Etiket Arşivleri: Chromatography

Liquid Chromatography – Mass Spectroscopy (LC-MS) ( Md Akbar Siddiq Khan )

LIQUID CHROMATOGRAPHY – MASS SPECTROSCOPY (LC-MS)

Presented by
Md Akbar Siddiq Khan

M.Pharm

Nizam College Of Pharmacy

Hyderabad – A.P

What is LC-MS?

It is the combination of liquid chromatography and the mass spectrometry.

• In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS.

• In the most of the cases the interface used in LC-MS are ionization source.

PROBLEMS IN COMBINING HPLC AND MS

HPLC

• Liquid phase operation

• 25 – 50 deg. C

• No mass range limitations

• Inorganic buffers

• 1 ml/min eluent flow is equivalent to 500 ml/min of gas

MS

• Vacuum operation

• 200 – 300 deg. C

• Up to 4000 Da for quadrupole MS

• Requires volatile buffers

• Accepts 10 ml/min gas flow

PARTS OF LC-MS

 Two key components in this process are the ion source, which generates the ions, and the mass analyzer, which sorts the ions.

Several different types of ion sources are commonly used for LC/MS.

MOBILE PHASE:-

The mobile phase is the solvent that moves the solute through out column.

 General requirements:-

(1)low cost, uv transperancy,high purity.

(2)low viscosity, low toxicity, non flammability.

(3)non corrosive to LC system component.

 Solvent strength and selectivity:- it is the ability of solvent to elute solutes from a column.

COLUMN:-

 Column type:-

 Specialized mode:-

 The use of di-functional or tri-functional silanes to create bonded groups with two or three attachement points leading to phases with higher stability in low or higher pH and lower bleed for LCMS

 Most widely used columns for LCMS are:-

(1) fast LC column. the use of short column. (15-50mm)

(2) Micro LC column. the use of large column. ( 20 150mm)

Thin Layer Chromatography – TLC ( Mr. Shaise Jacob )

THIN LAYER CHROMATOGRAPHY (TLC)

by

Mr. Shaise Jacob

Faculty, Nirmala College of Pharmacy

Muvattupuzha
Kerala, India

Chromatography

• There are two basic types of chromatography
– Gas
– Liquid
• Liquid includes TLC and high performance liquid chromatography (HPLC)

Introduction

• TLC is a form of liquid chromatography consisting of:
– A mobile phase (developing solvent) and
– A stationary phase (a plate or strip coated with a form of silica gel)
– Analysis is performed on a flat surface under atmospheric pressure and room temperature

• Michael Tswett is credited as being the father of liquid chromatography. Tswett developed his ideas in the early 1900’s.

TLC

• The two most common classes of TLC are:
– Normal phase
– Reversed phase

Normal Phase

• Normal phase is the terminology used when the stationary phase is polar; for example silica gel, and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.

Reversed Phase

• Reversed phase is the terminology used when the stationary phase is a silica bonded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase.

THIN LAYER CHROMATOGRAPHY

Chromatography is used to separate mixtures of substances into their components. Similar to P.C, except that a thin layer of some inert material, i.e. Aluminium oxide, mag.oxid. , sili.oxide is used instead of paper.
• A layer of any one of these oxide is made from a slurry of power in a suitable inert solvent.
• Slurry is spread over a flat surface ( glass, metal or rigid plastic ) & dried


Laboratory‎ > ‎Paper Chromatography and Beer’s Law

PAPER CHROMATOGRAPHY and BEER’S LAW

THEORY

PAPER CHROMATOGRAPHY

Chromatography is a method for analyzing complex mixtures (such as ink) by separating them into the chemicals from which they are made. Chromatography is used to separate and identify all sorts of substances. Drugs from narcotics to aspirin can be identified in urine and blood samples, often with the aid of chromatography. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed.

Paper chromatography is one method for testing the purity of compounds and identifying substances. Paper chromatography is a useful technique because it is relatively quick and requires small quantities of material. Separations in paper chromatography involve the same principles as those in thin layer chromatography. In paper chromatography, like thin layer chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. Components of the sample will separate on the stationary phase according to how strongly they adsorb to the stationary phase versus how much they dissolve in the mobile phase.The R f value for each spot should be calculated. R f stands for "ratio of fronts" and is characteristic for any given compound. Hence, known R f values can be compared to those of unknown substances to aid in their identifications.