This lecture presents an introduction to the beginner user on the usage of high-performance liquid chromatography. The main topics are; selecting a buffer solution, and the stationary & mobile phases.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and schematic of an HPLC system. It also summarizes key differences between thin layer chromatography (TLC) and HPLC. The document then covers HPLC theory, proposed reverse phase mechanisms, column selection guidelines, buffers, ion pair reagents, common stationary phase properties, detectors, and system suitability parameters.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and setup of an HPLC instrument. It also compares HPLC to thin layer chromatography. The document explains reverse phase and normal phase HPLC, proposed mechanisms, column selection, mobile phase preparation including buffers, ion pair reagents, column properties, and common detectors. It provides guidelines for handling HPLC including preparation of samples, standards and mobile phases, and assessing system suitability.
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).
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography and describing the basic principles of HPLC. It then discusses the types of HPLC separations based on modes, principles, elution techniques, scale of operation, and type of analysis. The document also covers the principles, types, and advantages of liquid chromatography. It provides details on the instrumentation of HPLC including solvent reservoirs, degassing, pumps, injectors, columns, detectors, and data handling. In summary:
HPLC is a type of column chromatography used to separate mixtures by distributing components between a stationary and mobile phase. It can be used for qualitative and quantitative analysis. The document outlines the various components of an HPLC system
High Performance Liquid Chromatography (HPLC) is a technique used to separate compounds dissolved in solution using high pressure to push a mobile phase through a column containing a stationary phase. HPLC instruments consist of pumps to deliver the mobile phase, an injector to introduce the sample, a separation column, and detectors. Compounds are separated based on differences in how they partition between the mobile and stationary phases. HPLC provides high resolution separation of complex mixtures and is characterized by its reproducibility, high pressure operation, and ability to use small particle sizes in the column.
High performance liquid chromatography (HPLC) is a technique used to separate, identify, and quantify compounds in a sample. It works by pumping a sample mixture through a column containing chromatographic packing material under high pressure. The discovery of HPLC in the 1970s allowed for higher pressures and improved performance over traditional liquid chromatography. HPLC is now widely used in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and schematic of an HPLC system. It also summarizes key differences between thin layer chromatography (TLC) and HPLC. The document then covers HPLC theory, proposed reverse phase mechanisms, column selection guidelines, buffers, ion pair reagents, common stationary phase properties, detectors, and system suitability parameters.
This document provides information about handling and operating high performance liquid chromatography (HPLC). It discusses the basic components and setup of an HPLC instrument. It also compares HPLC to thin layer chromatography. The document explains reverse phase and normal phase HPLC, proposed mechanisms, column selection, mobile phase preparation including buffers, ion pair reagents, column properties, and common detectors. It provides guidelines for handling HPLC including preparation of samples, standards and mobile phases, and assessing system suitability.
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).
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography and describing the basic principles of HPLC. It then discusses the types of HPLC separations based on modes, principles, elution techniques, scale of operation, and type of analysis. The document also covers the principles, types, and advantages of liquid chromatography. It provides details on the instrumentation of HPLC including solvent reservoirs, degassing, pumps, injectors, columns, detectors, and data handling. In summary:
HPLC is a type of column chromatography used to separate mixtures by distributing components between a stationary and mobile phase. It can be used for qualitative and quantitative analysis. The document outlines the various components of an HPLC system
High Performance Liquid Chromatography (HPLC) is a technique used to separate compounds dissolved in solution using high pressure to push a mobile phase through a column containing a stationary phase. HPLC instruments consist of pumps to deliver the mobile phase, an injector to introduce the sample, a separation column, and detectors. Compounds are separated based on differences in how they partition between the mobile and stationary phases. HPLC provides high resolution separation of complex mixtures and is characterized by its reproducibility, high pressure operation, and ability to use small particle sizes in the column.
High performance liquid chromatography (HPLC) is a technique used to separate, identify, and quantify compounds in a sample. It works by pumping a sample mixture through a column containing chromatographic packing material under high pressure. The discovery of HPLC in the 1970s allowed for higher pressures and improved performance over traditional liquid chromatography. HPLC is now widely used in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
HPLC (High Performance Liquid Chromatography) is a separation technique used to separate, identify, and quantify compounds in mixtures. It works by injecting samples into a column with a stationary phase and passing a liquid mobile phase through under high pressure. Compounds are separated based on how they partition between the mobile and stationary phases. HPLC is useful for pharmaceutical analysis, clinical applications, chemical separations, and purification of compounds due to its high resolution, sensitivity, repeatability, and ability to separate both volatile and non-volatile compounds.
