Docstoc

SPECTROPHOTOMETRY

Document Sample
SPECTROPHOTOMETRY Powered By Docstoc
					SPECTROPHOTOMETRY

  Department of Biochemistry
         UERMMMC
        Spectrophotometry
• Determines concentration of a
  substance in solution
  – Measures light absorbed by solution at a
    specific wavelength
       Spectrophotometry
• One of the simplest and most widely
  used methods to determine the amount
  of protein or nucleic acid present in a
  given solution
       Spectrophotometry
• Proteins do not absorb in visible
  wavelength region unless they have a
  prosthetic group (e.g., Fe2+), or an
  unnatural amino acid
       Spectrophotometry
• The amino acids tryptophan, tyrosine &
  cytosine absorb light in the UV
  wavelength
• Aromatic rings in the bases of nucleic
  acids also absorb light in the UV range
         Spectrophotometry
• Visible region: low energy electronic transition
  due to:
  a. Compounds containing transition metals
  b. Large aromatic structures & conjugated
  double bond systems (vitamin A, retinal,
  heme)
• UV region (200-400 nm):
  a. Small conjugated ring systems (Phe, Tyr,
  Trp)
       Spectrophotometry

                        Io             I


                                            A = 0.012
                             l

Lamp    Monochromator                      Detector
                             Cuvette
         Spectrophotometers
•   Light source (Lamp)
•   Optical filters or prism
•   Tube or cuvette
•   Photocell or photomultiplier tube
       Light source (Lamp)
• Visible region = tungsten or tungsten-
  halogen
• UV light = deuterium or hydrogen lamp
       Optical filters/prisms
• To limit light to a certain wavelength
• Monochromator can isolate a specific
  wavelength of white light and allow it to
  pass through the solution being
  analyzed
        Tubes or cuvettes
• Visible range = glass cuvette
• UV range = quartz cuvette
               Photocell
• To detect transmitted light
          Spectrophotometry
• Beer-Lambert’s Law

                       lo   g Io = cl
                            I

Where:        Io = intensity of incident light
              I = intensity of transmitted light
               = molar extinction coefficient
              c = concentration of the absorbing species (mol/L)
              l = path length of the light-absorbing sample (cm)
       Beer-Lambert’s Law
• The fraction of the incident light
  absorbed by a solution at a given
  wavelength is related to
     a. thickness of the absorbing layer
  (path length) and
     b. concentration of the absorbing
  species
       Beer-Lambert’s Law
• Assumes that the incident light is
  parallel and monochromatic
• Monochromator isolates a specific
  wavelength of light and allows it to pass
  thru the solution being analyzed
          Spectrophotometry
•    Light
    1. Frequency (n) = # of wave crests /unit
       time
    2. Wavelength (l) = distance
    3. Velocity (V) = distance travelled /unit time

                    V=lxn
                     l = V/n
            EMR spectrum
     Radiant energy       Wavelength (nm)
Cosmic rays           0.1 - 0.001
Gamma rays            0.1
X-rays                1
Ultraviolet (UV)      180 (200-400)
Visible light         390 (400-700)
Infrared (IR)         780 (700-900)
Microwave             400,000
    Visible region wavelength
Color           Wavelength (nm)
Ultraviolet     400 and under
Violet          400 - 450
Blue            450 - 500
Green           500 - 570
Yellow          570 - 590
Orange          590 - 620
Red             620 - 650
Infrared        750 & over
         Beer-Lambert’s Law
• Concentration  amount of light absorbed

      A = abc = log(100/%T)

Where A = absorbance
     a = absorptivity of the compound under standard
             conditions
     b = light path of the solution
     c = concentration of the compound
     %T = percent transmittance
         Beer-Lambert’s Law
• Absorbance

            A = K x C = Log10Io
                              I
Where: Io = amount of light absorbed by the solution
            expressed as absorbance or optical density
       K = constant
       C = concentration of the substance
            Transmittance
• Defined as the ratio of the intensity of
  light emerging from the solution (I) to
  that of incident light entering (Io)

