Chromatographic Separation of Hemoglobin from Phenol Red

Chromatographic Separation of Hemoglobin from Phenol Red Syed Ibrahim Chromatographic Separation of Hemoglobin from Phenol Red by Gel Filtration Introduction From catalysis to transport, proteins take part in countless cellular processes (Nelson Cox, 2008). However, due to their small size, proteins can be challenging to work with in the lab. To overcome this hurdle, scientists have developed many fractionation techniques including centrifugation, precipitation, dialysis, and chromatography (Alberts et al., 2008). Chromatography is one of the most versatile laboratory techniques as it is used in many fields of study (Partridge, 1952; Silva et al., 2014). There are several types of chromatography, and each separates molecules based on different properties. Size-exclusion gel filtration is a type of column chromatography which resolves molecules based on their size (Duong Gabelli, 2013). In the column, there is a cross-linked polymer matrix with pores of a predetermined size (Scott, 2003). Small molecules are more likely to spend time in the pore, so they migrate slower and thus elute in later fractions (Nelson Cox, 2008). Conversely, larger molecules are less likely to enter the pore, so they migrate faster and thus elute in earlier fractions (de Jong et al., 2015). There are numerous types of column matrices, such as Sephadex G (composed of dextran and epichlorohydrin) and Sepharose (consisting of agarose) (GE, 2015). Each matrix has different sized pores and thus different ranges of molecular sizes that it can separate, called fractionation ranges (de Jong et al., 2015). In this lab, Sephadex G-25, which has a fractionation range of 1,000-5,000Da, was the column matrix (de Jong et al., 2015). Hemoglobin and phenol red were also used. Hemoglobin is a protein found primarily found in mammalian red blood cells, and is used to transport oxygen from an organism’s lungs to its tissues (Seeley, 2014). Phenol red is a chemical compound that changes color depending on the pH of the chemical environment, and so it is typically used as a pH indicator (Klein, 2012). Hemoglobin has a molecular weight of 64,500Da while phenol red has a molecular weight of 354.38Da (de Jong et al., 2015). It was expected that hemoglobin would be completely excluded from the pores of the Sephadex G-25 resin, allowing it to elute in early fractions. As well, it was expected that phenol red would be completely included in the gel, making it elute in later fractions. Purpose The goal of this lab was to separate a mixture containing hemoglobin and phenol red using size-exclusion gel filtration. As well, the aim was to compare the standard and literature wavelengths of maximum absorption of hemoglobin and phenol red. Results The results from this experiment are shown in Table 1 and Table 2. Table 1. Fraction volumes from size-exclusion gel filtration using Sephadex G25. The size-exclusion gel filtration separated a mixture of hemoglobin and phenol red. In theory, fraction A contained void volume, fraction B contained hemoglobin, fraction C contained the buffer only, and fraction D contained phenol red. Table 2. Peak absorbance and wavelength of standard hemoglobin and phenol red. Using a Beckman spectrophotometer, the wavelength profile between 380nm and 600nm of two standard samples was determined. The standard hemoglobin sample contained 250ÃŽ ¼L of 0.8% hemoglobin, while the standard phenol red sample consisted of 20ÃŽ ¼L of 0.2% phenol red. Based on the graph, the maximum absorbance values and the corresponding wavelengths were determined. Questions and Research Question 1: Search Engine: Web of Science Search Terms: hemoglobin spectroscopy Reference: DiFeo, T. J., Addison, A. W., Stephanos, J. J. (1990). Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects. Biochem. J, 269, 739-747. Search Engine: Web of Science Search Terms: hemoglobin Soret band absorptivity (sorted by relevance) Reference: Akuwudike, A. R., Chikezie, P. C., Chilaka, F. C. (2010). Absorption spectra of normal adult and sickle cell haemoglobins treated with hydrogen peroxide at two pH values. Adv. Biomed. Res, 1, 55-60. Search Engine: Google Search Terms: phenol red maximum wavelength absorption Reference: LobaChemie. (2015). Phenol Red. Laboratory Reagents and Fine Chemicals. Retrieved February 21, 2015, from http://www.lobachemie.com/pH-Indicator-05180/PHENOL-RED-CASNO-143-74-8.aspx Based on the graph from Blackboard, the wavelengths of maximum absorbance (ÃŽ »max) are shown in Table 2. For the standard hemoglobin sample ÃŽ »max = 410nm, and for the standard phenol red sample ÃŽ »max = 560nm. Based on the literature, hemoglobin and related proteins are known to give three bands of maximum absorption: ÃŽ ±-band, ÃŽ ²-band, and Soret band (DiFeo et al., 1990). For normal adult hemoglobin (HgA), the wavelength of maximum absorbance in the Soret band is ÃŽ »max = 415nm (Akuwudike et al., 2010). The wavelengths of maximum absorbance of phenol red changes based on pH. At a pH of 8.2, phenol red has ÃŽ »max = 557-560nm while at a pH of 6, phenol red has ÃŽ »max = 430-435 (LobaChemie, 2015). This change in maximum absorbance with respect to pH is understandable because phenol red is a common pH indicator, meaning it changes color based on pH (Nelson Cox, 2008). The literature and experimental wavelengths of maximum absorbance are summarized in Table 3. The experimental and literature values were fairly close, and their variance may be accounted for due to difference in the buffer and pH of the solvent. Table 3. Literature and experimental wavelengths of maximum absorbance. The experimental wavelengths of maximum absorbance (ÃŽ »max) were obtained from Table 2. The literature ÃŽ »max of hemoglobin (HgA) was based on Akuwudike et al (2010). The literature ÃŽ »max of phenol red at pH = 8.2 was based on LobaChemie (2015). Question 2: Based on the data collected in this experiment, it is impossible to determine whether the eluted hemoglobin is free of phenol red. To determine its purity, spectroscopy of the eluted hemoglobin (fraction B) would need to be conducted in order to draw a wavelength-vs-absorbance graph between 380nm and 600nm (similar to the one posted on Blackboard). If the eluted hemoglobin curve has only one peak (ÃŽ »max = 410-415nm), it contains only hemoglobin. However, it the curve has two peaks (ÃŽ »max = 410-415nm and ÃŽ »max = 557-5605nm), then the eluted fraction contains both hemoglobin and phenol red. Question 3: Search Engine: Web of Science Search Terms: size-exclusion gel filtration (sorted by relevance) Reference: Wang, Q., et al. (2005). Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods. Carbohydrate research, 340(17), 2637-2644. In this experiment, size-exclusion gel filtration was carried out, so hemoglobin and phenol red were separated based on size or more quantitatively based on molecular weight (Nelson Cox, 2008; Wang et al., 2005). Smaller molecules, such as phenol red, spend more time in the pores and are eluted in later fractions (Nelson Cox, 2008). Conversely, larger molecules molecules, such as hemoglobin, spend almost no time in the pores and are eluted in earlier fractions (Nelson Cox, 2008). Question 4: Search Engine: Web of Science Search Terms: chromatography theory (sorted by relevance) Reference: Lee, W. C., Tsai, G. J., Tsao, G. T. (1993). Analysis of chromatography by plate theory. Separations Technology, 3(4), 178-197. Search Engine: PubMed Search Terms: chromatography theory column length (sorted by relevance) Reference: Bedani, F., et al. (2006). A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatographyÃâ€" liquid chromatography. Journal of Chromatography A, 1133(1), 126-134. Based on the plate theory of chromatography, the resolution (R) is influenced by numerous factors including the plate number (N) (Lee et al., 1993). The plate number is affected by many elements of the column including column length and inner column radius (Bedani et al., 2006). Based on mathematical relationships, it has been determined that longer columns lead to increased resolution (Lee et al., 1993; Nelson Cox, 2008). Thus longer chromatography columns will separate molecules better than shorter columns. Question 5: In order to separate ÃŽ ²1,3-galactosidase (75,000Da) and ÃŽ ²1,2-xylosidase (MW 100,000Da) the Sephadex