SYNTHESIS OF 1,2-O-CYCLOHEXYLIDENE-𝛼-D-GLUCOFURANOSE

• Heat a solution of 20 g (0.06 mol) of l,2:di-0-cyclohexylidene-𝛼-D-glucofuranose in 100ml of aqueous acetic acid (75% v/v) in a round-bottomed flask immersed in a hot-water bath held at 70-80 °C for 90 minutes with intermittent shaking; then remove the solvent under reduced pressure on a rotary evaporator. 

• To the residual syrup add 20 ml of hot water, then sufficient solid sodium hydrogen carbonate to neutralise the remaining acetic acid, and finally 90 ml of heptane. 

• Heat the heterogeneous liquid mixture until two clear layers are obtained and then remove the upper heptane layer by careful decantation (1). 

• Cool the aqueous layer to 0°C, filter off the crystals of 1,2-O-cyclohexylidene-𝛼-D-glucofuranose which separate and recrystallise from water to give the pure product, m.p. 149-150 °C, [𝛼]ᴅ²⁰ + 5.9° (c1 in Me₂CO). The yield is 11.5 g (75%).

Notes to keep in mind: 

1.  The solid which separates from the cooled heptane layer may be shown to be unchanged starting material by t.l.c. analysis on silica gel plates using methanol-benzene (4:96) as the developing solvent.

Cognate preparation: 3-O-Benzyl-l,2-O-cyclohexylidene-𝛼-D-glucofuranose

• Dissolve 100g (0.23 mol) of 3-O-benzyl-l,2:5,6-di-O-cyclohexylidene-𝛼-D- glucofuranose in 400 ml of aqueous acetic acid (75% v/v) maintained at 70-80 °C for 3 hours, remove the solvent under reduced pressure and dissolve the residual oil in 500 ml of dichloromethane. 

• Wash this solution with aqueous sodium hydrogen carbonate and with water, dry over calcium sulphate and remove the dichloromethane by evaporation under reduced pressure with a rotary evaporator. 

• Remove the last traces of solvent using an oil rotary immersion pump, transfer the warm fluid yellow syrup to the retort of a molecular still and distil using a vapour diffusion pump to give a pale yellow glass, b.p. 195-200 °C/2 x 10⁻³ mmHg, [𝛼]ᴅ²⁰ -36.4° (c4 in CHC1₃). 

• The yield is 76 g (94%), and the product is pure enough for most purposes. 

• However t.l.c. analysis on silica gel plates (solvent system; benzene-methanol 9:1) reveals one major and two minor components. 

• Purification may be effected by either of the two methods described below.

Method 1: Chromatographic purification of 3-O-benzyl-l ,2-O-cyclohexylidene-𝛼-D-glucofuranose. (The chromatographic column should be set up in a fume cupboard.)

• Prepare a silica gel column using benzene as a solvent; use 10 g of adsorbent for each 1 g of monosaccharide derivative to be chromatographed. 

• Dissolve the latter in the smallest volume of benzene and transfer the solution to the chromatographic column with a pipette. 

• Elute the column with benzene and collect suitable-sized fractions; evaporate the solvent from each fraction and weigh the residues which consist of 3-O-benzyl-l,2:5,6-di-O-cyclohexylidene-𝛼-D-glucofuranose. 

• When all of this has been eluted continue the development with methanol which elutes the required product. 

• Evaporate the methanol and distil the residual syrup using a molecular still; about 50 per cent recovery of the purified product may be expected.

Method 2: Purification of 3-O-benzyl-l,2-O-cyclohexylidene-𝛼-D-glucofuranose by benzoylation

• Dissolve 5 g of the crude product in 10 ml of pure dry pyridine and add 5 g of benzoyl chloride. 

• Leave the reaction mixture overnight at room temperature, pour it on to ice and stir thoroughly. 

• Extract the oil which separates into 50 ml of dichloromethane and wash the dichloromethane solution successively with 30 ml of ice-cold dilute aqueous hydrochloric acid (2 m), 30 ml of saturated aqueous sodium hydrogen carbonate, and 2 x 30 ml of water. 

• Dry over sodium sulphate and evaporate the dichloromethane. 

• The viscous oil crystallises on trituration with methanol and is recrystallised from methanol to give the pure derivative 5,6-di-O-benzoyl-3-O-benzyl- 1,2-O-cyclohexylidene-𝛼-D-glucofuranose, m.p. 104-106 °C,  [𝛼]ᴅ²⁰ — 26.4° (c1 in CHC1₃), in a yield of 3.6 g (57%). 

• Remove the benzoyl groups by dissolving 3 g of the foregoing product in 20 ml of methanol and adding 20 ml of a solution of sodium methoxide in methanol (0.5%). 

• After 2 hours neutralise the solution by adding ion exchange resin Zeolite 225 (H⊗), filter and evaporate. 

• Distil the resulting colourless oil in a molecular still to obtain chromatographically pure 3-O-benzyl-l,2-O-cyclohexylidene-𝛼-D-glucofuranose; the yield is 1.6 g (70% from the dibenzoate).

