05 Nov

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Published by Shwetapharma

CONTENT

▪ WHAT IS HPLC

▪ DISCOVERY OF HPLC

▪ TYPES OF HPLC

▪ PRINCIPLE OF HPLC

▪ PROCEDURE OR MECHANISM

▪ PARTS OF HPLC

▪ ADVANTAGES OF HPLC

•WHAT IS HPLC-

•

High-performance liquid chromatography(HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).

•DISCOVERY OF HPLC

•The term HPLC, coined by the Prof. Csaba Horváth for his 1970 Pittcon paper, originally indicated the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns.

• In the beginning, pumps only had a pressure capability of 500 psi [35 bar]. This was called high pressure liquid chromatography, or HPLC. The early 1970s saw a tremendous leap in technology.

• These new HPLC instruments could develop up to 6,000 psi [400 bar] of pressure, and incorporated improved injectors, detectors, and columns.

• HPLC really began to take hold in the mid-to late-1970s. With continued advances in performance during this time [smaller particles, even higher pressure], the term HPLC remained the same , but the name was changed to high performance liquid chromatography.

•High performance liquid chromatography is now one of the most powerful tools in analytical chemistry.

•

• It has the ability to separate, identify, and quantitative the compounds that are present in any sample that can be dissolved in a liquid. Today, compounds in trace concentrations as low as parts per trillion (ppt) may easily be identified.

•

• Now according to its performance and modernization of its part and the able to give precise result, that’s why today it is also called high precession liquid chromatography and UPLC.

•TYPES OF HPLC

1)Normal Phase HPLC:

This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform , diethyl ether , and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.

•2)Reverse Phase HPLC:

The stationary phase is non polar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.

PRINCIPLE OF HPLC

VAN DEEMTER EQUATION

H= HETP=A+ B/ ū + C x ū

A= EDDY’S DIFFUTION,

B= LONGITUDINAL DIFFUTION,

C= RESISTANCE TO MASS TRANSFER,

ū = AVARAGE MOBILE PHASE VELOCITY,

•HUBER AND KNOX EQUATION

• Huber pointed out that in relating plate height to mass transfer kinetic factors, need to add an additional term to account for mass transfer in both the stationary phase and mobile phase.

•H = A + B/ū + Cm ū + Cs ū

•

• Then KNOX develop an experimental equation for liquid chromatography that contain a term useful for correcting the deviation from the VAN DEEMPTER equation, containing the third root of velocity. The KNOX equation is usually expressed in the dimensionless reduce from since the physical meaning of the term is not clear.

•h = Av1/3 + B/v + C x v

•

• ▪ The term A is typically 1 or 2, with a large value representing a poorly bed; B is about 1.5 and C is 0.1. So a typical good column follows:

•h = v1/3 + 1.5/v + 0.1 x v

PROCEDURE OF HPLC

• The sample is introduced through an injection system into the entrance to the column

• The stationary phase is porous silica.

• Mobile phase can either be aq. Or org. solvent.

• In Interaction btw mobile and stationary phase, two types of scenarios can result.

PARTS OF HPLC

•SOLVENT RESERVOIR

•

Mobile Phase in NP-HPLC: –

Typically non polar solvents such as hexane , heptane , iso-octane are used in combination with slightly more polar solvents such as Isopropanol , ethyl acetate , or chloroform. Retention increases as the amount of non polar solvent increases in the mobile phase.

▪ Mobile phase in RP-HPLC: –

In reverse phase water is usually the base solvent. Other polar solvent such as Methanol , Acetonitrile or Tetrahydrofuran are added . pH is adjusted by buffers to modify separations of ionizable solutes.

•DEGASSER ▪

•

•Air in the pump will cause the pump to stop delivering mobile phase to the column. If only an occasional bubble is present, the flow rate will be erratic, causing retention time problems.

• Air in an optical detector, such as an ultraviolet (UV), fluorescence, or refractive index detector, will scatter light passing through the flow cell, causing noise spikes in the chromatogram or an off-scale signal.

•To get rid of these problems eliminated if the air is removed from the mobile phase is necessary. And that’s why degasser is use.

•SOLVENT DELIVERY SYSTEM (PUMP) ▪

•The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in millilitres per min (mL/min).

• Normal flow rates in HPLC are in the 1-3 mL/min range, pressure is about 2000 psi.

•Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar).

• During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

•INJECTOR

• Manual Injector:

1. User manually loads sample into the injector using a syringe

2. and then turns the handle to inject sample into the flowing mobile phase … which transports the sample into the beginning (head) of the column, which is at high pressure.

Auto sampler:

▪ User loads vials filled with sample solution into the auto sampler tray (100 samples) and the auto sampler automatically measures the appropriate sample volume, injects the sample, then flushes the injector to be ready for the next sample, etc., until all sample vials are processed.

•COLUMN

▪ The Columns are called “the heart of HPLC”

• Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.

There are several types of column use in HPLC according to their work:

A) Normal phase columns: Pours silica gel, polymer gel.

B) Reverse phase columns: Silica gel is coated with ODS, C18.

•DETECTORS ▪

The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column.

▪ Commonly used detectors are:

A) UV detector: If a compound can absorb ultraviolet light, a UV-absorbance detector is used.

B) Fluorescence Detector: Fluorescence detectors are very specific and selective among the others optical detectors. Use in the measurement of specific fluorescent species in samples.

C) Refractive Index Detector: Refractive index detector (RI or RID) is a detector that measures the refractive index of an analyte relative to the solvent. They are often used as detectors for high-performance liquid chromatography and size exclusion chromatography

•RECORDER

•A recorder, is a device that draws the chromatogram that results from a chromatographic process onto chart paper and provides a visual representation of the separation that has been achieve.

• The time scale of the chart movement normally ranges from about 1 cm per second to 1 cm per hour which can also be selected to suit the separation that is being carried out. Most modern chromatographs no longer employ recorders to present the chromatogram and the results are automatically processed by a computer and presented on the computer monitor or printed out as required.

APPLICATIONS OF HPLC

•HPLC can be used in both qualitative and quantitative applications, that is for both compound identification and quantification.

• ▪ Pharmaceutical Applications

•1. To control drug stability.

•2. Tablet dissolution study of pharmaceutical dosages form.

•3. Pharmaceutical quality control.

• ▪ Environmental Applications

•1. Detection of phenolic compounds in drinking water.

•2. Bio-monitoring of pollutants

•▪ Food and Flavour

•1. Measurement of Quality of soft drinks and water.

• 2. Sugar analysis in fruit juices.