1. Spectrophotometry & Chromatography
Biotechniques 410
Spectrophotometry & Chromatography

2. Spectrometry Spectro-metry Spectra – Range Meter – Measurement
Infinite values along a continuum
Meter – Measurement
Quantifiable data
Measures values along a given range

3. Types of Spectrometry Mass Spectrometry Spectrophotometry Spectroscopy
Measurement along the spectra of mass
Spectrophotometry
Measurement along the spectra of light
Spectroscopy
Interaction of matter and radiated energy
Reflectance

4. How Mass Spectroscopy Works
Used to determine the mass of material components
Molecules
Compounds
Elements
Samples are vaporized
Separating molecular components
Components are deflected, based on properties (mass, charge, etc.)

9. Spectrophotometry Spectra-photo-metry Spectra – Range Photo – Light
Infinite values along a continuum
Photo – Light
Most commonly visible light, UV & IR
Metry – Measurement
Quantifiable data
Absorbance/Transmittance

10. How Spectrophotometry Works
Spectrophotometry focuses on light
Part of the energy spectrum
Light energy moves a varying wavelengths
Eliciting a response from molecules it interacts with
Lights can be:
Reflected
Absorbed
Transmitted

11. Energy Spectrum Wavelengths (nanometers) Light is energy
Sound is energy
Heat is energy
Smaller wavelengths = more energy
1m=1,000,000,000

12. Visible light
Visible light is a part of energy spectrum that our eyes respond to
Animals can use energy above and below visible light
Reptiles use infrared energy
Insects use Ultra-violet
1m=1,000,000,000

13. Visible light
Color is the result of a portion of visible light energy bouncing off of a surface
Some surfaces reflect a potion of the spectrum
Absorb the rest
Some surfaces reflect all (White)
Others absorb all (Black)
1m=1,000,000,000

14. The Red t-shirt absorbs light from the entire visible spectrum; except RED, which is reflected
The Blue t-shirt absorbs light from the entire visible spectrum; except B:UE, which is reflected
The Black t-shirt absorbs light from the entire visible spectrum; and reflects NO Light
The White t-shirt absorbs no light from the visible spectrum; and reflects ALL

16. Energy Spectrum
Plants absorb light from either end of the visible spectrum
Reflect light in green & yellow range
Plants “appear” green

17. Light absorption Sunlight contains the full visible spectrum
All absorbed, by chloroplasts, except green
Similar to your t-shirt, the visible color of a piece of clothing comes from the reflection of light, all other visible light is absorbed
In chloroplasts some green is transmitted, meaning it passes through the chloroplast
When something absorbs light energy it can be transformed, often into heat
this is why white cars do not get as hot as black cars on a sunny day. They reflect most of the light that hits them, while balck or dark color cars absorb more of the light which is converted into heat energy.

18. Spectrophotometer
Full Spectrum light is shown through a diffraction grate, or prisim
To separate the individual color wavelengths
Light then passes through a slit, so only specific wave lengths reach the sample

19. Spectrophotometer
Light is either absorbed, or transmitted through the sample
Reaching the detctor
So the detector registers
No light (100% absorbance)
Full Light (100% Transmittance)

20. Spectrophotometer

21. Absorption Spectrum
Range of visible light shown through chloroplast solution.
All visible spectrum absorbed, EXCEPT green
Green is transmitted

22. Absorbance/Transmittance
Inverse scales
As absorption increases, transmission decreases
And vice versa
Absorbance
Transmittance (%)
100%
0.1
79%
0.25
56%
0.5
32%
0.75
18%
0.9
13% 1 1% 2
0.1% 3
0.01%

24. Meter
Wavelength
Select
Sample
Holder
Calibrate (0.00)
On/Off

25. Cuvettes Standardized sample holder for spectrophotometry
Fill above where the light source passes through
~2-3ml
Cuvette must be wiped with kimwipe
Remove fingerprints, which can affect samples.

