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Mr. K. Selvaraj B. Pharm, M. Tech
THANDALAM- 602 105




Classification of methods – calibration of instrumental methods – electrical
components and circuits – signal to noise ratio – signal – noise enhancement.


General design – sources of radiation – wavelength selectors – sample containers –
radiation transducers – types of optical instruments – Fourier transform measurements.


Measurement of transmittance and absorbance – beer's law – spectrophotometer
analysis – qualitative and quantitative absorption measurements - types of spectrometers –
UV – visible – IR – Raman spectroscopy – instrumentation – theory.


Thermo-gravimetric methods – differential thermal analysis – differential scanning


Introduction to chromatography – models – ideal separation – retention parameters –
van – deemter equation – gas chromatography – stationary phases – detectors – kovats
indices – HPLC – pumps – columns – detectors – ion exchange chromatography – size
exclusion chromatography – supercritical chromatography – capillary electrophoresis
Classification of methods – calibration of instrumental methods – electrical
components and circuits – signal to noise ratio – signal – noise enhancement.

Analytical Chemistry deals with methods for determining the chemical composition
of samples of matter. A qualitative method yields information about the identity of atomic or
molecular species or the functional groups in the sample; a quantitative method, in contrast,
provides numerical information as to the relative amount of one or more of these
Analytical methods are often classified as being either classical or instrumental. This
classification is largely historical with classical methods, sometimes called wet-chemical
methods, preceding instrumental methods by a century or more.
Classical Methods
Separation of analytes by precipitation, extraction, or distillation.
Qualitative analysis by reaction of analytes with reagents that yielded products that
could be recognized by their colors, boiling or melting points, solubilities, optical
activities, or refractive indexes.
Quantitative analysis by gravimetric or by titrimetric techniques.

1. Gravimetric Methods – the mass of the analyte or some compound produced from the
analyte was determined.
2. Titrimetric Methods – the volume or mass of a standard reagent required to react
completely with the analyte was measured.
Instrumental Methods
Measurements of physical properties of analytes, such as conductivity, electrode
potential, light absorption, or emission, mass to charge ratio, and fluorescence, began to be
used for quantitative analysis of a variety of inorganic, organic, and biochemical analyte.
Highly efficient chromatographic and electrophoretic techniques began to replace distillation,
extraction, and precipitation for the separation of components of complex mixtures prior to
their qualitative or quantitative determination. These newer methods for separating and
determining chemical species are known collectively as instrumental methods of analysis.
Instrumentation can be divided into two categories: detection and quantitation.
1. Quantitation
Measurement of physical properties of analytes - such as conductivity, electrode
potential, light absorption or emission, mass-to-charge ratio, and fluorescence-began to be
employed for quantitative analysis of inorganic, organic, and biochemical analytes.
2. Detection
Efficient chromatographic separation techniques are used for the separation of
components of complex mixtures.
Table 1. Classification of instrumental methods based on different analytical signals

Signal Instrumental Methods

Emission of radiation Emission spectroscopy (X-ray, UV, visible,
electron, Auger); fluorescence,
phosphorescence, and luminescence
(X-ray, UV, and visible)
Absorption of radiation Spectrophotometry and photometry (X-ray,
UV, visible, IR); photoacoustic
spectroscopy; nuclear magnetic resonance
and electron spin resonance spectroscopy
Scattering of radiation Turbidimetry; nephelometry; Raman
Refraction of radiation Refractometry; interferometry
Diffraction of radiation X-Ray and electron diffraction methods
Rotation of radiation Polarimetry; optical rotary dispersion;
circular dichroism
Electrical potential Potentiometry; chronopotentiometry
Electrical charge Coulometry
Electrical current Polarography; amperometry
Electrical resistance Conductometry
Mass-to-charge ratio Mass spectrometry
Rate of reaction Kinetic methods
Thermal properties Thermal conductivity and enthalpy
Radioactivity Activation and isotope dilution methods


A calibration curve is one approach to the problem of instrument calibration; other
approaches may mix the standard into the unknown, giving an internal standard.
The calibration curve is a plot of how the instrumental response, the so-called
analytical signal, changes with the concentration of the analyte (the substance to be
measured). The operator prepares a series of standards across a range of concentrations near
the expected concentration of analyte in the unknown. The concentrations of the standards
must lie within the working range of the technique (instrumentation) they are using (see
figure). Analyzing each of these standards using the chosen technique will produce a series of
measurements. For most analyses a plot of instrument response vs. analyte concentration will
show a linear relationship. The operator can measure the response of the unknown and, using
the calibration curve, can interpolate to find the concentration of analyte.
Calibration curve

Figure 1.Limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).

How to create a calibration curve

The data - the concentrations of the analyte and the instrument response for each
standard - can be fit to a straight line, using linear regression analysis. This yields a model
described by the equation y = mx + c, where y is the instrument response, m represents the
sensitivity, and c is a constant that describes the background. The analyte concentration (x) of
unknown samples may be calculated from this equation.
Many different variables can be used as the analytical signal. For instance, chromium
(III) might be measured using a chemiluminescence method, in an instrument that contains a
photomultiplier tube (PMT) as the detector. The detector converts the light produced by the
sample into a voltage, which increases with intensity of light. The amount of light measured
is the analytical signal.
Most analytical techniques use a calibration curve. There are a number of advantages
to this approach. First, the calibration curve provides a reliable way to calculate the
uncertainty of the concentration calculated from the calibration curve (using the statistics of
the least squares line fit to the data). [1]
Second, the calibration curve provides data on an empirical relationship. The
mechanism for the instrument's response to the analyte may be predicted or understood
according to some theoretical model, but most such models have limited value for real
samples. (Instrumental response is usually highly dependent on the condition of the analyte,
solvents used and impurities it may contain; it could also be affected by external factors such
as pressure and temperature.)
Many theoretical relationships, such as fluorescence, require the determination of an
instrumental constant anyway, by analysis of one or more reference standards; a calibration
curve is a convenient extension of this approach. The calibration curve for a particular
analyte in a particular (type of) sample provides the empirical relationship needed for those
particular measurements.
The chief disadvantages are that the standards require a supply of the analyte material,
preferably of high purity and in known concentration. (Some analytes - e.g., particular
proteins - are extremely difficult to obtain pure in sufficient quantity.)
Analysis of concentration
Verifying the proper functioning of an analytical instrument or a sensor device such as an
ion selective electrode
Determining the basic effects of a control treatment (such as a dose-survival curve in
clonogenic assay)
Standard addition method
The method of standard addition is used in instrumental analysis to determine
concentration of a substance (analyte) in an unknown sample by comparison to a set of
samples of known concentration, similar to using a calibration curve. Standard addition can
be applied to most analytical techniques and is used instead of a calibration curve to solve the
matrix effect problem.
This graph is an example of a standard addition plot used to determine the
concentration of calcium in an unknown sample by atomic absorption spectroscopy. The
point at zero concentration added Ca is the reading of the unknown, the other points are the
readings after adding increasing amounts ('spikes') of standard solution. The absolute value
of the x-intercept is the concentration of Ca in the unknown, in this case 1.69E-6 g/mL.

Standard addition is frequently used in atomic absorption spectroscopy and gas


Ohm's Law
For many conductors of electricity, the electric current which will flow through them
is directly proportional to the voltage applied to them. When a microscopic view of Ohm's
law is taken, it is found to depend upon the fact that the drift velocity of charges through the
material is proportional to the electric field in the conductor. The ratio of voltage to current is
called the resistance, and if the ratio is constant over a wide range of voltages, the material is
said to be an "ohmic" material. If the material can be characterized by such a resistance, then
the current can be predicted from the relationship:
Kirchhoff's Current Law (KCL)
The current entering any junction is equal to the current leaving that junction. i1 + i4 =
i2 + i3.This law is also called Kirchhoff's first law, Kirchhoff's point rule, Kirchhoff's junction
rule (or nodal rule), and Kirchhoff's first rule.
Power law
The general form of the law is
where I is the magnitude of the physical stimulus, ψ(I) is the psychophysical function relating
to the subjective magnitude of the sensation evoked by the stimulus, a is an exponent that
depends on the type of stimulation and k is a proportionality constant that depends on the type
of stimulation and the units used.


Direct Current.(DC) Is one that flows always in the same direction. Most electronic
devices need Direct Current because they require a steady flow of electrons that always head
in the same direction. A battery is Direct Current. Alternating Current (AC) is changed to
Direct Current (DC) with the use of Diode Rectifiers. You cannot use a transformer with
Direct Current.

Series Circuit
A Series circuit is one in which all components are connected in tandem. The current
at every point of a series circuit stays the same. In series circuits the current remains the same
but the voltage drops may vary.
Parallel Circuit
Parallel circuits are those in which the components are so arranged that the current
divides between them. In parallel circuits the voltage remains the same but the current may
vary. The circuits in your home are wired in parallel.

Digital voltmeters (DVM)

Digital voltmeters usually employ an electronic circuit that acts as an integrator,

linearly ramping output voltage when input voltage is constant (this can be easily
realized with an opamp). The dual-slope integrator method applies a known reference
voltage to the integrator for a fixed time to ramp the integrator's output voltage up, then
the unknown voltage is applied to ramp it back down, and the time to ramp output
voltage down to zero is recorded (realized in an ADC implementation). The unknown
voltage being measured is the product of the voltage reference and the ramp-up time
divided by the ramp-down time. The voltage reference must remain constant during the
ramp-up time, which may be difficult due to supply voltage and temperature variations.
Part of the problem of making an accurate voltmeter is that of calibration to check its
accuracy. In laboratories, the Weston Cell is used as a standard voltage for precision
work. Precision voltage references are available based on electronic circuits.Digital
voltmeters, like vacuum tube voltmeters, generally exhibit a constant input resistance of
10 megohms regardless of set measurement range.
The amplitude or peak value of the sinusoidal variation we shall represent by Vm and
Im, and we shall use V = Vm/21/2 and I = Im/21/2 without subscripts to refer to the RMS values.
For an explanation of RMS values, see Power and RMS values.
So for instance, we shall write:
v = v(t) = Vm sin (ωt + φ)
i = i(t) = Im sin (ωt).
where ω is the angular frequency. ω = 2πf, where f is the ordinary or cyclic frequency. f is the
number of complete oscillations per second. φ is the phase difference between the voltage
and current. We shall meet this and the geometrical significance of ω later.
Resistors and Ohm's law in AC circuits
The voltage v across a resistor is proportional to the current i travelling through it.
Further, this is true at all times: v = Ri. So, if the current in a resistor is
i = Im . sin (ωt) , we write:
v = R.i = R.Im sin (ωt)
v = Vm. sin (ωt) where
Vm = R.Im
So for a resistor, the peak value of voltage is R times the peak value of current. Further, they
are in phase: when the current is a maximum, the voltage is also a maximum.
(Mathematically, φ = 0.) The first animation shows the voltage and current in a resistor as a
function of time.
Impedance and reactance
Circuits in which current is proportional to voltage are called linear circuits. (As soon
as one inserts diodes and transistors, circuits cease to be linear, but that's another story.) The
ratio of voltage to current in a resistor is its resistance. Resistance does not depend on
frequency, and in resistors the two are in phase, as we have seen in the animation. However,
circuits with only resistors are not very interesting.
In general, the ratio of voltage to current does depend on frequency and in general there is a
phase difference. So impedance is the general name we give to the ratio of voltage to current.
It has the symbol Z. Resistance is a special case of impedance. Another special case is that in
which the voltage and current are out of phase by 90°: this is an important case because when
this happens, no power is lost in the circuit. In this case where the voltage and current are out
of phase by 90°, the ratio of voltage to current is called the reactance, and it has the symbol
Capacitors and charging
The voltage on a capacitor depends on the amount of charge you store on its plates.
The current flowing onto the positive capacitor plate (equal to that flowing off the negative
plate) is by definition the rate at which charge is being stored. So the charge Q on the
capacitor equals the integral of the current with respect to time. From the definition of the
vC = q/C, so

we have a sinusoidal current i = Im . sin (ωt), so integration gives

(The constant of integration has been set to zero so that the average charge on the capacitor is
Now we define the capacitive reactance XC as the ratio of the magnitude of the voltage to
magnitude of the current in a capacitor. From the equation above, we see that X C = 1/ωC.
Now we can rewrite the equation above to make it look like Ohm's law. The voltage is
proportional to the current, and the peak voltage and current are related by
Vm = XC.Im.
RC Series combinations
When we connect components together, Kirchoff's laws apply at any instant. So the
voltage v(t) across a resistor and capacitor in series is just
vseries(t) = vR(t) + vC(t)
However the addition is complicated because the two are not in phase. The next animation
makes this clear: they add to give a new sinusoidal voltage, but the amplitude is less than
VmR(t) + VmC(t). Similarly, the AC voltages (amplitude times 21/2) do not add up. This may
seem confusing, so it's worth repeating:
vseries = vR + vC but
Vseries > VR + VC.
This should be clear on the animation and the still graphic below: check that the voltages v(t)
do add up, and then look at the magnitudes. The amplitudes and the RMS voltages V do not
add up in a simple arithmetical way.
Here's where phasor diagrams are going to save us a lot of work. Play the animation again
(click play), and look at the projections on the vertical axis. Because we have sinusoidal
variation in time, the vertical component (magnitude times the sine of the angle it makes with
the x axis) gives us v(t). But the y components of different vectors, and therefore phasors, add
up simply: if
rtotal = r1 + r2, then
ry total = ry1 + ry2.
So v(t), the sum of the y projections of the component phasors, is just the y projection of the
sum of the component phasors. So we can represent the three sinusoidal voltages by their
phasors. (While you're looking at it, check the phases. You'll see that the series voltage is
behind the current in phase, but the relative phase is somewhere between 0 and 90°, the exact
value depending on the size of VR and VC.
All of the variables (i, vR, vC, vseries) have the same frequency f and the same angular
frequency ω, so their phasors rotate together, with the same relative phases. In this series
circuit, the current is common. (In a parallel circuit, the voltage is common, so I would make
the voltage the horizontal axis.)

