HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

BYSAHITHI. GADDE

PHARMACEUTICS

(H.P.L.C)

• 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

SEPARATION TECHNIQUE

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

HPLCGC

ADVANTAGES OF HPLC OVER GC

Not limited by sample volatility or thermal stabilitySeparates both polar andnon 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

TYPES OF HPLC

1) BASED ON MODE OF SEPARATION Normal phase chromatography Reverse-phase chromatography Ion-pair chromatography Gel permeation chromatography Chiral chromatography2) BASED ON ELUTION TECHNIQUE Isocratic Gradient

3) BASED ON SCALE OF OPERATION Analytical HPLC Preparative HPLC4)BASED ON TYPE OF ANALYSIS Qualitative Quantitative

PRINCIPLE OF HPLC Differences in the interactions

between the solutes and stationary and mobile phases enable separation.

STATIONARY PHASES

• NORMAL PHASE CHROMATOGRAPHY (POLAR)

Silica, alumina

• REVERSED PHASE CHROMATOGRAPHY ( NON- POLAR) Octa Decyl Silica, C18, C8 etc…

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

INSTRUMENTATION

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)

GRADIENT ELUTION

low pressure gradient systems

high pressure gradient systems

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/minPerformance 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.

The different types of pumps used are

Syringe type pumps

Constant pressure pumps

Reciprocating piston pump

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

Manual injectorA fixed-volume loop of between 1 – 200 l (20 l is often used as standard)

Auto

samplers

Injection volumes of < 1uL to >1 mL is possible

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

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 columns3) narrow-bore columns4)Short , fast columns

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.

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 PARTICLEMACRO 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.

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

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)

SELECTION OF DETECTORS

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.

APPLICATIONSHPLC 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 bacteriaAssessment of toxicitiesFORENSICS:Identification of anabolic steroids in serum, urine, sweat and hair.Forensic analysis of textile dyes.Quantification of psychotherapeutic drugs in human plasma.

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.

ADVANTAGES1) 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,

THANK YOU…