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HPLC System Selection

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Gamal Abdel Hamid

This document provides an overview of the key components of a high-performance liquid chromatography (HPLC) system, including the pump, solvent rack, autosampler, column, column compartment, and detector. It discusses the different types of pumps (isocratic, gradient, dual gradient), solvent racks, autosamplers (split loop, pulled loop), and columns. It also provides details on the configuration options and specifications for these various components. The goal is to help users select the appropriate components for their HPLC system based on their application needs.

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Gamal A. Hamid11
Gamal A. Hamid2 Contents • Introduction • Instrument • Pump • Solvent rack • Autosampler • Column • Column compartment • Detector • Applications • Food applications
Gamal A. Hamid3 Chromatography Chromatography Is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. ( IUPAC)
Gamal A. Hamid4 HPLC High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography), • Is a form of liquid chromatography where the high pressure used to separate the mixture components, then identify and quantify.
Gamal A. Hamid55
Gamal A. Hamid6 High pressure liquid chromatograph HPLC instruments consist of: I. Solvent rack, II. Pump, III. Injector, IV. Separation column, V. Detector.
Gamal A. Hamid7 Basic Systems Basic manual system Basic automated system Isocratic pump + manual sample injection + detector. A simple, expandable system where modularity makes anything possible. Isocratic or quaternary or binary pump 620 bar + integrated autosampler +100 Hz detector
Gamal A. Hamid8 Standard Systems The system model named according the used pump:- • UltiMate 3000 Standard Binary System • UltiMate 3000 Standard quaternary System • UltiMate 3000 Standard dual System
Gamal A. Hamid9 Rapid separation systems Rapid Separation (RS) Systems • Pressures up to 1034 bar at flow rates up to 5 mL/min. • Pressures of 800 bar at flows from 5 – 8 mL/min. • Binary, Quaternary, and Dual-Gradient Pumps. • Column Temperatures from 5 – 110°C. • Optional switching valves for advanced workflows. • 200 Hz DAD, MWD, VWD, FLD
Gamal A. Hamid10 Benefits of UHPLC  Faster Analysis (typically 10X faster) • Increased productivity (more samples/day) • Higher instrument ROI (more samples/system) • Faster results (e.g. decreased time to market) • Increased data content (more data in the same time)  Reduced Solvent Consumption (typically 10X reduction) • Lower solvent acquisition costs • Lower solvent disposal costs • Reduced environmental impact
Gamal A. Hamid11 Select your system SD or RS
Gamal A. Hamid12 Pumps Autosamplers Column compartments Detectors The UltiMate® 3000 LC Systems With ValvesStandard Quaternary Dual-GradientBinary VWD/MWD/DAD Fluorescence Standard Thermostatted +FractionationBasic Isocratic Coulo chemCorona
Gamal A. Hamid13 I. Pump A device designed to deliver the mobile phase at a controlled flow-rate to the separation system. • Isocratic analysis in which the eluent composition remains unchanged during the analysis. solvent must be pre-mixed • A gradient analysis allows the composition of the eluent to be changed during the analysis. The heart is the Strongest pump ever
Gamal A. Hamid14 The UltiMate® 3000 SD & RS pumps • First select a pump that match with your applications. • Four different Standard “SD” pumps, • And three Rapid Separation “RS” pumps.
Gamal A. Hamid15 Pump components No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 LPG-3400SD and RS: Capillary mixer LPG-3400BM: Capillary from purge to inline filter 7 4-channel vacuum degasser 8 Pump lights 9 LPG-3400SD and RS: Static mixer LPG-3400BM: Inline filter 10 Purge unit with purge valve and pressure transducer for the system pressure 11 Pump block status LED 12 4-channel proportioning valve
Gamal A. Hamid16 1. Isocratic pump ISO The mobile phase, either a pure solvent or a mixture, remains the same throughout during the run, Solvents must be pre-mixed. Without degasser.
Gamal A. Hamid17 2. High-pressure gradient pump HPG • High-pressure gradient pump with integrated mixing chamber and "2 from 4" solvent selectors Two pumps are used. • The eluents are mixed after pumping. Accurate gradient and minimized delay volume. • Without degasser. • Response of the gradient is superior because of the small volume from the mixing unit to the column.
Gamal A. Hamid18 High-pressure gradient pump HPG HPG-3400 HPG-3200
Gamal A. Hamid19 3. Low pressure gradient pump LPG • One pump is used for mixing, Up to four eluents can be mixed. • With degasser.
Gamal A. Hamid20 4. Dual gradient pump DGP • Two separate pumps with integrated mixing chambers in one enclosure (2x3 solvents). • Two different pumps with different solvents and columns. Without degasser. • Independently-optimized mobile phase composition for 2D separations.
Gamal A. Hamid21 Flow rate & pressure
Gamal A. Hamid22 Eluents flow diagram
Gamal A. Hamid23 RS & SD Pumps
Gamal A. Hamid24 Pump Chromeleon page
Gamal A. Hamid25 II. Solvent Rack • Second select your Solvent rack that match with your pump selection. • How many channel you have in your pump? • Is your selected pump has degasser or not?
Gamal A. Hamid26 Solvent Rack “SR” 1. SR-3000 Solvent Rack Without vacuum degasser typically for use with a LPG-3400, ISO-3100BM, or HPG-3200BX 2. SRD-3200 Solvent Rack With analytical 2-channel vacuum degasser typically for use with the following pumps: - one HPG-3200 (SD or RS) - one ISO-3100SD
Gamal A. Hamid27 SRD “Solvent Rack Degasser” 3. SRD-3400 Solvent Rack • With analytical 4-channel vacuum degasser typically for use with the following pumps: - one HPG-3400 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG- 3200 (SD or RS) or ISO-3100SD if you want to degas the solvents and the wash solution of autosampler 4. SRD-3600 Solvent Rack • With analytical 6-channel vacuum degasser typically for use with the following pumps: - one DGP-3600 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG- 3200 (SD or RS) and one HPG-3400 in a two-stack system - one HPG-3400 if you want to degas the solvents and the wash solution of autosampler.
