Final Exam Flashcards
(180 cards)
1
Q
technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by any one of several reverse transcriptases. This cDNA is then amplified, and particular targets are identified using a method known as Quantitative PCR (qPCR) or real-time PCR.
A
Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
2
Q
RT-qPCR is technology by which __ molecules are converted into their __ __ (__) sequences by any one of several __ __. This __ is then __, and particular targets are identified using a method known as __ __ (__) or __ __.
A
- RNA
- complementary DNA (cDNA)
- reverse transcriptase
- cDNA
- amplified
- Quantitative PCR (qPCR)
- real-time PCR
3
Q
used to detect and quantify RNA.
A
Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)
4
Q
In RT-qPCR what is used as the template for the quantitative PCR or real-time PCR reaction (qPCR)?
A
complementary DNA (cDNA)
5
Q
What methods are combined in RT-qPCR (Reverse Transcription Quantitative PCR)? (3)
A
- Reverse Transcription: Converts RNA into DNA.
- Real-Time PCR: Amplifies and quantifies DNA in real-time.
- Fluorescence Spectrophotometry: Measures fluorescence signals.
6
Q
What are the applications of RT-qPCR? (5)
A
- Measure the levels of gene expression.
- Validate RNA interference for gene loss of function studies.
- Identify pathogens to diagnose infectious diseases.
- Analyze gene expression or detect viral mRNA.
- Detect genetically modified organisms (GMOs).
7
Q
types of RT-qPCR (2)
A
- One-step RT-qPCR
- Two-step RT-qPCR
8
Q
A type of RT-qPCR that combines reverse transcription and PCR in a single tube and buffer, using a DNA polymerase.
Only utilizes sequence-specific primers.
A
One-step RT-qPCR
9
Q
A type of RT-PCR that is faster,
less pippeting steps, minimizes possible contamination, improves data reproducibility, and compatible with high-throughput.
A
One-step RT-qPCR
10
Q
Since __ __ __ __ can withstand the higher temperatures required for sequence-specific primer annealing, they are frequently used in one-step RT-qPCR experiments.
A
Genetically modified reverse transcriptase
11
Q
A type of RT-qPCR where reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.
A
Two-step RT-qPCR
12
Q
A type of RT-qPCR where that may employ random hexamers, oligo-dT primers, or gene-specific primers.
A
Two-step RT-qPCR
13
Q
What primers may be employed in Two-step RT-qPCR? (3)
A
- Random hexamers
- oligo-dT primers
- gene-specific primers.
14
Q
How does Two-step RT-qPCR enhance amplification efficiency?
A
Separate buffers and reagents allow for optimal selection of enzymes and reagents.
15
Q
What advantage does the first step of Two-step RT-qPCR offer?
A
More stable cDNA can be concentrated, purified, and stored for multiple gene quantification
16
Q
Steps in RT-qPCR (4)
A
Extraction of RNA → Reverse transcription → Amplification → Quantification
17
Q
Steps in Extraction of RNA (qRT-PCR) (3)
A
Sample collection and lysis → Separation of RNA from DNA and other cellular components → RNA Purification
18
Q
Steps in Reverse transcription (RT-qPCR) (2)
A
Reverse transcription
Priming → Reverse Transcriptase in Action
19
Q
Types of Primers in Reverse Transcription (4)
A
- Standard oligo dT
- Anchored oligo dT
- Random primers
- Gene-specific primers
20
Q
Steps in the Amplification (RT-qPCR) (3)
A
Denaturation through increased temperature → Annealing of primers → Extension of primer by DNA polymerase
21
Q
What is the role of the oligo dT primer in RT-PCR?
A
The oligo dT primer initiates cDNA synthesis by binding to the poly A tail of mRNA.
22
Q
Why do scientists often use a combination of oligo dT and random hexamer primers in RT-PCR?
A
slows down the processivity of the reverse transcriptase enzyme.
23
Q
How do normal PCR and RT-PCR differ in terms of templates and primers used?
A
Template:
- Normal PCR: template DNA
- RT-PCR: mRNA.
Primers:
- Normal PCR: forward and reverse primers
- RT-PCR: oligo dT, anchored oligo dT, random primers, and gene-specific primers.
24
Q
What are the optimal working temperatures for Taq polymerase and reverse transcriptase?
A
- 72°C
- 50°C
25
Steps in the Quantification (RT-qPCR) (3)
Addition of Fluorescent reporters like Sybr green dye or Taqman probe → Monitoring fluorescent signals per cycle → Noting the CT value
26
How does PCR monitor DNA amplification?
PCR uses fluorescent signals to monitor DNA amplification as the reaction progresses.
27
What are the two main types of fluorescence used in qPCR? (2)
1. Intercalating dyes (like SYBR Green)
2. hydrolysis probes (like TaqMan).
