Liquid Chromatography Lab

runniness Chromatography Laboratory 18 Introduction We ar using fluid chromatography to secernate the colo departure substances in grape-flavored drinks. We separate the part sullys, and and so we separate the flavorings and citric stingings. Background Chromatography is a process that is employ to separate a substance into its comp starnt move. The separation occurs between the nonmoving and pathetic anatomy of the lab. The moving kind consists of a fluid and the stationary grade consists of a potent. The sort we ar trying to fragmented up is integrated into the peregrine var..When the erratic degree interacts with the substantive variety, the components of the mixture argon attracted to the solid state phase in varying forms. Components with higher levels of attraction for the supple phase result in a red-hot speed of transport doneout the solid phase. Components with higher levels of attraction for the solid phase result in a slow-moving speed of tra nsport throughout the solid phase. These differing levels of attraction block off up in separation of the mixture into component sights, which electric receptacle the governance as distinct liquids. pic semiliquid chromatography labs are composed of six important parts . A separation tug filled with a fine-grain solid. b. A resolving power ( officious phase) that moves through the separation pillar. c. An drop by the waysideion system to transport the root to the separation chromatography tugboat. d. A pump to force the root through the separation column. e. A detector to crack when the components expiry the separation column. f. A recorder pic Although in most chromatography labs the solid phase is polar and the mobile phase is unionized, we are using Reverse Phase Liquid Chromatography, where the mobile phase is polar and the solid phase is nonpolar.Also read Fluorenol PolarityWhen the mobile phase interacts with the solid phase, the polar parts of the mobile pha se are attracted to distributively other, so they wash through the column quickly. The less polar components of the mobile phase are attracted to the nonpolar solid in the column, so they wash through the column slowly. This results in a separation of the components, whose degree is measured as the re resolve. pic Pre-lab Questions 1. What is the process of chromatography handling for? 2. In the chromatography, components of a mixture pass themselves between the stationary phase and the mobile phase.Explain how the components cease be separate with these 2 phases. 3. In the liquid chromatography column employ in this experiment, the solid has a C18 hydrocarbon bonded to it. Would a C18 hydrocarbon be polar or nonpolar? Explain. 4. Below are the typical information for this experiment. 1 mL of a Kool-Aid solution was loaded on a Sep-Pak C18 Column. The red and blue soils were eluted from the column with a uninterrupted rise of 18% isopropyl inebriant. The eluted solution was put in in a 10 mL gradational cylinder. The piles of eluant were recoreded at the number one and end of severally color tie. pic Information The jump step in calculating the selectivity and resultant role of the system is determining the good deals of eluant corresponding to the roach widths and band come tos for to apiece one eluted dye. a. mintwidth W is the volume in mL of eluant containing distributively dye as it emerges from the column. Calculate the bandwidth W for each dye for each of the three runs and then determine the fair(a) bandwidth W ordinary for each dye. b. Center of band, called just remembering Volume V spurt corresponds to the center of each band.The intermediate retention volume is mensural by taking the sightly starting volume for each band and adding one half the corresponding fair(a) band width. V rant = V start + (? ) W ave Calculate the average retention volume volume V Rave for the red and blue dyes. c. For each dye, a capacity ingredient k pile be calculated. This term is a relation measure of the attraction of the dye for the stationary phase as compared to its attraction for the mobile phase. The equation fo capacity factor is k = (V Rave V M)/V M here V Rave is the average retention volume for each dye and V M is mobile phase or eluant volume in the cartridge. V M so-and-so be estimated to be one half the cartridge volume, with the stationary phase occupying the other half. For the Sep-Pak cartridges, this V M grade is . 