Determining the Status of an Independent Contractor and Taxes Essay

Determining the Status of an Independent Contractor and Taxes - Essay Example Avoiding taxes is not the primary goal of an independent contractor but they know if they are classified as employees they suffer some consequences. Some of the consequences one suffers are that they might not be hired by the hiring firm since they these firms will be forced to pay additional expenses of treating them as employees. The aims of the firms to maximise their profit and to ensure they achieve their goals, they have to ensure that they minimise their operational expenses. For this case, if an independent contractor is classified as an employee he will never be hired by the hiring firms. (Fishman, 2006) The other reason as to why the independent contractors do not need to be classified as employees is because they will add additional tax burden to themselves by being subjected to tax. Their pay will be deducted, something they would not be experiencing if they were independent contractor. (Fishman, 2006) According to Fishman (1997), the U.S Bureau of Labour Statistics shows that in U.S.A alone, there are eight million independent contractors and in the next ten years, this number is expected to double. The use of independent contractors is beneficial companies that cannot afford to hire permanent employees especially small and medium ones. This is especially so for those companies that can not be able to employ a permanent employee for specialized function. For instance, a company engaged in international commerce can hire an attorney who has specialized in international trade as an independent contractor to provide international legal advice. This staffing approach is more affordable than employee a permanent employee and for this case due to the fact that this attorney's salary will not be taxed, then it does not mean he want to avoid paying the tax. 1 Permanent employees have been given a great deal of job security by European laws and because of these laws, the economic uncertainty has forced employers to use short-term contracts than using permanent employment. These short-term contracts are the use of independent contract and statistics shows that this tread is on the increase. In every five employees in France, one is on part-time contract; 30 percent of workforce in Britain is on temporary employment; in Spain, for every ten jobs created, seven of them are on temporary basis; and the ban on private temporary employment agencies has been lifted in Germany. These statistics shows that there is great rise in the use of independent contractors in many European countries. The use of independent contractors has become popular since it reduces costs and legal requirements imposed on termination or lay off of employment in Europe. Due to the fact that the temporary employees hired by companies under temporary employment does not mea n that they are avoiding paying taxes since they employment is being dictated by circumstances. (Templeman, 1996) Independent contractors results in cost savings which include: reduced book keeping and payroll preparations costs, avoidance of taxes, reduced fringe benefits, elimination of worker's compensation benefits, elimination of overtime pay, decreased administrative burdens and reduction in capital and maintenance costs if the independent contractors provides their own tools and equipment. (Bureau of National Affairs, 1994; Stalnaker, 1993). Even though independent contr

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

Essay on A Woman Bound by Society in Steinbecks The Chrysanthemums

A Woman Bound by Society in John Steinbeck's "The Chrysanthemums"  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   When John Steinbeck's short story "The Chrysanthemums" first appeared in the October 1937 edition of Harper's Magazine (Osborne 479), Franklin D. Roosevelt had just been reelected president. The country was recovering from the Great Depression, unions were developing, and child labor in manufacturing was terminated (Jones 805-6). The first female cabinet member in American history, Frances Perkins, was appointed the Secretary of Labor (Jones 802). She was one of the few women in her time to gain equality in a male-dominated society. For most women, liberation was a bitter fight usually ending in defeat. In "The Chrysanthemums," this struggle for equality is portrayed through Steinbeck's character Elisa Allen. According to Stanley Renner, "The Chrysanthemums" shows "a strong, capable woman kept from personal, social, and sexual fulfillment by the prevailing conception of a woman's role in a world dominated by men" (306). Elisa's appearance, actions, and speech depict the frustration w omen felt in Steinbeck's masculine world of the 1930's. "Steinbeck's world," observes Charles A. Sweet, Jr., "is a man's world, a world that frustrates even minor league women's liberationists" (214). This frustration is evident when Elisa is first introduced. Her figure is described as "blocked and heavy" because she is wearing heavy gloves, heavy shoes, a "man's black hat," and a big apron that hides her printed dress (Steinbeck 330). Her home has the masculine qualities of being "hard-swept" and hard-polished" (Steinbeck 330). Elisa is bored with her husband and with her life (McMahan 455). Obviously, Elisa is unhappy with the traditional female role and is attempti... ...et al. America and Its People: Volume Two From 1865. London: Scott, Foresman, 1989. McMahan, Elizabeth E. "'The Chrysanthemums': A Study of Woman's Sexuality." Modern Fiction Studies 14 (1968-69): 453-8. Marcus, Mordecai. "The Lost Dream of Sex and Childbirth in 'The Chrysanthemums.'" Modern Fiction Studies 11 (1965): 54-8. Osbourne, William R. "The Texts of Steinbeck's 'The Chrysanthemums.'" Modern Fiction Studies 12 (1966-67): 479-84. Renner, Stanley. "The Real Woman Inside The Fence In 'The Chrysanthemums.'" Modern Fiction Studies 31 (1985): 305-17. Steinbeck, John. "The Chrysanthemums." Literature and the Writing Process. Ed. Elizabeth McMahan, Susan Day, and Robert Funk. 2nd ed. New York: Macmillan, 1989. 330-6. Sweet, Charles A., Jr. "Ms. Elisa Allen and Steinbeck's 'The Chrysanthemums.'" Modern Fiction Studies 20 (1974): 210-14.

