Human Resources Strategies Essay Example | Topics and Well Written Essays - 1250 words

Human Resources Strategies - Essay Example Job applicants who take on-site interviews without seriously considering the prospective employer emerge to be ethically incorrect. Such candidates in most cases do not attend the interviews or written test as it is a requirement of any organization before employment (Kleiman, 2009). Ethical job applicants are prepared at all times and when unable to attend the interviews or written tests due to an important reason, they always inform their employer well in advance and ask him or her to make another arrangement if possible. Unethical job applicants who only take the on-site interviews knowing very well that they are not considering the organization a potential employer makes the organization to lose both financially and man hours in preparing interviews and written test. Job applicants who engage in unethical behaviors pose a great negative impact to the production of the organization. Such behaviors as laziness hastiness, being late for work and leaving early or not taking responsibilities of the new job seriously gives a strong negative impression to the employer. Candidates of this kind are less productive and do not perform to the standards expected by the employer. As such a conflict is likely to arise between the employer and the employee. This can often lead to organization failure in achieving its goals (Kleiman, 2009). There are job applicants who engage in unethical behavior such as bribery in order to get employed. Job recruiters when confronted by such situations may fail to make rational decision and avoid errors and this ending up in an unfair recruitment. This significantly lowers the reputation and tarnishes the name of the organization. Unethical job applicants may lead to hiring of employees and over staffing due to overoptimistic p rojections. When this projection are not met, unpleasant measures by the organization are employed in order to please the stakeholders and keep in pace with the competitors. These are measures in an attempt to cutback, restructure and downsize the organization are undertaken (Kleiman, 2009). Such an undertaking harms the future recruitment efforts of the organization, as potential employees will develop a perception of job insecurity in the particular organization. Furthermore, over staffing directly leads to underutilization of the talented recruits that leads to dissatisfaction. As such the corporate loyalty of the organization is impacted negatively. Each member of the human resource is therefore faced with the challenge of developing convincing ideas and priorities in trying to come up with the solutions to the problem. Furthermore, unethical job applicants leads to a decrease in morale among the workers in an organization. The middle class managers and those who are actually in volve in bringing the positive change to the organization become pessimistic about the possibility of sustained future change. This way, they start questioning their ability to make reasonable profit to the organization. Such a situation calls for the each member of the human resource to see into ways of totally transforming the organization. The corporate culture, knowledge base, skills level, style of leadership and team orientation would have to undergo change, for all the employees. The human each member of th

Summative Assessment In Education Research Paper

Summative Assessment In Education - Research Paper Example Why is it important to use authentic materials when teaching the English language? (3 marks) 2. Name six of the materials that can be used to supplement a course book when teaching English. (3 marks) 3. Discuss four methods which can be used to make a lesson more lively (4 marks) 4. Mention five advantages of using a course book while teaching (5 Marks) 5. Mention five best practices associated with the use of a course book (5 Marks) 6. Discuss the four options for use of a course book (8 marks) 7. Give four reasons why it is important to plan a lesson (2 marks) Section B: Lesson plan (30 marks) 1. With the news article and blank lesson plan provided, devise a lesson plan that can be used to teach a certain aspect of grammar. You are free to choose any aspect of your interest, but the lesson plan should include all the stages. Make sure all spaces are filled. Teacher: Room: Observer: Expected numbers: Date & Time: Class Level: Context: Teaching Aids: Learner Objectives: Personal Aims : Anticipated problems for students: Solution: Anticipated problems for teacher: Solution: Procedure Phase Timing Interaction (â€Å"Online TESOL / Online TEFL ITTT Answers†, 2009) Section C: Essay questions (40 marks) Write an essay of about 500 words to discuss one of the following concepts 1. Use a course book in lesson planning to maximize effects. 2. An ideal lesson plan. 3. Benefits of using a lesson plan. Note: your essay must contain a brief introduction and a conclusion. An assessment Triangle and cognitive Learning model In creating my summative assessment, I relied on the concept of the assessment triangle and the cognitive learning model. According to Selah & Khine (2011), the mentioned can provide a guide which helps, when an assessment instrument to support reasoning from evidence is designed, which is my objective. At this point, I feel that it is necessary to apply the concept of an assessment triangle where I considered all its elements. One of the elements o f an assessment triangle that I critically analyzed is cognition. Cognition refers to the theory, data, and a set of assumptions about how student present their knowledge and develop competency in a subject matter domain (Bernholt, 2012). Using the concept I try to align my summative assessment in the best possible way to make it test the level of competency my students have developed during the learning process. To understand this cognitive behavior fully, I decided to use the cognitive learning model. According to Busemeyer & Diederich (2010), this model has a goal of scientifically explaining processes used by the human brain to accomplish complex tasks. Using this model, I was able to understand the cognitive behavior of my students. This has helped me a lot in designing this summative assessment by showing me how the students behaviors interact with the goals we want to achieve through this summative assessments. Another element of as assessment triangle that I have considered is the beliefs element.

