Performing Thin-Layer Chromatography to analyze organic samples

Summary

In this instructional video, Dr. Frank demonstrates how to perform thin-layer chromatography (TLC) in a lab setting at the University of Ottawa. The video covers the necessary materials, including a developing jar, eluent, TLC plate, and samples. It outlines the steps for preparing the TLC plate, making a baseline, and applying sample spots. Dr. Frank also explains the importance of using a proper capillary for sample application and adjusting spot concentration. The video further details the development process, emphasizing the need to keep the baseline above the eluent level and concludes with analyzing the plate under UV light to determine compound purity and reaction progress.

Takeaways

• 🧪 **TLC Overview**: The video is a tutorial on thin-layer chromatography (TLC), a technique used in laboratories, particularly for undergraduates at the University of Ottawa.
• 📝 **Preparation**: Before starting, ensure familiarity with TLC concepts by watching part one of the series and gather necessary materials like a developing jar, eluent, TLC plate, capillaries, and samples.
• 💧 **Eluent Setup**: Pour a small amount of eluent into the developing jar, seal it, and swirl to allow solvent vapors to fill the jar before proceeding.
• 🔍 **Plate Inspection**: Check the TLC plate for smoothness and minimal chipping, as imperfections can affect solvent migration and spot analysis.
• 📏 **Baseline Marking**: Use a sharp pencil to lightly draw a baseline on the plate, which is where samples will be spotted and start their migration.
• 🧬 **Sample Preparation**: Dissolve the sample or reference compound in a volatile solvent to create a homogeneous solution for spotting.
• 🌡️ **Capillary Selection**: Choose a clean capillary that is neither too thin nor too wide, ensuring a smooth end for precise spotting.
• 💧 **Spotting Technique**: Dip the capillary in the sample solution, wick off excess, and deposit a small spot on the baseline with minimal contact time.
• 🔬 **UV Visualization**: Examine the spotted plate under UV light before development to assess the quality of the spots and avoid wasting time on poor samples.
• 📈 **Spot Adjustment**: Adjust the spotting based on the initial UV visualization, either respotting or diluting/concentrating the solution as needed.
• 🧑‍🔬 **Development Process**: Insert the plate into the developing jar with the eluent level below the baseline, and allow the solvent to migrate the compounds upwards.
• 🌟 **Final Analysis**: After development, mark the solvent front, use UV light to reveal compounds, and calculate Rf values to analyze the plate's results.

Q & A

What is the purpose of the video by Dr. Frank?

The purpose of the video is to demonstrate how to perform thin-layer chromatography (TLC) in an undergraduate lab setting at the University of Ottawa.

What is assumed about the viewer before watching this video?

It is assumed that the viewer is familiar with the technique of TLC and its terminology, and has watched part one of the series for foundational concepts.

What are the essential items needed to perform TLC as described in the video?

The essential items needed include a developing jar, eluent mixture, TLC plate, clean capillaries, a pencil, tweezers, a ruler, sample and reference compounds, and a solvent to dissolve them.

How much eluent should be poured into the developing jar and why?

Approximately 10 milliliters of eluent should be poured into the developing jar to allow the solvent vapors to fill the jar, which is important for the TLC process.

Why is it important for the TLC plate to be smooth and homogeneous?

A smooth and homogeneous TLC plate is important because imperfections like creases or chipping can affect the migration of the solvent and thus the spots' resolution.

How should the baseline be marked on the TLC plate?

The baseline should be marked lightly with a sharp pencil across the short axis of the plate, approximately one centimeter from the bottom, without chipping the silica.

What is the typical amount of compound used for preparing the solution in TLC?

About five milligrams of the compound is typically used, which corresponds to the tip of a small spatula for solids or one or two drops for liquids.

How should the capillary be prepared for spotting the sample on the TLC plate?

The capillary should be clean, of good quality, not too thin or wide, and the extremity used for dipping should be smooth, which can be achieved by rubbing it with brown paper.

What is the correct method for depositing a spot on the TLC plate?

The spot should be deposited by quickly dipping the capillary into the solution, wicking off excess liquid, and touching the plate very briefly to leave a small spot, roughly a millimeter or two in diameter.

Why is it recommended to visualize the TLC plate under UV light before developing it with the eluent?

