CC LE 3

T OR F Support takes up any of the substances that are being separated.

FALSE Does not take up any of the substances that are being separated.

Methods Used for Detection Colorless Substances ● ___ ● ___ ● __

Fluorescence Optical Absorbance pH

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY THE DETECTION METHODS USED

Reaction with Chemical Agents Charring the Separated Solute UV Radiation

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC enumerate 1. ___ 2. SEPARATION OF___ 3. DETECTION OF ___ IN URINE 4. ___ TECHNIQUES

THERAPEUTIC DRUG MONITORING & SCREENING TRICYCLIC ANTIDEPRESSANTS COCAINE AND HEROIN IMMUNOASSAY

Ion-Exchange Chromatography Important factors to take note of: ● ___ of stationary phase ● __ group ● Charge __ ● Stationary phase ___ ●___ of ions in the mobile phase ● Mobile phase ___

Type Ionic density matrix Type and concentration pH

EARLY METHODS OF STERIC EXCLUSION 2. Gel Permeation Chromatography Uses ___ of ___ with a ___ mobile phase

hydrophobic gel heads polystyrene non-aqueous

EARLY METHODS OF STERIC EXCLUSION enumerate

Gel Filtration Gel Permeation Chromatography

DETECTION Done to monitor the ___ for solutes to be separated

illusion fractions

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' DETECTORS enumerate

Thermal Conductivity (TC) Flame Ionization

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS PEAK AREA The peak is proportional to the ___ of the compound that produced the peak

concentration

CHROMATOGRAPHIC METHODS 6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) ● Used for quantitating ___ and___ and for confirming drug identification such as __ and/or its ___ ● Separation is based much like in __ on the ability of each compound to adsorb to the ___ phase, which virtually depends on the ___ of the compound in the gas versus the __ phase

serum drug levels therapeutic drug monitoring cocaine metabolites TLC stationary relative solubilities liquid

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RETENTION TIME ● Another means for __ the results for HPLC is thru the use of Retention time ● ___ required for the sample to elute from the column and observed by the detector ● Expressed in ___ ● __ may use retention time rather than volume for ____

recording Time minutes Chromatogram compound identification

MODES OF SEPARATION 1. ADSORPTION CHROMATOGRAPHY Common Adsorbents ENUMERATE

1. Alumina 2. SIlica Gel 3. Carbon 4. Magnesia 5. Magnesium Silicate 6. Magnesium Carbonate 7. Calcium Carbonate 8. Aluminum Silicate 9. Starch 10. Sugar & CaPO4

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS TYPES OF PUMPS FOR HPLC Take note that pressure used is ___ psi, and the peristaltic and diaphragm type pumps are suitable only for ___ pressure chromatography. ● The ____ pump on the other hand, which is also the most widely used, or the syringe type pumps - operate with pressures from 1000 - 3000 psi.

1000 - 3000 low mechanical reciprocating piston

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS METHODS OF DETECTION ___ MEASUREMENT ___ MEASUREMENT ___ DIFFERENCES ___ DETECTION

ABSORBANCE FLUORESCENCE REFRACTIVE INDEX ELECTROCHEMICAL

CLASS ACCORDING TO POLAR FUNCTION GROUPS The mixture is typically separated into classes according to polar function groups. ENUMERATE STATIONARY PHASE

Acidic Basic Non-Polar

MODES OF SEPARATION ____ ____ ___ Exclusion ___ Chromatography

Adsorption Partition Steric Ion-Exchange

Basic Components of Chromatography Mobile Phase - ___ the sample through the chromatographic system and may be in a ___ form Stationary Phase - Through which the mobile phase ___ and can be in ____ form. Column - Holds the ____ phase Eluate The ____ from the sample

Carries - gas or liquid flows -solid or liquid stationary separated components

__ ● The most important apparatus requirement for paper chromatography ● To maintain ___ between the ___ phases This is also to prevent __ of the solvent from the paper as not to cause ___ results.

Chamber or tank equilibrium liquid and the vapor evaporation erratic and less optimum

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC 1. THERAPEUTIC DRUG MONITORING & SCREENING examples enumerate

Chloramphenicol Barbiturates

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY DETECTION METHODS FOR TLC 1.) ___ with ___ different solvent, resulting in characteristic pattern for each drug 2.)__ , wherein the strip is subjected to UV light to ___ the fluorescence inside the compounds

Color reagents 3 UV Light excite

PROPERTIES OF A GOOD ADSORBENT ● ___ - --- in the ___ used ● Does not ___ with the substance to be separated nor ___ of the substances to be separated ● ____ ● The powder used should be sufficiently ___ in ___ to give a ___ surface area.

