Thin Layer Chromatography Essay

There are millions of different organic compounds. Most of them are found in mixtures and in order to achieve a pure form they need to be separated, isolated, and purified. However, there are endless numbers of possible mixtures, which make it impossible to have a pre-designed procedure for every mixture. So chemists often have to make their own procedures. The purpose of this experiment was to prepare the student to the real world by them designing their own procedure which will help them understand the techniques of separation and purification better. The goal was to extract two of the components of the

1. Explain why you can’t fully test the lipase activity in tube 5. _Measurement of lipase activity uses a decrease in pH. Because the pH in Tube #5 is already very low, it is hard to tell if fatty acids are released.__

In table three in Tube 1L there was no lipase action present. Also there was not any fat present in mixture. In tube 2L there was also no lipase action nor any lipase present in mixture. In tube 3L there was no lipase action. Lipase was denatured by boiling. In tube 4L there was Lipase action present. All conditions were favorable. In tube 5L there was not any lipase action because the temperatures were too low. In tube 4B Lipase action was present. All conditions were favorable and bile salts accelerated the digestion. In Tube 5B there was no lipase action because the temperatures were too

Brianda Mendez Lab 05 Report Introduction: Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for identifying compounds, determining their purity and following the progress of a reaction. Thin-layer chromatography or TLC, is a solid-liquid form of chromatography, it involves the distribution between two phases. The stationary phase is a polar adsorbent, it is coated on a glass slide or plastic sheet creating a thin layer of the particular stationary phase. The mobile phase, a liquid or a gas flows through the stationary phase and carries the components of the mixture with it.

In this experiment concerning the activity of enzymes we tested the different effects of various concentrations, pH, Temperature, and Inhibitors over intervals of time. For the effects of concentration of enzyme extract, or peroxidase we mixed six of seven tubes to get three different concentrations of extract. By doing this we wanted to know whether the concentration would positively or negatively affect the activity of the enzyme, in which we predicted that the concentration would increase the activity. The three extract concentrations being 0.5ml from the mixing of tubes 2 and 3, 1.0ml from tube 4 and 5, and 2.00ml from 6 and 7, while the first tube of the seven that wasn’t mixed was the control containing zero extract. The control and the mixed tubes together made 8ml, and was poured into cubettes to be placed in the spectrometer at an absorbance of 500nm.

Protein concentration needed to be determined to assure that SDS-PAGE would work effectively since too high or too low protein concentration of each sample would result in erroneous gels. Before measuring the protein concentration in the acetone powder solutions, a BSA curve needed to be created to find a conversion factor. In the BSA curve, there appeared to be a positive correlation between absorbance (A) and the amount of BSA protein (μg) when observing the best fit line (Figure 1). This was confirmed with a slope or conversion factor of 0.0255 A595/μg (Figure 1).

Two pieces of chromatography paper are trimmed so they are exactly 20.5 cm by 11 cm. The paper is handled by its edges only to avoid contamination from finger oils.

This experiment has allowed the determination and calculation of different kinetic parameters for alkaline phosphatase. First, it was necessary to generate a standard curve for PNP presenting the absorbance as a function of the concentration of PNP since the remaining of the experiment is based on measuring absorbance values of PNP. This standard curve is then a good tool to convert those absorbance values into PNP concentration values that can be used to study the kinetics of the alkaline phosphatase enzyme. Once the standard curve was generated, an assay optimization was performed to determine the best enzyme to be used. This was done through catalyzing the reaction of conversion of PNPP to PNP by using different alkaline phosphatase concentrations.

To determine how different temperatures and how they can directly affect the outcome of enzyme activity, we will need to look at the actual temperatures used in the study as well as the specific type of enzyme used. In remembering that enzymes; Proteins speed up the rates of chemical reactions in the types of living organisms used in the lab experiment this report is based upon. With our groups’ hypotheses, including prediction: The determination factor being can a higher temperature decrease the enzyme activity? In this study we as a lab group needed to be able to form conclusions with knowing at what temperatures did we see the results?