Cashier Training manual Essay

Addictions Do addictions really exist? People constantly seem to say that they are addicted to nicotine, alcohol and drugs, but there are continuous debates whether this is just a necessity or a general want. Most people start using these substances out of depression as they convince themselves that it will make them feel better about themselves and the difficult life their living. However, others begin with these substances due to social surroundings as they feel the need to fit in around the harsh public. This then turns into a habit. There are 3 main types of addictions; psychological, mental and physical. Every case has a different type of addiction and some are overly exaggerated. After doing some research on addictions, I found that the term â€Å"addictions† has been proven to exist by many different scientists on quite a few websites. However I believe it does exist but more over it depends on the way each human beings mind works as some people have greater insecurities than others which cause them to feel the need to take such substances and over use them while others are mentally strong and take these substances once in a way for entertainment. Once you start experimenting with your bodies system and allowing these chemicals in, you should be strong in your mind to stop whenever but if you’re weak and allow your mind to take over, this will become an addiction. As proven by science, physical addictions exist. This has been mentioned by scientists on treatment websites, as many addicts have tried to stay clean for days and they experience â€Å"withdrawal symptoms† which means; â€Å"the person has developed a physical dependence.† Sometimes addicts give up as they find the withdrawal period harder than anything and feel the need to start the usage of drugs again. According to the National Institute of Health; â€Å"the addict’s whole life revolves around the drug whether they are harming themselves severely or not†. (http://advancedpaintreatment.com/types.asp) There are certain drugs which can be physically addicting to certain human beings; this is has been proven as an addiction to some degree. But can some  drugs and alcohol can be serious addictions? It is always in the mind of the one using these substances as they are mentally insecure and dependable human minds. In the case of ciggerets it is the nicotine in cigarettes causes the addiction. Nowadays the percentage of smokers have increased, people have found more reasons to continue smoking, allowing it to become a mental addiction. People may think it helps them focus or fills them up as this is what they want to believe. However, people who say nicotine is a necessity have it in their minds and cause their body to go through this mind set addiction, due to insecurities or depression. Nicotine brings a general sense of fulfillment to these said to be â€Å"addicts†. Smokers need to have the will power to let go of nicotine. Due to the weak mentality of human beings, it has been proven that the majority death toll is due to the tobacco in the nicotine. According to the National Institute of Health; â€Å"Tobacco use killed approximately 100 million people during the 20th century and, if current smoking trends continue, the cumulative death toll for this century has been projected to reach 1 billion.† (http://drugabuse.gov/scienceofaddiction/health.html) On the other hand, alcohol damages the brain and the way in which the stimulants in the brain work. Many people end up in rehab as they are â€Å"alcoholics†, but then again do those exist? To me alcoholics are just like â€Å"nicotine addicts† it is set in their minds as it brings a sense of joy and serenity. Many sorts of psychological issues cause this. Some people tend to push themselves to drink every day, as they feel the need for it due to weak minds as they have no control over their actions and give up on themselves, some with no reason at all. Alcoholics think they physically need the alcohol in their systems but truly they are just mentally addicted to it as they lose control over their lifestyle. There are way too many different types of drugs; each one has a different effect but not all of them become physical or mental addictions. Marijuana is a drug and said to be herbal, however it does affect the way in which your mind works and reacts to certain situations, but not for long. It could never be an addiction to any human being, well that’s how I would see it as it is just a different way of enjoyment for certain people. But this wouldn’t be a necessity to anybody and if the  person calls themselves an addict to this drug it proves that the person cannot accept their life and feels the need for something which cannot be a true addiction. This would be a psychological problem rather than addiction. Then again, drugs such as; heroin, cocaine, prescription medications and chemicals, like LSD, MDMA, Ecstasy , etc. are psychologically addictive and can cause serious damages to the body and brain if over used. Many people find it very easy to fall into an addiction with such drugs as their body becomes weak and they feel emotionally attached to these chemicals as they highly affect the stimulants in your brain. They feel a sense of relaxation which they appreciate and then think they need. When these drugs are excessively used they cause hallucinations which affect the human brain to see what is real and what isn’t, people who are physically addicted to such drugs lose sense in what reality truly is and do not realize that they aren’t sober nor in their senses. I believe this is a true addiction which builds up in fragile minds. There have been many people who have recently started to abuse prescription medications without any illnesses. The national institute of health posted that these medications can be addictive as well as poisonous, â€Å"This has been proven to not only be addictive but also lethal. Commonly abused classes of prescription drugs include painkillers, sedatives, and stimulants.† The certain stimulants in the brains of such people cause them to carry on taking these chemicals into their system; this is more of a psychological addiction which soon leads to a mental addiction. (http://drugabuse.gov/scienceofaddiction/health.html) In conclusion, I find that addictions do exist to an extent. People, who find themselves in the trap of requiring such substances, should try gaining inner self confidence and face reality rather than depend on drugs, alcohol or nicotine to help them through life. However heavy drugs such as heroin, pharmaceuticals, cocaine and chemicals, when used excessively can cause physical addictions, while alcohol and nicotine are moreover mental and psychological addictions in certain cases and for some people these are just socially used and aren’t said to be addictions at all as they can face reality with or without them. References: National Institute Of Health. National Institute On Drug Abuse. What are some effects of specific abused substances? Retrieved November 16th, 2011, from http://drugabuse.gov/scienceofaddiction/health.html Advanced Pain Treatment & Diagnostics Group. Are you tired? Tired of the shame, tired of hiding? Retrieved November 16th, 2011, from http://advancedpaintreatment.com/types.asp

