Chromatography PDF

Analytical instrumentation: Chromatography What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyse, identify, purify, and/or quantify the mixture or components.  Uses Analyse Identify Purify Quantify ...

Analytical instrumentation: Chromatography What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyse, identify, purify, and/or quantify the mixture or components.  Uses Analyse Identify Purify Quantify  Mobile phase, buffer or gas (for solubilisation and movement of components) Column Stationary phase, matrix  Mobile phase B interacts Sample strongly with the matrix A+B (Stationary B phase) than A B A, hence B takes longer Packed to get to the A B detector. column This interaction can be A chemical or physical B To detector T0 T1 T2 T3 T4 Detector signal T0 T1 T2 T3 T4 time  Preparative chromatography refers to the isolation or purification of target molecules. In contrast, analytical chromatography uses the separation of molecules to identify and quantify components of mixtures. A bit of theory and maths  Theory, concentration versus elution time TR Detector signal TM Wh Wb Injection time TR: retention time TM: void Wb: baseline width of the peak in time units Wh: half-height width of the peak in time units The separation of compounds depends on: A difference in the retention of solutes A sufficient width of the solute peaks A B Detector signal T0 T1 T2 T3 T4 time We can use elution volume instead of elution time, in this case the volume of the mobile phase that it takes to elute a peak off the column is referred to as retention volume (VR) and the amount of mobile phase that it takes o elute a non-retained component is referred to as the void volume (VM) Solute retention The solute retention time or volume is related to the strength of the solute’s interaction with the mobile phase and stationary phases The retention on a particular column depends on the size of the column and the flow rate average migration rate, V=L/TR with L being the column length and TR the retention time Capacity (retention) factor Useful for comparisons of results from different systems Capacity factor, K’=(TR – TM )/ TM Or K’=(VR – VM )/ VM The fundamental definition of K’=moles A stationary phase /moles A mobile phase If K’≤ 1.0, separation is poor If K’> 30, separation is low If K’ = 2-10 separation is optimum Why? Separation factor, ratio of the K’ for 2 peaks Asymmetry of the peak  Efficiency – number of theoretical plates (N) Compares the efficiency of a system for compounds that have different retention times N=(TR/σ)2 , σ is related to the width of the peak from a Gaussian bell shape Theoretical plate The column Resolution, RS Resolution Efficiency Experimentally efficiency is related to the solute’s peak width, if it is good the peaks will be narrow, this means smaller difference in interactions in order to separate solutes Theoretically efficiency is related to many kinetic processes in solute retention and transport in the column Height equivalent of theoretical plates (HETP) H = L/N, L: column length and N: number of theoretical plates H can link various parameters such as flow rate, particle size, and more to the kinetic process of peak broadening The bands spread due to: eddy diffusion The bands spread due to: mobile phase mass transfer, The bands spread due to: stationary phase mass transfer, The bands spread due to: longitudinal diffusion Same sample, analysed with same stationary phase and temperature but with different mobile phases (same gradient). Same sample, analysed with different stationary phases (columns) but always same temperature, mobile phase and gradient. Same sample, analysed with same stationary and mobile phase, same gradient but different temperatures.  Methods and instrumentation Column chromatography is a method commonly used to separate molecules in complex mixtures. The stationary phase or resin is packed into a column. The mobile phase is then passed through the packed stationary phase to achieve separation. The purpose of this separation can be analytical or preparative. Both gas chromatography and liquid chromatography separations are performed on columns.  Column Chromatography Systems Column chromatography is performed on a packed, 3- dimensional stationary phase inside a glass, plastic, or metal column and can be used for both preparative and analytical purposes.  Gas chromatography Gas chromatography, the mobile phase is a gas: Gas-Liquid (the stationary phase is a liquid-coted support) Gas-Solid (the stationary phase is a solid underivatised support) It separates molecules based on their boiling points and their interaction with the stationary phase. Mixtures to be separated are heated past the boiling of their least volatile component. They are then swept across the solid phase by an inert gas, hence the name gas chromatography. The stationary phase in gas chromatography is the inside surface of a long capillary column coated with a liquid or polymer through which the gas mixture flows. Although GC can be used for preparative purposes, it is most commonly used for analytical work. Gas chromatography It involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.  