Instrumental analysis in research

INSTRUMENTAL
METHODS IN
RESEARCH
METHODOLOGY

By
Dr. M. Gopikrishna
Reader, PG Dept. of Rasashastra
SJG Ayurvedic Medical College, Koppal, Karnataka
email: rasashastra@rediffmail.com

TO IDENTIFIE NEW THINGS-

TO THROUGH MODERN LIGHT ON
OLD FACTS-

• TO UNDERSTAND ALL THESE WE NEED
FEW PARAMETERS TO ASSES THEM
HENCEFORTH FEW OF THE
INSTRUMENTS ARE MENTIONED TO
KNOW THE ANALYSIS OF THE DRUGS.EG;-
ROOT PRESSURE IN VARIOUS SEASONS
VARY AS BILVA IN GRISHMA RITU IS RICH
IN TANINS OR COLOURING AGENT IS
MORE,TO IDENTIFIE WE NEED
INSTRUMENTS LIKE PHYTOCHEMICAL
ANALYSIS AND MICROSCOPIC ANALYSIS.

PHYTOCHEMICAL ANALYSIS:-
• -TO ASSES AND ANALYSIS.
• -WATER SOLUBLE MATERIALS WITH MANY
COMPOUNDS ARE OBSERVED AND
IDENTIFIED.
• -SEPARATE CHEMICAL AND ISOLATE IT
AND ASSES THE DIFFERENT BONDING WITH
DIFFERENT ADVANCE TECHNIQUES.

• -BIO-SYNTHETIC PATHWAY CAN BE
ASSESED OF THE ISOLATED CHEMICAL.FOR
THIS WE NEED TO-
• * QUALITATIVE ANALYSIS.
• *QUANTITATIVE ANALYSIS.
• SEMIQUALITATIVE AND
SEMIQUANTITATIVE
EG:-WATER EXTRACTS ETC

METHODS NEEDED FOR
DOING ALL THESE-

1)INSTRUMENTAL
–BY THIS WE CAN KNOW
PHYSICOCHEMICAL NATURE.

2)NON INSTRUMENTAL
-AN ORGANOLEPTIC EVALUATION.

1)INSTRUMENTAL ANALYSIS:
• INSTRUMENTS PERFORM DIFFERENT FUNCTION EG;-
REFRACTIVE INDEX OF OILS,OR SOLUBILITY OF ANY
SUBSTANCE,TO KNOW ANY OF SUCH FACTORS WE NEED
INSTRUMENTS TO CONFIRM THE FACTORS.
• THE CONTACT OF THE PLANT TO A INSTRUMENT , ITS
REACTION TO THE SAMPLE AND COMPARED TO DESIGN
THIS GRASPED SIGNAL IN A MEASURABLE UNITS I.E TO
CONVERT THE ORIGINAL SIGNALS TO A CONVENTIONAL
SIGNAL.CONVERT THE VARIOUS ENERGIES TO A ELECTRIC
ENERGY AND CAN BE ASSESED IN A
GALVANOMETERETC.(IT’S A MEASURABLE
FORM).SOMETIMES TRANSFORMING SIGNALS IS
DIFFICULT THERE IN SUCH PLACES WE NEED AMPLIFIERS.
• PRESENTATION OF DATA IN DIFFERENT WAYS IS
TRANSFORMED,AMPLIFIED,AND GENERATED .I.E THESE
OBSERVATIONS ARE DIRECTLY PRAPORTIONAL TO THE
DATA.

ADVANTAGES:-
-TO ANALYSE A SAMPLE SMALL
AMOUNT OF DRUG IS ENOUGH.
-DETERMINATION OF QUALITATIVE
AND QUANTITATIVE ANALYSIS IN
SHORT TIME.
-COMPLEX MIXTURES CAN ALSO BE
ANALYSED WITH OR WITHOUT
ISOLATION.
-IT IS WITH SUFFICIENT RELIABILITY
AND ACCURATE.

DISADVANTAGES:-

-TOO COSTLY.

-MAINTANANCE OF INSTRUMENT IS DIFFICULT.

-CHEMICAL CLEANLINESS IS ALSO COSTLY.

-SENSITIVITY DEPENDS ON ADVANCEMENT OF
INSTRUMENT THEREFOR UPGRADE IT REGULARLY.

-SPECIALISED TEST FOR HANDLING OR USING IS
NEEDED.

-FREQUENT NEED OF CHECKING DRUGS IS
NECESSARY THEREFORE FREQUENTLY CHECK THE
INSTRUMENT.

• EXTRACTION OF DRUGS:-
• PHANTA,HIMA,SATWA,ARE EXTRACTS
ONLY
• -IN MODERN THE METHODS OF
EXTACTIONS ARE.
• *1)MACERATION
• *2)PERCOLATION.
• *3)DECOCTION.

*1)MACERATION:-
KEEPING THE POWDERED DRUG IN
SUITABLE SOLVENT FOR FEW HOURS
LIKE IN
WATER,ETHER,METHENOL,WITHOUT
APPLING HEAT TO DISSOLVE ITS
SOLUBLE PORTION IN IT CALLED AS
“ISOLATION MARC” AND INSOLUBLE
PART CALLED AS “MARC”.
SUB. NEEDED:-CONICAL
GLASS,FILTER PAPER,EVOPARATING
DISH,GLASS FUNNEL,HOT WATER
BATH,ELECTRIC OVEN,ANALYTICAL
WEIGHING
BALANCE,PIPPETE,VOLUMETRIC
FLASK,SOLVENTS,THE TEST DRUG.

PROCEDURE:TAKE 5GMS OF TEST DRUG POWDER I.E.AIR
DRIED DRUG POWDER ,ADD 100ML OF WATER TO THIS AND
PLACE IT IN A CONICAL FLASK SHAKE THE MIXTURE NOW
AND THEN FOR 18HRS THEN FILTER IT.SEPERATE THE
RESIDUE.FROM THE SOLVENT COLLECT 20ML AND PLACE
IT IN A PORCELINE DISH AND DRY IT ON A HOT WATER
BATH AND COLLECT THE RESIDUE AND WEIGH IT AND
CALCULATE THE % OF THE EXTRACTIVE.

PERCOLATION:-
IT IS THE METHOD OF EXTRACTION
OF ALCOLOIDS ETC.BY PASSING A
LIQUID THROUGH A COARS POWDER
DRUG.
THE LIQUID IS MADE TO PASS THE
DRUG AND THE SOLVENT IS
COLLECTED BY THE OPENING OF
THE STOP CORK,ALSO THE SOLVENT
IS MADE TO PASS THE COTTON PLUG
AT THE NECK TO FILTER AND TO
GAIN ONLY THE SOLLUBLE
EXTRACT ONLY.

• USE OF PERCOLATE: COLLECT THE
PERCOLATE OF 5MIN INTERVAL PF 4-5
BATCHES OF SAME SOLVENT AND ASSES
FOR THE RICH ALCOLOID SAMPLE.
• EG;IF GOOD %IN FIRST SAMPLE OR 3RD
SAMPLE OR 5TH SAMPLE,THAT PARTICULAR
SAMPLE CAN BE GIVEN TO THE PATIENT
AND STANDARDISE THE SAMPLE.

• DECOCTION:-
• IT IS A METHOD OF EXTRACTING OF
EXTRACTIVES,HERE FIRE OR HEAT TREATMENT IS
USED TO EXTRACT THE SOLVENTS,HERE THE
COARSE POWDER IS TAKEN IN A APPARATUS AND
A ARRANGEMENT IS MADE TO PASS THE HOT
LIQUID THROUGH THE DRUG AND RECYCLE IT ,AS
IT PASSES THROUGH THE DRUG IT IS FILTERED
THROUGH A COTTON PLUG AND IS COLLECTED
AT THE CHAMBER AT THE BASE WHICH IS
HEATED AND THE VAPOUR IS MADE TO PASS
THROUGH A PIPE INTO THE CONDENCER AREA
AND MADE TO LIQUID FORM AND AGAIN PASS
THROUGH THE DRUG.REPEAT THE PROCESS AS
PER REQUIREMENT AND STANDARDISE IT..

SOXLET APPARATUS
IS DESIGNED FOR THE
SAME PURPOSE.

