1
Q
What is spectrophotometry?
A
A scientific method based on the absorption of light by a substance
2
Q
Define spectrophotometer.
A
An apparatus for measuring intensity of light in a part of the spectrum, as transmitted or emitted by particular substances
3
Q
What does Lambert’s Law state?
A
The proportion of light absorbed by a medium is independent of the intensity of incident light
4
Q
Express Lambert’s law mathematically.
A
I/Io=T, where I = Intensity of transmitted light, Io = Intensity of the incident light, T = Transmittance
5
Q
What is Beer’s Law?
A
The absorbance of light is directly proportional to both the concentration of the absorbing medium and the thickness of the medium
6
Q
What is the standard pathlength for cuvette-based instruments?
A
10 mm
7
Q
How does using a shorter pathlength affect absorbance measurements?
A
It reduces the absorbance, increasing transmittance and reducing the incident light required to achieve reliable results
8
Q
What is the formula for absorbance in basic terms?
A
Absorbance = Concentration × Pathlength
9
Q
What wavelengths are typically used for nucleic acid measurements?
A
260 nm and 280 nm
10
Q
What is the A260/A280 ratio used for?
A
To check the purity of DNA and RNA samples, with a good level of purity being ≥ 1.8 for DNA and ≥ 2.0 for RNA
11
Q
What does an A260/A230 ratio of 2 or above indicate?
A
A pure sample
12
Q
What is background correction in spectrophotometry?
A
A process of measuring absorption at a point unrelated to the sample and subtracting it from the peaks
13
Q
What is the significance of measuring at 320 nm?
A
It helps to measure background absorption and correct for light scatter or contamination
14
Q
What are the criteria for meaningful nucleic acid measurements?
A
- Abs260 > approx. twice Abs230 (A260/A230 Ratio=2) * Abs260 = 0.1 or more
15
Q
At what wavelength do proteins absorb light for concentration measurement?
A
280 nm
16
Q
Which amino acids are primarily responsible for protein absorption at 280 nm?
A
- Tyrosine * Tryptophan
17
Q
What is the conversion factor for bovine serum albumin (BSA)?
A
1.551
18
Q
How is protein concentration calculated using absorbance?
A
Concentration (µg/ml) = Abs280 × Factor
19
Q
What are the common colorimetric methods for protein quantification?
A
- BCA * Biuret * Bradford * Lowry * 2-D Quant Kit
20
Q
What is the purpose of the A260/A280 ratio in protein analysis?
A
It serves as a guide to assess protein purity
21
Q
True or False: Dilution of samples is necessary for all absorbance measurements.
A
False
22
Q
Fill in the blank: The absorbance of light is directly proportional to the concentration of the absorbing medium and the _______.
A
thickness of the medium
23
Q
What factors can affect the UV absorbance of proteins?
A
- Temperature * pH * Ionic strength * Presence of detergents
24
Q
What is the significance of the 230 nm reading in nucleic acid measurements?
A
Higher readings can indicate contaminants in the sample
25
What factors can affect the ability of aromatic residues to absorb light at 280 nm?
Temperature, pH, ionic strength, presence of detergents
## Footnote
These conditions can alter the protein's extinction coefficient.
26
What is the conversion factor for bovine serum albumin (BSA)?
1.551
## Footnote
This factor is used for calculating protein concentration.
27
How is protein concentration calculated from absorbance at 280 nm (A280)?
c = A280 × Factor
## Footnote
Where c is the concentration in µg/ml.
28
What does the A260/A280 ratio indicate?
Purity of the sample
## Footnote
A higher ratio suggests contamination with nucleic acids.
29
What is the formula for calculating protein concentration in mg/ml?
c = A280/(E280,1 mg/ml) × l
## Footnote
E280,1 mg/ml varies between proteins.
30
What is E280,1 mg/ml and how can it be determined?
Absorbance coefficient for a 1 mg/ml solution of the protein; determined by measuring absorbance of known concentration or theoretical calculation.
## Footnote
E280, 1 mg/ml = (5500nTrp + 1490nTyr + 125nS-S)/M.
31
How can light scattering correction be applied to A280 value?
A280 = A280 (measured) – 1.929 × A330 (measured)
## Footnote
This correction accounts for scattering effects.
32
What is the estimated protein concentration formula when nucleic acids are present?