The phasor diagram below shows us a simple way to calculate the series voltage. The
components are in series, so the current is the same in both. The voltage phasors (brown for
resistor, blue for capacitor in the convention we've been using) add according to vector or
phasor addition, to give the series voltage (the red arrow).

From Pythagoras' theorem:

V2mRC = V2mR + V2mC
If we divide this equation by two, and remembering that the RMS value V = Vm/21/2, we also
Now this looks like Ohm's law again: V is proportional to I. Their ratio is the series
impedance, Zseries and so for this series circuit,

Note the frequency dependence of the series impedance ZRC: at low frequencies, the
impedance is very large; because the capacitive reactance 1/ωC is large (the capacitor is open
circuit for DC). At high frequencies, the capacitive reactance goes to zero (the capacitor
doesn't have time to charge up) so the series impedance goes to R. At the angular frequency ω
= ωo = 1/RC, the capacitive reactance 1/ωC equals the resistance R. We shall show this
characteristic frequency on all graphs on this page.
Remember how, for two resistors in series, you could just add the resistances: Rseries = R1 + R2
to get the resistance of the series combination. That simple result comes about because the
two voltages are both in phase with the current, so their phasors are parallel. Because the
phasors for reactances are 90° out of phase with the current, the series impedance of a resistor
R and a reactance X are given by Pythagoras' law:
Zseries2 = R2 + X2
Ohm's law in AC
We can rearrange the equations above to obtain the current flowing in this circuit.
Alternatively we can simply use the Ohm's Law analogy and say that I = V source/ZRC. Either
way we get
where the current goes to zero at DC (capacitor is open circuit) and to V/R at high
frequencies (no time to charge the capacitor).

From simple trigonometry, the angle by which the current leads the voltage is
tan-1 (VC/VR) = tan-1 (IXC/IR)
= tan-1 (1/ωRC) = tan-1 (1/2πfRC).
However, we shall refer to the angle φ by which the voltage leads the current. The voltage is
behind the current because the capacitor takes time to charge up, so φ is negative, ie

φ = tan-1 (1/ωRC) = tan-1 (1/2πfRC).

At low frequencies, the impedance of the series RC circuit is dominated by the
capacitor, so the voltage is 90° behind the current. At high frequencies, the impedance
approaches R and the phase difference approaches zero. The frequency dependence of Z and
φ are important in the applications of RC circuits. The voltage is mainly across the capacitor
at low frequencies, and mainly across the resistor at high frequencies. Of course the two
voltages must add up to give the voltage of the source, but they add up as vectors.
V2RC = V2R + V2C.
At the frequency ω = ωo = 1/RC, the phase φ = 45° and the voltage fractions are V R/VRC =
VC/VRC = 1/2V1/2 = 0.71.
So, by chosing to look at the voltage across the resistor, you select mainly the high
frequencies, across the capacitor, you select low frequencies. This brings us to one of the very
important applications of RC circuits, and one which merits its own page: filters, integrators
and differentiators where we use sound files as examples of RC filtering.

RL Series combinations
In an RL series circuit, the voltage across the inductor is aheadof the current by 90°,
and the inductive reactance, as we saw before, is XL = ωL. The resulting v(t) plots and phasor
diagram look like this.

RLC Series combinations

Now let's put a resistor, capacitor and inductor in series. At any given time, the
voltage across the three components in series, vseries(t), is the sum of these:
vseries(t) = vR(t) + vL(t) + vC(t),
The current i(t) we shall keep sinusoidal, as before. The voltage across the resistor, v R(t), is in
phase with the current. That across the inductor, vL(t), is 90° ahead and that across the
capacitor, vC(t), is 90° behind.
Once again, the time-dependent voltages v(t) add up at any time, but the RMS voltages V do
not simply add up. Once again they can be added by phasors representing the three sinusoidal
voltages. Again, let's 'freeze' it in time for the purposes of the addition, which we do in the
graphic below. Once more, be careful to distinguish v and V.
Look at the phasor diagram: The voltage across the ideal inductor is antiparallel to that of the
capacitor, so the total reactive voltage (the voltage which is 90° ahead of the current) is V L -
VC, so Pythagoras now gives us:
V2series = V2R + (VL - VC)2
Now VR = IR, VL = IXL = ωL and VC = IXC= 1/ωC. Substituting and taking the common
factor I gives:

where Zseries is the series impedance: the ratio of the voltage to current in an RLC series ciruit.
Note that, once again, reactances and resistances add according to Pythagoras' law:
Zseries2 = R2 + Xtotal2
= R2 + (XL  XC)2.
Remember that the inductive and capacitive phasors are 180° out of phase, so their reactances
tend to cancel.
Now let's look at the relative phase. The angle by which the voltage leads the current is
φ = tan-1 ((VL - VC)/VR).
Substiting VR = IR, VL = IXL = ωL and VC = IXC= 1/ωC gives:

The dependence of Zseries and φ on the angular frequency ω is shown in the next figure. The
angular frequency ω is given in terms of a particular value ωo, the resonant frequency
(ωo2 = 1/LC), which we meet below.
The next graph shows us the special case where the frequency is such that VL = VC.

Because vL(t) and vC are 180° out of phase, this means that vL(t) =  vC(t), so the two reactive
voltages cancel out, and the series voltage is just equal to that across the resistor. This case is
called series resonance.

The signal is what you are measuring that is the result of the presence of your analyte.
Noise is extraneous information that can interfere with or alter the signal. It can not be
completely eliminated, but hopefully reduced. True Noise is considered random.
Signal-to-noise ratio (often abbreviated SNR or S/N) is an electrical engineering concept,
also used in other fields (such as scientific measurements, biological cell signaling), defined
as the ratio of a signal power to the noise power corrupting the signal.
In less technical terms, signal-to-noise ratio compares the level of a desired signal (such as
music) to the level of background noise. The higher the ratio, the less obtrusive the
background noise is.
In analog and digital communications, signal-to-noise ratio, often written S/N or SNR, is a
measure of signal strength relative to background noise. The ratio is usually measured in
decibels (dB).If the incoming signal strength in microvolts is Vs, and the noise level, also in
microvolts, is Vn, then the signal-to-noise ratio, S/N, in decibels is given by the formula
S/N = 20 log10(Vs/Vn)
If Vs = Vn, then S/N = 0. In this situation, the signal borders on unreadable, because the noise
level severely competes with it. In digital communications, this will probably cause a
reduction in data speed because of frequent errors that require the source (transmitting)
computer or terminal to resend some packets of data.
Ideally, Vs is greater than Vn, so S/N is positive. As an example, suppose that V s = 10.0
microvolts and Vn = 1.00 microvolt. Then
S/N = 20 log10(10.0) = 20.0 dB
which results in the signal being clearly readable. If the signal is much weaker but still above
the noise -- say 1.30 microvolts -- then
S/N = 20 log10(1.30) = 2.28 dB
which is a marginal situation. There might be some reduction in data speed under these
If Vs is less than Vn, then S/N is negative. In this type of situation, reliable communication is
generally not possible unless steps are taken to increase the signal level and/or decrease the
noise level at the destination (receiving) computer or terminal.
Communications engineers always strive to maximize the S/N ratio. Traditionally, this has
been done by using the narrowest possible receiving-system bandwidth consistent with the
data speed desired. However, there are other methods. In some cases, spread spectrum
techniques can improve system performance. The S/N ratio can be increased by providing the
source with a higher level of signal output power if necessary. In some high-level systems
such as radio telescopes, internal noise is minimized by lowering the temperature of the
receiving circuitry to near absolute zero (-273 degrees Celsius or -459 degrees Fahrenheit). In
wireless systems, it is always important to optimize the performance of the transmitting and
receiving antennas.
Types of Noise
 Chemical Noise
 Chemical reactions
 Reaction/technique/instrument specific
 Instrumental Noise
 Germane to all types of instruments
 Can often be controlled physically (e.g. temp) or electronically (software
Instrumental Noise
 Thermal (Johnson) Noise:
 Thermal agitation of electrons affects their “smooth” flow.
 Due to different velocities and movement of electrons in electrical
 Upon both temperature and the range of frequencies (frequency bandwidths)
being utilized.
 Can be reduced by reducing temperature of electrical components.
 Eliminated at “absolute” zero.
 Considered “white noise” because it is independent of frequency (but
dependent on frequency bandwidth or the range of frequencies being measured).

 Shot Noise:
 Occurs when electrons or charged particles cross junctions (different
materials, vacuums, etc.)
 Considered “white noise” because it is independent of frequency.
 It is the same at any frequency but also dependent on frequency bandwidth
 Due to the statistical variation of the flow of electrons (current) across some
 Some of the electrons jump across the junction right away
 Some of the electrons take their time jumping across the junction

 Flicker Noise
 Frequency dependent
 Significant at frequencies less than 100 Hz
 Magnitude is inversely proportional to frequency
 Results in long-term drift in electronic components
 Can be controlled by using special wire resistors instead of the less expensive
carbon type.
 Environmental Noise
 Unlimited possible sources
 Can often be eliminated by eliminating the source
 Other noise sources can not be eliminated!!!!!!
 Methods of eliminating it…
 Moving the instrument somewhere else
 Isolating /conditioning the instruments power source
 Controlling temperature in the room
 Control expansion/contraction of components in instrument
 Eliminating interferences
 Stray light from open windows, panels on instrument
 Turning off radios, TV’s, other instruments



Lock-in amplifier
A lock-in amplifier (also known as a phase-sensitive detector) is a type of amplifier
that can extract a signal with a known carrier wave from extremely noisy environment (S/N
ratio can be as low as -60 dB or even less . It is essentially a homodyne with an extremely low
pass filter (making it very narrow band). Lock-in amplifiers use mixing, through a frequency
mixer, to convert the signal's phase and amplitude to a DC—actually a time-varying low-
frequency—voltage signal.
Basic principles
Operation of a lock-in amplifier relies on the orthogonality of sinusoidal functions.
Specifically, when a sinusoidal function of frequency ν is multiplied by another sinusoidal
function of frequency μ not equal to ν and integrated over a time much longer than the period
of the two functions, the result is zero. In the case when μ is equal to ν, and the two functions
are in phase, the average value is equal to half of the product of the amplitudes.
In essence, a lock-in amplifier takes the input signal, multiplies it by the reference
signal (either provided from the internal oscillator or an external source), and integrates it
over a specified time, usually on the order of milliseconds to a few seconds. The resulting
signal is an essentially DC signal, where the contribution from any signal that is not at the
same frequency as the reference signal is attenuated essentially to zero, as well as the out-of-
phase component of the signal that has the same frequency as the reference signal (because
sine functions are orthogonal to the cosine functions of the same frequency), and this is also
why a lock-in is a phase sensitive detector.

More basic principles

Lock-in amplifiers are used to measure the amplitude and phase of signals buried in
noise. They achieve this by acting as a narrow bandpass filter which removes much of the
unwanted noise while allowing through the signal which is to be measured.
The frequency of the signal to be measured and hence the passband region of the filter is set
by a reference signal, which has to be supplied to the lock-in amplifier along with the
unknown signal. The reference signal must be at the same frequency as the modulation of the
signal to be measured.
A basic lock-in amplifier can be split into 4 stages: an input gain stage, the reference
circuit, a demodulator and a low pass filter.
Input Gain Stage: The variable gain input stage pre-processes the signal by amplifying it to
a level suitable for the demodulator. Nothing complicated here, but high performance
amplifiers are required.
Reference Circuit: The reference circuit allows the reference signal to be phase shifted.
Demodulator: The demodulator is a multiplier. It takes the input signal and the reference and
multiplies them together. When you multiply two waveforms together you get the sum and
difference frequencies as the result. As the input signal to be measured and the reference
signal are of the same frequency, the difference frequency is zero and you get a DC output
which is proportional to the amplitude of the input signal and the cosine of the phase
difference between the signals. By adjusting the phase of the reference signal using the
reference circuit, the phase difference between the input signal and the reference can be
brought to zero and hence the DC output level from the multiplier is proportional to the input
signal. The noise signals will still be present at the output of the demodulator and may have
amplitudes 1000 times as large as the DC offset.
Low Pass Filter:
As the various noise components on the input signal are at different frequencies to the
reference signal, the sum and difference frequencies will be non zero and will not contribute
to the DC level of the output signal. This DC level (which is proportional to the input signal)
can now be recovered by passing the output from the demodulator through a low pass filter.
The above gives an idea of how a basic lock-in amplifier works. Actual lock-in amplifiers are
more complicated, as there are instrument offsets that need to be removed, but the basic
principle of operation is the same.