This document discusses high performance liquid chromatography (HPLC). It begins by providing background on the founder of liquid chromatography, Mikhail Tsvet. It then describes the basic concepts of HPLC including qualitative and quantitative analysis using retention time and peak area/height comparisons. The document outlines the types of HPLC including partition, adsorption, ion exchange, size-exclusion, and affinity chromatography. It also describes the various components of an HPLC system including the solvent system, injection valve, column, and detector.
High performance liquid chromatography (HPLC) is described. HPLC is a chromatographic technique used to separate mixtures of compounds. It involves pumping a pressurized mobile phase through a column containing a stationary phase. Compounds in a sample injected into the mobile phase are separated as they are carried through the column at different rates depending on their interactions with the stationary phase. HPLC is useful for both qualitative and quantitative analysis and has advantages such as high resolution, sensitivity, accuracy, and reproducibility. Common applications and components of an HPLC system are also outlined.
The document discusses High Performance Liquid Chromatography (HPLC). It begins by defining chromatography and describing the basic principles of HPLC. It then discusses the types of HPLC separations based on factors like the stationary and mobile phases used. The document also outlines the typical instrumentation used in HPLC, including solvent reservoirs, pumps, columns, detectors, and data collection systems. It highlights advantages of HPLC like high resolution, sensitivity, and reproducibility.
High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by pumping a sample mixture through a column containing chromatographic packing material at high pressure. The sample components interact differently with the stationary phase in the column, causing them to elute out at different rates and allowing separation. HPLC has many applications in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics. It provides a powerful analytical tool for identifying and quantifying compounds in samples.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, history, types, instrumentation, procedure, advantages, disadvantages and applications of HPLC. Key points include that HPLC uses high pressure to separate compounds faster and more efficiently than other chromatography techniques. Silica is commonly used as the stationary phase because it can withstand pressures over 300-400 atmospheres. The major components of HPLC instrumentation are the solvent reservoir, pump, injector, column, detector and data collector. HPLC has various applications in fields like analytical chemistry, pharmaceuticals, forensics and environmental analysis.
HPLC Principles,Operations, Maintenance and troubleshoting.pdfBALASUNDARESAN M
This document discusses the principles and operation of high performance liquid chromatography (HPLC). It covers basic HPLC instrumentation configuration and separation modes, including reversed phase and normal phase systems. Methods for converting between normal and reversed phase systems are provided. Various HPLC detectors are outlined, including UV-Vis, PDA, and ELSD detectors. The document also discusses HPLC maintenance strategies and preventative maintenance for individual modules. Good laboratory practices for HPLC are reviewed, such as solvent preparation, filtration, and buffer preparation. Finally, common problems and their solutions are addressed.
HPLC is a liquid chromatography technique used to separate compounds in a solution. It works by exploiting differences in how compounds partition between a stationary phase and mobile phase. There are four main types: partition, ion exchange, size exclusion, and affinity chromatography. HPLC systems consist of solvent reservoirs, pumps, injectors, columns, detectors, and data acquisition components. HPLC is used for research, quality control, environmental monitoring, and regulatory purposes to analyze complex mixtures and isolate compounds.
This document discusses high performance liquid chromatography (HPLC) for analyzing oils and fats. It explains that HPLC involves separating components based on how they are distributed between a liquid mobile phase and solid stationary phase. There are two main types of mobile phases used: isocratic elution with a fixed composition and flow rate, and gradient elution where the composition and flow rate change over time to improve separation. Reverse phase HPLC is most commonly used for oil and fat analysis, where nonpolar stationary phases are used and hydrophilic compounds elute more quickly than hydrophobic compounds.
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. High-performance liquid chromatography (HPLC) uses high pressure to pass a solvent or solvent mixture through a column containing a stationary phase to separate components in a mixture. HPLC consists of several major components including a pump, injector, column, column compartment, detector, and degasser. The injector introduces the sample into the mobile phase which passes through the column, allowing separation based on interactions between components and the stationary phase. A detector then measures and records separated components as they elute from the column.
