High Performance Liquid Chromatography HPLC dr.hayder obayes hashim University of babylon College of pharmacy

Chromatography Mikhail Tsvet in 1900 plant pigments such as chlorophyll, carotenes, and xanthophylls. from Greek  chroma which means "color" and  graphein "to write.

chromatography The term chromatography is employed to describe a wide verity of separation techniques but all of these techniques share the same principle , this principle can be described as the difference in interaction properties of the analyte to two phases . One of these phases is relatively static and called stationary phase and the other is relatively mobile and called mobile phase All the chromatography type that have a liquid mobile phase are called liquid chromatography or LC. Mobile ph. Stationary ph Type of chromatography gas Gas- gas chromatography liqued Gas- liquid chromatography Solid Gas- solid chromatograpy Liquid Liquid – liquid chromatography solid Liquid- solid chromatography Solid – solid chromatography

Conventional LC system called low pressure chromatography in this method the movement of the mobile phase occur solely by gravity or by aid of low pressure pump . The simplest system is composed from: Mobile phase reserver The column A suitable detection method to monitor the content of the post column mobile phase .(usually it is UV/vis detector)

General View Of The Method Assume that we have a mixture of three compound as the following Compound A which has no interaction with the stationary phase Compound B which has a little interaction with stationary phase Compound C has a higher degree of interaction with the stationary phase

The action moods of stationary phase Adsorption Chromatography Normal phase (silica ) Reverse phase (C18, C8 , etc) Ion-exchange chromatography (IEC) Affinity chromatography Size-exclusion chromatography (SEC) Ion paring

THE CHROMATOGRAM AND ITS PURPORT Qualitative and quantitative values the retention factor or k value (may be better than retention time )

THE CHROMATOGRAM AND ITS PURPORT Two components in a mixture cannot be separated unless they have different k values, the means of assessment being provided by the separation factor, α, formerly known as the relative retention

THE CHROMATOGRAM AND ITS PURPORT Resolution is a term used to describe the degree of separation between neighboring solute bands or peaks. N, the number of theoretical plates, is one index used to determine the performance and effectiveness of columns, and is calculated using equation

Chromatographic efficiency Both of these parameters ( the long retention time and narrow peak width ) can be enhanced by increasing the surface area of the stationary phase

The born of HPLC The best way to increase the surface area of the stationary phase is by using a stationary phase composed of small particles . But the usage of small particles stationary phase develop a very high resistance to the movement of the mobile phase Hagen–Poiseuille equation To overcome this resistance a high pressure pump is used to force the mobile phase through this small particles stationary phase column . This kind of chromatography is called high pressure liquid chromatography or high performance liquid chromatography and abbreviated to HPLC .

Isocratic and gradient mobile phase Isocratic mobile phase: mean that the composition of the mobile phase ( type , concentration ) is unchanged in all over the chromatographic procedure . Gradient mobile phase : mean that the mobile phase composition ( type , concentration ) is changed during the chromatographic procedure . time min mobile A mobile B 30 70 2 5 80 20 15

HPLC System

HPLC systems Can be classified according to column diameters 1- Micro HPLC ( column diameters <2mm) 2- Analytical HPLC ( column diameters 3-5 mm) 3- Semi-Preparative HPLC ( column diameters 6-30 mm) 4- Preparative ( column diameters >30mm)

Analytical and Preparative HPLC systems

HPLC Detectors UV-VIS Detector : the most commonly used detector. Its response is specific to a particular compound or class of compounds depending on the presence of light absorbing functional groups of eluting molecules. It is cheap and versatile but unspecific .

HPLC Detectors Fluorescence Detector Greater sensitivity than a UV-VIS detector. However, the number of naturally fluorescent compounds is smaller in comparison to light absorbing compounds. This limitation is overcome by post or pre column derivatization.

HPLC Detectors Mass Spectroscopic Detector Very high sensitivity and selectivity. Detection is based on fragmentation of molecules by electric fields and separation on basis of mass to charge ratios of fragmented molecules. LC –MS technique has opened up new application areas due to advantages of resolution and sensitivity. Expensive and sophisticated

HPLC Detectors Diode Array Detector used to record the ultraviolet and visible (UV-vis) absorption spectra of samples. This enables qualitative information to be gathered about the samples. the ability to select the best wavelength for analysis. Spectra matching

Peak purity by diode array detector If a peak is pure all UV-visible spectra acquired during the peak’s elution or migration should be identical.

The Capability of HPLC We can detect the presence of certain compound or compounds in the sample . We can not detect unknown compound in the sample by conventional HPLC (UV, DAD , etc) The unknown compounds can be detected by LC-MS or preparative HPLC then diagnosed by other techniques . We can separate the compounds in the sample We can check the sample purity .

HPLC method We can develop and validate our method . this can be tedious and costly . The best choice is to use previously validated method . We have to choose the method according to our need ,material and devices availability .

What are the information that we have to know from the previously validated method to do our detection and separation by HPLC ? Sample treatment before the injection some samples need only filtration while the other sample need a specific extraction or derivatization or other treatment . The chromatography conditions : 1- The amount of the sample that have to be injected . 2- The mobile phase type , concentration ,gradient or isocratic , flow rate ,( we have to keep the linear flow rate ) Volumetric flow rate = Linear flow rate * Column cross area The column parameters 1- type of stationary phase 2- The size of the stationary phase particles And its pores size . 3- column length 4- the internal diameter of the column (ID) 5- the column temperature Detector parameters 1- wave length 2- sampling rate 3- constant time 4- time scheduled program Linearity , LOD ,LOQ

Qualitative HPLC analysis

Qualitative HPLC analysis

Quantitative Analysis External Standard Calibration

Internal standard calibration X axis values :Concentration of Ex st/concentration of (IS) Y axis values : The peak area of Ex st / peak area of IS

Internal standard calibration The Good internal standards compound have to be : Elute near to, but are well resolved from, the analyte of interest chemically and physically similar to the analyte Not present in the original sample mixture Unreactive towards any of the sample components Available in highly pure form Added in the concentration range 0.3 - 0.5 of the expected MAXIMUM analyte concentration .

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