Application to signal measurements in a noisy environment

The essential idea in signal recovery is that noise tends to be spread over a wider
spectrum, often much wider than the signal. In the simplest case of white noise, even if the
root mean square of noise is 106 times as large as the signal to be recovered, if the bandwidth
of the measurement instrument can be reduced by a factor much greater than 106 around the
signal frequency, then the equipment can be relatively insensitive to the noise. In a typical
100 MHz bandwidth (e.g. an oscilloscope), a bandpass filter with width much narrower than
100 Hz would accomplish this.
In summary, even when noise and signal is indistinguishable in time domain, if signal
has a definite frequency band and there is no large noise peak within that band, noise and
signal can be separated sufficiently in the frequency domain.If the signal is either slowly
varying or otherwise constant (essentially a DC signal), then 1/f noise typically overwhelms
the signal. It may then be necessary to use external means to modulate the signal. For
example, in the case of detection of small light signal against a bright background, the signal
can be modulated either by a chopper wheel, acousto-optical modulator, photoelastic
modulator at a large enough frequency so that 1/f noise drops off significantly, and the lock-
in amplifier is referenced to the operating frequency of the modulator. In the case of an
atomic force microscope, in order to achieve nanometer and piconewton resolution, the
cantilever position is modulated at a high frequency, to which lock-in amplifier is again
When the lock-in technique is applied, care must be taken in calibration of signal,
because lock-in amplifiers generally detect only the root-mean-square signal of the operating
frequency only. For a sinusoidal modulation, this would introduce a factor of between the
lock-in amplifier output and the peak amplitude of the signal, and a different factor for a
modulation of different shape. In fact, in the case of extremely nonlinear systems, it may be
advantageous to use a higher harmonic of reference frequency because of frequency-doubling
that take place in a nonlinear medium.

Chopper amplifiers
One classic use for a chopper circuit and where the term is still in use is in chopper
amplifiers. These are DC amplifiers. Some types of signal that need amplifying can be so
small that an incredibly high gain is required, but very high gain DC amplifiers are much
harder to build with low offset and 1/f noise, and reasonable stability and bandwidth. It's
much easier to build an AC amplifier instead. A chopper circuit is used to break up the input
signal so that it can be processed as if it were an AC signal, then integrated back to a DC
signal at the output. In this way, extremely small DC signals can be amplified. This approach
is often used in electronic instrumentation where stability and accuracy are essential; for
example, it is possible using these techniques to construct pico-voltmeters and Hall sensors.

Signal Averaging
(one way of controlling noise)

 Ensemble Averaging

 Collect Multiple Signals Over The Same Time Or Wavelength (For Example)
 Easily Done With Computers
 Calculate The Mean Signal At Each Point In The Domain
 Re-Plot The Averaged Signal
 Since Noise Is Random (Some +/ Some -), This Helps Reduce The Overall Noise By

 Boxcar Averaging

 Take an average of 2 or more signals in some domain

 Plot these points as the average signal in the same domain
 Can be done with just one set of data
 You lose some detail in the overall signal
Polynomial Smoothing

 Like Boxcar Averaging

 Multipoint digital data averaging
 Results in loss of some data at the beginning and the end of the data set.
Physical Optical Domain Electrical Domain

Power Source: spectrochemical encoding system

Sample: must be in form suitable for analysis, may involve a separation or speciation
Sample cell: cuvette for UV-VIS, flame for atomic spectroscopy
Wavelength Disperser: an information sorting system, spreads light out spatially according to
its wavelength
Photodetector: radiation transducer changing optical info into electrical info
Readout: digital (ADC), meter, strip chart recorder

Continuum Sources
Ar Lamp VAC UV
H2 or D2 Lamp UV
Tungsten Lamp UV-Near IR
Nernst Glower UV-VIS-Near IR-IR
Nichrome Wire Near IR-Far IR
Globar Near IR-Far IR
Hollow Cathode Lamp UV-VIS
Lasers UV-VIS-Near IR

Radiation Sources
Sources may be continuous or pulsed in time
Continuum sources
- Continuum sources are preferred for spectroscopy because of their relatively
flat radiance versus wavelength curves
- Nernst glower (b) W filament (c) D2 lamp (d) arc (e) arc plus reflector
- produce broad, featureless range of wavelengths
- black and gray bodies, high pressure arc lamps
Line sources
- produce relatively narrow bands at specific wavelengths generating structured
emission spectrum
- lasers, low pressure arc lamps, hollow cathode lamps
Line plus continuum sources
- contain lines superimposed on continuum background
- medium pressure arc lamps, D2 lamp
Black body sources
 Nernst glowers (ZrO2, YO2), Globars (SiC)
 1000-1500 K in air - max lies in IR
 relatively fragile
 low spectral radiance (B~10-4 W·cm-2·nm-1·sr-1)
Arc sources
 Hg, Xe, D2 lamps
 AC or DC discharge through gas or metal vapor
- 20-70 V, 10 mA-20 A
Line sources
 Generally not much use for molecular spectroscopy
 useful for luminescence excitation, photochemistry experiments
 where high radiant intensity at one q required
Arc lamps
 Low pressure (<10 Torr) with many different fill vapors
 Hg, Cd, Zn, Ga, In, Th and alkali metals
 Excellent wavelength calibration sources

Hollow cathode lamps (HCL)

 primary line sources in atomic spectroscopy
 low gas pressure (<10 mtorr)
o linewidths ~ 0.01 Å
o high currents (>few mA) reduces lifetime and broadens lines
 single or multi-element cathodes
 moderate radiance B~10-2 W·cm-2·nm-1·sr-1
Electrodeless discharge lamps (EDL)
 contain a microwave or RF-excited plasma
 need ignition pulse to start plasma
 electric field of RF or microwave drives ions and electrons in plasma
 no electrodes
 gas pressures and temperatures relatively low
 slight pressure broadening
 line widths are not as narrow as the HCL (<1Å)
 moderate radiance B~10-1 W·cm-2·nm-1·sr-1
 intense (radiance B>104 W·cm-2·nm-1·sr-1)
 nearly monochromatic (0.01-0.1 Å)
 coherent (temporally and spatially)
 directed (small divergence)
 pulsed or continuous
 stable Continuous wave (cw) lasers
 operate continuously in time and are continuously pumped
 lifetime of the upper lasing state (j) must be longer than that of
 the lower lasing state (i) to maintain a population inversion
 low gain systems, typically just above threshold
 require high reflectivity mirrors
 Average powers of up to a few tens of watts are possible
Pulsed lasers
 operate intermittently in time
 single pulses
 repetitive pulse trains
 lifetime of level j is shorter than level i
 population inversion cannot be sustained indefinitely
 high gain systems
 can still lase with poor quality mirrors or with no mirrors
Peak and Average Power for Pulsed Laser
 Peak output power (energy per pulse divided by duration of pulse)
 may be MW or GW (109 W)
 Average output power (energy per pulse multiplied by pulse repetition rate)
 May be much more modest (few W).
 different because of short duty cycle of pulsed laser
Q-switched lasers
 Pulsed and cw lasers operate close to threshold - as soon as threshold
 is exceeded, lasing starts
- if delay lasing while pumping, larger population inversion created
 Q-switch works like pulsed lasers but contain an additional cavity
 component to prevent lasing action (cavity spoiling) Cavity spoiling
 slightly move one of mirrors
 add a saturable absorber to the cavity (for example, a dye)
 during pumping dye absorbs a considerable fraction of the photons traversing the
 population inversion is continually created
 when dye is saturated becomes transparent
 allows an intense burst of laser light
Mode-locked lasers
 force random cavity modes to be phase locked
 In free-running multimode laser, individual cavity modes not synchronized to each
 show a time varying range of phases and hence amplitudes
 Forcing the laser to operate with the phases of the cavity modes
 fixed (or locked) means that a pulse train is produced for each regular pulse
 Each component of pulse train may be picoseconds (10-12 s) or shorter
 Mode locking is achieved by rapid time-varying absorber in the cavity (up to 100
- commonly an electro-optic modulator in addition to Q-switch
- when modulator absorbs, the beam is spoiled, no lasing occurs
- when modulator becomes transparent, cavity modes established
 within a short period of time
 Most electro-optic modulators are based on the Pockels effect (Pockels cell)
 KDP crystal is birefringent when V applied
 rotates polarization of light
 apply sinusoidally varying voltage for mode-locking
Laser types
Solid state lasers
 contain solid-state crystal as lasing media
 single crystal rods with parallel mirrored ends
 flashlamp or continuously pumped
 ruby laser (Cr-doped alumina) (red 694 nm, 500 ns)
 Nd:YAG laser (Nd-doped yttrium aluminum garnet) (IR 1064 nm,10 ns)
Semiconductor diode lasers
 a type of solid state laser currently undergoing rapid development
 no optical pumping
 usually operated in cw mode
 current through semiconductor pn junction forces recombination of electrons and
 variety of 's can be produced by changing band-gap of semiconductor (for example
in AlGaAs)
 average powers up to a few tens of W (if cooled)
 small and relatively cheap to manufacture
Gas lasers
 include gas or gas mixture as lasing medium (He-Ne)
 pumped by an electrical discharge - fraction He atoms excited (ionized)
 electronically excited metastable states in the He (optically forbidden transitions long-
lived) transfer energy in a nearly resonant
 collisional process to Ne atoms
 632 nm line is a 3s 2p transition
 CO2 (contains N2, CO2 & He):
 electrical discharge excites N2(v=1)
 multiple rotational transitions can be involved
 many discrete lines in the region of 10.6-9.6 m
 may be pulsed or cw and can produce high peak and average powers
 creation of excited state dimers (excimer) between a noble gas atom and halogen
 excimer is only stable in the electronically excited state
 dissociates rapidly in ground state
 intense (up to >500 mJ·pulse-1) on 10 ns timescale
 somewhat tunable - few nm - lifetime broadened
 gas lifetime limited by reaction of the halogen with cavity materials
Dye lasers
 based on fluorescent dyes (rhodamine, coumarin, fluorescein)
 pumped by a flashlamp or another laser (often excimer or N2 for pulsed operation or
Ar+ for cw operation)
 four-level systems - emission from the ground vibrational state of some electronically
excited state to some (high-lying)
 vibrationally excited state of the ground electronic state
- fluorescence in liquid is solvent-broadened
- a wide range of wavelengths is produced
- narrower band of wavelengths selected by intracavity diffraction
 grating
- only 's supported by the diffraction grating are amplified
- tunable over a 40-50 nm

Wavelength selector
A. Filters are used to pass a band of wavelengths
 Absorption filters
 Interference filters
 Monochromators - one color - pass a narrow band of wavelnegths

B. Prisms
Dispersing prisms
 Separation of wavelengths due to differences in index of refraction of
the glass in the prism with each different wavelength. This leads to
constructive and destructive interference.
 Dispersion is angular (nonlinear). Single order is obtained. The larger
the focal length, the better the dispersion.
Reflecting prisms
 Designed to change direction of propagation of beam, orientation, or
Polarizing prisms
 Made of birefringent materials

C. Gratings
Can be considered as a set of slits at which diffraction occurs and
destructive/constructive interference occurs that yields a diffraction pattern. Grooves
patterns are now generated by machine. They are manufactured by ruling a piece of
glass or metal. Replica gratings are then produced by laying down a polymer film
over it to copy the groove pattern. Replicas are what are actually used in instruments
due to the great difficulty and cost of achieving high quality gratings. Holographic
gratings are also used but are not as efficient. Never touch a grating with your fingers.

D. Types of Mounts
1) Littrow: autocollimating
2)Czerny-Turner: two mirrors used to collimate and focus.
3) Fastie-Ebert: single mirror used to collimate and focus
4) Rowland Circle: used in polychromators
5) Echelle: uses prism to sort orders from a grating

E. Performance Characteristics
Resolving power

R= / =nN
where n = diffraction order and N =lines of the grating illuminated from the entrance
slit. Therefore depends on
1) Physical size of dispersing element
2) Order of hv being observed to get better resolution either
1) Increase N
2) Increase n (cost now is in lessened intensity)
If R = 100 Poor quality
If R = 106 High quality
Number of orders detectable is proportional to N
Higher orders yield greater resolution but poorer intensity
The quality of the slits is also important.
Some light is also lost in reflection (n = 0, zero order)

Reciprocal Linear Dispersion

Rd or D-1 =1/D l, nm/mm of wavelength intervals (e.g., nm) contained in each
interval of distance (e.g., mm) along the focal plane.

