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Does rotovapping ensure the highest purity you can actually get?
Rotovapping removes solvents but doesn’t separate compounds with similar boiling points, eliminate impurities, or ensure full dryness. Further purification is often needed for high purity.
What do we do in order to purify solids even further past rotovapping? Explain the process of what we have to do and how it achieves higher purity.
Recrystallization is the process by which a solid is dissolved in a hot solvent, and as the mixture cools, the molecule of interest begins to form crystals. As the crystals form, they exclude any impurities such as other compounds or solvent molecules.
What are the five steps of recrystallization?
Solvent selection.
Dissolving a solid.
Gravity filtration.
Crystallization.
Isolation of crystals.
What are the three criteria for a choice of solvent in recrystallization?
The choice for solvent depends on:
Cost.
Disposal.
Most importantly - solubility of solid in the solvent.
In regards to hazards, what should you note about handling organic solvents and recrystallization?
Most organic solvents are flammable and/or toxic. Take caution when heating solvents near boiling point or else you may inhale it. Use in fume hood.
Sometimes there may be no single suitable solvent to use. In these circumstances, what must be done, and why?
It may be necessary to use a mixture of two miscible solvents.
Good solvent – Dissolves the substance well, but won’t let it fully crystallize when cooled.
Poor solvent – Doesn’t dissolve much, but helps force the substance to crystallize.
By mixing them, you balance solubility—dissolving when hot and crystallizing when cooled.
How do you determine the right solvent to use to dissolve the solid of interest?
To do this, a small amount of the impure sample is added to a small volume of solvent. If the solid does not dissolve at RT, the mixture is then heated to see if it will dissolve when heated. The appropriate solvent to use for a recrystallization is one that will not dissolve the impure solid at room temperature but will dissolve the solid when heated.
The impurities can be dissolved or not.
Explain the procedure for solvent selection.
Prepare a hot water bath, setting the hot plate stirrer to 150ºC. It will take a lot of water.
Deposit a very small amount of sample to the unknown into four large dry test tubes.
Place approximately 1mL of solvent into each of the four tubes.
Record the solubility observations at room temperature. For low solubility, heat the test tube gently to see if it will be dissolved. Choose the solvent with complete solubility only once heated.
Explain the procedure for dissolving the solid in the chosen solvent.
Place your solid and a stir bead into a size-appropriate Erlenmeyer flask.
Add enough of the proposed crystallizing solvent to barely cover the mixture. Some solid may float, so approximate the solvent necessary to cover the solid.
Heat the solvent and unknown mixture using a hot plate to dissolve the entire compound. If the unknown compound does not fully dissolve, carefully add some more solvent and heat again.
Explain the procedure for gravity filtration.
Once the solid has dissolved, there may be insoluble impurities remaining in solution. To remove insoluble impurities, hot gravity filtration is the answer. When filtering a hot saturated solution, everything must be done quickly to prevent premature crystallization.
To ensure the whole setup is kept hot, a (1) stemless funnel and (2) pure solvent are also heated simultaneously, along with the flask with the (3) recrystallization mixture.
When the gravity filtration is to be performed, a hot funnel is placed over the ring clamp and aligned with the mouth of a clean flask.
Fluted filter paper is placed into the stemless funnel which is wet with pure, hot solvent.
Quickly pour the recrystallization solution onto the fluted filter paper, all into a clean flask.
Put a watch glass over the funnel to prevent heat and solvent loss.
Explain the procedure for crystallization. Ensure to discuss what slow cooling does, what small crystals mean, what very large crystals mean, and what it means if no crystals are formed.
Once the solution has been filtered into the receiving flask, the solution cools to RT and flask is further cooled in an ice-water bath.
Slow cooling with no agitation of the solution will often lead to large crystals.
Small crystals are formed if the solution is rapidly immersed in cold water. This is highly undesired because of the increase in solid surface area. Tiny crystals collectively have more surface exposed than a single big crystal. This increased surface area can retain some of the impurities.
Vary large crystals are undesired as well because they may occlude solvent and impurities within the crystals. If the solution is cool but no crystals have formed, this could be a supersaturated solution.
Crystallization can occur by adding a small crystal of the original solid (seeding).
Alternatively, the inside walls of the flask can be scratched and little slivers of glass act as seeds for the crystals to grow upon.
If neither works, there was too much solvent. Try rotovapping.
Explain the process of crystal isolation.
Crystals should have formed after cooling the solution ni an ice-water bath. The desired crystals can be isolated using a vacuum or suction filtration technique.