Reflective Thinking. Easy Jet on airline markets. PESTEL And SWOT Essay

Reflective Thinking. Easy Jet on airline markets. PESTEL And SWOT Analysis - Essay Example This step involves revisiting experiences and knowledge with respect to the topic in question and why and how one performs the task. It involves examination of values, beliefs, assumptions and attitudes. Reflective thinking helps clarify and recognise the connection between what one knows and learns (TLC, 2008). Figure 1: The Thinking Process (Source: TLC, 2008) The reflective report prepared aims to demonstrate my knowledge derived from the projects on Easy Jet and various sections like business environment, PESTEL, Porters Five Forces and other relevant theories which were used in the project. Findings from the Report Based on the project made on Easy Jet, I have found that Easy Jet is regarded as a low-cost operator and is performing well in Europe. It operates internationally and also in a domestic airline market. It aims to becomes the best and the cheapest airline operator in the world. The airline faces tough challenges from its competitors such as Ryanair, British Airlines an d others. One great impact which I have noticed is that with the coming up of low-cost airlines, travellers prefer to travel by air rather than by bus, train or other means of transport. However, it should also be noted that along with low prices the airline should also be able to provide a level of comfort for the passengers so that the passengers feel comfortable while travelling, and I think the airline will be able to retain its customers in a positive way. I have analysed the business environment with the help of tools like PESTEL analysis, SWOT Analysis, Porters Five Forces along with operational management, supply chain and micro economic factors. In the process of using Porter’s Five Forces – a tool taking into consideration five important forces and helping determine the level of attractiveness in a particular industry – it was learned that the airline industry cannot be considered to be attractive because of the many unfavourable economic conditions pr evailing in the airline industry. The cost of entering is high and maintenance of airline is also a costly affair (on the other hand, it reduces the threat from new entrants in the airline industry). The bargaining power of buyers has the potential to result in a threat because of the competitive price offered by other airlines. It has also been seen that a threat of substitute is pretty high, as with the existence of other transport means such as buses, trains, cars, etc, passengers prefer to travel rather than to spend almost the same or a slightly higher amount on more expensive means of transport. Reflective Statement While conducting the analysis, I have learned that the above tools form an integral part of any analysis and have helped me understand the impact of various factors on the organisation. The first framework which I have used to analyse the airline industry is PESTEL analysis; this is because PESTEL takes into account the industry as a whole and analyzes political fa ctors which might impact the industry, as well as economic, social, technological, environmental and, lastly, legal factors. Why PESTEL Analysis? PESTEL gives an overview of the industry and is necessary to conduct, as it helps organisation to take the advantage of the opportunities; it also has the potential to minimise the threats. In the process of conducting the project, I have understood that PESTEL analysis can be carried out in relation to any form of business and industry, and it is one of the most important tools through which the companies evaluate the external environment and make necessary changes to fit the competitive environment and to survive in it.

Summative Assessment In Education Research Paper

Summative Assessment In Education - Research Paper Example Why is it important to use authentic materials when teaching the English language? (3 marks) 2. Name six of the materials that can be used to supplement a course book when teaching English. (3 marks) 3. Discuss four methods which can be used to make a lesson more lively (4 marks) 4. Mention five advantages of using a course book while teaching (5 Marks) 5. Mention five best practices associated with the use of a course book (5 Marks) 6. Discuss the four options for use of a course book (8 marks) 7. Give four reasons why it is important to plan a lesson (2 marks) Section B: Lesson plan (30 marks) 1. With the news article and blank lesson plan provided, devise a lesson plan that can be used to teach a certain aspect of grammar. You are free to choose any aspect of your interest, but the lesson plan should include all the stages. Make sure all spaces are filled. Teacher: Room: Observer: Expected numbers: Date & Time: Class Level: Context: Teaching Aids: Learner Objectives: Personal Aims : Anticipated problems for students: Solution: Anticipated problems for teacher: Solution: Procedure Phase Timing Interaction (â€Å"Online TESOL / Online TEFL ITTT Answers†, 2009) Section C: Essay questions (40 marks) Write an essay of about 500 words to discuss one of the following concepts 1. Use a course book in lesson planning to maximize effects. 2. An ideal lesson plan. 3. Benefits of using a lesson plan. Note: your essay must contain a brief introduction and a conclusion. An assessment Triangle and cognitive Learning model In creating my summative assessment, I relied on the concept of the assessment triangle and the cognitive learning model. According to Selah & Khine (2011), the mentioned can provide a guide which helps, when an assessment instrument to support reasoning from evidence is designed, which is my objective. At this point, I feel that it is necessary to apply the concept of an assessment triangle where I considered all its elements. One of the elements o f an assessment triangle that I critically analyzed is cognition. Cognition refers to the theory, data, and a set of assumptions about how student present their knowledge and develop competency in a subject matter domain (Bernholt, 2012). Using the concept I try to align my summative assessment in the best possible way to make it test the level of competency my students have developed during the learning process. To understand this cognitive behavior fully, I decided to use the cognitive learning model. According to Busemeyer & Diederich (2010), this model has a goal of scientifically explaining processes used by the human brain to accomplish complex tasks. Using this model, I was able to understand the cognitive behavior of my students. This has helped me a lot in designing this summative assessment by showing me how the students behaviors interact with the goals we want to achieve through this summative assessments. Another element of as assessment triangle that I have considered is the beliefs element.