Measure of the light gathering power of the monochromator that emerges from the
entrance slit
Where F = focal length of the collimating mirror or lens
and d = diameter of collimating mirror or lens
The light-gathering power of an optical device increases as the inverse square of the
f/number, therefore an f/2 lens gathers four times more light than an f/4 lens.
The f/numbers of many monochromators lie in the 1 to 10 range

F. Slits

Slits are used to limit the amount of light impinging on the dispersing element as well
as to limit the light reaching the detector.
There is a dichotomy between intensity and resolution.

Wide slits Narrow slits

Throughput High Low
Resolution Low High
Quant Good Poor
Qual Poor Good

Atomic lines are not infinitely narrow due to types of broadening

1) Natural
2) Doppler:
3) Stark
4)Collisional broadening
The use of entrance and exit slits convolutes this broadening as a triangular function -
the slit function.
Spectral bandpass, s, is the width at half-height of the wavelength distribution as
passed by the exit slit
s = Rd W
where Rd is the reciprocal linear dispersion and W is the slit width.
The slit-width-limited resolution s is
= 2s = 2 Rd W
Required of all spectroscopic methods except emission spectroscopy
Must be made of material that is transparent to the spectral region of interest

UV Fused silica
VIS Plastic, glass
 High sensitivity
 High S/N
 Constant response over range of wavelengths
 Fast response
 Zero output in absence of illumination
 Electrical signal directly proportional to radiant power

Photomultiplier Tubes

• Sensitivity: Significantly more sensitive than simple phototube

• Process of Multiplication: Electrons emitted from cathode surface and accelerated
towards dynode (each successive dynode is 90 V more positive than preceding
• Construction
- Photocathode: made of alkali metals with low work functions
- Focusing electrodes
- An electron multiplier (dynodes) amplification by factor of 106 to 107 for each
- An electron collector (anode)
- Window: borosilicate, quartz, sapphire, or MgF2
• Spectral response
- Depends on photocathodic material
- Conversion efficiency varies with
- Lower cutoff determined by window composition


- An "electrical photographic plate"

- Detect differences in light intensity at different points on their photosensitive
- Fabricated from silicon using semiconductor technology
- Originally conceived as television camera sensing elements
- Placed at focal plane of polychromator in place of the exit slit
- Sensitive for detection of light in 200-1000 nm range
- Major advantage is simultaneous detection of all wavelengths within range
 SIT : silicon intensifier target
 PDA : photodiode array
 CCD : charge-coupled device
 CID : charge injection device

 Usually 1-3 cm long; contains a few hundred photodiodes (256 - 2048) in a linear
 Partitions spectrum into x number of wavelength increments
Each photodiode captures photons simultaneously
Measures total light energy over the time of exposure (whereas PMT measures
instantaneous light intensity)
 Each diode in the array is reverse-biased and thus can store charge like a capacitor
 Before being exposed to light to be detected, diodes are fully charged via a transistor
 Light falling on the PDA will generate charge carriers in the silicon which combine
with stored charges of opposite polarity and neutralize them
 The amount of charge lost is proportional to the intensity of light
 Amount of current needed to recharge each diode is the measurement made which is
proportional to light intensity
 Recharging signal is sent to sample-and-hold amplifier and then digitized
Array is however read sequentially over a common output line
Use minicomputer to handle data
 Must have fast data storage system
 High dark noise
 Must cool PDA to well below room temperature
 Diode saturates within a few seconds integration time
 Resolution not good, limited by # diodes/linear distance
 Stray radiant energy (SRE) is a killer
Used as detectors in Raman, fluorescence, and absorption
 Two-dimensional arrays of silicon integrated circuits, postage-stamp-size
 Typical pixel dimensions are 20 x 20 µm

Both CCDs and CIDs accumulate photogenerated charges in similar ways but differ in the
way accumulated charge is detected
A. Charge Injection Devices (CID)

• A CID sensing element can be thought of as two electrodes side by side

• One of the electrodes is biased so as to create a potential well near it
• When an incident photon creates an electron-hole pair in the sensor region,
one member of the pair will be attracted to the well and held there
n-doped Si used as charge storage region
• After exposure to light accumulated charge is moved from one electrode to
the other
• Potential change caused by the change in charge stored on second electrode is
• Potential change is proportional to amount of stored charge and thus
proportional to integrated light flux
• Charge sensing may be done non-destructively therefore can take repeated
readings of same accumulated charge to improve S/N
Charge-Coupled Devices (CCD)
• Potential well formed by an electrode as in CID
p-type material, however, used to store charges as electrons after exposure to light
charge packets are transferred along the row to special low-capacitance readout diode
• Passage of charge induces a voltage change proportional to amount of charge
• Advantages over CID include
A. An increased voltage change and
B. Lower reading noise
C. Small pixels are not well-suited to ordinary dispersive spectroscopy
• "Binning"
o Aggregates charges formed in several detector elements into one element prior
to readout
o Yields increased detector sensitivity at a cost of resolution but elements are
very small so loss of resolution can be minimized

• Summation is done on the chip rather than in memory after the readout, thus only one
read operation required for all the pixels to be summed, thus lower readout noise per
pixel is achieved
Used in astronomy and low light situations: fluorometry, Raman, CZE, HPLC
Thermal Transducers
• phototransducers not applicable in IR due to low energy
• Thermocouples
• Bolometers
• Pyroelectric Transducers



Spectroscopy is the study of the interaction between radiation (electromagnetic radiation, or

light, as well as particle radiation) and matter. Spectrometry is the measurement of these
interactions and an instrument which performs such measurements is a spectrometer or
spectrograph. A plot of the interaction is referred to as a spectrum.

Historically, spectroscopy referred to a branch of science in which visible light was used for
the theoretical study of the structure of matter and for qualitative and quantitative analyses.
Recently, however, the definition has broadened as new techniques have been developed that
utilise not only visible light, but many other forms of radiation.

Spectroscopy is often used in physical and analytical chemistry for the identification of
substances through the spectrum emitted from or absorbed by them. Spectroscopy is also
heavily used in astronomy and remote sensing. Most large telescopes have spectrometers,
which are used either to measure the chemical composition and physical properties of
astronomical objects or to measure their velocities from the Doppler shift of their spectral

Classification of spectroscopic methods

Nature of radiation measured

The type of spectroscopy depends on the physical quantity measured. Normally, the quantity
that is measured is an amount or intensity of something.

 Optical Spectroscopy (Electromagnetic Spectroscopy) involves interactions of matter

with electromagnetic radiation or light. Ultraviolet-visible spectroscopy is an
 Electron Spectroscopy involves interactions with electron beams. Auger spectroscopy
involves inducing the Auger effect with an electron beam.
 Mass spectroscopy involves the interaction of charged species with magnetic and/or
electric fields, giving rise to a mass spectrum. The term "mass spectroscopy" is
deprecated in favor of mass spectrometry, for the technique is primarily a form of
measurement, though it does produce a spectrum for observation.

Measurement process

Most spectroscopic methods are differentiated as either atomic or molecular based on

whether or not they apply to atoms or molecules. Along with that distinction, they can be
classified on the nature of their interaction:

 Absorption spectroscopy uses the range of the electromagnetic spectra in which a

substance absorbs. This includes atomic absorption spectroscopy and various
molecular techniques, such as infrared spectroscopy in that region and nuclear
magnetic resonance (NMR) spectroscopy in the radio region.
 Emission spectroscopy uses the range of electromagnetic spectra in which a substance
radiates (emits). The substance first must absorb energy. This energy can be from a
variety of sources, which determines the name of the subsequent emission, like
luminescence. Molecular luminescence techniques include spectrofluorimetry.
 Scattering spectroscopy measures the amount of light that a substance scatters at
certain wavelengths, incident angles, and polarization angles. The scattering process
is much faster than the absorption/emission process. One of the most useful
applications of light scattering spectroscopy is Raman spectroscopy.

Common types of spectroscopy

Spectrum of light from a fluorescent lamp showing prominent mercury peaks.

Fluorescence spectroscopy

Fluorescence spectroscopy uses higher energy photons to excite a sample, which will then
emit lower energy photons. This technique has become popular for its biochemical and
medical applications, and can be used for confocal microscopy, fluorescence resonance
energy transfer, and fluorescence lifetime imaging.

X-ray spectroscopy and X-ray crystallography When X-rays of sufficient frequency

(energy) interact with a substance, inner shell electrons in the atom are excited to outer empty
orbitals, or they may be removed completely, ionizing the atom. The inner shell "hole" will
then be filled by electrons from outer orbitals. The energy available in this de-excitation
process is emitted as radiation (fluorescence) or will remove other less-bound electrons from
the atom (Auger effect). The absorption or emission frequencies (energies) are characteristic
of the specific atom. In addition, for a specific atom small frequency (energy) variations
occur which are characteristic of the chemical bonding. With a suitable apparatus, these
characteristic X-ray frequencies or Auger electron energies can be measured. X-ray
absorption and emission spectroscopy is used in chemistry and material sciences to determine
elemental composition and chemical bonding.

X-ray crystallography is a scattering process; crystalline materials scatter X-rays at well-

defined angles. If the wavelength of the incident X-rays is known, this allows calculation of
the distances between planes of atoms within the crystal. The intensities of the scattered X-
rays give information about the atomic positions and allow the arrangement of the atoms
within the crystal structure to be calculated.

Flame Spectroscopy

Liquid solution samples are aspirated into a burner or nebulizer/burner combination,

desolvated, atomized, and sometimes excited to a higher energy electronic state. The use of a
flame during analysis requires fuel and oxidant, typically in the form of gases. Common fuel
gases used are acetylene (ethyne) or hydrogen. Common oxidant gases used are oxygen, air,
or nitrous oxide. These methods are often capable of analyzing metallic element analytes in
the part per million, billion, or possibly lower concentration ranges. Light detectors are
needed to detect light with the analysis information coming from the flame.

Atomic Emission Spectroscopy - This method uses flame excitation; atoms are excited
from the heat of the flame to emit light. This method commonly uses a total
consumption burner with a round burning outlet. A higher temperature flame than
atomic absorption spectroscopy (AA) is typically used to produce excitation of
analyte atoms. Since analyte atoms are excited by the heat of the flame, no special
elemental lamps to shine into the flame are needed. A high resolution polychromator
can be used to produce an emission intensity vs. wavelength spectrum over a range of
wavelengths showing multiple element excitation lines, meaning multiple elements
can be detected in one run. Alternatively, a monochromator can be set at one
wavelength to concentrate on analysis of a single element at a certain emission line.
Plasma emission spectroscopy is a more modern version of this method. See Flame
emission spectroscopy for more details.

Atomic absorption spectroscopy (often called AA) - This method commonly uses a pre-
burner nebulizer (or nebulizing chamber) to create a sample mist and a slot-shaped
burner which gives a longer pathlength flame. The temperature of the flame is low
enough that the flame itself does not excite sample atoms from their ground state. The
nebulizer and flame are used to desolvate and atomize the sample, but the excitation
of the analyte atoms is done by the use of lamps shining through the flame at various
wavelengths for each type of analyte. In AA, the amount of light absorbed after going
through the flame determines the amount of analyte in the sample. A graphite furnace
for heating the sample to desolvate and atomize is commonly used for greater
sensitivity. The graphite furnace method can also analyze some solid or slurry
samples. Because of its good sensitivity and selectivity, it is still a commonly used
method of analysis for certain trace elements in aqueous (and other liquid) samples.

Atomic Fluorescence Spectroscopy - This method commonly uses a burner with a round
burning outlet. The flame is used to solvate and atomize the sample, but a lamp shines
light at a specific wavelength into the flame to excite the analyte atoms in the flame.
The atoms of certain elements can then fluoresce emitting light in a different
direction. The intensity of this fluorescing light is used for quantifying the amount of
analyte element in the sample. A graphite furnace can also be used for atomic
fluorescence spectroscopy. This method is not as commonly used as atomic
absorption or plasma emission spectroscopy.

Plasma Emission Spectroscopy In some ways similar to flame atomic emission

spectroscopy, it has largely replaced it.

Direct-current plasma (DCP)

A direct-current plasma (DCP) is created by an electrical discharge between two

electrodes. A plasma support gas is necessary, and Ar is common. Samples can be
deposited on one of the electrodes, or if conducting can make up one electrode.

