How To Calculate Solubility BY HPLC: Learn Easily in 11 Minutes

Solubility is the ability of a substance (called a solute) to form a solution with another substance (called a solvent). It is the unique property of any pharmaceutical, which is why it plays a vital role in bioequivalence, chemical research, analytical research, and quality control in managing quality, safety, and efficacy. In this article, you will learn about solubility, its classification, the solubility principle, solubility calculation by HPLC, solubility curve, different factors affecting solubility, solvent selection for pharmaceutical solubility, applications, and different solubility terms with case studies and FAQs.

Solubility

Solubility is the ability of a substance (called a solute) to form a solution with another substance (called a solvent).

Example: The solubility of glucose in water at 20°C is 2000 grams per litre, meaning at most 2000 grams of sugar can dissolve in one litre of water at 20°C.

Solubility Classification as per USP: Very soluble, Freely soluble, Soluble, Sparingly soluble, Slightly soluble, Very slightly soluble, Practically insoluble

USP classifies solubility without considering the solvent used. The following are the different solubility classifications as per USP:

*Note: The needed amount of the solvent (in ml) per gram of the dissolved substance

You may like: How to Calculate Relative Response Factor (RRF)?

How is solubility classified?

The following steps are used to confirm the solubility class:

What are the units of Solubility?

Generally, mg/ml or mcg/ml or ppm is used as a Unit of Solubility in the pharmaceutical industries. But other unit can also be considered

What is the Solubility Principle?

The Solubility Principle states that “like dissolves like.” Solvents that have similar polarity or charge to the solute tend to dissolve it more readily. It refers to a set of guidelines that help predict whether a given ionic compound will dissolve in water to form a solution. The rules are based on the general behaviour of different types of ions and the nature of their interactions with water molecules.

Note: Solubility can be influenced by factors like temperature, pressure, and other ions in the solution.

How to choose the solvent/solvents for the solubility of a given compound?

The following 11-step components are widely used while selecting solvents for pharmaceutical solubility:

1. Polarity/Like dissolves like: Solvents with similar polarity to the pharmaceutical compound will likely dissolve it better. If the compound is nonpolar (hydrophobic), you’ll typically use nonpolar solvents like hexane, acetone, chloroform or oil. You may use polar solvents like water, aqueous buffers, ethanol, or methanol if the compound is polar (hydrophilic).
2. Solubility Profile of the Drug: Review any data available (such as literature report or published data) on the compound’s solubility profile.
3. Polarity Index (Solvent Strength): The polarity index gives an idea of the solvent’s ability to dissolve different types of compounds. For example, water (polarity index = 9.0) is highly polar, while toluene (polarity index = 2.4) is less polar.
4. Viscosity: Try to use solvents with lower viscosity for faster dissolution
5. Safety and Environmental Impact: Do not use toxic, harmful or class-I solvents such as Carbon tetrachloride and Benzene. Always pick solvents that align with safety/regulatory guidelines.
6. Boiling Point and Volatility: Use a solvent with a high boiling point if you are conducting solubility tests at high temperatures. Try to avoid low-volatile solvents since errors may arise solubility testing due to evaporation.
7. Miscibility and Solvent Mixtures: Some pharmaceuticals are more soluble in a mixture of solvents than in a single solvent. For example, if a pharmaceutical is poorly soluble in water, it might dissolve better in a water-alcohol mixture or in a mix of polar and non-polar solvents.
8. pH Sensitivity: Some pharmaceuticals have pH-dependent solubility. Therefore, you may need to test in buffers at various pH levels, or use acidic, neutral, or basic solvents to assess solubility in different conditions.
9. Pharmaceutical Formulation Considerations: For dosage forms (e.g., oral tablets, injections) solvent should be decided considering the excipients or the formulation medium (e.g., PEG (Polyethylene glycol), DMSO, or surfactants, etc.).
10. Experimental Design/Solubility Testing Conditions: Consider the temperature, agitation, and the presence of other solutes (such as salts or surfactants) in the system. Some pharmaceuticals may only dissolve at elevated temperatures or with stirring.

How to Calculate Solubility By HPLC?

