What GCMS users Want ?

Detection Limit- Sensitivity

•          Depends on: Mass Analyzer, Vacuum Pump, Detector

Qualitative/Quantitative Study

•           Selection of Mass Analyzer

Mode of Analysis

•           SIM/Full Scan

•          Specific Applications

•          Budget

•          Training

•          Service Support

•          Application Support

Detection Limit: Normally mass analyzers plays important role or you can say heart of GC-MS.

There are analyzers who have sensitivity who have signal to noise ratio : 850:1

Best solution providers are Agilent Technologies, Perkin Elmer, Shimadzu.

Types of GCMS

        1. Time of Flight

        2. Quadrupole

        3. Ion-trap

        4. IRMS

        Each of these are applied in different fields

        Based on the requirements of analysis and conclusions to be drawn.

FLAME IONISATION DETECTOR IN GAS CHROMATOGRAPHY

• Mass sensitive detector.
• Operates by mixing hydrogen with carrier gas and burning it at a jet in a stream of air.
• A collector electrode is placed above the flame.
• P.D. is set up between the jet and the collector electrode.
• The jet is at -ve potential w.r.t. the collector.
• Organic compounds produce ions and electrons when burnt.                                                                     
• Electrical current is set up whose magnitude is proportional to the mass flow of the sample.

Detectors used in HPLC

•   MS – provides additional information of molecular weight and structure by fragmentation pattern
•  Photodiode Array (PDA) – Like a UV detector but gives UV traces for all wavelengths from 210 nm to 500 nm at the same time
•  Refractive Index – used for samples that don’t have any light absorbing characteristics, typically very insensitive
•  Electrochemical – detects oxidation/reduction of sample, extremely sensitive but difficult to use
•  Any detector can be added on at any time, modular.

How to choose a column ?

• Suitability for a specific application
• The column must do the job
• If already using a different column the new column should provide a benefit in performance or durability
• Reproducibility of the result from column to column and Lot to Lot
•  Reliable performance
• Stability & Life time of the column
• Less column changes will cause less instrument down time and therefore more sample can be run
• Compliance with official methods that might need to be followed
•  In case the customer needs to follow official methods like Pharmacopoeia, EPA, DIN or other local
• LC/MS suitability (for LC/MS user)
• Low Bleed of a column to reduce background signal/interference. This makes the identification in the MS easier.

Waters column

Columns

• Solid Support - Backbone for bonded phases.    
• Usually 10µ, 5µ or 3µ silica or polymeric particles.
• Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support.            
• Extremely stable
• Reproducible
• Analytical - Performs the separation.
• Guard - Protects the analytical column
• Particles
• Interferences
• Prolongs the life of the analytical column

Pumps for HPLC

Ideal pumps:

1)      Ability to generate high pressure

2)      Pulse-free output

3)      Accurate control of flow

4)      Corrosion resistant

Role: Deliver the mobile phase

Two groups of pumps:

1)      Constant pressure

2)      Constant volume

Three types of pumps are available:

1)      Reciprocating pumps (90% of Commercial HPLC; produce pulse flow)

2)      Displacement pumps (produce flow that are independent of viscosity and back pressure)

3)    Pneumatic pumps (cannot do gradient and pressure less than 2000psi)

Waters 1525 pump

Reversed-phase Chromatography (RPC)

• Reversed phase
• Most popular since it can handle the broadest variety of sample types
• Opposite of normal phase – stationary phase is non-polar and mobile phase is polar
• C18 columns (e.g. octadecyl, -C₁₈H₃₇)
• Eluents used – aqueous solution of organic solvents e.g. MeOH, ACN, THF, etc.
• Applications
• Amino acids, homologs, herbicides, etc.

Normal-phase or Liquid-solid Chromatography (LSC)

• Normal phase
• Used polar stationary phase packings and low polarity solvents
• Non-polar compounds elute out first followed by increasingly polar compounds
• Eluents used – low to moderate polarity e.g. hexane, CH₂Cl₂, CHCl₃, etc.
• Application
• Isomers, compound class separations, etc.
• Ideal for samples with low/moderate polarity, water insoluble and non-ionic

Liquid Chromatographic Modes

• Normal-phase or Liquid-solid (NP, LSC)
• Separation based on adsorption/desorption of the analyte onto a polar surface (silica)
• Reversed-phase (RPC)
• Separation based on analytes’ partition coefficients between the mobile phase and the bonded stationary phase
• Ion-exchange (IEC)
• Separation based on ion-exchanging with the counter-ions and ionic interaction with the bonded ionic group
• Size-exclusion (SEC or GPC)
• Separation based on analyte’s molecular size and sieving action of the column packing