Glow discharge-optical emission spectrometry (GD-OES)

Inductively coupled plasma-atomic emission spectrometry (ICP-AES)

Laser Induced Breakdown Spectroscopy (LIBS) (LIBS), also called Laser-induced

plasma spectrometry (LIPS)

 Microwave-induced plasma (MIP)

Spark or arc (emission) spectroscopy - is used for the analysis of metallic elements in solid
samples. For non-conductive materials, a sample is ground with graphite powder to make it
conductive. In traditional arc spectroscopy methods, a sample of the solid was commonly
ground up and destroyed during analysis. An electric arc or spark is passed through the
sample, heating the sample to a high temperature to excite the atoms in it. The excited analyte
atoms glow emitting light at various wavelengths which could be detected by common
spectroscopic methods. Since the conditions producing the arc emission typically are not
controlled quantitatively, the analysis for the elements is qualitative. Nowadays, the spark
sources with controlled discharges under an argon atmosphere allow that this method can be
considered eminently quantitative, and its use is widely expanded worldwide through
production control laboratories of foundries and steel mills.
Visible spectroscopy

Many atoms emit or absorb visible light. In order to obtain a fine line spectrum, the atoms
must be in a gas phase. This means that the substance has to be vaporised. The spectrum is
studied in absorption or emission. Visible absorption spectroscopy is often combined with
UV absorption spectroscopy in UV/Vis spectroscopy.

Ultraviolet spectroscopy

All atoms absorb in the UV region because these photons are energetic enough to excite outer
electrons. If the frequency is high enough, photoionisation takes place. UV spectroscopy is
also used in quantifying protein and DNA concentration as well as the ratio of protein to
DNA concentration in a solution. Several amino acids usually found in protein, such as
tryptophan, absorb light in the 280nm range and DNA absorbs light in the 260nm range. For
this reason, the ratio of 260/280nm absorbance is a good general indicator of the relative
purity of a solution in terms of these two macromolecules. Reasonable estimates of protein or
DNA concentration can also be made this way using Beer's law.

Infrared spectroscopy

Infrared spectroscopy offers the possibility to measure different types of interatomic bond
vibrations at different frequencies. Especially in organic chemistry the analysis of IR
absorption spectra shows what types of bonds are present in the sample.

Raman Spectroscopy

Raman spectroscopy uses the inelastic scattering of light to analyse vibrational and rotational
modes of molecules. The resulting 'fingerprints' are an aid to analysis.

Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy analyzes the magnetic properties of certain atomic
nuclei to determine different electronic local environments of hydrogen, carbon, or other
atoms in an organic compound or other compound. This is used to help determine the
structure of the compound.
Photoemission spectroscopy

Mössbauer spectroscopy

Transmission or conversion-electron (CEMS) modes of Mössbauer spectroscopy probe the

properties of specific isotope nuclei in different atomic environments by analyzing the
resonant absorption of characteristic energy gamma-rays known as the Mössbauer effect.

Less frequently used / combined spectroscopy

 Photoacoustic Spectroscopy measures the sound waves produced upon the absorption
of radiation.
 Photothermal Spectroscopy measures heat evolved upon absorption of radiation.
 Circular Dichroism spectroscopy
 Raman Optical Activity Spectroscopy exploits Raman scattering and optical activity
effects to reveal detailed information on chiral centers in molecules.
 Terahertz spectroscopy uses wavelengths above infrared spectroscopy and below
microwave or millimeter wave measurements.
 Inelastic neutron scattering works like Raman spectroscopy, with neutrons instead of
 Inelastic electron tunneling spectroscopy uses the changes in current due to inelastic
electron-vibration interaction at specific energies which can also measure optically
forbidden transitions.
 Auger Spectroscopy is a method used to study surfaces of materials on a micro-scale.
It is often used in connection with electron microscopy.
 Cavity ring down spectroscopy
 Fourier transform is an efficient method for processing spectra data obtained using
interferometers. The use of Fourier transform in spectroscopy is called Fourier
transform spectroscopy. Nearly all infrared spectroscopy (FTIR) and Nuclear
Magnetic Resonance (NMR) spectroscopy are performed with Fourier transforms.
 Spectroscopy of matter in situations where the properties are changing with time is
called Time-resolved spectroscopy.
 Mechanical spectroscopy involves interactions with macroscopic vibrations, such as
phonons. An example is acoustic spectroscopy, involving sound waves.
 Time-resolved spectroscopy
 Spectroscopy using an AFM-based analytical technique is called Force spectroscopy.
 Dielectric spectroscopy
 Thermal infrared spectroscopy measures thermal radiation emitted from materials and
surfaces and is used to determine the type of bonds present in a sample as well as their
lattice environment. The techniques are widely used by organic chemists,
mineralogists, and planetary scientists.

Background Subtraction

Background subtraction is a term typically used in spectroscopy when one explains the
process of acquiring a background radiation level (or ambient radiation level) and then makes
an algorithmic adjustment to the data to obtain qualitative information about any deviations
from the background, even when they are an order of magnitude less decipherable than the
background itself.

Background subtraction can effect a number of statistical calculations (Continuum, Compton,

Bremsstrahlung) leading to improved overall system performance.


In physics, spectrophotometry is the quantitative study of electromagnetic spectra. It is more

specific than the general term electromagnetic spectroscopy in that spectrophotometry deals
with visible light, near-ultraviolet, and near-infrared. Also, the term does not cover time-
resolved spectroscopic techniques.

Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a

photometer (a device for measuring light intensity) that can measure intensity as a function of
the color, or more specifically, the wavelength of light. There are many kinds of
spectrophotometers. Among the most important distinctions used to classify them are the
wavelengths they work with, the measurement techniques they use, how they acquire a
spectrum, and the sources of intensity variation they are designed to measure. Other
important features of spectrophotometers include the spectral bandwidth and linear range.

Perhaps the most common application of spectrophotometers is the measurement of light

absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even
the emission half of a luminescence instrument is a kind of spectrophotometer.

There are two major classes of spectrophotometers; single beam and double beam. A double
beam spectrophotometer measures the ratio of the light intensity on two different light paths,
and a single beam spectrophotometer measures the absolute light intensity. Although ratio
measurements are easier, and generally stabler, single beam instruments have advantages; for
instance, they can have a larger dynamic range, and they can be more compact.

Historically, spectrophotometers use a monochromator to analyze the spectrum, but there are
also spectrophotometers that use arrays of photosensors and. Especially for infrared
spectrophotometers, there are spectrophotometers that use a Fourier transform technique to
acquire the spectral information more quickly in a technique called Fourier Transform

The spectrophotometer measures quantitatively the fraction of light that passes through a
given solution. In a spectrophotometer, a light from the lamp is guided through a
monochromator, which picks light of one particular wavelength out of the continuous
spectrum. This light passes through the sample that is being measured. After the sample, the
intensity of the remaining light is measured with a photodiode or other light sensor, and the
transmittance for this wavelength is then calculated.

In short, the sequence of events in a spectrophotometer is as follows:

1. The light source shines through the sample.

2. The sample absorbs light.
3. The detector detects how much light the sample has absorbed.
4. The detector then converts how much light the sample absorbed into a number.
5. The numbers are either plotted straight away, or are transmitted to a computer to be
further manipulated (e.g. curve smoothing, baseline correction)

UV and IR spectrophotometers

The most common spectrophotometers are used in the UV and visible regions of the
spectrum, and some of these instruments also operate into the near-infrared region as well.
Visible region 400-700nm spectrophotometry is used extensively in colorimetry science. Ink
manufacturers, printing companies, textiles vendors, and many more, need the data provided
through colorimetry. They usually take readings every 20 nanometers along the visible
region, and produce a spectral reflectance curve. These curves can be used to test a new batch
of colorant to check if it makes a match to specifications.

Traditional visual region spectrophotometers cannot detect if a colorant has fluorescence.

This can make it impossible to manage color issues if one or more of the printing inks is
fluorescent. Where a colorant contains fluorescence, a bi-spectral fluorescent
spectrophotometer is used. There are two major setups for visual spectrum
spectrophotometers, d/8 (spherical) and 0/45. The names are due to the geometry of the light
source, observer and interior of the measurement chamber. Scientists use this machine to
measure the amount of compounds in a sample. If the compound is more concentrated more
light will be absorbed by the sample; within small ranges, the Beer-Lambert law holds and
the absorbance between samples vary with concentration linearly.

Samples are usually prepared in cuvettes; depending on the region of interest, they may be
constructed of glass, plastic, or quartz.

IR spectrophotometry

Spectrophotometers designed for the main infrared region are quite different because of the
technical requirements of measurement in that region. One major factor is the type of
photosensors that are available for different spectral regions, but infrared measurement is also
challenging because virtually everything emits IR light as thermal radiation, especially at
wavelengths beyond about 5 μm.

Another complication is that quite a few materials such as glass and plastic absorb infrared
light, making it incompatible as an optical medium. Ideal optical materials are salts, which do
not absorb strongly. Samples for IR spectrophotometry may be smeared between two discs of
potassium bromide or ground with potassium bromide and pressed into a pellet. Where
aqueous solutions are to be measured, insoluble silver chloride is used to construct the cell.

Spectroradiometers, which operate almost like the visible region spectrophotometers, are
designed to measure the spectral density of illuminants in order to evaluate and categorize
lighting for sales by the manufacturer, or for the customers to confirm the lamp they decided
to purchase is within their specifications.

Components: 1. The light source shines onto or through the sample. 2. The sample transmits
or reflects light. 3. The detector detects how much light was reflected from or transmitted
through the sample. 4. The detector then converts how much light the sample transmitted or
reflected into a number.


Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV/ VIS)

involves the spectroscopy of photons and spectrophotometry. It uses light in the visible and
adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy space
molecules undergo electronic transitions.


UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of

transition metal ions and highly conjugated organic compounds.

 Solutions of transition metal ions can be coloured (i.e., absorb visible light) because d
electrons within the metal atoms can be excited from one electronic state to another.
The colour of metal ion solutions is strongly affected by the presence of other species,
such as certain anions or ligands. For instance, the colour of a dilute solution of
copper sulphate is a very light blue; adding ammonia intensifies the colour and
changes the wavelength of maximum absorption (λ_max).
 Organic compounds, especially those with a high degree of conjugation, also absorb
light in the UV or visible regions of the electromagnetic spectrum. The solvents for
these determinations are often water for water soluble compounds, or ethanol for
organic-soluble compounds. (Organic solvents may have significant UV absorption;
not all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly
at most wavelengths.)
 While charge transfer complexes also give rise to colours, the colours are often too
intense to be used for quantitative measurement.

The Beer-Lambert law states that the absorbance of a solution is directly proportional to the
solution's concentration. Thus UV/VIS spectroscopy can be used to determine the
concentration of a solution. It is necessary to know how quickly the absorbance changes with
concentration. This can be taken from references (tables of molar extinction coefficients), or
more accurately, determined from a calibration curve.

A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an

analyte gives a response which can be assumed to be proportional to the concentration. For
accurate results, the instrument's response to the analyte in the unknown should be compared
with the response to a standard; this is very similar to the use of calibration curves. The
response (e.g., peak height) for a particular concentration is known as the response factor.

Beer-Lambert law

The method is most often used in a quantitative way to determine concentrations of an

absorbing species in solution, using the Beer-Lambert law

where A is the measured absorbance, I0 is the intensity of the incident light at a given
wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the
concentration of the absorbing species. For each species and wavelength, ε is a constant
known as the molar absorptivity or extinction coefficient. This constant is a fundamental
molecular property in a given solvent, at a particular temperature and pressure, and has units
of 1 / M * cm or often AU / M * cm.

The absorbance and extinction ε are sometimes defined in terms of the natural logarithm
instead of the base-10 logarithm.

The Beer-Lambert Law is useful for characterizing many compounds but does not hold as a
universal relationship for the concentration and absorption of all substances. A 2nd order
polynomial relationship between absorption and concentration is sometimes encountered for
very large, complex molecules such as organic dyes (Xylenol Orange or Neutral Red, for

The instrument used in ultraviolet-visible spectroscopy is called a UV/vis

spectrophotometer. It measures the intensity of light passing through a sample (I), and
compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is
called the transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is
based on the transmittance:

A = − log(%T)

The basic parts of a spectrophotometer are a light source (often an incandescent bulb for the
visible wavelengths, or a deuterium arc lamp in the ultraviolet), a holder for the sample, a
diffraction grating or monochromator to separate the different wavelengths of light, and a
detector. The detector is typically a photodiode or a CCD. Photodiodes are used with
monochromators, which filter the light so that only light of a single wavelength reaches the
detector. Diffraction gratings are used with CCDs, which collects light of different
wavelengths on different pixels.

A spectrophotometer can be either single beam or double beam. In a single beam instrument
(such as the Spectronic 20), all of the light passes through the sample cell. Io must be
measured by removing the sample. This was the earliest design, but is still in common use in
both teaching and industrial labs.