• IF IT IS A COMPLEX COMPOUND SEPARATE THE
EXTRACTS WITH ANY METHODS ,AS ONE METHOD
IS-
• BATCH EXTRACTION:-
• IT IS A LIQUID LIQUID EXTRACTION,OR
EXTRACTION OF EXTRACTIVES FROM THE
LIQUIDS.
• TAKE A LIQUID SUBSTANCE IN A FUNNEL WITH
CORKS ON EITHER SIDES,ALLOW IT FOR FEW
HOURS AND LATER DISTINGUISH IT IN RESPECT
TO ITS COLOUR OR CONSISTANCY,OF
VISCOSITY,SEPARATE THEM AS PER
REQUIREMENT INTO A BEAKER.
•

• Separatory Funnel Extraction Procedure
• 1. Inspect your separatory funnel.
• The organic chem teaching labs have acquired
several different types of sep funnels over the past
years, with different types of stopcocks and
stoppers, as illustrated in the photo below. The
Teflon stopcocks work better than the ground glass
stopcock; if you have a sep funnel with a ground
glass stopcock, you may exchange for a Teflon
style.

The sep funnel on the left is a 60 mL size, the
others are 125 mL. The organic chem teaching
labs are downscaling slowly to this smaller size.

There are two different styles of stopper, too. Some
students swear by the plastic style, some by the
ground glass style. The disadvantage of the ground
glass style is that it can lodge permanently in the
sep funnel if it is not removed and stored separately
after use. Whichever style you have, make sure that
the stopper fits snugly in the top of the flask.

2. Support the separatory
funnel in a ring on a
ringstand.
The rings are located on
the back shelves and they
come in many sizes. Test
to make sure that you
haven’t chosen too large a
ring before setting the
funnel in it. You can add
pieces of cut tygon tubing
to the ring to cushion the
funnel.
Make sure the stopcock of the separatory funnel is closed!

• 3. Add the liquid to the separatory funnel.
• Place a stemmed funnel in the neck of the separatory
funnel. Add the liquid to be extracted, then add the
extraction solvent. The total volume in the
separatory funnel should not be greater than three-
quarters of the funnel volume. Insert the stopper in
the neck of the separatory funnel.

pour in liquid to be extracted . . .

pour in the solvent . .
.

add a stopper

4. Shake the separatory funnel.
Pick up the separatory funnel with the stopper in
place and the stopcock closed, and rock it once
gently. Then, point the stem up and slowly open
the stopcock to release excess pressure. Close the
stopcock. Repeat this procedure until only a small
amount of pressure is released when it is vented.

Now, shake the funnel vigorously for a few seconds. Release
the pressure, then again shake vigorously. About 30 sec total
vigorous shaking is usually sufficient to allow solutes to come
to equilibrium between the two solvents.
Vent frequently to prevent pressure buildup, which can cause the
stopcock and perhaps hazardous chemicals from blowing out. Take
special care when washing acidic solutions with bicarbonate or carbonate
since this produces a large volume of CO2 gas.

5. Separating the layers.

Let the funnel rest undisturbed until the layers
are clearly separated.

While waiting, remove the
stopper and place a beaker or
flask under the sep funnel.

Carefully open the stopcock and allow
the lower layer to drain into the flask.
Drain just to the point that the upper
liquid barely reaches the stopcock.

If the upper layer is to be
removed from the funnel,
remove it by pouring it out of the
top of the funnel.

• 6. Perform multiple extractions as necessary.
• Often you will need to do repeat extractions with fresh
solvent. You can leave the upper layer in the separatory
funnel if this layer contains the compound of interest. If the
compound of interest is in the lower layer, the upper layer
must be removed from the separatory funnel and replaced
with the drained-off lower layer, to which fresh solvent is
then added.
• Yes, it can be confusing! Plus, the beginning student often
does not know in which layer resides the compound of
interest. The best advice: Always save all layers until the
experiment is completely finished!

7. Store your separatory funnel with the cap (stopper)
separate from the funnel!

Please, remove the stopper before
storage!!

• DECANTATATION:-
• ALLOW THE SOLID PART TO SETTAL AND
ONLY THE LIQUID PART TO BE REMOVED
OR SEPERATED ITS DECANTATION,THIS
CAN BE DONE EVEN WITH A CENTRIFUGAL
MACHINE AND SEPERATED.

SEPERATION OF
VOLATILE SUBSTANCES
FROM A DRUG IN A
DISTILATION
APPARATUS.

CHROMATOGRAPHY:-
IT IS A TECHNIQUE TO SEPARATE
INDIVIDUAL COMPONENTS IN
A MIXTURE,IN A SPECIFIC MOBILE AND
STATIONARY PHASE.

CHROMA= COLOUR.

GRAPHY= WRITING .

• 1ST INVENTED BY M.TSWETT IN 1906 BY A
BOTONIST,HE USED CACO3 CRYSTALS IN A
COLOUM AND POURED EXTRACTIVES IN
COLOUM,ALSO POURED TEST SOUTION IN
IT AND DEVELOPED COLOURED BANDS IN
CACO3 .THEREFORE THIS SYSTEM WAS
NAMED AS “SYSTEM OF COLOURED
BANDS”.LATER NAMED AS
CHROMATOGRAPHY WHICH TOOK
TREMENDAROUS CHANGES TODAY TO
SEPARATE ALMOST ANY SUBSTANCE IN A
COMPLEX SOLVENT.

• IN 1930-FEW VARITIES OF TLC AND ION
EXCHANGE CROMATOGRAPHY WAS
INTRODUCED.
• IN 1941 PARTITION AND PAPER
CHROMATOGRAPHY WAS DEVELOPED.
• IN 1952 GAS CHROMATOGRAPHY WAS
INTRODUCED.
• TLC IS A TECHNIQUE PURELY BASED ON
RATE OF MOUNT OF COMPONENT THROUGH
A MEDIUM AND A STATIONARY
PHASE,BINDING CAPACITY OF A MIXTURE
IS SEPERATED .

2 PHASES IN
CROMATOGAPHY.
*STATIONARY PHASE -SOLID
FORM OR A LIQUID IN SOLID
FORM.
*MOBILE PHASE- EITHER
LIQUID OR GAS TECHNIQUE
IN MOBILE PHASE PASSES
THE STATIONARY PHASE
TRANSPORTS THE SEPARATE
COMPONENT AT DIFFERENT
SPEED AT DIRECTION OF
FLOW OF MOBILE PHASE.

• PRINCIPLE:
• SEPERATION OF SINGLE COMPONENT FROM A
MIXTURE IN STABLE AND MOBILE PHASE.
• 2 PHASES NEEDED-

• 1)STATIONARY PHASE -USING SILICA GEL ALSO
CELLULOS POWDER ETC.
• -2)MOBILE PHASE-
ETHANOL,BENZINE,CARBONTETRACHLORIDE,ETC
. CAN BE USED.

• APPLICATIONS OF TLC:-
• *IT IS MUCH BENIFICIAL WITH INDIVIDUAL COMPONENTS
IN A MIXTURE.
• *FOR CHECKING PURITY OF THE SAMPLE,ALSO FOR THE
PURIFICATION PROCESS.
• *HELPFUL IN CHEMISTRY LAB TO IDENTIFIE THE
REACTION.
• *FOR IDENTIFICATION OF INDIVIDUAL COMPONENTS.
• *STANDERD PARAMETER USEFUL FOR STANDERDISATION
OF PHARMACEUTICAL PROCEDURE IN INDUSTRIES.
• *ISOLATION OF MANY ORGANIC COMPOUNDS LIKE
ALCOLOIDS,AMIDES,ACIDS ARE POSSIBLE.
• *ALSO IN BIOCHEMICAL ANALYSIS IN METABOLITES LIKE
PLASMA,SERUM ANALYSIS,URINE ANALYSIS.

• ADVANTAGES:
• *SIMPLE AND EASY TEST IN STANDERDISATION.
• *TIME IS 20-40MIN ,ITS VERY FAST.
• *SEPARATE INDIVIDUAL COMPONENTS FROM
SMALL AMOUNT OF SAMPLE.
• *ITS HIGHLY SEPERATION METHOD IN
INDIVIDUAL COMPONENTS.
• *LESS EXPENSIVE.
• *VERY EASY FOR DETECTION OF SAMPLE.
• DIS ADVANTAGES:
• NOT POSSIBLE TO SEPARATE THE COMPONENTS
IN LARGE SCALE.

Procedure for TLC
1. Prepare the developing container.
The developing container for TLC can be
a specially designed chamber, a jar with a
lid, or a beaker with a watch glass on the
top:

In the teaching labs,
we use a beaker with
a watch glass on top.