HPLC is a useful tool for accurately determining the solubility of a pharmaceutical or an API, particularly when you want to measure how well a substance dissolves in a given solvent at a particular temperature. Solubility can be calculated as one of the following methods:

1. Direct Method
2. Calibration Method

Direct Standard Method

Chemicals and Equipment Needed:

• HPLC with UV detector
• Magnetic stirrer
• APIs (Active Pharmaceutical Ingredients) Standard (with Certificate of Analysis)
• API
• Volumetric flask of 100 ml capacity
• Balance
• HPLC method

1. Standard Preparation (100 mcg/ml standard solutions) for Solubility Calculation by Direct Method
   • Weigh about 10mg of ana API accurately and transfer in a 100 ml volumetric flask. Add about 20 ml of diluent and shake to dissolve. Make up the volume up to mark.
     • Note: Standard diluent may not be the same as sample solvent. The standard must be soluble in that solvent.
2. Dissolving solvent for Sample: Water or buffer (as per experiment need)
3. Number of Replicates: N = 3
4. Equilibration Time: 24 hours
5. Solubility Experiment Set up or Sample Preparation
6. Weigh about 600mg of the concerned API and transfer to a 100 ml volumetric flask. Make up the volume up to mark with the solvent. Stir the solution for 24 hours at 25⁰C (or as per experiment) using a magnetic bead and magnetic stirrer.
   • Note: The Sample preparation procedure can be modified as per requirement
7. Sample Filtration
   • Filter the solution: Filter the sample solution through a fine filter (0.45 μm or finer) before injecting it into the HPLC system.
8. HPLC Method
   • Use the existing assay or impurity profile method, chromatographic condition for solubility determination. If require new method can be developed.
9. Procedure
   • Inject system suitability solution, blank, standard solution and sample solution and record the chromatogram. Calculate the solubility of the API (in mg/ml) by the following Formula:

Solubility of API by HPLC using Standard Calibration Method

• Prepare at least five standard solutions from two different stock solutions 10mcg/ml to 150mcg/ml
• Generate the chromatograms for each standard concentration for the main analyte using the test method
• Calculate slope (m), intercept (c) and correlation coefficient (R²) using MS-Excel.
• Calculate concentration in the sample solution using the following formula :

y= mx + c and hence x = (y-c)/m

where: m is slope, x is the API sample concentration (to be calculated), c is the intercept, and y is the area response

Example: Suppose the slope (m) is 30914.1 and the intercept (c) is 1.4 ( Calculated from the linearity curve). API peak response in three different replicates of sample solutions (in water) are 6182800, 6182100 and 6189000.

Therefore water solubility of the above API is 0.2003/mg/ml (average of the above 3 results)

What are the common Solvents for Pharmaceutical Solubility Testing?

The following solvents are widely used in pharmaceutical solubility testing:

1. Water: It is a common choice for all types of pharmaceuticals, but is widely used for hydrophilic drugs.
2. Methanol, Ethanol or Isopropyl alcohol (IPA): Used for both hydrophilic and lipophilic compounds.
3. Acetone: A good solvent for non-polar and slightly polar compounds.
4. Dimethyl Sulfoxide (DMSO) or Dimethyl Formamide (DMF) : Often used to dissolve a wide range of pharmaceuticals, especially those with low solubility in water.
5. Chloroform: Used for lipophilic compounds.
6. Toluene: Another option for lipophilic drugs.
7. n-Hexane: A natural choice, especially for aliphatic hydrocarbon.

Note:

1. Always consider the stability of the pharmaceutical in the solvent. Some solvents might degrade or cause the drug to break down.
2. Use the QBD (Quality by design) approach: Test multiple solvents in a stepwise approach, starting with water, then progressing through common solvents (e.g., alcohols, acetone), and perhaps even mixed solvents, to identify the best one for the solubility of the compound.

What Are The-Factors That Affect Solubility?

The following factors affect the solubility:

1. Effect of Temperature
2. Effect of Pressure
3. Effect of pH
4. Effect of salts and counter-ions
5. Effect of co-solvents
6. Effect of surfactants
7. Effect of complexing agents
8. Effect of surface area (Dissolution rate)
9. Effect of surface energy (Nanoparticles)
10. Concentration of the solute
11. The polarity of the solute and the solvent

What is the Solubility Curve?

A solubility curve is a graph that shows the relationship between the solubility of a substance/pharmaceutical and temperature. It indicates how much of the substance can dissolve in a solvent at various temperatures, expressed as the amount of solute in a certain amount of solvent (usually grams of solute per 100 grams of solvent). Solubility curves help in predicting how much solute will dissolve at a particular temperature and are useful in processes like crystallisation or formulating solutions in chemistry.

1. X-axis: The temperature of the solvent
2. Y-axis: The solubility of the solute, often in grams of solute per 100 grams of solvent.

The curve typically rises for most solids, meaning that as temperature increases, the solubility of the solid in the solvent increases. However, the shape of the curve can vary depending on the solute. For some substances, the solubility may decrease with increasing temperature.

