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What is the purpose of analytical techniques and instrumentation in a modern clinical chemistry laboratory?

They provide the foundation for all measurements made in a modern clinical chemistry laboratory.

What is a spectrophotometer used for?

A spectrophotometer is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.

What does Beer's Law state?

Beer's Law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.

What are the two main types of light sources used for work in the visible and near-infrared region?

The two main types of light sources used are the incandescent tungsten or tungsten-iodide lamp and the deuterium-discharge lamp. Signup and view all the answers

What is the main purpose of a monochromator in a spectrophotometer?

The purpose of a monochromator is to isolate individual wavelengths of light. Signup and view all the answers

What are the two main types of detectors used in spectrophotometers?

The two main types of detectors used are photomultipliers and photodiodes. Signup and view all the answers

What is the role of the sample cell or cuvette in a spectrophotometer?

The sample cell or cuvette holds the sample solution through which the light beam from the spectrophotometer passes. Signup and view all the answers

Which of the following are some of the limitations of using a spectrophotometer?

Interference from other substances in the sample that may absorb light at similar wavelengths. Signup and view all the answers

What is the name of the technique that measures the concentration of specific elements in a sample by measuring the absorption of light by atoms of a particular element?

The technique you're looking for is called Atomic Absorption Spectrophotometry (AAS). Signup and view all the answers

What are the main parts of an atomic absorption spectrophotometer?

The main parts of an atomic absorption spectrophotometer include a light source, an atomizer, a monochromator, and a detector. Signup and view all the answers

What is the purpose of the atomizer in AAS?

The atomizer converts the sample into a fine mist or vapor of atoms. Signup and view all the answers

Which of the following are some of the limitations of atomic absorption spectroscopy?

It's limited to elemental analysis, meaning it cannot analyze molecules or compounds. Signup and view all the answers

What does the acronym "ESI" stand for, and how is this method used in mass spectrometry?

ESI stands for Electrospray Ionization. This method is used in mass spectrometry to convert a liquid sample into ions, which are then analyzed by the mass spectrometer. Signup and view all the answers

What is the goal of tandem mass spectrometry (MS/MS) in terms of analysis?

Tandem mass spectrometry (MS/MS) is a powerful technique that allows for greater selectivity and lower detection limits. It involves linking multiple mass analyzers in series, usually three quadrupoles, to achieve more detailed and precise analyses of complex mixtures. Signup and view all the answers

What is the main purpose of Fluorometry?

Fluorometry measures the fluorescence emitted by a substance. Signup and view all the answers

What is the basic principle of fluorometry?

The basic principle of fluorometry involves stimulating a sample with light of a specific wavelength (excitation wavelength), causing the molecules to absorb the energy and emit light at a different wavelength (emission wavelength). Signup and view all the answers

What are some of the limitations of fluorometry?

Fluorometry has some limitations: it is not suitable for all molecules, rapid scans are not possible, the sample and reference solution cannot be analyzed at the same time, it is not useful for identification, dilute samples are less stable, pH changes and oxygen can affect fluorescent intensity, UV absorption can chemically change the sample, and not all substances can be converted to fluorescent compounds. Signup and view all the answers

How is fluorometry being used in the medical and pharmaceutical sciences?

In the medical and pharmaceutical sciences, fluorometry is often used for drug discovery purposes, analyzing the binding affinity of potential drug candidates to target proteins, monitoring the efficacy of enzymes, and identifying vitamins, such as thiamin and riboflavin in food. Signup and view all the answers

What is the core principle of electrochemistry?

The core principle of electrochemistry revolves around the relationship between chemical reactions and electricity. Signup and view all the answers

Give three key principles of electrochemistry.

The three key principles of electrochemistry are Faraday's Law of Electrolysis, the Nernst Equation, and the Butler-Volmer Equation. Signup and view all the answers

List the main parts of an electrochemical system.

The main parts of an electrochemical system include electrodes, electrolyte, an electrochemical cell, a power source, and measurement instruments. Signup and view all the answers

What are Ion-Selective Electrodes (ISEs) used for?

ISEs are sensors that measure the concentration of specific ions in a solution. Signup and view all the answers

What is the principle behind Ion-Selective Electrodes (ISEs)?

The principle behind ISEs is based on the Nernst equation, which relates the potential difference across the electrode membrane to the concentration of the ion being measured. Signup and view all the answers

What are the five main parts of an ISE?

The five main parts of an ISE include an ion-selective membrane, an internal reference electrode, an external reference electrode, an electrode body, and a connector or cable. Signup and view all the answers

What are some of the limitations of Ion-Selective Electrodes?

Limited selectivity and sensitivity. Signup and view all the answers

How is electrophoreses used in a lab?