Gamal A. Hamid28 Specifications
Gamal A. Hamid29 3. Injector Instrumentation • A device by which a liquid, solid or gaseous sample is introduced into the mobile phase or the chromatographic bed. • The injector must also be able to withstand the high pressures of the liquid system. • Each type is equipped with six-port valves, so that a sample can be injected into the flow path at continuous pressure.
Gamal A. Hamid30 In-line Split-Loop autosamplers “SL” • The in-line split-loop (SL) (flow-through) analytical autosampler supports pressures up to 62 MPa (9000 psi) at analytical and micro flow rates. • Within in-line split-loop autosamplers, the sample loop, needle, and needle seat are integral parts of the high pressure fluidic path. • Autosampler aspirates only the sample volume injected onto the column. The needle and sample loop are constantly rinsed with mobile phase, achieving the lowest carryover.
Gamal A. Hamid31 Pulled-Loop Autosamplers “PL” • In pulled-loop autosamplers, the needle is not an integral part of the high pressure fluidic path. • Therefore, all autosampler parts in contact with the sample can be made of inert material. • High injection precisions are achieved in full- loop injections at a fixed-sample loop volume. • Variable injection volumes are accessible in partial loop injection mode.
Gamal A. Hamid32 Features and Benefits SL BL • High injection volume flexibility • Near zero carry over • No sample loss • Low sample dispersion • Excellent injection precision and linearity • Very short cycle times • Highest versatility and product • Lowest injection volumes down to the nL range • Biocompatible flow paths*1 • Microliter pick-up – zero sample loss for pulled-loop injections • Excellent injection precision even at low volume injections – at 20 nL a precision < 1% can easily be achieved. • Optional fractionation capabilities
Gamal A. Hamid33 Autosampler configurations • T Thermostatted • SL Split Loop • PL Pull Loop • B Bio compatible • A Analytical • RS Rapid Separation • FC Fraction Collector • X MS
Gamal A. Hamid34 Definitions T The sample thermostatting option allows sample cooling using peltier by max. 22 °C from the ambient temperature. The lower temperature limit is +4 °C. In addition, the samples can be warmed to max. +45 °C. SL In-Line Split Loop flow autosampler the needle is an integral part of the high pressure fluidic path. PL Pulled Loop autosampler the needle is not an integral part of the high pressure fluidic path. B Biocompatible Autosampler perfectly matches the requirements for injections of complex samples containing biomolecules, such as recombinant proteins or monoclonal antibodies. Almost all descriptions of the standard autosamplers apply also to the biocompatible versions. RS UHPLC Rapid Separation the 103 MPa (15,000 psi) in-line split-loop (flow-through) FC The autosampler is equipped with one 2-position, 6-port injection valve and one 2- position,6-port fractionation valve, With this configuration, injection, fraction collection, and reinjection can be fully automated on one system. X The 125 MPa (18,130 psi) in-line split-loop (flow-through) UHPLC autosampler for MS.
Gamal A. Hamid35 Autosampler Chromeleon page
Gamal A. Hamid36 4. Column Instrumentation • Is The tube and the stationary phase contained within, through which the mobile phase passes. • The heart of the chromatograph. • The column’s stationary phase separates the sample components of interest using various physical and chemical parameters. • The pump push hard to move the mobile phase through the column and this resistance causes a high pressure.
Gamal A. Hamid37 Column types Name Description 1 Acclaim® Reversed-Phase Col. RSLC, PepMap, Polar Advantage 2 Acclaim Multi-Mode Columns Trinity™ P-1 3 Acclaim Specialty Columns Organic acids, surfactants, explosive 4 ProSwift® Columns Monolith RP and ion exchange 5 PepSwift™ Columns RP nano, capillary and micro columns 6 DNASwift™ Columns Monolith SAX
Gamal A. Hamid38 Thermostatted Column Compartment TCC • Accommodation of up to 12 columns, the maximum column length is 300 mm. • Column switching valves make the TCC the appropriate choice for almost all applications in HPLC and UHPLC. • Homogeneous temperature distribution via a fan-based, forced-air design • Column identification system and comprehensive system wellness features • Titanium and PEEK valves are available for different bio-UHPLC and bio-HPLC applications. • Pre-column heater brings the solvent and the sample to the column temperature before they enter the column. This avoids temperature gradients in the column. • Post-column cooler (only TCC-3000RS) cools the column eluate to a temperature that is appropriate for the subsequent detection. Post-column cooling minimizes the noise values and thus leads to optimal detection results.
Gamal A. Hamid39 Column Compartment TCC-3000 RS • The Rapid Separation (RS) Thermostatted • Wide temperature range from 5 (maximum 18 °C below ambient)–110 °C with excellent accuracy and precision • Optional column switching valves • Post column cooling ensures lowest detector noise and drift • Column identification system for up to four columns.
Gamal A. Hamid40 Column Compartment TCC-3000 SD • The Standard (SD) Thermostatted Column • Compartment is a cost-effective choice for • conventional HPLC as well as bio chromatography separations. • Up to 80 °C and precolumn heating. • TCC 3000 SD similar to TCC 3000 RS in the most specifications except the temp. range.
