HPLC

High-performance liquid chromatography (high-pressure liquid

chromatography)

HPLC is a technique in analytical chemistry used to

separate, identify, and quantify each component in a mixture. It relies on
pumps to pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material.

Each component in the sample interacts slightly differently with the

adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as they flow
out of the column.

Chromatography can be described as a mass transfer process

involving adsorption. HPLC relies on pumps to pass a pressurized liquid
and a sample mixture through a column filled with adsorbent, leading to the
separation of the sample components.

The active component of the column, the adsorbent, is typically a granular

material made of solid particles ,silica, 2–50 μm in size. The components of
the sample mixture are separated from each other due to their different
degrees of interaction with the adsorbent particles. The pressurized liquid is
typically a mixture of solvents (e.g., water, acetonitrile and/or methanol)
and is referred to as a "mobile phase".

Its composition and temperature play a major role in the separation process

by influencing the interactions taking place between sample components
and adsorbent.

These interactions are physical in nature, such as hydrophobic

(dispersive), dipole–dipole and ionic, most often a combination..

The schematic of a HPLC instrument typically includes a Degasser,

Sampler,

Pumps,

Detector.
The sampler brings the sample mixture into the mobile phase stream
which carries it into the column.

The pumps deliver the desired flow and composition of the mobile phase
through the column.

The detector generates a signal proportional to the amount of sample

component emerging from the column, hence allowing
for quantitative analysis of the sample components.

A digital microprocessor and user software control the HPLC instrument

and provide data analysis. Some models of mechanical pumps in a HPLC
instrument can mix multiple solvents together in ratios changing in time,
generating a composition gradient in the mobile phase. Various detectors
are in common use, such as UV/Vis, photodiode array (PDA) or based
on mass spectrometry. Most HPLC instruments also have a column oven
that allows for adjusting the temperature at which the separation is
performed.

Operation

The sample mixture to be separated and analyzed is introduced, in a

discrete small volume (typically microliters), into the stream of mobile
phase percolating through the column.

The components of the sample move through the column at different

velocities, which are a function of specific physical interactions with the
adsorbent (also called stationary phase).

The velocity of each component depends on its chemical nature, on the

nature of the stationary phase (column) and on the composition of the
mobile phase.

The time at which a specific analyte elutes (emerges from the column) is
called its retention time. The retention time measured under particular
conditions is an identifying characteristic of a given analyte.
Common mobile phases used include any miscible combination
of water with various organic solvents (the most common
are acetonitrile and methanol). Some HPLC techniques use water-free
mobile phases (see normal-phase chromatography below).

The chosen composition of the mobile phase (also called eluent) depends
on the intensity of interactions between various sample components
("analytes") and stationary phase (e.g., hydrophobic interactions in
reversed-phase HPLC).

Depending on their affinity for the stationary and mobile phasesanalytes

partition between the two during the separation process taking place in the
column.

This partitioning process is similar to that which occurs during a liquid–

liquid extraction but is continuous, not step-wise. In this example, using a
water/acetonitrile gradient, more hydrophobic components will elute (come
off the column) late, once the mobile phase gets more concentrated in
acetonitrile (i.e., in a mobile phase of higher eluting strength).

In hplc two types of instruments are used aglient and shimadu .In these two
various components are analysed based on their concentration .low
concentration components are analysed in aglient and milligram level
components are analysed in shimadu.

Aglient Shimadu

Preservatives Aflatoxins B1,B2,G1,G2

Fat soluable vitamins Aflatoxin M1

Water soluble vitamins Orchotoxin A

Synthetic colours Sudan dyes

Hyperine Capasicin
 Deoxyvineynol Curcumin

Surcalose Antioxidants

Sweetners Carotenoids

Citric acid

Agaric acid

Histamine

Coumarine

Caffeine

Ethoxyquin

Formaldehyde

Indole

Piperin

ION CHROMOTOGRAPHY

In ion exchange chromatography the ions such as bromate,nitrate ,are

analysed in water samples .

