Analysis of Sulfonamides

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The Drug Substance

Analysis of Sulfonamides


In the science of rational drug design, log P value of a compound is important to determine its extent of capability to pass through cell membrane. In determining log P of sulfonamides, Thin-layer Chromatography (TLC), ‘shake-flask' method and High Performance Liquid Chromatography (HPLC) were used. Chemical purity of an unknown sulfonamide, C26 was determined using HPLC. Straight phase TLC is not suitable in determine the partition coefficient of sulphonamides due to the reason it is unable to relate its partition and retention factor. HPLC was found to be the best method in determining log P as it is a reliable and accurate method widely used in pharmaceutical industries. The Ptrue values obtained for sulfathiazole in pH 1 and pH9 using ‘shake-flask' method were 1.47 and 3.1859 respectively. The log P value obtained from HPLC for C26 is 3.300 and chemical purity value was 68%.


log P, sulfonamides, TLC, ‘shake-flask', HPLC


Sulfonamides are antibacterial drugs used to treat infections caused by microorganisms. Sulfonamide antibiotics target on enzyme dihydropteroate synthase(DHPS) which plays an important role in catalysing the bacterial production. They work by inhibit the activity of DHPS which eventually inhibits the synthesis of folic acid and DNA (Foye et al, 1995). Sulfonamides imitate p-aminobenzoic acid (PABA) and compete for the active site. In this way, bacteria could not multiply as there are insufficient of folates available for their growth. In manufacturing the required sulfonamide(C26), an amine which is known as 4-aminoacetophenone is required to obtain the desired final compound.

Sulfonamides' relative distribution in an n-octanol/water mixture can be done experimentally by measuring the hydrophobic character of the compound. Partition coefficient, P is one of the most crucial factors in controlling the drug action in biological systems. The partition coefficient is an important measure in predicting the absorption, distribution and elimination of drugs in the body. Not only that, it can be used to relate the biological activity of a drug to it properties. Log P, which is known as octanol-water partition coefficient, is used in rational drug design to measure the hydrophobicity of a compound.

It is important to test the hydrophobic character of a drug as it will affect the extent the drug crosses cell membranes easily. The different substituents on a drug have different hydrophobic character and eventually have different biological activity (Patrick 2009). Hydrophobic compounds have a high P value and vice versa for hydrophilic compounds.

Log P can be measured with different methods. Namely, ‘shake-flask' method, thin-layer chromatography, and High Partition Liquid Chromatography (HPLC) can be used to measure log P. HPLC and TLC are known to be yielding a hydrophobicity parameter rather than a partition coefficient (Takács-Novák and Avdeef, 1996).

The purpose of this study was to investigate the properties of sulphonamides, especially the hydrophobicity of the compounds. Log P was determined using various methods and the advantages and disadvantages of each method used were investigated. Chemical purity of the compound was investigated using the modern HPLC system.


Establishing the log P of sulfonamides using TLC

In the separation of sulfonamides, TLC was used to establish the log P of the sulphonamides. Sulfonamide solutions were prepared by dissolving 40mg of sulfonamide in acetone and the solutions were made up to 10mL in a volumetric flask which will give a concentration of 0.4% w/v. Then, each individual sulfonamide solution was transferred to a glass capillary tube. After that, the solutions contained in the glass capillary tube were spotted on a plate with silica coated and aluminium backed size at 12cm × 7cm. The plate was then placed in a chromatographic chamber containing mobile phase butan-1-ol for about 60 minutes. When 75% of the mobile phase moves up the plate, the plate was removed from the chromatographic chamber and the mobile phase front was marked. Hot air was washed to dry the plate and the sulfonamides were visualized under short wave UV and the center of each detected spot was marked. The distances from the spots to the mobile phase front and to the center of the detected spot of each sulfonamides were measured using a ruler. The retention factor, Rf value for each sulfonamides was then calculated by following formula:

Rf = Distance moved by spot

Distance moved by solvent

Exploring the partitioning of sulfonamide under ionising and non-ionising conditions using the ‘shake-flask' method

A 25µg mL-1 solution in 0.5M NaOH( Solution A) and 10µg mL-1 solution in water(Solution B) were prepared using a stock solution containing 0.02%w/v of sulfonamide in water. A range of calibration standards which contain 2.3, 5, 7.5,10, 12.5, and 15µg mL-1 of sulfonamide in 0.5M NaOH was prepared using Solution A. The maximum absorbance of 1.0774 was determined at the wavelength of 257nm using the 15µg mL-1 standard. The standards' absorbance was read at the λmax of 1.0774 with NaOH as the blank. A graph of absorbance versus concentration for the sulfonamide was plotted using the readings.