Pour solvent
into the beaker
to a depth of
just less than
0.5 cm.

To aid in the
saturation of the
TLC chamber with
solvent vapors, line
part of the inside of
the beaker with
filter paper.

Cover the beaker
with a watch glass,
swirl it gently, and
allow it to stand
while you prepare
your TLC plate.

2. Prepare the TLC plate.
TLC plates used in the
organic chem. teaching
labs are purchased as 5 cm
x 20 cm sheets. Each large
sheet is cut horizontally
into plates which are 5 cm
tall by various widths; the
more samples you plan to
run on a plate, the wider it
needs to be.

Plates will usually be cut and ready for
you when you come to lab.
Handle the plates carefully so that you do
not disturb the coating of adsorbent or get
them dirty.

Measure 0.5 cm from the
bottom of the plate. Take
care not to press so hard with
the pencil that you disturb
the adsorbent.
Using a pencil, draw a line
across the plate at the 0.5
cm mark. This is the
origin: the line on which
you will "spot" the plate.

Under the line, mark lightly the
name of the samples you will spot on
the plate, or mark numbers for time
points. Leave enough space between
the samples so that they do not run
together, about 4 samples on a 5 cm
wide plate is advised.
Use a pencil and do not press down
so hard that you disturb the surface
of the plate. A close-up of a plate
labeled "1 2 3" is shown to the right.

• 3. Spot the TLC plate
• The sample to be analyzed is added to the plate in a process
called "spotting".
• If the sample is not already in solution, dissolve about 1 mg
in a few drops of a volatile solvent such as hexanes, ethyl
acetate, or methylene chloride. As a rule of thumb, a
concentration of "1%" or "1 gram in 100 mL" usually works
well for TLC analysis. If the sample is too concentrated, it
will run as a smear or streak; if it is not concentrated
enough, you will see nothing on the plate. The "rule of
thumb" above is usually a good estimate, however,
sometimes only a process trial and error (as in, do it over)
will result in well-sized, easy to read spots.

add a few drops of
solvent . . .

. . . swirl until
dissolved

The solution is applied to the TLC plate with a 1µL microcap.

Microcaps come in plastic
vials inside red-and-white
boxes. If you are opening a
new vial, you will need to
take off the silver cap,
remove the white styrofoam
plug, and put the silver cap
back on. A small hole in the
silver cap allows you to
shake out one microcap at a
time. Microcaps are very
tiny; the arrow points to one,
and it is hard to see in the
photo.

• Take a microcap and dip it into the solution of the
sample to be spotted. Then, touch the end of the
microcap gently to the adsorbent on the origin in the
place which you have marked for the sample. Let all
of the contents of the microcap run onto the plate.
Be careful not to disturb the coating of adsorbent.
dip the microcap into solution - the arrow points to the
microcap, it is tiny and hard to see

make sure it is filled -
hold it up to the light if
necessary

touch the filled microcap
to TLC plate to spot it -
make sure you watch to
see that all the liquid has
drained from the microcap

rinse the microcap with clean
solvent by first filling it . . .

do this rinse process 3 times!

. . . and then draining it by
touching it to a paper
towel

here's the TLC plate, spotted and ready to be
developed

• 4. Develop the plate.
• Place the prepared TLC plate in the developing
beaker, cover the beaker with the watch glass, and
leave it undisturbed on your bench top. Run until the
solvent is about half a centimeter below the top of
the plate (see photos below).

place the TLC plate in the developing
container - make sure the solvent is not
too deep

The solvent will rise up the TLC
plate by capillary action. In this
photo, it is not quite halfway up the
plate.

In this photo, it is about
3/4 of the way up the
plate.

The solvent front is about half a cm
below the top of the plate - it is
now ready to be removed

Remove the plate from the beaker.

quickly mark a line across the plate
at the solvent front with a pencil

Allow the solvent to evaporate
completely from the plate. If the
spots are colored, simply mark
them with a pencil.

• 5. Visualize the spots
• If your samples are colored, mark them before they fade by
circling them lightly with a pencil.
• Most samples are not colored and need to be visualized with
a UV lamp. Hold a UV lamp over the plate and mark any
spots which you see lightly with a pencil.
• Beware! UV light is damaging both to your eyes and to your
skin! Make sure you are wearing your goggles and do not
look directly into the lamp. Protect your skin by wearing
gloves.
• If the TLC plate runs samples which are too concentrated,
the spots will be streaked and/or run together. If this
happens, you will have to start over with a more dilute
sample to spot and run on a TLC plate.

this is a UV lamp

here are two proper sized spots, viewed
under a UV lamp
(you would circle these while viewing
them)

The plate to the left shows three compounds run at
three different concentrations. The middle and right
plate show reasonable spots; the left plate is run too
concentrated and the spots are running together, making
it difficult to get a good and accurate Rf reading.

Here's what overloaded plates look like compared to well-spotted
plates. The plate on the left has a large yellow smear; this smear
contains the same two compounds which are nicely resolved on the
plate next to it. The plate to the far right is a UV visualization of
the same overloaded plate.

• DETECTION OF THE ISOLATED
COMPONENT:-
• 1)non specific method:-
• By colours-florensent phase
• Iodine chamber.
• H2so4 spray.
• u.v chambers.
• Color spray reagents can be used.

• 2)specific method:-
• For different types of components-
• i)phenolic compounds and tannins-ferric chloride
spray is advised.
• ii)for alcoloids-dragandroffs reagent.
• iii)for amino acids-ninhydrin in acetone.
• iv)for cardiac glycosides-spray with 3,5dinitro
benzoic acid.

FURTHER EVALUATION OF SEPERATED
COMPONENTS:-
• QUALITATIVE AND QUANTITATIVE-
• QUALITATIVE ANALYSIS:-
• -By visual assessment by observing
size,density,number of spots with different reagents
of components can be identified.
• -separately measure the spot in mm is directly
proportional to substance present in that spot.

• RETARDATION OR RETENTION FACTOR:‐Rf
• Measuring Rf values
• measurements are often taken from the plate in
order to help identify the compounds present.
These measurements are the distance travelled by
the solvent, and the distance travelled by individual
spots.
• When the solvent front gets close to the top of the
plate, the plate is removed from the beaker and the
position of the solvent is marked with another line
before it has a chance to evaporate.

These measurements are then taken:

The Rf value for each dye is then worked out using the formula:

For example, if the red component travelled 1.7 cm from the base
line while the solvent had travelled 5.0 cm, then the Rf value for
the red dye is:

• If you could repeat this experiment under exactly the
same conditions, then the Rf values for each dye
would always be the same. For example, the Rf
value for the red dye would always be 0.34.
However, if anything changes (the temperature, the
exact composition of the solvent, and so on), that is
no longer true. You have to bear this in mind if you
want to use this technique to identify a particular
dye

• QUANTITATIVE ANALYSIS:-
• Carried out in 2 ways-
• Direct method:-
• a) on the plate i.e., after spray of different reagents.
• b)by assessing the density of elute.
• c)measurement of spot area in mm is proportion to amount of quantity
more in the sample.
• d)densitometer:- method where intensity of color of substance is
measured in chromatogram –in situ method.
• E)densitometer-method where optical density of separated spots is
measured.
• f)spectrophotometer-instrument which gives qualitative and
quantitative analysis. by wave length of maximum absorption of
different spots and compared with standard spots.

Indirect method:-
• Components scraped is assessed further with
different test or different chromatograms etc. in
different titrations.
• Microanalysis can be performed by colorimeter
,electroporosis.etc.,

HPTLC:-HIGH PERFORMANCE THIN
LAYER CROMATOGRAPHY-
• In this a pre coated stationary phase is used and the
chemicals used are extremely small sized particles
so that adsorbent capacity is highly active.
• Instead of maneuver samples the standard samples
are available here for spotting and a new type of
development chamber which requires less amount of
solvent for development, more efficacy in separation
and shorter analysis time because of advance type of
densitometer scanner and improved data possessing
capacity by which you will know the readings in
computer.

PAPER CROMATOGRAPHY:-
• Technique in which analysis of unknown substance
with the help of the mobile phase and a stationary
phase is specially designed filter paper also known
as whattman chromatography paper, all principals of
chromatography holds good.