Insoluble- solvents react catalyze decomposition Colorless small particle size large

Different forms of separation ENUMERATE

Ion-exchange chromatography Partition chromatography Adsorption chromatography Size-exclusion chromatography

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY ● ___ chromatography ● The separation of solutes is based on____ in the ___ solvent and ____ solvent ● The solid adsorbent is replaced by the ___ . The stationary liquid is only ___ miscible with the flowing liquid as mobile phase. ○ The stationary phase may be quoted on an ____ ● Performed on a ___ in its ___ form. ○ Unlike adsorption chromatography where the governing factor is only to find the ___ on the proper __ , in partition chromatography,___ is the governing factor.

Liquid-liquid relative solubility organic/non-polar aqueous/polar stationary liquid partially inert chromatographic support separatory funnel simplest solvent polarity solubility and the two liquids/phases

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' STATIONARY PHASE enumerate These materials are __ bonded onto the ___ of the support particles or onto the __ of the column. These are more preferred due to their ___

Methyl Silicone Polymers Substituted SIlicone Polymers Silicone Polyesters chemically surface walls stability

Carrier gas used in gas chromatography can be:

Nitrogen Argon Helium Hydrogen

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) DIFF PHASES ENUMERATE

Normal Phase Reverse Phase

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY PROCESS > MODERN PARTITION CHROMATOGRAPHY PARTITION SYSTEMS ENUMERATE

Normal Phase Reverse Phase Support

STRENGTH OF ADSORBENTS ___ of an adsorbent ● Measured by the ___ in which a zone ___ in an ____ ○ "The greater the rate, the ___ the adsorbent" ● ___ the ___ of mixture into its components.

Power rate travels elution experiment weaker Facilitate separation

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ● Makes use of ____ equipment. ● A ___ of TLC (thin layer chromatography). ○ It has the ability to quantitate ____ ● Uses ____ for___ , ___ ,___ , and ___. ● It can run in ____ temperatures. ○ It is similar to ___ chromatography. Except that the mobile phase is ___ rather than ___

Semi-automated refinement separate compounds. pressure fast separations controlled temperature in-line detectors gradient elution techniques ambient gas liquid liquid gas

MOST COMMONLY USED SOLID SUPPORTS ENUMERATE

Silica Gels Cellulose Powder Diatomaceous Earth

FACTORS THAT INFLUENCE STRENGTH OF ADSORBENTS 1. ___ used 2.____ a. "The greater the surface area, the greater the ___ capable of being adsorbed."

Solvent Surface Area amount of solute

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY SYSTEM ● Gas cylinder -where the___ are stored ● Sample injector - otherwise known as the ____ is located ___ the column ● Column holds the __ phase ● Detector connected to the ____ ●Sample - sample injected through the __ must be injected as a __ or the temperature of the injection port must be ___ boiling point of the components so that they __ upon injection - Remember: The more volatile solute molecules or those with the __ boiling points tend to be swept most ___ along the carrier gas

carrier gases septum, heater, or injector port above stationary recorder or data system septum gas above vaporize lowest rapidly

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS INTERNAL STANDARD ● Has a similar ___ to the analyte ● Added to __ sample to compensate for variations in the ___ ,___ and other___

composition each size of sample injected solvent evaporation possible irregularities

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' QUANTIFICATION - Calculation of the concentration of the analyte in the samples by comparing the ____ ,___ , or ___ This is then used to calculate the ___ of the analyte and the samples. A reference solution called __ or ___ is prepared

curve of peak height peak area, spot density vs. the calibrator concentration constructed concentration internal calibration internal standardization

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS METHODS OF DETECTION 3. REFRACTIVE INDEX DIFFERENCES Detects only the compounds with refractive index ___ from that of the solvent ● It is a means of detecting ___

different alcohols, polyethylene glycols, salts and sugars

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS METHODS OF DETECTION 4. ELECTROCHEMICAL DETECTION ● Measurement of the ability of the ___ to conduct ___ producing __ reaction ● It is ___ in monitoring the separation of ___

eluate electric current redox sensitive catecholamines

CHROMATOGRAPHIC METHODS 6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) DETECTION ● Main parts include the ___ and the ___ ● Similar to the ___ procedure, a sample goes through the __ and is carried through a stationary phase by a __ but instead of being analyzed directly, it goes through a mass spectrometer where it is bombarded with ions which gives the compound a specific __ ratio ● Then the patterns or fragmentograms are then produced and analyzed by a ___

gas chromatography oven mass spectrometer or mass spectrophotometer Gas Chromatography injector port carrier gas mass-to-charge computer

