Post-Lab ( Finals )

CHROMATOGRAPHY

a process in which a solution of mixture containing inert materials, drug principles and impurities is separated

Mobile phase

solvent moving through the column

Stationary phase

substance that stay fixed inside the column

Eluent

fluid entering the column

Eluate

fluid exiting the column.

Analytical

determine chemical composition of a sample

Preparative

purify and collect one or more components of a sample

mobile phase
attractive forces

There are two classification schemes

Partition chromatography

solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support.

Adsorption Chromatography

Separation based on their adsorption onto the surface of solid (stationary phase).

for polar non-ionic compounds

Adsorption Chromatography is for

Normal

analyte is nonpolar organic; stationary phase MORE polar than the mobile phase

Reverse

analyte is polar organic; stationary phase LESS polar than the mobile phase

Ion Exchange Chromatography

Use ionic stationary phase- ions separated on the basis of their tendency to displace counter ions adsorbed on stationary phase

Anionic stationary phases

used for cation separation

Cationic stationary phases

for anion separation

Size Exclusion Chromatography

Separation is a result of “trapping” of molecules in the pores of the packing material

Liquid Chromatography

separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

Gas Chromatography

separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads

Beer – Lambert or Beer –

Bouguer’ s Law

is a combination of the above law and relates the power of the incident and the transmitted radiant beam to the thickness and concentration of the solution containing the absorbing chemical species.

Beer lambert’s law

_____ states that the power of a transmitted radiant beam decreases exponentially as the concentration of the solution containing the absorbing chemical species arithmetically.

Bouguer’ s Law

_____ states that the power of a transmitted radiant beam decreases exponentially as the thickness of the solution containing the absorbing chemical species increases arithmetically.

ABSORBANCE

the negative logarithm of transmittance.

TRANSMITTANCE

the ratio of the amount of light transmitted to the amount of light that initially fell on the surface.

Chromophore

functional group which absorbs radiant energy in the uv or vis regions

Colorimetry

branch of spectrophotometry in which absorption takes place in the vis region

220 – 380 nm

UV wavelength

380 – 780 nm

Visible wavelength

780 – 3000 nm

IR

3.0 – 15 um

Near Medium

15 – 300 um

Far IR

Frequency

is the number of waves passing through any point per second.

Wavelength

is the distance between the consecutive peaks or crests

Spectrophotometry

is a branch of spectrometry which embraces the measurement of the absorption by chemical species of radiant energy of definite and narrow wavelength monochromatic approximating radiation.

Spectrometry

is a method of analysis which deals with the measurement of spectra.

Yellow Spot Acids

Bromocresol Green Spot Color Compounds Present

Yellow/Red Spot Aldehydes, Ketones

2,4 dinitrophenyl hydrazine Spot Color Compounds Present

Fluorescent Amino Acids, Amines

Ninhydrin Spot Color Compounds Present

Yellow Brown Spots Alkaloids

Mercuric Nitrate Spot Color Compounds Present

Light Green Spot Lipids

Bromothymol Blue pot Color Compounds Present

Gray Black Spots Carbohydrates

Aniline phthalate

Separation of ink dyes

To compare ink dyes use in any company.

Botanist/herbalist

To isolate plant pigment from root and leaves

Food coloring

To differentiate coloring agent used in food product such as : M&M, Smarties dan Reese candies.

Thin Layer Chromatography

is similar to paper chromatography, but the stationary phase is a thin layer of a solid such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic.

30 minutes or less

Achieving separations in a relatively short time of about ______

20 mg of material

Accomplishing a complete analysis with as little as

Silica Gel G and Alumina

are the most widely used adsorbents.

iodine crystals.

Another method is the placing of the chromatogram in a close container holding a few

brown spots.

The organic component reacts with iodine and forms a

• UV absorbance
• Refractive index detectors

Detectors

• Hydrogen gas lamp

• Mercury lamp

UV spectro Light source

• tungsten lamp

Visible spectro Light source

Pharmaceutical Company

Real-life examples of uses for chromatography:
determine amount of each chemical found in new product

Hospital

Real-life examples of uses for chromatography: detect blood or alcohol levels in a patient’s blood stream

Law Enforcement

Real-life examples of uses for chromatography: to compare a sample found at a crime scene to samples from suspects

Environmental Agency

Real-life examples of uses for chromatography: determine the level of pollutants in the water supply

Manufacturing Plant

Real-life examples of uses for chromatography: to purify a chemical needed to make a product

fluorescence

The third method is the use of UV radiation for organic compounds