In a double-beam instrument, the light is split into two beams before it reaches the sample.
One beam is used as the reference; the other beam passes through the sample. Some double-
beam instruments have two detectors (photodiodes), and the sample and reference beam are
measured at the same time. In other instruments, the two beams pass through a beam chopper,
which blocks one beam at a time. The detector alternates between measuring the sample
beam and the reference beam.

Samples for UV/Vis spectrophotometry are most often liquids, although the absorbance of
gases and even of solids can also be measured. Samples are typically placed in a transparent
cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an
internal width of 1 cm. (This width becomes the path length, L, in the Beer-Lambert law.)
Test tubes can also be used as cuvettes in some instruments. The best cuvettes are made of
high quality quartz, although glass or plastic cuvettes are common. (Glass and most plastics
absorb in the UV, which limits their usefulness to visible wavelengths.)

Ultraviolet-visible spectrum

An ultraviolet-visible spectrum is essentially a graph of light absorbance versus wavelength

in a range of ultraviolet or visible regions. Such a spectrum can often be produced directly by
a more sophisticated spectrophotometer, or the data can be collected one wavelength at a time
by simpler instruments. Wavelength is often represented by the symbol λ. Similarly, for a
given substance, a standard graph of the extinction coefficient (ε) vs. wavelength (λ) may be
made or used if one is already available. Such a standard graph would be effectively
"concentration-corrected" and thus independent of concentration. For the given substance, the
wavelength at which maximum absorption in the spectrum occurs is called λmax, pronounced

The Woodward-Fieser rules rules are a set of empirical observations which can be used to
predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic
compounds such as dienes and ketones.

This spectrum can be used qualitatively to identify components in a sample as each

component has their own unique absorbance spectrum (like a fingerprint).


Infrared spectroscopy (IR spectroscopy) is the subset of spectroscopy that deals with the
infrared region of the electromagnetic spectrum. It covers a range of techniques, the most
common being a form of absorption spectroscopy. As with all spectroscopic techniques, it
can be used to identify compounds or investigate sample composition. Infrared spectroscopy
correlation tables are tabulated in the literature.


The infrared portion of the electromagnetic spectrum is divided into three regions; the near-,
mid- and far- infrared, named for their relation to the visible spectrum. The far-infrared,
approximately 400-10 cm-1 (1000–30 μm), lying adjacent to the microwave region, has low
energy and may be used for rotational spectroscopy. The mid-infrared, approximately 4000-
400 cm-1 (30–1.4 μm) may be used to study the fundamental vibrations and associated
rotational-vibrational structure. The higher energy near-IR, approximately 14000-4000 cm-1
(1.4–0.8 μm) can excite overtone or harmonic vibrations. The names and classifications of
these subregions are merely conventions. They are neither strict divisions nor based on exact
molecular or electromagnetic properties.

Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they
rotate or vibrate corresponding to discrete energy levels. These resonant frequencies are
determined by the shape of the molecular potential energy surfaces, the masses of the atoms
and, by the associated vibronic coupling. In order for a vibrational mode in a molecule to be
IR active, it must be associated with changes in the permanent dipole. In particular, in the
Born-Oppenheimer and harmonic approximations, i.e. when the molecular Hamiltonian
corresponding to the electronic ground state can be approximated by a harmonic oscillator in
the neighborhood of the equilibrium molecular geometry, the resonant frequencies are
determined by the normal modes corresponding to the molecular electronic ground state
potential energy surface. Nevertheless, the resonant frequencies can be in a first approach
related to the strength of the bond, and the mass of the atoms at either end of it. Thus, the
frequency of the vibrations can be associated with a particular bond type.

Simple diatomic molecules have only one bond, which may stretch. More complex molecules
have many bonds, and vibrations can be conjugated, leading to infrared absorptions at
characteristic frequencies that may be related to chemical groups. For example, the atoms in a
CH2 group, commonly found in organic compounds can vibrate in six different ways:
symmetrical and antisymmetrical stretching, scissoring, rocking, wagging and twisting:

The infrared spectra of a sample are collected by passing a beam of infrared light through the
sample. Examination of the transmitted light reveals how much energy was absorbed at each
wavelength. This can be done with a monochromatic beam, which changes in wavelength
over time, or by using a Fourier transform instrument to measure all wavelengths at once.
From this, a transmittance or absorbance spectrum can be produced, showing at which IR
wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details
about the molecular structure of the sample.
This technique works almost exclusively on samples with covalent bonds. Simple spectra are
obtained from samples with few IR active bonds and high levels of purity. More complex
molecular structures lead to more absorption bands and more complex spectra. The technique
has been used for the characterization of very complex mixtures.

Sample preparation

Gaseous samples require little preparation beyond purification, but a sample cell with a long
pathlength (typically 5-10 cm) is normally needed, as gases show relatively weak

Liquid samples can be sandwiched between two plates of a high purity salt (commonly
sodium chloride, or common salt, although a number of other salts such as potassium
bromide or calcium fluoride are also used). The plates are transparent to the infrared light and
will not introduce any lines onto the spectra. Some salt plates are highly soluble in water, so
the sample and washing reagents must be anhydrous (without water).

Solid samples can be prepared in two major ways. The first is to crush the sample with a
mulling agent (usually nujol) in a marble or agate mortar, with a pestle. A thin film of the
mull is applied onto salt plates and measured.

The second method is to grind a quantity of the sample with a specially purified salt (usually
potassium bromide) finely (to remove scattering effects from large crystals). This powder
mixture is then crushed in a mechanical die press to form a translucent pellet through which
the beam of the spectrometer can pass.

It is important to note that spectra obtained from different sample preparation methods will
look slightly different from each other due to differences in the samples' physical states.

The last technique is the Cast Film technique.

Cast film technique is used mainly for polymeric compound. Sample is first dissolved in
suitable, non hygroscopic solvent. A drop of this solution is deposited on surface of KBr or
NaCl cell. The solution is then evaporated to dryness and the film formed on the cell is
analysed directly. Care is important to ensure that the film is not too thick otherwise light
cannot pass through. This technique is suitable for qualitative analysis.
Typical method

Typical apparatus

A beam of infrared light is produced and split into two separate beams. One is passed through
the sample, the other passed through a reference which is often the substance the sample is
dissolved in. The beams are both reflected back towards a detector, however first they pass
through a splitter which quickly alternates which of the two beams enters the detector. The
two signals are then compared and a printout is obtained.

A reference is used for two reasons:

 This prevents fluctuations in the output of the source affecting the data

 This allows the effects of the solvent to be cancelled out (the reference is usually a
pure form of the solvent the sample is in)

Uses and applications

Infrared spectroscopy is widely used in both research and industry as a simple and reliable
technique for measurement, quality control and dynamic measurement. The instruments are
now small, and can be transported, even for use in field trials. With increasing technology in
computer filtering and manipulation of the results, samples in solution can now be measured
accurately (water produces a broad absorbance across the range of interest, and thus renders
the spectra unreadable without this computer treatment). Some machines will also
automatically tell you what substance is being measured from a store of thousands of
reference spectra held in storage.
By measuring at a specific frequency over time, changes in the character or quantity of a
particular bond can be measured. This is especially useful in measuring the degree of
polymerization in polymer manufacture. Modern research machines can take infrared
measurements across the whole range of interest as frequently as 32 times a second. This can
be done whilst simultaneous measurements are made using other techniques. This makes the
observations of chemical reactions and processes quicker and more accurate.

Techniques have been developed to assess the quality of tea-leaves using infrared
spectroscopy. This will mean that highly trained experts (also called 'noses') can be used
more sparingly, at a significant cost saving.[1]

Infrared spectroscopy has been highly successful for applications in both organic and
inorganic chemistry. Infrared spectroscopy has also been successfully utilized in the field of
semiconductor microelectronics[2]: for example, infrared spectroscopy can be applied to
semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous
silicon, silicon nitride, etc.

Isotope effects

The different isotopes in a particular species may give fine detail in infrared spectroscopy.
For example, the O-O stretching frequency of oxyhemocyanin is experimentally determined
to be 832 and 788 cm-1 for ν(16O-16O) and ν(18O-18O) respectively.

By considering the O-O as a spring, the wavelength of absorbance, ν can be calculated:Where

k is the spring constant for the bond, and μ is the reduced mass of the A-B system:

(mi is the mass of atom i).

The reduced masses for 16O-16O and 18O-18O can be approximated as 8 and 9 respectively.

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy is a measurement technique for

collecting infrared spectra. Instead of recording the amount of energy absorbed when the
frequency of the infra-red light is varied (monochromator), the IR light is guided through an
interferometer. After passing the sample the measured signal is the interferogram. Performing
a mathematical Fourier transform on this signal results in a spectrum identical to that from
conventional (dispersive) infrared spectroscopy.

FTIR spectrometers are cheaper than conventional spectrometers because building of

interferometers is easier than the fabrication of a monochromator. In addition, measurement
of a single spectrum is faster for the FTIR technique because the information at all
frequencies is collected simultaneously. This allows multiple samples to be collected and
averaged together resulting in an improvement in sensitivity. Because of its various
advantages, virtually all modern infrared spectrometers are FTIR instruments.

Two-dimensional infrared spectroscopy

Two-dimensional infrared correlation spectroscopy analysis is the application of 2D

correlation analysis on infrared spectra. By extending the spectral information of a perturbed
sample, spectral analysis is simplified and resolution is enhanced. The 2D synchronous and
2D asynchronous spectra represent a graphical overview of the spectral changes due to a
perturbation (such as a changing concentration or changing temperature) as well as the
relationship between the spectral changes at two different wavenumbers.

Nonlinear two-dimensional infrared spectroscopy[3][4] is the infrared version of correlation

spectroscopy. Nonlinear two-dimensional infrared spectroscopy is a technique that has
become available with the development of femtosecond infrared laser pulses. In this
experiment first a set of pump pulses are applied to the sample. This is followed by a waiting
time, where the system is allowed to relax. The waiting time typically lasts from zero to
several picoseconds and the duration can be controlled with a resolution of tens of
femtoseconds. A probe pulse is then applied resulting in the emission of a signal from the
sample. The nonlinear two-dimensional infrared spectrum is a two-dimensional correlation
plot of the frequency ω1 that was excited by the initial pump pulses and the frequency ω3
excited by the probe pulse after the waiting time. This allows the observation of coupling
between different vibrational modes. Because of its extremely high time resolution it can be
used to monitor molecular dynamics on a picosecond timescale. It is still a largely unexplored
technique and is becoming increasingly popular for fundamental research.
Like in two-dimensional nuclear magnetic resonance (2DNMR) spectroscopy this technique
spreads the spectrum in two dimensions and allow for the observation of cross peaks that
contain information on the coupling between different modes. In contrast to 2DNMR
nonlinear two-dimensional infrared spectroscopy also involve the excitation to overtones.
These excitations result in excited state absorption peaks located below the diagonal and
cross peaks. In 2DNMR two distinct techniques, COSY and NOESY, are frequently used.
The cross peaks in the first are related to the scalar coupling, while in the later they are
related to the spin transfer between different nuclei. In nonlinear two-dimensional infrared
spectroscopy analogs have been drawn to these 2DNMR techniques. Nonlinear two-
dimensional infrared spectroscopy with zero waiting time corresponds to COSY and
nonlinear two-dimensional infrared spectroscopy with finite waiting time allowing
vibrational population transfer corresponds to NOESY. The COSY variant of nonlinear two-
dimensional infrared spectroscopy has been used for determination of the secondary structure
content proteins.[5]


Raman spectroscopy is a spectroscopic technique used in condensed matter physics and

chemistry to study vibrational, rotational, and other low-frequency modes in a system.[1] It
relies on inelastic scattering, or Raman scattering of monochromatic light, usually from a
laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with
phonons or other excitations in the system, resulting in the energy of the laser photons being
shifted up or down. The shift in energy gives information about the phonon modes in the
system. Infrared spectroscopy yields similar, but complementary information.

Typically, a sample is illuminated with a laser beam. Light from the illuminated spot is
collected with a lens and sent through a monochromator. Wavelengths close to the laser line,
due to elastic Rayleigh scattering, are filtered out while the rest of the collected light is
dispersed onto a detector.

Spontaneous Raman scattering is typically very weak, and as a result the main difficulty of
Raman spectroscopy is separating the weak inelastically scattered light from the intense
Rayleigh scattered laser light. Raman spectrometers typically use holographic diffraction
gratings and multiple dispersion stages to achieve a high degree of laser rejection. In the past,
PMTs were the detectors of choice for dispersive Raman setups, which resulted in long
acquisition times. However, the recent uses of CCD detectors have made dispersive Raman
spectral acquisition much more rapid.

Raman spectroscopy has a stimulated version, analogous to stimulated emission, called

stimulated Raman scattering.

Basic theory

Energy level diagram showing the states involved in Raman signal. The line thickness is
roughly proportional to the signal strength from the different transitions.

