Spectrophotometry Equipment

Question 1

what is spectrophotometry?

Answer 1

is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. The light passing through the solution is detected by the photo-detector, generating an electrical current proportional to the intensity of the light, which is then converted into a reading.

Question 2

what does a spectrophotometer do?

Answer 2

measure the intensity of light at a certain wavelength

Question 3

what is the blank solution?

Answer 3

it is as similar as possible but without the analyte that is being measured

Question 4

what are the 2 main types of light source for general spectroscopy

Answer 4

tungsten
deuterium
sometimes lasers

Question 5

tungsten lamps

Answer 5

cover the visible spectral range reasonably well
tends to have higher intensity in the red region of the spectrum
cheap
not good for UV

Question 6

deuterium lamps

Answer 6

D2 lamps
deuterium: isotopic hydrogen (abundance ~1 in 6000 H atoms)
deuterium arc light produced mainly UV light (so invisible to the eye)
expensive
relatively short lifetime

Question 7

for optimal analytical performance:

Answer 7

• Incident light beam is parallel and of a constant wavelength (monochromatic)
• Incident light beam is of the wavelength which gives the maximum absorption (minimum transmission) of the light
• To achieve this spectrophotometers use a prism or diffraction grafting to isolate a portion of spectrum of white light from the bulb

Question 8

what is a collimator?

Answer 8

a lens to produce a near parallel beam of light. monochromators only work if beam is collimated.

Question 9

examples of monochromators:

Answer 9

• coloured filters- simplest and cheapest but less frequently used
• prism- light separated by refraction (bending by passing through transparent medium). target wavelength selected by rotating the prism. more frequently used.
  -diffraction grating-also frequently used, when light is separated by diffraction (bending of light at the edge of an opaque surface)

Question 10

how are wavelengths calibrated

Answer 10

holmium oxide

Question 11

what is holmium oxide

Answer 11

a rare earth lanthanide element, it forms oxide Ho2O3

Question 12

why is holmium oxide used for wavelength calibration?

Answer 12

it has well define peaks across the UV/visible range

Question 13

what are the properties of a cuvette

Answer 13

known path length
optically inert
no absorption in the region of interest
no internal reflection or scatter

Question 14

3 main types of cuvettes

Answer 14

polystyrene
glass
quartz

Question 15

which is the best cuvette for UV

Answer 15

quartz

Question 16

which cuvettes are mainly used in analysers?

Answer 16

polystyrene

Question 17

what are the 2 main types of detectors used?

Answer 17

photomultiplier tube
diode array

Question 18

what do photomultiplier tubes do?

Answer 18

v high sensitivity
cascade system of dynodes
convert light to electron beams and then to electric current
relatively expensive

Question 19

what does a diode array do

Answer 19

the array contains several light detectors
several wavelengths of light can be measured simultaneously

Question 20

what can affect absorbance

Answer 20

all components of the cuvette and contents

Question 21

how do we blank the instrument?

Answer 21

measure the absorbance of a solution before adding the analyte if possible
measure the absorbance prior to the addition of reagents (if producing a coloured product)
use a dual beam spectrophotometer

Question 22

how does a dual beam spectrophotometer work

Answer 22

the incident beam is split by a mirror
allows correction for drift and power variations in the light source

Question 23

what are the other forms of spectrophotometry other than absorbance

Answer 23

reflectance
scatter
fluorescence
luminometry

Question 24

what is reflectance spectrophotometry

Answer 24

diffuse light reflected from a surface is measured
a comparison is made with a reference surface
used un dry-reagent chemistry systems e.g. vitros and in some POCT applications e.g. bilirubinometer (doesnt require a blood sample)

Question 25

what can increase analytical sensitivity in immunoturbidimetry?

Answer 25

attaching antibodies to latex/polystyrene particles

Question 26

nephelometry

Answer 26

more sensitive 1-10mg/L
needs more specialist equipment
measures light scattered at 90 degrees

Question 27

what is fluorescence?

Answer 27

occurs when a molecule absorbs light at one wavelength and re-emits light at a lower wavelength
emitted light is always lost at a longer wavelength (lower energy) than the excitation light

Question 28

what is stokes shift?

Answer 28

difference between λ max of excitation light and emitted fluorescence light

Question 29

what is the name of a fluorescent molecule

Answer 29

fluorophore

Question 30

what is the energy state when a molecule emits light?

Answer 30

lower energy state

Question 31

where is fluorescence used in the clinical lab?

Answer 31

immunoassay detection systems (e.g. DEFLIA)
in some polarisation methods (FPIA e.g, for TDMs)

Question 32

what is luminescence?

Answer 32

generation of light from a chemical reaction (chemiluminescence)
exteremely sensitive

Question 33

what are the applications of spectrophotometry?

Answer 33

enzymatic reactions (e.g. glucose)
chemical reactions (e.g. creatinine)
dye binding (e.g. mg, ca)
transition metal complexes (e.g. phosphate)