Visualizing the plate under UV light before development helps to identify if the plate is already showing spots, which can save time and resources by not developing a useless plate.

How should the TLC plate be inserted into the developing jar during the elution process?

The plate should be inserted into the jar vertically as possible with the baseline below the level of the liquid eluent to ensure the solvent drags the compound upwards.

What is the significance of marking the solvent front on the TLC plate after elution?

Marking the solvent front is significant as it provides a reference point for calculating the Rf values of the spots, which is crucial for the analysis of the plate.

What is the Rf value and how is it calculated?

The Rf value is a measure of the distance a compound travels relative to the solvent front. It is calculated using the formula: Rf = (distance of the compound from the baseline) / (distance of the solvent front from the baseline).

Outlines

00:00

🔬 Introduction to Thin-Layer Chromatography (TLC) Setup

Dr. Frank introduces the process of thin-layer chromatography (TLC) in an undergraduate lab setting at the University of Ottawa. He assumes viewers are familiar with the technique and its terminology. The necessary materials for TLC are listed, including a developing jar, eluent mixture, TLC plate, capillaries, a pencil, tweezers, a ruler, and samples. The eluent is prepared by pouring a small amount into the developing jar, which is then sealed and swirled to allow solvent vapors to fill the jar. The TLC plate should be smooth and free of creases or chipping. A baseline is drawn on the plate using a pencil, approximately one centimeter from the bottom, which will be the starting point for the samples' migration. The preparation of the sample solution is discussed, including dissolving the compound in a volatile solvent and using a capillary to transfer a small amount of the solution onto the plate. The importance of using a good quality capillary and ensuring a smooth end for precise spotting is emphasized. The video also advises checking the TLC plate under UV light before development to assess the quality of the spots.

05:01

🧪 Executing the TLC Elution Process and Analysis

The video script details the steps for eluting a TLC plate. The process begins with inserting the spotted plate into the developing jar, ensuring the eluent level is below the baseline to prevent the compound from dissolving in the solvent. The elution process typically takes about five minutes, during which time the lab technician can perform other tasks. After elution, the plate is removed and the solvent front is marked on the plate to indicate the height reached by the solvent. The plate is then analyzed under UV light, which reveals the compounds as dark purple spots. Each spot is circled, and the distances from the baseline to the solvent front and each spot are measured. The Rf value for each spot is calculated using a provided formula. The video concludes by highlighting the efficiency and utility of TLC as a lab technique for quickly assessing compound purity and reaction progress with minimal equipment and time investment.

Keywords

💡Thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) is a widely used laboratory technique in analytical chemistry for the separation and identification of compounds. It involves the use of a stationary phase (the TLC plate) and a mobile phase (the eluent) to separate components of a mixture. In the video, TLC is the main focus, and the script provides a step-by-step guide on how to perform this technique, highlighting its utility in assessing compound purity and monitoring chemical reactions.

💡Eluent

An eluent is the solvent or mixture of solvents used in chromatography to carry the sample through the stationary phase. In the context of the video, the eluent is poured into the developing jar and is crucial for the TLC process as it facilitates the migration of the sample compounds across the TLC plate. The script mentions pouring a small amount of eluent into the jar and ensuring it covers the bottom to allow for proper vapor saturation.

💡TLC plate

A TLC plate is a glass, plastic, or aluminum sheet coated with a thin layer of adsorbent material, typically silica gel, used as the stationary phase in thin-layer chromatography. The script emphasizes the importance of a smooth and homogeneous TLC plate, free from creases and chipping, as these can affect the migration of the solvent and the quality of the chromatographic separation.

💡Capillary

A capillary is a thin, narrow tube used in laboratories for various purposes, including the transfer of small volumes of liquid. In the video script, capillaries are used to deposit precise spots of the sample solution onto the TLC plate. The script advises on selecting a capillary of good quality and ensuring its tip is smooth for accurate spotting.

💡Baseline

The baseline in TLC refers to the line drawn across the bottom of the plate where the samples are spotted. It is the starting point for the chromatographic run. The script describes drawing a baseline with a pencil and cautions against chipping the silica when doing so, as this could impact the migration of the solvent and the separation of compounds.

