Separation of Aspirin

One of the most frequently used pain relievers is acetylsalicylic acid, which is commonly called aspirin. An aspirin tablet contains more than aspirin, however. Manufacturers mix aspirin with starch, which keeps the tablets from falling apart and makes them large enough for easy handling. Furthermore, aspirin can break down into salicylic acid and acetic acid over time. Therefore, an aspirin tablet is a mixture of at least four substances aspirin, starch, salicylic acid, and acetic acid. 

What is the difference between a heterogeneous mixture and a homogeneous mixture  

Classify the following mixtures as heterogeneous or homogeneous salt and water, sand and water, nitrogen and oxygen in air. 

Which technique—distillation, crystallization, or filtration— is most useful for separating a heterogeneous mixture composed of a solid and a liquid  

What technique is used to separate the substances in a solution based on differences in their boiling points  

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As mentioned in the historical synopsis (Section 5.1), Levine121 perfected the compendial partition column procedure in which aspirin in chloroform is first trapped in an immobile phase of sodium bicarbonate on a column of siliceous earth (celite) then eluted with a solution of acetic acid in chloroform and measured spectrophotometrically. This has been also used for separation in combination products.80 For the determination of salicylic acid in presence of aspirin by this method, see Section 5.61. Ion exchange columns filled with strongly or weakly basic anion exchange resin in the acetate or chloride cycle have also been used for separation of aspirin in combination products. 122 123/l2lf This has also been adapted for a student experiment.125 A Sepha-dex-G25 column has been used for the separation of aspirin from salicylic acid.126... 

Aspirin can be separated from salicylic acid by ionophoresis at a pH of 4-5.1 8 Separation of aspirin in combination products has been achieved with paper strip electrophoresis, using buffers at pH 2-8 and a 200 V. applied potential.11 9 Aspirin was separated from metabolites by paper electrophoresis in a phthalate buffer of pH 3.2 and an ionic strength of 0.0125-0.0500.150... 

Nikelly (49) performed a similar separation without the need for derivatives. The column was 0.25% Carbowax 20M/0.4% isophthalic acid on acid-washed microbeads. Included in Kern s work (39) was a description of a separation of aspirin and salicylic acid as the TMS derivatives on 3% OV-17 on Chromosorb W-HP at 130 C°as well as the separation of phenacetin, antipyrine, and aminopyrine without derivatization on SE-30, 3% on VarAport 30 at 180°C. 

One example that illustrates this is the GPC separation of aspirin and propyl paraben. These two compounds have identical molecular weights (MW = 180) but can be separated using GPC (e.g., on a 100-A jiStyragel columns). From the structures (Fig. 11-3) it can be hypothesized that propyl paraben acts as a larger molecule in solution than does aspirin. Since the side chain on propyl paraben would give the molecules a larger end-to-end size than the more compact aspirin molecule, propyl paraben should elute before aspirin. 

A. Figure 13-1 shows an example chromatogram for the separation of aspirin, phenacetin, and caffeine on a low-efficiency column. Figure 13-2 shows the example chromatograms used in the construction of the phenacetin calibration curve (Fig. 13-3). 

FIGURE 14-3. Separation of aspirin reaction mixture on a low-efficiency column. Column Bondapak C18/Porasil B (37-75 p.m) 2 mm ID x 61 cm. A-H are times noted on the chromatogram. Mobile phase MeOH/4% HOAc in H2O (35/75). Flow rate 1 mL/min. Detector UV at 254 nm, 0.2 AUFS. Sample 10 p,L of aspirin solution. (Note Actual separation will depend upon the quality of the mobile phase and column packing). 

Prelab Exercise Prepare a detailed flow sheet for the separation of aspirin, caffeine, and acetaminophen from an analgesic tablet. 

As examples the separation of aspirin, caffeine, and phenacetin (APC tablets) is accomplished by a mobile phase of methanol-acetic acid-ether-benzene (1 18 60 120) and nitroglycerine, a vasodilator, present in heart medication capsules can be separated from the other capsule materials by benzene. 

Hawkey CJ, Hawthorne AB, Hudson N, Cole AT, Mahida YR, Daneshmend TK (1991) Separation of aspirin s impairment of haemostasis from mucosal injury in the human stomach. Clin Sci 81 565-573... 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

Since reproducibility of the flow system is critical to obtaining reproducibility, one approach has been to substitute lower-performance columns (50-to 100-p packings) operated at higher temperatures.1 Often, improvements in detection and data reduction can substitute for resolution. Chemometric principles are a way to sacrifice chromatographic efficiency but still obtain the desired chemical information. An example of how meaningful information can be derived indirectly from chromatographic separation is the use of system or vacancy peaks to monitor chemical reactions such as the titration of aniline and the hydrolysis of aspirin to salicylic acid.18... 