Adsorption Chromatography
Adsorption chromatography
is probably one of the oldest
types of chromatography
around. It utilizes a mobile
liquid or gaseous phase that
is adsorbed onto the surface
of a stationary solid phase.
The equilibriation between
the mobile and stationary
phase accounts for the
separation of different
solutes.

Partition Chromatography
This form of
chromatography is based
on a thin film formed on the
surface of a solid support by
a liquid stationary phase.
Solute equilibriates
between the mobile phase
and the stationary liquid.

Types of
Chromatography
- Radial chromatography
- Ascending
chromatography
- Descending
chromatography

Radial Chromatography
In this type of
chromatography, as the
pigment separates, the
different colours move
outwards.

Ascending
Chromatography
The solvent moves
upwards on the
separating media

Descending
Chromatography
The solvent moves
downwards on the
separating media.

Ion Exchange
Chromatography
In this type of
chromatography, the use of
a resin (the stationary solid
phase) is used to covalently
attach anions or cations
onto it. Solute ions of the
opposite charge in the
mobile liquid phase are
attracted to the resin by
electrostatic forces.

• Column Chromatography
• Procedure for Microscale Flash Column
Chromatography
• In microscale flash chromatography, the column
does not need either a pinchclamp or a stopcock at
the bottom of the column to control the flow, nor
does it need air-pressure connections at the top of
the column. Instead, the solvent flows very slowly
through the column by gravity until you apply air
pressure at the top of the column with an ordinary
Pasteur pipet bulb.

(1) Prepare the column.
The column is packed using a simple dry‐pack method.

Plug a Pasteur pipet with a
small amount of cotton; use
a wood applicator stick to
tamp it down lightly. Take
care that you do not use
either too much cotton or
pack it too tightly. You just
need enough to prevent the
adsorbent from leaking out.

Add dry silica gel
adsorbent, 230-400
mesh -- usually the jar
is labeled "for flash
chromatography." One
way to fill the column is
to invert it into the jar of
silica gel and scoop it
out . . .

. . . then tamp it down before Another way to fill the column is
scooping more out. to pour the gel into the column
using a 10 mL beaker.

Whichever method you use to When properly packed, the silica
fill the column, you must tamp it gel fills the column to just below
down on the bench top to pack the indent on the pipet. This
the silica gel. You can also use leaves a space of 4–5 cm on top
a pipet bulb to force air into the of the adsorbent for the addition
column and pack the silica gel. of solvent. Clamp the filled
column securely to a ring stand
using a small 3-pronged clamp.

• (2) Pre-elute the column.
• The procedure for the experiment that you are doing
will probably specify which solvent to use to pre-
elute the column. A non-polar solvent such as
hexanes is a common choice.

• Add hexanes (or other specified solvent) to the top
of the silica gel. The solvent flows
slowly down the column; on the
column above,it has flowed down
to the point marked by the arrow.

Monitor the solvent level,
both as it flows through the
silica gel and the level at
the top. If you are not in a
hurry (or busy doing
something else), you can
let the top level drop by
gravity, but make sure it
does not go below the top
of the silica. Again, the
arrow marks how far the
solvent has flowed down
the column.

Speed up the process by using
a pipet bulb to force the solvent
through the silica gel - this puts
the flash in microscale flash
chromatography. Place the
pipet bulb on top of the column,
squeeze the bulb, and then
remove the bulb while it is still
squeezed. You must be careful
not to allow the pipet bulb to
expand before you remove it
from the column, or you will
draw solvent and silica gel into
the bulb.

When the bottom
solvent level is at the
bottom of the column,
the pre-elution process
is completed and the
column is ready to load.

If you are not ready to load
your sample onto the
column, it is okay to leave
the column at this point.
Just make sure that it does
not go dry -- keep the top
solvent level above the top
of the silica (as shown in
the picture to the left) by
adding solvent as
necessary.

(3) Load the sample onto the
silica gel column.
Two different methods are used
to load the column: the wet
method and the dry method: wet
and dry. Below are illustrations
of both methods of loading a
crude sample of ferrocene onto
a column.
In the wet method, the sample
to be purified (or separated into
components) is dissolved in a
small amount of solvent, such
as hexanes, acetone, or other
solvent. This solution is loaded
onto the column.

• Wet loading method
• The column is being loaded by the wet method.
Follow the thumbnails below to see close‐up details
of the sample as it is allowed to sink into the
column. Once it's in the column, fresh eluting
solvent is added to the top and you are ready to
begin the elution process (see step 4).

• Sometimes the solvent of choice to load the sample
onto the column is more polar than the eluting
solvents. In this case, if you use the wet method of
column loading, it is critical that you only use a few
drops of solvent to load the sample. If you use too
much solvent, the loading solvent will interfere with
the elution and hence the purification or separation
of the mixture. In such cases, the dry method of
column loading is recommended.

Dry loading method
First dissolve the sample to be
analyzed in the minimum
amount of solvent and add
about 100 mg of silica gel. Swirl
the mixture until the solvent
evaporates and only a dry
powder remains. Place the dry
powder on a folded piece of
weighing paper and transfer it to
the top of the prepared column.
Add fresh eluting solvent to the
top ‐‐ now you are ready to
begin the elution process (see
step 4).

• (4) Elute the column.
• Force the solvent through the column by
pressing on the top of the Pasteur pipet with a
pipet bulb. Only force the solvent to the very top
of the silica: do not let the silica go dry. Add
fresh solvent as necessary.

The photo at the left shows the solvent being
forced through the column with a pipet bulb.
The series of 5 photos below show the
colored compound as it moves through the
column after successive applications of the
pipet bulb process.
The last two photos illustrate collection of the
colored sample. Note that the collection
beaker is changed as soon as the colored
compound begins to elute.
The process is complicated if the compound
is not colored. In such experiments, equal
sized fractions are collected sequentially and
carefully labeled for later analysis.

• (5) Elute the column with the second elution
solvent.
• If you are separating a mixture of one or more
compounds, at this point you would change the
eluting solvent to a more polar system, as
previously determined by TLC. Elution would
proceed as in step (4).

• (6) Analyze the fractions.
• If the fractions are colored, you can simply
combine like-colored fractions, although TLC
before combination is usually advisable. If the
fractions are not colored, they are analyzed by
TLC (usually). Once the composition of each
fraction is known, the fractions containing the
desired compound(s) are combined.

• Gas Chromatography: Procedure
• (1) Add the sample to be injected to the
syringe.
• A 25µL glass Hamilton syringe is used to inject
the GC samples. Only 2-4 µL of sample is
injected onto the column, which means that you
fill only a small part of the barrel with sample.
Examine the syringe carefully before you fill it.
The divisions are marked "5 - 10 - 15 - 20 - 25".

This is a 25 µL glass Hamilton syringe.
You only inject 2.5 µL, so it will NOT be
filled to the top.

Place the tip of the needle in the liquid. Slowly draw up a small amount
of liquid by raising the plunger, then press on the plunger to expel the
liquid back into the liquid. This serves to “rinse” the syringe with your
sample, ensuring that what you will measure in the GC run is the
composition of your mixture. Repeat the rinse process one or two times.
Then, draw up the plunger slowly again while the needle is in the liquid
and carefully fill the syringe with liquid about halfway to the “5”.

It is often hard to see the liquid in the
syringe. If the syringe is clogged, the
plunger will be in the correct position but the
barrel of the syringe will be filled with only
air, as in the bottom syringe in the photo to
the left.
The best thing to do is to carefully examing
the syringe after you think that you have
filled it. Hold it up to the light to get a better
view.
Small air bubbles in the syringe will not
affect the GC run (middle syringe in the
photo to the left). As long as there is enough
liquid in the syringe, the GC run will work
fine. If you keep getting bubbles, just pull
the plunger up a bit past the "halfway to the
5" mark to compensate.
If you have a VERY large air bubble, you
will not have enough liquid to show a
reading on the GC (e.g., the bottom syringe
in the photo).

• (2) Inject the sample into the injector port.
• You are need to do two things sequentially and quickly, so
make sure you know where the injection port is and where
the start button on the recorder is.
• Push the needle of the syringe through the injection port
and immediately press the plunger to inject the sample,
then immediately press the start button on the recorder.
• You will feel a bit of resistance from the rubber septum in
the injection port; this is to be expected and you should be
prepared to apply some pressure to the syringe as you
force the needle into the instrument all the way to the
base of the needle.

Push the needle of the filled
syringe through the injector (as
far as it will go) and quickly
push the plunger.