1. The document discusses high performance liquid chromatography (HPLC), including its principles, types, instrumentation, and applications. HPLC is a technique used to separate compounds in a mixture using high pressure to force the mixture through a column packed with a stationary phase.
2. The key components of an HPLC system are the solvent reservoirs, pump, injector, column, and detector. HPLC can be used for both analytical and preparative purposes to separate, purify, identify, and quantify compounds.
3. Common applications of HPLC include separation of volatile and non-volatile compounds, qualitative and quantitative analysis, and determination of retention times. Reversed phase HPLC using C18 columns is frequently utilized.
HPLC is a form of liquid chromatography that uses pumps to pass a pressurized mobile liquid phase through a column packed with solid particles. This allows the components of a dissolved sample to be separated as they are transported through the column at different rates depending on their interactions with the stationary and mobile phases. HPLC instruments consist of a pump, injector, column, and detector. Separation is based on the partitioning of compounds between the mobile and stationary phases, and detectors are used to measure separated components as they exit the column. HPLC provides efficient, sensitive, and high-pressure separations of sample mixtures.
Chromatography is a technique used to separate mixtures by distributing compounds between a stationary and mobile phase. High-performance liquid chromatography (HPLC) is commonly used and separates compounds using a column with a stationary phase and liquid mobile phase. HPLC can identify, detect, quantify, and purify individual components in a mixture using an apparatus including a pump, injector, column, detector, and recorder. The separation occurs as the compounds interact differently with the stationary phase in the column.
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography as a technique used to separate mixtures into their individual components using both a stationary and mobile phase. It then describes some key aspects of HPLC, including that it uses high pressure to force the mobile phase through a column with small particle sizes for better separation. The document outlines the basic components of an HPLC system, including the pump, injector, column, detectors, and computer. It also discusses some common terms and uses for HPLC, such as separating and analyzing compounds in research, quality control, and environmental monitoring.
The document provides an introduction and history of high performance liquid chromatography (HPLC). It discusses the components of an HPLC system including the pump, injector, column, detectors, and data collection. It describes the different types of chromatography, stationary and mobile phases used, and parameters for evaluating chromatographic separation such as capacity factor, resolution, asymmetry factor, and efficiency.
This document discusses various chromatography techniques including high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), ultra-performance liquid chromatography (UPLC), and rapid resolution liquid chromatography (RRLC). HPLC uses pumps to pass a pressurized liquid through a column to separate sample components. FPLC is a modified HPLC used for protein separations using aqueous buffers and resins. UPLC uses smaller particle columns (<2μm) than HPLC to improve resolution, speed, and sensitivity. RRLC also uses sub-2μm particles and high flow rates to achieve faster analysis times than HPLC while maintaining resolution.
1. Chromatography involves separating mixtures based on differences in how components distribute themselves between a stationary and mobile phase.
2. Common stationary phases include silica gel, alumina, and ion exchange resins. The interaction between the stationary phase and components leads to separation as they travel through the column at different rates.
3. Types of chromatography covered include liquid column chromatography, high performance liquid chromatography (HPLC), and ion exchange chromatography. Key applications are separating pharmaceuticals, proteins, food/industrial chemicals, and pollutants.
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High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
High performance liquid chromatography (HPLC) is summarized as follows:
HPLC is a technique used to separate mixtures by distributing the components between a stationary and mobile phase. It can be used for both qualitative and quantitative analysis. HPLC utilizes high pressure pumps to pass a mobile phase through a column packed with adsorbent particles, allowing separation of components based on differences in their partitioning behavior between the mobile and stationary phases. Common detectors used in HPLC include UV/Vis, refractive index, fluorescence, and mass spectrometry.
High performance liquid chromatography (HPLC) is described, including the basic principles and components of HPLC systems. HPLC uses high pressure to pass a liquid mobile phase through a column packed with solid adsorbent particles or porous beads. This allows for separation of mixtures based on differences in how components partition between the stationary and mobile phases. Key components reviewed are the solvent reservoirs, pump, injector, column, and detectors. Common applications of HPLC mentioned are qualitative and quantitative analysis of both volatile and non-volatile compounds.
HPLC (High Performance Liquid Chromatography) is a separation technique used to separate, identify, and quantify compounds in mixtures. It works by injecting samples into a column with a stationary phase and passing a liquid mobile phase through under high pressure. Compounds are separated based on how they partition between the mobile and stationary phases. HPLC is useful for pharmaceutical analysis, clinical applications, chemical separations, and purification of compounds due to its high resolution, sensitivity, repeatability, and ability to separate both volatile and non-volatile compounds.
