Elution Chromatography

What is Elution Chromatography?

Elution chromatography is a method that involves a column where the solutes from the given solution migrate through the entire system followed by solute detection as it emerges from the column. In elution chromatography, elute is the analyte material that passes out from the column in chromatography that specifically includes the analytes and solutes passing through the column. 

In elution chromatography the indicator continuously measures the solutes which are also termed eluting, emerging from the mobile-phase stream and transduces the signal in form of voltage in a strip chart recorder in the form of peaks. The recorder traces where the solute is absent and treats it as its baseline. 

Elution Chromatography Graph

When a variation of solute concentration is plotted along the migration coordinate of the chromatogram’s development, a peak of the same solute is obtained. These plots of solutes collectively form concentration profiles and are in the form of a bell, normal or error curves (all of these curves collectively known as Gaussian). 

From the exact moment when the solution is injected into the mobile phase stream of the column to the time required for the solute to migrate or elute from the column is known as its retention time. This retention time is the measured point at which the solution is put into the mobile phase stream to the point where the maximum peak is obtained. The adjusted retention time is measured by the impression of unretained solute present at the outlet. In order to rule out the dependence of these times on the flow rate, the retention volume in the elution chromatography is taken into consideration. This retention volume is given by the retention times and the volumetric flow rate of the mobile phase stream. The basic formula for the calculation of retention volume in  adsorption elution chromatography is given as:

log R0 (ml/g) = log Va + α(S0 - e0 At)

Where Ve and α are the relative parameters of the adsorbent.

               α is the adsorbent activity

               Va is the adsorbent surface volume (ml/g)

               S0 is the sample adsorption energy

               At is the effective molecular area of the given sample

               e0 is the solvent strength and an important parameter of eluent

The relevance of elution chromatography is described with a graphical diagram given below.

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What is Elution?

In organic chemistry, elusion meaning is a process that is established and carried out to separate one material from the other present in the solution by washing it with a solvent. For, example in an ion exchange resin solution, washing of ion exchange resin is done in order to remove captured ions. Thus in simple words, elution meaning in chemistry is the removal of an adsorbed substance from a solid adsorbed medium with the help of a solvent by the chromatographic process. 

In biological terms, it means the removal of an antibody from its antigen to which it is attached like red blood cells. It is called antibody elution. For example, in liquid chromatography, an analyte is adsorbed or bonded to an adsorbent. This absorbent is generally in the solid phase and is in powder form that is coated on a solid surface for support. Depending on the composition of the adsorbent, its affinity to hold on to the analyte molecules varies. Now when the analyte is either absorbed or is bonded with the adsorbent at a molecular level, then the process of elution begins. 

Elution is done by passing a solvent (eluent in chromatography) past the analyte-adsorbent complex in order to separate the analyte from the adsorbent. Thus, the solvent molecule while passing through the chromatographic column can pass the adsorbent and the analyte that emerges in the chromatographic column which is termed as elute. Alternatively, the solvent can displace the elute by binding the adsorbent together in one place. Now the analyte that is separated out is taken as a sample for further analysis.

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Elution Time and Volume

Elution time is defined as the time of separation of the analyte, that is, the time it enters the chromatographic column to the time it starts to elute in the column. Also, an elution volume is the volume of the eluent required to start the elution process in a chromatographic column. 

For a known mixture of solutes under a definite condition, the elution volume becomes enough to identify the solutes in the mixture. For example, as amino acids are separated by the ion exchange method, at a particular condition, the amino acids will elute in the same volume and in the same order. Thus, a relationship between the elution time and elution volume is established by a new technology named as UTK-DTE generator (Ultra Technekow Dry Ship Top Elute 99mTc generator) and the mathematical expression is established as follows:-

Elute volume, V(ml) = 0.3594 + 0.1889t - 0.0009t2 where t is the elute time in seconds. This equation is suitable for 10ml of solute vial. 

Types of Elution Chromatography

Elution chromatography is carried out in three different conditions. In the first condition, kI is constant, in the second condition kI is changing continuously and in the third one, kI is changing discontinuously. So by keeping the composition of the elution buffer constant, the capacity factor also becomes constant. This is known as isocratic elution. 

Now, for the smaller molecules, the mobile-phase composition is not very large, thus the variation of the retention factor of the elute is very small for smaller molecules as compared to the variation of retention factor for the larger molecules. Thus the smaller molecules use isocratic elution. 

For gradient elution, as the mobile-phase composition changes continuously with the increase or the decrease of ionic strength, as a result, kI also changes continuously. So where kI differs too much because of the presence of bigger molecules, gradient elution is applicable as isocratic elution is not feasible for a huge change in kI value. As the mobile-phase composition switches discontinuously, the kI value also changes momentarily. Therefore in order to desorb the components of the extreme difference in kI value, step up elution is used. Now based on these factors elution chromatography has been broadly classified into two groups, namely,

  1. Column Elution Chromatography: Elution chromatography by column method is generally followed to isolate elements from the solvent before they are sent for TIMS or ICP-MS analysis. For example, for column elution chromatography, the wet slurry of ion exchange resins are initially placed on the column for its water to drain out completely. Then the sample in the mobile phase is passed through the stationary phase and is either driven by gravity or is pumped in at a certain flow rate. Now the components in the sample solution that do not interact with the functional groups present in the raisins flow out at the speed of the mobile phase. But the components that do interact with the functional group of the resin are delayed by a certain rate, depending on their nature of the interaction. Thus the speed of different components in the sample solution is determined by the amount of time they spend in the mobile and the stationary phase interacting with the resin. Thus the elution of different components will take place at different times and they will separate out. Therefore, the distribution coefficient of the components directly depends on the time they had spent in the mobile phase.