Red Riding Hood Essay

How would you categorize the point of view [e. g. , first-person, second-person (i. e. , â€Å"you†), third-person limited, third-person omniscient]? * Is the point of view consistent throughout the story (told from the same perspective), or does it shift at any points in the narrative? If so, make note of when and how those changes occur. ) * How does point of view shape your reading of the work? In what ways does it contribute to or detract from your reading of the work? * How does point of view relate to the story’s themes or content? Your initial post should be at least 150 words in length. Support your claims with examples from the text, and properly cite any references. Respond to at least two of your classmates’ posts by Day 7. I chose to analyze â€Å"Little Red Riding Hood. The point of view is third person limited. The story is narrated as though the writer was watching over and retelling the story as it takes place. The point of view is consistent throughout its entirety. There is quoted dialogue from each character, especially when Little Red Riding Hood arrives at the Grandmother’s house and is comparing the features of the Wolf to the Grandmother. I struggled to remove the images I have stored in my mind from the storybook that I read of this over and over as a small child. I remember that in the picture-book the Wolf was drooling over Little Red Riding Hood because he was planning to eat her. The innocence of Little Red Riding Hood prevails as she continues to keep questioning Wolf about his features like the size of his hands, ears and eventually his mouth. I don’t know if it seems to change POV during this part of the story or if it because in my mind I no long hear a narrator’s voice. It may also be due to this being the climax of the story. I just felt it was important to note that instance as I read the story. I feel that third person limited point of view is a perfect way to tell this type of story as long as the author is able to portray the character’s nature well before the story is underway. For instance, if the reader is not informed that in some way that Little Red Riding hood is an innocent and compliant little girl (protagonist) and that the nature of a wolf (antagonist) is to kill and eat his prey by any means available, then the entire meaning may be misconstrued.