THIN LAYER CHROMATOGRAPHY (TLC) OR ORGANIC SPOTTING

(Organic Spotting)

Exercise:
Using a greatly magnified diagram of a TLC plate, locate or define each of the following terms: 1. Origin 2. Stationary Phase 3. Mobile Phase 4. Solvent Front 5. Component “spot” 6. R f value 7. Development 8. Visualization of spots 9. What is meant by differential partitioning between stationary and mobile phase? 10. What is the recommended procedure for cleaning a TLC spotting capillary?

Introduction

Chromatography is the separation of two or more compounds or ions by the distribution between two phases, one which is moving and the other which is stationary. These two phases can be solid-liquid, liquid-liquid or gas-liquid. Although there are many different variations of chromatography, the principles are essentially the same. If you took Chem 15 at University Park, you may remember using paper chromatography to separate inks and food dyes. The cellulose paper was the stationary or solid phase and the 1-propanol/water mixture was the mobile or liquid phase. This chapter explores a very similar microscale technique for separating organic molecules, thin-layer chromatography.

Thin-layer chromatography or TLC, is a solid-liquid form of chromatography where the stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or combination of solvents. TLC is a quick, inexpensive microscale technique that can be used to:

•    determine the number of components in a mixture

•    verify a substance’s identity

•    monitor the progress of a reaction

•    determine appropriate conditions for column chromatography

•    analyze the fractions obtained from column chromatogrpahy

In paper chromatography, the stationary phase is a specially manufactured porous paper. The samples are added to one end of the sheet of paper and dipped into the liquid or mobile phase. The solvent is drawn through the paper by capillary action and the molecules are distributed by partition between the mobile and stationary phase. The partition coefficient, k, similar to the distribution coefficient for extraction, is the equilibrium constant for the distribution of molecules between the mobile phase and the stationary phase. It is this equilibrium that separates the components. Different inks and dyes, depending on their molecular structures and interactions with the paper and mobile phase, will adhere to the paper more or less than the other compounds allowing a quick and efficient separation.

TLC works on the same principles. In thin-layer chromatography, the stationary phase is a polar absorbent, usually finely ground alumina or silica particles. This absorbent is coated on a glass slide or plastic sheet creating a thin layer of the particular stationary phase. Almost all mixtures of solvents can be used as the mobile phase. By manipulating the mobile phase, organic compounds can be separated.

Theory

To thoroughly understand the process of TLC, as well as all types of chromatography, we must travel to the molecular level. All forms of chromatography involve a dynamic and rapid equilibrium of molecules between the two phases. As shown in Figure, there are:

1. free - completely dissolved in the liquid or gaseous mobile phase and
2. absorbed - stuck on the surface of the solid stationary phase.

 Mixture of A & B free in mobile phase and absorbed on the stationary phase.

Molecules are continuously moving back and forth between the free and absorbed states with millions of molecules absorbing and millions of other molecules desorbing each second. The equilibrium between the free and absorbed states depends on three factors:

• the polarity and size of the molecule
• the polarity of the stationary phase
• the polarity of the solvent

Thus, one has three different variables to change in chromatography. The polarity of the molecules is determined by their structures. By selecting different stationary and mobile phases, one can change the equilibrium between the free and absorbed states. It is important to understand chromatography at this molecular level because this allows one to choose mobile and stationary phases that will separate just about any mixture of molecules.

Different molecules partition differently between the free and absorbed state, that is the equilibria between these two states is not the same. In Fig. below, molecule A is weakly absorbed, its equilibrium lies in the direction of the free state and there is a higher concentration in the mobile phase. Molecule B, on the other hand, is strongly absorbed, its equilibrium lies in the direction of the absorbed state, and has a higher concentration on the stationary phase.

Dynamic equilibrium between A & B and the mobile and stationary phase.

Simply adding your mixture to a combination of a mobile phase and a stationary phase will not separate it into its pure molecular components. For this to happen, the mobile phase must flow past the stationary phase as depicted in Fig. Since the A molecules spend more time in the mobile phase, they will be carried through the stationary phase faster and move farther in a given amount of time. Since B is absorbed to the stationary phase more than A, B molecules spend less time in the mobile phase and therefore move through the stationary phase particles more slowly. The B molecules don’t move as far in the same amount of time.

The consequences of this flowing mobile phase is that A is gradually separated from B by moving ahead in the flow. This separation process is depicted in Figure

In TLC, the stationary phase is typically alumina (Al2O3.xH2O)n or silica gel (SiO2.xH2O)n. The covalent network of these absorbents create very polar materials. The structure of silica is shown below.

Structure of Silica (SiO2.xH2O)n.