49 mL. Calculate k for each dye. d. A selectivity or separation factor, of import, can now be calculated. This is the ratio of the k determine for each dye, with the bigger value in the numerator. For good separation, a mobile phase is usually chosen that gives an alpha value between 2 and 10. Calculate alpha for this separation alpha = (k depressed)/(k Red) e.The resultant role R, a measure of how well the twain dyes are separated by the column and eluant, is heady by th e equation R = 2(V Rave Blue V Rave Red)/(W Blue + W Red) where the numerator is the volume between the band centers and the denominatory represents the average band width. The greater the selectivity, the larger the numerator and therefore the greater the resolution. The resolution can also increase as the might of the column increases, since this results in a level average band width. Calculate R for this separation. Materials Isopropyl Alcohol, 70% 50 mLIsopropyl Alcohol, 28%, 10 mL Isopropyl Alcohol, 18% 50 mL Isopropyl Alcohol, 5% 10 mL graduate Cylinder, 10mL Graduated Cylinder, 25 mL Distilled Water, ccc mL Grape Koolaid Solution, 20 mL Sep-Pak C18 Cartridge 10 mL syringe w/ male Luer tip Beaker, hundred mL, 3 Beaker, 50 mL, Safety Precautions Isopropyl intoxicantic beverageic beverage is inflammable and a fire hazard. Do not conduct this laboratory in the presence of flames. This inebriantic beverage is slightly cyanogenetic by ingestion and inhalation. Chemical-res istant goggles, gloves, and aprons are required. muffle and rinse hands thoroughly with lather and urine after conducting the lab.Procedure single out 1 Isocratic breakup (Constant rate of flow and solvent concentration) Pretreatment of the Sep-Pak C18 Cartridge 1. Cut off the exit tube/shorter end of the cartridge at the point where it meets the body of the cartridge. 2. Load the syringe with 10mL of 70% isopropyl alcohol. 3. Connect the tip of the syringe to the long end of the Sep-Pak cartridge. 4. Pump the isopropyl alcohol through the syringe cartridge at a rate of 5-10 mL/minute. 5. invite the alcohol in a 10 mL graduated cylinder to varan flow rate. 6. Repeat previous go with distilled water. pattern scene . practise 10 mL syringe to slowly inject 1 mL of Kool-Aid solution onto the column. 2. cast aside the sewer water that washes out. 3. Remove the cartridge from the syringe. 4. dampen the syringe with 10 mL of distilled water 3 times to erase Kool-Aid residue. Sample Elution 1. Fill the syringe with 18% isopropyl alcohol eluant and attach the syringe to the Sep-Pak Cartridge. 2. Pump the alcohol through the cartridge with a flow rate of 5-10 mL/min. 3. stash away outflowing in 10 mL graduated cylinder. 4. Record volume of effluent smooth as first and last of drab drops of each of the dyes exit.If separation is imperfect, record data for beginning/end of intermediate proud bands. Center of the purple band acts as the end of the first band and beginning of the last. Column Regeneration Repeat measurements two more times. Between injections, wash the column with 10 mL of distilled water at the same flow rate of 5-10 mL/min. If colored residue remains, repeat preatreatment. set forth 2 Step slope Separation Now, we change composition of the eluting liquid. We first use a polar solvent, and then we trend the polarity of the solid phase by adding isopropyl alcohol.Through this, we wash out citric acid and flavoring oils in addition. Pret reatment of the Sep-Pak C18 Cartridge come out the pretreatment in Part 1. Sample Injection and Component Elution 1. Inject 1 mL of Kool-Aid solution into the column. 2. Elute polar components of the mixture (citric acid and sugar) by passing 5 mL of distilled water through the column. 3. perk effluent in the first low beaker. 4. Elute the red dye by passing 10 mL of 5% isopropyl alcohol through the column. 5. Collect effluent in the second crushed beaker. 6. map 10 mL of the 28% isopropyl alcohol to elute blue dye. 7. Collect effluent in the third small beaker. . Use 10 mL of 70% isopropyl alcohol to elute nonpolar flavor oils and additives. 9. Collect effluent in the foursometh small beaker. 10. Record the color of each effluent. go away the solvents and examine the components. 1. Allow the solutions to evaporate and go steady them overnight in the fume street fighter until next lab period. Label solutions properly. 2. come after and describe contents of each of the beak ers. handbill using color, odor, and appearance. Data Table Part 1 Isocratic Separation Red dyestuff Blue Dye Run 1 Run 2 Run 3 Run 1 Run 2 Run 3 Start of Band (mL) End of Band (mL) W (mL) Vrave (mL) K Part 2 Step Gradient Separation Beaker Eluant Observations 1 H2O 2 5% isopropyl alcohol 3 28% isopropyl alcohol 4 70% isopropyl alcohol Calculations Determine the following values and show up calculations. Refer to question six in the Pre-Lab Questions. Enter results in the Part 1 data table. 1. Bandwidth W for each dye. 2. Average Retention Volume V Rave for each dye. 3. Capacity Factor k for each dye. 4. Selectivity alpha for the two dyes with this isocratic separation. 5. Resolution R for the two dyes with this isocratic separation.Post-Lab Questions 1. What is meant by polarity of molecules? What causes differences in polarity? 2. In discussing solubility, the rule standardized dissolves like is frequently used. W hat does this mean? 3. snuff it the structural formula of isopropyl alcohol. Explain how it differs in polarity from water. 4. For good separation of the dyes, the resolution should be greater than one. What was the value you calculated? Did the two dyes overlap as they emerged from the column, or was the separation a good one? 5. In the step gradient separation, four separate fractions were collected. How were these related to the polarities of the column and of the eluting solvent?