Two partitioning samples were prepared by adding 10ml of Solution B, 10mL 0.1M HCl and 20mL octanol in separating funnel (i) and 10mL of Solution B, 10mL pH 9.0 buffer and 20mL octanol in separating funnel (ii). The funnels were shaken at frequent intervals for 30 minutes so that the layers were separated fully. It is important to make sure the funnels were not shaken too vigorously to avoid an emulsion to be formed. Then, the aqueous layer was carefully run off and 20mL 0.5M NaOH was added using a pipette to the remaining octanol in the funnel. The funnel was shaken again for 5 minutes for separation and the absorbance of the aqueous layer was measured at the λmax of 1.0774. The concentration of the sulfonamides in the 0.5M NaOH was calculated from the calibration curve.

Determination of log P values using HPLC

The sample solution was injected into HPLC (Agilent 1120 Compact LC) with column Zorbax C8 measured at 150 mm X 4.6 mm ID. The wavelength is set at 254 nm, flow rate at 1 mL min-1, temperature at 40oC, and injection volume of 20 µL. The running time of the system is set to be 120 minutes. The ratio of mobile phase used in system is 85:15 (0.1% CH3COOH:MeOH)% v/v.

Chemical Purity Determination

A sample solution of C26 at a concentration of 0.1 mg mL-1 was prepared in a 100mL volumetric flask. 7.2mL of methanol was added to dissolve the sample and mobile phase was added to make up to 100mL. 20µL of the sample solution was injected to the HPLC-DAD system with column C18 Phenomenex Luna 5µ measured at 150 mm X 4.6 mm ID using a single injection mode and the method ‘Chempur 1'. The flow rate of the system was set at 1 mL min-1. The ratio of the mobile phase used in the HPLC system is 85:15 (CH3COOH:MeOH) %v/v.

Results and Discussion

Establishing the log P of sulfonamides using thin-layer chromatography


Log Papp

Log Ptrue


Log Rf







-1.3 at pH 7.5




















-0.4 at pH 7.5




Table 1: Log P and Log Rf values of sulfonamides

The literature values for log P of the sulphonamides were listed in the table above. A graph log Rf versus log P was drawn to compare estimate the extent of partitioning against the literature values of log P. Based on Figure 3, it was noticed that there was no relationship between log Rf and log P as the regression coefficient, R2 value was 0.1017. This small value of R2 of 0.1017 shows that only 10.17% of variation in biological activity was accounted for by the parameters of log P and log Rf (Patrick, 2009). However, R value should not be relied on too much as the value obtained does not take any account of the number of compounds that were involved in the study. This means that a higher value of R2 could be obtained if there were more compounds incorporated in the study.

This could be due to straight phase TLC was used in the experiment. The stationary phase in the experiment is the silica layer which will interact with hydrophilicity of a compound. Sulfonamides are hydrophobic compounds so it is best to apply a stationary phase which will interact with the hydrophobicity property of a compound. To improve the results, reversed phase TLC should be considered in determining log P value which is the main concern of hydrophobicity of a compound. In reversed phase TLC, the silica plate has increased amount of alkyl groups which will enable hydrophobic-hydrophobic interaction between the stationary phase and the compound. It will also increase the time of the compound travelling in the mobile phase.

The advantage of using TLC is that log P of many compounds can be determined on one plate. Small amount of sample is required to undergo TLC process. This method is cheaper compared to other methods in determining log P of the compounds. TLC also has the advantage that all constituents of a sample can be visualised easily, especially under UV light. In contrast, the quantitation from the spots obtained from the plate is not easy as TLC is more suitable for the separation of involatile compounds.

