Chromatography

What is full scan mode

(more specific): instead of programming in specific ions, you program in you're range of ions. Here you're not looking for something in particular (full fingerprint) .Identifies as many substance

Why do we extract

1. To PURIFY a sample All extractions do this 2. Some used To CONCENTRATE a sample Some extractions do this

Describe the quantitation of paper chromatography

1. visual estimation 2. cut and analyze 3. densitometry (photometric by transmitted or reflected light)

List separation techniques

Crystalization Distillation Electrophoresis Extraction Chromatography

List the two major types of HPLS

Normal and reverse (refers to stationary and mobile)

Describe Cyclosporin by H.P.L.C column

these columns deal with the very complex matrix of whole blood.

Describe partition in gas chromatography

(GLC: gas liquid chromatography) - The stationary phase is a liquid that thinly coats the inert support phase (i.e.: 3% OV17 (liquid) on Gas Chrom Q (support); 3% SE30). This type of chromatography is also known as GAS-LIQUID CHROMATOGRAPHY. The more soluble a substance= the longer it will take to pass through a column.

List the different columns/ applications used for sample prep. columns

Delta aminolevulonic acid Cyclosporin by H.P.L.C. Drug Screening

What is the mobile phase in paper chromatography

Mobile Phase: wide variety of choices but aqueous systems are more common

What is the basic theory for gas chromatography

The measure of peak sharpness is the number of theoretical plates, N. The greater the peak sharpness, the better the separating ability of the column.

What are the types of stationary phases in gas chromatography

absorption column, partition and capillary columns

What type of things does paper chromatography test

amino acids and many organic compounds

How do you develop paper chromatography

developed in covered container saturated with the mobile phase. Allow solvent front to travel most of the length of the paper, remove paper, and dry.

what subsatance will distinctively appear as a peak on the recorder but w/ a different retention time than any of the other substances under analysis

internal standard

What are the disadvantages of thermal conductivity in gas chromatography

too sensitive

How is the TLC better then simple chromatography

10 to 100 times as sensitive as paper chromatography More versatile than paper chromatography Can separate more compounds and more color reactions are possible

Describe free vs salt form of drugs

Free form of drug: More Soluble in organic layer- (salt to salt or acid to acid) Salt form of drug (ionized form): More soluble in aqueous layer (acid and basic)

What do sample preparatory columns do?

Used to clean up a specimen, removing possible interference's from sample matrix, drugs, or other substances

True or false: the further out the peak, the more significant it is

true

With chromatography can you measure more than one thing at once?

yes

What are the types of ion exchange chromatography

1. Cationic: Negatively charged groups are covalently bonded to the support phase. 2. Anionic: Positively charged groups are covalently bonded to the support phase.

How does ion exchange chromatography work?

A matrix of fixed (stationary) ions is balanced by free counter ions. These counter ions can be displaced by ions in a sample under the right conditions (ie pH). Compounds are separated by virtue of charge and charge magnitude.

Describe Acid drugs

Acidify sample. Drug is now in the free form and readily goes into the organic solvent. For aqueous analysis, back-extract into base.

What can be used as a "ruler" to correct for any variations in injections.

Adding same amount of internal standard to each sample

What are the two types of simple chromatography

Adsorption type: Stationary phase and support phase are one and the same thing Partition type: Support phase is coated with a thin layer of liquid (liquid is the stationary phase)

What is another name for sample preparatory columns? What are they replacing?

Also called clean-up columns, or solid phase extraction columns. These columns are replacing many liquid:liquid extractions

What are the uses for ion exchange chromatography

Amino acid analysis, proteins, nucleotides, inorganic ions, delta- amino-levulenic acid, some H.P.L.C. ion exchange columns available.

What is peak area

Best way to quantify. Area under curve represents the total amount of a compound measured. A cut-and-weigh technique can be used, or, integration by computer of area under the curve.

What does sample preparatory columns use?

Can use gravity or be used in conjunction with a vacuum manifold

What are the stationary phases of ion exchange composed of?

Cationic columns: Resin co-polymers (Dowex 50, Amberlite), silicates, carboxymethylcellulose, and dextran derivatives Anionic columns: Quaternary ammonium groups (Dowex 1, Amberlite 400), aminoehtylcellulose, and dextran derivatives (different ones than used in cationic colums)

What are the subclasses of liquid chromatography

Column L.C.: Adsorption or Partition HPLC (high pressure): Partition "Flat":T.L.C. or Paper Gel Filtration/ Gel Permeation: Adsorption Ion Exchange

Describe the process of a sample preparatory column

Column is first rinsed with a pretreatment solution, then the specimen is added. The analyte binds to the column. The column is then repeatedly washed with a solution that will elute off interfering substances, but not the analyte. And finally, an elution reagent is added to elute the analyte.

