Amorphous Preparation Method:

1. Melting- Quenching Technique:

Melting and quenching cooling of a crystalline drug to produce an amorphous product is a relatively straightforward technique. When the molten drug is cooled below its freezing point, the molecular motion slows down. If the cooling process occurs quickly enough, the molecules do not have time to rearrange into a crystalline lattice, resulting in a more disordered state and preventing crystallization.^15,17 Conversely, if the liquid is cooled slowly, there is more time for the molecules to explore different configurations at each temperature, leading to crystallization. One drawback of this method is the potential degradation of the drug substance during the melting step due to the high temperatures involved. Therefore, this technique is only suitable for thermally stable compounds.^13,15,17

2. Freezing and drying technique (Lyophilization):

Freeze-drying is a process that removes moisture from a substance by first crystallizing the water and then sublimating the water vapor from the solid state under reduced pressure. Depending on the cooling rate, some solutes may crystallize during the freezing process. Those solutes that do not crystallize become amorphous solids when the temperature drops below the glass transition temperature (Tg) of the maximally concentrated solute. At the end of the freeze-drying process, once the solvent has been completely removed through sublimation, it is important to note that while lyophilization can be performed using organic solvents, this is generally not preferred. This is because achieving complete removal of the solvent from the system can be challenging. Therefore, the lyophilization technique is best suited for water-soluble drug substances.^14,17,25

3. Spray Drying:

The spray drying technique is effective for obtaining the amorphous form of a drug. In this process, crystalline drugs are dissolved in a suitable solvent to create a concentrated solution. This concentrated solution is then pumped to an atomizing device, where it is broken into small droplets. These droplets encounter a stream of hot air, causing them to rapidly lose their solvent while remaining suspended in dry air.^15,18When the droplets reach their solubility limit on the surface, the components of the concentrated solution may not crystallize immediately. As a result, a fully or partially amorphous solid is formed. It is important to note that drug substances have a low glass transition temperature (Tg), which increases the likelihood that the final product may still contain a crystalline portion. This method can present challenges for drug substances with low Tg, as it may not be ideal for preventing crystallization.^24-30

4. Micronisation:

Milling, also known as grinding, is generally considered a method for reducing particle size. There are various types of milling equipment available for Micronisation, which can be categorized into three main groups based on how energy is transferred to the material being processed: ball mills, shear action mills, and shock action mills. In ball mills, energy is imparted to the material through the movement of mill bodies or impellers, exposing the material to both shear and normal stresses. In contrast, shear action mills grind the material using crushing elements—solid surfaces that move relative to each other. Shock action mills, on the other hand, transfer energy directly to the milled material through the direct collision of particles. While the primary objective of milling is to reduce particle size, the process can also lead to unintended effects, such as changes in morphology, crystallinity, polymorphism, glass transition temperatures, chemical stability, and melting behavior during subsequent storage. Milling is an energy-intensive process, and the high energy input can lead to the degradation of both the milled substance and the milling equipment. Additionally, the high-energy collisions that occur during milling can damage the crystal lattice of the material, resulting in the transformation of crystalline substances into amorphous forms.^25.28

5. Dehydration of Crystalline Hydrates:

Dehydrating crystalline hydrates has been shown to be an effective and gentle method for achieving the amorphous state of organic solids. In this process, crystalline drug substances are heated to remove their hydrate or solvate content. This results in a crystalline loss and the substance's conversion into a fully or partially amorphous phase.¹⁹

Amorphous Content Determination Method:

1. X-ray Diffraction technique

X-ray diffraction (XRD) techniques are primarily used in the pharmaceutical industry to analyze powder samples and determine crystal structures. This non-destructive method is valuable for evaluating the degree of crystalline, characterizing polymorphs and solvates, and studying phase transitions. One specific application of X-ray powder diffraction (XRPD) is identifying non-crystalline solid forms, known as amorphous forms. This determination is achieved by observing the loss of distinct XRPD peaks that indicate crystalline order, and the emergence of a general halo pattern associated with amorphous structures, where no diffraction peaks are visible.³⁴By analyzing these patterns, crystalline and amorphous fractions can be estimated using several methods, depending on the sample's nature. For samples containing crystalline and amorphous fractions with different chemical compositions, the quantities of each crystalline phase can be estimated using appropriate standard substances.^{26, 27}

The amorphous fraction is then calculated indirectly through subtraction. In samples containing one crystalline phase and one amorphous phase, either in a one-phase or two-phase mixture with the same elemental composition, the amount of the crystalline phase, referred to as the "degree of crystallinity," can be estimated by measuring three areas of the diffractogram.²⁹

The formula to calculate % Crystallinity is as follows:

% Crystallinity = (100A) / (A + B - C)

Where:

A is the sum of the areas of all peaks from the crystalline diffraction of the sample.