The Microsoft Antitrust Case essays

The Microsoft Antitrust Case essays The case against Microsoft was brought buy the U.S. Department of Justice, as well as several state Attorneys General. Microsoft is accused of using and maintaining monopoly power to gain an unfair advantage in the market. The case has been under observation for a long time, but the Justice department is having trouble coming up with substantial evidence against Microsoft. Specifically, the Department must prove: That Microsoft has monopoly power and is using it to gain unfair leverage in the market. And that Microsoft has maintained this monopoly power through exclusionary or predatory acts(Rule). Some say that Microsoft is only taking advantage of its position in the market and using innovative marketing strategies to attract new customers. They have chosen to implement a market development strategy to attract new customers who are looking for a system that has Internet capability. Microsoft feels that by integrating their Internet Explorer web browser technology into Windows, they are only improving its overall functionality available to the customer. Microsoft began expanding into the browser area because of increasing threat from Netscape and Java. Java is the programming language used to make Netscape. Programs that are written in Java can work on any PC, whether it has Windows on it or not. That is why there is a great threat to the Windows environment. The more Netscape is used, the more other vendors will begin writing Netscape compliant programs and the more Java will be used, which puts a damper on Windows. So Windows introduced their Internet explorer to combat the increasing Netscape usage. It did not do this to create a monopoly, but to protect itself. If people realize that Java programs can be run on ANY PC, then they will realize that they do not need to buy Windows. Some say that Microsoft began its illegal̶...

The Caucasian Chalk Circle Essays

The Caucasian Chalk Circle Essays The Caucasian Chalk Circle Essay The Caucasian Chalk Circle Essay Research investigation: What Verfremdungs Effekts does Brecht script in Caucasian Chalk Circle and how effective are these techniques in the dramatic movement of transformation? United World College in Mostar Student: Selmir Klicic Teacher: Melissa Ann Reed Subject: Theater Block: E First factor I would like to mention when it comes to this kind of topic is audience. The audience was always to big extent demanding for an author and a director to send the message throughout a work of art, a theater play which would be understood in proper way. Mostly concerning this problem, world’s biggest play writers developed a theory being practiced in their plays in order for better understanding of the idea by the audience. One of these theories is today called Verfremdungs Effekt or alienation effect. This significant influence was initiated by Shakespeare at first, who, using his drama plays applied V – effect for better understanding of his, usually very complicated plot. The theory kept being developed since it was first invented. A person who contributed to development of V – effect the most was Bertolt Brecht by inventing a completely new type of theater, nowdays called Brechtian Theater. His contribution was mostly based on Shakespeares theory, and it was just used as a pose and strategy to bring about the change by engaging the imagination and creative critisism. „We need a type of theatre which not only releases the feelings, insights and impulses possible within the particular historical field of human relations in which the action takes place, but employs and encourages those thoughts and feelings which help t ransform the field itself. – Brecht From the quote above we could conclude that what was important for Brecht wasnt the fiction actors would make by their performance, but the message that is contained in plot. As an example of this theory, and as my research investigation task I will take Brechts play Caucasian Chalk Circle, as well as the goal of this essay would be based on the same play. The actual goal of the research investigation is to conclude how Verfremdungs Effekt contributes to the undestranding of the idea and message being sent by the play. As it would be too long to analyze a complete piece, my research will be based on one scene from Caucasian Chalk Circle. The certain scene is the one in which two mothers are fighting about a child just like two farmers are fighting about the land. By this scene it would be possible to present the idea of emphasizing the important issues of society Brecht was trying to make us be aware of. The method that I am planning to use in the experiment is performing this scene in front of the audience two times. First time it would be without using V – effect and the second time should be performed with it. The significant thing is the reaction of audience and their opinion, as long as the research investigation is based on proper understanding of idealism being contained in a drama piece, for which audience is vital. To know the understanding of the audience I should ask questions concerning their perception of the plot. So the questions should take under the consideration both parts of the experiment. Knowing that the audience is more or less educated and familiar with the idea of V – effect, question should be prescribed as simple. 1. According to you, what was the idea of this scene? (this question should be asked after performing the first part, which has no V – effect in its performance) 2. What kind of the impression do the characters leave on you by their act in the first part, and what kind of the impression do they leave in second part? 3. Did V – effect change your understanding and prescription in second scene? If yes, please evaluate. I think that a conclusion could be properly finalized after these questions answered by the audience, are analyzed afterwards.