Gas chromatography Instrumental components Carrier gas: The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. Sample injection port: The sample should not be too large, and should be introduced onto the column as a "plug" of vapour. The temperature is usually about 50°C higher than the boiling point of the least volatile component of the sample. The injector contains a heated chamber which the sample is injected through the septum. The carrier gas enters the chamber. The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet.  Gas chromatography Instrumental components Columns: There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns. Fused Silica Open Tubular (FSOT) column. These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity. Column temperature: As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.  Gas chromatography Instrumental components Detectors. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. The signal from a concentration dependant detector is related to the concentration of solute in the detector. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample. Gas chromatography Detector Type Support gases Selectivity Detectability Dynamic range Flame ionization Mass flow Hydrogen and air Most organic cpds. 100 pg 107 (FID) Thermal conductivity Concentration Reference Universal 1 ng 107 (TCD) Halides, nitrates, nitriles, Electron capture Concentration Make-up peroxides, anhydrides, 50 fg 105 (ECD) organometallics Nitrogen-phosphorus Mass flow Hydrogen and air Nitrogen, phosphorus 10 pg 106 Sulphur, phosphorus, tin, Flame photometric Hydrogen and air Mass flow boron, arsenic, germanium, 100 pg 103 (FPD) possibly oxygen selenium, chromium Aliphatics, aromatics, ketones, esters, aldehydes, Photo-ionization Concentration Make-up amines, heterocyclics, 2 pg 107 (PID) organosulphurs, some organometallics Hall electrolytic Halide, nitrogen, Mass flow Hydrogen, oxygen conductivity nitrosamine, sulphur Liquid chromatography It separates compounds dissolved in a liquid mobile phase by passing the liquid over a solid stationary phase, the chromatography media, or resin, with which the compounds have differing degrees of interaction. Individual components of the mobile phase are thus more or less retarded in their passage through the chromatography media. LC is further subdivided into planar chromatography and column chromatography. When liquid chromatography is carried out in a single plane it is referred to as planar liquid chromatography; here, the liquid mobile phase passes through a solid stationary phase such as a strip of paper (paper chromatography) or silica gel that is immobilized on a glass slide (thin-layer chromatography/TLC). Liquid chromatography Liquid chromatography, the mobile phase is a liquid. Chromatography stationary phases vary widely in composition. One or more media types may be used depending on the properties of the molecules of interest to be separated: Ion exchange (the stationary phase is a support containing fixed charges) Size Exclusion (the stationary phase is a porous support) Partition (the stationary phase is a liquid-coated or derivatised support) Liquid – Solid Adsorption (the stationary phase is a solid underivatised support) Affinity (the stationary phase is a support with inmobilised ligand) Hydrophobicity (hydrophobic interaction chromatography) Paper, Thin layer The support material can be different too What happens inside the column? Time t Separation tr2-tr1 Peak width Wb1,2  Affinity Chromatography (AC) AC exploits specific interactions among molecules, for example, the binding of a protein to its in vivo binding partner or an antibody. Proteins of interest can be genetically fused to affinity tags such as polyhistidine or glutathione S-transferase (GST), or modified post-translationally with biotin or other nonpeptide tags. Chromatography resin can then be functionalized with molecules that specifically bind these tags, for example, reduced glutathione (GSH) to bind GST, or avidin/streptavidin to bind biotin. When a complex protein mixture is passed over a functionalised resin, the protein of interest is immobilised on the resin but contaminating proteins are not bound. The molecule of interest can then be released from the resin using a buffer with a high salt concentration, a pH shift, or a competing ligand. Tagged proteins may be removed by enzymatic tag cleavage or other methods, and the protein can be repurified using an affinity column to capture the cleaved tags while allowing free protein to pass through. Applications: affinity chromatography can be used as an initial capture step, an intermediate purification step, or a final polishing step. Affinity Chromatography (AC) Immobilized Metal Ion Affinity Chromatography (IMAC) A special subcategory of peptide tag–based affinity chromatography, immobilized metal ion affinity chromatography (IMAC), utilizes the interaction of histidine residues with divalent metal ions such as Ni2+, Cu2+, Zn2+, and Co2+. A myriad of commercially available expression vectors can be used to add polyhistidine tags to proteins of interest to facilitate histidine-tagged recombinant protein purification. Applications: IMAC can be used as an initial capture, intermediate purification, or final polishing step.  