💡Spotting

Spotting in TLC is the process of applying the sample solution onto the baseline of the TLC plate. The script provides detailed instructions on how to spot the sample using a capillary, emphasizing the need for a small, precise spot to ensure optimal separation and analysis of the compounds.

💡Solvent front

The solvent front is the boundary between the mobile phase (eluent) and the stationary phase on the TLC plate. It represents the furthest point reached by the eluent during the chromatographic process. The script instructs viewers to mark the solvent front with a pencil once the elution process is complete, as this is necessary for calculating the Rf values of the compounds.

💡Rf value

The Rf value, or retention factor, is a measure of how far a compound travels up the TLC plate relative to the solvent front. It is calculated using the distance from the baseline to the compound spot divided by the distance from the baseline to the solvent front. The script mentions calculating Rf values as part of the analysis process after the TLC run is complete.

💡UV light

UV light, or ultraviolet light, is used in the video to visualize compounds on the TLC plate after the chromatographic run. Many compounds fluoresce under UV light, which makes them visible as dark spots against a lighter background. The script advises checking the TLC plate under UV light before and after elution to assess the quality of the separation and to reveal the compounds.

💡Volatile solvent

A volatile solvent is a liquid that evaporates readily at room temperature. In the context of the video, a volatile solvent is used to dissolve the sample before spotting it onto the TLC plate. The script mentions dissolving the sample in one or two milliliters of a volatile solvent to ensure a homogeneous solution that can be easily applied to the plate.

Highlights

Introduction to thin-layer chromatography (TLC) technique and its terminology.

Overview of materials needed for TLC, including developing jar, eluent, TLC plate, and more.

Instructions on preparing the developing jar with eluent and sealing it for solvent vapor saturation.

Importance of a smooth and homogeneous TLC plate for accurate solvent migration.

Technique for drawing a baseline on the TLC plate without damaging the silica layer.

Preparation of sample solution for analysis, including dissolving the compound in a volatile solvent.

Selection and preparation of a clean capillary for precise spotting of the sample.

Method for sampling the solution and depositing a small spot on the TLC plate's baseline.

Advantage of visualizing the TLC plate under UV light before development to assess spot quality.

Guidelines for adjusting the sample spot based on its initial appearance under UV light.

Procedure for inserting the TLC plate into the developing jar and ensuring the eluent level is below the baseline.

Time required for the elution process and the importance of not disturbing the plate during this time.

Cleaning and preparing TLC capillaries for reuse with acetone and brown paper.

Marking the solvent front on the TLC plate after elution to assist in subsequent analysis.

Use of UV light to reveal compounds on the TLC plate, which appear as dark purple spots.

Measurement of the distance from the solvent front and each spot to the baseline for Rf calculation.

Conclusion on the utility of TLC as a quick and inexpensive method for assessing compound purity and reaction status.

Transcripts

Hi everyone, Dr. Frank here to show you  how to run thin-layer chromatography,  

or TLC, when in the undergraduates lab here at  the University of Ottawa. This video assumes  

you are familiar with the technique and  its terminology; if you haven't already,  

please make sure to watch part one of this series  which explains many of the concepts behind the  

technique. First, an overview of what you will  need: a developing jar, your eluent mixture,  

a TLC plate, clean capillaries, a  pencil, tweezers, a ruler and, of course,  

your sample and reference compounds, along  with some appropriate solvent to dissolve them.  

First, start by pouring a small amount of  your eluent in the clean developing jar;  

approximately half a centimeter of liquid  at the bottom, or roughly 10 milliliters,  

then seal the jar and give it a quick swirl.  Let it rest while you proceed to the next step;  

this allows the solvent vapors to fill the  jar. For best results, the TLC plate should  

be as smooth and homogeneous as possible: this  means no creases in the plate and little to no  

chipping in the silica powder, which would  reveal the aluminum plate underneath. These  

imperfections affect the migration of the solvent  and thus your spots. Minimal chipping is common  

and acceptable; if excessive chipping is found  on a single side, the plate can still be used,  

assuming the baseline is drawn near the extremity  in good condition. Using a sharp pencil, lightly  

draw a line across the short axis of the plate  approximately one centimeter from the bottom.  

