Lecture 9 Introduction to Chromatography “The Science of 9讲介绍色谱学”
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Preparative column : industrial
scale (for 1 kg of material, volume 300 L)
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
10
2.2. Open Tubular vs. Packed Columns
Lecture 7 – HPLC, 23-10-2019
5
1.2. HPLC versus classical LC
1. High resolution (i.e. better separation).
2. Small diameter (4.6 mm) stainless steel, glass or titanium columns.
2. Describe in detail the set-up of an HPLC system.
3. Determine equivalence in polarity among different mobile phase compositions.
© Dr. Rasha Hanafi, GUC
•As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.
3. Pharmaceutical development: developing dosage forms with optimized delivery and stability profiles for clinical supplies and final products.
4. Drug metabolism/pharmacokinetics (DMPK): evaluating the metabolic and pharmacokinetic profiles of the drug candidates in animal models and human clinical studies.
Protection:
1. by periodically renewed guard columns containing the same stat. phase like main column.
2. by passing solvents and samples through filters (0.5 m).
1. Drug discovery: finding new chemical entities (NCE) for adoption as new drug development candidates.
2. Chemical development: developing viable synthetic routes and scale-up processes for synthesizing active pharmaceutical Ingredients (API).
3. Column packing of stationary phase with very small (3, 5 and 10 μm) particles.
4. Relatively high inlet pressures and controlled flow of the mobile phase.
Lecture 7 High Pressure Liquid Chromatography
HPLC
Dr. Rasha Hanafi
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
1
Objectives
1. Define High Performance Liquid Chromatography (HPLC).
Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use
much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography UPLC" systems work at up to
100 MPa (15,000 lbf/in²), or about 1000 Bar .
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
8
2.1. Reciprocating Pump (in 90% of systems)
to column
Lecture 7 – HPLC, 23-10-2019
2
Realistic Set-Up of your HPLC
(location: instrumental analysis laboratory, B7-303)
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
5. Continuous flow detectors capable of handling small flow rates and detecting very small amounts.
6. Rapid analysis.
These characters refer of course to HPLC, can you compare these characters to the ones related to classic column chromatography that you performed in the lab.?
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
9
2.2. The Column
Analytical column:
• length: 5 – 30 cm.
• ID: 1 – 5 mm.
• made of steel or plastic.
• expensive (>2000 L.E.), easily damaged by dust or particles in the sample or solvent.
Requirements of pumping system:
1. Pulse free output.
2. Flow rates from 0.1-10 mL/min.
3. Flow control and flow reproducibility of a maximum error of
0.5%.
to pulse damper
ball check valves
Disadvantage:
pulsed flow, thus damping necessary
solvent
Advantages:
small internal volume (35 – 400 L), high output pressure (10,000 psi) adaptability for gradient elution, constant flow rates (largely independent of column back-pressure and solvent viscosity).
Packed column
Typically used in HPLC
Diffusion of liquids is ~ 100 times slower than diffusion in gases.
3
1. 1. What is HPLC?
•HPLC is one mode of chromatography, one of the most used analytical techniques. •HPLC utilizes a liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid phase. These components are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture separates into its components. studyhplc/hplcinstrument.php •The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases.
Gradient construction:
1. High-pressure gradient
2. Low-pressure gradient (more common) where pro-portioning of the solvents (up to 4) through a four-way valve at low pressure takes place, then pumping the mixture at high pressure into the column is performed.
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
4
HPLC in pharmaceutical analysis from drug discovery to quality control
HPLC is the dominant technique for pharmaceutical analysis used in research, development, and quality control with the following functions:
© Dr. Rபைடு நூலகம்sha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
6
2. Schematic Set-Up of HPLC
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
7
2.1. Pumps
The quality of a pump is measured by how steady and reproducible a flow it can produce. A fluctuating flow creates detector noise that obscures weak signals.
4. Corrosion resistant (stainless steel & Teflon).
Does high pressure in HPLC constitute an
5. Generation of pressure up to 6000 psi (400 Bar).
explosion hazard? Why?
5. Quality control (QC): assessing the quality of the final manufactured products against published specification for product release.