Gamal A. Hamid41 Column heating Reproducibility • If temperature varies, then it is difficult to assign “peaks” to specific compounds in the chromatogram and the peak areas/heights may vary Solubility • Certain chemical compounds may have low solubility in the HPLC mobile phase • If they are injected into the flow stream they may precipitate or other difficulties may arise Stability • Certain chemical compounds, especially biological compounds such as enzymes or proteins, may not be stable at room temperature or higher • The temperature needs to be much lower down to 4°C
Gamal A. Hamid42 Home page Chromeleon
Gamal A. Hamid43 5. Detector Instrumentation A device that measures the change in the composition of the eluent by measuring physical or chemical properties. The desirable features of a detector are: • Sensitivity towards solute over mobile phase • Low cell volumes to minimize memory effects • Low detector noise • Low detection limits • Large linear dynamic range
Gamal A. Hamid44 UV/UV-VIS Detectors (variable λ detector) • The Thermo Scientific™ Dionex™ UltiMate™ 3000 VWD-3000 is a variable wavelength detector (VWD) series for industry leading UV-VIS detection. • flow cells ensure optimal performance over a flow rate range of five orders of magnitude. 1. Lamps cover 2. Flow cell 3. Flow cell drainage 4. Leak sensor
Gamal A. Hamid45 VWD Principle • A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm. • If components are to be detected at wavelength longer than this, a UV-VIS detector is used, which employs an additional tungsten lamp (W lamp). • By monitoring the reference light divided from the light in front of the flow cell, the difference in light intensity can be determined between the back and front of the flow cell, and this is output as absorbance.
Gamal A. Hamid46 VWD Features and highlights • Data collection rate: Single wavelength up to 100 Hz (VWD-3100) Single wavelength up to 200 Hz (VWD-3400RS) Multiple wavelength up to 5 Hz (VWD-3400RS). • Light source: Deuterium lamp, tungsten lamp (Tungsten lamp optional on VWD-3100) Temperature control for both lamps • Flow cells: wide range of available ensure optimal performance over a flow rate range of five orders of magnitude, Analytical flow cell, 11 μL, stainless steel • Wavelength range: 190–900 nm with combined use of deuterium and tungsten lamp on one optical axis. • Temperature control of optics and electronics for data acquisition independent of ambient conditions. • The detector’s large linearity range of up to 2.5 AU is ideal for applications with widely varying analyte concentrations. • The VWD-3000 series uses a fast-turning grating monochromator placed in front of the flow cell. • No. of Channels: 1 channel (VWD -3100 SD) Up to 4 channels (VWD-3400 RS)
Gamal A. Hamid47 UV Detector Chromeleon page
Gamal A. Hamid48 Diode array detector (DAD, PDA) • Photodiode arrays (semiconductor devices) are used in the detection unit. • The Thermo Scientific™ Dionex™ UltiMate™ DAD 3000 detector is a high-resolution, 1024- element diode array detector (DAD) available in Rapid Separation and Standard versions. • Optical System, Single-beam, reverse optics design with concave holographic grating. • The detector is also available as a multiple wavelength detector (MWD) in Standard and Rapid Separation versions, MWD does not support the acquisition of 3-D spectra .
Gamal A. Hamid49 PDA Principle • The polychromatic beam passes the flow cell. • The grating splits up the polychromatic beam to different wavelengths, the intensities of which are measured by an array or photodiodes. • A photodiode is a semiconductor device that converts light into current. • The current is generated when photons are absorbed in the photodiode. 1. VIS Lamp 2. D 2 Lamp 3. Flow cell Adaptor 4. Flow cell
Gamal A. Hamid50 DAD Features and highlights • Data collection rate: Diode array detector (DAD) available in Rapid Separation (200 Hz) using a maximum of eight single-wavelength data channels and one 3-D field (and Standard (100 Hz) versions. • Light source: Deuterium lamp, tungsten lamp Flow cells: Flow cell range covers semi-micro, semi- analytical, analytical, and semi-preparative flow rate applications, in stainless steel and biocompatible versions. • Wavelength range: The tungsten and deuterium lamps emits light in UV and visible ranges (190 -800) nm. • Temperature control of optics and electronics for data acquisition independent of ambient conditions. • A typical detector linearity up to 2.0 AU. • Temperature control for both lamps. • No. of Channels: Up to 8 channels (DAD-3000 SD) Up to 8 channels (DAD-3000 RS)+ 3D
Gamal A. Hamid51 Fluorescence Detector • Xenon lamp Light source for the UV to near- infrared wavelength range. • PMT Photomultiplier tube (PMT) - Converts the emitted light to a measureable current signal. • Fluorescence detection offers 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 column Derivatization
Gamal A. Hamid52 FL Principle • FL is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. Such a substance has specific wavelengths of light that it absorbs (excitation wavelengths) and emits (emission wavelengths). • While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction orthogonal to the exciting light. • A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. However, some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength.
Gamal A. Hamid53 FLD Features and highlights • Data collection rate: Single wavelength: up to 200 Hz (FLD-3400RS) Multiple wavelength: up to 4 Hz (FLD-3400RS) Single wavelength: up to 100 Hz (FLD-3100). • Light source: High-intensity xenon flash lamp with smart control features • Flow cells: 8 μL analytical flow cell 2 μL micro flow cell. • Wavelength range: Wavelength range 200 – 650 nm Wavelength range 200 – 900 nm (dual). • Long lamp life (> 15,000 h in long life mode). • Flow Cell temp: Thermostatting: Ambient + 15 °C to 50 °C. • Spectrum Scanning Modes: 2D or 3D excitation, emission • No. of Channels: Up to 4 channels (FLD-3400RS) 1 channel (FLD-3100)
Gamal A. Hamid54 FL Detector Chromeleon page
Gamal A. Hamid55 Refracted index Detector • The response is dependent on changes in refractive index of eluting compounds in the mobile phase. • The mobile phase itself should have refractive index different from the sample. • Gradient programming is not possible due to resulting changes in refractive index of mobile phase. • Temperature control is necessary as it has high temperature sensitivity. • Any component in the eluate can be detected; thus, the RI detector is often called a “universal detector”. • Typical applications are in Size Exclusion Chromatography. • The detector is less sensitive than UV-VIS detector.
Gamal A. Hamid56 RI Principle • RI detector detects components based on the refraction of light in solution. • The reference-side cell is filled with eluate, and the column eluate is introduced into the sample-side cell through the changed flow channel. • When components are eluted from the column, the chemical composition changes in the sample-side solution, which changes its photorefractive level. As a result, the amount of light received by the light- receiving section changes, showing a peak which can be detected.