Figure 5 Separation of pharmaceuticals, including amines, on strong cation exchange. Column 0.46 x 15 cm Merckosorb SI-60-SCX, 5 p. Eluent 50 mM aqueous ammonium formate-10% ethanol, pH 4.8. Flow 1 ml/min. Temperature 50°C. The peaks are (1) aspirin, (2) paracetamol, (3) phenacetin, (4) caffeine, (5) phenylephrine, (6) salbutamol. (Reproduced with permission of Elsevier Science from Cox, G. B., Loscombe, C. R., Slucutt, M. J., Sugden, K., and Upheld, J. A., /. Chromatogr., 117, 269, 1976).

Cellulose anion-exchange paper chromatography with 5% acetic acid-n-propanol (5 1) gave good separation, detected by U.V. light, of aspirin (Rf. 0.07) from salicylic acid (Rf. 0.51).107... 

The chromatogram and bar graph show results of a study of aspirin metabolism in a rat. Aspirin is converted into salicylic acid by enzymes in the bloodstream. To measure the conversion rate, aspirin was injected into a rat and dialysate from a microdialysis probe in a vein of the rat was monitored by liquid chromatography. If you simply withdrew blood for analysis, aspirin would continue to be metabolized by enzymes in the blood. Microdialysis separates the small aspirin molecule from large enzyme molecules. 

In the present experiment, we measure the amount of the active ingredient, acetylsalicylic acid (see also Experiment 35), in common aspirin pills. Companies use different fillers and in different amounts, but the active ingredient, acetylsalicylic acid, must be the same in every aspirin tablet. We separate the acetylsalicylic acid from the filler based on their different solubilities. Acetylsalicylic acid is very soluble in ethanol, while neither starch, nor other polysaccharides, or even mono- and disaccharides used as a fillers, are soluble in ethanol. Some companies may use inorganic salts as fillers but these too are not soluble in ethanol. On the other hand, some specially formulated aspirin tablets may contain small amounts of ethanol-soluble substances such as stearic acid or vegetable oil. Thus the ethanol extracts of aspirin tablets may contain small amounts of substances other than acetylsalicylic acid. 

Umapathi et al. used spectrofluorimetric methods for the estimation of aspirin and dipyridamole in pure admixtures and in dosage forms [36]. The methods are described for the determination of the two drugs in pharmaceutical preparation without prior separation from each other... 

The assay, introduced by Bom (1962a,b), has become a standard method in clinical diagnosis of platelet function disorders and of aspirin intake. Furthermore, the method is used in the discovery of antiplatelet drugs with the advantage of rapid measurement of a functional parameter in intact human platelets. However, processing of platelets during the preparion of PRP, washed or filtered platelets from whole blood results in platelet activation and separation of large-size platelets. 

FIGURE 5-25. Separation of phenylpropanolamine and aspirin with and without the use of a paired ion. (a) Mobile phase contains no ion-pair reagent. (b) Mobile phase contains ion-pair reagent (5 mM heptanesulfonate). 

Small differences in the structure of the molecules result in changes in the elution volumes. Note the difference that shortening the side chain from propyl to methyl has on the elution volume of the paraben. Even such a minor difference is sufficient to permit the separation of these two compounds. The same effect can be observed if aspirin (acetylsalicylic acid) is hydrolyzed to salicylic acid. Reduction in molecular size increases the elution volume. 

Aspirin, caffeine, and phenacetin are very polar compounds. Normal-phase separations of polar compounds like these are generally not successful. Reverse-phase chromatography is usually a better method. 

Weigh 4.5 and 5.5 grains of aspirin (acetyl salicylic acid) and place each into separate 100-mL volumetric flasks. Dilute to mark with methanol. 

Make two separate 10-/aL injections, each for the 4.5 and 5.5 grain 100-mL standard solutions of aspirin. These were the solutions prepared in Activity B-3. The spectrophotometric detector should be set to 0.2 AUFS (254 nm). 

B. Results of the separation of the aspirin reaction mixture under the conditions of this experiment are presented in the series of chromatograms in Figure 14-3A through 14-3H for the separation on a low-efficiency column. Figure 14-4 shows example chromatograms of the reaction products at various times during the reaction on a high-efficiency column. 

Fig. 3.11. HPLC of the hydroxylated products of salicylate (from Halliwell et al., 1988) 2,3-DHB, 2,3-dihydroxybenzoate 2,5-DHB, 2,5-dihydroxybenzoate RS, resorcinol SA, salicylate SU, salicylurate (a major metabolite of aspirin) CA, catecholamines, (a) Separation of a standard mixture of 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate and resorcinol (internal standard) (b) Separation of an extract of a plasma sample from a healthy individual not consuming aspirin (c) Separation of an extract of a plasma sample from a healthy individual consuming aspirin (the large peaks to the left of the chromatogram are probably catecholamines - marked CA).

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