Source : Laboratoire du Dr B. Jill Venton – University of Virginia
Chromatographie d’échange ionique est un type de chromatographie qui sépare les analytes issu des frais. Une colonne est utilisée qui est remplie d’une phase stationnaire chargée sur un support solide, appelé une résine échangeuse d’ions. Chromatographie échangeuse de cations forts sépare préférentiellement les cations en utilisant une résine chargée négativement, tandis que la chromatographie échangeuse d’anions forts choisit préférentiellement des anions à l’aide d’une résine chargée positivement. Ce type de chromatographie est apprécié pour la préparation de l’échantillon, par exemple dans le nettoyage des protéines ou des acides nucléiques des échantillons.
Chromatographie d’échange ionique est un processus en deux étapes. Dans un premier temps, l’échantillon est chargé sur la colonne dans un tampon de charge. La liaison de l’échantillon chargée à la résine de colonne est basée sur des interactions ioniques de la résine pour attirer l’échantillon de charge opposée. Ainsi, les échantillons chargés de polarité opposée à la résine sont fortement liés. Autres molécules qui ne payent pas ou sont de charge opposée ne sont pas liés et sont lavés par le biais de la colonne. La deuxième étape consiste à éluer l’analyte qui est lié à la résine. Ceci est accompli avec un gradient de sel, où la quantité de sel dans la mémoire tampon est lentement augmentée. Fractions sont encaissées à la fin de la colonne que l’élution se produit et l’échantillon purifié d’intérêt peut être récupéré dans l’une de ces fractions. Une autre technique, telles que la spectroscopie, peut être nécessaire d’identifier quelle fraction contient l’échantillon. Chromatographie d’échange ionique est particulièrement utile dans les études de protéine, d’isoler les protéines d’intérêt qui ont une charge spécifique ou la taille, car la taille permet de déterminer le nombre d’interactions avec la résine.
Chromatographie d’échange ionique est une technique de séparation plus générale que la chromatographie d’affinité, qui est aussi souvent utilisée dans la préparation des échantillons de protéine, où un anticorps est liée à une colonne pour lier un analyte spécifique. Une nouvelle colonne d’affinité doit être achetée pour chaque analyte, tandis que le même type de colonne échangeuse d’ions, souvent atteints de différents troubles élution, peut servir à nettoyer les nombreuses protéines ayant la même charge. Chromatographie échangeuse d’ions peut également servir en conjonction avec d’autres types de chromatographie qui séparent basé sur d’autres propriétés. Par exemple, chromatographie d’exclusion stérique se sépare selon la taille et pourrait être utilisée avant la chromatographie échangeuse d’ions pour choisir composés de seulement une taille donnée.
Ion-exchange chromatography is widely used in biochemistry to isolate and purify protein samples. Proteins have many amino acids with functional groups that are charged. Proteins are separated based on net charge, which is dependent on pH. Some proteins are more positively charged while others are more negatively charged. In addition, peptide tags can be genetically added to a protein to give it an isoelectric point that is not in the range of normal proteins, making it possible to separate completely. Ion-exchange chromatography is useful for separating multimeric protein complexes, as different configurations would have different amounts of charge and different interactions.
Another major application of ion-exchange chromatography is water analysis. Anion-exchange chromatography can be used to measure the concentration of anions, including sulfates, nitrates, nitrites, fluoride, and chloride. Cation-exchange chromatography is used to measure the concentration of cations such as sodium, potassium, calcium, and magnesium. A type of ion-exchange chromatography is also used in water purification, as most water softeners filter out magnesium and calcium ions in hard water by binding them to a resin, which releases bound sodium. Heavy metals, such as copper or lead, can also be removed from water using ion-exchange chromatography.
Ion-exchange chromatography is also useful in metal purification. It can be used to purify actanides, such as plutonium, and remove it from spent nuclear reactor fuel rods. It can also be used to scavenge uranium and remove it from water or other environmental samples.
Ion-exchange chromatography is widely used in the separation and isolation of charged compounds, particularly large biomolecules.
This type of liquid chromatography uses a column of packed stationary-phase beads, called resin. The technique separates analytes based on their affinity with the charged resin.
There are two main types of this technique. In cation-exchange chromatography, negatively-charged resin is used to bind positively-charged analytes. Similarly, in anion-exchange, negatively-charged analytes bind to positively-charged resin. The unbound compounds are washed through the column, and the analyte can then be collected in a separate container.
This video will introduce the basics of ion-exchange chromatography, and demonstrate the technique by separating a protein mixture in the laboratory.
The stationary phase is a key component to a successful separation. Strong cation-exchange resins typically feature strong acid functional groups, such as sulfonic acid. Weak cation-exchange resins feature weak groups, such as carboxylic acids.