The electropositive character of the aluminum or silicon and the electronegative oxygen create a very polar stationary phase. Therefore, the more polar the molecule to be separated, the stronger the attractive force to the stationary phase. In some sense, the old adage “like-dissolves-like” can be applied here. The polar stationary phase will more strongly attract like or polar molecules. The equilibrium will be shifted as the molecules remain on the stationary phase. Nonpolar molecules will have a lower affinity for the stationary phase and will remain in the solvent longer. This is essentially how the partitioning separates the molecules. The equilibrium governs the separation, but the component’s attraction to the stationary phase versus the mobile phase determines the equilibrium. In general, the more polar the functional group, the stronger the bond to the stationary phase and the more slowly the molecules will move. In an extreme situation, the molecules will not move at all. This problem can be ovecome by increasing the polarity of the mobile phase so that the equilibrium between the free and absorbed state is shifted towards the free.

Although alumina and silica are the most common stationary phases used for TLC, there are many different types. They range from paper to charcoal, nonpolar to polar, and reverse phase to normal phase. Several different types of stationary phases are listed according to polarity in Figure

Now that we understand how the stationary phase operates, and assuming we are using a polar absorbent, how can we determine the elution sequence for our particular mixture? As mentioned previously, the more polar compounds will adhere more strongly to the stationary phase. Figure lists several common compound classes according to how they will elute from silica or alumma.

You may be able to see a trend developing. In general, the more nonpolar the compound, the faster it will elute (or the less time it will remain on the stationary phase) and the more polar the compound the slower it will elute (or more time on the stationary phase). It is important to become familiar with the trend so that you will not have to rely on the chart. You should be able to recognize the functional groups and easily determine which one is more polar than another. However, it should be noted that chromatography is not an exact science. This chart should be used to help predict the order of elution, but only performing the experiment will give clear answers.

As mentioned earlier, the mobile phase polarity can also bevaried to effect the chromatographic separation. Figure lists some common mobile phases according to increasing polarity.

Finding a good solvent system is usually the most difficult part of TLC. If the mobile phase has not been previously determined, start with a nonpolar solvent such as ligroin and observe the separation. If the mixture’s components do not move very far, try adding a polar solvent such as ether or ethyl acetate to the ligroin. Compare the separation to the previous plate. In most cases a combination of these two solvents is the best solution. If the spots stay at the bottom of the plate, add more of the polar solvent. If they run with the solvent front (go to the top too fast) than add a more nonpolar solvent. Unfortunately, rules are not full proof and some trial and error is involved in determining which solvent system is the best.

Spotting the TLC Plate

One advantage TLC has over other separation methods, is that it is truly a microscale technique. Only a few micrograms of material in solution is necessary to observe the solute on a TLC plate. Dissolve a few milligrams of material in a volatile solvent creating a dilute solution. Choose a volatile solvent that completely dissolves the sample. However, if it is partially soluble, since such only low concentrations are needed, normally you will be able to observe the compound.

Once the sample is prepared, a spotting capillary must be used to add the sample to the plate. The spotting capillaries must be extremely small. In fact, the opening at the end of a regular Pasteur pipet is too big for spotting a TLC plate. CG capillary columns donated by Restek, a chromatography company located in Bellefonte, are used to spot the plates. These columns are very small bore and can be cut into three inch sections to provide very good TLC spotting tubes. The solution can be drawn up the tube by capillary action (hence the name) and spotted on the plate at the hash mark labeled in pencil. This is known as the origin and is shown in Figure. Since a TLC plate can run three, if not four mixtures at one time, it is very important to properly label the plate. Notice that pencil is always used to mark a TLC plate since the graphite carbon is inert. If organic ink is used to mark the plate, it will chromatograph just as any other organic compound and give incorrect results.

To spot the plate, simply touch the end of the capillary tube to the coated side of the plate. The solvent should evaporate quickly leaving your mixture behind on the plate. You may have to spot the plate a couple of times to ensure the material is present, but do not spot too much sample. If too much solute is added to the plate, a poor separation will result. Smearing, smudging and spots that overlap will result making identification of separated components difficult.

Development

Once the dilute solution of the mixture has been spotted on the plate, the next step is the development. Just like paper chromatography, the solvent must be in contact with the stationary phase.

The bottle is filled with a small amount of the mobile phase and capped with a cork. In addition, a piece of filter paper is put in the bottle to help create an atmosphere saturated with solvent. Use your tweezers to place the plate in the development chamber; oils from your fingers can sometimes smear or ruin a TLC plate. Also make sure the origin spots are not below the solvent level in the chamber. If the spots are submerged in the solvent, they are washed off the plate and lost. Once the solvent has run within a centimeter of the top of the plate, remove it with tweezers. Using a pencil, immediately draw a line across the plate where the solvent front can be seen. The proper location of this solvent front line will be important for later calculations

Visualization

Some organic compounds are colored. If you are fortunate enough to be separating organic molecules that are colored such as dyes, inks or indicators, then visualizing the separated spots is easy. However, since most organic compounds are colorless, this first method does not always work.

In most cases observing the separated spots by UV light works well. TLC plates normally contain a fluorescent indicator which makes the TLC plate glow green under UV light of wavelength 254 nm. Compounds that absorb UV light willquench the green fluorescence yielding dark purple or bluish spots on the plate. Simply put the plate under a UV lamp, and the compounds become visible to the naked eye. Lightly circle the spots, so that you will have a permanent record of their location for later calculations.