G-100 resin would be ideal. This is because the molecular weight of both enzymes is within the fractionation range of this resin (4,000-150,000Da) (de Jong et al., 2015). The heavier protein (ÃŽ ²1,2-xylosidase) will elute before the lighter protein (ÃŽ ²1,3-galactosidase). Question 6: The fractionation range of Sephadex G-100 is 4,000-150,000Da (de Jong et al., 2015). Alcohol dehydrogenase (MW 140,000Da) falls within this range, while aldehyde dehydrogenase (MW 200,000Da) is above the range. Since both enzymes are not on the same extreme of the fractionation range, aldehyde dehydrogenase will elute very quickly and alcohol dehydrogenase will elute shortly after. Thus, Sephadex G-100 can be used to resolve alcohol dehydrogenase and aldehyde dehydrogenase. However, it would be ideal to use Sephadex G-200 because the molecular weight of both enzymes would be within the fractionation range of 5,000-600,000Da (de Jong et al., 2015). Question 7: Search Engine: PubMed Search Terms: â€Å"fast protein liquid chromatography† (sorted by relevance) Reference: Sheehan, D., O’Sullivan, S. (2004). Fast protein liquid chromatography. In Protein purification protocols (pp. 253-258). Humana Press. Search Engine: PubMed Search Terms: â€Å"fast protein liquid chromatography† (sorted by relevance) Reference: Goke, B., Keim, V. (1992). HPLC and FPLC. Recent progress in the use of automated chromatography systems for resolution of pancreatic secretory proteins. International Journal of Pancreatology, 11(2), 109-116. Search Engine: PubMed Search Terms: â€Å"fast protein liquid chromatography† (sorted by relevance) Reference: Lavanya, G., et al. (2011). Protein Chromatography. J Biomed Sci and Res, 3(3), 424-438. Search Engine: Google Search Terms: fast protein liquid chromatography Reference: Bio-Rad. (2015). Fast Protein Liquid Chromatography. Bio-Rad Life Science Research.Retrieved February 22, 2015, from http://www.bio-rad.com/en-ca/applications-technologies/fast-protein-liquid-chromatography Search Engine: PubMed Search Terms: fast protein liquid chromatography use Reference: Verbeke, K., Verbruggen, A. (1996). Usefulness of fast protein liquid chromatography as an alternative to high performance liquid chromatography of 99m Tc-labelled human serum albumin preparations. Journal of pharmaceutical and biomedical analysis, 14(8), 1209-1213. Fast protein liquid chromatography (FPLC) is an automated chromatography technique that uses columns with a small inner diameter (Goke Keim, 1992; Sheehan O’Sullivan, 2004). By means of pumps and valves, the FPLC system regulates how fast the liquid mobile phase moves down the agarose stationary phase a constant, and maintains a constant flow rate (Lavanya et al., 2011). As well, based on the readings of the UV detector and conductivity meter, the FPLC system is able to control how and when the eluted fractions should be separated (Bio-Rad, 2015). There are variations of FPLC that allow proteins to separated base on size, charge or hydrophobicity (Verbeke Verbruggen, 1996). FPLC, as its name suggests is quite useful in analyzing and purifying protein mixtures (Lavanya et al., 2011). For example, FPLC has been used to identify zinc-containing proteins in rat saliva; and it has also been used to separate and analyze pancreatic juice in order to examine disease-causing proteins (Lavanya et al., 2011). Conclusion In this lab, a mixture of hemoglobin and phenol red was resolved using sizeexclusion gel filtration. Since Sephadex G-25 (fractionation range 1,000-4,000Da) was used as the column matrix, it was expected that hemoglobin would be completely excluded from the matrix pores, allowing it to elute in early fractions. As well, it was anticipated that phenol red would be completely included in the gel, making it elute in later fractions. The hemoglobin/phenol red mixture was separated into four fractions and their respected volumes are shown in Table 1. In theory, fraction A contained void volume, fraction B contained hemoglobin, fraction C contained the buffer only, and fraction D contained phenol red. Due to time and budget constraints, the absorbance of the eluted fractions was not analyzed. However based on