This document discusses high performance liquid chromatography (HPLC). It begins by providing background on the founder of liquid chromatography, Mikhail Tsvet. It then describes the basic concepts of HPLC including qualitative and quantitative analysis using retention time and peak area/height comparisons. The document outlines the types of HPLC including partition, adsorption, ion exchange, size-exclusion, and affinity chromatography. It also describes the various components of an HPLC system including the solvent system, injection valve, column, and detector.
High performance liquid chromatography (HPLC) is described. HPLC is a chromatographic technique used to separate mixtures of compounds. It involves pumping a pressurized mobile phase through a column containing a stationary phase. Compounds in a sample injected into the mobile phase are separated as they are carried through the column at different rates depending on their interactions with the stationary phase. HPLC is useful for both qualitative and quantitative analysis and has advantages such as high resolution, sensitivity, accuracy, and reproducibility. Common applications and components of an HPLC system are also outlined.
The document discusses High Performance Liquid Chromatography (HPLC). It begins by defining chromatography and describing the basic principles of HPLC. It then discusses the types of HPLC separations based on factors like the stationary and mobile phases used. The document also outlines the typical instrumentation used in HPLC, including solvent reservoirs, pumps, columns, detectors, and data collection systems. It highlights advantages of HPLC like high resolution, sensitivity, and reproducibility.
High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by pumping a sample mixture through a column containing chromatographic packing material at high pressure. The sample components interact differently with the stationary phase in the column, causing them to elute out at different rates and allowing separation. HPLC has many applications in fields like pharmaceuticals, environmental analysis, food and flavors testing, clinical testing, and forensics. It provides a powerful analytical tool for identifying and quantifying compounds in samples.
This document provides an overview of high performance liquid chromatography (HPLC). It discusses the principle, history, types, instrumentation, procedure, advantages, disadvantages and applications of HPLC. Key points include that HPLC uses high pressure to separate compounds faster and more efficiently than other chromatography techniques. Silica is commonly used as the stationary phase because it can withstand pressures over 300-400 atmospheres. The major components of HPLC instrumentation are the solvent reservoir, pump, injector, column, detector and data collector. HPLC has various applications in fields like analytical chemistry, pharmaceuticals, forensics and environmental analysis.
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This document discusses the principles and operation of high performance liquid chromatography (HPLC). It covers basic HPLC instrumentation configuration and separation modes, including reversed phase and normal phase systems. Methods for converting between normal and reversed phase systems are provided. Various HPLC detectors are outlined, including UV-Vis, PDA, and ELSD detectors. The document also discusses HPLC maintenance strategies and preventative maintenance for individual modules. Good laboratory practices for HPLC are reviewed, such as solvent preparation, filtration, and buffer preparation. Finally, common problems and their solutions are addressed.
HPLC is a liquid chromatography technique used to separate compounds in a solution. It works by exploiting differences in how compounds partition between a stationary phase and mobile phase. There are four main types: partition, ion exchange, size exclusion, and affinity chromatography. HPLC systems consist of solvent reservoirs, pumps, injectors, columns, detectors, and data acquisition components. HPLC is used for research, quality control, environmental monitoring, and regulatory purposes to analyze complex mixtures and isolate compounds.
This document discusses high performance liquid chromatography (HPLC) for analyzing oils and fats. It explains that HPLC involves separating components based on how they are distributed between a liquid mobile phase and solid stationary phase. There are two main types of mobile phases used: isocratic elution with a fixed composition and flow rate, and gradient elution where the composition and flow rate change over time to improve separation. Reverse phase HPLC is most commonly used for oil and fat analysis, where nonpolar stationary phases are used and hydrophilic compounds elute more quickly than hydrophobic compounds.
Chromatography is a technique used to separate mixtures by distributing components between a stationary and mobile phase. High-performance liquid chromatography (HPLC) uses high pressure to pass a solvent or solvent mixture through a column containing a stationary phase to separate components in a mixture. HPLC consists of several major components including a pump, injector, column, column compartment, detector, and degasser. The injector introduces the sample into the mobile phase which passes through the column, allowing separation based on interactions between components and the stationary phase. A detector then measures and records separated components as they elute from the column.