Forever Free

Forever Free Exhibit Essay Forever Free, an exhibit held at the Reagan Library in Simi Valley, California and inspired by Mr. Abraham Lincoln’s promotion and assertion of Emancipation Proclamation, is much related to the written account of Frederick Douglass’ â€Å"narrative of his life as an American black slave† with special regards and attention to its both focus and objectives. Body At the Forever Free exhibit, the implicit objective was to stimulate ambiance of America’s bludgeoning, sadistic, and historical past. For the visitors to commemorate the beginning of the â€Å"anti-black slavery,† Mr. Abraham Lincoln distinguished and proclaimed the pressing need for the aforementioned act, Emancipation Proclamation to denounce and suppress the proliferating Black slavery. Moreover, the exhibit included several sentimental stuffs like: letters made and signed by Mr. Abraham, and the contents of his proclaimed act, the handkerchief signed and belonging to Lincoln’s wife, Ms. Mary Lincoln, and some photographs taken depicting the long time slavery in America. Inspired by Lincoln’s provision of welfare among the Blacks, the theme of the exhibit was carried out successfully (Giller, 2008). On the other hand, the â€Å"Narrative of the Life of Frederick Douglass, An American Slave,† tackled the same insatiable thirst for freedom against the injustice, discrimination, and evil acts suffered by the Black Americans (Douglass, 1997). Conclusion The Forever Free exhibit rendering commemoration to Abraham Lincoln’s pursuit for the liberation of the slaves (Giller, 2008), together with the striking and overwhelming account of Frederick Douglass’ personal traumatic experience of the unjust slavery, both presents the truth of the need for liberation, love, and respect for all human race as everyone is a son of one Divine (Douglass, 1997). References Douglass, F. (1997). Narrative of the life of Frederick Douglass, an American slave. Retrieved October 31, 2008, from, http://sunsite. berkeley. edu/Literature/Douglass/Autobiography/01. html Giller, M. (2008). Forever free-Abraham Lincoln and the emancipation proclamation. Retrieved October 31, 2008, from http://www. reaganlibrary. com/pressrelease. asp? press_id=135

Physico-chemical Processes that Occur During Freezing

Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio Physico-chemical Processes that Occur During Freezing Physico-chemical Processes that Occur During Freezing 1. Introduction Lyophilization respectively freeze-drying is an important and well-established process to improve the long-term stability of labile drugs, especially therapeutic proteins.[1] About 50% of the currently marketed biopharmaceuticals are lyophilized, representing the most common formulation strategy.[2] In the freeze-dried solid state chemical or physical degradation reactions are inhibited or sufficiently decelerated, resulting in an improved long-term stability.[3] Besides the advantage of better stability, lyophilized formulations also provide easy handling during shipping and storage. [1] A traditional lyophilization cycle consists of three steps; freezing, primary drying and secondary drying.[1] During the freezing step, the liquid formulation is cooled until ice starts to nucleate, which is followed by ice growth, resulting in a separation of most of the water into ice crystals from a matrix of glassy and/or crystalline solutes.[4-5] During primary drying, the crystalline ice formed during freezing is removed by sublimation. Therefore, the chamber pressure is reduced well below the vapor pressure of ice and the shelf temperature is raised to supply the heat removed by ice sublimation.[6] At the completion of primary drying, the product can still contain approximately 15% to 20% of unfrozen water, which is desorbed during the secondary drying stage, usually at elevated temperature and low pressure, to finally achieve the desired low moisture content.[7] In general, lyophilization is a very time- and energy-intensive drying process.[8]   Typically, freezing is over within a few hours while drying often requires days. Within the drying phase, secondary drying is short (~hours) compared to primary drying (~days).[1, 4] Therefore, lyophilization cycle development has typically focused on optimizing the primary drying step, i.e., shortening the primary drying time by adjusting the shelf temperature and/or chamber pressure without influencing product quality.[5, 9] Although, freezing is one of the most critical stages during lyophilization, the importance of the freezing process has rather been neglected in the past.[10]   The freezing step is of paramount importance. At first, freezing itself is the major desiccation step in lyophilization [6] as solvent water is removed from the liquid formulation in the form of a pure solid ice phase, leading to a dramatic concentration of the solutes.[11-12] Moreover, the kinetics of ice nucleation and crystal growth determine the physical state and morphology of the frozen cake and consequently the final properties of the freeze-dried product.[11-13] Ice morphology is directly correlated with the rate of sublimation in primary and secondary drying.[14] In addition, freezing is a critical step with regard to the biological activity and stability of the active pharmaceutical ingredients (API), especially pharmaceutical proteins.[1] While simple in concept, the freezing process is presumably the most complex but also the most important step in the lyophilization process.[10] To meet this challenge, a thorough understanding of the physico-chemical processes, which occur during freezing, is required. Moreover, in order to optimize the freeze drying process and product quality, it is vital to control the freezing step, which is challenging because of the random nature of ice nucleation. However, several approaches have been developed to trigger ice nucleation during freezing. The purpose of this review is to provide the reader with an awareness of the importance but also complexity of the physico-chemical processes that occur during freezing. In addition, currently available freezing techniques are summarized and an attempt is made to address the consequences of the freezing procedure on process performance and product quality. A special focus is set on the critical factors that influence protein stability. Understanding and controlling the freezing step in lyophilization will lead to optimized, more efficient lyophilization cycles and products with an improved stability. 