an absorbance-vs-wavelength graph, the wavelengths of maximum absorbance (ÃŽ »max) for standard hemoglobin (ÃŽ »max = 410nm) and standard phenol red (ÃŽ »max = 560nm) were determined, as described in Table 2. These values were also compared to literature values, as shown in Table 3. Normal adult hemoglobin (HgA) is known to have maximum absorbance at ÃŽ »max = 415nm, while phenol red is known to have ÃŽ »max = 557-560nm. The experimental and literature values were fairly close to one another, and their slight variance may be accounted for due to difference in the buffer used and pH of the solvent. For further research, spectroscopy of the eluted hemoglobin (fraction B) would need to be conducted in order to draw a wavelength-vs-absorbance graph between 380nm and 600nm. This would help to clarify whether the eluted hemoglobin is free of phenol red. References Akuwudike, A. R., Chikezie, P. C., Chilaka, F. C. (2010). Absorption spectra of normal adult and sickle cell haemoglobins treated with hydrogen peroxide at two pH values. Adv. Biomed. Res, 1, 55-60. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2008).Molecular Biology of the Cell(5th ed.). New York: Garland Science. Bedani, F., et al. (2006). A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatographyÃâ€" liquid chromatography. Journal of Chromatography A, 1133(1), 126-134. Bio-Rad. (2015). Fast Protein Liquid Chromatography. Bio-Rad Life Science Research. Retrieved February 22, 2015, from http://www.bio-rad.com/en-ca/applications-technologies/fast-protein-liquid-chromatography de Jong, M., Botelho, R., Antonescu, C., Wakarchuk, W. (2015).Laboratory Manual: Biochemistry- BCH261(Winter 2015 ed.). Toronto: Ryerson University. DiFeo, T. J., Addison, A. W., Stephanos, J. J. (1990). Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects. Biochem. J, 269, 739-747. Duong-Ly, K. C., Gabelli, S. B. (2013). Gel filtration chromatography (size exclusion chromatography) of proteins. Methods in enzymology, 541, 105-114. GE. (2015). Product Catalog. GE Healthcare Life Sciences. Retrieved February 21, 2015, from http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/ GELifeSciences/products/products_by_technology/ Goke, B., Keim, V. (1992). HPLC and FPLC. Recent progress in the use of automated chromatography systems for resolution of pancreatic secretory proteins. International Journal of Pancreatology, 11(2), 109-116. Klein, D. R. (2012). Organic chemistry (2nd ed.). Hoboken, N.J.: John Wiley. Lavanya, G., et al. (2011). Protein Chromatography. J Biomed Sci and Res, 3(3), 424-438. Lee, W. C., Tsai, G. J., Tsao, G. T. (1993). Analysis of chromatography by plate theory. Separations Technology, 3(4), 178-197. LobaChemie. (2015). Phenol Red. Laboratory Reagents and Fine Chemicals. Retrieved February 21, 2015, from http://www.lobachemie.com/pH-Indicator-05180/PHENOL-RED-CASNO-143-74-8.aspx Nelson, D., Cox, M. (2008). Lehningers Principles of Biochemistry (5th ed.). New York, N.Y.: WH Freeman and Company. Partridge, M. W. (1952). Chromatography and its applications in pharmacy. Journal of Pharmacy and Pharmacology, 4(1), 217-230. Scott, R. P. (2003). Principles and practice of chromatography. Chrom-Ed Book Series, 1. Seeley, R. (2014). Seeleys Anatomy Physiology (10th ed.). New York, NY: McGraw-Hill. Sheehan, D., O’Sullivan, S. (2004). Fast protein liquid chromatography. In Protein purification protocols (pp. 253-258). Humana Press. Silva, J., Teodoro, J., Afonso, R., Aquino, S., Augusti, R. (2014). Photolysis and photocatalysis of ibuprofen in aqueous medium: characterization of byà ¢Ã¢â€š ¬Ã‚ products via liquid chromatography coupled to highà ¢Ã¢â€š ¬Ã‚ resolution mass spectrometry and assessment of their toxicities against Artemia Salina. Journal of Mass Spectrometry, 49(2), 145-153. Verbeke, K., Verbruggen, A. (1996). Usefulness of fast protein liquid chromatography as an alternative to high performance liquid chromatography of 99m Tc-labelled human serum albumin preparations. Journal of pharmaceutical and biomedical analysis, 14(8), 1209-1213. Wang, Q., et al. (2005). Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods. Carbohydrate research, 340(17), 2637-2644.

