Chromatographic Separation of Hemoglobin from Phenol Red

chromato chartical Separation of hemoglobin from Phenol RedSyed IbrahimChromatographic Separation of Hemoglobin from Phenol Red by gelatin Filtration admissionFrom catalysis to transport, proteins take part in countless cellular processes (Nelson Cox, 2008). However, due to their small size, proteins butt be challenging to work with in the lab. To bounce back this hurdle, scientists collapse developed many splitation techniques including centrifugation, precipitation, dialysis, and chromatography (Alberts et al., 2008).Chromatography is one of the most various(a) laboratory techniques as it is implement in many fields of survey (Partridge, 1952 Silva et al., 2014). on that point argon several types of chromatography, and each separates molecules found on different properties. Size-censure 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 hyaloplasm with pores of a p exitetermine size (Scott, 2003). Small molecules be more than likely to spend clock sequence in the pore, so they migrate slower and thus wash in later(prenominal) components (Nelson Cox, 2008). Conversely, larger molecules are less likely to enter the pore, so they migrate profuseer and thus rinse in earlier fractions (de Jong et al., 2015).There are numerous types of column matrices, such(prenominal) as Sephadex G (composed of dextran and epichlorohydrin) and Sepharose (consisting of agarose) (GE, 2015). individually matrix has different sized pores and thus different sends of molecular(a) sizes that it gutter separate, called fractionation cathode-ray oscilloscopes (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 oxybenzene red were also used. Hemoglobin is a protein institute primarily found in mammalian red blood cells, and is used to transport oxygen from an organisms 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 haemoglobin 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.PurposeThe goal of this lab was to separate a mixture containing haemoglobin and phenol red development size-exclusion gel filtration. As well, the aim was to study the threadbare and literary works wavelengths of grievous bodily harmimum absorption of hemoglobin and phenol red.ResultsThe results from this taste are shown in get across 1 and flurry 2.Table 1. ele ment volumes from size-exclusion gel filtration using Sephadex G25.The size-exclusion gel filtration separated a mixture of hemoglobin and phenol red. In possible action, 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 visibleness between 380nm and 600nm of two standard samples was determined. The standard hemoglobin sample contained 250L of 0.8% hemoglobin, while the standard phenol red sample consisted of 20L of 0.2% phenol red. Based on the graph, the supreme absorbance values and the corresponding wavelengths were determined. doubtfulnesss and exploreQuestion 1 calculate railway locomotive electronic network of science calculate legal injury hemoglobin spectroscopy citation DiFeo, T. J., Addison, A. W., Stephanos, J. J. (1990). Kinetic and spectroscopic s tudies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects. Biochem. J, 269, 739-747. take care locomotive Web of ScienceSearch Terms hemoglobin Soret mob absorptivity (sorted by relevance) file name extension Akuwudike, A. R., Chikezie, P. C., Chilaka, F. C. (2010). assiduousness spectra of normal adult and sickle cell haemoglobins treated with hydrogen bleach at two pH values. Adv. Biomed. Res, 1, 55-60.Search Engine GoogleSearch Terms phenol red uttermost wavelength absorptionReference LobaChemie. (2015). Phenol Red. science laboratory Reagents and delicately Chemicals. Retrieved February 21, 2015, from http//www.lobachemie.com/pH-Indicator-05180/PHENOL-RED-CASNO-143-74-8.aspxBased on the graph from Blackboard, the wavelengths of oozeimum absorbance (max) are shown in Table 2. For the standard hemoglobin sample max = 410nm, and for the standard phenol red sample max = 560nm.Based on the literature, hemoglobin and think proteins are cognise to give th ree bands of maximum absorption -band, -band, and Soret band (DiFeo et al., 1990). For normal adult hemoglobin (HgA), the wavelength of maximum absorbance in the Soret band is 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 max = 557-560nm while at a pH of 6, phenol red has max = 430-435 (LobaChemie, 2015). This change in maximum absorbance with respect to pH is understandable because phenol red is a crude 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 most, and their variance may be accounted for due to residuum in the buffer and pH of the solvent.Table 3. Literature and experimental wavelengths of maximum absorbance.The experimental wavelengths of maximum absorbance (max) were obtained from Table 2. The literature max of hemoglobin (HgA) was based on Akuwudike et al (2010). The literature max of phenol red at pH = 8.2 was based on