Remove the syringe immediately . . .

. . . and quickly press the
start button on the
integrating recorder or the
start recording button on
the computer

Here's a close-up of the
integrating recorder.

• MECHANISM:-
• Gas is used as a mobile phase and solid or liquid
coated on a solid support is used as a stationary
phase. The test mixture is converted to vapor and to
this vapor the mobile phase is passed, hence the
components through stationary phase with help of
mobile phase and separated components which is
less soluble in stationary phase travels faster and
which is greater soluble capacity in stationary phase.
Here with help of some gas , if you heat the test
sample it travels and one with higher affinity will react
here and one with less affinity will move far, we have
detectors and amplifiers and lastly the recorder in
different forms.

• Important criteria in GC-
• 1) Mobile phase as gas.
• 2) Test mixture heated and evaporated which
should be inert to mobile phase.
• 3) To vaporize mixture if they posses volatile oil
or volatile mixture are separated in technique.
• 4) Mobile should not mix with vapor in
stationary phase.
• 5) Mixture sample should not change by heat
i.e. thermostable, as we heat the mixture.
•

• Here mobile gas as
hydrogen,helium,nitrogen,and argon are used,
among these helium is expensive.
• Result is in form of curves, individual peaks is
of individual components and retention time is
the time between injection of sample to the time
to get maximum peak of components, retention
factor of standard is time of maximum peak.i.e,
rf.
• Compare the test sample curve with standard
curve asses it with considering the retention
time even.

• (3) Sit back and wait.
• Observe the recorder. Within several minutes, it should record
several peaks.
• (4) End the GC run.
• When you have seen all of the peaks which you suspect are in
the mixture, or when the recorder has shown a flat baseline for
a few minutes or so, press stop on the recorder.
• When you press stop, the recorder will print out the peaks, the
retention times, and the areas under the peaks. When it is done
printing, you can press “enter” a couple times to advance the
paper.
• Carefully tear the paper off the recorder. The paper is not
perforated, so do not try to pull up and expect it to pop out of
the recorder. Instead, pull it down to start a tear from one edge,
and then continue the tear until the paper is cut and free.

Uses:
• mainly for detecting steroids,food components
,identification of various substances.

• When a beam of light pass through a object , this
object absorbs some amount of light and transmits the
light to some extent.
• I0 = I0 + IR + IT.
• Incidence of light = incidence of absorbance +
incidence of refractence + incidence of transmitted.
• Thickness of the object and concentration is much
important for change in transmitted light.i.e., in various
frequency depends on thickness.

• LAMBERTS LAW:-
• When a beam of light is allowed to pass
through a transparent media , the rate of
decrease of intensity with the thickness of
medium is directly proportional to the intensity
of light.
• If intensity is more. The frequency varies or
• If thickness is more. Changes.

Beers law:-
Intensity of beam of light decreases with
the increase in the concentration of
absorbing substance.

1 cm

X y

2 cm

x
Y1 y

• Similarly a concentration of solution with the
light ray through them , the greater or lesser
amount of concentration energy.

»
»

• Standard incidence energy = Is
• Absorbance                            = As
• Transmittance                         = Ts
• Suppose
Is = 230
As = 4 of given standard.
Ts = 6 of a given drug.
Then
4     ‐ 230
6     ‐ ?
Calculate   6  X  230
4 =  345

•
• Colorimeter Definition
• A colorimeter is a device used in the practice of
colorimetery, or the science of color.
Colorimetry has also been termed the
quantitative study of color perception. A number
of instruments are available to determine the
concentration of a solution. The simplest of
those instruments is a colorimeter.

• Function
– A colorimeter is a device used to measure the
absorption of a specific wavelength of light by a
solution, which can in turn be used to determine the
concentration of a solute in a solution.
• Components
– The critical parts of a colorimeter are a light source
(commonly a low-voltage filament lamp), adjustable
aperture, colored filters, solution cuvette, a
transmitted light detector (such as a photoresistor)
and an output display meter. Some colorimeters
might also contain a regulator to prevent damage
related to the machine from voltage fluctuations as
well as a second light path to allow comparison
between two solutions.

• Filters
– A filter is used to select and measure the wavelength of light
that the solution absorbs the most. The measurements are
done in nanometers (nm). Typically, the wavelength used is
between 400nm and 700nm.
• Results
– Following the reading, the colorimeter will provide data in
either an analog or digital formation, depending upon the
machine. The data can be shown as transmittance on a
linear percentage scale between 0 percent and 100 percent.
It can also be shown as absorbance on a logarithmic scale
between zero and infinity. Typically, the range of absorbance
is from 0 to 2 with the ideal range being between 0 to 1.

• History
– The colorimeter was invented by Jan Szczepanik and
applies the Beer‐Lambert law when determining the
concentration. The Beer‐Lambert law states that
absorbance is proportional to the concentration of a
solute.
• Different Parts of a Colorimeter
• A colorimeter is a tool to measure the ability of a
solution to absorb a specified wavelength of light.
This absorption is used to determine the
concentration of a substance in the solution.

• Components
– The basic colorimeter is made up of a low‐voltage light
source that shines through the solution held in a
cuvette. The cuvette fits in a small receptacle and, as the
light shines through it, a detector measures the light
that is passed through. The detector's readings are
shown on an LCD display.
• Options
– Colored filters are inserted in front of the cuvette
depending on which wavelength of light you need. This
is determined by knowing which wavelength of light the
solute you are measuring absorbs the best. Field and lab
manuals will have lists of these wavelengths for
reference.

• Function
– Once you select and insert the filter for the wavelength
you need, you must analyze a blank and two to three
standards (minimum) using the colorimeter. The blank is
typically pure water. Standards are solutions of various
concentrations of the solute you are measuring in your
sample. The readings from the blank and the standards
are used to set up a chart from which you determine
your sample concentration after you analyze it.

• Colorimeter Type
• Colorimeters measure the perception of color.
• Colorimetry is a technique to describe and quantify
the human perception of color by focusing on the
physical aspects of color. A colorimeter measures
the amount of color from a given medium. Various
different applications of colorimeters exist today to
quantify color, ranging from laboratories to the
electronic industry.

• Tristimulus Colorimeter
• Tristimulus colorimeters are often used in the
application of digital imaging. The tristimulus
colorimeter measures color from light sources such
as lamps, monitors and screens. By taking multiple
wideband spectral energy readings along the visible
spectrum, this colorimeter can profile and calibrate
specific output devices. The measured quantities
can approximate tristimulus values, which are the
three primary colors needed to match a test color

• Densitometer
– A densitometer measures the density of light passing
through a given frame. Density can be characterized as
the level of darkness in film or print. When an image is
printed, the ink pigments block light naturally when
deposited by the printing process. Graphics industry
professionals use densitometers to help control color in
the various steps of the printing process.

• Spectroradiometer
– Spectroradiometers quantify the spectral power
distribution emitted from a given light source. In other
words, the spectroradiometer measures the intensity of
color. Characteristically similar to spectrophotometers,
spectroradiometers are used to evaluate lighting for
sales within manufacturing and for quality control
purposes. Other applications include confirming a
customer's light source specifications and calibrating
liquid crystal displays for televisions and laptops.

• Spectrophotometer
– A spectrophotometer is an analytical tool that measures
the reflection and transmittance properties of a color
sample. Using functions of light wavelengths, the
spectrophotometer passes a beam of light through the
sample to record both absorbance and transmittance.
The instrument does not require human interpretation
and is much more complex than a standard colorimeter.
Common applications for the spectrophotometer
include color formulation and industry research and
development.

• What Is Absorbance When Dealing With a Colorimeter?
• Absorption is measured by color intensity.
• Absorbency is crucial in determining concentration of a
substance in a sample through colorimeter analysis.
Colorimeters measure the intensity of color and light
transmittance by the sample to achieve the concentration.
• Function
– A colorimeter works by shining a white light through an
optical filter into a cuvette that holds the sample. By
adding a color reagent to the sample, the substance will
darken in color depending on the amount of
concentration. The darkening (intensity) of the color is
measured by how much light is absorbed by the sample.
The higher the intensity, the higher the concentration.