UV LIGHT CHAMBER

Another useful visualizing technique is an iodine (I2) chamber. Iodine sublimes and will absorb to organic molecules in the vapor phase. The organic spots on the plate will turn brown and can be easily identified. Also circle these observed spots, since the color stain will eventually fade from the plate. Sometimes, a combination of both a UV lamp and iodine is needed to observe all the spots. Some compounds are not “UV active”, that is, they do not absorb light at the wavelength of 254 nm. Using both methods will ensure correct identification of all the spots on the TLC plate.

Rf Values

In addition to qualitative results, TLC can also provide a chromatographic measurement known as an Rf value. The Rf value is the “retardation factor” or the “ratio-to-front” value expressed as a decimal fraction.

Rf	=	distance spot travels
		distance solvent travels

This number can be calculated for each spot observed on a TLC plate. Essentially it describes the distance traveled by the individual components. If two spots travel the same distance or have the same Rf value then it might be concluded that the two components are the same molecule. For Rf value comparisons to be valid; however, TLC plates must be run under the same exact conditions. These conditions include the stationary phase, mobile phase, and temperature. Just as many organic molecules have the same melting point and color, many can have the same Rf value, so idential Rf values doesn’t necessarily mean identical compounds. Additional information must be obtained before this conclusion can be made. It is important to restate that this number is only significant when the same chromatographic conditions are used. Shows a diagram of a typical TLC plate and how the distances are measured to calculate the Rf value.

Preparative Plates

TLC can be used on a microscale to monitor a reaction and determine if the product or products were successfully produced using only microgram quantities of materials. It is difficult to separate gram quantities using TLC and therefore column chromatography is used at this scale . However, larger TLC plates, called a Preparative Plates, can be used for separations of milligram quantities of materials because they are coated with thick layers (1-3 mm) of stationary phase. Once the plate is developed, the spots are scraped off the plate along with the absorbent. Each separate component is then extracted from the stationary phase with a polar solvent.

The polarity arguments discussed in this chapter can be applied to many different types of chromatography including: Column Chromatography ,Gas Chromatography and High Performance Liquid Chromatography (HPLC). You will be applying these principles to column chromatography in the next chapter.

TLC Experiment

Analgesics are substances that relieve pain. The most common of these is aspirin, a component of more than 100 nonprescription drugs. In the present experiment analgesic tablets will be analyzed by thin-layer chromatography to determine which analgesics they contain and whether they contain caffeine, which is often added to counteract the sedative effects of the analgesic.


In addition to aspirin and caffeine the most common components of analgesics are, at present, acetaminophen and ibuprofen (Motrin). Phenacetin, the P of the APC tablets and a former component of Empirin, has been removed from the market because of deleterious side effects. In addition to one or more of these substances each tablet contains a binder, often starch or silica gel. To counteract the acidic properties of aspirin, an inorganic buffering agent is added to some analgesics. Inspection of labels will reveal that most cold remedies and decongestants contain both aspirin and caffeine in addition to the primary ingredient.

To identify an unknown by TLC, the usual strategy is to run chromatograms of known Standards substances (the standards) that are likely to be present in the unknown and the unknown at the same
time. If the unknown has one or more spots that correspond to spots with the same Rf’s as the standards, then those substances are probably present.
Proprietary drugs that contain one or more of the common analgesics and sometimes caffeine are sold under the names of Bayer Aspirin, Anacin, Datril, Advil, Excedrin, Extra Strength Excedrin, Tylenol, and Vanquish.

Procedure 1•• TLC Separation of Analgesics

Choose one of the analgesics and prepare a sample by crushing 1/4 of a tablet, adding this powder to a reaction tube or small vial along with a few drops of ethanol, and then mixing the suspension. Not all of the tablet will dissolve, but enough will go into solution to spot the plate. The binder, starch or silica, will not dissolve.

From the side shelf, acquire just a few drops of the 1% or 2% solutions of the four standard analgesics: aspirin, acetaminophen, ibuprofen, and caffeine in shorty vials or corked small test tubes, properly labeled. Work with the three other students on your side of the bench so that each of you get one vial of a standard and share it with the others.

Using a lead pencil (not a pen) and a ruler, mark two plates as shown below. First, draw a light pencil line across the plate about 1 cm from the bottom of two TLC plates. Make three equally spaced vertical dashes on this line. Then label the lanes at the top of the plate.

Using separate spotting capillary tubes from your desk, practice spotting on a paper towel using a pure solvent such as dichloromethane. After filling the capillary by dipping it in the liquid, touch it quickly to the towel so that the spot is no larger than 1 to 2 mm diameter. (The smaller the spot, the better the final TLC analysis.) After the solvent evaporates, you can apply more material in the same spot by, again, quickly touching the surface at the same place.

Now spot each of the plates on the middle vertical dash with your unknown tablet extract and on the outside with two standards as shown. Make each spot as small as possible, preferably less than 2 mm in diameter. Examine the plate under the UV light to see that enough of the compound has been applied by observing a visible dark purple dot; if not visible, spot more.

