RP-HPLC, EFFICIENCY PARAMETERS, RESOLUTION, COMPARISON OF SENSITIVITY, SELECTIVITY AND FIELD OF APPLICATIONS OF DETECTORS

Mr. Shrishail Pattadakal

Contents
 Introduction
 Types of HPLC  RP-HPLC  HPLC Parameters  Resolution  Conclusion  HPLC Detectors
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Introduction
H P L C
= High = Pressure (Performance) = Liquid = Chromatography

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What is HPLC ?
HPLC is a form of column chromatography used frequently in biochemistry and analytical chemistry. To separate, identify and quantify compounds. Separation technique involving mass-transfer between stationary and mobile phase.

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Types of HPLC
HPLC

NP-HPLC

RP-HPLC

Aq. NP-HPLC

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Normal Phase HPLC

    Using normal phase solvents : Most retained compounds are polar Least retained compounds are less polar More non-polar solvents make compounds retain longer

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Aqueous NP Chromatography
Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reverse-phase solvents.

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Reverse Phase HPLC

Solvents using : Most retained compounds are least polar Least retained compounds are most polar More polar solvents make compounds retain longer.

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Principle
Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)

Difference in analyte sorption affinities results in their separation

More polar analytes retained less Analytes with larger hydrophobic part are retained longer Almost no separation of structural isomers
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Parameters Used In HPLC

1) Retention parameter 2) Column efficiency parameter 3) Peak symmetric parameter

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Retention Parameters

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Efficiency Parameters

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Peak symmetry
S : Symmetry factor ( T : Tailing factor )

h x 0.05 f W0.05 W0.05 S= 2f

S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading

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Resolution

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Selectivity
Selectivity (a) is equivalent to the relative retention of the solute peaks and, unlike efficiency, depends strongly on the chemical properties of the chromatography medium. The selectivity, a, for two peaks is given by = k2 /k1 = V2 - V0/V1 V0 = V2/V1
where V1 and V2 are the retention volumes, and k2 /k1 are the capacity factors, for peaks 1 and 2 respectively, and V0 is the void volume of the column.

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Eluent Composition Effect on Selectivity

25 20

15

10

90% MeCN
0 1 2 3 4 5 6 7

18 16 14 12 10 8 6 4 2 0

80% MeCN
0 1 2 3 4 5 6 7

16 14 12 10 8 6 4 2 0

70% MeCN
0 1 2 3 4 5 6 7 8 9

12

10

60% MeCN

10

12

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Sensitivity
sensitivity was considered to define how small a mass or concentration of a solute could be unambiguously identified by a detector. This concept of sensitivity still persists, although it has been replaced in some cases by minimum detectable mass (MDM) or, alternatively, minimum detectable concentration (MDC). MDM or MDC are classically defined as the mass or concentration of solute passing through the detector that will give a signal equivalent to twice that of the noise. Originally the signal given by a detector for unit mass or unit concentration change was termed the detector response.

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HPLC Detectors

Requirements for an HPLC detector

Good sensitivity (high signal, low noise) No interference from mobile phase Must be able to work in a liquid phase environment

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Types of Detectors
Refractive Index UV-Visible Fluorescence Conductivity (for ion chromatography) Mass Spectrometry

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Types of HPLC Detectors

Fixed P Variable P UV-Vis Solute Property Electrochemical Photodiode Array Amperometry Pulse Amperometry Fluorescence Voltammetry Coulometry

Deflectance Type Refractive Index Bulk Property Conductivity Non-suppressed Reflectance Type Suppressed

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UV-Vis Absorbance Detector

UV Detectors measure the ability of a sample to absorb light. This can be accomplished at one or several wavelength.
Fixed wavelength measures at one wavelength, usually 254 nm.

Variable wavelength measures at one wavelength at a time, but can detect over a range of wavelengths.

 Diode Array measures a spectrum of wavelengths simultaneously.

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UV-Vis Absorbance Detector

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Fluorescent Detectors
Fluorescent detectors measures the ability of a compound to absorb then re-emit light at given wavelength. Each compound has a characteristic fluorescence. The excitation source passes through the flow-cell to a photo detector while a monochromator measures the emission wavelengths.
Has sensitivity limit of 10-9 to 10-11 gm/ml.
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Fluorosence

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Electrochemical Detector
It measure compounds that undergo oxidation or reduction reactions. Usually accomplished by measuring gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential. Has sensitivity of 10-12 to 10-13 gm/ml.
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Optical Detectors
There are mainly 4 types of detectors, 1) Circular Dichroism Chiral Detector. 2) Chiral Detector. 3) Chemiluminiscence Detector. 4) Refractive Index Detector.
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Circular Dichroism Chiral Detector

Simultaneous acquisition of UV factor signals. Direct determination of optical isomer separation & purity. Spectral scanning capability.

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Chiral Detectors
These are specially designed for optically active compounds.

Chemiluminescence Detector
Detection limits as demand increases for detection of trace amounts of lipids, nucleotides, nitrogen oxides & catachol amines.
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Refractive Index
Monitors are the closest to being universal HPLC detectors, as nearly all dissolved solutes alter the refractive index of the mobile phase.

They are differential detectors, generating a signal that depends on the difference between the RI of the pure mobile phase and the modified value caused by the dissolved solute.
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Instrumentation of Refractive Index

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Conclusion
HPLC is probably the most universal type of analytical procedure; its application areas include, quality control, process control, forensic analysis, environmental monitoring and clinical testing. In addition HPLC also ranks as one of the most sensitive analytical procedures and is unique in that it easily copes with multi-component mixtures. It has achieved this position as a result of the constant evolution of the equipment used in LC to provide higher and higher efficiencies at faster and faster analysis times with a constant incorporation of new highly selective column packings.

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Bibliography
 Separation Chemistry
international(p) Limited. R.P.Budhiraja, New age

 Analytical Chromatography

Gurudeep R. Chatwal Edited By Madhu Arora, Himalaya Publishing House

 www.google.com

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