Column Packing Materials

• Support Type
• Silica (or alumina) or polymer (cross-linked polystyrene)
• Bonded groups
• C18, C8, C4, amino, cyano, phenyl
• Diethylaminoethyl (DEAE), sulfonate, quaternary ammonium
• Particle size (d_p): 3-, 5-, 7,- 10- or 20 μm
• Efficiency is inversely proportional to d_p
• Column pressure is inversely proportional to (d_p)²
• Pore size (d_pore): 60-300A
• Wide pore materials (300 A) are used for biomolecules or polymers
• Surface area: 90-400 m²/g
• High surface area maximizes solute interaction with bonded groups

Advantages of HPLC over conventional HPLC

• Rapid and precise quantitative analysis
• Typical analysis time of 5-20 min, precision <0.5-1% RSD
• Automated analysis
• Using autosampler and data system for unattended analysis and report generation
• High sensitivity detection
• Detection limits of ng to pg
• Quantitative sample recovery
• Preparative technique from mg to kg quantities
• Amenable to diverse samples
• Can handle >60% of all existing compounds vs. 15% for GC
• Can analyze samples with little or minimal preparation

Lower one is HPLC,upper is conventional

Basic Hardware Components of HPLC

• Solvent Delivery System (Pump)
• Injector (introduce samples)
• Column guard
• Column (containing stationary phase)
• Detectors (“eyes” )
• Waste Collector
• Recorder (Data Collection)

Terms used in Chromatography

• Analyte : The substance / compound to be separated
• Preparative chromatography :  Purify sufficient quantities of a substance for further use, rather than analysis.
• Analytical chromatography is used to determine the existence and 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
• Stationary Phase : The immobilized phase that is on the support particles or the inner walls of the column tubing
• Mobile phase : The phase that moves through the stationary phase in a definite direction and at a defined speed. Liquid – LC, CE, Gas in GC, SFC.

History of Chromatography

• Russian-Italian scientist Mikhail Tsvet in 1900
•  Separation of plant pigments such as chlorophyll, carotenes, and xanthophylls
• Chroma –mans colour and graphy means study
• 1940s and 1950  Archer John Porter Martin and Richard Laurence Millington Synge worked on partition chromatography
• It was not confined to coloured materials and could be applied for varied separations
• Advances continuously being done which helps in separation of increasingly similar components

Applications of Uv-Vis Spectrophotometer

• Structure elucidation of organic compounds

Location of peaks and combination of peaks, it can be concluded that whether the compound is saturated or unsaturated, hetero atoms are present or not etc.

• Quantitative analysis
• A = log I₀ / I_t = log 1/ T = – log T = εbc 
• Qualitative analysis

Can characterize those types of compounds which absorbs UV radiation. Identification is done by comparing the absorption spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic compounds and aromatic olefins.

•  Chemical kinetics
  Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed.
• Quantitative analysis of pharmaceutical substances
  Many drugs are either in the form of raw material or in the form of formulation. They can be assayed by making a suitable solution of the drug in a solvent and measuring the absorbance at specific wavelength.
•  Molecular weight determination
  Molecular weights of compounds can be measured spectrophotometrically by preparing the suitable derivatives of these compounds.
• As HPLC detector

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.

• Detection of Impurities

 Perkin Elmer Uv-Vis

Role of sales Person while selling Analytical Instrument

 1.       DISPLAY RIGHT ATTITUDE.

2.       ENSURE CUSTOMER SATISFACTION.

3.       CUSTOMER  RELATION  MANAGEMENT.

4.       UNDERSTAND THE MARKET.

5.       UNDERSTAND THE CUSTOMER.

6.       GAINING THE TRUST OF THE CUSTOMER.

7.       COMMUNICATION TO CUSTOMER.

8.       PROD. KNOWLEDGE.

9.       SOLN. ORIENTED SELLING.

10.   ACCOUNT OWNERSHIP.

11.   BRIDGE   BTN  CUSTOMER  AND  COMPANY.

12.   RESPONSIBILITY FOR  RESPONSE (TIME).

13.   UNDERSTANDING CUSTOMER NEEDS.

14.   ESTABLISH BRAND WITH CUSTOMER.

15.   UNDERSTANDING CUSTOMER BUSINESS PLANS.

16.   TRANSPARENT , HONEST , ETC .

17.   COMPETITIVE  INFORMATION.

18.   GET , KEEP , & GROW  CUSTOMERS .

19.   REVENUE& PROFIT GENERATOR FOR COMPANY.

20.   THE BOUNDARY SPANNER FOR  THE COMPANY.

Chromatography

Chromatography:
Chroma means colour ; technique is used for the separation of different Analytes in sample.
This technique is used from many years . Very popular in Pharmacy industries for various purposes.

Chromatography is of various types.
Thin paper chromatography.
High Pressure chromatography.

Depending on type of sample we choose method in chromatography.

What you mean by Analytical Instruments? Where we use them?

Various types of instruments we use in day today life. But have you ever thought about analytical instruments?

You can say there is another world which depends on Analytical instruments
Analytical instruments basically made for calculation of various parameters which are important for quality purpose.

Analytical instruments are of various types:
1.Chromatography
2.Material characterisation
3.Inorganic