Gamal A. Hamid57 FLD Features and highlights • Data collection rate: Single wavelength: up to 200 Hz (FLD-3400RS) Multiple wavelength: up to 4 Hz (FLD-3400RS) Single wavelength: up to 100 Hz (FLD-3100). • Light source: High-intensity xenon flash lamp with smart control features • Flow cells: 8 μL analytical flow cell 2 μL micro flow cell. • Wavelength range: Wavelength range 200 – 650 nm Wavelength range 200 – 900 nm (dual). • Long lamp life (> 15,000 h in long life mode). • Flow Cell temp: Thermostatting: Ambient + 15 °C to 50 °C. • Spectrum Scanning Modes: 2D or 3D excitation, emission • No. of Channels: Up to 4 channels (FLD-3400RS) 1 channel (FLD-3100)
Gamal A. Hamid58 Corona Detector • The Corona CAD detector provides the ability to measure virtually any nonvolatile and many semi volatile analytes: • Positive, negative, or neutral, with or without a chromophore, all can be detected routinely with charged aerosol detection. • The Corona CAD can readily detect compounds present in single digit nano gram quantities. • Corona CAD’s unique method of detection allows quantitation across a range that exceeds four orders of magnitude
Gamal A. Hamid59 Corona principle • The Corona CAD “Charged Aerosol Detector “ measures charge that is imparted to analyte particles, with the charge being in direct proportion to the amount of the analyte in the sample. • Samples nebulized by air or nitrogen then dried, dried particles combined with the charged gas (charged using high voltage), pass through the collector where the charged particles are measured using sensitive electrometer.
Gamal A. Hamid60 Corona Applications • Pharmaceuticals • Proteins • Lipids • Steroids • Oligosaccharides • Surfactants • Carbohydrates • Polymers • Peptides Corona CAD is appropriate for any nonvolatile and many semi volatile compounds,
Gamal A. Hamid61 Systems variations Item Nano/Cap Basic automated / Standard RSLC Pump Max. Pressure 800 bar 620 bar 1034 bar Pump. Max. Flow 50 µL/min 10 mL/min 5 mL/min @ 1034 bar 8 mL/min @ 800 bar Autosampler Max. pressure 1000 bar 620 bar 1034 bar Column Oven Max. Temperature 80 °C 50 oC (Basic) 80 °C (Standard) 110 °C Column Oven Max pressure 1000 bar 620 bar 1034 bar Detector Data Collection Rate 200 Hz* 100 Hz 200 Hz*
Gamal A. Hamid6262
Gamal A. Hamid63 Fit the system to the applications The UltiMate 3000 systems are fully modular, allowing you to choose common system configurations or design the most suitable system for your application needs.
Gamal A. Hamid64 Applications fields • Environmental applications • Food applications • Pharmaceutical applications • Bioanalytical applications • Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) • Salts like sodium chloride and potassium phosphate • Proteins like egg white or blood protein • Organic chemicals like polymers (e.g. polystyrene, polyethylene) • Heavy hydrocarbons like asphalt or motor oil • Many natural products such as ginseng, herbal medicines, plant extracts • Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
Gamal A. Hamid65
Gamal A. Hamid66 Carbohydrates Analysis https://tools.thermofisher.com/content/sfs/brochures/AI-71469-Chromatography-Foods-Beverages-Carbohydrates-AI71469-EN.pdf
Gamal A. Hamid67 Sugar Substitutes http://tools.thermofisher.com/content/sfs/brochures/AI71470-Chromatography-Foods-Beverages-Sugar-Substitutes-AI71470-EN.pdf
Gamal A. Hamid68 Fats and Oils Analysis https://tools.thermofisher.com/content/sfs/brochures/AI-71471-Chromatography-Foods-Beverages-Fats-Oils-AI71471-EN.pdf
Gamal A. Hamid69 Flavors, Colorants and Additives http://tools.thermofisher.com/content/sfs/brochures/AI-71472-Chromatography-Foods-Beverages-Flavors-Colorants-Additives-AI71472- EN.pdf
Gamal A. Hamid70 Supplements Analysis http://tools.thermofisher.com/content/sfs/brochures/AI-71473-Chromatography-Foods-Beverages-Supplements-AI71473- EN.pdf
Gamal A. Hamid71 Profiling Beverages https://tools.thermofisher.com/content/sfs/brochures/AI-71474-Chromatography-Profiling-Beverages-AI71474-EN.pdf
Gamal A. Hamid72 Adulteration and Authentication https://tools.thermofisher.com/content/sfs/brochures/AI-71475-Chromatography-Foods-Beverages-Adulteration-Authentication- Applications-Notebook-AI71475-EN.pdf
Gamal A. Hamid73 Contaminants http://tools.thermofisher.com/content/sfs/brochures/AI-71476-Chromatography-Foods-Beverages-Contaminants-Applications- Notebook-71476-EN.pdf
Gamal A. Hamid74 Antifoaming Agents, Emulsifiers and Surfactants http://tools.thermofisher.com/content/sfs/brochures/AI-71477-Chromatography-Foods-Beverages-Antifoaming-Emulsifiers- Surfactants-AI71477-EN%201214S.pdf
Gamal A. Hamid75 Vitamin and Antioxidant https://tools.thermofisher.com/content/sfs/brochures/AI-71478-Chromatography-Foods-Beverages-Vitamins-Antioxidants- AI71478-EN.pdf
Gamal A. Hamid76 Visit thermoscientific.com/AppsLab • Access GC, IC, LC, GC-MS, and LC-MS application notes in a single online location. • Download one-click workflows: immediately ready to run in Chromeleon CDS. • Compare results and identify optimum chromatographic separation conditions quickly and easily. • Visible to all users. Register once for free and download complete separation methods. https://appslab.thermofisher.com/
Gamal A. Hamid77
Gamal A. Hamid7878

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HPLC System Selection

  • 2. Gamal A. Hamid2 Contents • Introduction • Instrument • Pump • Solvent rack • Autosampler • Column • Column compartment • Detector • Applications • Food applications
  • 3. Gamal A. Hamid3 Chromatography Chromatography Is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. ( IUPAC)
  • 4. Gamal A. Hamid4 HPLC High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography), • Is a form of liquid chromatography where the high pressure used to separate the mixture components, then identify and quantify.