C (mg/ml) = 1.55 × A280 - 0.76 × A260
## Footnote
This method is less accurate.
33
What is the principle behind colorimetric methods for protein concentration measurement?
Dye binds specifically to proteins; absorption of standards is measured to create a standard curve.
## Footnote
Unknown samples are compared to this curve.
34
What does the BCA method rely on for protein quantification?
Reaction of cupric ions (Cu2+) with peptide bonds to form cuprous ions (Cu+) detected with BCA.
## Footnote
Absorbance peak maximum at 562 nm.
35
What is the working range for the Bradford method?
1 to 1500 µg/ml
## Footnote
This method is resistant to interference.
36
What is the principle of the Lowry method?
Reaction of Folin-Ciocalteu’s phenol reagent with tyrosyl residues.
## Footnote
Absorbance is measured at 750 nm.
37
What is the purpose of the 2-D Quant Kit?
To determine protein concentration in samples for high resolution electrophoresis techniques.
## Footnote
It quantitatively precipitates proteins, leaving interfering substances in solution.
38
What are the advantages of the A280 Direct UV method?
* Simple and direct UV measure
* Wide working range
* Minimizes need for dilution
## Footnote
Useful for identifying protein on column fractions.
39
What are the disadvantages of the Bradford assay?
* Absorbance spectra overlap
* Stains labware
* Susceptible to high detergent concentrations
## Footnote
Precipitates can form.
40
What is the sensitivity level of the Micro BCA method?
Very sensitive
## Footnote
Less susceptible to interference from common buffer substances.
41
What is the working range of the Biuret method?
1000 to 15,000 µg/ml
## Footnote
It is easy to use with a single reagent.
42
What type of cuvettes are suitable for UV/vis spectrophotometry?
Specialist plastics or quartz glass
## Footnote
Normal plastics and glass absorb UV light and are unsuitable for DNA analyses.
43
What is the effect of buffers and detergents on spectrophotometric analysis?
They can absorb light and interfere with analysis
## Footnote
Choosing non-absorbing buffers is crucial.
44
What is the significance of the beam height in a spectrophotometer?
It affects the optical clarity and pathlength accuracy
## Footnote
Common beam heights include 8.5 mm, 15 mm, and 20 mm.
45
What are the two types of cuvettes based on usage?
* Disposable cuvettes
* Reusable cuvettes
## Footnote
Each has its own advantages and disadvantages.
46
What does the absorbance spectrum vary in terms of molecule measurement?
It can vary from very narrow (<1 nm) to broad (>3 nm)
## Footnote
This affects the precision of measurements.
47
How is bacterial cell culture density typically measured?
By analyzing light scatter at 600 nm
## Footnote
More cells scatter more light, affecting the detector's readings.
48
What type of buffer should be chosen for sample preparation to avoid interference?
A non-absorbing buffer or one that will not interfere with the sample analysis
## Footnote
This is crucial to obtain accurate results.
49
Which detergents have been found to have a lesser effect on direct UV results?
Brij 35, CHAPS, Tween 20
## Footnote
These may yield better results compared to Igepal or Triton x-100.
50
What is the typical pathlength range for low volume spectrophotometers?
0.1 mm to 1 mm
## Footnote
These instruments may rely on the sample itself to form the cuvette.
51
What pathlength should be selected for volumes below 2 µl with NanoVue?
0.2 mm
## Footnote
For volumes of 2 µl or above, a pathlength of 0.5 mm should be selected.
52
What should always be switched on when using low volume instruments with background correction capability?
Background correction
## Footnote
This corrects for drop misplacement and sample background values.
53
What does a reading at 320 nm indicate?
A contaminant or faulty drop placement
## Footnote
DNA should ideally have zero absorbance at this wavelength.
54
What are common sources of interference in low volume spectrophotometry?
* Poor drop placement
* Air bubbles in sample
* Particles in sample
## Footnote
Each of these can affect light transmission and absorption readings.
55
What specifications should be considered when choosing a spectrophotometer?
* Data recording requirements
* User accessibility and experience
* Applications routinely run
* Sensitivity level needed
* Pharmacopoeia compatibility
## Footnote
Balancing these specifications is crucial for optimal performance.
56
What is the wavelength range of the Ultrospec 9000 spectrophotometer?