28
- a phase which does not move with the sample
- This is the substance within the column that the sample components interact with, causing separation.
stationary phase
29
- a phase which moves with the sample.
- a chemically inert gas that serves to carry the molecules of the analyte through the heated column.
mobile phase
30
a common stationary phase: Commonly used for separating hydrocarbons and other nonpolar compounds. Examples include dimethylpolysiloxane.
Nonpolar phase
31
A common stationary phase: Used for separating polar compounds such as alcohols, acids, and amines. Examples include polyethylene glycol.
Polar phase
32
a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify, and purify the individual components of the mixture.
High-pressure/performance liquid chromatography (HPLC)
33
Basic principles of HPLC (3)
- Separation
- Identification
- Quantification
34
It separates compounds based on their interactions with a stationary phase (a solid or liquid supported on a solid) and a mobile phase (a liquid solvent that flows through the stationary phase).
High-pressure/performance liquid chromatography (HPLC)
35
How does HPLC identify compounds?
Each compound interacts differently with the stationary and mobile phases, causing them to elute at different times. This difference in elution times allows for identification.
36
How does HPLC quantify compounds?
The amount of each compound is determined by measuring the area under the peaks in the chromatogram.
37
Components of the HPLC System (5)
1. Gradient pump
2. Injector
3. Column
4. Detector
5. Data system
38
Types of pumps for the HPLC System (3)
1. Reciprocating pumps
2. Syringe pumps
3. Constant pressure pumps
39
A type of pump for the HPLC system that use a piston mechanism to generate high pressure. These are the most common due to their ability to deliver consistent flow rates and handle high pressures.
Reciprocating pumps
40
A type of pump for the HPLC system that provide very precise flow rates and is often used for applications requiring exact volume deliveries.
Syringe pumps
41
A type of pump for the HPLC system that maintain a constant pressure rather than a constant flow rate, which can be useful in specific analytical situations.
Constant pressure pumps
42
The HPLC pump is crucial for maintaining a consistent flow of the __ __ through the system. It operates at high pressures, typically between __ to __ psi (pounds per square inch), to push the __ and sample through the densely packed __.
- mobile phase
- 1,000 to 10,000 psi
- mobile phase
- column
43
In the HPLC pump, the __ introduces the sample mixture into the mobile phase stream. It must be precise and reproducible to ensure consistent sample volumes are __ each time.
- injector
- injected
44
Two main types of injector in HPLC pump (2)
- Manual injectors
- Auto-samplers
45
A type of injector that the sample is injected manually using a syringe.
manual injectors
46
A type of injector that automatically inject samples from vials loaded in a sample tray. This allows for high throughput analysis and reduces human error.
auto-samplers
47
the heart of the HPLC system where separation occurs. It is packed with fine particles (stationary phase) that interact differently with the components of the sample. It varies in length, diameter, and the nature of the stationary phase material.
Column
48
Columns vary in length, diameter, and the nature of the stationary phase material. Key considerations include: (3)
- Length and diameter
- Particle size
- Stationary phase
49
How does column length and diameter affect HPLC analysis?
Shorter columns provide faster analysis times, while longer columns offer better separation.
50
How does particle size influence HPLC performance?
Smaller particles offer greater surface area for interactions, leading to better separation, but require higher pressure.
51
What factors determine the type of stationary phase used in HPLC?
The stationary phase can be polar or non-polar, depending on the type of HPLC (normal-phase or reverse-phase).
52
A component in HPLC that identifies and quantifies the separated components as they elute from the column. Different types of these are used based on the properties of the analytes and the requirements of the analysis.
Detector
53
Different types of detectors in HPLC (4)
1. UV-Vis Detectors
2. Fluorescence Detectors
3. Refractive Index Detectors
4. Mass Spectrometers (LC-MS)
54
Type of detector in HPLC that measure absorbance of ultraviolet or visible light by the analyte. Widely used due to their sensitivity and ease of use.
UV-Vis Detector
55
Type of detector in HPLC that detects fluorescence emitted by analytes. They are more sensitive than UV-Vis detectors but require analytes that fluoresce or can be derivatized to fluoresce.
Fluorescence Detector
56
Type of detector in HPLC that measures changes in the refractive index of the eluent. Useful for compounds without UV absorption but less sensitive and more susceptible to temperature changes.
Refractive Index Detectors
57
Type of detector in HPLC that provides highly sensitive and specific detection by identifying compounds based
on their mass-to-charge ratio.