Develop your spotted TLC plates in one of the 250-mL wide-mouth bottles found in your desk. Make sure it is clean and dry. Use a large piece of filter paper in it to saturate the atmosphere with solvent vapor. Pour the 50:50 hexane:ethyl acetate developing solvent into the bottle to a depth of about 1/2 cm. Using forceps, carefully place the two TLC plates into the bottles so that they are leaning against the wall of the bottle, but not touching each other. Be sure that the spots are not below the solvent level or they will wash away into the solvent.

After the solvent has risen to the top of the plate or at least within a cm of the top, remove the plate with forceps and immediately mark the solvent front with a pencil. Allow the solvent to completely evaporate. Examine the plate under the UV lamp to see the components as dark spots against a bright orange or green-blue background. Outline the spots with a pencil. The spots can also be visualized by putting the plate in an iodine chamber which can be found on the side shelf. After a few minutes sitting inside the closed bottle, compound spots turn brown. Mark your spots with a pencil even after development in iodine vapor because the iodine color fades with time. Calculate the Rf value for the spots and identify the components in the unknown as shown in

NOTE: If the higher Rf compounds run into the broad band of “grunge” at the top of the plate, use 60:40 hexane:ethyl acetate. Don’t be disappointed if your TLC analysis doesn’t come out perfect the first time and you have to repeat the procedure. This is common. With practice, good results can be easily obtained.

Tape the properly labeled TLC plates in your notebook using the wide sticky tape available on the side shelf. Cover the whole plate with tape.

Cleaning up. Solvents should be placed in the appropriate organics waste container in your hood. Return unused iodine to the side-shelf supply bottle. The spotting capillaries are not
disposable!! They may be cleaned by dipping the ends into acetone and blotting the ends with a paper towel. The acetone will move by capillary action (pardon the pun), carrying the residue
of solute(s) with it. Gently blow a stream of nitrogen through the capillary to ensure complete drying. Store them safely in the test tube labeled for spotting capillary storage in your drawer for
use in other experiments.

SYNTHESIS OF 1,2:5,6-DI-O-CYCLOHEXYLIDENE-𝛼-D-GLUCOFURANOSE

• Fit a 3-litre flange flask with a multiple socket head carrying a mechanical stirrer capable of effecting vigorous agitation, a calcium chloride guard-tube, a 100-ml dropping funnel and a stoppered opening wide enough to allow for the addition of solid. 

• Immerse the flask in a large plastic or metal container filled with an intimate mixture of ice and salt. 

• Add 1000 g (1050 ml, 10mol) of redistilled cyclohexanone to the flask and cool to °C. 

• Charge the separatory funnel with 62.5 ml of concentrated sulphuric acid and run the acid slowly into the vigorously stirred cyclohexanone; the final solution should be a light straw colour. 

• Add slowly and portionwise with continued vigorous stirring 450 g (2.5 mol) of finely powdered dried 𝛼-D-glucose (1). 

• Remove the cooling bath and allow the reaction mixture to reach ambient temperature with continual stirring; over a period of 8 hours the reaction mixture becomes progressively more viscous and finally sets into a solid off-white crystalline mass. 

• Some caution should be exercised to prevent the stirrer motor from being overstrained. 

• Allow the reaction mixture to stand at room temperature overnight, break up the crystalline mass, add 750 ml of heptane and a solution of 124 g of sodium carbonate in 375 ml of water, and heat on a boiling water bath with vigorous stirring. 

• Decant as much of the upper heptane layer as possible from undissolved solid. 

• Add a further 750 ml portion of heptane to the residue and heat under reflux until the remainder of the solid dissolves; decant the clear heptane layer and cool the combined heptane extracts in the refrigerator. 

• Filter off the crystalline material, m.p. 121-124 °C, and recrystallise from heptane (2) using decolourising charcoal to clear the hot solution of traces of dark oily droplets. 

• The purified l,2:5,6-di-0-cyclohexylidene-oe-D-glucofuranose has m.p. 131-132 °C, [𝛼]ᴅ²⁰-2.2° (cl.8 in EtOH); the yield is 380 g (47%).

Notes to keep in mind:

1.  If adequate cooling and stirring is not employed the final solution is dark yellow; furthermore the addition of glucose leads to an unacceptable local rise in temperature, and the final appearance of the reaction mixture is a dark red intractable oily mass. The powdered glucose should be dried in a vacuum desiccator over phosphorus pentoxide, not in an oven which apparently causes changes on the surface of the glucose particles which render them unreactive.

2.  Another solvent for recrystallisation is methylcyclohexane (0.17g/ml).

Cognate preparations: 1,2:4,5-Di-O-cyclohexylidene-D-fructopyranose

• Add 200 g (1.1 1 mol) of finely powdered dry D-fructose with vigorous stirring to 419 g (440 ml, 4.49 mol) of ice-cooled cyclohexanone containing 30 ml of concentrated sulphuric acid; the reaction mixture becomes solid within 30 minutes. 

• Leave the mixture overnight at room temperature, dissolve the product in 500 ml of chloroform and wash the solution with dilute aqueous sodium hydroxide, dilute hydrochloric acid and water and finally dry and evaporate. 