Electrophoresis is a laboratory technique used to separate and analyze charged particles, such as DNA, RNA, proteins, and other molecules, based on their size and charge. Signup and view all the answers

What are the three principles of electrophoresis?

The three principles of electrophoresis are electrophoretic mobility, electroosmosis, and sieving. Signup and view all the answers

What are the main parts of an electrophoresis system?

The main parts of an electrophoresis system include the electrophoresis chamber, a power supply, electrodes, a gel or capillary medium, sample wells or an injection system and a detection system. Signup and view all the answers

What are some of the limitations of electrophoresis?

It's a time-consuming and labor-intensive process. Signup and view all the answers

What is osmometry used for?

Osmometry is a laboratory technique used to measure the osmotic pressure or concentration of solutes in a solution. Signup and view all the answers

On what principle is osmometry based?

Osmometry is based on the osmotic pressure equation, which relates the osmotic pressure to the concentration of solutes, the ideal gas constant, and the temperature of the solution. Signup and view all the answers

What are the main components of an osmometer?

The main components of an osmometer include an osmotic cell, a semi-permeable membrane, a pressure sensor, a temperature control unit, and a data acquisition system. Signup and view all the answers

What is chromatography, and what is its main purpose?

Chromatography is a group of techniques used to separate complex mixtures based on different physical interactions between the individual compounds and the stationary phase of the system. Signup and view all the answers

Which of the following are modes of separation used in chromatography?

Adsorption Signup and view all the answers

What is the main advantage of high-performance liquid chromatography (HPLC)?

HPLC uses pressure for fast separations, controlled temperature, inline detectors, and gradient elution techniques. Signup and view all the answers

What are the main components of an HPLC system?

The main components of an HPLC system include a reservoir, a pump, a column, a detector, and a recorder. Signup and view all the answers

In HPLC, what is the purpose of the pump?

The pump forces the mobile phase through the column at a much greater velocity than that accomplished by gravity flow columns. Signup and view all the answers

In HPLC, what is the role of the column?

The column is responsible for packing the stationary phase and facilitating the separation of the components in a sample based on their interactions with the stationary phase. Signup and view all the answers

What is the purpose of the detector in HPLC?

HPLC detectors monitor the eluate and produce electronic signals based on the concentration of each component. These signals are then used to generate a chromatogram, which is a visual representation of the separation process. Signup and view all the answers

What is the main purpose of the recorder in HPLC?

The recorder produces a chromatogram that shows detector response versus the time taken for the mobile phase to pass through the instrument. Signup and view all the answers

What is gas chromatography (GC) used for?

Gas chromatography (GC) is a method used to separate volatile compounds from nonvolatile mixtures. Signup and view all the answers

What are the main differences between gas chromatography (GC) and high-performance liquid chromatography (HPLC)?

The main differences between GC and HPLC lie in the mobile phase used. GC utilizes a gas as the mobile phase, while HPLC employs a liquid mobile phase. Signup and view all the answers

What is mass spectrometry (MS), and for what purpose is it employed?

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. It helps identify the amount and type of chemicals present in a sample by ionizing chemical compounds to generate charged molecules or molecule fragments and then measuring their mass-to-charge ratios. Signup and view all the answers

Describe the steps involved in mass spectrometry for analyzing a sample.

The sample in MS is first volatilized and then ionized to form charged molecular ions and fragments. These ions are then separated according to their mass-to-charge ratio (m/z) and measured by a detector. Signup and view all the answers

What is the main purpose of the mass analyzer in MS?

The mass analyzer in MS is responsible for separating ions based on their m/z values, allowing for the identification and quantification of each ion. Signup and view all the answers

What is the purpose of a detector in MS?

The detector in MS measures the intensity of the ion current for each species, allowing for the quantification of each specific ion. Signup and view all the answers

What is the main goal of tandem mass spectrometry (MS/MS)?

Tandem mass spectrometry (MS/MS) aims to increase the selectivity and sensitivity of mass spectrometry analysis by linking multiple mass analyzers in series. Signup and view all the answers

What is the main advantage of using high-resolution mass spectrometry?

High-resolution mass spectrometry offers increased precision in mass measurements, allowing for the analysis of complex mixtures with high accuracy. Signup and view all the answers

What is full width at half maximum (FWHM), and how does it relate to mass spectrometer resolution?

FWHM is a measure of the peak width of a mass spectrum at half of the peak's maximum height. A higher FWHM indicates a lower resolution, meaning that the mass spectrometer has a harder time distinguishing between ions with similar mass-to-charge ratios. Signup and view all the answers

Study Notes

Analytical Techniques

Analytical techniques form the foundation of measurements in modern clinical chemistry laboratories.
Key techniques include spectrophotometry, fluorometry, electrochemistry, and chromatography.