Mass Spectrometers (LC-MS)
58
In HPLC, it consists of computer software that collects, processes, and analyzes data from the detector. It generates a chromatogram, which is a graphical representation of the detector response (y-axis) over time (x-axis).
data system
59
How HPLC works 1/2 The separation principle of HPLC relies on the distribution of the __ (__) between a mobile phase (__) and a __ __ (column packing material). The analyte's chemical structure causes the molecules to be delayed as they pass through the __ __. The specific interactions between the sample molecules and the packing material determine their time "__-__." Consequently, different sample components are eluted at different times, achieving the separation of the sample's ingredients. A __ __ identifies the analytes as they exit the column. The signals are then converted and recorded by a data management system (computer software) and displayed in a __.
- analyte (sample)
- eluent
- stationary phase
- stationary phase
- on-column
- detection unit
- chromatogram
60
How HPLC works 2/2 After passing the detection unit, the __ __ can be directed to additional detectors, a fraction collection unit, or waste. Typically, an HPLC system includes the following components: a solvent reservoir, a pump, an injection valve, a column, a detector unit, and a data processing unit. The __ delivers the solvent (__) at high pressure and a constant rate through the system. To minimize detector signal drift and noise, maintaining a constant, pulseless flow from the pump is essential. The analyte (__) is introduced into the __ via the __ __
- mobile phase
- pump
- eluent
- sample
- eluent
- injection valve
61
a quantitative analysis useful for determining the macronutrient content of a substance, used often in the food industry
proximate analysis
62
used for estimation of the quantitative of food and food substance including moisture, crude protein, total fat, total carbohydrate, and dietary fiber.
proximate analysis
63
Proximate analysis has several procedures to detect each macronutrient. The commonly determined ones are tests for: (5)
- Moisture - Water content of the sample
- Ash content - Weight of inorganic matter in sample
- Crude protein - Protein content
- Crude lipid - Fat content
- Crude fiber - Fiber content
64
Method for the Determination of Moisture Content %
1. Containers (dishes/pans) are weighed and heated to dry completely, then weighed again after drying
2. A solid sample is ground up fine and weighed into a covered dish, at least 3 replicates
3. The sample is heated in an oven to 105-115 Celsius for 16-24 hours, depending on the sample
4. Sample is taken out of oven and cooled in a desiccator
5. Weight is taken and recorded
6. Moisture content is computed
65
Moisture content is computed with the equation:
Moisture % = (S-X)/(S-C) * 100
S = weight of sample and container prior to drying
C = weight of the container
X = weight of sample and container after drying
S - C = weight of sample
S - X = weight of sample after drying
66
Method for the Determination of Ash Content% (8)
1. Porcelain dish is weighed and heated to dry completely, then weighed again after drying
2. A solid sample is ground up fine and weighed into a covered dish, at least 3 replicates
3. The sample is heated in an oven at 105 Celsius for 3 hours to dry.
4. The dried sample is set in a muffle furnace at 500-600 Celsius until grayish white.
5. If repeats are necessitated (grayish white not achieved, some parts of sample are unevenly heated), moisten sample with distilled water and repeat step 4 and 3, moistening slightly in
between repeats until sample is grayish white
6. Sample is taken out of oven and cooled in a desiccator
7. Weight is taken and recorded
8. Ash content is computed
67
Determination of Crude Protein % (Digestion)
1. Sample is ground and placed in a Kjeldahl digestion flask
2. Concentrated sulfuric acid is added to the flask. Catalysts such as mercury (I) sulfate or copper (II) sulfate could be added to speed up the reaction. This will convert nitrogen from proteins in the sample into ammonium sulfate.
3. The flask is heated until the solution is clear, generally around 370 Celsius. Clarity of the solution indicates the completion of digestion. It could be cooled and reheated to ensure total reaction.
68
Determination of Crude Protein % (Digestion) 1/2:
1. Sample is ground and placed in a __ __ __
2. __ __ __ is added to the flask. Catalysts such as __ __ or __ __ could be added to speed up the reaction. This will convert __ from __ in the sample into __ __.
3. The flask is heated until the solution is clear, generally around __ Celsius. __ of the solution indicates the completion of digestion. It could be cooled and reheated to ensure total reaction.
1) - Kjeldahl digestion flask
2) - Concentrated sulfuric acid
- mercury (I) sulfate
- copper (II) sulfate
- nitrogen
- proteins
- ammonium sulfate
3) - 370
- Clarity
69
Method for the Determination of Ash Content%:
1. __ __ is weighed and heated to dry completely, then weighed again after drying
