High Performance(Pressure) Liquid Chromatography

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Contents
Liquid chromatography HPLC Principle of HPLC Instrumentation Stationary Phase Mobile phase Columns Detectors Applications Strengths Limitations Summary References

Liquid chromatography
Type of chromatography in which mobile phase is liquid. Carried out either in column or plane The placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) Where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.

Liquid Chromatography cont

Separation mechanism interactions. Collection of separated component at the exit of column Identification of separated components e.g by physical and chemical

Spectrophotometer
By another device that can measure their amount.

HPLC
HPLC is a form of liquid chromatography Used to separate compounds that are dissolved in solution. Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at different rates Due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

HPLC
o Improved performance
o High pressure HPLC- Separation is accomplished by partitioning b/w a M.P & Stationary column material. Packing material Small, uniform particle Gives high column efficiencies High pressure To achieve desired flow rates

Principle

Loading of analyte Stainless steel column containing particles of stationary phase

Separation of components

Monitoring of effluents

By variety of Detectors

Liquid mobile phase

Length of time spent in stationary phase

HPLC system

Instrumentation
o o o o o o o o A solvent reservoir Solvent Delivery System (Pump) Injector Sample Column Detectors (Diode Array) Waste Collector Recorder (Data Collection)

Instrumentation

Stationary Phase

Examples of stationary phases

ODS silica gel Octylsilane and butylsilane silica gels Phenyl silane silica gel Silica gel Aminopropyl silica gel Cyanopropyl silica gel Strong cation exchanger(SCX) Strong anion exchanger(SAX)

Mobile Phase

Types of High Performance Liquid Chromatography (HPLC):

Partition Adsorption (liquid-solid)

Ion exchange
Size exclusion

Gel permeation

Types of HPLC techniques

Based on Modes of chromatography 1. Normal phase mode: S.P is polar M.P is non polar 2. Reverse phase mode: S.P is non polar M.P is polar

Based on elution technique Isocratic separation Gradient separation Based on scale of operation
Analytical HPLC Preparative HPLC

Based on the type of Analysis

Qualitative analysis Quantitative analysis

Particle size of the S.P material plays a crucial role in HPLC Micro particulate column packing : Silica particles uniform, porous, with spherical or irregular shape Diameter 3.5 to 10 m

HPLC columns
The column is one of the most important component of the HPLC chromatograph. Separation of the sample components is achieved when those components pass through the column. Made up of stainless steel tubes Diameter of 3 to 5mm Length ranging from 10 to 30cm.

HPLC Column Cont

Normally, columns are filled with silica gel Particle shape, Surface properties, Pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, Inert to most compounds And has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible.

Picture of HPLC column

Elution of Neutral Compounds

Balance between its polarity and lipophilicity Will determine the time it takes for it to elute from an HPLC column. The pH of the mobile phase does not play a part. For reverse-phase column For polar columns

Elution of Neutral Compounds

HPLC Chromatogram

Elution Order of Steroids

Control of Elution Rate of Ionisable Compounds by Adjustment of pH of Mobile Phase

Only applicable to compounds in which the degree of ionization is dependent on pH This covers a majority of commonly used drugs The pH of the mobile phase can only be set within the range of ca 2-8.5 pH units The tendency for extremes of pH to dissolve silica gel and break the bonds between silanecoating agents and the silica gel support.

Effect of pH on Ibuprofen Separation

Acidic drug pKa=4.4 Stationary phase ODS Acetonitrile /0.1 M acetate buffer(60:40) Retention time at pH 4.2 23.32 mints Retention time at pH 5.2 12.23

Effect of pH on Bupivacaine Separation

Basic drug pKa 8.1 Stationary phase ODS Mobile phase acetonitrile/ TRIS buffer pH 8.4(40:60) Retention time at pH 8.4 17.32 min Retention time at pH 7.4 6.18 min

Detectors
1. UV DETECTOR : Based on UV light ab.
o fixed WL detector (254nm)

2. REFRACTIVE INDEX DETECTOR :

o Non specific / Universal detector o sensitivity & specificity

3. PHOTODIODE ARRAY DETECTOR (PDA)

o Similar to UV detector, non destructive o 190-600 nm for quantification & identification o Spectra is 3D, Response vs time vs WL

Photodiode Array Detector

Detectors cont
Flourimetric detector Excitation & emission WL can be selected sensitive than UV Disadvantage: Some comp. are not fluorescent Conductivity detector Based on electrical conductivity

Detectors cont
Evaporative Light Scattering Detector(ELSD): Based on the scattering of a beam of light by particles of compound remaining after evaporation of the mobile phase. For measuring Pharmaceuticals with Cl - and Na+ Sensitive to 10ng of analyte

Quantitative Analysis Using HPLC

1. Preparation of stock solution of Standard for the analyte. 2. Preparation of dilutions from the stock to produce a calibration series of solutions.