2. Physico-chemical fundamentals of freezing The freezing process first involves the cooling of the solution until ice nucleation occurs. Then ice crystals begin to grow at a certain rate, resulting in freeze concentration of the solution, a process that can result in both crystalline and amorphous solids, or in mixtures.[11] In general, freezing is defined as the process of ice crystallization from supercooled water.[15] The following section summarizes the physico-chemical fundamentals of freezing. At first, the distinction between cooling rate and freezing rate should be emphasized. The cooling rate is defined as the rate at which a solution is cooled, whereas the freezing rate is referred to as the rate of postnucleation ice crystal growth, which is largely determined by the amount of supercooling prior to nucleation.[16-17] Thus, the freezing rate of a formulation is not necessarily related to its cooling rate.[18] 2.1 Freezing phenomena: supercooling, ice nucleation and ice crystal formation In order to review the physico-chemical processes that occur during freezing of pure water, the relationship between time and temperature during freezing is displayed in figure 1. When pure water is cooled at atmospheric pressure, it does not freeze spontaneously at its equilibrium freezing point (0 °C).[19] This retention of the liquid state below the equilibrium freezing point of the solution is termed as â€Å"supercooling†.[19] Supercooling (represented by line A) always occurs during freezing and is often in the range of 10 to 15 °C or more.[12, 18] The degree of supercooling is defined as the difference between the equilibrium ice formation temperature and the actual temperature at which ice crystals first form and depends on the solution properties and process conditions.[1, 6, 11, 20] As discussed later, it is necessary to distinguish between â€Å"global supercooling†, in which the entire liquid volume exhibits a similar level of supercooling, and â€Å"lo cal supercooling†, in which only a small volume of the liquid is supercooled.[14] Supercooling is a non-equilibrium, meta-stable state, which is similar to an activation energy necessary for the nucleation process.[21] Due to density fluctuations from Brownian motion in the supercooled liquid water, water molecules form clusters with relatively long-living hydrogen bonds [22] almost with the same molecular arrangement as in ice crystals.[11, 15] As this process is energetically unfavorable, these clusters break up rapidly.[15] The probability for these nuclei to grow in both number and size is more pronounced at lowered temperature.[15] Once the critical mass of nuclei is reached, ice crystallization occurs rapidly in the entire system (point B).[15, 21-22]   The limiting nucleation temperature of water appears to be at about -40 °C, referred to as the â€Å"homogeneous nucleation temperature†, at which the pure water sample will contain at least one spontaneously f ormed active water nucleus, capable of initiating ice crystal growth.[11] However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is â€Å"heterogeneous nucleation†, meaning that ice-like clusters are formed via adsorption of layers of water on â€Å"foreign impurities†.[6, 11] Such â€Å"foreign impurities† may be the surface of the container, particulate contaminants present in the water, or even sites on large molecules such as proteins.[23-24] Primary nucleation is defined as the initial, heterogeneous ice nucleation event and it is rapidly followed by secondary nucleation, which moves with a front velocity on the order of mm/s through the solution. [14, 25] Often secondary nucleation is simply referred to as ice crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.[14] Once stable ice crystals are formed, ice crystal growth proceeds by the addition of molecules to the interface.[22] However, only a fraction of the freezable water freezes immediately, as the supercooled water can absorb only 15cal/g of the 79cal/g of heat given off by the exothermic ice formation.[12, 22] Therefore, once crystallization begins, the product temperature rises rapidly to near the equilibrium freezing point.[12, 26] After the initial ice network has formed (point C), additional heat is removed from the solution by further cooling and the remaining water freezes when the previously formed ice crystals grow.[12] The ice crystal growth is controlled by the latent heat release and the cooling rate, to which the sample is exposed to.[22] The freezing time is defined as the time from the completed ice nucleation to the removal of latent heat (from point C to point D). The temperature drops when the freezing of the sample is completed (point E).[21] The number of ice nuclei formed, the rate of ice growth and thus the ice crystals` size depend on the degree of supercooling.[14, 20] The higher the degree of supercooling, the higher is the nucleation rate and the faster is the effective rate of freezing, resulting in a high number of small ice crystals. In contrast, at a low degree of supercooling, one observes a low number of large ice crystals.[14, 19] The rate of ice crystal growth can be expressed as a function of the degree of supercooling.[23]   For example for water for injection, showing a degree of supercooling of 10 °C +/- 3 °C, an ice crystal growth rate of about   5.2cm/s results.[23] In general, a slower cooling rate leads to a faster freezing rate and vice versa. Thus, in case of cooling rate versus freezing rate it has to be kept in mind â€Å"slow is fast and fast is slow†. Nevertheless, one has to distinguish between the two basic freezing mechanisms. When global supercooling occurs, which is typically the case for shelf-ramped freezing, the entire liquid volume achieves a similar level of supercooling and solidification progresses through the already nucleated volume.