Performing Basic Experiments with Chromatography Consumables & Solutions

Chromatography is a powerful analytical technique used to separate, identify, and quantify components in a mixture. From quality control in manufacturing to cutting-edge research, chromatography consumables and solutions are essential tools for both routine and novel experiments. This guide will overview some of the fundamentals of chromatography and provide a walkthrough of basic experimental procedures using common chromatography supplies.

Introduction to Chromatography

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Chromatography encompasses a diverse family of laboratory techniques that leverage the differential affinities of analytes between a mobile phase and a stationary phase to achieve separation. The basic principles governing all chromatographic separations are:

• A stationary phase which provides a fixed matrix through which the mobile phase can flow. The stationary phase can take the form of a solid adsorbent, a liquid immobilized on a solid support, or a gel. The specific chemical properties of the stationary phase material are chosen to interact with analytes in the mixture of interest.
• A mobile phase chosen to flow over the stationary phase. The mobile phase may be a liquid solvent or solvent mixture, or an inert gas. The polarity, pH, and other properties of the mobile phase can be tailored to influence the migration rates of analytes.
• Differential partitioning or adsorption of the analytes occurs between the stationary and mobile phases. Components that have greater affinity for the stationary phase will move more slowly, while components that favor the mobile phase will migrate faster. This partitioning behavior is based on the unique chemical properties of each analyte.
• The differential migration results in the separation of the mixture into discrete zones or 'bands' containing the individual components. The separation power stems from compounds spending different amounts of time interacting with the stationary phase versus being carried by the mobile phase flow.
  
  

There are numerous specific chromatographic techniques that share these common principles but utilize different physical formats. Some of the most widely used and powerful chromatographic methods include:

• Column chromatography - Employs a vertical glass or metal column packed with a powdered solid stationary phase such as silica gel or alumina particles. A liquid mobile phase is flowed through the column via gravity or low pressure.
• Thin layer chromatography (TLC) - Uses a stationary phase coated as a thin uniform layer on a flat inert backing like glass, plastic, or aluminum. The plate is developed in a sealed chamber with a shallow pool of mobile phase solvent.
• Gas chromatography (GC) - Utilizes an inert carrier gas mobile phase flowing through a heated column containing either a solid adsorbent or liquid stationary phase chemically immobilized on solid support particles.
• High performance liquid chromatography (HPLC) - Pumps a liquid mobile phase at extremely high pressures of up to 10,000 psi through a stainless steel column densely packed with very fine, uniform particles of stationary phase typically 3-5 μm in size.
• Ion exchange chromatography - Employs an ion exchange resin as the stationary phase which contains fixed charged functional groups. It separates compounds based on differential ionic interactions with the resin and the aqueous buffer mobile phase.
• Affinity chromatography – Uses a stationary phase matrix covalently modified with a ligand designed to have specific binding affinity for the analyte(s) of interest. Highly selective purification can be achieved.
• Chiral chromatography – Employs a chiral stationary phase containing enantiomerically pure selector compounds. Capable of separating chiral isomers including enantiomers and diastereoisomers.

While there are countless chromatography instruments, stationary phases, and experimental configurations, the core principles of differential partitioning between the mobile and stationary phases remain consistent. This makes chromatography universally applicable for countless analytical separations and purification challenges in research and industry.

Equipment and Consumables

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To perform chromatography, certain equipment and consumables are required. The specific materials will depend on the technique, but some common supplies include:

Chromatography Columns

• Glass or plastic columns are needed to contain the stationary phase bed. For column chromatography, glass columns from 150-300 mm tall x 10-25 mm diameter are typical. Larger diameter columns can be used for preparative separations.
• The column attaches to a stand via clamps and adjustable height ring. A stopcock at the bottom controls mobile phase flow.
• Shorter glass jars or square glass tanks are used in thin layer chromatography. The tanks are sealed during development.

Stationary Phases

• Packing materials like silica gel, alumina, Florisil, and celite for column chromatography. Particle sizes from 40-63 μm are common. Smaller particles improve resolution but slow flow.
• Reverse phase packings like C18 bonded silica for retention of non-polar analytes.
• Bonded phases for ion exchange, chiral, and affinity separations.
• TLC plates coated with uniform layers of silica gel, alumina, cellulose on plastic or aluminum backing. C18 reverse phase TLC plates also available.

Mobile Phases

• Liquid solvents: Hexane, dichloromethane, ethyl acetate, acetone, acetonitrile, methanol, water, buffers. HPLC grade solvents are required.
• Gas supplies like nitrogen, helium, and hydrogen for gas chromatography. Ultra high purity gases needed.
• Deionized water for buffer preparation. Inline vacuum degassing essential for HPLC.

Sample Application

• Micropipettes for precise liquid sample application onto stationary phase. Digital adjustable pipettes for volumes from 0.1 μL to 1000 μL are standard.
• Microsyringes for direct sample injection into GC and HPLC injector ports. 10 μL syringes common for capillary columns.
• TLC capillary tubes for sample spotting. Disposable 1 μL tubes ideal. Automated TLC spotters available.

Fraction Collection

• Erlenmeyer flasks, vials, tubes to collect column fractions. Bulb-type drip collectors allow fractionation without stopcock adjustment.
• Capillary tubes or micropipette tips used to sample TLC band spots.
• Automated fraction collectors available for preparative HPLC.