Spectrophotometry
Fluorometry
Electrochemistry
Chromatography

Spectrophotometric Instruments

Spectrophotometer measures transmitted light to determine the concentration of a light-absorbing substance.
Beer's Law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.

Transmittance

% Transmittance = (T/1) x 100
T is defined as 100% T.
T = (Sample beam signal / Blank beam signal) x 100

Absorbance

Absorbance (A) = -log(I/I₀)
A = εbc
ε is the molar absorptivity, b is the path length, and c is the concentration.

Example Calculation

Incident light intensity: 0.50 W/m²
Intensity entering detector: 0.36 W/m²
Calculate transmittance:
- %T = [(0.36 W/m²) / (0.50 W/m²)] x 100 = 72%
Determine absorbance
- A = -log(0.72) = 0.14

Example Calculation 2

Solution concentration (c): 0.25 M.
Path length (b): 1 cm.
Absorbance (A): 0.075
Calculate molar absorptivity (ε):
- ε = A / (bc) = 0.075 / (1 cm x 0.25 M) = 0.3 M^-1cm^-1

Components of a Spectrophotometer

Light Source:
- Incandescent tungsten or tungsten-iodide lamps for visible and near-infrared.
- Deuterium-discharge lamp and mercury-arc lamp for ultraviolet (UV) work.
Monochromators: isolates individual wavelengths of light.
Sample Cell: cuvets (round or square), discard scratched ones.
Photodetector:
- Photocell: light-sensitive material (selenium) on a plate of iron.
- Phototube: photosensitive material gives off electrons when light strikes.
- Photomultiplier (PM) tube: detects and amplifies radiant energy.

Limitations of a Spectrophotometer

Potential interference from other sample substances absorbing light at similar wavelengths
Biological samples can be complex matrices, affecting measurement accuracy
The stability and calibration of light sources, detectors, and optical components also affect accuracy.

Clinical Applications of Spectrophotometer

Biochemical analysis: measuring enzyme activities, metabolites, etc.
Clinical diagnostics: quantifying hormones, vitamins.
Drug monitoring: ensure therapeutic ranges.
Quality control: ensuring reliability of results.
Research applications: studying biochemical pathways, etc.

Atomic Absorption Spectrometry

Atomic absorption spectrometry determines the concentration of specific elements in a sample.
It works by measuring absorbance of light by atoms of a particular element.
Components include:
- Light source(hollow cathode lamp): emits light specific to the element.
- Atomizer (flame or graphite furnace): converts sample into atoms.
- Monochromator: isolates the specific wavelength of light absorbed by the element.
- Detector: measures transmitted light intensity.

Atomic Absorption Spectrometry—Applications

Metal Toxicity Assessment: Measuring lead levels in blood to diagnose and monitor lead poisoning (especially in children).
Assessing mercury levels in blood/urine to assess exposure to this toxic metal.
Diagnosing deficiencies and excess of essential metals like copper, zinc, iron, magnesium.
Analyzing trace elements like chromium, selenium, manganese which are metabolically important.
Monitoring therapeutic levels of metal-based drugs like lithium and platinum-based anticancer agents.
Detect and quantify metals in biological samples for forensic investigations of poisoning cases.
Analyze food samples in dietary intake assessments.

Limitations of Atomic Absorption Spectrometry

Limited to elemental analysis; cannot directly analyze molecules or compounds.
Matrix effects; presence of other elements can interfere with the analysis.
Instrumentation is complex and requires skilled operators.

Fluorometry

Fluorometry measures fluorescence emitted from a sample.
Fluorescence is the release of light from a substance that has absorbed light or electromagnetic energy (at a different wavelength).
Applications are in various fields, including pharmacology, biochemistry, molecular biology, environmental research, and more. Used to measure protein, nucleic acid, and environmental pollutant concentration.

Components of a Fluorometer

Components include excitation source (lamp or laser), excitation monochromator, sample cell, emission monochromator, detector, recorder/data acquisition system.
Specific components such as filter systems may be used.

Applications of Fluorometry

Biochemical analysis: analyzes concentration, structure, interactions among compounds like proteins and nucleic acids.
Environmental sciences: detects pollutants like polycyclic aromatic hydrocarbons (PAHs), heavy metals, other contaminants in water, soil, and air, etc.
Medical and pharmaceutical sciences: screening and characterizing potential drugs, assessing binding affinity of compounds to target proteins.

Limitations of Fluorometry

Analyze only fluorescent molecules, not all.
Difficult to measure excitation and emission spectra simulatenously
Solutions of dilute sample compounds are less stable
pH changes and presence of oxygen affect fluorescent intensity.
Chemical changes can occur when samples are subjected to UV absorption.
Not all compounds can be analyzed by fluorometry.