2. A solid sample is ground up fine and weighed into a covered dish, at least __ replicates
3. The sample is heated in an oven at __ Celsius for __ hours to dry.
4. The dried sample is set in a muffle furnace at __-__ Celsius until ____.
5. If repeats are necessitated (__ not achieved, some parts of sample are unevenly heated), moisten sample with __ __ and repeat step 4 and 3, moistening slightly in between repeats until sample is grayish white
6. Sample is taken out of oven and cooled in a __
7. Weight is taken and recorded
8. Ash content is computed
1) Porcelain dish
2) 3
3) - 105
- 3
4) - 500-600
- grayish white
5) - grayish white
- distilled water
6) desiccator
70
Method for the Determination of Moisture Content %:
1. Containers (dishes/pans) are weighed and heated to dry completely, then weighed again after drying
2. A solid sample is ground up fine and weighed into a covered dish, at least 3 replicates
3. The sample is heated in an oven to __-__ Celsius for __-__ hours, depending on the sample
4. Sample is taken out of oven and cooled in a desiccator
5. Weight is taken and recorded
6. Moisture content is computed
3) - 105-115
- 16-24
71
Ash content is computed with the equation:
Ash % = (x-c)/(s-c) * 100
S = weight of sample and container prior to ashing
C = weight of the container
X = weight of sample and container after ashing
S - C = weight of sample
S - X = weight of sample after ashing
72
Determination of Crude Protein % (Digestion) 1/2- Chemical equation
Protein + H2SO4 --> CO2 + SO2 + H2O + (NH4)2HSO4
73
Method for Determination of Crude Protein % (Distillation and Titration) 2/2 (3)
1) Sample is transferred to a Kjeldahl apparatus. The end of the condenser is dipped into a known volume of a weak acid like boric acid.
2) Sodium hydroxide (NaOH) is then added to the digested solution to produce ammonia (NH3).
Ammonia is then boiled off into the boric acid at the end of the condenser, this reacts with the weak acid back into ammonium.
3) The ammonium is titrated using a strong acid like hydrochloric acid (HCl) or sulfuric acid (H2SO4).
74
Method for Determination of Crude Protein % (Distillation and Titration) 2/2
1) Sample is transferred to a __ __. The end of the __ is dipped into a known volume of a __ __ like boric acid.
2) __ __ (__) is then added to the digested solution to produce __ (__).
__ is then boiled off into the __ __ at the end of the __ , this reacts with the weak acid back into __.
3) The __ is titrated using a __ __ like hydrochloric acid (HCl) or sulfuric acid (H2SO4).
1) - Kjeldahl apparatus
- condenser
- weak acid
2) - Sodium hydroxide (NaOH)
- ammonia (NH3)
- Ammonia
- boric acid
- condenser
- ammonium
3) - ammonium
- strong acid
75
Step 3 in Determination of Crude Protein % (Distillation and Titration): Crude protein % equation
Crude protein % = (T * 14.01 * N * 100 * 6.25)/Weigh of sample * 100
T = Final volume after titration (Titre)
N = Normality of acid
14.011000 = Constant
6.25 = Protein conversion factor
76
Step 2 in Determination of Crude Protein % (Distillation and Titration): Sodium hydroxide (NaOH) is then added to the digested solution to produce ammonia (NH3) - show the chemical reaction:
(NH4)2HSO4 + 2NaOH --> NA2SO4 + 2H2O + 2NH3
77
Method for Determination of Crude Lipid %
1. The previously weighed sample is ground up and placed in a __ __ with __ __, placed in a __, and dried in an oven at 105 Celsius for 6 hours.
2. The __ containing the sample is placed inside a __ __. The beaker is rinsed 3 times with a solvent (hexane or ethyl ether) and placed into the __ __.
3. The __ __ is heated, evaporating the __ and condensing back to the __. The __ then washes away the fat back into the __ __ through a __
4. Fat extract is placed on a pre-weighed __ __, and rinsed with more solvent.
5. The sample is placed on a __ __ to take out the solvent from the extracted lipid. In the absence of a __, the evaporating dish is placed in a fume hood with the fan on until there is no discernible smell in the fume hood, a sign that all the solvent has evaporated.
6. Dish is dried in an oven at 105 Celsius, cooled in a desiccator, and weighed.
7. Crude lipid content is computed
1) - thimble lined
- filter paper
- beaker
2) - thimble
- Soxhlet extractor
- extraction flask
3) - extraction flask
- solvent
- thimble
- solvent
- extraction flask
- siphon
4) evaporation dish
5) - rotary evaporator
- rotary evaporator
78
Equation for Crude lipid %
Crude lipid % = (W2-W1)/(Weight of sample*) *100
*Weight of sample before initial drying prior to fat extraction
W1 = Weight of empty evaporating dish
W2 = Weight of evaporating dish + contents after drying
W2 - W1 = Weight of extracted fat
79
Method for Determination of Crude Fiber%:
1. Place ground, pre-weighed sample (usually 5 grams) into a 1L __ __
2. Add pre-heated __ __ (usually around 0.182 M of H2SO4) into the flask and boil
3. Filter out the __, leaving the sample behind, and rinse with __ __ 3 times
4. Repeat step 2 and 3 but substitute acid with __ (usually uses 0.313 M of NaOH). Weigh residue afterwards.
5. The collected fiber is dried in an oven to dry, temperature should be at least 130 Celsius (sources vary) for at least 1 hour then weigh afterwards.