Quantitative Analysis Cont

3. The concentration of analyte which is expected in a
diluted extract from the sample is at approximately the mid-point of the range of concentrations prepared in the calibration series. 4. Injection of the calibration solutions into the HPLC system. 5.Preparation of the formulation for extraction, e.g. powder tablets, and weigh accurately portions of the prepared material.

Quantitative Analysis Cont

Extraction of the formulation. Filtration of the sample extract. Dilute this until its concentration falls at approximately the mid-point of the calibration series prepared using the analytical standard.

 Injection of the diluted sample solution into the HPLC system. Replicates of the sample preparation and of the injection of the sample in HPLC may be carried out; Sample preparation procedures are more likely to give rise to imprecision than instrumental variation

Quantitative Analysis Cont

Plotting of a calibration curve.

Quantitative Analysis Cont

Determine the concentration of the diluted sample extract from the calibration curve by substituting the area of its chromatographic peak into the equation for the calibration line.

Analysis of Paracetamol Tablets Using Calibration Curve

Tablets Tablets contain paracetamol 500 mg, phenylpropanolamine 5 mg.

Analysis of Paracetamol Cont

1. Weigh out 125 10 mg of the paracetamol standard and transfer it to a 250 ml volumetric flask made up to volume with acetic acid (0.05 M) and shake well (stock solution). 2. Prepare a series of solutions from the stock solution containing 0.5, 1.0, 1.5, 2.0 and 2.5 mg/100 ml of paracetamol. 3. Weigh and powder 20 tablets. 4. Weigh out tablet powder containing 125 mg 10 mg of paracetamol.

Analysis of Paracetamol Cont

5. Shake the tablet powder sample with ca 150 ml of acetic acid (0.05 M) for 5 min in a 250 ml volumetric flask and then adjust the volume to 250 ml with more acetic acid (0.05 M). 6.Filter ca 50 ml of the solution into a conical flask and then transfer a 25 ml aliquot of the filtrate to 100 ml volumetric flasks and adjust the volume to 100 ml with acetic acid (0.05 M).

Analysis of Paracetamol Cont

7. Take 10 ml of the diluted extract and transfer to a further 100 ml volumetric flask and make up to volume with 0.05 M acetic acid. Analyze the standards and the extract using the chromatographic conditions specified earlier.

8.

Analysis of Paracetamol

Analysis of Paracetamol

Analysis of Paracetamol

Strengths
Easily controlled and precise sample introduction ensures quantitative precision. HPLC has seen the most intensive development in recent years leading to improved, columns, detectors and software control. The variety of columns and detectors means that the selectivity of the method can be readily adjusted. Compared to GC there is less risk of sample degradation. Readily automated.

Limitations
There is still a requirement for reliable and
inexpensive detectors which can monitor compounds that lack a chromophore. Drugs have to be extracted from their formulations prior to analysis. Large amounts of organic solvent waste is generated, which is expensive to dispose of.

Summary
HPLC is a separation technique
The combination of HPLC with monitoring by UV/visible detection provides an accurate, precise and robust method for quantitative analysis of pharmaceutical products and is the industry standard method for this purpose. Monitoring of the stability of pure drug substances and in drugs in formulations with quantification of any degradation products. Measurement of drugs and their metabolites in biological fluids. Determination of partition coefficients and pKa values of drugs and of drug protein binding.

References
Pharmaceutical Analysis by DG Watson http://192.215.107.101/ebn/942/tech/techfocus/1071main.html http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998. http://weather.nmsu.edu http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/ HPLCHP1090/HPLCINJ.HTM http://testequipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/A nalytical_Instruments/Chromatographs/HPLC_Columns http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lccol.htm