• Significance
– Absorbance can be defined as a logarithmic measurement of the
amount of light at a particular wavelength taken in by sample.
This measurement is known as the Beer‐Lambert Law or Beer's
Law. The law states that absorbance is equal to the concentration
multiplied by the path length of light and the molar extinction
coefficient (how strongly the solution absorbs color).
• Color Importance
– The color of the sample depends on the transmittance of light,
not absorption. For example, a sample is seen as red because it
absorbs blue and purple wavelengths. Therefore in that example,
the wavelength of light used to determine absorbance will be in
the blue region of the visible color spectrum.

• How to Use Colorimeters
• A colorimeter is any device that measures the color of a
particular substance. It's a generic term that may refer to a
range of devices such as a spectrophotometer, which is a
specific type of colorimeter typically used to measure a
solution's absorbance of a particular wavelength of light. This
allows the concentration of a known solute in the solution to be
calculated with considerable accuracy. This type of colorimeter
is a standard piece of equipment in analytical chemistry.
• Instructions
Things You'll Need
• Colorimeter
• Reference solution
• Test solution

–1
• Determine the wavelength of light that the solution absorbs
most strongly. A colorimeter has a set of changeable filters that
can show which color of light to examine for the greatest
accuracy. Set the colorimeter to this wavelength.
–2
• Calibrate the colorimeter. Turn the unit on and wait 30 minutes
for the colorimeter to warm up before taking any
measurements. Remove the container (cuvette) from its
chamber.
–3
• Read the colorimeter. The meter is extremely sensitive, and
you must use the correct measurement. Most models have a
reflective surface under the needle and you should look at the
needle so that you can't see its reflection in the mirror.

–4
• Fill the reference cuvette with the test solution as
indicated by the colorimeter model you're using. Clean
the reference cuvette carefully to ensure it doesn't
have any smudges and place it in the chamber. Read
the absorbance and adjust the colorimeter to show an
absorbance reading of 0.
–5
• Put the test solution in the specimen cuvette and clean
the outside of the cuvette. Place it in the chamber and
read the absorbance of the test solution. You will
typically record this value and use it in calculations to
determine the solution's concentration.

• Colorimeter Analysis
• Colorimeters plot concentration and absorbance on a
line graph.
• Using a colorimeter is one of the fastest and easiest
ways to measure unknown concentrations of a sample
substance. Measuring absorbency is crucial for
colorimetry analysis since it is in a linear relationship
with concentration.
• Function
– Substances in a sample absorb light, and color
pigments also absorb light at different wavelengths.
Colorimeters use color and light at different
wavelengths to measure the intensity of color and
how much light that color absorbs in a sample.

• Beer-Lambert Law
– To obtain the concentration of a substance in a sample,
colorimeters use the Beer-Lambert law to gain results. The
mathematical formula states that concentration is equal to
the absorbance divided by the path length of light and the
molar extinction coefficient (how strong the solution absorbs
color).
• Considerations
– To get the curve for the Beer-Lambert law, standards of
known concentration are measured for their absorbance of
color and light and then plotted on a graph. This graph uses
the y = mx + b formula to achieve a straight line with
concentration on the x-axis and absorbance on the y-axis.
Unknown samples will then be measured using this curve.
For example, if y = .301, then the absorbance (determined
by the colorimeter) multiplied by .301 will equal the
concentration.

• Application of Colorimeter
• Essentially, a colorimeter is a scientific instrument that
measures the amount of light passing through a solution
relative to the amount that passes through a sample of
pure solvent. Colorimeters have many applications in the
fields of biology and chemistry.
• Beer‐Lambert Law
• The Beer‐Lambert Law states that the concentration of a
dissolved substance, or solute, is proportional to the
amount of light that it absorbs. A common application of a
colorimeter is therefore to determine the concentration of
a known solute in a given solution

• Biological Culture
– In biology, a colorimeter can be used to monitor the
growth of a bacterial or yeast culture. As the culture
grows, the medium in which it is growing becomes
increasingly cloudy and absorbs more light.
• Bird Plumage Coloration
– A colorimeter can also be used to eliminate subjectivity,
or personal opinion, from the assessment of color in bird
plumage. Modern colorimeters provide highly accurate
results, under standard, repeatable conditions and have
proved to be very reliable.
•

• What is the Difference Between a Colorimeter and a
Spectrophotometer?
• Composed of many different wavelengths and colors,
perceived light can be deconstructed using colorimeters
and spectrophotometers in different ways.
• Since the advent of the XYZ color system established by the
Commission Internationale de l'Eclairage (CIE) in 1931,
many devices have been invented to measure and
interpret light ‐‐‐ a science known as spectroscopy. Two of
these machines ‐‐‐ colorimeters and reflectance
spectrophotometers ‐‐‐ have made homes in laboratories
and design studios, but their similar quantitative outputs
can be confusing despite their very different functionality
and design.

• Color Measurement
– In 1931 the Commission Internationale de l'Eclairage
attempted to quantify the light that humans perceive by
matching the three primary colors that make up all colors
with three values, called the tristimulus values --- x, y and z -
-- which approximately correspond to red, blue and green.
Any visible color can be quantified using these three values,
and this allowed for objective measuring and comparing of
colors.
• Colorimeters
– Relying on exactly that system laid out by the CIE,
colorimeters measure the color of a sample compared to a
white control surface and output data for x, y and z values.
Used to color-match or color-mix, these relatively simple
devices can often be found in the textile, paint and design
industries. Colorimeters are generally inexpensive and
rugged devices.

• Colorimeters in the Lab
– Known concentrations of a sample can be tested with a
colorimeter and plotted on a graph to create a
calibration curve. Using this data, a sample with an
unknown concentration can be tested and compared
against the graph to ascertain its concentration.

• Reflectance Spectrophotometers
– Similar in the respect that reflectance spectrophotometers can
also be used to determine a sample's color, these more complex
devices provide a greater amount of data, and are otherwise
completely different machines. Rather than just one broad
wavelength analysis, spectrophotometers analyze the intensities
of 16 or more narrow wavelengths of the sample light by
diffracting the light into its component wavelengths. Unlike
colorimeters, spectrophotometers typically include adjustments
for many variables such as observer angle and illuminant, and are
usually connected directly to a computer for data analysis. Some
spectrophotometers include a gloss trap to either include or
exclude the specular components of reflected light from either
xenon or tungsten lamps.

• Spectrophotometers in the Lab
– Reflectance spectrophotometers have a variety of
applications in many laboratory disciplines. Similar to
colorimeters, spectrophotometers can more accurately
determine the concentration of solute in a solution
against a graphed curve. But these more expensive
devices can also be used to, for example, measure the
rate of bacterial growth in a sample or calculate the
color‐changing effect of a chemical reaction in real time.

• Difference Between Colorimeter &
Spectrophotometer
• Colorimeters and spectrophotometers are used
in the medical, research, and scientific
industries to provide rapid results of unknown
samples through the use of color. Although both
the colorimeter and the spectrophotometer use
color to analyze samples, they operate
differently.
• Light
– Colorimeters use a colored light beam to measure
sample concentration. Spectrophotometers use a
white light that is passed through a slit and filter to
analyze samples.

– Wavelengths
– In colorimetry, colored light passes through an optical
filter to produce a single band of wavelengths. With
spectrophotometers, the white light is passed through a
special filter that disperses the light into many bands of
wavelengths.
• Measurement
– Both of these techniques use the Beer‐Lambert Law to
determine concentration. The difference is that
colorimeters measure the absorbency of light in a
sample while spectrophotometers measure the amount
of light that passes through it.

• Function
– The colorimeter uses psychophysical analysis,
comparing color in the same manner as human
eye-brain perception. The spectrophotometer uses
only physical analysis, using different wavelengths
of light to determine the reflection and transmission
properties of color.

The spectrophotometer is
an instrument which
measures the amount of
light of a specificed
wavelength which passes
through a medium.
According to Beer's law, the
amount of light absorbed by
a medium is proportional to
the concentration of the
absorbing material or solute
present.

Thus the concentration of a
colored solute in a solution may
be determined in the lab by
measuring the absorbency of
light at a given
wavelength. Wavelength (often
abbreviated as lambda) is
measured in nm. The
spectrophotometer allows
selection of a wavelength pass
through the solution. Usually,
the wavelength chosen which
corresponds to the absorption
maximum of the solute.
Absorbency is indicated with a
capital A.

To familiarize yourself
with the
spectrophotometer,
illustrate and label the
following features which
are important to its
proper use. You should
know the function
and/or significance of
each of these features
before you use the
instrument.