CHROMATOGRAPHIC METHODS 6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) DETECTION ● The separated compounds enter the___ (a special detector that measures the ___ pattern of ions) where they are subjected to bombardment by ____ ● Results in the ___ ions with specific ___ ratios which are ___ in a field and passed through an ___ field ● Only those ions with a ___ mass-to-charge ratio (m/c) will pass through a ___ field so that they strike the detector

mass spectrometer or spectrophotometer fragmentogram electrons creation molecule mass-to-charge accelerated electric quadrupole narrow tuned

COLUMN CHROMATOGRAPHY 1. Mixture to be separated is dissolved in the ___ 2. ___ is added throughout the process 3. Components ___ (__) 4. Each component is collected as it reaches the ___ of the column.

mobile phase Mobile phase separate - descending bottom

strongest absorption occurs from ___ . Weakest absorption occurs from ___ solvents. This is due to the fact that polar solvents tend to ___ polar solutes more ___ than nonpolar solutes.

nonpolar solvents highly polar dissolve readily

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY Based on ___ chromatography ● Variant of ___ chromatography ● For ___ ● Most commonly used as a ___ screening test ● Absorbance is measured with a ___ ● The___ is of great importance in Thin Layer Chromatography than in most other forms of chromatography because of the ___ sample size used

partition column drug screening and identification semi-quantitative densitometer purity of a solvent small

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) REVERSE PHASE HPLC Most ___ HPLC for ___ ● Effectively separates ___ easily, and these molecules are easily degraded by___ ● Stationary Phase: ___ coated with___ is used ● Mobile Phase: ○ Solvents are usually ___ -based and may contain either ___ compounds. ○ Often a mixture of __ , a mildly polar organic solvent, and an inorganic ___ is the combination used to facilitate the removal of material from our reverse phase column. ○ Common examples are: ■ ___ ■ ___ ■ ___

preferred and performed drug analysis unstable biochemical molecules harsher organic solvents Silica nonpolar material water organic or inorganic water salt Acetonitrile Methanol Water

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY PROCESS > MODERN PARTITION CHROMATOGRAPHY Uses ____ stationary phases that are ___ to the support of ___ polymers that are ___ in the mobile phase

pseudo-liquid chemically bonded high molecular weight insoluble

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY CONDITIONS FOR EFFECTIVE RESOLUTION ● Must be a __ of sufficient capacity for the solvent to complete the chromatogram without ___ ○ ___ - visible record or graph showing the result of separating the components of a mixture by chromatography. ● Solvent and vapor phases must always be in ___ ● Paper must be _____ ● ___ must be avoided

reservoir interruption Chromatogram equilibrium freely suspended Large temperature changes

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY ADVANTAGES OF GC 1. Most ___ chromatographic method 2. Amenable to ___ 3. Adaptable to ___

sensitive automation computer resolution

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY TOXI LAB THIN LAYER CHROMATOGRAPHY (TLC) A TLC kit supplied with discrete strips of ___ , ____ and ___ ● The specimens used are: ○ ___ - best specimen for analysis ○ ___ - for drugs taken orally ● The kit comes with a ___ book which Aids in identifying each drug by its ___ and characteristics____ in different reagents ○ (eg. heroin has a retention factor of ___ and has a characteristic ___ color in the first solvent that ___ in the second solvent which is water)

silicate extraction solvents color developing solutions Urine Gastric Juice reference pattern Rf value color change 0.14 red disappears

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY PROCEDURE 1. Plot sample as ___ spots near one edge of the plate 2. Repeat applications may be ___ upon each other with __ in between applications 3. Place plate in a ___ containing appropriate __ mixture ● The plate is then positioned to the applied sample along the lower edge, __ above the level of the mobile phase 4. As the solvent moves up the plate, the ___ also moves upward as well ● Separated by the ___ between the sorbent 5. When sorbent reaches a ___ height, weight is removed and dried

small superimposed , drying closed chamber , solvent slightly sample differing actions predetermined