Looking at “The Withered Arm” by Thomas Hardy Essay

Looking at â€Å"The Withered Arm† and at least two other short stories, comment on how Thomas Hardy uses the female characters to influence the reader’s response To prepare for this essay I have read a selection of Thomas Hardy’s short stories: â€Å"The Withered Arm†, â€Å"The Melancholy Hussar of the German Legion†, â€Å"The Distracted Preacher†, â€Å"Tony Kytes, the Arch Deceiver† and â€Å"Absent-mindedness in a Parish Choir†. The first three stories have been studied more closely than the latter two, and will be used to answer the essay title. Given that a reader’s response may be influenced by many factors, such as time of reading, gender, and personal values it is still clear how Hardy expects his readers to respond. Hardy has moulded the language in many ways, resulting in an intricately woven and complex idea of each character in the reader’s mind. There are certain similarities in each story. Hardy seems to use clever twists of coincidences which are not the fault of the unfortunate character/s involved. Tragedy and death also tend to characterise his stories, for example, Matthà ¯Ã‚ ¿Ã‚ ½us and Christoph are shot and Rhoda Brook’s son is hanged. The reputation of women plays a big part in all three of the short stories. â€Å"The Withered Arm† differs from the other two stories as it has two main women characters, who are naturally compared, in a subtle but effective manner. Rhoda Brook is first introduced into the story through another characters speech, as â€Å"she† and later in the sentence is described as a â€Å"thin worn milkmaid†. This gives the reader a clear picture of Rhoda before she even enters the story: she is shown as little more than a solitary victim of other people’s gossip. Hardy then presents Rhoda in her home, which is in the lonely and rural countryside. The house is made of mud-walls and is rather rundown and ramshackle: â€Å"in the thatch above a rafter showed like a bone protruding through the skin†. The house and Rhoda seem similar as their looks are both past their best. Rhoda is often presented in the context of a serious and lonely landscape: â€Å"thick clouds made the atmosphere dark†. Rhoda is never a happy character and Hardy shows this well, through descriptions of gloomy landscapes. Rhoda has broken social convention by having a son out of marriage and it seems as if she is being punished for this as the story unravels. Rhoda struggles to look after her son alone, and lives in near poverty. Rhoda does not seem particularly disturbed by her situation but it has cut her off from the rest of the small farming community. Hardy has cleverly associated Rhoda with sadness and superstition. Whenever Rhoda is being mentioned in the story the atmosphere is anxious, sad or tense, for example: â€Å"Brook felt like a guilty thing†. Initially, Hardy places Gertrude on the bright forefront, emphasizing her outgoing, carefree personality, while Rhoda is left as a figure in the background. Rhoda’s speech is limited but even when she does speak it is normally about Gertrude. Hardy uses Rhoda to show the harshness of social convention on women. Rhoda is eventually so excluded from her community that she moves away. Rhoda is threatened by her reputation as she does not want Gertrude to find out about her being the possible cause of the damaged arm. Overall, Rhoda’s situation provokes a sympathetic response from the reader. Gertrude is initially introduced into the story with the landscape in her favour: â€Å"the low sun was full in her face, rendering every feature, shade, and contour distinct†. She is shown as generous, caring, (helping those less fortunate than herself) gentle and innocent; emotionally and sexually. Her speech and behaviour are described with gentle language: â€Å"innocent young thing†. Gertrude changes considerably over time, and is slowly transformed into the spectre of Rhoda’s dream. Hardy prepares the reader by starting to taint Gertrude with Rhoda’s main associations, and gradually Gertrude becomes a darker and more sinister figure. This is shown clearly when Gertrude’s subconscious mind is praying: â€Å"O Lord, hang some innocent or guilty person soon†. She is described as â€Å"cunning†, â€Å"shrivelled† and â€Å"furtive†. Hardy shows what a monster an innocent young woman can become while trying to cope with the harsh world of the 1800’s when her marriage and reputation is all she has. Gertrude failed her marriage in two ways, firstly