1. The document discusses high performance liquid chromatography (HPLC), including its principles, types, instrumentation, and applications. HPLC is a technique used to separate compounds in a mixture using high pressure to force the mixture through a column packed with a stationary phase.
2. The key components of an HPLC system are the solvent reservoirs, pump, injector, column, and detector. HPLC can be used for both analytical and preparative purposes to separate, purify, identify, and quantify compounds.
3. Common applications of HPLC include separation of volatile and non-volatile compounds, qualitative and quantitative analysis, and determination of retention times. Reversed phase HPLC using C18 columns is frequently utilized.
HPLC is a form of liquid chromatography that uses pumps to pass a pressurized mobile liquid phase through a column packed with solid particles. This allows the components of a dissolved sample to be separated as they are transported through the column at different rates depending on their interactions with the stationary and mobile phases. HPLC instruments consist of a pump, injector, column, and detector. Separation is based on the partitioning of compounds between the mobile and stationary phases, and detectors are used to measure separated components as they exit the column. HPLC provides efficient, sensitive, and high-pressure separations of sample mixtures.
Chromatography is a technique used to separate mixtures by distributing compounds between a stationary and mobile phase. High-performance liquid chromatography (HPLC) is commonly used and separates compounds using a column with a stationary phase and liquid mobile phase. HPLC can identify, detect, quantify, and purify individual components in a mixture using an apparatus including a pump, injector, column, detector, and recorder. The separation occurs as the compounds interact differently with the stationary phase in the column.
This document discusses high performance liquid chromatography (HPLC). It begins by defining chromatography as a technique used to separate mixtures into their individual components using both a stationary and mobile phase. It then describes some key aspects of HPLC, including that it uses high pressure to force the mobile phase through a column with small particle sizes for better separation. The document outlines the basic components of an HPLC system, including the pump, injector, column, detectors, and computer. It also discusses some common terms and uses for HPLC, such as separating and analyzing compounds in research, quality control, and environmental monitoring.
The document provides an introduction and history of high performance liquid chromatography (HPLC). It discusses the components of an HPLC system including the pump, injector, column, detectors, and data collection. It describes the different types of chromatography, stationary and mobile phases used, and parameters for evaluating chromatographic separation such as capacity factor, resolution, asymmetry factor, and efficiency.
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Tags: Information Security, ISO/IEC 27001, ISO/IEC 42001, Artificial Intelligence, GDPR
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2. INTRODUCTION
Chromatography:
Is the separation and detection of certain compounds in a sample (Food, water, blood, urine,
chemicals, …).
based on their different chemical-physical properties
based on different physical interactions between two immiscible phases
Such separation may be accomplished in a liquid-liquid extraction
system or by its transfer through an immobilized stationary phase by a
carrier gas (GC) or a mobile phase (LC).
4. THE BASIC COMPONENTS OF
HPLC SYSTEM
MOBILE PHASE
COLUMN
DETECTOR
Sample loading with the mobile phase
(Pump role)
A
C
D
B
B
A
D C
Mobile phase
Stationary phase, column
Detector
At HPLC, The sample is partitioning between
the mobile phase and the stationary phase
(not just being carried by a carrier gas, GC).
5. THE BASIC COMPONENTS OF
HPLC SYSTEM
Pilar Campíns-Falcó; Rosa Herráez-Hernández; Pascual Serra-Mora, Liquid Chromatography-Instrumentation
MOBILE PHASE
COLUMN
DETECTOR
6. THE BASIC COMPONENTS OF
HPLC SYSTEM
HPLC separation
Adsorption
Polar St phase
Normal phase
Partition
Non polar St phase,
Reversed phase
size-exclusion Ion Exchange Chiral
7. THE BASIC COMPONENTS OF
HPLC SYSTEM
B
B
A
C
A
D
x
H
x
x
x
H
Adsorption partition size-exclusion Ion Exchange Chiral
C
E
B
A
A- Analytes are in an adsorption interaction with functional gps on surface of stationary phase
B-Analytes partitions between the mobile phase and the stationary phase depending on its solubility
B-Strong ads, late eluted
A- Weak ads, early eluted
10. Polar
Stationary phase
Non polar
Stationary phase
Analyte Highly polar Moderately to non
polar
Mobile phase for
loading
Low polar solvent High polar solvent
Mobile phase for
eluting
High polar solvent Low polar solvent STATIONARY
PHASE;
(RP // NP)
• Improvement of the obtained selectivity by using a specific
separation condition (NP or RP) was carried out firstly by
selecting proper mobile phase conditions and then changing the
stationary phase.