[12, 14] In contrast, directional solidification occurs when a small volume is supercooled, which is the case for high cooling rates, e.g. with nitrogen immersion. Here, the nucleation and solidification front are in close proximity in space and time and move further into non-nucleated solution. In this case, a faster cooling rate will lead to a faster freezing rate.[12, 14] Moreover, as ice nucleation is a stochastically event [6, 18], ice nucleation and in consequence ice crystal size distribution will differ from vial to vial resulting in a huge sample heterogeneity within one batch.[6, 14, 27] In addition, during freezing the growth of ice crystals within one vial can also be heterogeneous, influencing intra-vial uniformity.[5] Up to now, 10 polymorphic forms of ice are described. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of ice is limited to the hexagonal type, in which each oxygen atom is tetrahedrally surrounded by four other oxygen atoms.[23] The fact that the ice crystal morphology is a unique function of the nucleation temperature was first reported by Tammann in 1925.[28] He found that frozen samples appeared dendritic at low supercoolings and like â€Å"crystal filaments† at high supercooling. In general, three different types of growth of ice crystals around nuclei can be observed in solution[15]: i) if the water molecules are given sufficient time, they arrange themselves regularly into hexagonal crystals, called dendrites; ii) if the water molecules are incorporated randomly into the crystal at a fast rate, â€Å"irregular dendrites† or axial columns that originate from the center of crystallization are formed; iii) at higher coo ling rates, many ice spears originate from the center of crystallization without side branches, referred to as spherulites. However, the ice morphology depends not only on the degree of supercooling but also on the freezing mechanism. It is reported that â€Å"global solidification† creates spherulitic ice crystals, whereas â€Å"directional solidification† results in directional lamellar morphologies with connected pores.[12, 14] While some solutes will have almost no effect on ice structure, other solutes can affect not only the ice structure but also its physical properties.[19] Especially at high concentrations, the presence of solutes will result in a depression of the freezing point of the solution based on Raoults`s Law and in a faster ice nucleation because of the promotion of heterogeneous nucleation, leading to a enormously lowered degree of supercooling.[21] 2.2 Crystallization and vitrification of solutes The hexagonal structure of ice is of paramount importance in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal ice, when ice forms.[23] Consequently, the concentration of the solute constituents of the formulation is increased in the interstitial region between the growing ice crystals, which is referred to as â€Å"cryoconcentration†.[11-12] If this separation would not take place, a solid solution would be formed, with a greatly reduced vapor pressure and the formulation cannot be lyophilized.[23] The total solute concentration increases rapidly and is only a function of the temperature and independent of the initial concentration.[4] For example, for an isotonic saline solution a 20-fold concentration increase is reported when cooled to -10 °C and all other components in a mixture will show similar concentration increases.[4] Upon further cooling the solution will increase to a critical concentration, ab ove which the concentrated solution will either undergo eutectic freezing or vitrification.[7] A simple behavior is crystallization of solutes from cryoconcentrated solution to form an eutectic mixture.[19] For example, mannitol, glycine, sodium chloride and phosphate buffers are known to crystallize upon freezing, if present as the major component.[12] When such a solution is cooled, pure ice crystals will form first. Two phases are present, ice and freeze-concentrated solution. The composition is determined via the equilibrium freezing curve of water in the presence of the solute (figure 2). The system will then follow the specific equilibrium freezing curve, as the solute content increases because more pure water is removed via ice formation. At a certain temperature, the eutectic melting temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve will meet the solubility curve. Here, the freeze concentrate is saturated and eutectic freezing, which means solute crystallization, will occur.[7, 19] Only below Teu, which is defined as the lowest temperat ure at which the solute remains a liquid the system is completely solidified.[19] The Teu and Ceu are independent of the initial concentration of the solution.[7] In general, the lower the solubility of a given solute in water, the higher is the Teu.[19] For multicomponent systems, a general rule is that the crystallization of any component is influenced, i.e. retarded, by other components.[11] In practice, analogous to the supercooling of water, only a few solutes will spontaneously crystallize at Teu.[11] Such delayed crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more extreme freeze concentration.[11] Moreover, supersaturation can inhibit complete crystallization leading to a meta-stable glass formation, e.g. of mannitol.[12, 23] In addition, it is also possible that crystalline states exist in a mixture of different polymorphs or as hydrates.[11] For example, mannitol can exist in the form of several polymorphs (a, b and d) und under certain processing conditions, it can crystallize as a monohydrate.[11] The phase behavior is totally different for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.[11] The fact that sucrose does not crystallize during freeze-concentration is an indication of its extremely complex crystal structure.[11] The interactions between sugar -OH groups and those between sugar -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.[11] In this case, water continues to freeze beyond the eutectic melting temperature and the solution becomes increasingly supersaturated and viscous.[11] The increasing viscosity slows down ice crystallization, until at some characteristic temperature no further freezing occurs.[11] This is called glassification or vitrification.