m4 part 2 chromatography

GAS CHROMATOGRAPHY

Also known as “gas-liquid chromatography” or “gas-liquid partition chromatography”

gas chromatography

It is used in the analysis of gaseous and volatile substances

60; 10 to 12

GC ADVANTAGES

 Excellent separation, usually less than __ seconds.

 Sensitivity & speed are extraordinary, with __ gram sample being detectable for many substances.

 GC can be run 1,000 times faster than LC

 Larger preparative GLC’S can be used for purification of samples.

o Helium

o Nitrogen

o Hydrogen

o Argon

o Air

(MOBILE PHASE)

Carrier Gases Employed in GC

▪ Safety

▪ Purity

▪ Inertness

▪ Availability

▪ Cost

▪ Detector to be used

▪ Sample’s matrix

CONSIDERATIONS IN THE CHOICE OF CARRIER GAS (GC)

helium

most common carrier gas in GC (mobile phase)

STATIONARY PHASE

It functions in separating the different components of the sample

GC COLUMN

These are long tubes packed with a sorbent materia

o Packed columns

o Capillary columns

GC Column Types

carrier gas tank

A chamber made of stainless steel that supplies the carrier gas needed in the analysis

PRESSURE REGULATOR

A suitable two-stage diaphragm controlled pressure regulator that reduces the pressure level compatible with the requirement of the instrument

FLOW CONTROLLER

Contained within a thermostated chamber capable of maintaining a constant temperature as high as 400C

FLOW CONTROLLER

Controls gas flow rate, the gas flow rate is selected to compromise between the length of the analysis and the level of separation

FLOW CONTROLLER

The rate at which a sample passes through the column is directly proportional to the gas flow rate in the column

INJECTOR PORT

A small chamber where the sample is introduce into the system

INJECTOR PORT

The primary requirement of the injection system is that the sample be vaporized instantaneously so that a narrow band of vapor is introduced into the beginning of the column

INJECTOR PORT

It is equipped with a self-sealing septum made of rubber or silicone to prevent the sample from escaping

COC injector

(INJECTOR PORT)

Dissolved samples are introduced directly onto the column via __

S/SL

(INJECTOR PORT)

If a solvent matrix has to be vaporized and partially removed, a __ injector is used

gas switching valve system

(INJECTOR PORT)

Gaseous samples are usually injected using a __ system

COLUMN OR COLUMN OVEN

It is contained in an oven, the temperature of which is precisely controlled

COLUMN OR COLUMN OVEN

Its sole function is to maintain the constancy and uniformity of the column temperature at the desired value

airbath

COLUMN OR COLUMN OVEN

An __ is used to maintain this requirement

directly

COLUMN OR COLUMN OVEN

The rate at which a sample passes through the column is __ proportional to the temperature of the column.

Thermal Conductivity Detector [TCD]

Flame Ionization Detector [FID]

The most common detectors

RECORDER/INTEGRATOR

Used to graphically reproduce the output of the detector and record the resulting “chromatogram”

RETENTION TIME [RT]

The time required by an average molecule of component to pass from the injector port through the column to the detector

RETENTION VOLUME [RV]

The volume of the carrier gas necessary to carry an average molecule of the component from the point of injection to the detector

LIQUID CHROMATOGRAPHY

Can be carried out either in a column or a plane

High Performance Liquid Chromatography [HPLC]

Generally utilizes very small packing particles and a relatively high pressure

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Also known as “High Pressure Liquid Chromatography”

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

It is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify and quantify compounds

HPLC grade

HPLC Prerequisite

Sample must be soluble with solvent (must be __)

degassed

MOBILE PHASE

HPLC

One important requirement is that the solvents used must be __ first

STATIONARY PHASE

It is contained in the column and it consists of packing materials that are of two types

SOLVENT RESERVOIR

A glass or stainless steel container capable of holding up to a liter of mobile phase

INJECTION VALVE

A chamber where the solute is introduced through a self-sealing rubber or Teflon disc using a microliter syringe

PUMPS

A device needed to move the mobile phase through the column

INTEGRATORS

Used to graphically reproduce the output of the detector and record the result

DETECTOR

A device that measures the concentration of the sample injected on the column

Refractive index detector

(detector) universal detetor in HPLC

Conductivity detector

(detector) used in ionic compound

Conductivity detector

(detector) used in oxidizable/reducible compounds

Precolumn or Guard Column

A solid support coated with a high percentage of liquid phase than

the analytical column

Analytical Column

A stainless steel tube where the actual separation takes place

NORMAL PHASE HPLC

A method that separates analytes based on polarity and is useful for polar analytes

REVERSED PHASE HPLC

It is used for testing and qualifying drugs before their release in the market

ION-EXCHANGE HPLC

Used in purifying water

SIZE EXCLUSION HPLC

It is generally a low resolution chromatography and is often reserved for the final polishing step of purificatio

SIZE EXCLUSION HPLC

it is also useful for determining the tertiary structure and quaternary structure of purified proteins

nitrogen

carrier gas in GC (mobile phase)

high efficiency/slower flow rate

hydrogen

carrier gas in GC (mobile phase)

moist gas/ required special precautionary method

argon and air

carrier gas in GC (mobile phase)

lower efficiency

less used

packed columns

gc column type better for larger volume of samples

cappilary columns

gc column type that provide higher resolution

column or column oven

heart of column chromatography

poor

fast analysis time = __ separation

thermal conductivity detector (TCD)

universal detector for chromatography

flame ionization detector (FID)

highest detectivity detector