  • 6. Gamal A. Hamid6 High pressure liquid chromatograph HPLC instruments consist of: I. Solvent rack, II. Pump, III. Injector, IV. Separation column, V. Detector.
  • 7. Gamal A. Hamid7 Basic Systems Basic manual system Basic automated system Isocratic pump + manual sample injection + detector. A simple, expandable system where modularity makes anything possible. Isocratic or quaternary or binary pump 620 bar + integrated autosampler +100 Hz detector
  • 8. Gamal A. Hamid8 Standard Systems The system model named according the used pump:- • UltiMate 3000 Standard Binary System • UltiMate 3000 Standard quaternary System • UltiMate 3000 Standard dual System
  • 9. Gamal A. Hamid9 Rapid separation systems Rapid Separation (RS) Systems • Pressures up to 1034 bar at flow rates up to 5 mL/min. • Pressures of 800 bar at flows from 5 – 8 mL/min. • Binary, Quaternary, and Dual-Gradient Pumps. • Column Temperatures from 5 – 110°C. • Optional switching valves for advanced workflows. • 200 Hz DAD, MWD, VWD, FLD
  • 10. Gamal A. Hamid10 Benefits of UHPLC  Faster Analysis (typically 10X faster) • Increased productivity (more samples/day) • Higher instrument ROI (more samples/system) • Faster results (e.g. decreased time to market) • Increased data content (more data in the same time)  Reduced Solvent Consumption (typically 10X reduction) • Lower solvent acquisition costs • Lower solvent disposal costs • Reduced environmental impact
  • 11. Gamal A. Hamid11 Select your system SD or RS
  • 12. Gamal A. Hamid12 Pumps Autosamplers Column compartments Detectors The UltiMate® 3000 LC Systems With ValvesStandard Quaternary Dual-GradientBinary VWD/MWD/DAD Fluorescence Standard Thermostatted +FractionationBasic Isocratic Coulo chemCorona
  • 13. Gamal A. Hamid13 I. Pump A device designed to deliver the mobile phase at a controlled flow-rate to the separation system. • Isocratic analysis in which the eluent composition remains unchanged during the analysis. solvent must be pre-mixed • A gradient analysis allows the composition of the eluent to be changed during the analysis. The heart is the Strongest pump ever
  • 14. Gamal A. Hamid14 The UltiMate® 3000 SD & RS pumps • First select a pump that match with your applications. • Four different Standard “SD” pumps, • And three Rapid Separation “RS” pumps.
  • 15. Gamal A. Hamid15 Pump components No Description 1 Peristaltic pump 2 Detector of the rear seal wash system 3 Capillary guides 4 Pump head with working cylinder and equilibration cylinder 5 Leak sensor 6 LPG-3400SD and RS: Capillary mixer LPG-3400BM: Capillary from purge to inline filter 7 4-channel vacuum degasser 8 Pump lights 9 LPG-3400SD and RS: Static mixer LPG-3400BM: Inline filter 10 Purge unit with purge valve and pressure transducer for the system pressure 11 Pump block status LED 12 4-channel proportioning valve
  • 16. Gamal A. Hamid16 1. Isocratic pump ISO The mobile phase, either a pure solvent or a mixture, remains the same throughout during the run, Solvents must be pre-mixed. Without degasser.
  • 17. Gamal A. Hamid17 2. High-pressure gradient pump HPG • High-pressure gradient pump with integrated mixing chamber and "2 from 4" solvent selectors Two pumps are used. • The eluents are mixed after pumping. Accurate gradient and minimized delay volume. • Without degasser. • Response of the gradient is superior because of the small volume from the mixing unit to the column.
  • 18. Gamal A. Hamid18 High-pressure gradient pump HPG HPG-3400 HPG-3200
  • 19. Gamal A. Hamid19 3. Low pressure gradient pump LPG • One pump is used for mixing, Up to four eluents can be mixed. • With degasser.
  • 20. Gamal A. Hamid20 4. Dual gradient pump DGP • Two separate pumps with integrated mixing chambers in one enclosure (2x3 solvents). • Two different pumps with different solvents and columns. Without degasser. • Independently-optimized mobile phase composition for 2D separations.
  • 21. Gamal A. Hamid21 Flow rate & pressure
  • 22. Gamal A. Hamid22 Eluents flow diagram
  • 23. Gamal A. Hamid23 RS & SD Pumps
  • 24. Gamal A. Hamid24 Pump Chromeleon page
  • 25. Gamal A. Hamid25 II. Solvent Rack • Second select your Solvent rack that match with your pump selection. • How many channel you have in your pump? • Is your selected pump has degasser or not?
  • 26. Gamal A. Hamid26 Solvent Rack “SR” 1. SR-3000 Solvent Rack Without vacuum degasser typically for use with a LPG-3400, ISO-3100BM, or HPG-3200BX 2. SRD-3200 Solvent Rack With analytical 2-channel vacuum degasser typically for use with the following pumps: - one HPG-3200 (SD or RS) - one ISO-3100SD
  • 27. Gamal A. Hamid27 SRD “Solvent Rack Degasser” 3. SRD-3400 Solvent Rack • With analytical 4-channel vacuum degasser typically for use with the following pumps: - one HPG-3400 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG- 3200 (SD or RS) or ISO-3100SD if you want to degas the solvents and the wash solution of autosampler 4. SRD-3600 Solvent Rack • With analytical 6-channel vacuum degasser typically for use with the following pumps: - one DGP-3600 - two HPG-3200 (SD or RS) pumps in a two-stack system - one HPG- 3200 (SD or RS) and one HPG-3400 in a two-stack system - one HPG-3400 if you want to degas the solvents and the wash solution of autosampler.