Detection and Visualization

• TLC spray reagents for visualization like ninhydrin, vanillin, iodine. Heated plates accelerate development.
• TLC densitometers for quantitation. UV, fluorescence, and IR modes available.
• GC detectors: FID for organics, TCD for gases, ECD for organohalogens. Mass spec provides analyte identification.
• HPLC UV-Vis, fluorescence, and photodiode array detectors. Refractive index and ELSD for non-UV analytes.

Safety

• Gloves, lab coat, goggles minimum PPE for solvent handling. Chemical fume hoods for vapor containment.
• Safety screens for TLC tanks and HPLC detectors. Solvent-resistant tubing. Leak detectors.
• Flame arrestors, capabilities for inert operation on GC systems. Gas cylinder restraints.

Other Equipment

• Analytical balances for precise stationary phase and sample weighing. Moisture analyzers to determine water content.
• pH meters, electrode buffers, and chemical standards for mobile phase preparation and adjustments.
• Vacuum pumps, manifolds, flasks, and rotary evaporators for solvent concentration of fractions.
• Sonicators, hot plates, stirrers, and specialty glassware. Centrifuges for extract clarification.

The wide range of chromatography equipment and consumables available from IT Tech facilitates any analytical separation or purification challenge. Our technical experts can recommend optimal supplies tailored for your specific application needs.

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Experimental Procedures

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With the necessary equipment and supplies ready, we can go through more detailed examples of fundamental chromatography experiments. Proper safety precautions should always be followed when handling solvents and hazardous chemicals.

Column Chromatography

Column chromatography is a versatile technique used for both analytical small scale sample separations and preparative large scale compound purifications.

Steps:

1. Select an appropriate size glass column and secure it vertically to a ring stand using clamps. Funnel and lightly plug the column outlet with glass wool or cotton.
2. Prepare a slurry of the selected stationary phase (silica gel, alumina, etc.) in an appropriate solvent. Carefully pour the slurry into the column using a glass rod to minimize bubbles. Gently tap the column to settle the packing.
3. Rinse the stationary phase with at least 2 column volumes of solvent to remove fines. Allow solvent flow to compact the bed. Stop the flow before the bed runs dry.
4. Dry the packed column with a gentle stream of air or nitrogen. Tap to remove any residual solvent pockets.
5. Add a top layer of clean sand or solid stationary phase to protect the column bed. Rinse to level the sand layer.
6. Dissolve the sample mixture in a minimum volume of solvent selected for solubility and stationary phase compatibility. Carefully load onto the column without disturbing the bed.
7. Begin isocratic elution with starting mobile phase. Collect fractions - initially smaller volumes, then switch to larger volumes once separation is achieved.
8. Monitor fraction composition using TLC. Adjust solvent polarity and/or pH to optimize separation.
9. Pool and concentrate the purified fractions using rotary evaporation. Analyze for purity and identity.
10. For subsequent runs, regenerate the column by flushing with multiple volumes of new solvent to remove residual sample.

With careful selection of stationary phase, mobile phase, and fractionation parameters, excellent separations are possible. Scale the procedure up or down by adjusting the column size and sample amounts.

Thin Layer Chromatography

Thin layer chromatography (TLC) provides rapid analysis of reaction mixtures and evaluates the purity of compounds.

Steps:

1. Prepare a TLC developing chamber by lining with filter paper wetted with mobile phase solvent. Allow to equilibrate.
2. Using a capillary tube, spot the sample mixture onto a TLC plate about 1.5 cm from the bottom. Let dry completely.
3. Place the spotted TLC plate into the chamber. Seal and allow to develop until the solvent front reaches near the top of the plate.
4. Remove the plate and mark the solvent front with a pencil. Allow the plate to dry.
5. Visualize the separated spots by illumination under UV light or treatment with a chromogenic spray reagent.
6. For each spot, measure the migration distance from the origin and calculate the Rf value:
   Rf = Distance traveled by analyte / Distance traveled by solvent
7. Compare Rf values between sample and standard spots to identify compounds.

With practice, TLC can separate complex multicomponent mixtures for characterization. Multiple development sequences and 2D-TLC procedures can further improve resolution.

Ion Exchange Chromatography

Ion exchange chromatography leverages electrostatic interactions between charged analytes and immobilized ionic functional groups on resin beads. It is widely used for separation of proteins, peptides, nucleotides, and other ionic biomolecules.

Steps:

1. Prepare a glass econo column fitted with porous frits on the inlet and outlet to retain resin.
2. Equilibrate the selected ion exchange resin with starting buffer. Load into the column, allowing buffer flow to eliminate air.
3. Wash extensively with starting buffer until the column effluent pH and conductivity match the buffer.
4. Prepare the sample, adjust to starting buffer conditions, filter if needed, and load onto the column.
5. Begin isocratic elution with starting buffer. Collect fractions while monitoring effluent with a UV detector.
6. When sample bands stop eluting, switch to a higher ionic strength buffer and/or more extreme pH to elute bound analytes.
7. Wash again with starting buffer to re-equilibrate column for subsequent purifications
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With careful selection of the ion exchange resin, sample loading and wash steps, and elution buffers, even highly complex biological samples can be separated into purified components by ion chromatography.

Conclusion

From these examples, we can see the wide applicability of chromatography techniques for analytical separations. With the proper selection of equipment, stationary phases, mobile phases, and visualization strategies, researchers can solve countless separation challenges. IT Tech offers a full range of high quality chromatography consumables, solutions, and instruments to meet your needs. Our technical experts are also available to collaborate on developing novel chromatography methods tailored for your specific applications. Contact our customer service team today to see how IT Tech products can advance your research.

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