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The following diagram is the representation of ion exchange column elution chromatography in combination with the gravitational flow. The column is made up of quartz glass, PE, Teflon shrinking tubing etc. depending upon the chemical properties of the sample solution taken as mobile-phase. As the column method is carried out for the large batches in series it completes the ion exchange reaction. 

For example, the resin reservoir is divided into two sections with an arbitrary amount.  Now solute A is loaded onto the top section of resin and according to its Kd, it forms an equilibrium with the top section of resin as the Csolid / Cliquid remains constant. Thus the maximum concentration of solute A is adsorbed on the top section of resin. Now the depleted concentration of solute A will come down to the next section beneath the top layer of resin where another equilibrium of solute and the resin will form but the concentration of A will be very less as a maximum of it is adsorbed by the upper layer of resins.  Then the further depleted concentration of solute A will reach the third section to form equilibrium and the process continues until another solute is added to the resin bed on top in the form of the mobile phase where no concentration of solute A is present. 

Thus in order to maintain Kd, that is to keep  Csolid / Cliquid constant, desorption of solute A begins in order to maintain an equilibrium of the new solute with the resin. This process continues further down to the second and the third section till the sufficient solution is added in order for the sorption band to reach the end of the reservoir and then starts to elute. This is further explained by the graphical representation of the same process.

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Here VR denotes the retention volume, that is, the volume passed through the column until the elute reaches its maximum peak. 

  1. Gradient and Isocratic Elution Chromatography: Gradient elution in gradient elution chromatography is a technique by which the composition of the mobile phase is altered during the course of chromatographic progression. Thus in many HPCL separations, the isocratic elution condition is applied as in isocratic elution the mobile phase remains constant throughout the chromatographic progression. When a mixture of a large number of solutes with a wide range of capacity factors needs to be separated, gradient elution comes into play. Regardless of the mode of chromatography, the gradient elution is first initiated by a weak eluting solvent during a chromatographic run and later a high eluting solvent is added in the course of separation. This process allows a better and improved resolution of the weakly retained solutes as the strongly retained solutes elute much faster in a shorter period of time. The below graph shows the gradient and isocratic elution for the case of ionic strength gradient with conductivity detection.

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Efficiency of Column Elution Chromatography

The efficiency of the column in chromatography that is packed with permeable packings is expressed as the no. of theoretical plates denoted with N, with the formula:-

N = 16 (tr / w)2 

Where tr is the retention time when the complex solution is injected and w is the peak width obtained when tangents are drawn to the side of the Gaussian curve at the inflected point and then extrapolating the tangents for them to intercept the baseline. Thus, the distance between the intercepts is actually the value of peak width. Now if the peak is equal to the Gaussian distribution, then its width is measured by the standard deviation, σ and the formula is W = 4σ. Another method by which the column Chromatography can be characterised as HETP for packed permeable packing columns. HETP can be calculated by the experimental peak values using formula as follows:-

HETP = σ2 L / μ12

where σ2 is the peak variance, μ1 and  μ2 are the first and second moment of the peak and L is the length of the column. The following diagrammatic representation shows the experimental value of reduced HETP h = HEDP/dp as the function of the bed superficial velocity.

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FAQs (Frequently Asked Questions)

Q1. State the Difference Between Isocratic Elution and Gradient Elution?

Ans. The basic difference between isocratic and gradient elution is that isocratic elution uses single mobile phase composition that has single polarity whereas gradient elution has multiple single phase composition whose polarity varies gradually throughout the process of separation. Smaller molecules of the mobile phase composition use isocratic elution as their variation of retention factor is very small.  

Q2. What is Eluent in Chromatography?

Ans. In elution chromatography when a solute is injected into the chromatographic column it needs a career substance in order to move the solute also called analyte throughout the chromatography. Thus this career of the liquid phase  is known as eluent. For liquid chromatography, the eluent is a liquid for gas chromatography it is a gas carrier. 

Q3. Which Compound Elutes First in Column Chromatography?

Ans. the molecules of the analyte that have the minimum interaction with the compound in the mobile phase will elute first. For instance, a weak polar solvent will elute the less polar analyte first. So if hexane is present in the solute then it will elute first than any alkenes present as alkanes are less polar than the former. 

Q4. What  is the Use of Gradient Elution in Column Chromatography?

Ans. The main purpose of the gradient elution in chromatography is to elute the substances that are strongly retained by the mobile phase with a proper and clear resolution of the less retained organic components that are eluted first.