Spectrophotometer

The instrument used for quantitative measurement of absorbance or transmittance of UV/Vis light. Must contain five basic components 1- Light source: required to emit the wavelength of interest. 2- Monochromator : used to split the light into the different wavelengths. 3-A sample compartment : holds the sample to be analysed. 4-A detector. 5-The readout

How Do UV-Vis spectrometers work?

Types of spectrophotometers
Single-Beam Spectrophotometers:

Detector Monochromator
Sample cuvette

Light source

Amplifier

Meter

Components of spectrophotometer
Light source: - UV measurement: hydrogen or deuterium discharge lamp (190 375 nm) - Visible measurement: Tungsten lamp (350 1000 nm) Monochromator: Function: To select light beam of certain wavelength. - Filter - Prisms - Grating

Monochromator
The monochromator isolates a narrow band of wavelength (monochromatic radiation) from the all wavelengths emitted by the light source (polychromatic radiation). Monochromator consists of three elements: a- Entrance slit. b- A dispersing element. c- Exit Slit.

Monochromator
Dispersing element:

Dispersing element split the incident light from the entrance slit into monochromatic beam. It may be one of the following:
1- Filters: function via selective absorption of unwanted wavelength and transmitting the complementary color. It consists of colored glass, or dye suspended in gelatin and sandwiched between two glass plates. Disadvantages of filters: Not able to isolate a very narrow band of wavelengths. The number of colors that can be measured depends on the number of filters available.

Monochromator
2- Prisms:

Prisms have also been used to split light into different wavelengths. As white light pass from air through the glass of the prism it is bent. This spread is uneven or non linear with crowding of the colors at the longer wavelengths. Prisms: function via refraction of light.

Monochromator
Glass prisms are used for the visible range only since glass is opaque to ultraviolet wavelengths. For the UV range quartz prisms can be used.

Due to the nonlinear dispersion of the wavelengths, prisms are not often chosen for use in spectrophotometers, since this non-linear dispersion requires the use of expensive variable width exit slits to consistently isolate the same narrow band of wavelengths.

Monochromator
Gratings: Consist of large number of parallel ruled groves very close to each other on a highly polished surface, e.g. aluminium, or aluminized glass (600 groove/mm). Each ruled groove functions as a scattering center for light falling on its edge and through diffraction and interference the grating disperses the light beam into almost single wavelength.
Incident light

Diffracted light

Monochromator
Gratings are expensive as the production process is time consuming and need the exact precision and care in creating exactly parallel grooves that are 0.83 m wide. The linear dispersion of gratings allows the use of fixedwidth exit slits and relatively simple wavelength-setting mechanisms. For this reason, monochromators usually contain diffraction gratings rather than prisms. There are two types of gratings: Transmission gratings and Reflection gratings.

Sample Compartment
Sample Cell (Cuvette) :
Transparent Quartz for UV measurements Glass or Quartz cell for VIS measurements Sample cuvette Pathlength: usually 0.5, 1 or 1 cm The cuvettes should be kept very clean and free from dirt, lint, fingerprints and scratches, all of which will absorb, diffract, and diffuse light, and give an incorrect transmittance reading.

Detector
Detectors :
Receive light emerged from the sample, which excite electrons and generate an electric current that proportional to the received light intensity.
Electrons Anode (iron) Light beam Cathode (selenium)

Photocell

Photomultiplier tube

Readout Device
Readout devices include: 1- Digital displays. 2- Printers and computers They function to translate the received current (current intensity proportional to light emerged from sample) to signals on paper or computer (integration of data).

Types of spectrophotometers
Single-Beam Spectrophotometers:

Detector Monochromator
Sample cuvette

Light source

Amplifier

Meter

 1- Single beam-spectrophotometer: In single beam spectrophotometers, all the light from the monochromator goes to the sample In the same compartment, and then to the detector. The advantages Relatively inexpensive and simple. The disadvantage is that the blank has to be measured frequently when measuring a series of samples over a period of time (time consuming), diffecult to determine max
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Types of spectrophotometers
Double-Beam Spectrophotometers:
Blank cuvette