          T=I
            Io          Io              I
           Transmittance
• Inversely related to the concentration of
  the solution and is expressed in %

       % T = 1 x 100
             Io
          Transmittance
• 100% transmittance means no light is
  absorbed by the solution so that
  incident light is 100% transmitted
 Absorbance & Transmittance
• Absorbance  concentration



• Transmittance 1/  to concentration and
  absorbance
         Sample Problem
• Cytosine has a molar extinction
  coefficient of 6 x 103 mol-1 cm-1 at 270
  nm at pH 7. Calculate absorbance and
  % transmittance of 1 x 10-3 M cytosine
  solution in 1mm cell at 270 nm

          A = Log I0 = lc
                   I
          Sample Problem
• Solution:
1. A = lc = (6 x 103)x (0.1) x (1 x 10-3)
              = 6 x 10-1
              = 0.6 (O.D.)

O.D. between 0.1 and 2 are most reliable
         Sample Problem
• Solution:
2. % Transmittance = I
                     I0
          = 10-0.6
          = 0.25
          = 25%
        Spectrophotometry
• Clinical applications:
  1. Aromatic amino acids have
  characteristic strong absorbance of light
  at a wavelength of 280 nm ex.
  Tryptophan & tyrosine
Spectrophotometry
Spectrophotometry
         Spectrophotometry
• Clinical applications:
  2. Nucleic acids have their maximal
  absorption at 260 nm, which is used as the
  wavelength of choice for measurement of
  DNA or RNA concentrations
> Action spectrum of UV light for mutagenesis
  correlates well with this absorption spectrum
          Spectrophotometry
• Clinical applications:
  3. Mitochondrial cytochromes a, b & c are
  distinguished by differences in their light absorption
  spectra. To distinguish among closely related
  cytochromes of 1 type, exact absorption maximum is
  sometimes used in their names ex. Cytochrome b562
        cytochrome a = 600 nm
        cytochrome b = 560 nm
        cytochrome c = 550 nm
          Spectrophotometry
• Clinical applications:
  4. Cytochrome P450 family of heme proteins
       - usually in endoplasmic reticulum
       - involved in many hydroxylation reactions
       - carbon monoxide complex of its reduced form
         absorbs light strongly at 450 nm
          Spectrophotometry
• Clinical applications:
  5. NAD(P)+-dependent dehydrogenases undergo
  spectrophotometric assays. Reduced coenzymes
  NADH & NADPH (written as NAD(P)H) absorb light
  at a wavelength of 340 nm, while their oxidized forms
  (NAD(P)+) do not. For a dehydrogenase that
  catalyzes oxidation of NAD(P)H, a decrease in
  absorbance at 340 nm will be observed. The rate of
  change in optical density at 340 nm will be
  proportionate to the quantity of enzyme present
          Spectrophotometry
• Clinical applications:
  5. (cont.) Both NAD+ and NADH absorb strongly in
  the UV due to the adenine base. They also differ in
  their fluorescence. NADH in solution has an emission
  peak at 460 nm and a fluorescence lifetime of 0.4
  nanoseconds, while the oxidized form does not
  fluoresce.
          Spectrophotometry
• Clinical applications:
  6. Used to test for coproporphyrins and
  uroporphyrins, which are excreted in increased
  amounts in porphyrias. Absorption curve for
  hematoporphyrin (0.01% solution in 5% HCl) exhibits
  a sharp absorption band near 400 nm which is a
  distinguishing feature of the porphyrin ring and is
  characteristic of all porphyrins regardless of the side
  chains present (Soret band).
   Absorption Maxima of Hgb
Hgb           Abs peak 1   Abs peak 2 Abs peak 3