At the
spectrophotometer, you
should have two
cuvettes in a plastic
rack. Solutions which
are to be read are
poured into cuvettes
which are inserted into
the machine. One
should be marked "B"for
the blank and one "S"
for your sample.

• . A wipette should be available to polish them
before insertion into the cuvette
chamber. Cuvettes are carefully
manufactured for their optical uniformity and are
quite expensive. They should be handled with
care so that they do not get scratched, and
stored separate from standard test tubes.

• Try not to touch them except at the top of the
tube to prevent finger smudges which alter the
reading. For experiments in which minor
inprecision is acceptible, clean, unscratched 13
x 100 mm test tubes may be used.

WARM-UP:
1. Plug in and turn on (left hand front
dial, labeled ZERO in the illustration).
Allow about 30 minutes for warm up.

ZERO ADJUST:
2. With no cuvette in the
chamber, a shutter cuts off
all light from passing though
the cuvette chamber. Under
this condition therefore, the
machine may be adjusted to
read infinite absorbance
(zero% transmittance) by
rotating zero adjust knob
(front left on Spectronic 20).

Do not touch this knob
again during the rest of
the following procedure.

SELECT WAVELENGTH:
3. Select the desired
wavelength of light at which
absorbance will be
determined by rotating
wavelength selection
knob (top right knob) until
the desired wavelength in
nanometers appears in the
window. A nanometer (nm),
formerly millimicron, equals
10-9 meter.

BLANK ADJUST:
4. Fill the B (blank) cuvette with the solvent
used to dissolve specimen (often distilled
water). Polish to clean, insert into the cuvette
chamber, aligning mark to front. Close chamber
cover.

5. Rotate blank adjust
knob (front right knob) to
adjust absorbance to read
zero .
6. Remove blank
cuvette, place in plastic
test tube rack.

READ SPECIMEN:
7. Pour the sample into the S (specimen)
cuvette, polish and insert into the chamber,
aligning mark to the front.

8. Note that the scale for absorbance is
the lower scale on the dial, and should
be read from R to L .

For all readings of the dial, line up the
reflection of the needle in the mirror behind
the dial with the needle itself. Otherwise,
parallax error will occur, giving an erroneous
reading. The illustration shows the correctly
aligned dial with a reading of 0.116.

PARALLAX ERROR:
Here, the picture was taken of the identical
solution as in the previous image, but with the
point of view too far to the right. Note that
the needle reflection is to the right of the
needle. The apparent reading is 0.120.

PARALLAX ERROR:
Here, the picture was taken of the identical
solution as in the previous image but with
the point of view too far to the left. Note
that the needle reflection is to the left of the
needle. The apparent reading is 0.113.

9. If you read additional specimens, you
should confirm that the machine is still
zeroed and blanked out, as in steps 2, 4 and 5
for all readings.

• CLEAN UP:
10. Remove cuvette from machine, carefully
wash and store spectrophotometer cuvettes
keeping them separate from regular test tubes.
• Return spectophotometer to its storage
location.

INFRARED
SPECTROSCOPY

INFRARED
SPECTROSCOPY

• Infrared spectroscopy exploits the fact that
molecules absorb specific frequencies that are
characteristic of their structure. These
absorptions are resonant frequencies, i.e. the
frequency of the absorbed radiation matches
the frequency of the bond or group that
vibrates. The energies are determined by the
shape of the molecular potential energy
surfaces, the masses of the atoms, and the
associated vibronic coupling.

• 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.

Symmetrical Antisymmetrical Scissoring
stretching stretching

Rocking Wagging Twisting

• Uses and applications
• Infrared spectroscopy is a simple and reliable
technique widely used in both organic and inorganic
chemistry, in research and industry. It is used in
quality control, dynamic measurement, and monitoring
applications such as the long-term unattended
measurement of CO2 concentrations in greenhouses
and growth chambers by infrared gas analyzers.
• It is also used in forensic analysis in both criminal and
civil cases, for example in identifying polymer
degradation. It can be used in detecting how much
alcohol is in the blood of a suspected drink driver
measured as 1/10,000 g/mL = 100 μg/mL.

• A useful way of analysing solid samples without the
need for cutting samples uses ATR or attenuated total
reflectance spectroscopy. Using this approach,
samples are pressed against the face of a single
crystal. The infrared radiation passes through the
crystal and only interacts with the sample at the
interface between the two materials.
• 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).

• Infrared spectroscopy has also been
successfully utilized in the field of
semiconductor microelectronics,for example,
infrared spectroscopy can be applied to
semiconductors like silicon, gallium arsenide,
gallium nitride, zinc selenide, amorphous
silicon, silicon nitride, etc.
• The instruments are now small, and can be
transported, even for use in field trials.
• Infrared spectroscopy is also useful in
measuring the degree of polymerization in
polymer manufacture.

ULTRAVIOLET–VISIBLE SPECTROSCOPY

•Violet: 400 - 420 nm
•Indigo: 420 - 440 nm
•Blue: 440 - 490 nm
•Green: 490 - 570 nm
•Yellow: 570 - 585 nm
•Orange: 585 - 620 nm
Red: 620 - 780 nm

• A diagram of the components of a typical
spectrometer are shown in the following diagram.
The functioning of this instrument is relatively
straightforward. A beam of light from a visible
and/or UV light source (colored red) is separated
into its component wavelengths by a prism or
diffraction grating. Each monochromatic (single
wavelength) beam in turn is split into two equal
intensity beams by a half‐mirrored device. One
beam, the sample beam (colored magenta), passes
through a small transparent container (cuvette)
containing a solution of the compound being
studied in a transparent solvent.

• The other beam, the reference (colored blue),
passes through an identical cuvette containing only
the solvent. The intensities of these light beams are
then measured by electronic detectors and
compared. The intensity of the reference beam,
which should have suffered little or no light
absorption, is defined as I0. The intensity of the
sample beam is defined as I. Over a short period of
time, the spectrometer automatically scans all the
component wavelengths in the manner described.
The ultraviolet (UV) region scanned is normally
from 200 to 400 nm, and the visible portion is from
400 to 800 nm.

• Applications
• UV/Vis spectroscopy is routinely used in analytical chemistry for
the quantitative determination of different analytes, such as
transition metal ions, highly conjugated organic compounds,
and biological macromolecules. Determination is usually carried
out in solutions.
• Solutions of transition metal ions can be colored (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 sulfate is a very light blue;
adding ammonia intensifies the colour and changes the
wavelength of maximum absorption (λmax).

• 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.) Solvent polarity and pH
can affect the absorption spectrum of an organic
compound. Tyrosine, for example, increases in
absorption maxima and molar extinction coefficient
when pH increases from 6 to 13 or when solvent
polarity decreases.

• While charge transfer complexes also give rise to
colours, the colours are often too intense to be
used for quantitative measurement.

• named after C. V. Raman is a spectroscopic
technique used 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
molecular vibrations, 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 vibrational 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.

• Applications
• Raman spectroscopy is commonly used in
chemistry, since vibrational information is
specific to the chemical bonds and symmetry of
molecules. Therefore, it provides a fingerprint
by which the molecule can be identified.
• Another way that the technique is used to study
changes in chemical bonding
• 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
sample
• Raman spectroscopy is being investigated as a
means to detect explosives for airport security

• Nuclear magnetic resonance (NMR) is a physical
phenomenon in which magnetic nuclei in a
magnetic field absorb and re‐emit electromagnetic
radiation. This energy is at a specific resonance
frequency which depends on the strength of the
magnetic field and the magnetic properties of the
isotope of the atoms. NMR allows the observation
of specific quantum mechanical magnetic
properties of the atomic nucleus

• Many scientific techniques exploit NMR
phenomena to study molecular physics,
crystals, and non-crystalline materials through
NMR spectroscopy. NMR is also routinely used
in advanced medical imaging techniques, such
as in magnetic resonance imaging (MRI).
• All isotopes that contain an odd number of
protons and/or of neutrons (see Isotope) have
an intrinsic magnetic moment and angular
momentum, in other words a nonzero spin,
while all nuclides with even numbers of both
have a total spin of zero. The most commonly
studied nuclei are 1 H

• A key feature of NMR is that the resonance frequency
of a particular substance is directly proportional to the
strength of the applied magnetic field. It is this feature
that is exploited in imaging techniques
The principle of NMR usually involves two
sequential steps:
• The alignment (polarization) of the magnetic nuclear
spins in an applied, constant magnetic field H0.
• The perturbation of this alignment of the nuclear spins
by employing an electro-magnetic, usually radio
frequency (RF) pulse. The required perturbing
frequency is dependent upon the static magnetic field
(H0) and the nuclei of observation.

