUID-
SOLID), PARTITION (LIQUID-SOLID), AND AFFINITY OR DIFFERENCES
AMONG THEIR MOLECULAR WEIGHTS.
✗ BECAUSE OF THESE DIFFERENCES, SOME COMPONENTS OF THE
MIXTURE STAY LONGER IN THE STATIONARY PHASE, AND THEY
MOVE SLOWLY IN THE CHROMATOGRAPHY SYSTEM, WHILE OTHERS
PASS RAPIDLY INTO THE MOBILE PHASE, AND LEAVE THE SYSTEM
FASTER.
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5. STATIONARY PHASE
“THIS PHASE IS ALWAYS
COMPOSED OF A SOLID
PHASE OR A LAYER OF A
LIQUID ADSORBED ON THE
SURFACE SOLID SUPPORT”
EXAMPLE : CELLULOSE
SILICA
PRINCIPLE AND OPERATION
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MOBILE PHASE
“THIS PHASE IS ALWAYS
COMPOSED OF LIQUID OR
A GASEOUS COMPONENT”

6. CLASSIFICATION OF CHROMATOGRAPHY
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BASED ON PHYSICAL
STATE OF MOBILE PHASE
BASED ON PHYSICAL STATE
OF STATIONARY PHASE
GEL
CHROMATOGRAPHY
ION-EXCHANGE
CHROMATOGRAPHY
LIQUID
CHROMATOGRAPHY
CHROMATOGRAPHY
BASED ON TYPE OF
COLUMN
PARTITION
CHROMATOGRAPHY
ADSORPTION
CHROMATOGRAPHY
GAS
CHROMATOGRAPHY
COLUMN
CHROMATOGRAPHY
SHEET
CHROMATOGRAPHY

7. COLUMN CHROMATOGRAPHY
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8. COLUMN CHROMATOGRAPHY
✗ COLUMN CHROMATOGRAPHY IS A TECHNIQUE IN WHICH
SEPARATION IS CARRIED OUT IN THE COLUMN.
✗ THE COLUMN IS FILLED WITH A STATIONARY PHASE. THE
SAMPLE IS LOADED WITH THE MOBILE PHASE ON THE TOP OF
THE COLUMN.
✗ ONCE THE SAMPLE IS LOADED THE MOBILE PHASE IS
CONTINUOUSLY PASSED TO CAUSE SEPARATION.
✗ THE MOBILE PHASE MOVES DOWN TO THE COLUMN BECAUSE
OF GRAVITY.
✗ SEPARATION OF MOLECULES OCCURS DUE TO THEIR
DIFFERENCES IN INTERACTION WITH MOBILE PHASE AND THE
STATIONARY PHASE.
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9. COLUMN CHROMATOGRAPHY
✗ MOBILE PHASE – ELUENT
✗ SAMPLE LEAVING FROM THE COLUMN – ELUITE
✗ MOBILE PHASE LEAVING COLUMN – ELUATE
✗ THERE ARE TWO TYPES OF COLUMN PREPARATION METHOD
✗ WET METHOD – STATIONARY PHASE IS MIXED WITH A
MOBILE PHASE TO MAKE A SLURRY AND THEN PACKED IN
THE COLUMN.
✗ DRY METHOD – THE POWDER OF STATIONARY PHASE IS
DIRECTLY ADDED TO THE COLUMN .
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10. COLUMN CHROMATOGRAPHY
✗ APPLICATION OF COLUMN CHROMATOGRAPHY
✗ COLUMN CHROMATOGRAPHY IS USED TO ISOLATE ACTIVE
INGREDIENTS.
✗ IT IS VERY HELPFUL IN SEPARATING COMPOUND MIXTURES.
✗ IT IS USED TO DETERMINE DRUG ESTIMATION FROM DRUG
FORMULATIONS.
✗ IT IS USED TO REMOVE IMPURITIES.
✗ USED TO ISOLATION METABOLITES FROM BIOLOGICAL FLUIDS.
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11. PAPER CHROMATOGRAPHY
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12. PAPER CHROMATOGRAPHY
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✗ PAPER CHROMATOGRAPHY IS A CHROMATOGRAPHY TECHNIQUE,
THAT USES PAPER FOR THE SEPARATION PURPOSE.
✗ THE PAPER ACTS AS SOLID SUPPORTING PHASE.
✗ THE WATER MOLECULES IN THE PORES OF THE PAPER ACTS AS A
STATIONARY PHASE.
✗ MIXTURE OF DIFFERENT SOLVENTS CAN BE USED AS A MOBILE
PHASE.
✗ THERE ARE DIFFERENT MODES IN WHICH PAPER CHROMATOGRAPHY
IS CARRIED OUT, THESE ARE ASCENDING PAPER
CHROMATOGRAPHY, DESCENDING PAPER CHROMATOGRAPHY,
RADIAL PAPER CHROMATOGRAPHY AND 2D PAPER
CHROMATOGRAPHY.