she did not have children, and secondly, Farmer Lodge no longer found her attractive because of her withered arm (and this was the main motive for her wanting to cure it). Hardy showed that without a respectable marriage and children, many women felt that their lives were inadequate. Social convention and reputation ruled many women’s lives. This makes the reader feel sympathetic towards women living in that time, and highlights the amount of freedom women have today. â€Å"The Melancholy Hussar†, contains one main female character, Phyllis Grove, who is presented as a figure of the past. Throughout the story Phyllis is rarely described physically; she is an invisible character. Hardy describes Phyllis’ feelings extensively through direct authorial comment, as she says very little. Phyllis is also closely connected with the landscape, and is part of its timelessness. At the beginning of the story, Phyllis’ seclusion is shown through this: â€Å"secluded old manor houses and hamlets lie in the ravines†. In addition, the landscape at this stage in the story is very bleak, symbolising the bleakness of Phyllis’ lifestyle. Phyllis spends most of the story outside, and words describing her are most energetic outside: â€Å"climbs†, â€Å"walks† and â€Å"clambers†. When Phyllis becomes dislocated from what makes her happiest, she becomes dislocated from her landscape; Phyllis was indoors for weeks after she saw Matthà ¯Ã‚ ¿Ã‚ ½us and Christoph being shot. To highlight Phyllis’ shyness, she uses the fog and mist to hide her away and protect her. Hardy uses Phyllis to show women’s isolation, which can be due to men; Phyllis’ father is unsympathetic towards her situation and Phyllis’ only hope of having a better life would be through marriage. Phyllis’ life seems to be mapped out before the story unravels because she is connected with men right at the beginning of the story, in this case her father. Phyllis’ only escape from her desperate isolation seems to be to marry, suitable Humphrey Gould, and their engagement is used by Hardy to convey the fact that marriages were seen as respectable and even an accomplishment. Hardy uses the York Hussars as a contrast to â€Å"suitable† and â€Å"ordinary† Humphrey; the Hussars are shown with excitement and passion â€Å"crowds of admirers† and â€Å"foreign air†. Hardy then uses contrast once again regarding Matthà ¯Ã‚ ¿Ã‚ ½us Tina; this man is different and catches Phyllis’ attention. When Phyllis is with Matthà ¯Ã‚ ¿Ã‚ ½us she is especially alive, as her speech is direct and not shown through reported speech or authorial comment. Also, this seems to be the only time she can actually be heard. In addition, she is described using the most animated language when she is with Matthà ¯Ã‚ ¿Ã‚ ½us: â€Å"flushed†, â€Å"agitation†, and â€Å"shaked†. The reader wants Phyllis and Matthà ¯Ã‚ ¿Ã‚ ½us’ relationship to survive, as Phyllis is obviously so happy with him. This is what makes the ending so tragic. The stone wall is the place of Matthà ¯Ã‚ ¿Ã‚ ½us and Phyllis’ meetings, and it symbolises an important boundary between them. This figurative language used by Hardy is not only showing the physical but also the moral boundary (Phyllis’ engagement) between the two lovers. Phyllis has a quiet independence, which Hardy shows through her willingness to elope with Matthà ¯Ã‚ ¿Ã‚ ½us; she is prepared to defy social convention. Hardy shows Phyllis as a victim of circumstance and coincidence, because when she hears that Humphrey has bought her a present, the only thing that holds her back from eloping is her conscience and honesty. Here Hardy indicates that one small, seemingly insignificant moment can change one’s life forever. This can make the reader feel quite insecure and that the world is unjust; Phyllis suffered for the rest of her life simply because she was a good person. Irony is used when Phyllis is buried near Christoph and Matthà ¯Ã‚ ¿Ã‚ ½us, near the wall where Matthà ¯Ã‚ ¿Ã‚ ½us and her met. The most well known marriage vow contains â€Å"until death do us part†, though ironically they failed to be together in life, and death brought them together. It is also ironic because marriage was meant to be Phyllis’ escape from her secluded life but in fact it was marriage that prevented her freedom. Hardy’s harsh world also extends to men: for example in â€Å"The Withered Arm† when Rhoda Brooks’ son is hanged. In fact, Matthà ¯Ã‚ ¿Ã‚ ½us and Christoph were subtly shown as heroes because they took the blame for the