6. The fiber is then __ at 500-550 Celsius for 30 minutes to 2 hours depending on the sample.
7. Fiber ash residue is taken out and cooled in a desiccator to be weighed.
8. Weight is taken and recorded.
9. Ash content is computed
1) conical flask
2) acid solution
3) acid
- hot water
4) alkali
5) ashed
80
Crude fiber % equation
Crude Fiber % = (W1-W2)/(Weigh of sample) * 100
81
Why can the nutritional content of food or feed be inaccurate in broad categorization (crude protein)?
Broad categorization, such as Crude Protein, counts total nitrogen in a sample, but not all nitrogen is from protein alone.
82
How does accuracy impact the results of nutritional analysis?
The results are only as good as the sample collected, and environmental conditions like weather can impact results.
83
What nutrients are typically excluded in proximal analysis?
determination of vitamins and minerals
84
Applications of proximate analysis (5)
1) Food Industry
2) Pharmaceutical Industry
3) Environmental Science
4) Feed Industry
5) Fuel Industry
85
It is a special type of protein produced by immune cells (B cells) that circulates to the blood, recognizing antigens specified for each antibody and bind with it for the immune system to search
Antibodies
86
It is any substance that are from a foreign organism such as bacteria or viruses. It can be a protein, a molecule or carbohydrates.
Antigen
87
- A set of procedure/methods that utilizes the use of immunity to detect and quantify samples.
- It mainly detects/uses antigen and antibodies
Immunoassay
88
- It is a type of immunoassay that utilizes the mechanics of the immune system to detect and quantify a specific target from a sample.
- It usually detects the presence of antibodies or antigens from a sample and are detected by the use of an enzyme linked to an antibody. This causes a chemical reaction used for detection and identification.
Enzyme-Linked Immunosorbent Assay (ELISA)
89
It is a type of labeled immunoassay, which means a label is attached so that it can easily be detected, in this case, the enzyme
Enzyme-Linked Immunosorbent Assay (ELISA)
90
ELISA is widely used in various areas of bioresearch (4)
- Healthcare
- Food safety
- Immunology
- Microbiology
91
4 main components of ELISA (4)
1) Capture Antibody (attached to the walls of the microplate)
2) Target Agent (antigens from the sample/analyte)
3) Enzyme-linked Antibody (used for detection if the target exists)
4) Substrate (acting as a dye(color) for the enzyme)
92
Where are ELISA assays performed?
wells (holes) of microplates
93
How are the agents attached in ELISA microplate wells?
The agents are attached to the walls of the microplate wells via heat incubation.
94
How ELISA works (4)
1) Attach capture antibodies to microplate walls.
2) Apply sample; if target is present, it binds to antibodies.
3) Introduce labeled antibodies; wash away excess.
4) Add enzyme-compatible substrate; color develops if target is present, indicating binding.
95
What is the principle behind ELISA?
determine whether the target agent is present in the sample.
96
Why are microplates washed between steps in ELISA?
prevent interference and ensure accurate results.
97
Why are microplates incubated between steps in ELISA?
to facilitate absorption and maximize interaction between biochemicals.
98
How does ELISA serve as a positivity test?
If color develops after the substrate is placed, it indicates a positive result, confirming the presence of the target agent.
99
4 types of ELISA (4)
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
100
A type of ELISA where the sample is placed first in the microplate, followed by an enzyme-linked antibody. It does not require a capture antibody, and incubation is done to absorb the antigen-antibody complex. It is simple and cost-effective but uses only one type of antibody and has a high background of color.
Direct ELISA
101
A type of ELISA where an unconjugated antibody binds to the target first, followed by enzyme-linked antibodies that bind to the original antibody. It is more effective but has a risk of cross-reactions.
Indirect ELISA
102
A type of ELISA that requires a capture antibody to absorb through the walls via incubation. The sample’s target agent binds to the capture antibody, and an added enzyme-linked antibody binds on top of the complex. It offers high specificity and sensitivity, works on complex samples, but is time-consuming and harder to perform.
Sandwich ELISA
103
A type of ELISA that uses a capture antibody and a sample added to the plates. Instead of an enzyme-linked antibody, an antigen is used. The sample antigen and labeled antigen compete for the antibody, producing different results. It is used with smaller molecules and often combined with methods like light absorbance.
