PILM- Clinical chem

what are some analysis techniques used in clinical chemistry

• spectrophotometery

• thin layer chromatography

• gas chromatography

• liquid chromatography

• electrophoresis

• mass spectrometry

what is a spectrophotometer

a machine that measures the amount of light absorbed by a solution at a particular wavelength in order to perform an assay and determine the amount purity or function of a substance

• what wavelengths are absorbed depend on the substances chemical components

• amount of absorption is directly proportional to the concentration of a componenet

what is transmittance in spectrophotometer

the amount of light that is not absrobed or refeected by the component being analysed

• % of transmittance = transmitted intesity (light from solution)/. radial instesity (light from the spectrophotometer)

what is beers law

a law that states that there is a directly proportional relationship between absorption and concentration of a component

• absorbance = molar absorptivity (constant) x path length(depends on size of cuvet) x Concentration of solute

how to use a spectrophotometer

spectrophotometers can measure either absorbance of an exponential scale(0-infinte) or transmittance on a linear scale (0-100%)

• you will also use cuvet that are typically 1cm in width

• the solute solvent will always be water as it has 0 transmittance and absorption

protein assays using spectrophotometry

specifcally measures the concentration of proteins in a solution using a standrad curve

• maresure the absorbance of solutions with a know protein concentration to create a standad curve

• then measure absorbance of unknown and plots it on curve using the line to determine its concentration

• can also measure concentration of DNA and RNA

immunoassays using spectrophotometry

a technique used to dtermine the concentration of a subatnce like a hormone, protein, drug or pathogen

• uses a antibody attached to an enzyme to bidn to the substance (acts as an antigen)

• then adds a reactant or sort that triggers a chemical reactant that causes the colour of the solution to change

• the more of the”antigen present the dark / mroe intense the colour

what are immunoassay used in

• prenancy tests

• troponin tests (dteremines f heart attcak is occuring)

• vitamin D tests

• HIV or hepatitis tests

what are speration techniques

techniques that surround separting molecules in a mixture for further analysis which can be done through

• flitration

• centrifusion

• chromatography

• electrophoresis

what is chromatographty

a techniques used to seprate mixture into their base components to analyse, identify, purify and or quantify saud components

• seperates based on polarity or the natural attraction between elements and a mobile(non/less polar) and stationary (more polar) phase

• the mobile phase carries the component forward and the stationary phase aim to hold the element in its place

what is thin layer chromatography

uses a tin layer of an inert material on a rigid backing at which components move up with the aid of a solvent that soaks into the inert material

• inert material = stationary phase, solvent = mobile phase

• used in screening tests or in the seperation of amino acids, drugs or vitamins

• can also be used to determine purity of substance

what is gas chromatography

a techniques used to sperate volitile organic compounds by vapourising them and pushing them through a coloum lined with or filled with a stationary phase using an inert gas like helium as a mobile phase

• the column /tube is placed in an oven to ensure proper temp during process

• the speed at which the component moves trhough the tunnel dpeneds on its tendency to be a gas and it polarity as it interacts with either the stationary phase (slows down) or the mobile phase

• a detector is then placed at the end to record how quickly different components make it through the tube

what does volitile mean

something is volatile if it can be easily converted to a gas

what is gas chromatography used for

• the analysis of lipids

• detection of volitile short chain organic acids by anaerobic bacteria

• urine analysis in drug screening

• blood analysis also in drug screening

what is liquid chromatography (+ use)

a technique used to seperate non volitile substance dissolved in a solvent

• are often not volatile as they are very polar and heavy

• used to analyse toxins, drugs and metabolites

how does liquid chromatography work

1. a liquid sample is dissolved into a mobile phase

2. that solution is then pushed through a coloum contains and solid or liquid stationary phase

3. the speed at which the sample moves depends on how much it interacts with each phase

• slow moving = interacts more with stationary

4. components are identified and measured using the time it take for them to exit the tube

what is high performance liquid chromatogrphy

same as liquid chromatography but faster and it pushes the mobile phase + sample throguh the solid stationary phase containg coloumn used lots of pressure

• very commonly used

• aka HPCL

what are some strength of HPLC

• a very sensitive technique allowing for more accurate results

• good for seprating non-volatile components like amino acids, proteins, nucleic acids, carbs, drugs, antibiotics, steroids and vitamins

what is HPLC used for

• alcohol and rug screening

• diabetes monitoring

• vitamin level checking

• dteemiening the presence of catechol amines like hormones and neruotransmitters

what is electrophoresis

a techniques used to seperate charhed components by forcing them through a gel or liquid using an electrical current

• will spread out depending on differences in charge and size

• negatively charged molecule move away from negative eletrode and towards positive and positively charged molecule do the opposite

what are the two methods of electrophoresis

1. agrose gel electrophoresis which seperates DNA and RNA into different base pairs

• need to use UV light to tag the molecules

2. Polyacrylamide electrophoresis which separte proteins and small DNA molecules

• uses staining to display molecules

what is mass spectrometry

measures the mass to charge ration of substance and the abundance of gas phase ions to identify unknown compounds, molecular weight, quantify known substances and determine the presence of different chemical structures

• the sample is turned into a gas and ionised to create charge molecules that can be manipulated

• magnetic feils seperate the now ions based on their ratios in a vaccum

what is ionisation

the additon or removal or electron from a molecule giving it a positive or negative charge

what are the application of mass spectrometry

• identifying pathogens in microbiology

• drug and alcohol screening

• neonatal screening (detects metabolic disorders)

• therapeutic drug monitoring ( ensures that too much of a drug isn’t given)

• to study proteins or metabolites