13. PAPER CHROMATOGRAPHY
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✗ THE SAMPLE IS APPLIED ON PAPER USING A CAPILLARY.
✗ ONCE THE SAMPLE IS APPLIED THE CHROMATOGRAPHY CAN BE
CARRIED OUT IN DIFFERENT MODES.
✗ IN THE ASCENDING MODE THE MOBILE PHASE SOLVENT IS CAPPED
AT THE BOTTOM OF THE CHAMBER SMALL PORTION OF PAPER IS
IMMERSED IN THE MOBILE PHASE.
✗ THE SOLVENT IN THE MOBILE PHASE MOVES UPWARDS DUE TO THE
CAPILLARY ACTION DURING THIS PROCESS THE SAMPLE MOLECULES
GET SEPARATED SEPARATION OCCUR BECAUSE OF THE DIFFERENCE
IN THE SOLUBILITY OF MOLECULES IN THE MOBILE PHASE AND THE
STATIONARY PHASE.
✗ DRY PAPER AFTER EXPERIMENT IS CALLED AS CHROMATOGRAM.
✗ 𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
= 𝐸𝐴𝐶𝐻 𝑆𝑈𝐵𝑆𝑇𝐴𝑁𝐶𝐸 𝑀𝑂𝑉𝐸𝑆
𝑇𝑂 𝐼𝐷𝐸𝑁𝑇𝐼𝐹𝑌 𝐻𝑂𝑊 𝐹𝐴𝑅

14. PAPER CHROMATOGRAPHY
✗ APPLICATION OF PAPER CHROMATOGRAPHY
✗ SEPARATION OF AMINO ACIDS, PEPTIDES, ALKALOIDS, SUGARS,
LIPIDS ETC. IN BIOLOGICAL SAMPLES.
✗ TO LIMITED EXTENT AS A PREPARATIVE METHOD.
✗ TO STUDY THE STRUCTURE AND AMINO ACID COMPOSITION OF
PROTEINS.
✗ ANALYSIS OF BLOOD, HEMOGLOBIN, URINE ETC WHICH IS OF
GREAT DIAGNOSTIC VALUE.
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15. PHARMACEUTICAL SECTOR
✗ TO IDENTIFY AND ANALYZE SAMPLES FOR THE PRESENCE OF TRACE
ELEMENTS OR CHEMICALS.
✗ SEPARATION OF COMPOUNDS BASED ON THEIR MOLECULAR WEIGHT
AND ELEMENT COMPOSITION.
✗ DETECTS THE UNKNOWN COMPOUNDS AND PURITY OF MIXTURE.
✗ IN DRUG DEVELOPMENT.
APPLICATION OF CHROMATOGRAPHY
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FOOD INDUSTRY
✗ IN FOOD SPOILAGE AND ADDITIVE DETECTION
✗ DETERMINING THE NUTRITIONAL QUALITY OF FOOD

16. APPLICATION OF CHROMATOGRAPHY
CHEMICAL INDUSTRY
✗ IN TESTING WATER SAMPLES AND ALSO CHECKS AIR QUALITY.
✗ HPLC AND GC ARE VERY MUCH USED FOR DETECTING VARIOUS
CONTAMINANTS SUCH AS POLYCHLORINATED BIPHENYL (PCBS) IN
PESTICIDES AND OILS.
✗ IN VARIOUS LIFE SCIENCES APPLICATIONS
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FORENSIC SCIENCE
✗ IN FORENSIC PATHOLOGY AND CRIME SCENE TESTING LIKE
ANALYZING BLOOD AND HAIR SAMPLES OF CRIME PLACE.

17. APPLICATION OF CHROMATOGRAPHY
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MOLECULAR BIOLOGY STUDIES
✗ VARIOUS HYPHENATED TECHNIQUES IN CHROMATOGRAPHY SUCH
AS EC-LC-MS ARE APPLIED IN THE STUDY OF METABOLOMICS AND
PROTEOMICS ALONG WITH NUCLEIC ACID RESEARCH.
✗ HPLC IS USED IN PROTEIN SEPARATION LIKE INSULIN PURIFICATION,
PLASMA FRACTIONATION, AND ENZYME PURIFICATION AND ALSO IN
VARIOUS DEPARTMENTS LIKE FUEL INDUSTRY, BIOTECHNOLOGY, AND
BIOCHEMICAL PROCESSES.

18. Thanks!
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