Competitive ELISA
104
Direct ELISA is called “direct” as the labeled antibody __ __ to existing target agents for detection in the sample.
directly binds
105
In indirect ELISA, an __ __ (called as primary antibody) is introduced to the sample and bind to existing target agents. The __ __ (called as secondary antibody) will bind to the primary antibody instead of the target agent. It is called “indirect” as the labeled antibody does not interact with the target agent.
- unconjugated antibody
- enzyme-linked antibody
106
In sandwich ELISA, the target antigen is sandwiched with both the __, thus calling it “sandwich”. This interaction of both __ provide the best results in terms of __ __ and __ between both biochemicals.
- antibodies
- antibodies
- antigen specificity
- sensitivity
107
In competitive ELISA, if the sample antigen is __, then the result is __ __ making it near __ __. If many sample antigen is present, then the result is __ __, showing it is near to __ __. The vividness of the result is inversely proportional to the number of __ present.
- low
- more prevalent
- negative results
- less prevalent
- positive results
- antigens
108
- high sensitive and high selective analytical method.
- can provide information including excitation and emission spectrum, emission light intensity and measurement of life of emission light and polarization fluorescence etc.
Fluorescence Spectrophotometry
109
Fluorescence spectrophotometer has been used in various fields (6)
1) medical and clinical
2) pharmaceutical
3) biochemistry
4) food industry
5) pollution analysis
6) organic and inorganic chemistry
110
- father of fluorescence spectroscopy
- polarization of photoluminescence in solutions
- differentiated the transition moments between absorption and emission famous on his work, “__ __ __”
- Alexander Jablonski
- Jablonski Energy Diagram
111
a powerful analytical technique used to measure the fluorescent properties of substances. It involves exciting molecules in a sample with light and measuring the emitted fluorescence.
Fluorescence spectrophotometry
112
where molecules absorb photons of light energy. This absorption promotes electrons to higher energy levels.
Excitation
113
molecules return to their ground state, emitting photons of light. This emitted light has a longer wavelength and lower energy than the excitation light.
Fluorescence Emission
114
- difference in wavelength between the excitation and emission light.
- due to energy loss during relaxation processes
Stokes Shift
115
a measure of the efficiency of fluorescence emission. It represents the ratio of photons emitted to photons absorbed.
Quantum yield
116
Quantum yield (Φ) equation
Quantum yield (Φ) = N protons emitted / N protons absorbed
117
a plot of the intensity of emitted fluorescence light as a function of wavelength. It provides information about the composition and concentration of fluorescent molecules in the sample.
Fluorescence Spectrum
118
These spectra depict the wavelengths of light that cause excitation and the wavelengths of emitted fluorescence, respectively.
Excitation and Emission Spectra
119
Components in Fluorescence Spectrophotometry (6)
1. Light Source
2. Monochromators
3. Sample Holder/Cuvette
4. Optics
5. Data Display/Processor
6. Control Panel
120
What is the light source used in a fluorescence spectrophotometer?
A high-energy light source, such as a xenon lamp or a laser, is used to excite the sample.
121
A component in fluorescence spectrophotometry that selects the excitation wavelength of light, allowing for precise control of the light used to excite the sample.
monochromator
122
A component in fluorescence spectrophotometry that holds the sample solution and is designed to be optically transparent to minimize interference with light transmission.
sample holder, often a rectangular cuvette made of high-quality glass or quartz
123
A component in Fluorescence spectrophotometry that directs the excitation light onto the sample and collect the emitted fluorescence light, focusing the light and minimizing interference or stray light.
Optics, including filters, lenses, and mirrors
124
A component in fluorescence spectrophotometry that shows the measured absorbance values and allows for data analysis, such as wavelength scanning, calculation of concentration, or generation of calibration curves.
data display/processor
125
A component in fluorescence spectrophotometry that allows the user to set parameters such as wavelength, scan speed, and data acquisition settings using buttons, knobs, and a digital interface.
control panel
126
- refers to a sample that contains all the components of the analysis except the analyte itself.
- a control sample used to calibrate instruments, assess background levels, and correct for any interferences in the measurement process.
- crucial for ensuring the accuracy and precision of analytical results by providing a baseline reference point for comparison with samples containing the analyte of interest.
Blank
127
Analytical steps in fluorescence spectrophotometry (5)
1) calibration
2) blank measurement
3) sample preparation
4) measurement
5) data analysis
128
Analytical steps in FS: A __ __ is prepared using standard solutions with known concentrations of the analyte of interest.
calibration curve
129
Analytical steps in FS: A "__" measurement is taken using a solvent or reference solution without the analyte to establish a __.