LobaChemie (2015).Question 2Based on the data collected in this experiment, it is impossible to determine whether the eluted hemoglobin is costless 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 stick on on Blackboard). If the eluted hemoglobin curve has only one peak (max = 410-415nm), it contains only hemoglobin. However, it the curve has two peaks (max = 410-415nm and max = 557-5605nm), then the eluted fraction contains both hemoglobin and phenol red.Question 3Search Engine Web of ScienceSearch 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 ligh t scattering methods. wampum 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 4Search Engine Web of ScienceSearch Terms chromatography theory (sorted by relevance)Reference Lee, W. C., Tsai, G. J., Tsao, G. T. (1993). summary of chromatography by plate theory. Separations Technology, 3(4), 178-197.Search Engine PubMedSearch Terms chromatography theory column length (sorted by relevance)Reference Bedani, F., et al. (2006). A theoretical infrastructure for parameter selection and instrument design in comprehensive size-exclusion chromatography liquified chromatography. diary of Chromatography A, 1133(1), 126-134.Based on the plate theory of chromatography, the effect (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 roentgen (Bedani et al., 2006). Based on mathematical relationships, it has been determined that longer columns lead to increase resolution (Lee et al., 1993 Nelson Cox, 2008). Thus longer chromatography columns leave alone separate molecules better than shorter columns.Question 5In order to separate 1,3-galactosidase (75,000Da) and 1,2-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 (1,2-xylosidase) will elute before the lighter protein (1,3-galactosidase).Q uestion 6The 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 7Search Engine PubMedSearch Terms riotous protein fluid chromatography (sorted by relevance)Reference Sheehan, D., OSullivan, S. (2004). Fast protein liquifiable chromatography. In Protein purging protocols (pp. 253-258). Humana Press.Search Engine PubMedSearch Terms fast protein runny chromatography (sorted by relev ance)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 PubMedSearch Terms 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 GoogleSearch Terms fast protein liquid chromatographyReference 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-chromatographySearch Engine PubMedSearch Terms fast protein liquid chromatography useReference Verbeke, K., Verbruggen, A. (1996). Usefulness of fast protein liquid chromatography as an election to high performance liquid chromatography of 99m Tc-labelled charitable serum albumin preparations. Journal of pharmac eutical 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 OSullivan, 2004). By means of pumps and valves, the FPLC system regulates how fast the liquid mobile manikin moves down the agarose stationary phase a constant, and maintains a constant combine 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 localise zinc-containing proteins in rat saliva and it has also been used to separate and decompose pancreati c juice in order to examine disease-causing proteins (Lavanya et al., 2011).ConclusionIn 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 (max) for standard hemog lobin (max = 410nm) and standard phenol red (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 max = 415nm, while phenol red is known to have max = 557-560nm. The experimental and literature values were fairly close to one another, and their slight variance may be accounted for due to divagation 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.ReferencesAkuwudike, A. R., Chikezie, P. C., Chilaka, F. C. (2010). submerging spectra of normal adult and sickle cell haemoglobins treated with hydrogen hydrogen peroxide at two pH values. Adv. Biomed. Res, 1, 55-60.Alberts, B., earth-closetson, A., L ewis, 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 chromatography 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-chromatographyde 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 enz ymology, 541, 105-114.GE. (2015). Product Catalog. GE Healthcare Life Sciences. Retrieved February 21, 2015, from http//www.gelifesciences.com/webapp/wcs/stores/servlet/ catalogue/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.aspxNelson, D., Cox, M. (2008). Lehningers Principles of Biochemistry (5th ed.). New Yor k, 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 hand Series, 1.Seeley, R. (2014). Seeleys Anatomy Physiology (10th ed.). New York, NY McGraw-Hill.Sheehan, D., OSullivan, 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 byproducts via liquid chromatography coupled to highresolution mass spectrometry and assessment of their toxicities against genus 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 prepar ations. 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.