3 Tips for Producing Consistent Written Content

3 Tips for Producing Consistent Written Content 3 Tips for Producing Consistent Written Content 3 Tips for Producing Consistent Written Content By Mark Nichol There are many editorial strategies for making text easy to write, edit, and read. Here are a few guidelines for simplifying how your company, organization, or publication (even if it’s merely a personal blog) produces content. 1. Minimize House Style â€Å"House style† refers to treatment of specialized terminology and treatment of spelling, capitalization, numbers, or punctuation that differs from the norm. Before you decide to routinely spell a word in a variant or obsolete form (for example, writing archeology instead of archaeology), capitalize generic words (â€Å"The Company is dedicated to excellence†), use a numeral rather than spelling the number out (â€Å"We have 5 guiding principles†), or go against custom in formatting punctuation (for example, employing single quotation marks instead of double quotation marks), consider whether the divergence is worth the effort- and, if so, publicize and document the decision so that all content your organization produces is consistent. The more clear and thorough your house style is, the easier it is to maintain high-quality content. On the other hand, the less extensive and cumbersome your house style is, because there are fewer exceptions to attend to, the easier it is to maintain high-quality content. 2. Always Use the Serial Comma Many publications follow the Associated Press Style Book’s policy of omitting serial commas. (The serial comma is the last comma in a list such as â€Å"apples, oranges, and pears.†) Unfortunately, this modest effort to simplify by avoiding an optional punctuation mark actually complicates matters: When a list contains an element that includes a conjunction (â€Å"apples, oranges and tangerines and pears†), the sentence organization is compromised, so an exception must be made, which results in inconsistency. For the sake of uniformity and simplicity, always include a serial comma, the recommendation of The Chicago Manual of Style, the handbook of record for many book publishers and other content producers. On a related note, use semicolons for lists only when the presence of one or more commas within one or more list elements creates ambiguity, especially when one or more elements of the list is itself a list (â€Å"apples, oranges, and pears; milk and cheese; and bread†). The length of the list, and the presence of conjunctions within list elements, are not factors. 3. Capitalize Only When Necessary Capitalize proper names only, and capitalize job titles only before names. Generic abbreviations of entity names (â€Å"the company,† â€Å"the board,† â€Å"the department†) and references to concepts (â€Å"human resources†) are not proper names (though â€Å"Human Resources† is correct as the name of a specific department). Capitalization rules about art movements, medical and scientific terminology, geological and historical eras, and other scientific or cultural phenomena can seem (and sometimes are) arbitrary, so double-check reliable editorial resources. Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Writing Basics category, check our popular posts, or choose a related post below:100 Idioms About Numbers16 Misquoted Quotations10 Functions of the Comma

Organisation Theory and Behaviour Essay Example | Topics and Well Written Essays - 2250 words

Organisation Theory and Behaviour - Essay Example Everything that a human being needs for his/her survival, health and safety is dependant, either directly or indirectly, on the natural environment.   The concept of sustainability is based on this simple principle. Sustainability helps in creating and maintaining the environment under which nature and human can subsist in productive accord that allows satisfying the social and economic needs of present and future generations2. As there has been growing interest in the concept of sustainability in recent years, the focus of organizations has also shifted towards this concept. There are three assumptions as to why managers would show interest in sustainability. The first being, that most of the businesses have interest in creating value in the long-standing. Managers are very much concerned about the repute of the organization. With people being more aware about how an organization might harm the environment and nature has resulted in mangers being responsible socially and economica lly3. Second of these is that if the conduct of organization is destructive and is harmful to the social and natural environment, it will ultimately pressurize the managers in shape of direct or indirect costs. Formal penalties might be imposed by the government or damaged relationship with important stakeholders such as; shareholders, employees and customers as consequences of careless and irresponsible social behaviour may result in loss of value. Thirdly, new mercantile opportunities for business can be created by pursuing sustainable growth. This would help in developing and marketing new services and goods that would undoubtedly increase profits and assist in achieving the objective of sustainability4. COMMENTS, CRITIC, EVALUATE AND APPLICATION As more emphasis is placed on sustainability the leaders of businesses, inclusive of multinational organizations as well as the singularly owned companies, face the unprecedented and unique challenge of planning organization culture that cater to achieve sustainability. The organization design and culture should be such that, that it serves to a broad range of stakeholders and ensures that business is sustainable within their physical, market, social, and financial realms. The corporate board of directors must be able to provide strategic directions and an oversight that stretches further than short-term financial performance and recognize social, environmental and governance responsibility of the organization and are essential to its performance in the long-term sustainability5. This requires organizations to be tactically positioned within the distinctive intersection of corporate citizenship, environmental stewardship, financial strength, and product/service excellence. A great amount of attention is required to the intricacies of the internal and external environments of the organization for designing it to achieve and uphold a strategic outlook deftly balanced within the nexus of sustainability. In order to ac hieve this balance, leaders must seek to build up an incorporated perceptive of organization culture, design and sustainability by: Organization’s background and the cultural mind-sets of its employees must be explored in order to design effective, high quality, and sustainable work environments; cultural factors must be considered and used to impede and facilitate sustainable organization des