- blank
- baseline
130
Analytical steps in FS: The __ __ is prepared, ensuring that it is compatible with the solvent used in the calibration and blank measurements.
sample solution
131
Analytical steps in FS: The obtained absorbance values are compared to the __ __ to determine the concentration of the analyte in the sample.
calibration curve
132
A Fluorescent material
fluorophore
133
an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions.
mass spectrometry (MS)
134
Mass spectrometry (MS) is divided into two (2)
- Atomic or Elemental Mass Spectrometry
- Molecular Spectrometry
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This type of MS can determine nearly all the elements in the periodic table
- Detection limits are often several orders of magnitude better than optical methods
Atomic or elemental mass spectrometry
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This type of MS can provide information about the structures of inorganic, organic, and biological molecules
- Can determine the qualitative and quantitative composition of complex mixtures.
Molecular mass spectrometry
137
In MS, molecules are bombarded with a __ __ __ __, ionizing them and breaking them into many fragments, some of which are positive ions.
beam of energetic electrons
138
The __ __ is the mass-to-charge ratio of an ion. For most ions, the charge is one, so this simply the molecular mass of the ion.
m/e ratio
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Ions pass through __ and __ to reach the detector, where they are detected and signals are recorded to give mass spectra.
- magnetic and electric fields
140
A __ __ is a plot of relative abundance against the mass-to-charge ratio (m/e), used to determine the elemental or isotopic signature of a sample, the masses of particles and molecules, and to elucidate the chemical structures of molecules and other chemical compounds.
mass spectrum
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Steps in Mass Spectrometry (MS) (5)
1) Sample inlet
2) Ionization
3) Acceleration
4) Deflection
5) Detection
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Step 1 in MS: How do molecules enter the ionization chamber in mass spectrometry?
through a pinhole called "Molecular leak" from a large reservoir.
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Step 2 in MS: What happens during ionization in mass spectrometry?
Atoms are bombarded with electrons to form positive ions by knocking off one or more electrons.
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Step 4 in MS: What determines the degree of deflection of ions in mass spectrometry?
Deflection depends on the mass-to-charge ratio (m/e ratio) and the number of positive charges an ion carries.
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Step 5 in MS: How are ions detected in mass spectrometry?
Ions passing through the mass analyzer are detected based on their m/e ratio, and their charge is neutralized upon hitting the detector by electron transfer.
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- a chromatographic technique that can be used to separate volatile organic compounds.
- a type of partition chromatography.
Gas Chromatography (GC)
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Types of Gas Chromatography (2)
1) Gas-solid chromatography (GSC)
2) Gas-liquid chromatography (GLC)
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a technique used for separating and analyzing volatile compounds in a mixture based on their interaction with a stationary phase and a mobile phase, typically involving a gas carrier.
Gas Chromatography (GC)
149
a type of gas chromatography where the stationary phase is a solid material. Retention of analytes occurs through physical adsorption onto this solid phase.
Gas-solid chromatography (GSC)
150
a type of gas chromatography where the stationary phase is a liquid coated onto an inert solid support. It is particularly useful for separating ions or molecules dissolved in a solvent.
Gas-liquid chromatography (GLC)
151
Basic principles of Gas Chromatography
1) The sample solution injected into the instrument enters a __ __ which transports the sample into a separation tube known as the "__." (Helium or nitrogen is used as the so-called __ __.) The various components are separated inside the __
2) The __ measures the quantity of the components that exit the __. To measure a sample with an unknown concentration, a standard sample with a known concentration is injected into the instrument.
3) The standard sample __ __ __ (appearance time) and __ are compared to the test sample to calculate the concentration.
1) - gas stream
- column
- carrier gas
- column
2) - detector
- column
3) - peak retention time
- area
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Steps in Gas Spectrometry (7)
1) Sample injection
2) Carrier gas
3) Column
4) Separation
5) Temperature Control
6) Detector
7) Data analysis
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Step 1 in GS: Sample injection: The __, which can be a liquid or gas, is __ into the chromatograph. For liquids, it is vaporized in the injection port.
- sample
- injected
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Step 2 in GS: An inert __ __ (e.g. , helium, nitrogen, or hydrogen) transports the vaporized sample through the chromatographic column. The __ __ should not react with the sample components.
- carrier gas
- carrier gas
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Step 3 in GS: The heart of the gas chromatograph, the __ is typically a long, coiled tube made of glass or metal and filled with a stationary phase. These can be __ (filled with a solid stationary phase) or __ (with a liquid stationary phase coated on the inner wall).
- column
- packed
- capillary
156
Step 4 in GS: As the sample components travel through the __, they interact with the stationary phase. Components that interact __ __ with the stationary phase move __, while those that interact __ move __, leading to separation based on their different __ for the stationary phase.
- column
- more strongly
- slower
- less
- faster
- affinities
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Step 5 in GS: The column is housed in an oven with precise __ __. The temperature can be held constant (__) or varied (temperature programming) to optimize __.
