1. SPECTROPHOTOMETRY IN BIOTECHNOLOGY1

2. TOPICS Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers 2

3. BIOTECHNOLOGY PROCESS
Find gene that codes for useful protein
Isolate gene
Insert gene into vector
Insert vector into cells (transform/transfect cells)
Grow cells, cells manufacture protein product
Purify product
Sell product

4. BIOTECHNOLOGY PROCESS
Find gene that codes for useful protein
Isolate gene
Estimate DNA [ ]
Insert gene into vector
Check cell density
Insert vector into cells (transform/transfect cells)
Grow cells, cells manufacture protein product
Check protein
activity
Purify product
Check protein
concentration
Sell product
Check protein
purity

5. Light and its Interactions with Matter
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers

6. LIGHT IS A TYPE OF ELECTROMAGNETIC RADIATION
Imagine electromagnetic radiation like waves on a pond
But instead of water, electromagnetic radiation is energy moving through space
Distance from one crest to the next is the wavelength

7. WAVELENGTH AND COLOR
Different wavelengths of light correspond to different colors
All colors blended together is called white light
The absence of all light is black
Light of slightly shorter wavelengths is ultraviolet
Eyes do not perceive UV light

9. INTERACTION OF LIGHT WITH MATERIALS IN SOLUTION
When light shines on a solution, it may pass through – be transmitted – or
Some or all of the light energy may be absorbed

12. BIOLOGICAL SOLUTIONS Usually appear clear to our eyes – have no color
DNA, RNA, most proteins do not absorb any visible light
But they do absorb UV light, so UV spectrophotometers are useful to biologists
Example, can use a detector that measures absorbance at 280 nm, or 254 nm to detect proteins

13. Spectrophotometer Design
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers

14. SPECTROPHOTOMETERS
Are instruments that measure the interaction of light with materials in solution

16. Monochromator Separates Light into Its Component Wavelengths
Monochromator Separates Light into Its Component Wavelengths. Modern Specs Use Diffraction Gratings

17. Spectrophotometer Operation
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers

18. THE BLANK
Spectrophotometers compare the light transmitted through a sample to the light transmitted through a blank.
The blank is treated just like the sample
The blank contains everything except the analyte (the material of interest)
Contains solvent
Contains whatever reagents are added to the sample

20. WHEN OPERATING SPEC Blank is inserted into the spectrophotometer
Instrument is set to 100% transmittance or zero absorbance

21. PROPER SELECTION, USE, AND CARE OF CUVETTES
Cuvettes are made from plastic, glass, or quartz.
Use quartz cuvettes for UV work.
Glass, plastic or quartz are acceptable visible work.
There are inexpensive plastic cuvettes that may be suitable for some UV work.

23. 2. Cuvettes are expensive and fragile (except for “disposable” plastic ones). Use them properly and carefully.
a. Do not scratch cuvettes; do not store them in wire racks or clean with brushes or abrasives.
b. Do not allow samples to sit in a cuvette for a long period of time.
c. Wash cuvettes immediately after use.

24. 3. Disposable cuvettes are often recommended for colorimetric protein assays, since dyes used for proteins tend to stain cuvettes and are difficult to remove.
4. Matched cuvettes are manufactured to absorb light identically so that one of the pair can be used for the sample and the other for the blank.

25. 5. Do not touch the base of a cuvette or the sides through which light is directed.
6. Make sure the cuvette is properly aligned in the spectrophotometer.
7. Be certain to only use clean cuvettes.

28. Qualitative Spectrophotometry
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers

30. EXAMPLES Some examples of qualitative spectrophotometry
The absorbance spectra of various common solvents. Note that some solvents absorb light at the same wavelengths as DNA, RNA, and proteins
Hemoglobin bound to oxygen versus carbon monoxide
Native versus denatured bovine serum albumin (a protein commonly used in the lab)

33. Quantitative Spectrophotometry
Spectrophotometers in Biotechnology
Light and its Interactions with Matter
Spectrophotometer Design
Spectrophotometer Operation
Qualitative Spectrophotometry
Quantitative Spectrophotometry
UV Spectrophotometry of DNA, RNA and Proteins
Calibration of Spectrophotometers

35. OVERVIEW OF QUANTITIVE SPECTROPHOTOMETRY
A. Measure the absorbance of standards containing known concentrations of the analyte
B. Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis
C. Measure the absorbance of the unknown(s)
D. Determine the concentration of material of interest in the unknowns based on the standard curve

37. LINEAR RANGE
If there is too much or too little analyte, spectrophotometer cannot read the absorbance accurately

38. COLORIMETRIC ASSAYS
Quantitative assays of materials that do not intrinsically absorb visible light
Combine the sample with reagents that make the analyte colored
The amount of color is proportional to the amount of analyte present

39. BRADFORD PROTEIN ASSAY
A quantitative colorimetric assay
Used to determine the concentration, or amount, of protein in a sample

40. Prepare standards with known protein concentrations
Add Bradford Reagent to the samples and to standards
Read absorbances
Create a standard curve
Determine the concentration of protein in the samples based on the standard curve

41. MORE ABOUT THE CALIBRATION LINE ON A STANDARD CURVE
Three things determine the absorbance of a sample:
The concentration of analyte in the sample
The path length through the cuvette
The intrinsic ability of the analyte to absorb light at the wavelength of interest

43. BEER-LAMBERT LAW A =  B C Where:
A = absorbance at a particular wavelength
 = absorptivity constant – intrinsic ability of analyte to absorb light at a particular wavelength
B = path length through cuvette
C = concentration of analyte

44. APPLYING THE EQUATION Suppose you have a sample:
And you know the path length
And you know the absorptivity constant for the analyte of interest at a particular wavelength
Then, measure the sample’s absorbance at the specified wavelength

45. Can calculate the concentration of the analyte from the Beer-Lambert equation
A =  B C
But this is a shortcut that may give inaccurate results!

46. EQUATION FOR A LINE
A =  B C
y = m x

47. Y intercept should be zero because of the blank
Blank has no analyte (zero concentration) and is used to set transmittance to 100% = absorbance to zero

48. SLOPE
Slope relates to the absorptivity constant
A =  B C
y = m x

49. DETERMINATION OF THE ABSORPTIVITY CONSTANT
1. Prepare a calibration line based on a series of standards
Plot concentration on the X axis and absorbance on the Y axis
2. Calculate the slope of the calibration line:
Y2 – Y1
X2 - X1

50. Determine the path length for the system (assume 1 cm for a standard sample holder and cuvette)

51. Slope = absorptivity constant X path length
A =  B C
y = m x
Slope = absorptivity constant X path length
Absorptivity constant = slope
path length
(Observe that the constant has units that depend on how concentration was expressed in the standards)