Be careful not to chip the silica when doing  so: this will be your baseline, where you will  

spot your samples and where they will begin  their journey migrating across the plate.  

You then need to prepare a solution  of whatever you will be analyzing.  

If you are monitoring a reaction's progress, you  can typically use the reaction solution as-is.  

If you are analyzing either a crude or a pure  product that you've isolated, or a reference  

sample, you first need to dissolve it. To do so,  transfer about five milligrams of your compound  

into a small clean container: this corresponds  to either the tip of a small spatula for solids  

or one or two drops for liquids. In either cases,  precision is not too important, as you can always  

adjust later down the line. Then dissolve in  one or two milliliters of a volatile solvent:  

manually stir for a few seconds to ensure a  homogeneous solution. Then pick a clean capillary:  

as they are made in-house, the quality of  capillaries is batch dependent, so ensure  

that your capillary is of good quality first.  Too thin and they won't pull any liquids at all.  

Too wide and they won't allow for precise  spotting. Furthermore, ensure that the extremity  

you will use to dip in the solution is relatively  smooth: this can be easily achieved by delicately  

rubbing the jagged extremity with a piece of  brown paper until a smooth end is obtained.  

Proceed to sample some of your solution to  analyze by quickly dipping in the smooth  

end of your capillary; wick off any excess of  liquid very rapidly on a piece of brown paper  

then deposit a spot on the baseline of your plate  by touching the capillary very rapidly. Contact  

time between the capillary and the plate should  be very brief; a fraction of a second at most.  

The spot you deposit should be very small,  roughly a millimeter or two in diameter.  

A good idea is to visualize your TLC plate with  UV light BEFORE developing it with the eluent:  

you want to make sure that you are not wasting  precious time eluting what is ultimately a useless  

plate. The picture on the left and right show  the spotted plate before and after elution. The  

lane on the left is useless, as we can barely see  anything in the first place. On the other hand,  

the lane on the right is likely to be useless  as the spot is too big and too concentrated.  

The lane in the middle is just perfect, and  should provide optimal resolution. Now is  

a good time to adjust your plate: if your spot  is too weak, you may respot with your solution.  

You may or may not have to concentrate  your solution by adding compound to it.  

If your spot is too strong, either your  solution is too concentrated and you need  

to dilute it a bit, or your spot is too big and  you should re-spot while aiming for the smallest  

visible spot you can achieve. Once satisfied  with the spots, quickly open your developing jar,  

grab the top of the TLC plate with tweezers and  insert into the jar as vertically as possible,  

then seal the jar again. It is extremely important  here that the level of the liquid eluent be below  

that of your baseline, as you want the migrating  solvent to drag the compound upwards on the plate.  

Having the baseline touching the liquid  means the compound will simply dissolve  

in the solvent. Depending on the size of your  plate, the elution process should require roughly  

five minutes; you do not need to closely monitor  the process, so now would be a good time to assist  

your partner with a step or to clean some  glassware, such as your TLC capillaries.  

To do so, gently touch a piece of brown paper  to drain any leftover liquid onto the tissue,  

then gently dip the capillary into clean acetone  and drain it again on the paper. Repeat these  

steps two or three more times, finishing with a  drain capillary that is ready to be reused. Allow  

the elution of the solvent until it reaches half  a centimeter from the top of the plate, then open  

the jar and pull out the plate with your tweezers  and lay it down somewhere safe, white side  

pointing up. While the plate is still wet, gently  draw a line with a pencil to indicate the height  

reached by your solvent: this is the solvent  front and will be necessary to the analysis of  

the plate. Make sure to seal your TLC developing  jar, to avoid any evaporation of your eluent.  

Shine UV light on the plate to reveal  your compounds, which will appear as dark  

purple spots. Using your pencil, gently  circle the various individual spots.  

Using a ruler, measure the distance  separating the solvent front  

from the baseline and, for each spot, the  distance separating them from the baseline.  

For each spot, you can calculate an individual  Rf using the formula shown on the screen.  

Congratulations, you've successfully ran a TLC  plate. As you can now tell, TLC is a very useful  

technique in the lab: in less than 10 minutes,  you can obtain critical information regarding  

the purity of a compound and the status of an  ongoing reaction with simple, inexpensive tools.