other two people found in the boat and as a result were shot. The final story â€Å"The Distracted Preacher† contains one main female character, Lizzy Newberry. Lizzy completely strays from the social conventions of the time. Lizzy, like Rhoda, is introduced to the reader through someone else’s speech, but in a completely different manner: â€Å"she won’t have† is used when Mr Stockdale is being informed about her, telling you immediately that she is confident and assertive. Lizzy’s personality is also shown through her leading Mr Stockdale to various destinations and giving him instructions. Hardy makes it clear that Mr Stockdale cannot take her for granted as he is always the one waiting for her and how he looks forward to seeing her: Mr Stockdale â€Å"longed for the morrow†. He cannot control her life as she is so independent and unpredictable. The environment surrounding Lizzy is â€Å"snug and cheerful†, unlike the rural countryside of Rhoda’s and Phyllis’. Other environments associated with Lizzy are smugglers caves such as â€Å"Daggers Grave†; these show her life as exciting and exhilarating. Hardy makes Lizzy the first to speak between her and Mr Stockdale; she is confident as she is the one that approaches him. Lizzy being a widow puts her in a good position as she can be sexually experienced yet single, without it being a scandal. Hardy uses very sophisticated language to describe Lizzy, this shows her subtle superiority and sophistication. He also uses animated language such as â€Å"ascended the hill† and â€Å"tripped at a quickening pace† to show her freedom. Hardy shows Mr Stockdale as rather naà ¯Ã‚ ¿Ã‚ ½ve, by him automatically assuming that a conversation is about love or marriage, when Lizzy is talking to the miller. Also, it takes him a long time to understand what Lizzy does to make money, even after so many clues: Lizzy’s irregular sleeping patterns, her absence from her bedroom at night and him discovering a man’s freshly washed clothes in his room. Lizzy dresses like a man when going smuggling, symbolising her masculinity. In fact, Lizzy is like the man of the house; she makes all the choices and makes money. At one point, Lizzy handles money and this is very symbolic of her freedom. In the other two stories men are hurt physically and in this story Hardy uses this to show Lizzy as masculine; she was shot in the hand. In â€Å"The Withered Arm† Rhoda is jealous of Gertrude for being with Farmer Lodge, and in â€Å"The Melancholy Hussar† Phyllis stays inside for four weeks because she is so upset about Matthà ¯Ã‚ ¿Ã‚ ½us. In this story, however, Mr Stockdale gets upset about Lizzy. For a few days he becomes â€Å"sad† and â€Å"restrained† when he starts to suspect that there might be another man in Lizzy’s life. Also, there is a lack of choices for Mr Stockdale regarding his job â€Å"brought with a single eye to ministry†; in the previous stories the women tend to lack choices. Social convention expected women to centre their lives around men but Lizzy was not prepared to give up her life for a man. Lizzy even suggests that Mr Stockdale gives up his ministry to marry her. I think Hardy implies that money plays a big part in independence, because in the previous short stories women were restrained because of their lack of money, and in this story Lizzy is economically independent and has freedom. â€Å"Render unto Caesar the things that are Caesar’s† â€Å"†He’s dead,† she pouted.† This quote shows Lizzy’s rebelliousness but ironically, she ends up writing a book called â€Å"Render unto Caesar†. Lizzy’s marriage and apology to Mr Stockdale show that she eventually conformed to social convention. In the author’s note Hardy says he would have preferred Lizzy to marry Jim the smuggler, and emigrate to America, and that the other ending was only written because of the expectations of the time of publishing. So, even Hardy had to conform to the conventions of the day. The latter ending is more modern and clearly shows the influence of time on Hardy and his admiration of Lizzy. Throughout these short stories Hardy has shown a wide range of characters and has used different women in varying ways to achieve different purposes. Hardy is shown to have a good understanding of the women in that time through these stories. Even though all three of the short stories were written in the third person, Hardy has used language ingeniously. He has evoked many responses from the reader, but predominantly it is one of sympathy for the women.