Key points about solubility curves:

• Saturation: If you add more solute than the solubility curve indicates at a given temperature, the extra solute will not dissolve and will settle at the bottom of the container.
• Supersaturation: If a solution is cooled slowly without disturbance, it can sometimes hold more solute than the solubility curve suggests, creating a supersaturated solution.

What are the factors affecting the Solubility of Gases?

The solubility of gases refers to how much of a gas can dissolve in a liquid at a given temperature and pressure. It is governed by the following factors:

1. Nature of the Gas:
   • Polarity: Polar gases, such as ammonia (NH₃) and carbon dioxide (CO₂), are generally more soluble in water because water itself is polar and can form hydrogen bonds with these gases.
   • Size and Intermolecular Forces: Larger, non-polar gases like oxygen (O₂) or nitrogen (N₂) are less soluble in water because they do not interact as strongly with water molecules.
2. Temperature:
   • The solubility of gases in liquids usually decreases with increasing temperature. As the temperature rises, the kinetic energy of the gas molecules increases, making them more likely to escape from the liquid phase back into the gas phase.
3. Pressure:
   • According to Henry’s Law, the solubility of a gas in a liquid is directly proportional to the partial pressure of that gas above the liquid, provided the temperature is constant. This means that increasing the pressure of a gas above a liquid will increase the gas’s solubility.
     • C = k_P x P
       • Where: C is the concentration of the gas dissolved in the liquid. k_P is the Henry’s law constant (specific to each gas-solvent pair) and P is the partial pressure of the gas.
4. Nature of the Solvent:
   • Water is a common solvent for gases, but the solubility of gases will vary in different solvents. For example, gases tend to dissolve better in solvents that have similar polarity or are able to interact through hydrogen bonding.
5. Presence of Other Substances:
   • The presence of salts, alcohols, or other substances in a solution can sometimes change the solubility of a gas. For example, adding salt to water can reduce the solubility of gases, a phenomenon known as the salting-out effect.

Examples of Gas Solubility:

• Carbon dioxide (CO₂): CO₂ is highly soluble in water, and this solubility increases with pressure. It’s responsible for the effervescence in carbonated beverages.
• Oxygen (O₂): The solubility of oxygen in water is much lower compared to CO₂, which is why aquatic organisms rely on gills for oxygen extraction from water.

What is the difference between solubilisation and solubility?

Solubility is a property of a substance, indicating how much of it can dissolve in a solvent under specific conditions. Solubilization is a process that improves the solubility of a substance, often by adding agents like surfactants or using certain techniques.

Solubility is about how much of a substance can dissolve, and insolubility is the opposite, where a substance does not dissolve or dissolves only minimally. The following are the few examples of solubility and insolubility:

• Oil is insoluble in water because oil molecules are nonpolar, while water molecules are polar.
• Table salt (NaCl) dissolves in water. It has high solubility in water

Solubility examples

Solubility of Methane, Ethane and Propane in Water

Methane, ethane, and propane are non-polar molecules, thus, they are not soluble in water.

BSA Solubility in water

The solubility of BSA is 40mg/ml in water

Dibasic calcium phosphate Solubility in water

Dibasic calcium phosphate is very slightly soluble in boiling water and practically insoluble in ethanol.

What are the advantages of solubility pharmaceutical development ?

Solubility has many advantages in pharmaceutical development, such as:

• Formulation development
• Analytical research
• Chemical research
• Bioavailability

What are the different applications of Solubility?

Solubility plays a vital role in the following industries:

1. Pharmaceuticals and Drug Formulation
   • Pharmaceuticals Development/Synthetic Research: Solubility is crucial for designing effective medications. Drugs must dissolve in the body to be absorbed into the bloodstream. Poor solubility can lead to reduced bioavailability and therapeutic efficacy. Researchers focus on improving solubility to create more effective oral formulations.
   • Solubility-Enhanced Formulations: Techniques like solid dispersions or the use of surfactants are employed to increase the solubility of poorly water-soluble drugs.
2. Analytical Research:
   • Solubility plays a vital role in traditional analysis such as titration, spectroscopic analysis and chromatographic analysis such as HPLC and GC.
   • For example, in acid-base and redox titrations, the solubility of the substances involved can affect the accuracy of measurements. HPLC: The separation of compounds in HPLC depends on the solubility of substances in diluents.
3. Food and Beverage Industry:
   • Food Processing: Solubility plays a role in the formulation of beverages, sauces, and emulsions. Ingredients like sugars, salts, and flavourings must dissolve properly to achieve the desired taste and texture.
   • Preservatives: Solubility influences the use of preservatives in food. Some preservatives must dissolve in water to be effective in inhibiting microbial growth.
4. Cosmetics and Personal Care Products:
   • Formulation of Creams and Lotions: Solubility is important in designing products where active ingredients (such as vitamins or oils) must dissolve to penetrate the skin.
   • Fragrance: The solubility of aromatic compounds in solvents determines how fragrances are formulated and applied in perfumes, deodorants, and shampoos.
5. Biology and Medicine:
   • Blood Circulation: The solubility of gases, like oxygen and carbon dioxide, in blood is essential for respiration. Gases must dissolve in the blood plasma to be transported to tissues.
   • Enzyme Reactions: Enzymes often catalyse reactions in solutions, and the solubility of substrates and products affects the rate of the reaction.
6. Agriculture:
   • Pesticides and Fertilisers: Solubility affects how pesticides or fertilisers dissolve in water, impacting their absorption by plants and their effectiveness.
   • Soil Remediation: Solubility is important in the dissolution and removal of soil contaminants or in the formulation of soil amendments to optimise plant growth.
7. Environmental Science:
   • Pollution Control: Understanding the solubility of pollutants helps in managing environmental contamination. For example, the solubility of oils or heavy metals in water can determine their spread and toxicity in aquatic ecosystems.
   • Water Treatment: Solubility is a key factor in the treatment of wastewater. Chemicals are often added to precipitate contaminants from water, based on their solubility.
8. Chemical Engineering and Manufacturing:
   • Crystallisation: In processes like the production of sugar, salt, and other chemicals, controlling solubility is essential for crystallisation. Solubility curves help determine the conditions under which a substance will crystallise from a solution.
   • Solvent Selection: In chemical manufacturing, choosing the right solvent for dissolving reactants or controlling the solubility of a product can impact reaction rates and yield.

Solubility Related Terms: Solute, solvent and Solution

Solute:

• The solute is the substance that is dissolved in a solvent.
• It is typically present in a smaller amount in the solution.
• For example, in a saltwater solution, salt is the solute.

Solvent:

• The solvent is the substance that does the dissolving.
• It is usually present in a larger quantity in the solution.
• In the case of saltwater, water is the solvent because it dissolves the salt.

Solution:

• A solution is a homogeneous mixture formed when a solute dissolves in a solvent.
• It has the same composition throughout.
• In the case of saltwater, the mixture of salt (solute) and water (solvent) forms the solution.

What is the Common Ion Effect and its role in solubility?

The common ion effect refers to the shift in equilibrium that occurs when an ion that is already present in the solution is added again. This effect is based on Le Chatelier’s principle, which states that if a system at equilibrium is disturbed, it will adjust to counteract the disturbance and restore equilibrium.

In the context of the common ion effect, when a salt that shares a common ion with a substance already in solution is added, the concentration of that ion increases. This causes the equilibrium to shift in a way that reduces the concentration of that ion, often by precipitating out or reducing the dissociation of one of the compounds.

Example: Consider the dissociation of acetic acid (CH₃COOH) in water:

CH₃COOH(aq)⇌CH₃COO⁻(aq)+H⁺

If you add sodium acetate (CH₃COONa), which contains the acetate ion (CH₃COO⁻), the increased concentration of CH₃COO⁻ will shift the equilibrium to the left (according to Le Chatelier’s principle), reducing the dissociation of acetic acid. As a result, fewer H⁺ ions are produced, making the solution less acidic.

What is the Solubility Product?

The solubility product is an equilibrium constant that describes the solubility of a sparingly soluble ionic compound in water. It represents the product of the concentrations of the ions involved in the dissolution of the compound, each raised to the power of its stoichiometric coefficient in the balanced dissolution equation.
Let us consider an ionic compound AB dissolves in water to produce its ions A+ and B-
AB(s) = A⁺(aq) + B^–(aq)
Then the Solubility Product can be written as:
KSp = [A⁺][B^–]

Conclusion

Solubility plays a vital role at each step during pharmaceutical development. I hope this article has helped you understand Solubility and its importance. You may also want to check out other articles on my blog, such as

Related:

• Biopharmaceutics Classification System (BCS)

FAQs: Interview Questions On Solubility

What is the water solubility?

Water solubility refers to the ability of a substance to dissolve in water to form a homogeneous solution. It is usually expressed as the maximum amount of a substance that can dissolve in a specific amount of water at a given temperature, often in units like grams per liter (g/L) or moles per liter (mol/L) or ppm.

For example:

• High water solubility: Sodium chloride (table salt) dissolves easily in water.
• Low water solubility: Oil does not dissolve in water.

Further Reading

• Reference Tables: Description and Solubility