B is the total area under the diffractogram generated by the sample (excluding area A due to the amorphous halo pattern).

C is the area of the background noise resulting from air scattering. Once these areas are measured, the degree of crystallinity can be roughly estimated,

the % Amorphous can be calculated as:

% Amorphous = 100 - % Crystallinity (by XRPD).

2. FT-Raman:

Raman spectroscopy is a spectroscopic method used to observe a system's vibrational, rotational, and other low-frequency modes. It probes the properties of the molecule itself, and changes in the solid-state properties of a substance are inferred from changes in the molecular conformation and molecular environment.³¹ This is due to the different packing conditions of the molecules in various solid forms. Differences can then be seen as subtle changes in the peak positions and intensities in the Raman spectra. So, it needs a suitably different spectra for crystalline and amorphous forms. That’s the time raman spectroscopy was used for quantifications of amorphous form.³

3. DSC & Modulated DSC:

The most common measurement of amorphous structure involves the analysis of the glass transition by DSC. It is essential to examine the size of the transition in heat flow or heat capacity units and the temperature (Tg) at which it occurs. The transition size provides quantitative information about the amount of amorphous structure in the sample, and the temperature identifies the point where there is a dramatic change in physical properties. There is limited molecular mobility below the glass transition temperature; above Tg there is high mobility, which results in much lower viscosity and potentially much greater chemical interaction between components.^11-12

The increase in heat capacity or “ΔCp” at the glass transition is a quantitative indicator of the amount of amorphous material present. For many samples, it can be difficult to detect the glass transition by DSC even when the sample has a high amorphous content, due to interferences from other transitions that occur over the same temperature range. Modulated DSC, however, separates heat flow into the Reversing and non-reversing components, which can be more easily interpreted. The Reversing Heat Flow signal, (which is the heat capacity component of the Total signal), is extremely useful for measuring glass transitions in all types of difficult samples.

An alternative method for quantifying the amorphous content involves crystallization measurement. All compounds exhibit a known heat of crystallization (ΔHc), and the value can be calculated or sometimes directly measured. The ratio of a measured ΔHc to its literature value can be used to calculate the amorphous content of the sample. An amorphous compound can be crystallized by raising the temperature above its glass transition and crystallization temperature (Tc). Because many pharmaceutical materials are unstable and undergo thermal degradation at elevated temperatures, it is desirable to induce crystallization at lower temperatures to preserve the sample's integrity in many materials; this allows the sample to crystallize at temperatures well below the thermal decomposition point.

The amount of amorphous content can be determined by measuring the ΔHc and comparing it to the literature value. However, for materials with extremely low levels of amorphous material, the heat flows associated with this crystallization process are extremely small, increasing the sensitivity of the method by altering pan size, Sample weight, and heating rate. However, a very low quantity of amorphous content (less than 1%) that time sensitivity is not achieved in differential calorimetry analysis; hence, it is detectable only by microcalorimetry.^11-15

4. Microcalorimetry:

In microcalorimetry, the relative humidity (RH) is adjusted while temperature is held constant during a measurement. Increasing the RH effectively plasticizes the material, and the Tg is reduced. The sample crystallizes once the Tg is lower than the isothermal temperature, and ΔHc can be determined. The figure below shows a micronized substance's heat flow response as the RH is increased from 0 to 80 % at a constant rate over 25 hours. Several phases appear, which agree with the known behavior of a crystalline substance with amorphous regions. Three stages are seen: absorption, crystallization, and evaporation of excess water following crystallization. A fourth event is water condensation as the sample turns to solution. Since RH changes can be made slowly and the system is open, the sample is in a state of “near-equilibrium.” When the RH range is determined, the unknown sample can be analyzed.^10,11

Figure 2. Pharmaceutical Material Response to Changes in Relative Humidity

As per literature data, lactose amorphous form converted to crystalline form in 40% RH. Hence contains the data from a lactose sample during a series of RH ramps over the range of 30-40% RH. As humidity is initially ramped, the sample undergoes two simultaneous exothermic processes, absorption and crystallization, During the subsequent ramp down from 40-30%, the absorption is the only reversible process and the heat of absorption/desorption is measured, this is confirmed in the final RH ramp from 30-40%, in which the exothermic process corresponds to only the heat of absorption. Hence the ΔHc is calculated as by first cycle heat flow subtracted from second cycle heat flow. That difference was observed in heat floe due to amorphous contented converted to crystalline form. Based on this we can measure a very low amount of amorphous content by microcalorimetry.¹¹