Electrochemistry

Electrochemistry studies the relationship between chemical reactions and electricity.
Basic principles include Faraday's Law of Electrolysis, Nernst Equation, and Butler-Volmer Equation.

Parts of an Electrochemical System

Electrodes (anode and cathode)
Electrolyte (solution or gel)
Electrochemical cell
Power source (e.g., potentiostat or galvanostat)
Measurement instruments (e.g., voltammeter, ammeter)

Limitations of Electrochemical Techniques

Complex instrumentation & setup
Electrode fouling or degradation
Interference from electrolytes or sample matrix
Limited sensitivity and selectivity

Clinical Applications of Electrochemical Techniques

Diagnosis and monitoring of diseases (e.g., diabetes, cancer).
Biosensors for biomarker detection.
Electrochemical therapy for medical procedures (electrocautery, electroporation).
Implantable devices (e.g., pacemakers, cochlear implants).
Wound healing & tissue engineering.

Ion-Selective Electrodes

Ion selective electrodes (ISEs) measure the concentrations of specific ions in a solution.
The principle is based on the Nernst equation: E = E° + (RT/nF) * In(a)

Parts of an ISE

Ion-selective membrane
Internal reference electrode
External reference electrode
Electrode body
Connector or cable

Limitations of ISEs

Limited selectivity and sensitivity
Interference from other ions or substances
Temperature and pH dependence
Calibration requirements
Limited dynamic range

Clinical Applications of ISEs

Acid-base balance assessment
Diagnostic testing (e.g., kidney function, cardiac function, neurological disorders)
Monitoring patients (e.g., ICU patients, dialysis patients) with electrolyte imbalances.

Electrophoresis

Electrophoresis separates charged particles (DNA, RNA, proteins) based on size and charge.
Principles include electrophoretic mobility (movement influenced by charge, size, and shape), electroendosmosis (solvent movement affecting migration), and sieving (separation based on size as particles pass through medium pores).

Components of Electrophoresis System

Electrophoresis chamber
Power supply
Electrodes (anode and cathode)
Gel or capillary medium
Sample wells or injection system
Detection system (e.g. UV, fluorescence, staining)

Limitations of Electrophoresis

Time-consuming and labor-intensive
Specialized equipment needed
Limited resolution and sensitivity
Sample preparation may be challenging
Sample matrix/contaminants may present interference.

Clinical Electrophoresis Applications

DNA analysis (genotyping, sequencing, fingerprinting)
Protein analysis (identification, quantification, purification)
Genetic disorder diagnosis (e.g. sickle cell anemia)
Forensic analysis (DNA profiling)
Cancer research and monitoring.

Osmometry

Osmometry measures the osmotic pressure or concentration of solutes in a solution.
Principle: based on the osmotic pressure equation: Π = CRT
- Π is osmotic pressure, C is solute concentration, R is the ideal gas constant, and T is absolute temperature).

Components of an Osmometer

Osmotic cell
Semi-permeable membrane
Pressure sensor
Temperature control unit
Data acquisition system

Limitations of Osmometry

Limited accuracy and precision
Requires calibration
Sample preparation can be challenging
Interference from contaminants or sample matrix
Limited dynamic range

Clinical Applications of Osmometry

Diagnosis and monitoring of diseases (Diabetes insipidus, Diabetes mellitus).
Kidney diseases(e.g. nephrotic syndrome)
Liver diseases (e.g., cirrhosis)
Serum osmolality, urine osmolality, and blood glucose level measurements
Quality control of injectable solutions; stability testing of pharmaceuticals

Chromatography

Chromatography separates complex mixtures based on interactions between individual compounds and the stationary phase of the system.
Includes various types such as adsorption, partition, steric exclusion, gel filtration and gel permeation, ion-exchange, and thin-layer chromatography (TLC).

HPLC (High-Performance Liquid Chromatography)

Modern technique using pressure for fast separations, controlled temperature, inline detectors, and gradient elution techniques.
Components include reservoir, pump, column, detector, and display.

GC (Gas Chromatography)

Separation method for volatile compounds from non-volatile mixtures.
Components include gas source, injector, column, and detector.

Mass Spectrometry

Measures mass-to-charge ratio of ions to quantify chemical species.
Principle of separating ions based on their mass-to-charge ratio (m/z).
Includes Sample Introduction/Ionization(like Electron Ionization, Atmospheric Pressure Chemical Ionization and Electrospray Ionization) and Mass Spectrometer Analyzer (like Quadrupole, Ion Trap).
Commonly used in clinical applications for drug screening, diagnosis, and metabolic profiling.

Limitations

Many methods are labor intensive, time-consuming
Sophisticated equipment involved
Expertise/skilled operators needed

Clinical Applications

Various methods used in pharmaceuticals, to detect and quantify drugs and metabolites or contaminants, diseases detection, and forensic investigations.

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