Detector 1
Monochromator Amplifier
Beam splitter Sample cuvette

Meter

Light source

Detector 2

 2-Double beam spectrophotometer: Double beam spectrophotometers contains, beam splitter which splits the beam into two paths of equal intensity one directed to the reference (blank) and the other to the sample. Both beams are either directed to the same detector, or each has its own detector. By subtracting the reference form the sample, the difference in the signal is due to the substance in the sample.
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 The advantages of this system are the stability of the readings over time and the ability to simultaneously correct for any solvent or peripheral substance effects. The disadvantages are the expensive and the less sensitivity to measure highly absorbing samples due to equal spliting of light.
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Application of UV - VIS Spectrophotometry

I- Qualitative Analysis. II- Quantitative Analysis. III- Determination of the complexation ratio. IV- Detection of impurities. V- Determination of some physical constants.

I- Qualitative Analysis: Absorption spectrum and max are finger prints for each compound in specific solvent.
Amax

max

II- Quantitative Analysis: A- Using Transmittance data: Using semi-log paper: T = 10-abc B- Using Absorbance data: The absorbance (A) refers to the amount of light absorbed by the solution. It is a very convenient term because it is directly proportional to the concentration. A = abc

Using Absorbance Data:

1- Analysis of single compound: Prepare a solution of the sample in a suitable solvent. Determine the max by measuring a suitable concentration that gives an absorbance of 0.1 - 1 (good accuracy and precision) against reagent blank. Any unknown concentration can be calculated by measuring its absorbance at max by using one of the following:

 1- Using Standard Absorptivity Value: (non-accurate) A = abc c = A / a 2- Using Single point Standardization: ( non-accurate) Measure the absorbance of suitable known concentration of a reference standard material from which the unknown concentration is calculated as following: Cx Ax = Cs As Cx = Cs As / Ax
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 3- Using Standard curve: (more accurate) A standard curve is a plot of the Absorbance versus concentration of each standard. To construct a standard curve, at least five different dilutions of standard plus a reagent blank are recommended. The dilutions of the standard should cover the expected concentration range of the sample to be tested.

The absorbance value of each standard is plotted on the Y-axis against the concentration of that standard on the X-axis According to Beer's Law, absorbance and concentration are directly related. From the standard curve we can determine the concentration of the sample.

 2- Analysis of Binary mixtures If we have a binary mixture of X & Y At 1: A1 = A1x + A1y = a1x b cx + a1y b cy = a1x cx + a1y cy At 2: A2 = A2x + A2y = a2x cx + a2y cy Cx = a1y A2 a2y A1 / a1x a2y a2x a2y Cy = a1x A2 a2x A1 / a1x a2y a2x a2y
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III- Determination of the complexation ratio The complexation ratio between drug and reagent can be determined by using one of the following methods: 1- Molar Ratio Method. 2- Continuous variation (Jobs Method).

 IV- Detection of impurities: The same concentration of sample (x) and standard (s) are measured at two wave lengthes (1 & 2). The purity of sample is determined using: 1- The impurity index (I.I) I.I = (A1s /A2s) (A1x /A2x) For 100% purity, I.I must equal zero. 2- Spectrophotometric purity index (S.P.I) S.P.I = (A1s /A2s) / (A1x /A2x) For 100% purity, S.P.I must equal one.
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 V- Determination of some physical constants e.g. pka, pkb,.. HA = H+ + A- (in alk. med.) Ka = [H+] [A-] / [HA] -log Ka = - log [H+] + ( - log [A-] / [HA]) pKa = pH - log [A-] / [HA] At [A-] = [HA] pKa = pH
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Colorimetry
It is the method for quantitative analysis which depends on measuring the absorption of color VIS radiation. A compound can be analyzed colorimetrically if it falls in one of the following classes: 1- The compound is self-colored e.g. potassium dichromate, copper sulphate, cobalt chloride. 2- The compound can react with reagent to produce colored product, this reagent is called chromogen. e.g. Reaction of Fe++ with o-phenanthroline reagent to produce red colored complex .

3- The compound can be converted to a derivative which reacts with a reagent to produce a colored product. e.g. Reaction of esters with hydroxylamine. The formed hydroxamic acid derivative gives a red or purple color chelate with ferric ion.