Define Chromatography

Definition: Chromatography is a method of analysis in which the flow of solvent or gas promotes the separation of substances by differential migration from a narrow initial zone in a porous absorptive medium.

Describe quantitation for a tlc

Densitometry - as with paper chromatography Zonal scrapes - scrape and analyze

What are the compoenents of HPLC

Detectors - may be U.V. (most popular), fluorescent(right angle), refractive index, electrochemical Pump - most are capable of producing up to 3000 psi Injection Port - specialized injector places loopful of sample into the high pressure stream Recorder - chart recorder

What is the extraction ratio

Distribution ratio: KD = (solute, organic)/ (solute, aqueous)

What things does electron capture test for.

Excellent for detecting electronegative atoms (halides, organometallics, nitriles, polynuclear aromatics, and chlorinated pesticides.)

What are the subclasses of gaseous mobile phase chromatography

G.L.C.: Partition G.S.C.: Adsorption

What does HPLC stand for

High Pressure Liquid Chromatography or High Performance Liquid Chromatography

Describe the normal phase of HPLC

Inert support phase has covalently bound polar chemical groups which constitute the stationary phase. A non-polar solvent (i.e. - heptane, hexane, or other organic solvent) is used as the mobile phase. (polar substances pass through slower being attracted to the polar stationary phase)

List all of the indicators of TLC

Iodoplatinate (toxic) - colors many organic compounds show up with this Acid-Base indicators - Good for visualizing carboxylic acids Water - can be used as an indicator for hydrophobic compounds Iodine Vapors - will reveal most organics, especially unsaturated ones

What are the components of extraction

Liquid/liquid extraction/partitioning For example: salad dressing, extracting drugs Two distinct layers (immiscible) : organic and aqueous

Describe basic drugs

Make sample basic. Drug is now in the free form and goes into the organic layer. For aqueous analysis, back-extract into acid

What kind of struggle exists in chromatography

Mobile phase (Solvent or gas) Stationary phase (tries to hold onto compounds) Porous absorptive medium

Describe flame ionizing detector

Molecules of compounds are "burned" using a flame that is maintained by air plus hydrogen gas. The molecules are thereby ionized, and an electrical current can flow. Carrier gases possible: Helium, Nitrogen, or even Argon. Nitrogen, due to low cost, is often used, if the analysis permits. This detector has good sensitivity, and is the most popular one in use today..

Describe Electron capture in gas chromatography

N2 (carrier gas) is ionized by radioactive 63Ni or 3H. Slow electrons are thus formed, and a steady standing current is maintained, until an electron-capturing molecule is present. These molecules "steal" electrons, thereby reducing the standing current.

List ways to detect specimens on paper chromatography

Ninhydrin to visualize amino acids, amines U.V. Light (usually 250-260 nm). Many organic compounds absorb U.V. light. These would show up as spots of quenching, with the paper providing a faintly fluorescing background. Fluorescence with U.V. Light - i.e. - Quinine, Quinidine Chemical Reaction - atomizer, sprayer, or draw paper thru developing solution Enzymatic - usually to detect enzymes but sometimes to detect substrates. i.e. - to detect amylase, use starch substrate, and treat with iodine to develop. Biological - using microorganisms, suppressing or promoting their growth

What is the stationary phase in paper chromatography. What does it do

Paper (cellulose) Paper chromatography is an adsorption technique May be coated with an aqueous or hydrophobic solvent

What is relative retention time

Relative Retention Time = Distance traveled by peak of interest/ Distance traveled by internal standard

What is the Rf value

Rf value: The distance traveled by a given component divided by the distance traveled by the solvent front.

How does mass spec work?

Sample is bombarded with electrons (70eV) Produces many fragments of various masses and charges. (larger peak is more stable) Each particular compound has a characteristic distribution of these fragments Termed compound's MASS SPECTRA The quadrupole mass filter has 2 opposing rods with an Rf voltage and 2 with a D.C. voltage. Voltages are varied so that only molecular fragments with a particular mass/charge ratio can pass through the filter and others collide into the rods and are not counted. Then only fragments of the next higher mass/charge ratio can make it through, etc Information is tallied up and mass spectra for that compound is obtained Called ELECTRON IMPACT M.S. There is also chemical ionization M.S.

What are the uses of chromatography

Separate compounds of interest Qualitative - To identify, or help to identify, a compound Quantitative: -How much of the substance is present

How does filtration/ permeation work?

Separation in this technique is based mostly on particle size. Smaller particles get trapped in micro-pores (channels) of the stationary phase, while larger particles pass through faster.

Are all compounds colored in paper chromatography?

Some compounds are colored however most are not.

Describe amphoteric and neutral drugs

Sometimes difficult to deal with, but many will go into organic solvents Some can be extracted

What is the large and small peak in chromatography

Standard : large peak Smaller peak: drug

How would ion exchange chromatography work with amino acid analysis (process)

Starting with a low pH, all the amino acids bind to the column. As the pH is gradually raised, the more acidic amino acids are eluted off the cationic column first. Eventually, when the pH is high enough, the basic amino acids elute off the column. Amino acid methods by ion exchange chromatography are available both in column and T.L.C. forms.