Principle :

The most popular method for the purification of proteins and other charged
molecules is ion exchange chromotography. Conversely in anion exchange
chromotgraphy,negatively charged molecules are attracted towards a
positively charged solid support.

Introduction :

Ion chromotgraphy is a chromatography process that separates ions and

polar molecules based on their affinity to the ion exchange .it works on
almost any kind of charged molecule - including large proteins ,small
nucleotides and amnio acids.

As positively chargedactions flow across action resin beads , the actions

are exchanged for hydrogen ( H+) . Likewise ,as negatively charged anions
flow across anion resin beads ,the anions are exchanged for
hydroxyl( OH-).

Ion chromotgraphy is commonly used to separate charged biological

molecules such as proteins ,amino acids .the amino acids that make up
proteins are zwitterionic compounds that contain both positively and
negetivey charged chemical groups .

Ion chromatography is used for water chemistry analysis .ion

chromarographs are able to measure concentration of major anions such
as fluoride,chloride ,nitrate,and sulfate as well as major actions such as
lithium ,sodium ,ammonium ,potassium ,calcium,magnesium in the ppb
range .

SUPPRESSOR :

The thermo scientific dionex ERS 500 electrolytically regenerated

supperssor exhibits back pressure tolerance up to 900 psi ,has peak
efficiencies optimised for 4micrometer resin bead based columns and has
very high current efficiency and static capacity

Additionally, the suppressor is anintergal part of RFIC system ,where the

samples is determined using supperssed conductivity detection in the
lowest possible background of high purity water. Autosuppression means
ease of use ;the supperssor is constantly regenerated by the continuous
electrolysis of water derived from the cell effluent.

FTIR

The concentration of dispersed oil and grease (OG) is an important

parameter for water quality and safety
The absorbance measurements were performed using the PerkinElmer
Spectrum 400 FT-IR/FT-NIR spectrophotometer in mid-IR mode and
equipped with a DTGS detector . Other instrument models with similar
configurations1 such as the Spectrum One or Spectrum 100 can also be
used. The software used to acquire the spectra was Spectrum version 6.3.
Spectra were collected in transmission mode using a glass cell with 10 mm
pathlength. Spectra were acquired over the range 3200 - 2700cm-1 at 4
cm-1 resolution with ~1 minute acquisition time, and ratioed against a
spectrum of pure solvent. The peak maximum between 2930 and 2926 cm-
1 was determined and used in the linear regression described below. A
linear baseline fit through the points at 3100 and 2800 cm-1 was subtracted
before measuring the peak height.

Procedure:

The calibration reference oil was prepared by mixing iso-octane,

hexadecane and benzene in the ratio 3:3:2, and stored in a sealed
container to avoid evaporative loss. The calibration stock solution was
prepared by weighing about 1 gm of calibration reference oil into a clean
and dry 100 mL volumetric flask and diluting up to the mark with solvent,
i.e., carbon tetrachloride. From the calibration stock solution, a series of
standard solutions were prepared using the volumetric techniques in the
range 1-40 mg/L with 9 calibration points.

Sample Preparation: Samples were prepared as follows:

1. Acidification of 1 liter of sample using hydrochloric acid to pH 2.0.

2. E xtraction of above sample with 30 mL of carbontetrachloride three

times (i.e., 1 x 3 times)
3. Filtration of extract through 10 g of sodium sulfate and dilution of
combined collected extract up to 100 mL with solvent.

4. Measurement of the solution at the absorbance maximum near 2930 /cm

Results and Discussion

Calibration – Linearity

Over the calibration range excellent linearity was observed; with a

correlation coefficient (R2) of 0.9997. A standard error of prediction of 4
mg/L was obtained.

Spike recovery studies:

A recovery study has been performed at 6 mg/L concentration in three

replicates. the recoveries are excellent, ranging from 90 to 95 percent.
This indicates that the solvent extraction recovers nearly all of the OG and
introduces only a small negative bias to the reported result.