 General requirement for ideal chromogen: 1- It should be colorless or has no absorbance at max of the colored product. 2- It should be selective i.e. reacts with the analyte only. 3- It should produce one single colored product having a specified max. 4- The mechanism of color formation reaction should be known and the reaction has definite stoichiometry. 5- Color development must be rapid.
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 General requirement for colored product: 1- The colored product should be intensely colored (to increase the sensitivity of the method). 2- It should be soluble in the solvent used. 3- The colored product should be unaffected by pH (if it is pH dependent, its solution should be buffered at specified pH. The color should be stable within + one pH unit).
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4- The color formation reaction should have a definite stoichiometry, quantitative and rapid. 5- The colored solution should be stable within certain reasonable period of time and the measurement must be carried out within this specified time. 6- The colored solution should obey Beer's Lambert's law.

EMISSION (LUMINESCENCE) SPECTROSCOPY

The emission of radiation from a species after that species has absorbed radiation.
FLUORESCENCE

LUMINESCENCE SPECTROSCOPY

PHOSPHORESCENCE CHEMILUMINESCENCE

LUMINESCENCE SPECTROSCOPY

Absorption first -

Followed by emission in all directions, usually at a lower frequency

LUMINESCENCE SPECTROSCOPY
In favourable cases, luminescence methods are amongst some of the most sensitive and selective of analytical methods available. Detection Limits are as a general rule at ppm levels for absorption spectrophotometry and ppb levels for luminescence methods.

LUMINESCENCE SPECTROSCOPY
Collectively, fluorescence and phosphorescence are known as photoluminescence. A third type of luminescence Chemiluminescence - is based upon emission of light from an excited species formed as a result of a chemical reaction.

LUMINESCENCE SPECTROSCOPY
Most chemical species are not naturally luminescent. Derivatisation reactions are often available to form luminescent derivatives of nonluminescent compounds. However, this extra step lessens the attractiveness of luminescence methods.

Energy Level Diagram

SINGLET STATES TRIPLET STATES

s2

VIBRATIONAL RELAXATION

T2 T1
INTERSYSTEM CROSSING PHOSPHORESCENCE INTERNAL CONVERSION INTERNAL CONVERSION

s1

FLUORESCENCE

Ground State

Theory of Fluorescence and Phosphorescence

Theory of Fluorescence: In solution and at room temperature, the majority of molecules are in the lowest vibrational level of the ground state. During the process of excitation, most of the affected molecules gain vibrational as well as electronic energy. Then, the majority of the excited molecules tend to drop to the lowest vibrational level losing energy to the surrounding solvent molecules via collision, this process is called vibrational relaxation.

This radiationless process stops at the S1 excited singlet electronic level in which the molecules are able to return directly to the ground state by radiation of a photon (fluorescence process).

 Theory of Phosphorescence
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In phosphorescence, an intersystem crossing can take place readily from S1, to one of the vibrational levels of T1, state that has very nearly the same energy level. This is usually followed by non radiative decay to the T1 level. Intersystem crossing process involves a change in the spin of the excited electron and thus a change in spin multiplicity.

Differences between Fluorescence and Phosphorescence

Item Emission Fluorescence is always at shorter wavelength Phosphorescence is always at longer wavelength

Temperature

usually observed at room temperature

in liquid solution

usually observed at very low temp.

in rigid medium

Medium Life time

usually in the range usually in the range 10-7 10-9 sec 10-4 10 sec

Fluorescence Spectra
Instruments that measure the intensity of fluorescence are called fluorimeters. Those that measure the fluorescence intensity at variable wavelengths of excitation and emission, and are able to produce fluorescence spectra are called spectrofluorimeters. Fluorescence Spectrum involve: a- Excitation Spectra B- Emission Spectra (Fluorescence)

Advantages of Spectrofluorimetry
1-High sensitivity: Substances that are reasonably fluorescent may be determined at concentration up to 1000 times lower than those required for absorption spectrophotometry. 2- High Selectivity: Substances that are fluorescent are characterized by their wavelengths of maximum excitation and emission.

Factors Affecting Fluorescence Intensity

1- Concentration 2- Intensity of incident light : An increase in the intensity of light incident on the sample produces a proportional increase in the fluorescence intensity. The power of fluorescent radiation, F, is proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence: (F P0 ) 3- Path length (b): The symbol for path length (b) does not refer to the dimension of the sample cuvette but to the internal volume of sample solution. The effective path length viewed by the detector depends on both the excitation and emission slit widths.

