A brief introduction to HPLCX its principle of working , types of column , detectors and more importantly, the caliberation and validation of HPLC its column, detector , volume of injection , oven tempreture etc

2.  
GROUP Members
 Rashid ullah
 Saba sabir
 Aiman mohsin
 Maryam anwar
 Zahra batool
 Asmat naz

3. AFTER STUDYING THIS TOPIC STUDENT SHOULD BE
ABLE TO :
 DEFINE HPLC
 DESCRIBE HPLC PRINCIPLE
 EXPLAIN MAJOR COMPONENTS OF HPLC AND THEIR
FUNCTION
 EXPLAIN APPLICATION OF HPLC
 STEPS INVOLVED IN VALIDATION AND CALIBRATION OF
HPLC
 DESCRIBE ADVANTAGES & DISADVANTAGES OF HPLC

4.  CHROMATOGRAPHY: ANALYTICAL TECHNIQUE
 CHROMATOGRAPH: INSTRUMENT
 CHROMATOGRAM: OBTAINED “PICTURE”
 CHROMATOGRAPHER: PERSON

5.  THE ANALYTE IS THE SUBSTANCE TO BE SEPARATED
DURING CHROMATOGRAPHY. IT IS ALSO NORMALLY WHAT
IS NEEDED FROM THE MIXTURE.
 THE ELUENT IS THE SOLVENT THAT CARRIES THE ANALYTE.
 AN IMMOBILIZED PHASE IS A STATIONARY PHASE THAT IS
IMMOBILIZED ON THE SUPPORT PARTICLES, OR ON THE
INNER WALL OF THE COLUMN TUBING.
 THE MOBILE PHASE MOVES IN A DEFINITE DIRECTION. IT
IS THE SOLVENT THAT MOVES THE SAMPLE/ ANALYTE
THROUGH THE COLUMN.

6.  PREPARATIVE CHROMATOGRAPHY IS USED TO PURIFY
SUFFICIENT QUANTITIES OF A SUBSTANCE FOR
FURTHER USE, RATHER THAN ANALYSIS.
 THE 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.
 THE THEORITICAL PLATES IS THE EFFICIENCY OF
COLUMN
 RESOLUTION PROVIDES A QUANTITATIVE MEASURE OF
THE ABILITY OF A COLUMN TO SEPARATE TWO
ANALYTES

7.  CHROMATOGRAPHY IN WHICH THE MOBILE PHASE IS
A LIQUID.
o THE LIQUID USED AS THE MOBILE PHASE IS
CALLED THE “ELUENT”.
 THE STATIONARY PHASE IS USUALLY A SOLID OR A
LIQUID.
 IN GENERAL, IT IS POSSIBLE TO ANALYZE ANY
SUBSTANCE THAT CAN BE STABLY DISSOLVED IN THE
MOBILE PHASE.

8.  Higher degree of separation!
 Refinement of packing material (3 to 10 µm)
 Reduction of analysis time!
 Delivery of eluent by pump
 Demand for special equipment that can
withstand high pressures
The arrival of high performance liquid chromatography!

9.  HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC) IS BASICALLY A
HIGHLY IMPROVED FORM OF COLUMN LIQUID
CHROMATOGRAPHY.
 THE MAIN PURPOSES FOR USING HPLC ARE FOR
SEPERATION, IDENTIFYING, QUANTIFYING AND
PURIFYING THE INDIVIDUAL COMPONENTS OF
THE MIXTURE.

10.  PRINCIPLE:
HPLC OPERATE UNDER THE SAME BASIC PRINCIPLE;
SEPARATION OF A SAMPLE INTO ITS CONSTITUENT
PARTS BECAUSE OF THE DIFFERENCE IN THE RELATIVE
AFFINITIES OF DIFFERENT MOLECULES FOR THE
MOBILE PHASE AND THE STATIONARY PHASE USED IN
THE SEPARATION.

11.  .

