Spectrophotometer Basics and Instrumentation
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Spectrophotometer Basics and Instrumentation

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What is the primary purpose of a spectrophotometer?

  • To quantify the viscosity of a solution
  • To calculate the mass of a solid sample
  • To measure temperature changes in a solution
  • To detect the absorption spectrum of a compound (correct)

    • Which device is used in a spectrophotometer to split the light beam into its component wavelengths?

  • Photodetector
  • Radiant energy source
  • Cuvette
  • Monochromator (correct)

    • What type of cuvettes are typically used for samples in the ultraviolet region?

  • Plastic cuvettes
  • Metal cuvettes
  • Quartz cuvettes (correct)
  • Ceramic cuvettes

    • Which principle is most spectrophotometer detectors based on?

    <p>Photoelectric effect</p> Signup and view all the answers

    What are the two types of prisms commonly used in commercial spectrophotometers?

    <p>300 Littrow Prism and 600 Cornu Prism</p> Signup and view all the answers

    What is the range of wavelengths that a spectrophotometer's incident light can typically achieve?

    <p>1 to 2 nm</p> Signup and view all the answers

    What is the function of transport vessels in a spectrophotometer?

    <p>To hold the sample being analyzed</p> Signup and view all the answers

    What feature allows a prism to disperse light into its constituent wavelengths?

    <p>Its ability to reflect different wavelengths to different extents</p> Signup and view all the answers

    What is the formula for calculating DNA concentration?

    <p>DNA Concentration = Abs 260 x Dilution factor x 50</p> Signup and view all the answers

    Which type of cuvettes are appropriate for use in a UV spectrophotometer?

    <p>Quartz cuvettes</p> Signup and view all the answers

    What is an essential component of a PCR reaction that contributes to the synthesis of DNA?

    <p>dNTPs</p> Signup and view all the answers

    What is the optimal concentration range of MgCl2 typically used in PCR reactions?

    <p>0.5 to 2 mM</p> Signup and view all the answers

    How should water be sourced for PCR reactions to avoid contamination?

    <p>Chemical supply company water with analysis</p> Signup and view all the answers

    Why can't a single PCR protocol be applied to all situations?

    <p>Each primer pair has varying optimal reaction conditions.</p> Signup and view all the answers

    What is the purpose of the buffer mix in PCR?

    <p>To provide the optimal salt concentration, pH, and cofactors</p> Signup and view all the answers

    What is the common final concentration of dNTP in PCR reactions?

    <p>0.2 mM</p> Signup and view all the answers

    How much PCR buffer is required for a single reaction volume of 50 µl?

    <p>5 µl</p> Signup and view all the answers

    What is the total volume of dNTPs needed for 5 reactions?

    <p>2.0 µl</p> Signup and view all the answers

    Which component should be added last when setting up the master mix?

    <p>Taq Polymerase</p> Signup and view all the answers

    What is the purpose of adding 10% more components when preparing a master mix?

    <p>To account for pipetting errors</p> Signup and view all the answers

    What temperature and duration are set for the final extension in a standard PCR program?

    <p>72 °C for 5 min</p> Signup and view all the answers

    How much DNA template is required for a single PCR reaction?

    <p>250 ng</p> Signup and view all the answers

    What should you do with the tubes after aliquoting the master mix?

    <p>Add DNA template directly</p> Signup and view all the answers

    What is the initial denaturation temperature and time in a standard PCR cycle?

    <p>94 °C for 1 min</p> Signup and view all the answers

    At what pH does the change begin and complete in electrophoresis using TAE?

    <p>pH 4.6 and completes at pH 3.0</p> Signup and view all the answers

    Which buffer has a significantly higher buffering capacity compared to TAE?

    <p>TBE</p> Signup and view all the answers

    How does the migration speed of double stranded linear DNA fragments in TAE compare to TBE or TPE?

    <p>10% faster in TAE</p> Signup and view all the answers

    What is the primary reason for using TAE in Southern blots for complex genomes?

    <p>Better resolution of DNA fragments in complex mixtures</p> Signup and view all the answers

    What is the working solution composition of TAE?

    <p>40 mM Tris-acetate, 1 mM EDTA</p> Signup and view all the answers

    Which buffer is reported to have a slightly better resolving power for high-molecular-weight DNAs?

    <p>TAE</p> Signup and view all the answers

    Which component is NOT part of the 50x stock solution for TAE?

    <p>Boric acid</p> Signup and view all the answers

    What is the main disadvantage of using TAE for low molecular-weight DNAs?

    <p>Worse resolving power than TBE</p> Signup and view all the answers

    Which method is NOT commonly used for visualizing results on a TLC plate?

    <p>Visible light</p> Signup and view all the answers

    What does the Rf value represent in thin layer chromatography?

    <p>Retention factor of compounds</p> Signup and view all the answers

    Which of the following is a limitation of thin layer chromatography?

    <p>It cannot differentiate between enantiomers and some isomers</p> Signup and view all the answers

    Which application is NOT associated with thin layer chromatography?

    <p>Binary classification of organisms</p> Signup and view all the answers

    Which staining method is particularly useful for detecting carbohydrates in TLC?

    <p>Iodine Staining</p> Signup and view all the answers

    What is one of the advantages of using thin layer chromatography?

    <p>It has a short development time</p> Signup and view all the answers

    Why is it important to know Rf values beforehand in thin layer chromatography?

    <p>To identify specific compounds accurately</p> Signup and view all the answers

    Which of the following is NOT an advantage of thin layer chromatography?