Exploring the partitioning of sulphonamide under ionising and non-ionising conditions using the ‘shake-flask' method

In shake-flask method, the pKa values are required to calculate the partition coefficient ( Takács-Novák and Avdeef, 1996).

Calibration standards for sulfathiazole:

Concentration (µg mol-1)

Absorbance 1

Absorbance 2

Average absorbance





























Table 2: Concentrations and average absorbance for the calibration standards of sulfathiazole

Partitioning samples:



Absorbance 1

Absorbance 2

Average absorbance











Table 3: Absorbances obtained in different pH.

From Figure 4, the equation obtained from the linear line is y=0.0735x.

The absorbance value obtained for Funnel (i) is 0.219. The value was substituted in the equation as y to obtain x value which is the concentration of sulfathiazole for in 0.5M NaOH.

For Funnel (i);

y = 0.0735x

0.219 = 0.0735x

x = 2.9796 µg mL-1

For Funnel (ii);

0.014 = 0.0735x

x = 0.1905 µg mL-1



Concentration(µg mL-1)


Total sulfonamide amount in organic phase(µg)

Total sulfonamide amount in aqueous phase(µg)



















Table 4: Calculated Ptrue obtained from the concentration in different pH.

To calculate Ptrue from the concentration of the sulfonamide contained in 0.5M NaOH, total amount of drug in organic and aqueous phase, Papp and pH of the buffer are required. The working calculations are as below:

For Funnel (i) in pH 1;

Concentration of sulfonamide in organic phase = 2.9796 µg mL-1

1 mL of the organic phase contained 2.9796 µg of drug, this means that 20 mL of organic phase contained a total of 59.592 µg of sulfonamide.

Total sulfonamide in organic and aqueous phase = 10 mL X 10 µg mL-1

= 100 µg

Total sulfonamide in aqueous phase = 100 µg - 59.592 µg

= 40.408 µg

Papp = [sulfonamide] in organic phase / [sulfonamide] in aqueous phase

= (59.592/20)/(40.408/20)

= 1.47

In this case, Papp depends on pH of the solution.

log D at pH 1 = log P - log [1 + 10(pH-pKa)]

log Ptrue = log 1.47 + log [1 + 10(1-7.1)]

log Ptrue = 0.16731

Ptrue = 1.47

As for Funnel (ii) in pH 9,

Concentration of sulfonamide in organic phase = 0.1905 µg mL-1

20 mL of organic phase contained a total of 3.81 µg of sulfonamide

Total sulfonamide in aqueous phase = 100 µg - 3.81 µg

= 96.19 µg

Papp = (3.81/20)/(96.19/20)

= 0.039609

log Dat pH 9= log P - log [1 + 10(pH-pKa)]

log Ptrue = log 0.039609 + log [1 + 10(9-7.1)]

= 0.50323

Ptrue = 3.1859

Papp = Ptrue × funionised

0.039609 = 3.1859 × funionised

funionised = 0.012433

= 1.2433%

Sulfathiazole is a weak sulfonamide with pKa value of 7.1. Therefore, it is significantly ionised within a physiological pH range. In this ‘shake-flask' method, the effect of ionisation on the Papp of sulfathiazole can be investigated by measuring the amount of sulfathiazole being extracted into octanol (organic phase) from aqueous phase at different pH. In pH 1, it is obviously noticed that Papp = Ptrue which means that the drug is 100% unionised as the funionised = 1. From the calculations made from the experiment, the concentration in octanol layer for acidic medium (pH 1) is 2.9796µg mL-1. As for the alkali medium (pH 9), the concentration in octanol is 0.1905µg mL-1. The concentration difference in different pH is due to ionisation of the drug to form a salt which will eventually change the solubility profile. Papp value in pH 1 is expected to be higher than in pH 9 buffer so it is also expected that the absorbance is higher in acidic medium. There are some human errors occurring when preparing the standards. The standard absorbance is set to be more than 1.0 which is not ideal for absorbance measurements. In order to obtain a more accurate result, the absorbance should be set less than 1.0.