12.  BASED ON TYPES OF ANALYSIS
 QUANTITATIVE ANALYSIS
 QUALITATIVE ANALYSIS

13.  ADSORPTION CHROMATOGRAPHY
ADSORPTION CHROMATOGRAPHY IS BASED ON THE
INTERACTION BETWEEN THE SOLUTE MOLECULES AND ACTIVE
SITES ON THE STATIONARY PHASE. THIS ATTACHMENT OR
INTERACTION DEPENDS ON THE POLARITY OF SOLUTES.


14. BASED ON ION EXCHANGE
THE PRINCIPLE OF SEPERATION IS ION EXCHANGE WHICH IS
REVERSIBLE EXCHANGE OF FUNCTIONAL GROUP

15. SIZE EXCLUSION CHROMATOGRAPHY
SEPERATION BASED ON DIFFERENCE IN MOLECULAR SIZE
USING GEL THAT ACT AS MOLECULAR SEIVE

16.  AFFINITY CHROMATOGRAPHY
 AFFINITY OF SAMPLE WITH SPECIFIC STATIONARY PHASE

17.  CHIRAL CHROMATOGRAPHY
CHIRAL CHROMATOGRAPHY REFERS TO THE SEPARATION OF
ENANTIOMERS USING A CHIRAL HPLC COLUMN


18. Normal phase chromatography
STATIONARY PHASE
o POLAR
EXAMPLE
o SILICA, ALUMINA
MOBILE PHASE
o NON POLAR
EXAMPLE
o HEXANE ; HEPTANE mixed with
slightly polar solvent like
CHLOFORM OR ISOPROPANOL
etc
Reverse phase chromatography
Widely used in pharmaceutical
analysis
• STATIONARY PHASE
• NON POLAR
• EXAMPLE
• ODS SILICA GEL
• C18, C8
• MOBILE PHASE
• POLAR
• EXAMPLE
• WATER ; METHANOL; ACETONITRILE;
TETRAHYDROFURAN (THF)

19. Analytical hplc
 ONLY ANALYSIS OF
SAMPLE IS DONE
 RECOVERY OF SAMPLE
FOR RE USING IS NOT
DONE, SINCE THE
SAMPLE USED IS VERY
LOW.
Preparative hplc
 INDIVIDUAL COMPONENTS
OF PURE COMPOUNDS
CAN BE COLLECTED USING
FRACTIONAL COLLECTOR.
 THE COLLECTED SAMPLE
CAN BE REUSED

21. Isocratic
 A SEPARATION IN WHICH THE
MOBILE PHASE COMPOSITION
REMAINS CONSTANT
THROUGHOUT THE
PROCEDURE IS
TERMED ISOCRATIC.
 PEAK WIDTH INCREASES WITH
RETENTION TIME LINEARLY SO
LATE-ELUTING PEAKS GET
VERY FLAT AND BROAD.
gradient
 A SEPARATION IN WHICH
THE MOBILE PHASE
COMPOSITION IS
CHANGED DURING THE
SEPARATION PROCESS IS
DESCRIBED AS A GRADIENT
ELUTION.
 GRADIENT ELUTION
DECREASES THE RETENTION
OF THE LATER-ELUTING
COMPONENTS SO THAT THEY
ELUTE FASTER, GIVING
NARROWER (AND TALLER)
PEAKS FOR MOST
COMPONENTS.

23. 1. SOLVENT RESERVOIR
2. PUMPS
3. SAMPLE INJECTION SYSTEM
4. COLUMNS
5. DETECTORS
6. DATA PROCESSING
7. WASTE

24. • RESERVOIR FOR MOBILE PHASE THAT CARRY
SAMPLE INTO THE COLUMN

25.  PROBLEMS CAUSED BY DISSOLVED AIR IN THE
ELUENT
o UNSTABLE DELIVERY BY PUMP
o MORE NOISE AND LARGE BASELINE DRIFT IN
DETECTOR CELL
IN ORDER TO AVOID THESE PROBLEMS, THE ELUENT
MUST BE DEGASSED.