[18]   The temperature at which the maximal freeze-concentration (Cg`) occurs is referred to as the glass transition temperature Tg`.[11, 29] This point is at the intersection of t he freezing point depression curve and the glass transition or isoviscosity curve, described in the â€Å"supplemented phase diagram† [30] or â€Å"state diagram† (figure 2).[11] Tg ´ is the point on the glass transition curve, representing a reversible change between viscous, rubber-like liquid and rigid, glass system.[19] In the region of the glass transition, the viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.[19] Tg` depends on the composition of the solution, but is independent of the initial concentration.[4, 11, 27]   For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is reported.[31] In addition to Tg` the collapse temperature (Tc) of a product is used to define more precisely the temperature at which a structural loss of the product will occur. In general Tc is several degrees higher than Tg`, as the high viscosity of the sample close to Tg` will pre vent .[10] The glassy state is a solid solution of concentrated solutes and unfrozen, amorphous water. It is thermodynamically unstable with respect to the crystal form, but the viscosity is high enough, in the order of 1014 Pa*s, that any motion is in the order of mm/year.[4, 11, 29] The important difference between eutectic crystallization and vitrification is that for crystalline material, the interstitial between the ice crystal matrix consists of an intimate mixture of small crystals of ice and solute, whereas for amorphous solutes, the interstitial region consists of solid solution and unfrozen, amorphous water.[19, 23] Thus, for crystalline material nearly all water is frozen and can easily be removed during primary drying without requiring secondary drying.[19] However, for amorphous solutes, about 20% of unfrozen water is associated in the solid solution, which must be removed by a diffusion process during secondary drying.[19] Moreover, the Teu for crystalline material or the Tg` respectively Tc for amorphous material define the maximal allowable product temperature during primary drying.[19] Eutectic melting temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which resu lts in more efficient drying processes.[19] If the product temperature exceeds this critical temperature crystalline melting or amorphous collapse will occur, resulting in a loss of structure in the freeze-dried product, which is termed â€Å"cake collapse†.[11, 19] 2.3 Phase separation and other types of freezing behavior A characteristic property of multicomponent aqueous solutions, especially when at least one component is a polymer, is the occurrence of a liquid-liquid phase separation during freezing into two liquid equilibrium phases, which are enriched in one component.[11, 19] This phase separation behavior has been reported for aqueous solutions of polymers such as PEG/dextran or PVP/dextran but is also reported for proteins and excipients.[32-33] When a critical concentration of the solutes is reached, the enthalpically unfavorable interactions between the solutes exceed the favorable entropy of a solution with complete miscibility.[34] Another proposed explanation is that solutes have different effects on the structure of water, leading to phase separation.[35] Besides the separation into two amorphous phases, two other types of phase separation are stated in literature; crystallization of amorphous solids and amorphization from crystalline solids.[18] Crystallization of amorphous solids often occurs when metastable glasses are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that normally would crystallize during slower freezing is entrapped as an amorphous, metastable glass in the freeze-concentrate.[12, 23] However, with subsequent heating above Tg`, it will undergo crystallization, which is the basis for annealing during freeze-drying (see 3.3).[19] Without annealing, the metastable glass can crystallize spontaneously out of the amorphous phase during drying or storage.[18] Amorphization from crystalline solids, that can be buffer components or stabilizers, predominantly occurs during the drying step and not during the freezing step.[18, 36]   Additionally, lyotropic liquid crystals, which have the degree of order between amorphous and crystalline, are reported to form as a result of freeze-concentration. However, their influence on critical quality attributes of the lyophilized product are not clarified.[19] Moreover, clathrates, also termed gas hydrates, are known to form, especially in the presence of non-aqueous co-solvents, when the solute alters the structure of the water.[23] 3. Modifications of the freezing step As aforementioned, the ice nucleation temperature defines the size, number and morphology of the ice crystals formed during freezing. Therefore, the statistical nature of ice nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and homogeneous freezing process. Several methods have been developed in order to control and optimize the freezing step. Some of them only intend to influence ice nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true control of the nucleation at the desired nucleation temperature. 3.1 Shelf-ramped freezing Shelf-ramped freezing is the most often employed, conventional freezing condition in lyophilization.[37] Here, at first, the filled vials are placed on the shelves of the lyophilizer and the shelf temperature is then decreased linearly (0.1 °C/min up to 5 °C/min, depending on the capacity of the lyophilizer) with time.[37-38] As both water and ice have low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is limited, the shelf-ramped cooling rate is by nature slow.[11] In order to ensure the complete solidification of the samples, the samples must be cooled below Tg` for amorphous material respectively below Teu for crystalline material. Traditionally, many lyophilization cycles use a final shelf temperature of -50 °C or lower, as this was the maximal cooling temperature of the freeze-drier.[7] Nowadays, it is suggested to use a final shelf temperature of -40 °C if the Tg` or Teu is higher than -38 °C or to use a temper ature of 2 °C less than Tg` and Teu.