Li and Fung: Growth for a Supply Chain Specialist Essay

The case study for this paper is on a Hong Kong-Based Li and Fung Limited â€Å"Li and Fung†. This paper will try to answer the question of â€Å"What would Li and Fung do to safeguard the growth of its business? How could it achieve its target turnover of US$20 billion between 2008 and 2010?† Increase the efficiencies in its global value chain process and to ensure continued growth in net income. One of the primary strategies which set the stage for strong robust growth was the implementation of intranet and extranet information technologies. The internet technology standardized systems across the organization and linked the company’s offices and manufacturing sites throughout the world, encouraging easy tracking of orders and improved quality. The extranet technology linked the company directly to the customer and enabled it to meet and sometimes even exceed customer satisfaction requirements. The strong history of the company and the implementation of information systems and technologies enabled Li & Fung to orchestrate the whole value chain process in a virtual manufacturing environment. Lifung.com, studiodirect.com, ‘electronic stock offer’ and other systems and business processes were implemented to enable the firm to further penetrate into new buyer markets and also to discover opportunities in the supplier markets. This case analyzes the effectiveness of these information and technology systems and recommends steps that could be taken by Li & Fung to draw upon its traditional strengths and explore new opportunities for future growth.

The Values of a Warrior Society as Seen in Beowulf Essay

The Values of a Warrior Society As Seen in Beowulf When Nelson Mandela, the president of South Africa died, thousands of people gathered to say their goodbyes. It was not a happy and joyous occasion but rather one where people were mourning of the loss of a great man. They may not have personal known him but they know of the great things that he has done to make the world a better place. To them, it is what made his death difficult and sad. However, if we lived in a warrior society his death would not have something to mourn; instead we would celebrate his life. This is seen in the epic Beowulf as the people of Geatland lay him to rest and celebrate his life. The celebration reveals that the society knows what a great king is and that†¦show more content†¦Scyld’s funeral is not a somber occasion, but rather a joyous celebration. The people do not mourn that he is dead but rather rejoice for the life that he led. Their celebration shows that they do not grieve death. I n this warrior society death is seen as honorable. The one who has died has given the ultimate sacrifice, death, for their people. They may have followed their leader into battle and died or they may have died of old age as is the case with Scyld. This is confirmed later on when Beowulf is speaking Hrothgar on the death of his best thane. Beowulf states, Wise sir, do not grieve. It is always better to avenge dear ones than to indulge in mourning. For every one of us, living in this world means waiting for our end. Let whoever can win glory before death. When a warrior is gone, that will be his best and only bulwark (lines 1384-89). This statement solidifies what is hinted at earlier on in the epic during Scyld’s funeral. It is part of a heroic code. Death is what comes to people who have earned it in their dedication to a cause or great warriors who have earned it. Since death is found to be so honorable, the people do not mourn for the loss of their king but celebrate his de ath. Scyld’s funeral plays an important role in discovering what was important to the society at this time including honor in death and the qualities found in a greatShow MoreRelatedBeowulf Is The Oldest Recorded Poem1328 Words   |  6 Pagespoem, Beowulf, is the oldest recorded poem in English and at 3200 lines long, comprises roughly ten percent of the surviving poetry in Old English. Although written in this language, Beowulf focuses on the feats of the poem’s namesake Beowulf, a Geatish prince, and the invading Germanic tribes in Denmark. Understood early on in the poem, these tribes have a lengthy and powerful warrior culture; a culture heavily influenced by heroic virtues, blood vengeance, and paganism. Along with these values ofRead MoreGenerosity And Values Of The Epic Of Beowulf884 Words   |  4 Pagesthe Anglo-Saxons during this time period. One of the most well known epics Beowulf, celebrates the deeds of the legendary and heroic Geat warrior named Beowu lf. 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