• It is also preferred to use previously published conditions
(especially on the used column) and then modify it according to
your own need.
11. COLUMN
SPECIFICATION
C18, 250 mm, 4,6 mm, 5 µm, 300 A
Bonded phase
Particle size ,
Smaller PZ introduce
• higher surface area,
• higher peak resolution
• Shorter run time
• But, higher back
pressure too
Particle ‘s Pores size
• Below 60 A
• 60-150 A
• 150-300 A (proteins)
Depending on target
analytes and matrix
Inter diameter
Smaller ID
• Higher peak
resolution
• But, higher back
pressure too
Length
Increasing L
• Higher peak
resolution
• But, higher back
pressure too
Injection volume, Vinj;
Vinj < 0.14 𝐝2
𝐋 x dp
d, L; internal diameter and length of the used column
dp diameter of solid phase particles
High Vinj and analyst Conc.
May cause fronting of un-retained molecules
12. COLUMN
SPECIFICATION
C18, 5 µm, 150 mm × 4.6 mm column
C18, 3 µm, 150 mm × 4.6 mm column
C18, 2.7 µm, 100 mm × 4.6 mm
HPLC-FLD chromatograms of aflatoxins
15. MOBILE PHASE, KOW
THE ELUTION STRENGTH APPLING RP-HPLC ;
• HEXANE(3.13)>TERT-BUTANOL(0.54),ISOPROPANOL(0.25), ACETONE (0.11),
ETHANOL (-0.16)ACETONITRILE (-0.34), METHANOL(-0.77), WATER (-1.38).
first loading of sample
components in RP HPLC.
changing between two immiscible
solvents. It is also used for long
storage of the used columns
Kow Polarity
water and a miscible organic solvent are commonly used mobile phase
mixtures in HPLC since they give a new Kow that rapid the chromatographic
separation run.
Log Kow (A+B)= 1 − 𝑥 𝐿𝑜𝑔 𝐾𝑜𝑤 𝐴 + 𝑋 𝐿𝑜𝑔 𝐾𝑜𝑤 𝐵
A& B; used solvents.
16. MOBILE PHASE, KOW
Kow Polarity
• Acetone has a strong eluting properties in
RP-HPLC but it has a high UV cut-off
value.
• Methylene chloride, chloroform have
medium polarity but in RP HPLC it is
immiscible with water.
• Acetonitrile has a very low UV cut-off,
17. THE BASIC COMPONENTS OF
HPLC SYSTEM
Gradient elution results in; minimizing the run time, good shape for eluted peaks, and
cleaning the used column for each run.
includes three basic steps:
Short isocratic start (Solution A, lower elution)
Gradient program (gradually increase solution B, higher elution).
Short isocratic for cleaning the used column ( Highest percent of solution B).
Return to the initial conditions (column conditioning, very critical step).
18. MOBILE PHASE,
GRADIENT ELUTION
A (H2O:MeOH 5%, pH 4.6). B (MeOH)
Time
10 % B
0.01-5.00
% B
70
5.01-7.00
10 % B
7.01-10.00
BENZOIC AND SORBIC ACID
DETERMINATION IN JUICE, MILK
PRODUCTS,…
19. BUFFER, HPLC MOBILE PHASE
In RP- HPLC Solvent A is mainly water as it:
The highest polar solvent (weakest eluent), suitable for sample loading.
Buffers can be easily prepared in water at different concentrations.
The purity of water for HPLC is very important, especially when using MS/ MS.
It is preferred to use a percent (About 10 %) of a suitable organic solvent in water
(Solvent A);
To prevent algae formation.
Enhance the evaporation in ESI ionization unit (LC-MS/MS).
20. BUFFER, HPLC MOBILE PHASE
For separation of acidic or basic compounds, a buffer should be
used in solvent A to keep the ionizable compounds in neutral
form as possible. Where pH of the mobile phase A (using
buffer) should be acidic for basic compounds and the reverse.
Changing pH by 2 units (lower or higher than its pKa) create a
reverse partitioning condition for this compound.