[1] Moreover, complete solidification requires significant time.[11] In general, the time for complete solidification depends on the fill volume; the larger the fill volume the more time is required for complete solidification.[11] Tang et al.[1]   suggest that the final shelf temperature should be held for 1 h for samples with a fill depth of less than or equal to 1 cm or 2 h for samples with a fill depth of greater than 1 cm. Moreover, fill depth of greater than 2 cm should be avoided, but if required, the holding time should be increased proportionately. In order to obtain a more homogeneous freezing, often the vials are equilibrated for about 15 to 30 min at a lowered shelf temperature (5 °C 10 °C) before the shelf temperature is linearly decreased.[1] Here, either the vials are directly loaded on the cooled shelves or the vials are loaded at ambient temperature and the shelf temperature is decreased to the hold temperature. [1, 5, 9] Another modification of the shelf-ramped freezing is the two-step freezing, where a â€Å"supercooling holding† is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset lowered shelf temperature to about -5 to -10 °C for 30 to 60min hold. This leads to a more homogenous supercooling state across the total fill volume.[1, 5] When the shelf temperature is then further decreased, relatively homogeneous ice formation is observed.[5] In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is reached, ice crystal growth proceeds extremely fast, resulting in many small ice crystals.[9, 39] However, the ice nucleation cannot be directly controlled when shelf-ramped freezing is applied and is therefore quite random.[4] Thus, one drawback of shelf-ramped freezing is that different vials may become subject to different degrees of supercooling, typically about +/- 3 °C about the mean.[4] This results in a great variability in product quality and process performance.[4] Moreover, with the shelf-ramped freezing method it is not practical to manipulate the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling might not change within such a small range.[1, 14] 3.2 Pre-cooled shelf method When applying the pre-cooled shelf method, the vials are placed on the lyophilizer shelf which is already cooled to the desired final shelf temperature, e.g. -40 °C or -45 °C.[1, 13-14] It is reported that the placement of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5 °C) compared to the conventional shelf-ramped freezing (-13.4 °C).[14] Moreover, with this lowered degree of supercooling and more limited time for thermal equilibration throughout the fill volume, the freezing rate after ice nucleation is actually slower compared to shelf-ramped freezing.[40]   In addition, a large heterogeneity in supercooling between vials is observed for this method.[14] A distinct influence of the loading shelf temperature on the nucleation temperature is described in literature.[13-14] Searles et al.[14] found that the nucleation temperatures for samples placed on a shelf at -44 °C were several degrees higher than for samples placed on a -40 °C shelf. Thus, when using this method the shelf temperature should be chosen with care. 3.3 Annealing Annealing is defined as a hold step at a temperature above the glass transition temperature.[12] In general, annealing is performed to allow for complete crystallization of crystalline compounds and to improve inter-vial heterogeneity and drying rates.[1, 19] Tang et al.[1] proposed the following annealing protocol: when the final shelf temperature is reached after the freezing step, the product temperature is increased to 10 to 20 °C above Tg` but well below Teu and held for several hours. Afterwards the shelf temperature is decreased to and held at the final shelf temperature. Annealing has a rigorous effect on the ice crystal size distribution [17, 41] and can delete the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg`, the system pursues the equilibrium freezing curve and some of the ice melts.[12, 41] The raised water content and the increased temperature enhance the mobility of the amorphous phas e and all species in that phase.[12] This increased mobility of the amorphous phase enables the relaxation into physical states of lower free energy.[12] According to the Kelvin equation ice crystals with smaller radii of curvature will melt preferentially due to their higher free energy compared to larger ice crystals.[12, 37, 41] Ostwald ripening (recrystallization), which results in the growth of dispersed crystals larger than a critical size at the expense of smaller ones, is a consequence of these chemical potential driving forces.[12, 41] Upon refreezing of the annealed samples small ice crystals do not reform as the large ice crystals present serve as nucleation sites for addition crystallization.[41] The mean ice crystal radius rises with time1/3 during annealing.[37, 41] A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with increasing annealing time, as vials comprising smaller ice crystals â€Å"catch u p† with the vials that started annealing containing larger ice crystals.[12, 17, 37, 41] Searles et al.[41] found that due to annealing multiple sheets of lamellar ice crystals with a high surface area merged to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing opened up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the increased annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.[42] Above Tg` the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.[41] This is of importance when samples were frozen too fast a nd water capable of crystallization was entrapped as amorphous water in the glassy matrix. In addition, the phenomenon of annealing also becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.[11] 3.4 Quench freezing During quench freezing, also referred to as vial immersion, the vials are immersed into either liquid nitrogen or liquid propane (ca. -200 °C) or a dry ice/ acetone or dry ice/ ethanol bath (ca. -80 °C) long enough for complete solidification and then placed on a pre-cooled shelf.