By this point, the cooled mixture is comprise of the desired solid product and unwanted liquid. Collect the solid using an appropriate method. The Hirsch funnel can be used to remove the solid from a small volume of liquid, while the Buchner funnel is used for larger amounts of liquid.
What is a melting point? What happens to a solid when being heated? What happens to a solid when heat is removed?
The melting point is defined as the temp at which the solid and liquid phases are at eq with each other. When a solid is heated, the temperature of the solid rises until the melting temperature has been reached. Upon reaching the melting point, the temperature of the compound will remain constant until the entire solid has melted. If the heat was removed at the melting point, the temperature of the compound will remain constant until complete solidification and thereafter the compound temperature drops.
From melting, what determines if a pure solid crystalline sample has been obtained?
A pure solid crystalline sample has been obtained if there is a sharp melting point (1–2º range).
What do you compare the experimental melting point to?
What does this comparison mean if the mp was broad and lower than its comparative value?
What does it mean if the mp is higher than its comparison?
We compare the experimental melting point to the literature melting point.
If the exp. mp is broad and lower than the lit. value, then either the sample is contaminated or the heating was too rapid.
If the exp. mp is higher than the lit. value, then this is a sign of poor heat conduction which is the result of poor packing in the capillary tubes.
What do you do if you have two different compounds A and B with identical melting points?
If A and B were the same, then the mixture (of A and B) melting point would be the same as A and B. However, if the mixture melting point be depressed below the melting point of A or B and the range broadened, then A and B are not identical.
Explain how you would perform a melting point determination.
Using a spatula, powder some sample on a watch glass. A wet sample is not a pure sample.
Partially fill a melting point capillary tube (glass tube that is only open with one end) with sample by pushing the open end of the tube into the powdered sample.
Pack the material to the bottom of the tube by tapping the closed end on the bench. Aim for the layer of product at the bottom of the tube to be about 1–2mm deep.
Follow the instructions of the melting point apparatus on the side.
What are the onset point, meniscus point, and clear point? On that note, what is the melting point range representative of?
Onset point: Sample appearance changes. Start of melting.
Meniscus point: Equal volumes of solid and liquid. Liquid meniscus becomes visible.
Clear point: All solid becomes totally liquid.
Melting point range is represented by the onset and clear point values.
What concept do all chromatographic techniques have in common? How does it work?
Concept: All chromatographic techniques have one principle in common: a liquid or gaseous solution of the sample, called the mobile phase, moves through an adsorbent called the stationary phase.
How It Works: The different compounds in the sample move through the adsorbent at different rates because of physical differences and interactions with the stationary phase. Thus, the individual compounds in a sample become separated from one another as they pass through the adsorbent and can be either collected or detected, depending on the chromatographic technique and the quantity of sample used.
How does thin layer chromatography (TLC) generally work?
The stationary phase (silica gel) is thinly spread over a plastic or aluminum backing. A small spot of a solution of the substance to be tested is placed (known as "spotting") near one end of the plate using a capillary pipet. The plate is "developed" by placing ti ni a jar with a small amount of solvent. The mobile phase (eluent) wil slowly creep up the TLC plate and carry the sample spot up the plate.
What two types of information can you get from TLC analysis?
Purity: If a compound is impure, then the TLC plate will show multiple spots due to the contaminants having different Rf values from the desired product.
Identity: If compounds on plate are the same, they will have the same Rf value.
How do different adsorbents affect molecule separation in TLC, e.g. silica gel?
Different adsorbents attract different types of molecules. A highly polar adsorbent like silica gel adsorbs polar molecules strongly but does not have much attraction for nonpolar molecules.
What issue can arise when using organic solvents in TLC, and how does it affect the separation?
TLC solvents are organic chemicals and thus can be adsorbed by the adsorbent on the plate and compete with sample components for positions on the adsorbent.
What does an Rf value greater than 0.9 and less than 0.1 indicate about the eluent in chromatography?
What does it mean if smudges appear on a TLC plate, and how can it be fixed.
What does it suggest if the mobile phase takes a long time to reach the top of a TLC plate?
If the Rf > 0.9, then the eluent is too polar. If it is < 0.1, then the eluent is not polar enough.
If smudges are seen on the plate, too much material was placed on the plate. Remedy by diluting the sample solution and use a smaller deposition onto the plate.
If the mobile phase takes a long time to reach the top, evaporation can occur if the jar wasn’t sealed
What are the three steps of TLC?
Spotting.
Developing.
Visualizing.
Explain the procedure of spotting in TLC.
Prepping the plate
In a sample vial, dissolve about 1 mg of the sample (about a quarter of a match head size) in a few drops of acetone.