• Solidify the residue by m.p. 145-156 °C, [𝛼]ᴅ²⁰ - 133.5° (c 1 in CHCl₃). The yield is 142 g (37%).

1,2:5,6-Di-O-isopropylidene-𝛼-D-glucofuranose

• A suspension of 150g (0.83 mol) of dry D-glucose, 120 g (0.83 mol) of anhydrous zinc chloride and 7.5 g of phosphoric acid (88% v/v) in 1 litre of dry acetone is stirred at ambient temperature for 30 hours. 

• Unchanged glucose is removed by filtration, and inorganic salts are precipitated by the addition of a solution of 85 g of sodium hydroxide in 85 ml of water. 

• The resulting suspension is filtered, the residue washed with acetone and the acetone evaporated. The mass which remains is dissolved in 200 ml of water and extracted with five 100-ml portions of dichloromethane. 

• The organic phase is dried and evaporated on a rotary evaporator. 

• Recrystallisation from light petroleum (b.p. 80-100 °C) gives 70g of product, m.p. 109-110°C,[𝛼]ᴅ²⁰ -18.5° (c5 in H 2 0).

1,2:5,6-Di-O-isopropylidene-D-mannitol

• To a solution of 60 g of zinc chloride in 300 ml of acetone is added 10 g of finely powdered D-mannitol. 

• The mixture is stirred vigorously at 20°C until solution is complete (2-3 hours) and then allowed to stand for 16 hours. 

• The reaction mixture is then poured into a solution of 70 g of potassium carbonate in 70 ml of water which is covered with 300 ml of ether. 

• The mixture is stirred for half-an-hour when the organic layer is filtered from the agglomerated pellets of zinc carbonate. 

• The latter is washed with 100 ml of 1:1 acetone-ether solution, and the combined filtrates evaporated to dryness on a rotary evaporator. 

• The dry residue is successively extracted with five 250-ml portions of boiling light petroleum (b.p. 60-80°C) and the combined filtrates slowly cooled to give the product, 7.9 g (55%), having m.p. 119°C.

SYNTHESIS OF METHYL 𝛼-D-GALACTOPYRANOSIDE

• In a 2-litre flask fitted with a reflux condenser place 100 g (0.56 mol) of dry 𝛼-D-galactose and 700 ml of an anhydrous methanolic solution of hydrogen chloride (about 0.6 m) (1). 

• Heat the mixture under reflux for 14 hours, cool, add 1 50 ml of distilled water and treat the light brown solution with solid lead carbonate until all the acid has been neutralised (2). 

• Filter off the inorganic salts and remove the solvent on a rotary evaporator under reduced pressure. 

• Triturate the resulting brown syrup with absolute ethanol with cooling in ice to cause the product to crystallise and recrystallise it from the minimum quantity of absolute ethanol to obtain 62 g of crude material, m.p. 85-90 °C.

• Isolate the methyl a-D-galactopyranoside as the hydrate by dissolving the crude product in 30 ml of water and allowing the solution to stand for one day at room temperature and two days at 4 °C. 

• Repeat the recrystallisations several times using proportionate amounts of water until pure hydrated product, m.p. 109-110 °C, [𝛼]ᴅ²⁰ + 173.4° (c 1 in H 2 0), is obtained; the yield is 38g(35%)(3).

Notes to keep in mind: 

1. Dry hydrogen chloride gas is passed into dry methanol (contained in a flask protected by a calcium chloride guard-tube) until analysis of aliquot portions by titration with standard aqueous sodium hydroxide solution reveals the required concentration has been reached. It is usually more convenient to prepare initially a smaller volume of a more concentrated solution and dilute it to the

appropriate concentration with dry methanol. The aliquot portions (say 5 ml) should be diluted with distilled water (20 ml) before titration.

2. Universal indicator paper moistened with distilled water gives a satisfactory indication of neutralisation.

3. Pure methyl 𝛽-D-galactopyranoside (m.p. 1 77-180 °C) may be isolated from the combined aqueous filtrates of these several crystallisations by removal of water and recrystallisation of the residue from absolute ethanol.

SYNTHESIS OF METHYL 𝛽-D-GLUCOPYRANOSIDE

• In a 100-ml conical flask place 5.5 g (0.015 mol) of methyl 2,3,4,6-tetra-O-acetyl- 𝛽 -D-glucopyranoside, 50 ml of dry methanol and 10 ml of a solution of sodium methoxide in methanol previously prepared by the cautious addition of 0.1 g of sodium to 20 ml of methanol. 

• Stopper the flask and allow the solution to stand for 1 hour, then add sufficient ion exchange resin [Zeolite 225 (H🜨)] to render the solution neutral to moist universal indicator paper. 

• Remove the resin by filtration, wash with methanol and evaporate the combined filtrate and washings under reduced pressure (rotary evaporator). Triturate the colourless syrup with absolute ethanol to cause it to solidify and recrystallise from absolute ethanol. The pure methyl 𝛽-d-glucopyranoside has m.p. 108-109 °C, [𝛼]ᴅ²º-30.2° (c2.8 in H 2 0); the yield is 2.4 g (83%).

