Spectrophotometry

Machine used to measure the number of protons emitted to estimate the intensitiy of light spectron absorved and transmitted by a sample

Provides information on the amount of the compound present into a sample

Measure the concentration of certain analyte into our sample

Into the spectrophotometer we use light or radiant energy to measure the concentration of the substance

Radiant energy from short wavelength gamma rays to long wavelength radio waves

Photons of energy traveling in a wavelike manner

Types of Electromagnetic Energy

Transmitted via electromagnetic energy characterized by frequency and wavelength

Shows the relationship between wavelength and energy

The distance betwen peaks

Light absorbed by the molecule

Other term for Absorbed Light

Light that passed through

The highest part of a wave

The lowest part of a wave

The distance between the peak and trough

The distance between 2 peaks

The wave or vibration per second

The number of vibration

Light travel in a wavelike manner

Types of Light

Kinds of Wavelength

Visible Spectra

Invisible Spectra

Below 340 nm

Above 700 nm

Very important in Clinical Chemistry

The radiant energy is travelling in a wavelength matter

Leads to characteristic of light

The shorter the wavelength

The longer the wavelength

Type of region seen with the naked eye

Type of region that cannot be detected by the eye

Invisible Region - Below 340 nm (UV Region)

Invisible Region - Above 700 nm (Infrared Region)

Red

Orange

Yellow

Green

Blue

Indigo

Violet

Use of light to determine the concentration or presence or absence of analyte

2 primary considerations in colorimetric analysis

Kinds of Colorimetry

Uses the eyes in determining end point

2 types of photoelectric colorimetry

Measurement of light intensity in a much narrower wavelength

It uses a device (prism or gratings)

It uses a filter to isolate parts of the spectrum

Measurement of light intensity of multiple wavelengths

States that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of transmitted light

nm

1 nanometer equivalence

mM

1 millimicron equivalence

Principle followed by the spectrophotometer

Order of Light

Ratio of the radiant energy transmitted divided by the radiant energy incident on the sample

The amount of light absorbed

Proportional to the inverse log of transmittance

Mathematically derived from %T

Formula for Absorbance

Light that was able to pass through into a cuvette which has a sample

2nd formula for Absorbance

A=abc

A in A=abc

a in A=abc

b in A=abc

c in A=abc

Provides radiant energy in the form of visible or non-visible light that may pass through the monochromator

The light of proper wavelength will be made incident on the analytical cell

Commonly used light source in the visible and near infrared region (up to 1,200 nm)

LASER stands for

Factors for choosing a light source

Factor: free from stray light

Factor: depends on the type of light bulb

Factor: shelf-life

Light Source: produce energy wavelength (340 to 700 nm) visible region

Light Source: contains small amount of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament

Light Source: provides energy source with high output in the UV range (down to 165 nm)

Light Source: used above 800 nm

Examples of Infrared Energy Source

IES: an electrically heated rod of rare earth element oxides

IES: uses silicon carbide

Light Source: exits narrow bands of energy at well-defined places in the spectrum (UV and visible)

Light Source: consists of a gas-tight chamber containing anode, a cylindrical cathode, and insert gas such as helium of argon

Minimizes unwanted or stray light

Prevents the entrance of scattered light into the monochromator system

Causes systematic error and loss of linearity

Isolate specific wavelength of light

Light radiation of a single wavelength

Refine and select the wavelength used for our specimen

Wedge-shaped pieces of glass

Has small grooves cut at such as angle that each groove behave like a very small prisms

Monochromator: disperses white light into a continuous spectrum of colors based on variation of refractive index for different wavelength

Monochromator: separates white light into various color component

Monochromator: principle that wavelengths are bent as they pass a sharp corner

Monochromator: made of a glass that absorbs some portion od the electromagnanetic spectrum and transmit others

Monochromator: utilize the wave character of light to enhance the intensity of the desired wavelength by constructive interference and eliminates other by destructive interference and reflections

Controls the width of the light beam

Used to hold the solution in the instrument whose concentration is to be measured. Made up of glass, quartz, or plastic

Types of Cuvette

In a detectoer, the conversion is

Cuvette: for solutions that do not etch glass (acids can etch the glass)

Cuvette: does not absorb UV radiation at wavelength below 320 nm

Cuvette: good for 340 nm and above (visible)

Electron tube amplifying a current that can convert transmitted energy into an equivalent amount of electrical or photoelectric energy

Types of Detector

Other term of Barrier Layer Cell

Other term of Photoemissive tube

Composed of a film of light sensitive material like selenium on iron plate with transparent layer of silves

Require no external voltage sources (own source of electricity)

Has photosensitive material that gives off electron when light energy strikes it

Consist of 2 electrodes (cathode and anode) sealed in an evacuated glass to prevent scattering of photoelectrons by collision with gas molecules

Require an outside voltage for operation

Uses a series of electrodes to internally amplify the photo signal before leaving the tube

Most common type that meaasures visible and UV regions

Excellent sensitivity and rapid response

Must never be exposed to a room light

Simplest method of displaying output of the detection system

Other term for meter

Example of Meter

Used to determine whether the spectrophotometer is calibrated

Used to check wavelength accuracy

Verify absorbance accuracy on linearity

Splits monochromatic light into 2 components

This component of monochromatic light iuses 2 photodetectors for the sample and reference beam

This component of monochromatic light uses 1 photodetector and alternately passes the monochromatic light through the sample cuvette and then reference cuvette using a chopper

Monochromator: light energy is absorbed by dye compounds on the glass and is dissipated as heat