Heather Fraser, Mae Shaw and Paulo Freires View of Communitarianism Essay

Heather Fraser, Mae Shaw and Paulo Freires View of Communitarianism - Essay Example From the report findings it is clear a community represents a high level of employing an elementary preposition of political and social contribution. Neighbourhoods, families and churches all form part of the entire community despite the political and moral interpretations. Despite many differences across the globe, a community constitutes shared values and goals that coincide with the individual interests. In addition, community members have an instrumental value that built a personal relationship for different members to enjoy a sense of ownership. Most importantly, members of a community enjoy a sense of identity that allows them to enjoy various present conditions. The paper states that friendship has been an instrumental factor in defining different relationships. On other hand, this has failed in many instances, as it does not converge solidarity. Arguably, this is because of communal concerns and other issues such as sexual practices as well as understanding of the universe. Above all, communitarianism remains a viewpoint that has significantly affected the community. Despite going through change from one community to another, the term community probably remains an umbrella words that has not change meaning but only use. In his book, Four different approaches to community participation Heather Fraser presents a theoretical orientation of communitarianism from different approaches.

Economic history Assignment Example | Topics and Well Written Essays - 500 words

Economic history - Assignment Example The insatiable quest for many profits necessitated the involvement of many workers and extensive labor division. Meaning that employees could work in different locations, anonymous to each other, with the aim of producing various parts for the same commodity. This led to each producer developing a sense of isolation and loneliness, which in the end translated to producers emerging as egoistic. This state of egoism led to a natural state of war with each fighting solely against a myriad others. However, this state of war could only be controlled if there were a central party which every producer would submit to and in turn gain protection from the rest producers. This was explained by Hobbes in his writing where he stressed that it was only after submission to an absolute monarch that individuals would escape the conflict existing among them. Economic specialization, on the other hand, explained the co-existence of different producers in the market system to help each other survive. There was complete dependence on each other for successful functioning of the market. Economic specialization was important as it provided for a relatively free functioning market where producers assisted each other produce and in turn benefit (Hunt 128). Labor and economic specialization contradict each other in that for the former, producers worked in isolation that prompted a feeling of competition among each other. For the latter, however, producers work to benefit each other. There is no single producer that can exist without the other. In conclusion, human beings have a desire to achieve pleasure but avoid pain with his nature being competitive and egoistic. Specialization of labor is meant to maximize profits and speed up production in any market system. When producers are left to work independent, there is the development of an

Measuring the reactions of the enzymes catecholase in different levels Lab Report

Measuring the reactions of the enzymes catecholase in different levels of pH in different tempereture - Lab Report Example Temperature rise of about 10oC normally doubles or triples the rate of enzymatic reactions. However beyond the optimum temperature the enzyme activity decreases. If catecholase activity is minimally affected at different temperatures and pH then it can be hypothesized that it has a large range of optimal activity. The aim of this experiment was to test the effect of temperature and pH on the enzymatic activity of catecholase. The effect of catecholase enyme at different temperatures, 0 and 37oC were tested at different levels of pH 2, 4, 7 and 12. Three tubes for each pH were set up and labelled A, B and C. To three tubes in each pH 3ml of the pH solution was added and 2ml of banana extract (substrate). To two tubes 1ml of the enzyme (potato extract) was added while the third tube the enzyme was not added and acted as the control. The test tubes were then shaken and put in the appropriate temperature and the optical density measured after three minutes. The reaction rate was the highest at all temperatures at pH 7. At room temperature the reaction rate was the highest in the tube without the enzyme. pH 4 and 12 had the lowest reaction rates at all temperatures. The reaction rates were highest between 0oC and room temperature in the presence of catecholase and lowest at 0oC without the enzyme. Catechol oxidase (catecholase) is a polyphenol oxidase enzyme present in many plants (Aniszewski et al. 2008). It is responsible for catalysing the reaction between oxygen and hydroxylated benzenes leading to the production of quinines and water. The enzyme mediated reaction rate was the highest at physiological pH and at temperatures between zero and room temperature. This shows that the enzyme has a large range of optimal temperatures. However, it has a narrow range of optimal pH. The narrow pH range during which catecholase activity was the highest is due to the effect of pH on the redox