26. Chromatography Related to spectrometry
Examines components that give matter its “characteristics”
Spectraphometry
Spectroscopy
Relies on separation of components due to chemical/physical properties

27. Types of Chromatography
Paired with Mass Spectroscopy
Gas Chromatography
Liquid Chromatography
Capillary Electrophoresis
Ion Mobility
Paired with Spectrophotometry
Paper Chromatography
Thin Plate Chromatography

28. Gas & Liquid Chromatography
Both Gas & Liquid Chromatography separate compounds before entering the spectrometer
More accurate results for spectrographic readings

29. Paper Chromatography Paper Chromatography
Separate pigments (molecules) based on polarity
Use a solvent to get polar molecules in solution
Special polar paper
More polar molecules do not travel far
Less polar molecules travel vary far

30. Paper Chromatography
Once separated we can examine each pigment individually
To determine the absorbance/transmittance spectra

31. How to use the Spectrophotometer

32. How to use the Spectrophotometer
Turn on and let the spectrophotometer warm up.
Make sure the chamber is empty
Set Spectrophotometer to “SPEC D200”
When it says live interface in the upper right hit enter
Hit the right arrow until it says absorbance
Fill a square cuvette with 95% EtOH and cover with parafilm.
This is your Blank (Control)

33. How to use the Spectrophotometer
Place your blank into the spec., and hit “0.00”
This will adjust the spec to account for the plastic and alcohol of the sample.
Fill a second cuvette with your sample
Set the wavelength to 400nm using the dial
Once the number stops moving record the absorbance

34. How to use the Spectrophotometer
When done with a sample pour into the waste container and rinse with water.
Cuvettes can be reused once rinsed
It is wise to check the calibration with the blank ever few measurements.
Record absorbance for each sample at all 18 wavelengths.
Create a table in your notes, and add to class Data on Google Docs

35. Today’s Lab (Day 1)
Please use the same spectrophotometer for the entire experiment
Work in groups of THREE (3)
Measure absorbance of whole leaf homogenate
Spinach
2 Different color maple leaves
Paper Chromatography separation of pigment
And spectra reading

37. Spectrophotometry II Applications of spectrophotometry
Absorbance/Transmission of Pigments
Density (Turbidity)
Determination of material components
Using an indicator (Absorbance/Transmittance)

38. Pigment Determination
We have now seen how a spectrophotometer can be used to determine pigment (color) of material.
Chromatography to separate pigments; for individual analysis
Graphing spectrophotometer results
Color profiles

39. Graphing Color Profiles
We want to compare the whole color to the separate pigments
Chromatography pigments should line up with whole color
However, chromatography pigments are not as concentrated as original whole pigment
Must account for this on the graph
Two y-axes

44. Secondary Axis Graph data Select data to be graphed on secondary axis
Then right click
Select Format Data Series
Select Axis from left-hand menu
Then select Secondary

45. Turbidity Turbidity, or the amount of stuff that block light
Not by absorbance, but reflectance
Can be used to measure density of a sample
Bacteria Density (Next Lab)
DNA Density (Next Week)

46. Determination of Material Components
The materials that make up a solution can be detected with an indicator
Causes the components to change pigment
We can measure the intensity of pigment to quantify the relative concentration

47. Glucose Oxidase Trinder
Tests for the presence of glucose in solution
Glucose Trinder turns pink when added to solution containing Glucose

48. Disaccharide digestion produces glucose
Monosaccharide
We can test for the presence of glucose
Glucose oxidase trinder
Lactose (milk) & Sucrose (Veggies) solutions will not contain glucose
Until sugars are digested

50. Applications This is a common medical test. Glucose indicator in blood
Measure absorbance at a specific wavelength
Dictated by the pigment of the indicator reacting with the substance
Can measure decrease or increase over time

51. Applications Enzyme activity
The more active the enzyme; the less indicator interaction, over time

52. Applications Enzyme supplements For when you do not make enough
Or any at all

53. Lactaid

54. Square cuvettes Same idea as last time
Fill with 2-3ml (to about 1cm from the top)
Wipe the front and back window with kimwipe
Where the light passes through (arrow side)
Line up arrow (window) with the light source

55. This weeks Lab Examine absorbance of solutions containing a pigment
Create a standard curve
Calculate the amount of glucose and lactose in an unknown sample
Measure Enzyme-Substrate Specificity, and activity
Check minute intervals, for 10 minutes