Ion Exchange Chromatography (IEX) IEX exploits the electrostatic interaction between opposite charges to separate proteins based on their isoelectric points (pI). In a buffer with a pH greater than the pI of the protein of interest, the protein will have a net negative charge; thus, a positively charged anion exchange resin is chosen to capture the molecule of interest. The molecule is then eluted with a buffer with a pH lower than the protein pI, resulting in a net positive charge on the protein and a loss of attraction for the resin. Alternatively, an increasing gradient of negatively charged ions is used to displace the protein from the resin by competing with it for the positively charged groups on the resin. If the buffer pH is below the target molecule’s pI, the protein will carry a positive net charge and a negatively-charged cation exchange resin is chosen. The molecule is then eluted with a pH higher than the protein’s pI or with an increasing gradient of negatively charged ions. Applications: IEX can be used as an initial capture step, an intermediate purification step, or a final polishing step. Ion Exchange Chromatography (IEX) Size Exclusion Chromatography/Gel Filtration Size exclusion chromatography (SEC), also called gel filtration chromatography, separates molecules based on their sizes. SEC resins are gels that contain beads with a known pore size. When a complex mixture is passed over SEC resin, small molecules move through the bead pores, whereas molecules too large to fit into the pores move around the beads and through the void space between beads. Since the traveled distance through the pores is greater than the distance around the beads, molecules elute from SEC resins in order of. Applications: SEC is not suitable as a first purification step but is often used as a final polishing step for buffer exchange and to remove protein fragments and aggregates. Size Exclusion Chromatography/Gel Filtration Hydrophobic Interaction Chromatography Hydrophobic interaction chromatography (HIC) separates molecules based on their hydrophobicity. Hydrophobic amino acids (phenylalanine, tyrosine, and tryptophan) are generally buried within the three-dimensional structure of proteins, although some can be exposed on the protein surface. In an aqueous solvent, hydrophobic residues interact with each other via Van der Waals interaction, which is exploited for HIC. HIC resin is functionalised with aliphatic compounds that interact with and bind hydrophobic residues. Hydrophobic Interaction Chromatography This interaction can be modulated by differing salt concentration, pH, temperature, and organic solvent concentration. In particular, high ionic strength increases the interaction of hydrophobic residues with the HIC resin. For this reason HIC columns are generally loaded in high salt. Compounds are then eluted using a reverse salt (often ammonium sulfate) gradient or a gradient of organic solvent such as ethylene glycol. Applications: HIC is suitable for initial capture, intermediate purification, and final polishing steps. HIC is an excellent choice following ammonium sulfate precipitation and ion exchange chromatography due to its compatibility with high-ionic-strength buffers. Hydrophobic Interaction Chromatography Partition chromatography Absorption chromatography Multimodal or Mixed-Mode Chromatography Multimodal or mixed-mode chromatography (MMC) incorporates multiple modes of chromatography in a single resin. This enhances the selectivity of the resin because compounds can be separated based on several of their characteristics, rather than just a single one. MMC is of great value in cases where other resins cannot provide sufficient resolution. Applications: MMC is an excellent choice for initial capture, intermediate, and polishing steps.  Chromatography Media Selection 1. A good rule of thumb is to get to the required level of purity in the least number of steps. 2. When possible, choose column combinations that do not require buffer exchange or concentration steps. 3. Some affinity chromatography resins, conversely, are incompatible with high-ionic-strength buffers. Rather than choosing an IEX → buffer exchange → AC → HIC workflow, an IEX → HIC → AC combination would allow you to eliminate the buffer exchange step. 4. An important consideration when contemplating use of SEC resins is that samples often have to be concentrated unless their volume is already small. 5. Finally, features of the compound of interest are also important considerations. The high ionic strength required for HIC may cause the compound of interest to “salt out”, that is, precipitate from solution due to physicochemical effects of various salts.  