Chromatography is a separation technique in which the mixture to be separated is dissolved in a solvent and the resulting solution, often called the mobile phase, is then passed through or over another material, the stationary phase. The separation of the original mixture depends on how strongly each component is attracted to the stationary phase. Substances that are attracted strongly to the stationary phase will be retarded and not move alone with the mobile phase. Weakly attracted substances will move more rapidly with the mobile phase.

Thin layer chromatography (TLC) separates compounds, through migration, from a mixed solution with the assistance of a solvent and an absorbent strip of cellulose. The purpose of this lab is to allow students to determine an unknown amino acid by comparing results to six known amino acids (slowest to fastest: lysine, glycine, alanine, tyrosine, phenylalanine, and leucine) and properly separate a combined solution of amino acids using the TLC method. But to fully understand the lab, two terms must be understood: amino acid and protein. Amino acids are building blocks of proteins linked by peptide bonds that contain both carboxyl and amino functional groups.

Briefly, for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), cell lysate (20 ug protein per lane) samples were loaded onto 10-12 lane Tris-Glycine 4-20% mini-gels (Invitrogen) (Zhang et al., 2009). Following electrophoresis, separated proteins were laterally transferred to polyvinylidene fluoride (PVDF) membranes (iBlot semi-dry transfer system) at a constant voltage of 12 V for 9 min at ambient temperature. After electrotransfer, the membranes were blocked for 1 hr at ambient temperature in 5% non-fat milk prepared in tris-buffered saline containing 0.05% Tween-20 (TBST) followed by incubation at 4o C for 3 hr or overnight with primary antibodies [mouse anti-αII-spectrin (Biomol) or rabbit anti-GFAP (Abcam) at 1:2000 dilution in TBST-5% non-fat milk). This was followed by three washes with TBST, a 2 hr incubation at ambient temperature with a secondary antibody (goat anti-rabbit or anti-mouse IgG conjugated to alkaline phosphatase (Novagen) and developed with substrate BCIP-NBT reagent (KPL). Housekeeping proteins, β-actin and/or carbonic

Chromatography paper is used to separate mixtures and substances into different groups. In paper chromatography a mixture is dissolved and moves with water up a piece of paper. This mixture separates because the different colors(components) move up the paper at different speeds. You use chromatography paper to find different dyes in pens like a sharpie or a marker.

Introduction/Procedure: Thin Layer Chromatography (tlc) is a technique used to separate two or more compounds or ions, using a think later or material made out of silica gel on a glass or plastic plate. There are two phases that contribute, the mobile and stationary phase, the mobile phase is when the molecule in solution has to decide to stay in the solution and the stationary phase is when the solution decides to adhere to the solid. Thin layer chromatography is convenient and used for fast qualitative analysis and is very important in organic chemistry because it helps establish the number of components in a mixture and allows us to conclude if two compounds are identical. Chromatography is additionally a green method; the materials such as the tlc plates and the capillary tubes are inexpensive and easily accessible.

Caspase-3 enzyme activity was assessed applying a protocol organized by Movsesyan (2002). The evaluation is based on the hydrolysis of the p-nitroaniline (PNA) moiety by caspase-3. Briefly, experiment were examined on 96 well culture plate which 20 µl of SH-SY-5Y cells lysates were incubated with assay buffer containing (50 mmol/L HEPES, PH7.4;0.2% CHAPS, 20% Sucrose; 2 mmol/L EDTA and 10 mM dithiothreitol) and a 50 µ mol/L concentration chromogenic pNA specific substrate for caspase-3. Each test sample was taken to 100 µL volume and incubated for 3 h at 37℃. Finally, the amount of released p-nitroaniline was evaluated spectrophotometrically at 405 nm with a microplate reader (Bio Tek, USA). Values were