Cognate preparation: Methyl 𝛽 -D-galactopyranoside

• Use 3.6 g (0.01 mol) of methyl 2,3,4,6-tetra-0-acetyl-𝛽-D-galactopyranoside and proceed as above. After recrystallisation from absolute ethanol, 1.4 g (73%) of the methyl galactopyranoside, m.p. 174-175 °C, [𝛼]ᴅ²º+1.3° (cl in H 2 0), is obtained.

SYNTHESIS OF METHYL 2,3,4,6-TETRA-0-ACETYL-𝛽-d-GLUCOPYRANOSIDE

• In a 500-ml two-necked flask fitted with a mechanical stirrer and calcium chloride guard-tube place 110 ml of dry methanol, 110 ml of pure chloroform, 22 g of anhydrous calcium sulphate, 7.2 g of yellow mercury(n) oxide and 0.55 g of mercury(n) bromide. 

• Stir the suspension for 30 minutes and add 16.5 g (0.04 mol) of 2,3,4,6-tetra-O-acetyl-𝛼 -D-glucopyranosyl bromide in one portion. 

• The temperature of the mixture will rise to about 25-30 °C, the pH of the solution will fall from 7 to 2 and the yellow coloration of the mercury(n) oxide will disappear (1).

• Stir the suspension for a further 90 minutes, filter through a pad of Celite filter-aid and evaporate the filtrate on a rotary evaporator under reduced pressure. 

• Dissolve the viscous oil which remains in 10 ml of chloroform, remove the inorganic salts which are precipitated by filtration and wash the residue well with further portions of chloroform. 

• Evaporate the chloroform and triturate the resulting viscous oil with methanol until it solidifies. 

• Recrystallise from methanol to give pure methyl 2,3,4,6-tetra-O-acetyl-𝛼 -D-glucopyranoside, m.p. 104-105 °C, [𝛼]ᴅ²º    - 18.2° (c 1 in CHC1 3 ). The yield is 13.7g (95%).

Notes to keep in mind:

1.  The yellow coloration in the solution disappears within a few minutes of addition of the glucosyl halide and t.l.c. analysis (solvent system benzene-methanol, 98:2) reveals virtual completion of the reaction.

Cognate preparation: Methyl 2,3,4,6-tetra-O-acetyl-𝛽-D-galactopyranoside

•  Use 13.5 g (0.033 mol) of 2,3,4,6-tetra-O-acetyl-𝛼-D-galactopyranosyl bromide, 19 g of anhydrous calcium sulphate, 5.6 g of yellow mercury(n) oxide, 0.5 g of mercury(n) bromide, 90 ml of dry chloroform and 90 ml of dry methanol under the reaction conditions and subsequent isolation procedure described above; 7.5 g (63%) of methyl 2,3,4,6-tetra-0-acetyl-𝛽-D- galactopyranoside, m.p. 96-97 °C, [𝛼]ᴅ²º-28.0° (c2.5 in CHC1 3 ), is obtained after several recrystallisations from ethanol.

SYNTHESIS OF 2,3,4,6 TETRA-O-BENZOYL-𝛼-D-GLUCOPYRANOSYL BROMIDE

• In a 250-ml conical flask fitted with a ground glass stopper place 40 ml of 1,2-dichloroethane and 20 g (0.029 mol) of 𝛼-D-glucopyranose pentabenzoate (1). 

• When all the solid has dissolved add 40 ml (0.225 mol) of a solution of hydrogen bromide in glacial acetic acid (45% w/v HBr), stopper the flask and allow the reaction mixture to stand in the refrigerator overnight or at room temperature for about 2 hours. 

• Pour the mixture into ice-water, rinse the flask with 1,2-dichloroethane, separate the organic layer and shake it with several portions of a saturated aqueous solution of sodium hydrogen carbonate until no further effervescence occurs. 

• Wash the organic layer with water, dry over magnesium sulphate, filter and remove the 1,2-dichloro-ethane under reduced pressure on a rotary evaporator. 

• Dissolve the crystalline solid which remains in dry ether, heating to 35 °C, and slowly add with further heating light petroleum (b.p. 40-60 °C) until a slight persistent cloudiness develops; then add a little more ether to give a clear solution, which is left to cool slowly to room temperature and finally refrigerated. 

• Filter off the purified product and allow it to dry in the air; the yield is 16.5 g (88%), m.p. 129-130 °C, [𝛼]ᴅ²º + 125° (c2.0 in CHC1 3 ).

Notes to keep in mind:

1.  A mixture of anomeric glucose pentabenzoates such as might be obtained from a benzoylation reaction on glucose without careful temperature control gives equally good results.

SYNTHESIS OF 2.3.4.6-TETRA-O-ACETYL-𝛼-D-GLUCOPYRANOSYL BROMIDE (𝛼-Acetobromoglucose)

• Fit a 1 -litre three-necked flask located in the fume cupboard with a mechanical stirrer unit using a Kyrides seal, a dropping funnel and a thermometer to read the temperature of the reaction mixture. 