Column Chromatography Systems, LC Column chromatography is classified according to the type of fluid flow system used: Gravity chromatography Low-pressure chromatography Medium-pressure chromatography (including fast protein liquid chromatography) High-pressure/high-performance liquid chromatography (HPLC)  Gravity Chromatography Uses gravity to pass sample and buffers across the column resin. Although prepacked gravity columns are commercially available, it is not uncommon for users to pack their own columns. Small-volume columns (~1.5 ml) designed for quick flow through by spinning in a micro-centrifuge provide a convenient method for rapidly purifying many small samples. Elution from the column is often followed by quantification in collected fractions using fluorometry or spectrophotometry, typically in the UV-visible range. Gravity chromatography is a low-cost, simple method of preparative chromatography, however it does not afford high resolution. Applications: small-scale, rapid preparative chromatography.  Low-Pressure Chromatography Low-pressure chromatography systems are operated at less than 50 psi (0.35 MPa). These systems require a sample pump and are often equipped with fraction collectors, gradient capabilities, and detectors to monitor column elution. Low-pressure systems are compatible with prepacked columns and offer more flexibility and higher resolution than gravity chromatography. Nonetheless, low-pressure chromatography is not a high-resolution chromatography method suitable for complex protein purifications. Applications: medium-scale preparative chromatography.  Medium-Pressure Chromatography Medium-pressure chromatography, also referred to as fast protein liquid chromatography (FPLC), is more suitable for complex purifications and can also be used for analytical purposes. Medium-pressure chromatography is conducted at operating pressures that are actually rather high, up to 3,500 psi (24 MPa). These systems are compatible with a wide range of prepacked columns and resins and can therefore be used for simple recombinant protein purifications as well as for complex analytical purposes. Applications: preparative and analytical chromatography of a wide variety of molecules, ranging from nonvolatile organics to nucleic acids, peptides, and proteins.  High-Pressure Liquid Chromatography (HPLC) HPLC is conducted at very high pressures — up to 5,000 psi (34 MPa). HPLC is a powerful analytical tool providing high resolution and sensitivity, with the ability to detect concentrations down to parts per trillion while having very small sample requirements (in the microliter range). Because HPLC systems can be operated at higher pressure, the resin particle size can be decreased, thereby increasing the resin’s resolution. HPLC systems usually have several types of detectors. A common combination is a UV- absorbance detector in combination with an evaporative light scattering detector (ELSD) and/or a mass spectrometer (MS). The combination of HPLC and mass spectrometry, a technique for determining the mass-to-charge ratios of molecular ions created by fragmenting the analytes after elution using electrospray, electron-impact, or other ionisation methods, yields HPLC-MS, often abbreviated LC-MS.  High-Pressure Liquid Chromatography (HPLC) HPLC, like lower-pressure forms of chromatography, can be used to separate molecules based on size (size exclusion chromatography, or SEC), hydrophobicity (hydrophobic interaction chromatography, or HIC), or charge (ion exchange chromatography, or IEX). Unlike other forms of liquid chromatography, HPLC is not suitable for affinity chromatography. Applications: preparative and analytical chromatography of a wide variety of molecules, ranging from non-volatile organics to nucleic acids, peptides, and proteins.  Liquid Chromatography Workflow Most liquid chromatography protocols begin with a resin equilibration step. A buffer that is compatible with the molecule of interest and the resin of choice is passed over the column. A common practice is to equilibrate the column with 5–10 column volumes (CVs) of equilibration buffer. Sample Loading: After equilibration, the sample is loaded onto the column. Sample can be loaded manually or using a sample pump. Column Washing: Once molecules have been immobilised on the stationary phase, molecules that interact only weakly or nonspecifically with the resin are removed by washing the column with several column volumes of wash buffer. Sample Elution: After all non-specifically and weakly interacting proteins have been washed off of the resin, molecules that interact strongly with the resin are eluted from the column by changing the composition of the buffer that is passed over the resin.  Liquid Chromatography Workflow Final Column Washing: This step permits columns to be reused for future separations. Column Regeneration: After stripping the remaining compounds bound to the media, the column is then either saturated with equilibration buffer for subsequent reuse or filled with a storage buffer.  