    <p>In-depth molecular structure analysis</p> Signup and view all the answers

    Study Notes

    Spectrophotometer

    • A spectrophotometer splits an incident light beam into different wavelengths using a prism or grating.
    • Specific wavelengths can be manipulated to fall on a test solution.
    • The range of wavelengths of the incident light can be as low as 1 to 2nm.
    • The spectrophotometer measures the absorption of light by a solution at each wavelength, which is known as the absorption spectrum of a compound.

    Spectrophotometer Instrumentation

    • Materials excited by a high voltage electric discharge or by electrical heating can serve as excellent radiant energy sources.
    • A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands.
    • Prisms disperse polychromatic light from the source into its constituent wavelengths by virtue of their ability to reflect different wavelengths to a different extent.
    • Two types of prisms are usually employed in commercial instruments: 600 cornu quartz prism and 300 Littrow Prism.
    • Gratings are often used in the monochromators of spectrophotometers operating in ultraviolet, visible, and infrared regions.
    • Samples to be studied in the ultraviolet or visible region are usually glasses or solutions and are put in cells known as “CUVETTES”.
    • Cuvettes meant for the visible region are made up of either ordinary glass or sometimes Quartz.
    • Cuvettes for the ultraviolet range are made of Quartz or UV-transparent plastic.
    • Most detectors depend on the photoelectric effect, where the current is proportional to the light intensity and therefore a measure of it.
    • The following formula is used for calculating the concentration of DNA: DNA Concentration = Absorbance at 260 nm x Dilution factor x 50 (µg/ml or ng/µl).

    Polymerase Chain Reaction (PCR)

    • PCR is a widely used molecular biology technique with applications in diagnostics, tracing evolutionary relationships, cloning of genomic DNA, and cloning of expressed DNA sequences.
    • PCR reaction conditions can vary depending on the primer pair.

    PCR Reaction Components

    • Use only water that is purchased from a chemical supply company that does a full analysis of every batch of water.
    • The buffer mix provides the optimal salt concentration, pH, and cofactors required by the polymerase.
    • dNTPs are required in order for the polymerase to synthesize DNA.
    • MgCl2 is required as a co-factor for most DNA polymerases used in PCR reactions.
    • The optimal reaction concentration of MgCl2 is generally between 0.5 to 2 mM.

    Electrophoresis Buffers

    • TAE, TBE, and TPE are common electrophoresis buffers.
    • TAE is slightly less expensive than TBE and TPE but has a lower buffering capacity.
    • Double-stranded linear DNA fragments migrate ~10% faster through TAE than through TBE or TPE.
    • For analyzing complex genomes, Southern blots are generally derived from gels prepared in and run with TAE as the electrophoresis buffer.
    • The resolution of supercoiled DNAs is better in TAE than in TBE.
    • TBE is usually made and stored as a 5x or 10x stock solution.

    Basic PCR Reaction

    • A master mix is created by combining all the components except the DNA.
    • The water is added first to the master mix, followed by the enzyme last.
    • The DNA template is added to individual tubes containing the master mix.
    • Cycling conditions for PCR are application and primer dependent. Here is a relatively standard program:
      • 35 cycles of:
        • 94 °C for 1 min
        • Tm of primers +/- 2 °C for 1 min
        • 72 °C for 1 min
      • 1 cycle of:
        • 72 °C for 5 min (final extension)
      • Hold at 6 °C until products can be removed from the machine.
    • This is a protocol for setting up a PCR reaction containing 50 µl volume:
      • PCR Buffer (10X) - 5 µl
      • MgCl2 (50 mM)- 1.5 µl
      • dNTPs (25 mM) - 0.4 µl
      • Primer ‘F’ (20 µM) - 1.5 µl
      • Primer ‘R’ (20 µM) - 1.5 µl
      • Taq Polymerase (5 Units/µl)- 1.2 µl
      • DNA (50ng/µl) - 12.5 µl
      • H2O - 26.4 µl
      • Total - 50 µl

    Thin Layer Chromatography (TLC)

    • TLC is a technique used to separate compounds based on their differences in polarity.
    • The process involves applying a sample to a TLC plate coated with a stationary phase, developing the plate in a solvent, and visualizing the separated compounds.
    • Common techniques for visualizing the results of a TLC plate include:
      • UV light
      • Iodine Staining
      • KMnO4 stain
      • Ninhydrin Reagent

    Applications Of Thin Layer Chromatography (TLC)

    • Monitoring the progress of reactions
    • Identifying compounds present in a given mixture
    • Determining the purity of a substance
    • Analyzing ceramides and fatty acids
    • Detecting pesticides or insecticides in food and water
    • Analyzing the dye composition of fibers in forensics
    • Assaying the radiochemical purity of radiopharmaceuticals
    • Identifying medicinal plants and their constituents

    Advantages of Thin Layer Chromatography (TLC)

    • Simple process with a short development time
    • Easy visualization of separated compound spots
    • Isolation of most of the compounds
    • Faster separation process with higher selectivity
    • Easy assessment of purity standards
    • Cheaper chromatographic technique

    Limitations of Thin Layer Chromatography (TLC)

    • Cannot distinguish between enantiomers and some isomers
    • Requires prior knowledge of Rf values for identifying specific compounds
    • Limited separation length due to short stationary phases

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    Description

    This quiz covers the fundamentals of spectrophotometry, including how a spectrophotometer works and the role of prisms and monochromators in spectroscopic analysis. Understand the significance of absorption spectra and the precision of wavelength measurements in chemical applications.

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