• Immerse the flask in an ice-salt bath supported on a laboratory jack so that it may be easily removed if the reaction conditions so demand. 

• Place 432 g (400 ml, 4.24 mol) of acetic anhydride in the flask, cool to 4 °C and add dropwise and with stirring 2.4 ml of 60 per cent perchloric acid. 

• Remove the cooling bath and allow the reaction mixture to warm to room temperature; then add 100g (0.56 mol) of dry powdered 𝛼-D-glucose in portions with stirring so that the temperature of the reaction mixture is maintained at between 30 and 40 °C. 

• Cool to about 20°C and add 31 g (1 mol) of red phosphorus followed by 181 g (58ml, 2.26 mol) of bromine (CAUTION) dropwise at a rate that the temperature does not exceed 20 °C. 

• Then add 36 ml of water over a period of about half an hour, the stirring and cooling being continued and the temperature being maintained below 20 °C. 

• Allow the reaction mixture to stand for 2 hours at room temperature, transfer to a fume cupboard and dilute with 300 ml of dichloromethane, and filter through a large 60° glass funnel having a glass wool plug inserted not too tightly into the outlet (1). 

• Finally rinse the reaction flask and funnel with small portions of dichloromethane, transfer the filtrate and washings to a 3-litre separatory funnel and wash it rapidly by shaking vigorously with two 800 ml portions of iced water (2). 

• Run the lower dichloromethane layer from the second washing into 500 ml of a stirred saturated solution of aqueous sodium hydrogen carbonate to which has also been added some crushed ice. 

• When the vigorous evolution of carbon dioxide has subsided transfer the mixture to a separatory funnel, run the dichloromethane layer into a large flask containing 10 g of powdered activated silica gel and filter after about 10 minutes (the bulk of the solution may be decanted from the silica gel and the remainder filtered under reduced pressure using a sintered glass funnel). 

• Remove the solvent under reduced pressure using a rotary evaporator on a water bath maintained at 60 °C. 

• Towards the conclusion of this operation the syrupy mass crystallises as a thick layer around the inside of the flask. 

• At this stage remove the flask from the evaporator, break the crystalline cake away from the sides of the flask and remove the remaining solvent under reduced pressure without heating further. 

• Transfer portions of the solid to a mortar and grind with a 2:1 mixture of light petroleum (b.p. 40-60 °C) and dry ether. 

• Filter the combined slurry and wash the filter cake first with a light petroleum-ether solvent mixture and then with 50 ml of previously chilled (0°C) dry ether. 

• The crude product is obtained in a yield of 210 g (92%), and when recrystallised from ether-light petroleum (b.p. 40-60 °C) has m.p. 88-89 °C, [𝛼]ᴅ²⁰ + 197.5° (c2 in CHC1₃). 

• The glucosyl halide should be stored in a desiccator over sodium hydroxide pellets; whenever possible it should be used without delay.

Notes to keep in mind:

1.  If care is used most of the solution may be decanted from the solid deposit so that the glass wool does not become blocked with material and hence slow down the filtration process. This filtration is best conducted in a fume cupboard.

2.  All the isolation operations must be conducted with the minimum of delay and under conditions which reduce the contact of the solutions of unstable glucosyl halide with moisture. Solutions to be used for washing the organic layer should have been previously prepared and contain sufficient ice to ensure that the temperature of the liquid is approximately 4 °C. To obtain good yields and to ensure that vessels do not become unduly 'sticky' as the result of residual carbohydrate deposits, the separatory funnels, receiver vessels and aqueous extracts before being discarded should be rinsed with dichloromethane at each stage and these washings combined with the main

organic solution.

Cognate preparations: 2,3,4,6- Tetra-O-acetyl-𝛼-D-galactopyranosyl bromide

• Use 100 g (0.56 mol) of dry D-galactose under precisely the same conditions; the product is obtained in a yield of 202 g (88%). When recrystallised from ether-light petroleum (b.p. 40-60 °C) it has m.p. 84-85 °C, [𝛼]ᴅ²⁰ + 214° (c 1.2 in CHC1 3 ).

2,3,4-Tri-O-acetyl-𝛽-L-arabinopyranosyl bromide

• For this preparation use 10 g (0.067 mol) of l-( + )-arabinose, 40 ml (0.424 mol) of acetic anhydride, 0.24 ml of 60 per cent perchloric acid, 30 g (0.1 mol) of red phosphorus, 18.1 g (5.8 ml, 0.226 mol) of bromine and 3.6 ml of water. 

• The yellow syrup which is obtained after the appropriate isolation procedure gives 21 g of crude crystalline product. 

• Recrystallisation is effected by dissolving it in a mixture of benzene/ether (5:95) warming and adding light petroleum (b.p. 40-60 °C) until a slight cloudiness is apparent, and then allowing the solution to cool. The pure product is obtained in a yield of 11 g (48%), m.p. 136-138 °C, [𝛼]ᴅ²² +280° (c3.13 in CHC1 3 ).