Oxy Hgb       415          542        577

Reduced Hgb   431          555

Carboxy Hgb   420          539        600

Meth Hgb      406          506        630

Cyanmet Hgb   421          540
             Calculation

     Cu   = Cs x A(u) x D
                   A(s)
Where: Cs = concentration of standard
        Cu = concentration of unknown
        A(s) = absorbance of standard
        A(u) = absorbance of unknown
        D = dilution factor
              Calibration Curve
                                           Glucose Standard Calibration Curve
Glucose       Absorba
Std. Concn.   nce                    1.2
60 mg%        0.2                      1

                        Absorbance
                                     0.8
120 mg%       0.4                    0.6
                                     0.4
                                     0.2                                   Linear ( )
U             0.5
                                      0
                                             60     120     180    200
180 mg%       0.6
                                                    Mg% glucose
           References
• Henry: Clinical Diagnosis and
  Management by Laboratory Methods,
  20th ed. Chaps 3 & 24
Amylose




    Amylopectin
           Dietary Starch
• Amylose - consists of long, unbranched
  chains of D-glucose residues connected
  by (1-4) glycosidic linkages
• Amylopectin - highly branched; residues
  connected by (1-4) linked subunits,
  branch points are (1-6) linkages
          Salivary Amylase
• Hydrolyzes the internal glycosidic linkages of
  starch, producing short polysaccharide
  fragments or oligosaccharides
• Inactivated by low pH in the stomach
• Pancreatic amylase in the small intestine
  continues the breakdown process
 Colorimetric determination of
       reducing sugars
• Dinitrosalicylate
• Potassium ferric hexacyanid (Prussian
  blue)
• Nelson-Somogyi (molybdenum blue)
           DNS method
• Developed by Sumner & Sisler (1944)
  and modified by Miller (1959)
• Based on reduction of sugars by DNS
  under alkaline conditions to yield 3-
  amino-5-nitrosalicylate (brown color)
            DNS method
• Measured at 540 nm
• Quantity of reducing sugar is
  extrapolated from a calibration curve
  prepared with D-glucose
• Amylase-catalyzed reactions are
  typically buffered at pH5 using acetate
  or citrate
            DNS method
• Amylase-catalyzed reactions are
  typically buffered at pH 5 using acetate
  or citrate
• Citrate may interfere with DNS color
  development
              Principle
• Carbohydrates are essentially
  aldehydes or ketones that contain
  multiple hydroxyl (-OH) groups
• Monosaccharides can be aldoses
  (glucose) or ketoses (fructose
                Principle
• Both aldoses & ketoses occur in equilibrium
  between the open-chain forms and cyclic
  forms (chain lengths of C4)
• These are generated by bond formation
  between one of the (-OH) groups of the sugar
  chain with the C of the aldehyde or keto
  group to form a hemiacetal bond.
Principle
Principle
Principle
                Principle

• When salivary amylase is added to
  starch, a hydrolysis reaction is initiated
  in which water breaks bonds, releasing
  maltose
              Principle
• DNS tests for the presence of free
  carbonyl groups (C=O), the so-called
  reducing sugars
• Involves oxidation of the aldehyde
  functional groups in glucose and the
  ketone functional groups in fructose
               Principle
• Simultaneously, 3,5 DNS is reduced to
  3-amino, 5 nitrosalicylic acid under
  alkaline conditions
• As hydrolysis proceeds, more reducing
  sugar will be available to react with the
  3,5 DNS
                       Principle

                          oxidation
Aldehyde group                          carboxyl group
                        reduction

3,5 Dinitrosalicylic                  3-amino, 5 nitrosalicylic
 Standard Absorbance Curve
• Done by reacting know concentration of
  glucose with DNS then determining
  absorbance at 540 nm
• Plot absorbance vs. glucose
  concentration
            Absorbance
• Absorbance corresponds to
    0.1 ml of test = x mg of glucose

 10 ml contains = x (10 mg of glucose)
                 0.1
                = % of reducing sugars

				
DOCUMENT INFO