Describe the types of stationary and mobile phases for tlc's

Stationary phases: many different types are possible. Common are Al2O3 (alumina gel) and silica gel. Small particle size is necessary Mobile phase: many different systems possible

How do the mobile and stationary phase work (describe attractions)

The mobile phase wants to move the compounds along rapidly, but the stationary phase has a certain attraction for each compound. If these attractions vary in strength from one compound to another, a separation of the compounds can be achieved.

Describe absorption column in gas chromatography

The stationary phase is simply the uncoated support phase. An example of this is for volatile analysis (methanol, ethanol, acetone, isopropanol, and ethylene glycol), where a simple uncoated POROPAK S column can be used. This type of chromatography is also called - GAS-SOLID CHROMATOGRAPHY. More absorption= the more time it takes to go through the column

What are the detectors of gas chromatography? What do they do?

The type of detector used is what primarily determines the type of carrier gas that will be used. Thermal Conductivity Flame Ionization Detector Electron Capture

What is thermal conductivity of gas chromatography

This detector operates based on the fact that a hot body loses heat at a rate depending on the composition of the surrounding gases. This change in temperature causes a change in resistance, which is measurable. Helium or hydrogen are suitable as carrier gases.

Descibe Elution chromatography

This term applies not only to L.C. but also to G.C., H.P.L.C., etc

What is mass spec used in conjunction with? How

Used in conjunction with G.C. (G.C./M.S.). The G.C. separates the compounds and the M.S. (like a fingerprint) identifies them Also aids in identification of retention times.

What is peak height

Valid as an approximation of peak area if the number of theoretical plates is high (other words- if peaks are sharp.) Probably most widely used method of quantitation. Usually very good approximation of peak area.

What are the advatanges of HPLC

Very sensitive: often compounds quantifiable to nanograms Very flexible: Mobile phases are often multi-solvent systems, designed specifically to separate the compounds under observation Good method for heat labile, or non-volatile substances (G.C. cannot be used in these cases). However, substance must be soluble in the mobile phase.

Describe the uses for gel filtration

aqueous Hydrophilic uses Enzymes, antibodies, proteins, hemoglobins, nucleic acids

What are the types of paper chromatography

ascending, descending most commonly used. Horizontal and radial techniques also exist

What movement propels paper chromatography

capillary action

What are common stationary phases in filtration/ permeation

cross-linked Dextran (Sephadex), Polyacrylamide gel, and Agarose gel. May be used in an H.P.L.C. set up.

What does filtration/ permeation useful for?

desalting tests

What is retention time

expressed either in time or distance units The retention time is simply the measure time (or distance) from injection point to where the peak ( apex) comes out.

What do the number of theoretical peak measure? How?

measures peak sharpness X- distance where the peak initiates Y- baseline width of peak N=16(x/y)^2

Describe the uses of gel permeation

non-aqueous hydrophobic uses Triglycerides, fatty acids

Describe back extraction

organic to aqueous (need drug in salt form) so put it in unlike media.

Describe capillary columns of gas chromatography

part of partition or GLC. No support phase as the liquid coats the inner wall of the capillary tube. Have much better resolution (ability to see detail) than packed columns..

What are the qualitative considerations for GC/ HPLC

retention time, peak area and peak height

Describe displacement chromatography

sample is placed onto the head of the column and is displaced by a solute that is more strongly absorbed than the components of the original mixture.

What is selective ion monitoring

set the machine up to look for specific things Selective Ion Mode: partial finger print: scans through limited number of ions Disadvantage: you have to know what you're looking for

Describe the mobile phase of gas chromatography

the mobile phase is the carrier gas. It is, by convention, considered to be non-polar. Nitrogen is the most popular choice due to its low cost, however nitrogen may not be suitable in specific procedures. Helium and even Argon may be required.

What is a TLC

thin layer chromatography

What is a disadvantage of HPLC

tiny inner boar, therefore its more susceptible to contamination = requires constant cleaning

What is an example in thermal conductivity in gas chromatography

to aid in the identification of anaerobic bacteria by gas chromatographic analysis of organic acids produced..

true or false Conditions must be exactly the same as described in a specific method if Rf values are to be used.Even then, they are not very reliable. It is always best to run known standards, and compare migration distance directly with these.

true

true or false Samples injected into columns are small, can be less than a microliter

true

true or false Variations in amt of sample can vary due to different techniques of injection. Because of this, an internal standard is used to quantify chromatography results

true

Describe drug screening column

two different columns used to extract acid and basic drugs.

Describe Delta aminolevulonic acid column

two ion exchange columns, one anionic and one cationic, are used in tandem.