about hplc

1. HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
BY
SAHITHI. GADDE
PHARMACEUTICS
(H.P.L.C)

2. • HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
• Originally referred to as High-Pressure Liquid
Chromatography
• Now more commonly called High
Performance Liquid Chromatography
• HPLC is really the automation of traditional
liquid chromatography under conditions which
provide for enhanced separations during shorter
periods of time, utilizing very small particles,
small column diameters, and very high fluid
pressures.
INTRODUCTION

3. SEPARATION TECHNIQUE
I have two separation techniques:
HPLC and GC…… which
should I
use…………….????????????
HPLC
GC

4. ADVANTAGES OF HPLC OVER GC
Not limited by sample volatility or thermal
stability
Separates both polar and
non polar compounds
 Needs a small sample with a high accuracy and
precision
 Non-destructed sample during operation
compared to GC.
Room temperature analysis
Ease of sample recovery

5. TYPES OF HPLC
1) BASED ON MODE OF SEPARATION
 Normal phase chromatography
 Reverse-phase chromatography
 Ion-pair chromatography
 Gel permeation chromatography
 Chiral chromatography
2) BASED ON ELUTION TECHNIQUE
 Isocratic
 Gradient

6. 3) BASED ON SCALE OF OPERATION
 Analytical HPLC
 Preparative HPLC
4)BASED ON TYPE OF ANALYSIS
 Qualitative
 Quantitative

7. PRINCIPLE OF HPLC
Differences in the interactions between the
solutes and stationary and mobile phases
enable separation.

8. STATIONARY PHASES
• NORMAL PHASE CHROMATOGRAPHY
(POLAR)
 Silica, alumina
• REVERSED PHASE
CHROMATOGRAPHY ( NON- POLAR)
 Octa Decyl Silica, C18, C8 etc…

10. MOBILE PHASES
 NORMAL CHROMATOGRAPHY ( non- polar )
Hexane, Dichloromethane, Iso-propanol, Methanol
 REVERSED PHASE CHROMATOGRAPHY (
polar )
Water, Methanol, Aceto-nitrile, Tetra hydro
furan(THF)
Increasing strength
Increasing strength

11. INSTRUMENTATION

13. MOBILE PHASE DELIVERY
SYSTEM
• Isocratic elution
--- Eluent composition remains constant
--- Single solvent or single solvent mixture
• Gradient elution: ( mostly used)
--- Eluent composition (and strength)
changed
--- Increases separation efficiency
--- Decreases retention time
--- Peak shape is improved (Less tailing)

14. GRADIENT ELUTION
 low pressure gradient systems

15.  high pressure gradient systems

16. PUMPS
To produce an appropriate pressure to push solvent into
the sample.
A pump capable of pumping solvent up to a pressure of
4000 psi and at flows of up to 10 ml/min
Performance Requirements
Capacity to withstand high load pressures.
Pulsations that accompany pressure fluctuations are
small.
Flow rate does not fluctuate.
Solvent replacement is easy.
The flow rate setting range is wide and the flow rate is
accurate.

17. The different types of pumps used are
Syringe type pumps
Constant pressure pumps
Reciprocating piston pump

18. SAMPLE
INTRODUCTION
Performance Requirements
– No sample remaining in unit
– Minimal broadening of sample band
– Free adjustment of injection volume
– Minimal loss
– Superior durability and pressure resistance
Sample introducing systems are two
types:
– Manual injector system
– Auto-sampler

19. Manual injector
A fixed-volume loop of between 1 – 200 l (20 l is often used as standard)
Autosamplers Injection volumes of
< 1uL to >1 mL is
possible

20. COLUMNS
Columns are constructed from smooth bore
stainless steel tubing , heavy walled glass
tubing such as poly ether ether ketone(PEEK)
to with stand high pressures.
Columns are of two types:
• Guard columns
• Separation / analytical columns

21.  GUARD COLUMN:
it is introduced before the analytical column to increase the life of the analytical
column by removing not only particulate matter and contaminants from the solvents but
also sample components that bind irreversibly to the stationary phase.
Composition should be similar to the analytical column.
 SEPARATION / ANALYTICAL COLUMNS:
most of the columns range from 10 to 30 cm. straight columns are used.
Packed columns are also available. Different types of columns used are:
1)Standard columns ( id:4-5mm, particle diameter; 3-5um)
2)Radial compression columns
3) narrow-bore columns
4)Short , fast columns

22. Separation columns should be housed with a
stable system with temperature variations of
less than 0.1oc when temperature changes must
be avoided.
Circulating air baths or electrically heated
chambers are used to control the column
temperature.
Solvent is pre heated before entering the
separation column.