Similarly, strong anion-exchange resins utilize strong bases, like quaternary amines, while weak anion-exchange resins use secondary or tertiary amines. The selection of resin will depend on the properties of the sample mixture, and the analyte of interest.
The buffers used, collectively called the mobile phase, are also important to separation, particularly in terms of pH. For proteins, pH is selected based on its isoelectric point, or pI. At a pH equal to the protein’s pI, the protein is neutral. Above the pI, it will have a net negative charge, while below the pI, it will have a net positive charge. The buffer pH must be selected so the protein is properly charged and able to bind to the stationary phase.
Ion-exchange chromatography is generally a four-step process. First, a packed column containing either anion- or cation-exchange resin is equilibrated using buffer. For anion-exchange columns, this involves protonating the resin, ensuring it is positively charged.
Next, the sample is loaded on the column. The buffer must have low conductivity, as charged species can compete with the sample for interactions with the resin. Compounds of opposite charge bind to the resin. Molecules that are not charged, or carry the same charge, remain unbound.
In the third step, the column is washed with additional buffer to remove the unbound components from the column, leaving the bound behind.
Finally, the fourth step is the elution of the bound analyte. This is accomplished either by using a salt gradient, where the salt concentration is gradually increased, or using a high salt elution buffer.
Molecules that are weakly bound will be eluted first, as the low salt will most easily disturb their ionic bonding to the resin. Compounds that are more strongly bound will elute with higher salt concentrations.
Now that the basics of ion exchange chromatography have been outlined, lets take a look at its use in the separation of two proteins.
First, to prepare the protein mixture for separation, add 0.2 mL of binding buffer, and vortex to mix thoroughly. Then, centrifuge the mixture to remove any froth. To prepare the cation-exchange column, clamp it vertically onto a ring stand, and allow the resin to settle.
Open the top cap of the column, and then the bottom. Allow the buffer to drip out under gravity into a tube below.
To prepare the column, equilibrate it by loading a column-volume of buffer, in this case 0.3 mL. Let the buffer drip out of the column into a waste vial. After a column-volume of buffer has exited, repeat the equilibration step.
To run the experiment, place a 2-mL centrifuge tube labeled “Unbound 1” below the column. Carefully load 0.1 mL of the protein sample onto the top of the column.
Once the sample has been loaded, wash with a column-volume of buffer and allow it to flow all the way through. Repeat for a total of 5 washes. Collect each wash in its own tube, labeled “Unbound 1” through “5”. For the last 2 washes, centrifuge the column for 10 s to make sure that all unbound species wash off the column. Put the column in a new 2-mL centrifuge collection tube, and label it “Bound 1”. Load 1 column-volume of elution buffer on top of the column. Centrifuge for 10 s at 1,000 x g.
Repeat the elution step 2 more times to ensure collection of the bound analyte. Label the tubes “Bound 2” and “3”. Record any color changes or observations about the fractions.
In this example, hemoglobin and cytochrome C were separated. Hemoglobin has a pI of 6.8, while cytochrome C has a pI of 10.5. In the pH 8.1 buffer, hemoglobin is negatively charged and does not bind to the column. Conversely, cytochrome C is positively charged at pH 8.1 and binds to the column.
Hemoglobin, a brownish colored protein, was found in the unbound fractions, while cytochrome C, a reddish colored protein, was observed in the bound fraction.
There are many forms of liquid chromatography, each with different abilities to separate components of a mixture.
In this example, column chromatography was used to separate a mixture of single and double stranded DNA. Hydroxyapatite, or HA, is a crystalline form of calcium phosphate commonly use as a stationary phase due to its positively-charged calcium ions. In this case, the HA column was ideal for the separation of DNA as it can bind to DNA’s negatively-charged backbone.
Another form of column chromatography frequently used to separate proteins is immobilized metal affinity chromatography, or IMAC. In IMAC, the stationary phase possesses a ligand with a metal ion, which binds to a histidine tag on the protein of interest.
All other components of the mixture exit the column. The protein is then eluted with a solution of imidazole, which has a similar structure to histidine, and binds more strongly with the metal ion.
A common application of column chromatography is high performance liquid chromatography, or HPLC. HPLC is widely used in analytical chemistry for both the identification and separation of biological and non-biological compounds in a mixture.
HPLC is similar to the column chromatography demonstrated in this video, except that it is automated, and operated at very high pressures. This enables the use of smaller stationary-phase beads, with a higher surface area to volume ratio. Thus, improved interactions between the stationary phase and components in the mobile phase are possible.
You’ve just watched JoVE’s introduction to ion-exchange chromatography. You should now understand the concepts behind it, the 4 steps involved, and some related techniques.
Thanks for watching!