Figure 3. Amorphicity Determination by Microcalorimetry

5. Dynamic Vapor Sorption (DVS) Technique:

Amorphous solid sorbs relatively more water as compared with their crystalline counterpart. This is because both adsorption onto the surface, as well as absorption into the bulk, occurs in an amorphous solid. The term “sorption” is often used when both adsorption and absorption occur.³⁰Thus, in contrast to adsorption, where the amount of water taken up depends on the available surface area, uptake by amorphous solids is predominantly determined by the total mass of amorphous solids. schematic presentation of the phenomenon of adsorption (onto the surface) and absorption (into the bulk) of water vapors in crystalline and amorphous solids is shown in Figure 4 Based on this sorption principle, mainly three methods for amorphous content determination by DVS.³¹

Figure 4. Schematic presentation of the phenomenon of adsorption and absorption of water vapours in crystalline and amorphous solids

A. Equilibrium Moisture Uptake Method (Method-I):

In this method, the determination of equilibrium moisture sorbed by pure crystalline and amorphous forms, along with their physical mixtures, is performed. Water vapor sorption (g/g) is plotted versus known amorphous content at a particular RH and temperature. Excellent linearity can be observed over the broad range of percent amorphous content, and the detection limits are limited to mass differences obtained at humidities.^24,31

Figure 5. Moisture sorption profile of Amorphous Sample

B. Water Uptake Method (Method-II):

This method compares the relative mass difference at specific humidity levels before and after water vapor-induced crystallization. Assuming there is no formation of hydrates or solvates, the difference in uptake between partially amorphous and recrystallized materials is directly related to the amount of amorphous material present in the sample. The principle behind this method is that the weight gain observed in a crystalline standard is primarily due to surface adsorption, while for an amorphous standard, it results from both surface adsorption and bulk absorption. To analyze this, a moisture sorption profile for both amorphous and crystalline standards is created by incrementally raising the humidity in steps of 5% up to 95% relative humidity (RH), with equilibration at each step, as illustrated in Figure 6. This process provides insights into the difference in weight change between the amorphous and crystalline forms. At a chosen reference point within the moisture sorption profile, the mass uptake is calculated. The difference between the initial and final mass uptake at this reference point indicates the water uptake (or absorbed water) by the amorphous material, which is proportional to the amount of amorphous content in the sample. Subsequently, a calibration curve can be plotted to show the relationship between the amount of water absorbed (i.e., water uptake) at the reference point and the known amorphous content.^30,31

CONCLUSIONS:

Pharmaceutical materials range in complexity from crystalline, having long-range order in the crystal lattice, to amorphous, having short-range order. It is well established that amorphous regions can be generated in crystalline materials during the process. The level of amorphous materials can affect every step of pharmaceutical development, from formulation and processing to storage and stability. However, quantification of low levels of amorphous content still poses a considerable challenge. Amorphous solid dispersion is widely used to prepare oral poorly water-soluble drugs. This review mainly explains the preparative methods of amorphous and the characterization. Amorphous solids minimize the various disadvantages of the oral drug delivery system. Solubility enhancement of the drugs remains one of the most challenging aspects of drug development in the pharmaceutical field.

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Received on 24.02.2025 Revised on 29.03.2025

Accepted on 26.04.2025 Published on 06.05.2025

Available online from May 10, 2025

Asian Journal of Pharmaceutical Analysis. 2025; 15(2):123-130.

DOI: 10.52711/2231-5675.2025.00020

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Techniques Name	Advantages	Disadvantages	Typical Limit of Detection (LOD)
XRPD	‘Gold-standard’ for physical form characterization. Suited to powders.	Sensitive to crystalline structure, rather than the lack of it. The equipment is expensive. The detection limit is typically greater than 5% w/w.	<10%
Raman spectroscopy	Quick and use small sample mass. Does not change sample during measurement.	Need suitably different spectra for crystalline and amorphous forms. Small beam size can create sampling issues and the possibility of localized heating. The effect of particle size is not known.	<5%
Differential scanning calorimetry (DSC)	Fast and specific only a small amount of substance required.	limited to temperature-stable substances and low sensitivity	<2%
Microcalorimetry (μCal)	High sensitivity Simple sample preparation	requires suitable crystallization kinetics	<1%
DVS	Applicable to any sample if a suitable plasticizer is available. Loss in mass post-crystallization is a characteristic indicator of crystallization. Correlates to stability testing.	Difficult to compensate for sample wetting. Small sample masses are needed to ensure exposure to plasticizers. Experimental run-time can be long.	<1%
Solution calorimetry (SolCal)	high sensitivity	requires rapid dissolution	<1%