Use a non-mechanical pencil (not pen) to gently mark a start position (origin) on the TLC plate about 1.5 cm from the short end of the plate on the silica side.
Make sure there is about 5 mm between the spots to prevent the traveling spots from running into each other. Pens and markers have organic dyes and therefore wil also develop on the TLC plate, so avoid.
Be sure to spot the compounds high enough on the plate so that they will be above the solvent level in the developing jar. If the spots are below the solvent level, they will be dissolved away from the plate by the solvent.
Use capillary micropipettes to spot the sample on the TLC plate. The micropipettes are infinitely reusable by cleaning then with some acetone after use. To clean the micropipette, place acetone in a small sample vial and draw it up into the pipette and spot it onto a small piece of paper towel. Repeat this at least three times and the micropipette will be clean for use with the next sample.
When handling the TLC plate, be sure to not touch the silica gel surface with your fingers because your fingerprints on the TLC plate wil appear under UV light.
Spotting the plate
Practice dabbing the pipet on paper towel first to ensure that you can control the spot size (1-2 mm diameter).
Dip the end of a fresh micropipet into the sample solution, which rises into the pipet by capillary action. Touch the end of the pipet gently where the pencil mark was placed on the TLC plate, so that the solution runs out of the pipet onto the adsorbent.
To ascertain the adequacy of spot material, examine the plate under a UV lamp (wavelength 254 nm), the spots should appear dark. If the first spotting is not sufficient, a better spot is usually obtained by several applications of small amounts onto the same spot (once the previous spotting has evaporated) than by one application of a larger quantity.
What is the purpose of the "co-spot" in chromatography, and what does it indicate if two spots appear?
The co-spot helps identify differences in Rf values by showing two spots if the compounds have slightly different Rf values, even if they appear similar due to an uneven solvent front.
Explain the procedure of developing in TLC.
Retrieve a TLC chamber or make your own with a beaker, watch glass and filter paper. The filter paper in the jar increases the solvent vapour within the chamber.
Pour the appropriate solvent in (usually to a depth of less than 1 cm - the important thing is to keep the solvent height BELOW your TLC's origin). You must not pour a different solvent into the jar than what it's labeled with since you'll end up with a mixed eluent system.
The TLC plate will be placed into a "TLC chamber" which is a jar with an eluent and a sheet of filter paper. Place the TLC plate into the solvent chamber as carefully as possible to prevent the solvent front from running uneven. Do not move the TLC chamber when the plate is developing to minimize solvent loss, especially in the mixed solvent systems.
The solvent will rise rapidly up the adsorbent on the plate by capillary action. When the solvent front has risen almost to the top of the plate (about 1 cm from the end), open the jar, remove the plate, and quickly mark a line across the plate at the solvent front with a pencil.
How does the filter paper in the chromatography chamber affect the solvent?
The filter paper in the jar absorbs the solvent and, as it evaporates, releases solvent vapour into the air inside the jar. This keeps the chamber saturated with solvent vapour, which helps the solvent move up the paper or plate more evenly during chromatography, leading to better separation of compounds.
Briefly explain the procedure of visualizing in TLC.
Place the developed plate under a UV lamp and circle all the spots you see. Measure the distance the solvent front travelled as well as the spots.
What does the Rf value in chromatography represent, and what does it tell us about purity?
The Rf value is the distance that the spot of a particular compound moves up the plate relative to the distance moved by the solvent front. The Rf tell us nothing about purity. When all variables are held constant (temperature, solvent, adsorbent, thickness of adsorbent, and amount of compound on the plate), the Rf is constant.
How does the co-spot technique in TLC help determine if an unknown molecule is identical to a known molecule?
In the co-spot technique, if the unknown molecule (A) is identical to the known molecule (B), both will have the same Rf value, and only one spot will appear in the co-spot lane. If A is different from B, two spots with different Rf values will appear, indicating two different molecules.
How can a second TLC comparison with a different solvent help confirm the identity of two substances?
If two substances have the same Rf value, they are likely (but not necessarily) the same compound. A second TLC with a different solvent may show different Rf values. If the Rf values remain the same, the likelihood that the substances are identical increases. However, additional evidence, such as NMR, MS, or IR spectra, should be used for confirmation.
How does TLC analyze results from an organic reaction?
From the analysis of a reaction that is meant to convert a starting material to product, a co- spot of S and P is included (Lane C) as well as a lane to analyze the reaction mixture (Lane R). What is observed in Lane R is that there is the production of spots with an identical Rf for the individual starting material and product and two additional "side products.”
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