  • 29. Gamal A. Hamid29 3. Injector Instrumentation • A device by which a liquid, solid or gaseous sample is introduced into the mobile phase or the chromatographic bed. • The injector must also be able to withstand the high pressures of the liquid system. • Each type is equipped with six-port valves, so that a sample can be injected into the flow path at continuous pressure.
  • 30. Gamal A. Hamid30 In-line Split-Loop autosamplers “SL” • The in-line split-loop (SL) (flow-through) analytical autosampler supports pressures up to 62 MPa (9000 psi) at analytical and micro flow rates. • Within in-line split-loop autosamplers, the sample loop, needle, and needle seat are integral parts of the high pressure fluidic path. • Autosampler aspirates only the sample volume injected onto the column. The needle and sample loop are constantly rinsed with mobile phase, achieving the lowest carryover.
  • 31. Gamal A. Hamid31 Pulled-Loop Autosamplers “PL” • In pulled-loop autosamplers, the needle is not an integral part of the high pressure fluidic path. • Therefore, all autosampler parts in contact with the sample can be made of inert material. • High injection precisions are achieved in full- loop injections at a fixed-sample loop volume. • Variable injection volumes are accessible in partial loop injection mode.
  • 32. Gamal A. Hamid32 Features and Benefits SL BL • High injection volume flexibility • Near zero carry over • No sample loss • Low sample dispersion • Excellent injection precision and linearity • Very short cycle times • Highest versatility and product • Lowest injection volumes down to the nL range • Biocompatible flow paths*1 • Microliter pick-up – zero sample loss for pulled-loop injections • Excellent injection precision even at low volume injections – at 20 nL a precision < 1% can easily be achieved. • Optional fractionation capabilities
  • 33. Gamal A. Hamid33 Autosampler configurations • T Thermostatted • SL Split Loop • PL Pull Loop • B Bio compatible • A Analytical • RS Rapid Separation • FC Fraction Collector • X MS
  • 34. Gamal A. Hamid34 Definitions T The sample thermostatting option allows sample cooling using peltier by max. 22 °C from the ambient temperature. The lower temperature limit is +4 °C. In addition, the samples can be warmed to max. +45 °C. SL In-Line Split Loop flow autosampler the needle is an integral part of the high pressure fluidic path. PL Pulled Loop autosampler the needle is not an integral part of the high pressure fluidic path. B Biocompatible Autosampler perfectly matches the requirements for injections of complex samples containing biomolecules, such as recombinant proteins or monoclonal antibodies. Almost all descriptions of the standard autosamplers apply also to the biocompatible versions. RS UHPLC Rapid Separation the 103 MPa (15,000 psi) in-line split-loop (flow-through) FC The autosampler is equipped with one 2-position, 6-port injection valve and one 2- position,6-port fractionation valve, With this configuration, injection, fraction collection, and reinjection can be fully automated on one system. X The 125 MPa (18,130 psi) in-line split-loop (flow-through) UHPLC autosampler for MS.
  • 35. Gamal A. Hamid35 Autosampler Chromeleon page
  • 36. Gamal A. Hamid36 4. Column Instrumentation • Is The tube and the stationary phase contained within, through which the mobile phase passes. • The heart of the chromatograph. • The column’s stationary phase separates the sample components of interest using various physical and chemical parameters. • The pump push hard to move the mobile phase through the column and this resistance causes a high pressure.
  • 37. Gamal A. Hamid37 Column types Name Description 1 Acclaim® Reversed-Phase Col. RSLC, PepMap, Polar Advantage 2 Acclaim Multi-Mode Columns Trinity™ P-1 3 Acclaim Specialty Columns Organic acids, surfactants, explosive 4 ProSwift® Columns Monolith RP and ion exchange 5 PepSwift™ Columns RP nano, capillary and micro columns 6 DNASwift™ Columns Monolith SAX
  • 38. Gamal A. Hamid38 Thermostatted Column Compartment TCC • Accommodation of up to 12 columns, the maximum column length is 300 mm. • Column switching valves make the TCC the appropriate choice for almost all applications in HPLC and UHPLC. • Homogeneous temperature distribution via a fan-based, forced-air design • Column identification system and comprehensive system wellness features • Titanium and PEEK valves are available for different bio-UHPLC and bio-HPLC applications. • Pre-column heater brings the solvent and the sample to the column temperature before they enter the column. This avoids temperature gradients in the column. • Post-column cooler (only TCC-3000RS) cools the column eluate to a temperature that is appropriate for the subsequent detection. Post-column cooling minimizes the noise values and thus leads to optimal detection results.
  • 39. Gamal A. Hamid39 Column Compartment TCC-3000 RS • The Rapid Separation (RS) Thermostatted • Wide temperature range from 5 (maximum 18 °C below ambient)–110 °C with excellent accuracy and precision • Optional column switching valves • Post column cooling ensures lowest detector noise and drift • Column identification system for up to four columns.
  • 40. Gamal A. Hamid40 Column Compartment TCC-3000 SD • The Standard (SD) Thermostatted Column • Compartment is a cost-effective choice for • conventional HPLC as well as bio chromatography separations. • Up to 80 °C and precolumn heating. • TCC 3000 SD similar to TCC 3000 RS in the most specifications except the temp. range.