• History
• Nuclear magnetic resonance was first
described and measured in molecular beams
by Isidor Rabi in 1938,[1] and in 1944, Rabi was
awarded the Nobel Prize in physics for this
work.[2] In 1946, Felix Bloch and Edward Mills
Purcell expanded the technique for use on
liquids and solids, for which they shared the
Nobel Prize in Physics in 1952

• Theory of nuclear magnetic resonance
• Nuclear spin and magnets
• All nucleons, that is neutrons and protons, composing
any atomic nucleus, have the intrinsic quantum
property of spin. The overall spin of the nucleus is
determined by the spin quantum number S.
• . It is this magnetic moment that allows the
observation of NMR absorption spectra caused by
transitions between nuclear spin levels. Most nuclides
(with some rare exceptions) that have both even
numbers of protons and even numbers of neutrons,
also have zero nuclear magnetic moments, and they
also have zero magnetic dipole and quadrupole
moments. Hence, such nuclides do not exhibit any
NMR absorption spectra.

• NMR spectroscopy is one of the principal
techniques used to obtain physical, chemical,
electronic and structural information about
molecules due to either the chemical shift,
Zeeman effect, or the Knight shift effect, or a
combination of both, on the resonant
frequencies of the nuclei present in the sample.
It is a powerful technique that can provide
detailed information on the topology, dynamics
and three-dimensional structure of molecules in
solution and the solid state. Additional structural
and chemical information may be obtained by
performing double-quantum NMR experiments
for quadrupolar nuclei

• Applications
• Medical MRI
• The application of nuclear magnetic resonance best
known to the general public is magnetic resonance
imaging for medical diagnosis and magnetic
resonance microscopy in research settings, however,
it is also widely used in chemical studies, notably in
NMR spectroscopy such as proton NMR, carbon-13
NMR, deuterium NMR and phosphorus-31 NMR.
Biochemical information can also be obtained from
living tissue (e.g. human brain tumors) with the
technique known as in vivo magnetic resonance
spectroscopy or chemical shift NMR Microscopy.

• Chemistry
• By studying the peaks of nuclear magnetic
resonance spectra, chemists can determine the
structure of many compounds. It can be a very
selective technique, distinguishing among many
atoms within a molecule or collection of molecules
of the same type but which differ only in terms of
their local chemical environment

Electrophoresis is a molecular
separation technique that involves
the use of high voltage electric
current for inducing the movement
of charged molecules---proteins,
DNA, nucleic acids---in a support
medium.
The movement of charged
molecules is called mobility. The
mobility of molecules is towards
the opposite charge, for instance,
a protein molecule with a negative
charge moves toward the positive
pole of the support medium. The
medium may be a paper, a gel or a
capillary tube.

• HOW TO DEFINE ELECTROPHORESIS
• Electrophoresis is any process that uses
electricity to separate particles in a fluid. It's a
common method of identifying molecules
according to some criteria such as molecular
weight. Electrophoresis may also be used to
prepare a sample for some other scientific
technique. There are many different types of
electrophoresis and this term doesn't refer to a
specific process.

• ELECTRICAL CHARGE
• Electrical current is placed at one end of the gel, with
positive electrodes at the top of the gel -- closest to
the pits with the DNA inside -- and negative electrodes
at the bottom. Due to the fact DNA is negatively
charged, it will move through the gel toward the
bottom and the positive electrodes. The gel will offer
resistance for the DNA, so it will take larger strands a
longer time to pass through the gel than shorter
strands. The process is stopped at a predetermined
time, and wherever the DNA is in the gel, that will give
an indication of how large each of the strands were.
This final image, after the electrophoresis, is the DNA
"fingerprint."
•

• Capillary electrophoresis is an analytical technique
that separates ions based on their electrophoretic
mobility with the use of an applied voltage. The
electrophoretic mobility is dependent upon the charge
of the molecule, the viscosity, and the atom's
radius. The rate at which the particle moves is directly
proportional to the applied electric field--the greater
the field strength, the fast the mobility. Neutral
species are not affected, only ions move with the
electric field.

• If two ions are the same size, the one with
greater charge will move the fastest. For ions
of the same charge, the smaller particle has
less friction and overall faster migration
rate. Capillary electrophoresis is used most
predominately because it gives faster results
and provides high resolution separation. It is a
useful technique because there is a large range
of detection methods
Available.

• MICROSCOPIC ELECTROPHORESIS
• A technique in which the electrophoresis of
individual particles is observed
with the aid of a microscope or ultra-
microscope.

• The moving boundary method was the first to
be used by Tiselius to demonstrate the efficacy
of the electrophortic process. The apparatus
consisted of a U tube the horizontal lower
portion of the U tube being filled with a mixture
of the substances under examination dispersed
in a suitable buffer. The two vertical limbs were
filled solely with buffer and the cathode and
anode dipped into the buffer at the top of each
limb respectively.

ELECTROPHORESIS IN FREE SOLUTION
MOVING BOUNDARY ELECTROPHORESIS:
Arne Tiselius (1937) developed the
moving boundary technique for the electrophoretic
separation of substances,
for which, besides his work on adsorption analysis, he
received the Nobel prize in 1948. The sample, a mixture
of proteins
for example, is applied in a U-shaped cell filled with a
buffer solution
and at the end of which electrodes are immersed. Under
the influence

of the applied voltage, the compounds will
migrate at different velocities towards the anode
or the cathode depending on their charges. The
changes in the refractive index at the boundary
during migration can be detected at both ends
on the solution using Schlieren optics.

• ZONE ELECTROPORESIS:-
– a sophisticated instrument, which has a chamber ,
two separate containers for buffers, a stage and
electric supply.
watt man paper is taken onto the stage and a
small quantity of the test solution is placed over it
and switch on the instrument wherein due to the
electric supply and the capillary action of the
substance, the migration of the test solution
particles occur and separate bands are formed
where in the electrically charged particles can be
separated.

• TYPES:‐
2 types‐ a) vertical
b) horizontal.

Make up the gel which the DNA will be
put into

Buffer solution added to the tank

DNA samples loaded into wells

Electrical current applied to the
chamber

DNA is stained using ethidium bromide

• POINTS TO CONSIDER:‐
• Selection of buffer
• Temperature
• Electro osmosis
• U.v florescent chamber

• FLORIMETRIC ANALYSIS:-
• a large number of substances
absorb,transmits,reflects light-during this
process there is a production of heat of varied
quantum( less or more ) which form a new
wavelength or radiation called as
ELECTROMAGNETIC RADIATION .new light
is different from absorbed light which emit
longer electromagnetic radiation known as
LUMINESCENCE.
• IT IS OF TWO TYPES
-FLUORASCENCE
-PHOSPHOROSCENCE

• FLUORESCENCE:‐
• Means when a beam of light is incident on certain
substance they emit visible light or visible radiation
and this phenomena is called as fluorescence
substance ,this phenomena is instantaneous and it
starts immediately after absorption of light and
stops as soon as the incidence light cuts off

• PHOSPHOROSCENCE
• Means they emit light continuously even after
the incident light is cut off.
• in florescence the light emit 10-6 to 10-4 seconds
of absorption whereas in phosphorescence the
radiation of light re emit in 10-4 to 20 seconds or
longer than that , therefore it takes much time to
re emit radiation even after the incident light is
cut off.
• The florescent substance is proportional to the
concentration of the incident of light , the devise
to analyze such substance of intensity of
florescence is florimetry.

• Fluorimetry is the instrument to measure the
fluorescence of substance nature , routinely used in
lab to identifie the drugs of different fluorescence
substance , hormones , certain protines ,
components of haemostasis etc.
• Types:‐
• A) fluorimeter ( filters are used.)
• B) spectrofluorimeter.( prisms and gratings are
used.)

SCHEMATIC DIAGRAM OF FLUORIMETER

• APPLICATIONS:‐
• Useful in clinical laboratory to identify specific
proteins and also to determine uranium in salts ,
henceforth used in nuclear research .
• It is one of the sensitive analysis of many elements
like aluminum in alloys , boron in steel.vit B1 and
B2,analysis of food products, qualitative and
quantitative analysis of aromatic substance.

Instrumental analysis in research

Instrumental analysis in research

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