The Raman effect occurs when light impinges upon a molecule and interacts with the electron
cloud of the bonds of that molecule. The incident photon excites one of the electrons into a
virtual state. For the spontaneous Raman effect, the molecule will be excited from the ground
state to a virtual energy state, and relax into a vibrational excited state, which generates
Stokes Raman scattering. If the molecule was already in an elevated vibrational energy state,
the Raman scattering is then called anti-Stokes Raman scattering.

A molecular polarizability change, or amount of deformation of the electron cloud, with

respect to the vibrational coordinate is required for the molecule to exhibit the Raman effect.
The amount of the polarizability change will determine the intensity, whereas the Raman shift
is equal to the vibrational level that is involved.

Although the inelastic scattering of light was predicted by Smekal in 1923, it was not until
1928 that it was observed in practice. The Raman effect was named after one of its
discoverers, the Indian scientist Sir C. V. Raman who observed the effect by means of
sunlight (1928, together with K. S. Krishnan and independently by Grigory Landsberg and
Leonid Mandelstam).[1] Raman won the Nobel Prize in Physics in 1930 for this discovery
accomplished using sunlight, a narrow band photographic filter to create monochromatic light
and a "crossed" filter to block this monochromatic light. He found that light of changed
frequency passed through the "crossed" filter.

Subsequently the mercury arc became the principal light source, first with photographic
detection and then with spectrophotometric detection. Currently lasers are used as light


Raman spectroscopy is commonly used in chemistry, since vibrational information is very

specific for the chemical bonds in molecules. It therefore provides a fingerprint by which the
molecule can be identified. The fingerprint region of organic molecules is in the range 500-
2000 cm-1. Another way that the technique is used is to study changes in chemical bonding,
e.g. when a substrate is added to an enzyme.

Raman gas analyzers have many practical applications, for instance they are used in medicine
for real-time monitoring of anaesthetic and respiratory gas mixtures during surgery.

In solid state physics, spontaneous Raman spectroscopy is used to, among other things,
characterize materials, measure temperature, and find the crystallographic orientation of a

As with single molecules, a given solid material has characteristic phonon modes that can
help an experimenter identify it. In addition, Raman spectroscopy can be used to observe
other low frequency excitations of the solid, such as plasmons, magnons, and
superconducting gap excitations.
The spontaneous Raman signal gives information on the population of a given phonon mode
in the ratio between the Stokes (downshifted) intensity and anti-Stokes (upshifted) intensity.

Raman scattering by an anisotropic crystal gives information on the crystal orientation. The
polarization of the Raman scattered light with respect to the crystal and the polarization of the
laser light can be used to find the orientation of the crystal, if the crystal structure
(specifically, its point group) is known.

Raman active fibers, such as aramid and carbon, have vibrational modes that show a shift in
Raman frequency with applied stress. Polypropylene fibers also exhibit similar shifts.

The radial breathing mode is a commonly used technique to evaluate the diameter of carbon

Spatially Offset Raman Spectroscopy (SORS), which is less sensitive to surface layers than
conventional Raman, can be used to discover counterfeit drugs without opening their internal
packaging, and for non-invasive monitoring of biological tissue.[2][3]

Raman spectroscopy can be used to investigate the chemical composition of historical

documents such as the Book of Kells and contribute to knowledge of the social and economic
conditions at the time the documents were produced. This is especially helpful because
Raman spectroscopy offers a non-invasive way to determine the best course of preservation
or conservation treatment for such materials.

Raman microspectroscopy

Raman spectroscopy offers several advantages for microscopic analysis. Since it is a

scattering technique, specimens do not need to be fixed or sectioned. Raman spectra can be
collected from a very small volume (< 1 µm in diameter); these spectra allow the
identification of species present in that volume. Water does not interfere very strongly. Thus,
Raman spectroscopy is suitable for the microscopic examination of minerals, materials such
as polymers and ceramics, cells and proteins. A Raman microscope begins with a standard
optical microscope, and adds an excitation laser, a monochromator, and a sensitive detector
(such as a charge-coupled device (CCD) or photomultiplier tube (PMT)). FT-Raman has also
been used with microscopes.
In direct imaging, the whole field of view is examined for scattering over a small range of
wavenumbers (Raman shifts). For instance, a wavenumber characteristic for cholesterol could
be used to record the distribution of cholesterol within a cell culture.

The other approach is hyperspectral imaging or chemical imaging, in which thousands of

Raman spectra are acquired from all over the field of view. The data can then be used to
generate images showing the location and amount of different components. Taking the cell
culture example, a hyperspectral image could show the distribution of cholesterol, as well as
proteins, nucleic acids, and fatty acids. Sophisticated signal- and image-processing
techniques can be used to ignore the presence of water, culture media, buffers, and other

Raman microscopy, and in particular confocal microscopy, has very high spatial resolution.
For example, the lateral and depth resolutions were 250 nm and 1.7 µm, respectively, using a
confocal Raman microspectrometer with the 632.8 nm line from a He-Ne laser with a pinhole
of 100 µm diameter.

Since the objective lenses of microscopes focus the laser beam to several micrometres in
diameter, the resulting photon flux is much higher than achieved in conventional Raman
setups. This has the added benefit of enhanced fluorescence quenching. However, the high
photon flux can also cause sample degradation, and for this reason some setups require a
thermally conducting substrate (which acts as a heat sink) in order to mitigate this process.

By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of

microscopic regions of samples can be measured. As a result, the fluorescence of water,
media, and buffers can be removed. Consequently in vivo time- and space-resolved Raman
spectroscopy is suitable to examine proteins, cells and organs.

Raman microscopy for biological and medical specimens generally uses near-infrared (NIR)
lasers (785 nm diodes and 1064 nm Nd:YAG are especially common). This reduces the risk
of damaging the specimen by applying high power. However, the intensity of NIR Raman is
low (owing to the ω-4 dependence of Raman scattering intensity), and most detectors required
very long collection times. Recently, more sensitive detectors have become available, making
the technique better suited to general use. Raman microscopy of inorganic specimens, such as
rocks and ceramics and polymers, can use a broader range of excitation wavelengths.[5]

Several variations of Raman spectroscopy have been developed. The usual purpose is to
enhance the sensitivity (e.g., surface-enhanced Raman), to improve the spatial resolution
(Raman microscopy), or to acquire very specific information (resonance Raman).

 Surface Enhanced Raman Spectroscopy (SERS) - Normally done in a silver or

gold colloid or a substrate containing silver or gold. Surface plasmons of silver and
gold are easily excited by the laser, and the resulting electric fields cause other nearby
molecules to become Raman active. The result is amplification of the Raman signal
(by up to 1011). This effect was originally observed by Fleishman but the prevailing
explanation was proposed by Van Duyne in 1977.[6]

 Hyper Raman - A non-linear effect in which the vibrational modes interact with the
second harmonic of the excitation beam. This requires very high power, but allows
the observation of vibrational modes which are normally "silent". It frequently relies
on SERS-type enhancement to boost the sensitivity.

 Resonance Raman spectroscopy - The excitation wavelength is matched to an

electronic transition of the molecule or crystal, so that vibrational modes associated
with the excited electronic state are greatly enhanced. This is useful for studying large
molecules such as polypeptides, which might show hundreds of bands in
"conventional" Raman spectra. It is also useful for associating normal modes with
their observed frequency shifts.

 Spontaneous Raman Spectroscopy - Used to study the temperature dependence of

the Raman spectra of molecules.

 Optical Tweezers Raman Spectroscopy (OTRS) - Used to study individual

particles, and even biochemical processes in single cells trapped by optical tweezers.

 Stimulated Raman Spectroscopy - A two color pulse transfers the population from
ground to a rovibrationally excited state, if the difference in energy corresponds to an
allowed Raman transition. Two photon UV ionization, applied after the population
transfer but before relaxation, allows the intra-molecular or inter-molecular Raman
spectrum of a gas or molecular cluster (indeed, a given conformation of molecular
cluster) to be collected. This is a useful molecular dynamics technique.

 Spatially Offset Raman Spectroscopy (SORS) - The Raman scatter is collected

from regions laterally offset away from the excitation laser spot, leading to
significantly lower contributions from the surface layer than with traditional Raman


sketch of a typical TGA (Setaram TG-DTA 92 B type); the cooling water pipe was omitted

Thermogravimetric Analysis or TGA is a type of testing that is performed on samples to

determine changes in weight in relation to change in temperature. Such analysis relies on a
high degree of precision in three measurements: weight, temperature, and temperature
change. As many weight loss curves look similar, the weight loss curve may require
transformation before results may be interpreted. A derivative weight loss curve can be used
to tell the point at which weight loss is most apparent. Again, interpretation is limited without
further modifications and deconvolution of the overlapping peaks may be required.

TGA is commonly employed in research and testing to determine characteristics of materials

such as polymers, to determine degradation temperatures, absorbed moisture content of
materials, the level of inorganic and organic components in materials, decomposition points
of explosives, and solvent residues. It is also often used to estimate the corrosion kinetics in
high temperature oxidation.


The analyzer usually consists of a high-precision balance with a pan loaded with the sample.
The sample is placed in a small electrically heated oven with a thermocouple to accurately
measure the temperature. The atmosphere may be purged with an inert gas to prevent
oxidation or other undesired reactions. A computer is used to control the instrument.

Analysis is carried out by raising the temperature gradually and plotting weight against
temperature. After the data is obtained, curve smoothing and other operations may be done
such as to find the exact points of inflection.

Differential thermal analysis (or DTA) is a thermoanalytic technique, similar to differential

scanning calorimetry. In DTA, the material under study and an inert reference are heated (or
cooled) under identical conditions, while recording any temperature difference between
sample and reference.[1] This differential temperature is then plotted against time, or against
temperature (DTA curve or thermogram). Changes in the sample, either exothermic or
endothermic, can be detected relative to the inert reference. Thus, a DTA curve provides data
on the transformations that have occurred, such as glass transitions, crystallization, melting
and sublimation. The area under a DTA peak can be to the enthalpy change and it is not
affected by the heat capacity of the sample.


A DTA apparatus consist of a sample holder comprising thermocouples, sample containers

and a ceramic or metallic block; a furnace; a temperature programmer; and a recording
system. The key feature is the existence of two thermocouples connected to a voltmeter. One
thermocouple is placed in an inert material such as Al2O3, while the other is placed in a
sample of the material under study. As the temperature is increased, there will be a brief
deflection of the voltmeter if the sample is undergoing a phase transition. This occurs because
the input of heat will raise the temperature of the inert substance, but be incorporated as latent
heat in the material changing phase.


A DTA curve can be used only as a finger print for identification purposes but usually the
applications of this method are the determination of phase diagrams, heat change
measurements and decomposition in various atmospheres.

DTA is widely used in the pharmaceutical and food industries.

DTA may be used in cement chemistry, mineralogical research and in environmental studies.

DTA curves may also be used to date bone remains or to study archaeological materials.

Differential scanning calorimetry or DSC is a thermoanalytical technique in which the

difference in the amount of heat required to increase the temperature of a sample and
reference are measured as a function of temperature. Both the sample and reference are
maintained at nearly the same temperature throughout the experiment. Generally, the
temperature program for a DSC analysis is designed such that the sample holder temperature
increases linearly as a function of time. The reference sample should have a well-defined heat
capacity over the range of temperatures to be scanned. The basic principle underlying this
technique is that, when the sample undergoes a physical transformation such as phase
transitions, more (or less) heat will need to flow to it than the reference to maintain both at
the same temperature. Whether more or less heat must flow to the sample depends on
whether the process is exothermic or endothermic. For example, as a solid sample melts to a
liquid it will require more heat flowing to the sample to increase its temperature at the same
rate as the reference. This is due to the absorption of heat by the sample as it undergoes the
endothermic phase transition from solid to liquid. Likewise, as the sample undergoes
exothermic processes (such as crystallization) less heat is required to raise the sample
temperature. By observing the difference in heat flow between the sample and reference,
differential scanning calorimeters are able to measure the amount of heat absorbed or
released during such transitions. DSC may also be used to observe more subtle phase
changes, such as glass transitions. DSC is widely used in industrial settings as a quality
control instrument due to its applicability in evaluating sample purity and for studying
polymer curing.[1][2][3]

An alternative technique, which shares much in common with DSC, is differential thermal
analysis (DTA). In this technique it is the heat flow to the sample and reference that remains
the same rather than the temperature. When the sample and reference are heated identically
phase changes and other thermal processes cause a difference in temperature between the
sample and reference. Both DSC and DTA provide similar information; DSC is the more
widely used of the two techniques.[1][2][3]

DSC curves

The result of a DSC experiment is a curve of heat flux versus temperature or versus time.
There are two different conventions: exothermic reactions in the sample shown with a
positive or negative peak; it depends by the different kind of technology used by the
instrumentation to make the experiment. This curve can be used to calculate enthalpies of
transitions. This is done by integrating the peak corresponding to a given transition. It can be
shown that the enthalpy of transition can be expressed using the following equation:


where ΔH is the enthalpy of transition, K is the calorimetric constant, and A is the area under
the curve. The calometric constant will vary from instrument to instrument, and can be
determined by analyzing a well-characterized sample with known enthalpies of transition.[2]


Differential scanning calorimetry can be used to measure a number of characteristic

properties of a sample. Using this technique it is possible to observe fusion and crystallization
events as well as glass transition temperatures (Tg). DSC can also be used to study oxidation,
as well as other chemical reactions.[1][2][3]

Glass transitions may occur as the temperature of an amorphous solid is increased. These
transitions appear as a step in the baseline of the recorded DSC signal. This is due to the
sample undergoing a change in heat capacity; no formal phase change occurs.[1][3]

As the temperature increases, an amorphous solid will become less viscous. At some point
the molecules may obtain enough freedom of motion to spontaneously arrange themselves
into a crystalline form. This is known as the crystallization temperature (Tc). This transition
from amorphous solid to crystalline solid is an exothermic process, and results in a peak in
the DSC signal. As the temperature increases the sample eventually reaches its melting
temperature (Tm). The melting process results in an endothermic peak in the DSC curve. The
ability to determine transition temperatures and enthalpies makes DSC an invaluable tool in
producing phase diagrams for various chemical systems.