190-1100 nm
## Footnote
It is a high-performance dual-beam, variable bandwidth UV-Visible spectrophotometer.
57
What is the significance of bandwidth in spectrophotometry?
Bandwidth describes the width of a peak at half-light intensity
## Footnote
It affects sensitivity and resolution in measurements.
58
What type of light source does a dual beam spectrophotometer use?
It splits light into two paths, one for the sample and one for a reference
## Footnote
This provides more reliable results by correcting for variations.
59
What is the function of a monochromator in a UV/Vis spectrophotometer?
It transmits a mechanically selected band of wavelengths of light
## Footnote
It allows for precise wavelength selection for measurements.
60
Fill in the blank: Stray light is light that reaches the detector that is of a different wavelength than that being selected by the _______.
monochromator
## Footnote
Stray light can lead to inaccuracies in measurements.
61
What is the primary application of the GeneQuant 100 spectrophotometer?
It is designed for nucleic acids, proteins, and cell density measurements
## Footnote
It features built-in applications for various biological analyses.
62
What are the features of the NanoVue Plus spectrophotometer?
* No cuvettes needed
* Low volume measurements (0.5 to 5 µl)
* PC control available
## Footnote
It offers simplicity in drop measurement.
63
True or False: Deuterium lamps are used to provide stable light in the visible region of the spectrum.
False
## Footnote
Deuterium lamps are primarily used for the UV region, while tungsten lamps provide stable light in the visible region.
64
What is the purpose of using a sipper system in spectrophotometry?
Automated sample aspiration
## Footnote
It enhances the efficiency of sample measurement processes.
65
What is the significance of the pathlength in spectrophotometry?
It is the distance light travels through the sample to the detector
## Footnote
Standard cuvettes typically have a pathlength of 10 mm.
66
What is the standard pathlength of cuvettes used in spectrophotometry?
10 mm (1 cm)
## Footnote
GE Healthcare offers instruments covering pathlengths from 0.2 mm to 100 mm.
67
What is stray light in the context of spectrophotometry?
Light that reaches the detector at a different wavelength than selected by the monochromator
## Footnote
Stray light can be caused by diffraction patterns or light leaks.
68
What regions of the electromagnetic spectrum do GE Healthcare UV/Vis spectrophotometers measure?
UV (10-400 nm), visible (380-760 nm), and near infra-red (750-2250 nm)
## Footnote
This covers a range of 190-1100 nm.
69
What does the Greek letter lambda (λ) represent?
Wavelength
## Footnote
Wavelength measures the distance between successive peaks in a waveform.
70
Who authored the work 'Photometria sive de mensura et gradibus luminis, colorum et umbrae'?
J.H. Lambert in 1760
## Footnote
This work discusses the measurement of light, colors, and shades.
71
What is the primary focus of Beer’s 1852 study?
Determination of the absorption of red light in colored liquids
## Footnote
Published in Annalen der Physik und Chemie.
72
What method did Bradford develop in 1976?
A rapid and sensitive method for quantitation of microgram quantities of protein utilizing protein-dye binding
## Footnote
Published in Anal. Biochem.
73
Name one product for protein sample preparation offered by GE Healthcare.
Mag Sepharose™
## Footnote
Other products include MiniTrap™, MultiTrap™, SpinTrap™, and GraviTrap™.
74
What does the term 'chromatography media' refer to in protein purification?
Materials used in columns for separating proteins based on their properties
## Footnote
Examples include TALON®, HisTrap™, and GSTrap™.
75
True or False: GE Healthcare's spectrophotometer range is available from any supplier.
False
## Footnote
It is available from preferred suppliers.
76
Fill in the blank: Stray light can be caused by _______ patterns produced by the monochromator.
diffraction
77
What is the wavelength range for visible light?
380-760 nm
78
What is the purpose of the Hybond™ membranes?
Blotting and detection in biochemical assays
## Footnote
Part of the Amersham™ product line.
79
What is the significance of the term 'ECL' in GE Healthcare products?
Labeling and detection range in biochemical applications
## Footnote
ECL stands for Enhanced Chemiluminescence.
80
Who are the authors of the study regarding the isolation and crystallization of the fermentation enzyme enolase?
O. Warburg and W. Christian in 1942
81
What does the term 'UV/Vis' stand for?
Ultraviolet/Visible
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