4- pH : It is to be expected that alteration of the pH of a solution will have a significant effect on fluorescence if the absorption spectrum of the solute is changed. Many phenols, for example, are fluorescent in both dissociated and undissociated forms. Consequently, the fluorescence from a solution of the phenol will show two peaks, one being due to the ionic form, acidic solutions may be necessary to suppress the peak due to the ionic form.

 5- Adsorption: The extreme sensitivity of the method requires very dilute solution, 10-100 times, weaker than those employed in absorption spectrophotometry. Adsorption of the fluorescent substance on the container walls may therefore presents serious problems.
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 6- Photodecomposition: In absorption spectrophotometry, the intensity of the radiation passing through solution is weak by photochemical standards, although adequate for measurements; decomposition of the solute is therefore, not very likely. Spectrofluorimetry, on the other hand, requires high intensity illumination for irradiation, and the risk of photochemical change is thereby increased.
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7- Temperature and viscosity: Variation in temperature and viscosity will cause variations in the frequency of collision between molecules. Thus, an increase in the temperature or the decrease in the viscosity is likely to decrease the fluorescence by deactivation of the excited molecules by collision.

INSTRUMENTATION
SOURCE SAMPLE

EXCITATION WAVELENGTH SELECTOR EMISSION WAVELENGTH SELECTOR

DETECTOR

INSTRUMENTATION
The fluorescence is often viewed at 90 orientation (in order to minimise interference from radiation used to excite the fluorescence). The exciting wavelength is provided by an intense source such as a xenon arc lamp (remember F P0).

INSTRUMENTATION
Because An intense monochromatic light source is required ... Lasers are an almost ideal light source for fluorimetry (laser-induced fluorescence) but are too expensive and/or impractical for most routine applications. Two wavelength selectors are required filters (in fluorimeters) and monochromators (in spectrofluorometers).

Types of Fluorescent Molecules

Experimentally it is found that fluorescence is favoured in rigid molecules, eg., phenolphthalein and fluorescein are structurally similar as shown below. However, fluorescein shows a far greater fluorescence quantum efficiency because of its rigidity.

phenolphthalein

Types of Fluorescent Molecules

It is thought that the extra rigidity imparted by the bridging oxygen group in Fluorescein reduces the rate of nonradiative relaxation so that emission by fluorescence has sufficient time to occur.
Fluorescein

APPLICATIONS
A. Determination of polyaromatic hydrocarbons

Benzo[a]pyrene is a product of incomplete combustion and found in coal tar.

APPLICATIONS
Benzo[a]pyrene, is a 5ring polycyclic aromatic hydrocarbon that is mutagenic and highly carcinogenic It is found in tobacco smoke and tar The epoxide of this molecule intercalates in DNA, covalently bonding to the guanine base nucleotide

APPLICATIONS
Excitation and fluorescence spectra for benzo(a)pyrene in H2SO4. In the diagram the solid line is the excitation spectrum (the fluorescence signal is measured at 545 nm as the exciting wavelength is varied). The dashed line is the fluorescence spectrum (the exciting wavelength is fixed at 520 nm while the wavelength of collected fluorescence is varied).

Benzo(a)pyrene

APPLICATIONS
B. Fluorimetric Drug Analysis Many drugs possess high quantum efficiency for fluorescence. For example, quinine can be detected at levels below 1 ppb.

Quinine

APPLICATIONS
In addition to ethical drugs such as quinine, many drugs of abuse fluoresce directly. For example lysergic acid diethylamide (LSD) whose structure is:

APPLICATIONS
Because LSD is active in minute quantities (as little as 50 mg taken orally) an extremely sensitive methods of analysis is required. Fluorimetricaly LSD is usually determined in urine from a sample of about 5mL in volume. The sample is made alkaline and the LSD is extracted into an organic phase consisting of n-heptane and amyl alcohol. This is a "clean-up" procedure that removes potential interferents and increases sensitivity. The LSD is then back-extracted into an acid solution and measured directly using and excitation wavelength of 335 nm and a fluorescence wavelength of 435 nm. The limit of detection is approximately 1 ppb