26.  TO PRODUCE AN APPROPRIATE PRESSURE TO PUSH
SOLVENT INTO THE SAMPLE.
 A PUMP CAPABLE OF PUMPING SOLVENT UP TO A
PRESSURE OF 4000-6000 PSI AND AT FLOW RATE
RANGING FROM 0.1- 10 ML/MIN.
 MOSTLY PHARMACEUTICAL ANALYSIS IS CARRIED
OUT AT 5000PSI AND FLOWRATE OF 0.5-3ML/MIN.

27.  BY SYRINGE :, IT MAY BE
o MANUAL
o AUTOINJECTOR
 A FIXED-VOLUME LOOP OF BETWEEN 1 – 100 L (20
L IS OFTEN USED AS STANDARD) IS INJECTED VIA
SAMPLE VALVE .

28.  GUARD COLUMNS ARE INSTALLED BETWEEN THE
INJECTOR AND THE ANALYTICAL COLUMN OF A HPLC
SYSTEM, MAINLY TO PROTECT ANALYTICAL COLUMNS.
 GUARD COLUMNS ARE WIDELY USED AS A COST
EFFECTIVE FOR PROLONG HPLC COLUMN LIFE.

29. o STRAIGHT, 100 TO 250 MM IN LENGTH
o 2 TO 6 MM INNER DIAMETER.
o PACKING - SILICA GEL, ALUMINA, CELITE ETC
o COLUMN ARE SELECTED ACCORDING TO THE NATURE OF
COMPOUND TO BE ANALYZED AND THE MOBILE PHASE.
o COLUMN PERFORMANCE SHOULD BE EVALUATED TIME TO
TIME.
o C8 AND C18 COLUMNS ARE USED
o C-18 IS USUALLY BETTER FOR SEPARATING OUT COMPOUNDS
LIKE LONG CHAIN FATTY ACIDS (SAY OLEIC ACID), RETAIN NON
POLAR COMPOUNDS AS COMPARED TO A RELATIVELY SMALL
ORGANIC COMPOUND (SAY BUTYRIC ACID).

30. SPECIFIC DETECTORS
UV/VIS
FLUORESCENCE
MASS DETECTOR
DIODE ARRAY DETECTOR
IR DETECTOR
BULK PROPERTY
ELECTROCHEMICAL DETECTOR
OPTICAL ROTATION DETECTOR
REFRACTIVE INDEX

31.  PROCESSING IS DONE USING SPECIFIC SOFTWARE
THAT IS CONNECTED TO HPLC MACHINE.
 RECEIVE THE INFORMATION FROM HPLC MACHINE
AND PRESENT IT AS A GRAPH.
 THE GRAPH DESCRIBES ABOUT QUALITATIVE DATA
(RETENTION TIME) AND QUANTITATIVE DATA (AREA
UNDER CURVE).

33. DEFINITION
VALIDATION IS ESTABLISHING A DOCUMENTED
EVIDENCE WHICH PROVIDES A DEGREE OF
ASSURANCE THAT A SPECIFIC PROCESS WILL
CONSISTENTLY PRODUCE A PRODUCT MEETING ITS
PRE-DETERMINED SPECIFICATIONS AND QUALITY
ATTRIBUTE.

34. PURPOSE OF VALIDATION
THE PRINCIPLE PURPOSE OF ANALYTICAL
VALIDATION IS TO ENSURE THAT THE SELECTED
ANALYTICAL PROCEDURE WILL GIVE REPRODUCIBLE
AND RELIABLE RESULTS THAT ARE ADEQUATE FOR
THE INTENDED PURPOSE.

37.  DEFINITION (ICH)
o THE DEMONSTRATION THAT A PARTICULAR
INSTRUMENT OR DEVICE PRODUCES RESULTS
WITH IN SPECIFIED LIMITS BY COMPARISION
WITH THOSE PRODUCED BY A REFERENCE OR
TRACEABLE STANDARD OVER AN APPROPRIATE
RANGE OF MEASUREMENTS.

41. ADVANTAGES
 SEPARATION OF VOLATILE AND NON VOLATILE
COMPONENTS
 QUICK ANALYSIS
 HIGH RESOLUTION
 MORE REPRODUCIBILITY
DISADVANTAGES
 HIGH COST
 COMPLEX TO OPERATE