1305- spectrophotometry (3)

Beer-Lamberts Law states that:

the absorbance of light by a substance dissolved in a solvent is directly proportional to its [ ] (c) and the path length (l) which the light travels through the sample

Does spectrophotometry use Beers law?

yes

What is spectrophotometry?

Quantitative analytical technique that measures how much light a chemical substance absorbs/transmits at specific wavelengths

How does spectrophotometry determine the concentration of substances?

by analyzing light interaction (usually in UV, visible, or infrared spectrum)

Name 2 limitation of spectrophotometry

Used for dilute sltns, so higher concentrations will cause deviations (electrostatic interactions between molecules)

Name 2 requirements for spectrophotometry?

Monochromatic light used

Homogenous sltns used

Name the 6 parts of a spectrophotometer:

Light source

Monochromator

Collimator

Cuvette

Detector

Display/readout system

Describe lights path through a spectrophotometer:

Light goes from light source, through collimator lens, to monochromator (prism or grating), through wavelength selector (slit), so sample sltn (in cuvette), through detector, which displays results on a meter

collimator

A lens or mirror that converts diverging light from the source into a parallel (collimated) beam

Cuvette

A transparent container that holds the liquid sample.

What are cuvettes made of

made of glass or plastic (visible range) or quartz/silica (UV range)

detector

A device that converts transmitted light into an electrical signal, measuring its intensity

Give an example of a detector used in spectrophotometry:

photodiode

photo multiplier tube

Display/readout system works how?

electrical signal from the detector displays the data as absorbance or transmittance

common light sources

tungsten lamps for visible light 320 to 1000 nm and deuterium lamps for ultraviolet light 190 to 320 nm

A=Elc

A=absorbance

E=molar absorptivity

l=path length (cm)

c= concentration (mol/L)

The light source must:

provide stable, constant, broadband radiation

How is a monochromator structured?

It typically includes a prism or diffraction grating to disperse the light, along w/ an entrance/exit slits to select the desired wavelength.

What does a monochromator do?

Isolates an arrow brand of wavelengths from the light source

Extracts the interfering wavelengths before hitting the sltn

red light wavelength

longer, ~700nm

violet/blue light wavelength

400-500nm

Wavelength

physical distance between 2 identical points on consecutive waves: can be crest to crest or trough to trough

We calibrate with what substance

potassium dichromate

Why do we calibrate with potassium dichromate

to validate absorbance accuracy and linearity

Calibration, 7 simple steps

1) start spectrophotometer

2) select wavelength

3) zero the display

4) use dedicated filter, close cover

6) record reading

7) if data matches expected value, move to next calibration. If not, troubleshoot.

[Total protein] = (A x Ac)/ [calibrator]

What is A and Ac?

A= absorbance of the unknown

Ac= absorbance of the calibrator

How ofte is internal QC performed?

daily, if results fall outside +/- 2SD troubleshooting needs to be done

Give an example of an environmental control and why we'd use it

monitoring lab's humidity and ambient temperature to prevent detector drifts

What does cuvette maintenance consist of?

ensuring cuvette are clean, scratch-free, and appropriate for the wavelength (quartz-UV, glass for visible)

stray light is caused by what?

reflections of light from scratches on optical surfaces or dust particles in light path

OR

higher order spectra produced by diffraction gratings

What issue does stray light cause?

May cause a bias at a corresponding wavelength but doesn't originate from the sample.

absorbance of sample is altered by stray light

What QC do we run for stray light?

Use potassium chloride solution at the specific wavelength

QC is used to check what 3 things on a spectrophotometer

wavelength accuracy

stray light

linearity

Wavelength accuracy is ensured when?

when the wavelength on the dial is the actual wavelength of the light passing by the monochromator

stray light is?

any wavelength outside the band transmitted by the monochromator

What type of filter can check linearity over diff wavelengths?

Neutral density filters

Linearity

when there is a change in concentration results in a straight-line curve.

In what 3 situations should new curves be performed?

new reagent lot used

new mthodology used

major instrument

maintenance done (bulb change)

Principle behind turbidimetry

Measures the attenuation of transmitted light caused by scattering and absorption of suspended solids

2 applications of turbidimetry

Micro: [bacterial]

Chem: quantifying proteins (CRP, HBA1c

Turbidimetry instrumentation setup

A light source (tungsten lamp), passes through a cuvette to a detector placed in line with the light at 180°

Nephelometry principle

measures light scatter in a sltn- uses a nephelometer

What type of equipment does nephelometry use

light source (laser or tungsten lamp), collimator, cuvette and photoelectric detector

Detection angle of nephelometry

detectors are at 90° to the light source, provides higher sensitivity for dilute sltns

How is nephelometry used in antigen-antibody complex science?

by quantifying specific proteins (IgG, IgM, IgA, and complement components C3 and C4) in serum or body fluids

Principle behind absorbance spectrophotometry

molecules in a sample absorb photons, causing transitions between energy levels- electronic or vibrational

what applications does absorbance spectrophotometry have?

used to quantify substances (often), monitor chemical reactions, determine molecular structures

Is transmitted light proportional to the [ ] of colour intensity?

NO, it wont give a straight line on a graph

Does potentiometry consume the analyte?