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
At pH= 2,
-2.2= Log (
[𝐴−]
[𝐻𝐴]
), [HA]=166 [𝐴−
]
http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4
22. BUFFER, HPLC MOBILE PHASE
A buffer is
a solution of a weak acid (HA) and its conjugate base (𝐴−
) &
a solution of a weak base (B) and its conjugated acid (𝐵𝐻+
)
Such buffer system has the capability to resistance changing in pH upon the addition of
small amounts from a strong acid or base.
pH= pKa+ Log (
[𝐴−]
[𝐻𝐴]
)
Cbuf = [𝐴−] + [𝐻𝐴]
Buffer
(pH & Concentration)
23. BUFFER, HPLC MOBILE PHASE
Buffer capacity, β:
The number of added moles (dn) of a strong acid or a base that change the pH of one-liter
buffer solution.
β=
𝑑 𝑛
𝑝𝐻
Since, the addition of n moles from a base (NaOH) leads to the formation of [𝐴−
Na] or CNaA
β=[𝐴−
b]
𝑝𝐻
http://dx.doi.org/10.1016/B978-0-12-803684-6.00004-4
https://www.kisspng.com/png-bouncer-clip-
art-bodyguard-security-guard-royalty-
5953930/
β
25. BUFFER, HPLC MOBILE PHASE
• BUFFER CONCENTRATION IN HPLC UV IS USUALLY MADE WITH A CONCENTRATION OF 10-
200 MM. (LOWER CONCENTRATIONS FOR LC MSMS <50MM, VOLATILE SALTS ARE MORE
FAVORABLE).
• THE SOLUBILITY OF INORGANIC SALTS DEPENDS MAINLY ON THE NATURE OF THE CATION,
AND THEIR SOLUBILITY TREND IN ORGANIC SOLVENTS FOLLOWS (THE SAME AS IN
WATER): NH4+ >𝐾+>𝑁𝑎+.
• A HIGHER CONTENT OF ORGANIC PHASE SHOULD BE AVOIDED NOT TO PRECIPITATE THE
BUFFER SALTS.
_____________________________________________________
• FOR PREPARING A BUFFER AT PH 4.5 YOU SHOULD USE:
A WEAK ACID OF PKA CLOSE TO THE REQUIRED PH (……ACID PKA =….)
THE CONJUGATE BASE FOR THIS ACID WILL BE ITS (NA, K, AMMONIUM) SALT
WHAT IS THE TOTAL BUFFER CONCENTRATION AND HOW TO PREPARE ?
29. SOLVENT OF THE SAMPLE
The solvent of the sample should be with;
- Same polarity as the mobile phase (loading solvent, A)
or weaker (increase solvent compressing, enhance the
retention of solutes).
- pH close to that of the mobile phase (loading solvent, A)
These points are more critical, especially for higher injection volume
and for ionizable solutes solutes
Zorbax Eclipse XDB-
C18, 150 mm, 4.6 mm, 5 µm
with a mobile phase:
35% ACN and 65% H2O (0.2% H3PO4);
flow-rate: 2 mL/min.
31. COLUMN EFFICIENCY
Jesús Lozano-Sánchez; Isabel Borrás-Linares; Agnes Sass-Kiss; Antonio Segura-Carretero, Chapter 13 - Chromatographic
Technique: High-Performance Liquid Chromatography (HPLC)
Dead time, Void time
T0 = Time at which mobile phase pass through the chromatographic column
Depending on column length and flow rate
Actual Rt (X)= Obtained Rt (X)+ t0
32. COLUMN EFFICIENCY
Plate Number (N)
N= (𝑅𝑡/ )2
N= 16(𝑅𝑡/Wb)2
N= 5.45 (𝑅𝑡/W 0.5)2
Wb= 4*x Standard deviation ( )
Selectivity
=
𝑅𝑡 (𝑥)
𝑅𝑡 (𝑦)
Resolution
R =
2 [𝑅𝑡 𝑥 −𝑅𝑡 𝑦 ]
(𝑊𝑏 𝑥 +𝑊𝑏 (𝑦)
R=
Δ𝑅𝑡
Column efficiency:
High Plate N per unit length (N/L)
or
Height of the theoretical plate H= 𝐿/𝑁≃ 2dp
dp diameter of solid phase particles
Selectivity (Separation Factor)
A measurement for the separation
of two compounds X and Y
Resolution
Separation of apexes of two adjacent peaks
33. A. Prof. Sherif M. Taha
Tel: 01004724944
sherif2taha@gmail.com
Thank You