23. STRUCTURAL TYPES OF
COLUMN PACKINGS
The stationary phase may be either a totally porous particle ( macro porous
polymer) or a superficially porous support ( porous layer beads or pellicular
supports) either of these types may have a polymer bonded to its surface.
POROUS LAYER BEADS:
Particle dia: 30-40um, outer shell 1-3um thick may be silica gel layer, surface area
ranges from 5 to 15m2 /g.
POROUS PARTICLE
MACRO POROUS POLYMERS:
Macroporous styrene divinyl benzene polymers have large ion channels in addition
to micro pores which offers the ions easy access to the functional groups of the
exchanger.

24. OPTIMISATION OF COLUMN
EFFICIENCY
Effect of temperature (increased temperature results in decreased mobile phase
viscosity, increased mass transfer, increased sample solubility resulting in either better
resolution and faster analysis)
Pressure drop ( it varies inversely with retention time. Increase in pressure
increase maximum attainable plate number but generation of heat with in the
column at very high pressure degrades column efficiency)
Particle diameter of stationary phase ( it is directly proportional to analytical
performance)
Column length (plate count directly proportional to the column length)
Viscosity ( low viscosity is preferred. Increase in viscosity decreases the flow rate)
Extra column band broadening

25. DETECTORS
 Ultra violet / visible detector (UV-VIS)
 Photo diode array detector (PDA)
Fluorescence detector (FL)
 Conductivity detector (CDD)
 Refractive index detector (RF)
 Electro chemical detector (ECD)
 Evaporative light scattering detector (ELSD)
 Mass spectrometer detector (MS)

26. SELECTION OF DETECTORS

27. The mostly used detector is PDA detector
 It could analyze samples simultaneously at different wavelengths.
 Relatively robust to temperature and flow rate fluctuations.
 Compatible with gradient elution.

28. APPLICATIONS
HPLC is one of the most widely applied analytical separation techniques…
PHARMACEUTICAL:
Tablet dissolution of pharmaceutical dosages.
Shelf life determination of pharmaceuticals.
Identification of counterfeit drug products.
Pharmaceutical quality control.
ENVIRONMENTAL:
Phenols in drinking water.
Identification of diphenhydramine in sediment samples.
Toxicity of tetracycline's and tetracycline degradation products to
environmentally relevant bacteria
Assessment of toxicities
FORENSICS:
Identification of anabolic steroids in serum, urine, sweat and hair.
Forensic analysis of textile dyes.
Quantification of psychotherapeutic drugs in human plasma.

29. CLINICAL:
Analysis of antibiotics.
Increased urinary excretion of aquaporin 2 in patients with liver
cirrhosis.
Detection of endogenous neuro peptides in brain extracellular
fluids.
FOOD AND FLAVOUR:
Ensuring soft drink consistency and quality.
Analysis of vicinal diketones in beer.
Sugar analysis in fruit juices.
Polycyclic aromatic hydrocarbons in brazilian vegetable and fruits.
Stability of aspartame in the presence of glucose and vanillin.

30. ADVANTAGES
1) Separation fast and efficient.
2) Continuous monitoring of the column effluent.
3) Can be used for separation of various complex mixtures.
4)Accurate quantitative measurements.
5)Repetitive and reproducible analysis using same column.
6) Both aueous and non aqueous samples can be analyzed
with little or no sample pretreatment.
7) A variety of samples and column packing are available,
providing a high degree of selectivity for specific analysis.
8) It provides a means for determination of multiple
components in a single analysis,