  • 41. Gamal A. Hamid41 Column heating Reproducibility • If temperature varies, then it is difficult to assign “peaks” to specific compounds in the chromatogram and the peak areas/heights may vary Solubility • Certain chemical compounds may have low solubility in the HPLC mobile phase • If they are injected into the flow stream they may precipitate or other difficulties may arise Stability • Certain chemical compounds, especially biological compounds such as enzymes or proteins, may not be stable at room temperature or higher • The temperature needs to be much lower down to 4°C
  • 42. Gamal A. Hamid42 Home page Chromeleon
  • 43. Gamal A. Hamid43 5. Detector Instrumentation A device that measures the change in the composition of the eluent by measuring physical or chemical properties. The desirable features of a detector are: • Sensitivity towards solute over mobile phase • Low cell volumes to minimize memory effects • Low detector noise • Low detection limits • Large linear dynamic range
  • 44. Gamal A. Hamid44 UV/UV-VIS Detectors (variable λ detector) • The Thermo Scientific™ Dionex™ UltiMate™ 3000 VWD-3000 is a variable wavelength detector (VWD) series for industry leading UV-VIS detection. • flow cells ensure optimal performance over a flow rate range of five orders of magnitude. 1. Lamps cover 2. Flow cell 3. Flow cell drainage 4. Leak sensor
  • 45. Gamal A. Hamid45 VWD Principle • A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm. • If components are to be detected at wavelength longer than this, a UV-VIS detector is used, which employs an additional tungsten lamp (W lamp). • By monitoring the reference light divided from the light in front of the flow cell, the difference in light intensity can be determined between the back and front of the flow cell, and this is output as absorbance.
  • 46. Gamal A. Hamid46 VWD Features and highlights • Data collection rate: Single wavelength up to 100 Hz (VWD-3100) Single wavelength up to 200 Hz (VWD-3400RS) Multiple wavelength up to 5 Hz (VWD-3400RS). • Light source: Deuterium lamp, tungsten lamp (Tungsten lamp optional on VWD-3100) Temperature control for both lamps • Flow cells: wide range of available ensure optimal performance over a flow rate range of five orders of magnitude, Analytical flow cell, 11 μL, stainless steel • Wavelength range: 190–900 nm with combined use of deuterium and tungsten lamp on one optical axis. • Temperature control of optics and electronics for data acquisition independent of ambient conditions. • The detector’s large linearity range of up to 2.5 AU is ideal for applications with widely varying analyte concentrations. • The VWD-3000 series uses a fast-turning grating monochromator placed in front of the flow cell. • No. of Channels: 1 channel (VWD -3100 SD) Up to 4 channels (VWD-3400 RS)
  • 47. Gamal A. Hamid47 UV Detector Chromeleon page
  • 48. Gamal A. Hamid48 Diode array detector (DAD, PDA) • Photodiode arrays (semiconductor devices) are used in the detection unit. • The Thermo Scientific™ Dionex™ UltiMate™ DAD 3000 detector is a high-resolution, 1024- element diode array detector (DAD) available in Rapid Separation and Standard versions. • Optical System, Single-beam, reverse optics design with concave holographic grating. • The detector is also available as a multiple wavelength detector (MWD) in Standard and Rapid Separation versions, MWD does not support the acquisition of 3-D spectra .
  • 49. Gamal A. Hamid49 PDA Principle • The polychromatic beam passes the flow cell. • The grating splits up the polychromatic beam to different wavelengths, the intensities of which are measured by an array or photodiodes. • A photodiode is a semiconductor device that converts light into current. • The current is generated when photons are absorbed in the photodiode. 1. VIS Lamp 2. D 2 Lamp 3. Flow cell Adaptor 4. Flow cell
  • 50. Gamal A. Hamid50 DAD Features and highlights • Data collection rate: Diode array detector (DAD) available in Rapid Separation (200 Hz) using a maximum of eight single-wavelength data channels and one 3-D field (and Standard (100 Hz) versions. • Light source: Deuterium lamp, tungsten lamp Flow cells: Flow cell range covers semi-micro, semi- analytical, analytical, and semi-preparative flow rate applications, in stainless steel and biocompatible versions. • Wavelength range: The tungsten and deuterium lamps emits light in UV and visible ranges (190 -800) nm. • Temperature control of optics and electronics for data acquisition independent of ambient conditions. • A typical detector linearity up to 2.0 AU. • Temperature control for both lamps. • No. of Channels: Up to 8 channels (DAD-3000 SD) Up to 8 channels (DAD-3000 RS)+ 3D
  • 51. Gamal A. Hamid51 Fluorescence Detector • Xenon lamp Light source for the UV to near- infrared wavelength range. • PMT Photomultiplier tube (PMT) - Converts the emitted light to a measureable current signal. • Fluorescence detection offers 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 column Derivatization
  • 52. Gamal A. Hamid52 FL Principle • FL is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. Such a substance has specific wavelengths of light that it absorbs (excitation wavelengths) and emits (emission wavelengths). • While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction orthogonal to the exciting light. • A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. However, some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength.
  • 53. Gamal A. Hamid53 FLD Features and highlights • Data collection rate: Single wavelength: up to 200 Hz (FLD-3400RS) Multiple wavelength: up to 4 Hz (FLD-3400RS) Single wavelength: up to 100 Hz (FLD-3100). • Light source: High-intensity xenon flash lamp with smart control features • Flow cells: 8 μL analytical flow cell 2 μL micro flow cell. • Wavelength range: Wavelength range 200 – 650 nm Wavelength range 200 – 900 nm (dual). • Long lamp life (> 15,000 h in long life mode). • Flow Cell temp: Thermostatting: Ambient + 15 °C to 50 °C. • Spectrum Scanning Modes: 2D or 3D excitation, emission • No. of Channels: Up to 4 channels (FLD-3400RS) 1 channel (FLD-3100)
  • 54. Gamal A. Hamid54 FL Detector Chromeleon page
  • 55. Gamal A. Hamid55 Refracted index Detector • The response is dependent on changes in refractive index of eluting compounds in the mobile phase. • The mobile phase itself should have refractive index different from the sample. • Gradient programming is not possible due to resulting changes in refractive index of mobile phase. • Temperature control is necessary as it has high temperature sensitivity. • Any component in the eluate can be detected; thus, the RI detector is often called a “universal detector”. • Typical applications are in Size Exclusion Chromatography. • The detector is less sensitive than UV-VIS detector.
  • 56. Gamal A. Hamid56 RI Principle • RI detector detects components based on the refraction of light in solution. • The reference-side cell is filled with eluate, and the column eluate is introduced into the sample-side cell through the changed flow channel. • When components are eluted from the column, the chemical composition changes in the sample-side solution, which changes its photorefractive level. As a result, the amount of light received by the light- receiving section changes, showing a peak which can be detected.