© Dr. Rasha Hanafi, GUC
scale (for 1 kg of material, volume 300 L)
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
10
2.2. Open Tubular vs. Packed Columns
Lecture 7 – HPLC, 23-10-2019
5
1.2. HPLC versus classical LC
1. High resolution (i.e. better separation).
2. Small diameter (4.6 mm) stainless steel, glass or titanium columns.
2. Describe in detail the set-up of an HPLC system.
3. Determine equivalence in polarity among different mobile phase compositions.
© Dr. Rasha Hanafi, GUC
•As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.
3. Pharmaceutical development: developing dosage forms with optimized delivery and stability profiles for clinical supplies and final products.
4. Drug metabolism/pharmacokinetics (DMPK): evaluating the metabolic and pharmacokinetic profiles of the drug candidates in animal models and human clinical studies.
Protection:
1. by periodically renewed guard columns containing the same stat. phase like main column.
2. by passing solvents and samples through filters (0.5 m).
1. Drug discovery: finding new chemical entities (NCE) for adoption as new drug development candidates.
2. Chemical development: developing viable synthetic routes and scale-up processes for synthesizing active pharmaceutical Ingredients (API).
3. Column packing of stationary phase with very small (3, 5 and 10 μm) particles.
4. Relatively high inlet pressures and controlled flow of the mobile phase.
Lecture 7 High Pressure Liquid Chromatography
HPLC
Dr. Rasha Hanafi
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
1
Objectives
1. Define High Performance Liquid Chromatography (HPLC).
Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use
much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography UPLC" systems work at up to
100 MPa (15,000 lbf/in²), or about 1000 Bar .
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
8
2.1. Reciprocating Pump (in 90% of systems)
to column
Lecture 7 – HPLC, 23-10-2019
2
Realistic Set-Up of your HPLC
(location: instrumental analysis laboratory, B7-303)
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
5. Continuous flow detectors capable of handling small flow rates and detecting very small amounts.
6. Rapid analysis.
These characters refer of course to HPLC, can you compare these characters to the ones related to classic column chromatography that you performed in the lab.?
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
9
2.2. The Column
Analytical column:
• length: 5 – 30 cm.
• ID: 1 – 5 mm.
• made of steel or plastic.
• expensive (>2000 L.E.), easily damaged by dust or particles in the sample or solvent.
Requirements of pumping system:
1. Pulse free output.
2. Flow rates from 0.1-10 mL/min.
3. Flow control and flow reproducibility of a maximum error of
0.5%.
to pulse damper
ball check valves
Disadvantage:
pulsed flow, thus damping necessary
solvent
Advantages:
small internal volume (35 – 400 L), high output pressure (10,000 psi) adaptability for gradient elution, constant flow rates (largely independent of column back-pressure and solvent viscosity).
Packed column
Typically used in HPLC
Diffusion of liquids is ~ 100 times slower than diffusion in gases.
3
1. 1. What is HPLC?
•HPLC is one mode of chromatography, one of the most used analytical techniques. •HPLC utilizes a liquid mobile phase to separate the components of a mixture. The stationary phase can be a liquid or a solid phase. These components are first dissolved in a solvent, and then forced to flow through a chromatographic column under a high pressure. In the column, the mixture separates into its components. studyhplc/hplcinstrument.php •The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases.
Gradient construction:
1. High-pressure gradient
2. Low-pressure gradient (more common) where pro-portioning of the solvents (up to 4) through a four-way valve at low pressure takes place, then pumping the mixture at high pressure into the column is performed.
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
4
HPLC in pharmaceutical analysis from drug discovery to quality control
HPLC is the dominant technique for pharmaceutical analysis used in research, development, and quality control with the following functions:
© Dr. Rபைடு நூலகம்sha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
6
2. Schematic Set-Up of HPLC
© Dr. Rasha Hanafi, GUC
Lecture 7 – HPLC, 23-10-2019
7
2.1. Pumps
The quality of a pump is measured by how steady and reproducible a flow it can produce. A fluctuating flow creates detector noise that obscures weak signals.
4. Corrosion resistant (stainless steel & Teflon).
Does high pressure in HPLC constitute an
5. Generation of pressure up to 6000 psi (400 Bar).
explosion hazard? Why?
5. Quality control (QC): assessing the quality of the final manufactured products against published specification for product release.
© Dr. Rasha Hanafi, GUC