Gradient vs. Isocratic Conditions Isocratic: Mobile phase solvent composition remains constant with time Best for simple separations Often used in quality control applications that support and are in close proximity to a manufacturing process Gradient: Mobile-phase solvent composition increases with time Best for analysing complex Separation of Herbicides on ZORBAX StableBond-C18 Isocratic Elution Column: ZORBAX SB-C18 Gradient Elution 70% water/30% Acetonitrile 4.6 x 150 mm, 5 µm 20 – 60% Acetonitrile/water Mobile Phase: A: H2O with 0.1% TFA, pH 2 1,2 1,2 4 B: Acetonitrile 5 4 5 Flow Rate: 1.0 mL/min 4 4 55 Temperature: 35°C Sample: 1. Tebuthiuron 88 2. Prometon Note: 3. Prometryne last peak 4. Atrazine eluted in ~ 28 5. Bentazon minutes and it 6. Propazine 22 is sharper 33 7. Propanil 3 3 8. Metolachlor 66 11 66 Note: last peak eluted 8 8 77 7 7 in ~70 minutes 00 25 25 50 50 75 75 0 0 5 5 10 10 15 20 15 20 25 25 30 30 Tim e (m in) Tim e (m in) Time (min) Time (min)  Liquid Chromatography Considerations Resolution refers to the separation of peaks in a chromatogram. The purpose of chromatography is to separate molecules of interest. If the goal of chromatographic separation is purification then yield, defined as the amount of the desired protein fraction recovered, is an important consideration. Sample integrity is another key consideration for preparative chromatography. Lastly, sample purity is an important consideration. When developing a purification workflow it is wise to consider the sample purity that is required for the intended downstream applications because sample purity, integrity, and yield often display an inverse relationship. Absolute sample purity is essential in certain applications such as antibody production for diagnostic or therapeutic applications. Some enzymatic studies, however, may require only functional purity; proteins that do not interfere with or enhance the protein of interest’s activity may be tolerated as contaminants to maximize sample integrity and yield. Chromatography and pH  Polarity A molecule’s structure, activity, and physicochemical characteristics are determined by the arrangement of its constituent atoms and the bonds between them. This structure often determines whether the molecule is polar or non-polar. Polarity Normal and Reverse phase Chromatography Normal phase HPLC means the stationary phase is polar and the mobile phase is non-polar; Reversed phase means the stationary phase is non-polar and the mobile phase is polar. Today, because it is more reproducible and has broad applicability, reversed-phase chromatography is used for approximately 75% of all HPLC methods. Most of these protocols use as the mobile phase an aqueous blend of water with a miscible, polar organic solvent, such as acetonitrile or methanol. This typically ensures the proper interaction of analytes with the non- polar, hydrophobic particle surface. A C18–bonded silica [sometimes called ODS] is the most popular type of reversed-phase HPLC packing.  U(H)PLC UPLC (ultra performance liquid chromatography) systems were first introduced in 2004. By almost doubling the overall operating pressure (to 15,000 psi) in order to obtain more rapid flow rates, UPLC developers were able to achieve equal or better resolution LC separations in much shorter time frames. By contrast, UHPLC (ultra high performance liquid chromatography) operates in the 20,000 psi range. UPLC and UHPLC have been used for many purposes, including legal, pharmaceutical and medical. The systems are also important tools in the fields of research and manufacture.  Case: XBridge Columns for High pH and Method Development XBridge are built to withstand high pH, high temperatures , and other extreme operating conditions, including volatile eluents, for a wide range of small molecule and biomolecule LC- MS analysis (Pharm Industry). These columns are available in a broad range of sorbent particles and sizes. XP Columns (2.5 µm) for rapid analytical UHPLC separations, (3.5 and 5 µm) for reliable analytical method development and OBD Prep (5 and 10 µm) for purification chromatography. High pH stability and chemical resistance for reliability and longevity Maximum method development flexibility Seamless method transfer of analytical to preparative LC XBridge Columns Column Performance Benefit HPLC General purpose, reversed-phase chromatography, ideally suited for method Analytical to C18 development due to extreme pH stability and applicability to the broadest range of Prep compound classes. 3.5, 5, 10 μm Analytical to General purpose reversed-phase chromatography, ideally suited for method development C8 Prep due to extreme pH stability and applicability to the broadest range of compound classes. 3.5, 5, 10 μm Analytical to Alternate selectivity compared to straight chain C18, particularly with phenolic analytes. Shield RP18 Prep Compatible with 100% aqueous-phase composition. 3.5, 5, 10 μm Analytical to Excellent for method development for alternate selectivity, particularly in regard to Phenyl Prep polyaromatic compounds. Unique level of pH stability for a phenyl bonded phase. 3.5, 5 μm Excellent for retention of very polar, basic, water soluble analytes. Specifically designed Analytical to HILIC and tested for HILIC separations using mobile phases containing high concentrations of Prep organic solvent. 3.5, 5 μm Rugged HILIC stationary phase designed to separate a wide range of very polar Analytical to compounds. Especially good at separating carbohydrates (saccharides) using high Amide Prep concentrations of organic modifier, elevated temperature, and high pH. Compatible with 3.5, 5 μm all modern detectors including MS, ELSD, UV, and fluorescence.