NO

What is potentiometry capable of detecting?

Capable of detecting low concentrations of specific ions

Electrolyte

A medium containing ions that conducts electricity but is electronically insulating

Anode

electrode where oxidation occurs (electron loss)

Cathode

electrode where reduction occurs (electron gain)

Electrochemical cell

device that generates electricity from spontaneous redox reactions or uses electricity to drive non spontaneous reactions

Redox rxn

Process involving electron transfer where one substance is oxidized ( loses electrons) and another is reduced (gains electrons)

What is potentiometry used for?

Used for measuring ions like Na+, K+ and Cl-

What is the principle behind potentiometry?

Relies on the Nernst equation: Which relates the measured potential to the concentration (activity) of the analyte in the solution

What is electrochemistry?

Branch of physical chemistry studying the relationship between electrical potential difference and chemical change

What does electrochemistry focus on?

movement of electrons in oxidation-reduction reactions

Energy conversion between chemical and electrical forms

Measurement on a spectrophotometer is based on what?

is based on the reaction between the substance to be measured and a reagent to produce a colour (which depends on concentration; colour intensity is proportional to concentration)

Name 3 advantages to nephelometry

-rapid, reproducible, simple to operate

-common in high volume labs

-many uses in immunology (CRP, RF)

Give an example of an substance which could interfere with nephelometer machinery

Microbial contamination

Name 3 disadvantages to nephelometry

-high initial cost

-interfering substances cause erroneous results (and protein denaturation)

-spec turbidity/lipemia may exceed preset limits (may need clearing agent or ultrafuge)

Name a few common sources of error in standard curves

-bottom of y-axis didnt start at 0.000

-compressed axis

-uneven axis spacing

-incorrect labelling

-incorrect placement of regression line

-large point marks

-drawing curve point to point instead of avg

Is turbidimetry or nephelometry more sensitive to low levels of turbidity?

Nephelometry

Is absorbance spectrophotometry based on beers law?

Yessss

How can you ID molecular structures and quantify [protein]?

With absorbance spectrophotometry: pass electromagnetic radiation through the sample and it does all that fancy stuff

What does absorbance spectrophotometry measure?

Measures amount of light a sample absorbs at specific wavelength

Absorbance spectrophotometry, name the spectral regions:

UV, visible and infrared light

How does absorbance spectrophotometry work?

spectrometer passes light through sample (cuvette) and measures remaining transmitted light

Chromatography principle

Components with higher affinity for the stationary phase move slower, with those with affinity for mobile phase move faster = results in separation

Liquid chromatography is used for what?

Used for separating ions or molecules dissolved in a solvent

Gas chromatography is used for what?

Used for volatile components in the gas phase

Name the 4 components of a chromatographic system:

Stationary phase

Mobile phase

Eluent

Eluate

Stationary phase

the substance remains fixed in place (eg paper, silica on a plate, resin in a column)

Mobile phase

the solvent or gas that moves through the stationary phase, carrying the sample

Eluent

solvent used

Eluate

mixture of solvent and separated solutes exiting the system

Turbidimetry deals with what kind of light at what angle?

transmitted light @ 180 °

Nephelometry deals with what kind of light at what angle?

scattered light @ 90°

What is the purpose of chromatography

Purpose is to detect or quantitate a particular component or group of components to be assayed in their pure form

Gas chromatography is ideal for analyzing what kinds of compounds?

volatile, thermally stable compounds

Liquid chromatography is ideal for analyzing what kinds of compounds?

non-volatile, high MW substances

Liquid chromatography handles what kinds of substances?

Solvent, water, organic solvents

Gas chromatography handles what kinds of substances?

Inert gas (He, N2 H2)

Gas chromatography operates at what temps?

Operates at high temps (>100C) to vaporize samples

Liquid chromatography operates at what temps?

Lower temps, high pressure (5000-8000 psi) to move liquids through columns

Name a few applications of reflectance spectrophotometry:

evaluates tissue for diseases (measures hgb or O2)

can be used also in measuring paints/colours

identifies minerals, agricultural quality, food freshness

What fields use reflectance spectrophotmetry most?

Used mainly in dermatology, GI and surgical fields

Which POCT device uses reflectance spectrophotometry?

glucose monitors

Does reflectance spectrophotometry follow beers law?

No, it does not directly follow standard linear Beer-Lambert law

2 types of reflection in reflectance spectrophotometry

Specular (regular)

Diffuse

Specular reflection

occurs on smooth surfaces-angle of reflection = angle of incidence

Diffuse reflection

occurs on rough surfaces where light scatters in many directions.

Absorbance is directly proportional to?

to the [ ] of the sltn, SO:

As the [ ] of the coloured sltn increases, the amount of light absorbed increases and the percentage of light transmitted will increase

How do electronic devices express absorbance and transmittance?

%T

A

What is %T?

its the amount of light that passes through the coloured sltn, compared w/ the amount of light that passes through the blank or a standard

Light path in absorbance spectrophotometry

Transmitted through the sample

Light path in reflectance spectrophotometry

Reflected/scattered off the sample

typical samples in reflectance spectrophotometry

Solids, powders, rough surfaces, coatings