  • 57. Gamal A. Hamid57 FLD Features and highlights • Data collection rate: Single wavelength: up to 200 Hz (FLD-3400RS) Multiple wavelength: up to 4 Hz (FLD-3400RS) Single wavelength: up to 100 Hz (FLD-3100). • Light source: High-intensity xenon flash lamp with smart control features • Flow cells: 8 μL analytical flow cell 2 μL micro flow cell. • Wavelength range: Wavelength range 200 – 650 nm Wavelength range 200 – 900 nm (dual). • Long lamp life (> 15,000 h in long life mode). • Flow Cell temp: Thermostatting: Ambient + 15 °C to 50 °C. • Spectrum Scanning Modes: 2D or 3D excitation, emission • No. of Channels: Up to 4 channels (FLD-3400RS) 1 channel (FLD-3100)
  • 58. Gamal A. Hamid58 Corona Detector • The Corona CAD detector provides the ability to measure virtually any nonvolatile and many semi volatile analytes: • Positive, negative, or neutral, with or without a chromophore, all can be detected routinely with charged aerosol detection. • The Corona CAD can readily detect compounds present in single digit nano gram quantities. • Corona CAD’s unique method of detection allows quantitation across a range that exceeds four orders of magnitude
  • 59. Gamal A. Hamid59 Corona principle • The Corona CAD “Charged Aerosol Detector “ measures charge that is imparted to analyte particles, with the charge being in direct proportion to the amount of the analyte in the sample. • Samples nebulized by air or nitrogen then dried, dried particles combined with the charged gas (charged using high voltage), pass through the collector where the charged particles are measured using sensitive electrometer.
  • 60. Gamal A. Hamid60 Corona Applications • Pharmaceuticals • Proteins • Lipids • Steroids • Oligosaccharides • Surfactants • Carbohydrates • Polymers • Peptides Corona CAD is appropriate for any nonvolatile and many semi volatile compounds,
  • 61. Gamal A. Hamid61 Systems variations Item Nano/Cap Basic automated / Standard RSLC Pump Max. Pressure 800 bar 620 bar 1034 bar Pump. Max. Flow 50 µL/min 10 mL/min 5 mL/min @ 1034 bar 8 mL/min @ 800 bar Autosampler Max. pressure 1000 bar 620 bar 1034 bar Column Oven Max. Temperature 80 °C 50 oC (Basic) 80 °C (Standard) 110 °C Column Oven Max pressure 1000 bar 620 bar 1034 bar Detector Data Collection Rate 200 Hz* 100 Hz 200 Hz*
  • 63. Gamal A. Hamid63 Fit the system to the applications The UltiMate 3000 systems are fully modular, allowing you to choose common system configurations or design the most suitable system for your application needs.
  • 64. Gamal A. Hamid64 Applications fields • Environmental applications • Food applications • Pharmaceutical applications • Bioanalytical applications • Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) • Salts like sodium chloride and potassium phosphate • Proteins like egg white or blood protein • Organic chemicals like polymers (e.g. polystyrene, polyethylene) • Heavy hydrocarbons like asphalt or motor oil • Many natural products such as ginseng, herbal medicines, plant extracts • Thermally unstable compounds such as trinitrotoluene (TNT), enzymes
  • 66. Gamal A. Hamid66 Carbohydrates Analysis https://tools.thermofisher.com/content/sfs/brochures/AI-71469-Chromatography-Foods-Beverages-Carbohydrates-AI71469-EN.pdf
  • 67. Gamal A. Hamid67 Sugar Substitutes http://tools.thermofisher.com/content/sfs/brochures/AI71470-Chromatography-Foods-Beverages-Sugar-Substitutes-AI71470-EN.pdf
  • 68. Gamal A. Hamid68 Fats and Oils Analysis https://tools.thermofisher.com/content/sfs/brochures/AI-71471-Chromatography-Foods-Beverages-Fats-Oils-AI71471-EN.pdf
  • 69. Gamal A. Hamid69 Flavors, Colorants and Additives http://tools.thermofisher.com/content/sfs/brochures/AI-71472-Chromatography-Foods-Beverages-Flavors-Colorants-Additives-AI71472- EN.pdf
  • 70. Gamal A. Hamid70 Supplements Analysis http://tools.thermofisher.com/content/sfs/brochures/AI-71473-Chromatography-Foods-Beverages-Supplements-AI71473- EN.pdf
  • 71. Gamal A. Hamid71 Profiling Beverages https://tools.thermofisher.com/content/sfs/brochures/AI-71474-Chromatography-Profiling-Beverages-AI71474-EN.pdf
  • 72. Gamal A. Hamid72 Adulteration and Authentication https://tools.thermofisher.com/content/sfs/brochures/AI-71475-Chromatography-Foods-Beverages-Adulteration-Authentication- Applications-Notebook-AI71475-EN.pdf
  • 74. Gamal A. Hamid74 Antifoaming Agents, Emulsifiers and Surfactants http://tools.thermofisher.com/content/sfs/brochures/AI-71477-Chromatography-Foods-Beverages-Antifoaming-Emulsifiers- Surfactants-AI71477-EN%201214S.pdf
  • 75. Gamal A. Hamid75 Vitamin and Antioxidant https://tools.thermofisher.com/content/sfs/brochures/AI-71478-Chromatography-Foods-Beverages-Vitamins-Antioxidants- AI71478-EN.pdf
  • 76. Gamal A. Hamid76 Visit thermoscientific.com/AppsLab • Access GC, IC, LC, GC-MS, and LC-MS application notes in a single online location. • Download one-click workflows: immediately ready to run in Chromeleon CDS. • Compare results and identify optimum chromatographic separation conditions quickly and easily. • Visible to all users. Register once for free and download complete separation methods. https://appslab.thermofisher.com/