- temperature control
- isothermal
- separation
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Step 6 in GS: After separation, the components exit the __ and are detected by a detector. Common detectors include __ __ __ (__), __ __ __ (__), and __ __ (__). The detector provides a signal proportional to the amount of each component.
- column
- flame ionization detectors (FID)
- thermal conductivity detectors (TCD)
- mass spectrometers (MS)
159
Step 7 in GS: The detector signal is recorded as a __, a plot of __ __ (y-axis) versus __ (x-axis). Each peak in the chromatogram represents a different component, with the retention time (time at which a peak appears) used to identify the compound and the peak area used to quantify it.
- chromatogram
- detector response
- time
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Analytical components of Gas Spectrometry (7)
- Injection port
- Carrier gas
- Gas flow regulator
- Column
- Oven
- Detector
- Computer/Data analysis
161
Analytical components of Gas Spectrometry:
This is where the sample is introduced into the GC. It is heated to vaporize the sample.
Injection port
162
Analytical components of Gas Spectrometry: an inert gas that flows through the column and carries the vaporized sample
Carrier gas
163
Analytical components of Gas Spectrometry: device that controls the flow rate of the carrier gas
Gas flow regulator
164
Analytical components of Gas Spectrometry: Long, narrow tube that is packed with a stationary phase. This is where vaporized sample travels through where the components interact with the stationary phase to different extents.
Column
165
Analytical components of Gas Spectrometry: a chamber that houses the chromatographic column
Oven
166
Analytical components of Gas Spectrometry: Located at the end of the column. It senses the components of the sample as they escape from the column.
Detector
167
Analytical components of Gas Spectrometry: used to acquire and process the data from the detector. The data is typically displayed as a chromatogram, a graph with time (or flow) on the x-axis and detector signal intensity on the y-axis.
Computer/Data analysis
168
an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample
UV-Vis Spectrophotometry
169
Ultraviolet-visible (UV-Vis) spectroscopy is a widely used technique in many areas of science (4)
- bacterial culturing
- drug identification
- nucleic acid purity checks and quantitation
- quality control in the beverage industry and chemical research
170
Principle in UV-vis Spectrophotometry: When light interacts with matter, some of the light can be absorbed by the molecules in the sample. This absorption depends on the energy of the light (wavelength) and the electronic structure of the absorbing molecules.
Light-Matter Interaction
171
Principle in UV-vis Spectrophotometry: A law that states that the amount of light absorbed (A) is directly proportional to the concentration (c) of the absorbing species and the path length (l) that the light travels through the sample. This relationship can be expressed by the equation:
A = ε * l * c
where ε (epsilon) is the molar absorptivity coefficient.
Beer-Lambert Law
172
General components of UV-vis Spectrophotometry (5)
1) Light source
2) Monochromator
3) Sample cell
4) Detector
5) Readout and Data Processing Unit
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A UV-Vis spectrophotometer utilizes a light source that emits a broad spectrum of light, encompassing both __ and __ wavelengths. Common light sources include __ __ for the __ range and __ __ for the __range.
- ultraviolet (UV)
- visible
- Deuterium lamps
- UV
- Tungsten-halogen lamps
- visible
174
General component in UV-vis S: This component acts like a filter, selecting a specific wavelength of light from the broad spectrum emitted by the source. It allows scientists to analyze the interaction of the sample with light at various wavelengths one at a time.
Monochromator
175
What general component in UV-vis S? The sample solution is placed in a __, often made of quartz or plastic depending on the wavelength range used. The __ __ __, which is the distance the light travels through the sample, is a crucial factor in __ __ __ calculations
- Sample cell
- cuvette
- cuvette path length
- Beer-Lambert Law
176
General component in UV-vis S: A __ positioned opposite the light source measures the intensity of the light that passes through the sample. This allows the instrument to quantify the amount of light absorbed by the sample.
Detector
177
General component in UV-vis S: The instrument processes the data from the detector, converting it into an absorbance or transmittance spectrum. This spectrum displays the amount of light absorbed or transmitted at each measured wavelength.
Readout and Data Processing Unit
178
Analytical steps in UV-vis Spectrophotometry
1) Sample preparation
2) Instrument calibration
3) Blank measurement
4) Wavelength selection
5) Sample measurement
6) Data analysis
7) Quality control
8) Data interpretation
179
An analytical step in UV-vis Spectrophotometry that measures the absorbance or transmittance of a blank solution that does not contain the analyte. This accounts for any background absorption or scattering by the solvent or other components
Blank measurement
180
Simplified steps/components in UV-vis Spectrophotometry
Light source -(light)-> Wavelength selector -(light)-> Sample -(light)-> Detector -(Electric current)-> Computer for single processing and output