[9, 16] In this case the heat-transfer media is in contact with both the vial bottom and the vial wall [10], leading to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a lowered degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. Liquid nitrogen immersion has been described to induce less supercooling than slower methods [9, 37, 39] , but more precise this faster cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.[12, 14]   While it is reported that external quench freezing might be advantag eous for some applications [39], this uncontrolled freezing method promotes heterogeneous ice crystal formation and is not applicable in large scale manufacturing.[7] 3.5 Directional freezing In order to generate straight, vertical ice crystallization, directional respectively vertical freezing can be performed. Here, ice nucleation is induced at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.[9] In this case, the ice propagation is vertically and lamellar ice crystals are formed.[9] A similar approach, called unidirectional solidification, was described by Schoof et al. [43]. Here each sample was solidified in a gradient freezing stage, based on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology. 3.6 Ice-fog technique In 1990, Rowe [44] described an ice-fog technique for the controlled ice nucleation during freezing. After the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a flow of cold nitrogen is led into the chamber. The high humidity of the chamber generates an ice fog, a vapor suspension of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio

Imagery in Midnight in the Garden of Good and Evil :: Midnight Garden Good Evil

Imagery in Midnight in the Garden of Good and Evil One of the most stunningly powerful features of John Berendt's Midnight in the Garden of Good and Evil is the vivid imagery used hroughout the book. Berendt has a way of making everything he writes about come to life. The reader doesn't merely read about Savannah, he lives it. The characters that are represented in the book come to life as the book progresses. Their actions take form before the audience's eyes. The characters are not, however, the only things brought to life by Berendt's vivid style. Savannah itself becomes real to the reader. The detailed settings make the city more than just a background for the story. It is an integral part of the tale. All of these aspects come together to make Midnight less of a book and more of an experience. The story relayed by Berendt in Midnight in the Garden of Good and Evil is filled to the brim with interesting characters. What makes them even more interesting to the reader is the knowledge that they are, at least for the most part, actually real-life characters. His descriptions of these people and their characteristics are so detailed that after reading only two or three pages about someone, the reader begins to feel as if she knows the person. The vivid descriptions are extremely detailed. "She had both hands on her hips and a sassy half-smile on her face as if she had been waiting for me" (Berendt 96). This is just a small portion of Berendt's opening description of Chablis, the drag queen of the book. Berendt also brings these people to life through their behaviors. A perfect example of this method is the character of Danny Hansford. The descriptions of Danny's behavior are so vivid that it is hard to think of him as a character in a book. Everything from Danny's walk to his v iolent fits of rage are represented with great detail. The imagery associated with Savannah itself is nothing short of astounding. The squares that populate Savannah, the houses in the area, and even the town cemetery are presented with wonderful detail. At one point Berendt speaks of James Oglethorpe, the founder of Georgia, and the fact that Oglethorpe had the squares planned before he had set sail from England. The layout was to be "based on the design of a Roman military encampment. Imagery in Midnight in the Garden of Good and Evil :: Midnight Garden Good Evil Imagery in Midnight in the Garden of Good and Evil One of the most stunningly powerful features of John Berendt's Midnight in the Garden of Good and Evil is the vivid imagery used hroughout the book. Berendt has a way of making everything he writes about come to life. The reader doesn't merely read about Savannah, he lives it. The characters that are represented in the book come to life as the book progresses. Their actions take form before the audience's eyes. The characters are not, however, the only things brought to life by Berendt's vivid style. Savannah itself becomes real to the reader. The detailed settings make the city more than just a background for the story. It is an integral part of the tale. All of these aspects come together to make Midnight less of a book and more of an experience. The story relayed by Berendt in Midnight in the Garden of Good and Evil is filled to the brim with interesting characters. What makes them even more interesting to the reader is the knowledge that they are, at least for the most part, actually real-life characters. His descriptions of these people and their characteristics are so detailed that after reading only two or three pages about someone, the reader begins to feel as if she knows the person. The vivid descriptions are extremely detailed. "She had both hands on her hips and a sassy half-smile on her face as if she had been waiting for me" (Berendt 96). This is just a small portion of Berendt's opening description of Chablis, the drag queen of the book. Berendt also brings these people to life through their behaviors. A perfect example of this method is the character of Danny Hansford. The descriptions of Danny's behavior are so vivid that it is hard to think of him as a character in a book. Everything from Danny's walk to his v iolent fits of rage are represented with great detail. The imagery associated with Savannah itself is nothing short of astounding. The squares that populate Savannah, the houses in the area, and even the town cemetery are presented with wonderful detail. At one point Berendt speaks of James Oglethorpe, the founder of Georgia, and the fact that Oglethorpe had the squares planned before he had set sail from England. The layout was to be "based on the design of a Roman military encampment.