DSC may also be used in the study of liquid crystals. As matter transitions between solid and
liquid it often goes through a third state, which displays properties of both phases. This
anisotropic liquid is known as a liquid crystalline or mesomorphous state. Using DSC, it is
possible to observe the small energy changes that occur as matter transitions from a solid to a
liquid crystal and from a liquid crystal to an isotropic liquid.[2]
Using differential scanning calorimetry to study the oxidative stability of samples generally
requires an airtight sample chamber. Usually, such tests are done isothermally (at constant
temperature) by changing the atmosphere of the sample. First, the sample is brought to the
desired test temperature under an inert atmosphere, usually nitrogen. Then, oxygen is added
to the system. Any oxidation that occurs is observed as a deviation in the baseline. Such
analyses can be used to determine the stability and optimum storage conditions for a

DSC is widely used in the pharmaceutical and polymer industries. For the polymer chemist,
DSC is a handy tool for studying curing processes, which allows the fine tuning of polymer
properties. The cross-linking of polymer molecules that occurs in the curing process is
exothermic, resulting in a positive peak in the DSC curve that usually appears soon after the
glass transition.[1][2][3]

In the pharmaceutical industry it is necessary to have well-characterized drug compounds in

order to define processing parameters. For instance, if it is necessary to deliver a drug in the
amorphous form, it is desirable to process the drug at temperatures below those at which
crystallization can occur.

In food science research, DSC is used in conjunction with other thermal analytical techniques
to determine water dynamics. Changes in water distribution may be correlated with changes
in texture. Similar to material science studies, the effects of curing on confectionery products
can also be analyzed.

DSC curves may also be used to evaluate drug and polymer purities. This is possible because
the temperature range over which a mixture of compounds melts is dependent on their
relative amounts. This effect is due to a phenomenon known as freezing point depression,
which occurs when a foreign solute is added to a solution. (Freezing point depression is what
allows salt to de-ice sidewalks and antifreeze to keep your car running in the winter.)
Consequently, less pure compounds will exhibit a broadened melting peak that begins at
lower temperature than a pure compound.

In last few years this technology has been involved in metallic material study. The
characterization of this kind of material with DSC is not easy yet because of the low quantity
of literature about it. It is known that it is possible to use DSC to find solidus and liquidus
temperature of a metal alloy, but the widest application is, by now, the study of
precipitations, Guiner Preston zones, phase transitions, dislocations movement, grain growth

Chromatography (from Greek χρώμα:chroma, color and γραφειν:"graphein" to write) is the

collective term for a family of laboratory techniques for the separation of mixtures. It
involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which
separates the analyte to be measured from other molecules in the mixture and allows it to be

Chromatography may be preparative or analytical. Preparative chromatography seeks to

separate the components of a mixture for further use (and is thus a form of purification).
Analytical chromatography normally operates with smaller amounts of material and seeks to
measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.

.An analogy which is sometimes useful is to suppose a mixture of bees and wasps passing
over a flower bed. The bees would be more attracted to the flowers than the wasps, and
would become separated from them. If one were to observe at a point past the flower bed, the
wasps would pass first, followed by the bees. In this analogy, the bees and wasps represent
the analytes to be separated, the flowers represent the stationary phase, and the mobile phase
could be thought of as the air. The key to the separation is the differing affinities among
analyte, stationary phase, and mobile phase. The observer could represent the detector used in
some forms of analytical chromatography. A key point is that the detector need not be
capable of discriminating between the analytes, since they have become separated before
passing the detector.

Chromatography terms

• The analyte is the substance that is to be separated during chromatography.

• Analytical chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
• A bonded phase is a stationary phase that is covalently bonded to the support
particles or to the inside wall of the column tubing.
• A chromatogram is the visual output of the chromatograph. In the case of an optimal
separation, different peaks or patterns on the chromatogram correspond to different
components of the separated mixture.

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for
example obtained by a spectrophotometer, mass spectrometer or a variety of other
detectors) corresponding to the response created by the analytes exiting the system. In
the case of an optimal system the signal is proportional to the concentration of the
specific analyte separated.

• A chromatograph is equipment that enables a sophisticated separation e.g. gas

chromatographic or liquid chromatographic separation.
• Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary (stationary
phase) while the other (the mobile phase) moves in a definite direction.
• The effluent is the mobile phase leaving the column.
• An immobilized phase is a stationary phase which is immobilized on the support
particles, or on the inner wall of the column tubing.
• The mobile phase is the phase which moves in a definite direction. It may be a liquid
(LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid
chromatography, SFC). A better definition: The mobile phase consists of the sample
being separated/analyzed and the solvent that moves the sample through the column.
In one case of HPLC the solvent consists of a carbonate/bicarbonate solution and the
sample is the anions being separated. The mobile phase moves through the
chromatography column (the stationary phase) where the sample interacts with the
stationary phase and is separated.
• Preparative chromatography is used to purify sufficient quantities of a substance
for further use, rather than analysis.
• The retention time is the characteristic time it takes for a particular analyte to pass
through the system (from the column inlet to the detector) under set conditions. See
also: Kovat's retention index
• The sample is the matter analysed in chromatography. It may consist of a single
component or it may be a mixture of components. When the sample is treated in the
course of an analysis, the phase or the phases containing the analytes of interest is/are
referred to as the sample whereas everything out of interest separated from the sample
before or in the course of the analysis is referred to as waste.
• The solute refers to the sample components in partition chromatography.
• The solvent refers to any substance capable of solubilizing other substance, and
especially the liquid mobile phase in LC.
• The stationary phase is the substance which is fixed in place for the chromatography
procedure. Examples include the silica layer in thin layer chromatography.

Techniques by chromatographic bed shape

Column chromatography

A diagram of a standard column chromatography and a flash column chromatography setup

Column chromatography is a separation technique in which the stationary bed is within a

tube. The particles of the solid stationary phase or the support coated with a liquid stationary
phase may fill the whole inside volume of the tube (packed column) or be concentrated on or
along the inside tube wall leaving an open, unrestricted path for the mobile phase in the
middle part of the tube (open tubular column). Differences in rates of movement through the
medium are calculated to different retention times of the sample.

In 1978, W. C. Still introduced a modified version of column chromatography called flash

column chromatography (flash). The technique is very similar to the traditional column
chromatography, except for that the solvent is driven through the column by applying
positive pressure. This allowed most separations to be performed in less than 20 minutes,
with improved separations compared to the old method. Modern flash chromatography
systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the
cartridge. Systems may also be linked with detectors and fraction collectors providing
automation. The introduction of gradient pumps resulted in quicker separations and less
solvent usage.

In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a
packed bed. This allows omission of initial clearing steps such as centrifugation and
filtration, for culture broths or slurries of broken cells.

Planar Chromatography

Thin layer chromatography is used to separate components of chlorophyll

Planar chromatography is a separation technique in which the stationary phase is present as

or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography) or a layer of solid particles spread on a support such
as a glass plate (thin layer chromatography).

Paper Chromatography

Paper chromatography is a technique that involves placing a small dot of sample solution
onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer
of solvent and sealed. As the solvent rises through the paper it meets the sample mixture
which starts to travel up the paper with the solvent. Different compounds in the sample
mixture travel different distances according to how strongly they interact with the paper. This
paper is made of cellulose, a polar molecule, and the compounds within the mixture travel
farther if they are non-polar. More polar substances bond with the cellulose paper more
quickly, and therefore do not travel as far. This process allows the calculation of an Rf value
and can be compared to standard compounds to aid in the identification of an unknown

Thin layer chromatography

Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar

to paper chromatography. However, instead of using a stationary phase of paper, it involves a
stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat,
inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and
the choice between different adsorbents. Different compounds in the sample mixture travel
different distances according to how strongly they interact with the adsorbent. This allows the
calculation of an Rf value and can be compared to standard compounds to aid in the
identification of an unknown substance.

Techniques by physical state of mobile phase

Gas chromatography

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is

a separation technique in which the mobile phase is a gas. Gas chromatography is always
carried out in a column, which is typically "packed" or "capillary" (see below) .

Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid

stationary phase (often a liquid silicone-based material) and a mobile gas (most often
Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a
capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely
used in analytical chemistry; though the high temperatures used in GC make it unsuitable for
high molecular weight biopolymers or proteins (heat will denature them), frequently
encountered in biochemistry, it is well suited for use in the petrochemical, environmental
monitoring, and industrial chemical fields. It is also used extensively in chemistry research.
Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid.
Liquid chromatography can be carried out either in a column or a plane. Present day liquid
chromatography that generally utilizes very small packing particles and a relatively high
pressure is referred to as high performance liquid chromatography (HPLC).

In the HPLC technique, the sample is forced through a column that is packed with irregularly
or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid
(mobile phase) at high pressure. HPLC is historically divided into two different sub-classes
based on the polarity of the mobile and stationary phases. Technique in which the stationary
phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the
stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite
(e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary
phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase"
has fewer applications and RPLC is therefore used considerably more.

Specific techniques which come under this broad heading are listed below. It should also be
noted that the following techniques can also be considered fast protein liquid chromatography
if no pressure is used to drive the mobile phase through the stationary phase. See also
Aqueous Normal Phase Chromatography.

Affinity chromatography

Affinity chromatography is based on selective non-covalent interaction between an analyte

and specific molecules. It is very specific, but not very robust. It is often used in biochemistry
in the purification of proteins bound to tags. These fusion proteins are labelled with
compounds such as His-tags, biotin or antigens, which bind to the stationary phase
specifically. After purification, some of these tags are usually removed and the pure protein is

Supercritical fluid chromatography

Supercritical fluid chromatography is a separation technique in which the mobile phase is a

fluid above and relatively close to its critical temperature and pressure.
Techniques by separation mechanism
Ion exchange chromatography

Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is

usually performed in columns but the mechanism can be benefited also in planar mode. Ion
exchange chromatography uses a charged stationary phase to separate charged compounds
including amino acids, peptides, and proteins. In conventional methods the stationary phase is
an ion exchange resin that carries charged functional groups which interact with oppositely
charged groups of the compound to be retained. Ion exchange chromatography is commonly
used to purify proteins using FPLC.

Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel permeation chromatography

(GPC) or gel filtration chromatography and separates molecules according to their size (or
more accurately according to their hydrodynamic diameter or hydrodynamic volume).
Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute,
whereas larger molecules are excluded from the pores and elute faster. It is generally a low
resolution chromatography technique and thus it is often reserved for the final, "polishing"
step of purification. It is also useful for determining the tertiary structure and quaternary
structure of purified proteins, especially since it can be carried out under native solution

Special techniques
Reversed-phase chromatography

Reversed-phase chromatography is an elution procedure used in liquid chromatography in

which the mobile phase is significantly more polar than the stationary phase.

Two-dimensional chromatography

In some cases, the chemistry within a given column can be insufficient to separate some
analytes. It is possible to direct a series of unresolved peaks onto a second column with
different physico-chemical (Chemical classification) properties. Since the mechanism of
retention on this new solid support is different from the first dimensional separation, it can be
possible to separate compounds that are indistinguishable by one-dimensional

Pyrolysis gas chromatography

Fast protein liquid chromatography

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography

techniques which are used to purify proteins. Many of these techniques are identical to those
carried out under high performance liquid chromatography.

Countercurrent chromatography

Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both

the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing
them to settle into layers and then separating the layers.

Chiral chromatography
Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers,
these have no chemical or physical differences apart from being three dimensional mirror
images. Conventional chromatography or other separation processes are incapable of
separating them. To enable chiral separations to take place, either the mobile phase or the
stationary phase must themselves be made chiral, giving differing affinities between the
analytes. Chiral chromatography HPLC columns (with a chiral stationary phase) in both
normal and reversed phase are commercially available