The advantage of ‘shake-flask' method is that log P can be directly obtained using this method. One of the disadvantages of ‘shake-flask' method is that it is time consuming to make sure that the aqueous and organic layers are separated fully. This method requires good laboratory skills such as skills in shaking the separating funnels. Separating funnels should be made sure not to be shaken too vigorously to prevent emulsion from forming. Laboratory skill such as running off the bottom layer in the funnels is important to avoid the organic layer from running off together with the aqueous layer.

Determination of Log P values using HPLC




k = (tr-t0)/t0

Log k

Log P





































Table 5: Log P values obtained using HPLC

Figure 5: Graph of log k versus log P in determining log P values using HPLC.

Based on Figure 5, the equation obtained was y =0.4818x + 0.3096.

As C26 is an unknown compound, the log P is unknown too. Therefore, the log P for C26 could be calculated based on the equation obtained.

y=0.4818x + 0.3096

1.8998= 0.4818x + 0.3096

x= 3.300

Log P value for C26 is 3.300

The data follows a straight line with R2 of 0.8782. This straight line indicates that log k increases with log P. This shows that the retention time( time taken to elute from HPLC column) in HPLC is directly affected by the log P of the compound. The higher value of log P causes the longer time the compound stays in the column due to hydrophobic-hydrophobic interaction.

Sulfadiazine and sulfathiazole have Log P values of 1.3 and -0.4 respectively at specific pH of 7.5. This indicates that the log P values are known as Papp which is affected by the ionisation to form a salt. Papp values changed due to the charge as the compound become ionised. The line of a graph would be severely affected with decreasing values of R2 value if Papp values were used to plot the graph. Therefore, Papp should be converted to Ptrue to ensure the linearity of the graph's line.

There are problems in obtaining Log P for unknown sulphonamide using the old HPLC model system. I could not get any results even though I have waited for more than two hours. This might due to the reason that C26 is a very hydrophobic compound compared to other sulphonamides. C26 has a Log P of 3.300, therefore it is a very hydrophobic drug. This is proven due to the high retention time of 127.257 obtained from the new model of HPLC system .

Polarity of mobile phase can affect the separation of the compound as the retention in reversed-phase liquid chromatography is regulated by interactions in the mobile phase. Low polarity of mobile phase will increase the retention time of the compound as hydrophobic-hydrophobic interaction in the column longer than expected. In contrast, higher polarity of mobile phase will decrease the retention time of the compound. For instance, retention time and separation factor will decrease if the concentration of methanol is increased. This is due to the increased amount of adsorbed organic compound will cause weakening of hydrophobic interaction between the solute and adsorbent (Ching, 2000).

Improvements could be made to the experiment to improve the results. Repeated measurements such as replicate injections of the same solution can be done to obtain accurate results.

The advantages of HPLC method in determining P is that HPLC requires a small amount of sample which does not necessary to have 100% purity. HPLC is more sensitive in detecting the signals even in a small change of concentration. It is easy to operate and is a reliable system. In contrast, it is expensive compared to TLC and ‘shake-flask' method. Only one sample can be tested at a HPLC system at a time.

Of all the methods, I think that HPLC method is the best method in determining the log P of sulphonamides. The time taken to prepare the sample for HPLC assay is usually short. HPLC can provide a large number of quantitative and qualitative results with a single analytical run (Vogeser et al, 2008).

Determination of chemical purity







Table 6: Areas obtained from HPLC for chemical purity assays

X 100%

= X 100%

= 67.9975 %

≈ 68%

C26 has a chemical purity of 68%. Since C26 is an unknown compound, there is no standard required. The chemical purity value of C26 could not be compared with any literature value due to the fact it is an unknown compound.


The partition coefficient of sulphonamides can be measured by different methods in chemistry laboratory such as TLC, ‘shake-flask' method, and HPLC. From this experiment, it can be concluded that HPLC is the best method in determining the properties of sulphonamides due to the fact that it advantages in obtaining accurate results.


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2. Donald Cairns, 2003. Essentials of Pharmaceutical Chemistry. Second Edition. Pharmaceutical Press. Chapter 2. Page 29-36.

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3. Michael Vogeser and Christoph Seger, 2008. A decade of HPLC-MS/MS in the routine laboratory - Goals for further developments. Clinical Biochemistry. 41. 649-662.

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