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY Retention factor values of the components of the sample are compared with the retention factor values of ___ on the same plate ● Good practice to run standards ___ with the unknown samples Since it is fairly difficult to maintain exactly the same experimental conditions ● And since Rf values may ___ for some components, Further identifying information is obtained by spraying ___ stains on the dried plate and comparing the colors to that of the standards ● Rf value is ___ for each substance when chromatographed in a certain solvent system ● More ____ result may be obtained by scraping the spots off the plates by ___ the colored compound ● Optical density is read in a __ or ___

standards along overlap different characteristic quantitative eluting spectrophotometer densitometer

MODES OF SEPARATION 4. ION-EXCHANGE CHROMATOGRAPHY The partition systems in ion exchange chromatography are as follows: Sorbent or Adsorbent - acts as the ___ phase, consists of polymers with ___ ions and as such it is ___ in water. We usually make use of ___ consisting of large polymers of ___ , ___

stationary covalently-bound insoluble resin substituted benzene silicates or cellulose derivatives

Different forms of separation 1. Ion-exchange chromatography is described as the separation based on exchange of ions between___ 2. Partition chromatography Separation is based on ___ partitioning between two liquid phases 3. Adsorption chromatography Separation is due to a series of___ steps. 4. Size-exclusion chromatography Separation is based on___

surface and eluents solute adsorption/desorption molecular size

Ion-Exchange Chromatography more highly charged molecules are more ___ or have a ___ to the stationary phase, and therefore travel ___ through the chromatographic system and are eluted ___ . The less charged molecules bind ___ to the stationary phase and therefore travel ___ and are eluted ___ . The moderately charged molecules, however, equilibrate between the ____

tightly bound greater affinity slowly later less strongly rapidly sooner resin and the moving buffer

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' DETECTORS 1. Thermal Conductivity (TC) ● Detector contains ___ that change ___ with change in ___ ● ___ is ___ to the concentration of the analyte ● ___ - carrier gas

wires electrical resistance temperature Electrical change proportional Helium

Packing techniques Dry Packing - __ alone is introduced into the column WET Packing - ___ and ____ is poured into the column

Adsorbent Slurry adsorbent solvent mixture

Adsorbents used in column chromatography ● ___ (Most commonly used) ● ___ ● __ (Magnesium dioxide) ● ___ - has low water content

Alumina Silica gel MgO2 Charcoal

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY ADSORBENTS use ENUMERATE

Alumina Silica gel Crossed-linked dextran Cellulose

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY PROCEDURE 4. ● ___ carries the volatile compounds though the column to a detector ● The compound that are soluble in the liquid phase are ___ for a period of time in the liquid, and therefore movement is ___ ● While the compounds with ___ boiling points will spend __ time in the stationary phase and move __ through the column ● The more ___ solute molecules or those with the ___ boiling points, tend to be swept most ____ along the carrier gas ● Volatile compounds that are present mainly in the ___ phase will have ___ and move ___ to the column ● Remember: Both volatility & solubility differences affect the rate of ___ of the sample molecules through the gas chromatography column 5. Compound interacts with the ___ creating electrical signal whose ___ is proportional to the amount of material at the detection site recorder

Carrier gas retained slow higher more slowly volatile lowest rapidly gas low partition coefficients quickly flow detection system intensity

MODES OF SEPARATION 4. ION-EXCHANGE CHROMATOGRAPHY Mobile Phase - ____ Exchange Resins - Change in ___ and ___ allows separation of ____ In the mobile phase, cation or anion exchange resins are used. The solutes are eluted with a solution of ____ containing ___ ● A change in pH and ionic concentration allows separation of organic and inorganic ions and in some cases, there is adjustment of the pH to decrease the __ _ of bound ions or in the ion exchange surface.

Cation/Anion pH ionic concentration organic and inorganic ions low to medium salt concentration competing counter ions. charge

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY DETECTION METHODS 1. __ with Reagents wherein the paper is exposed to ___ 2. ___ scanning 3. ___ wherein there is ___ of ___ with the ___ layer and ___ with a ____ . In this method, the solutes appear as ___ against a fluorescing background. 4. ___ Absorption 5. ___ Absorption

Color Reaction iodine vapor Radioactivity Fluorescence incorporation phosphor- sorbent irradiation - UV lamp black spots Ultraviolet (UV) Infrared

Methods Used for Detection ENUMERATE FOR

Colored Substances Colorless Substances

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' SUPPORT MEDIUM OR INERT MATERIALS 1 . ___ 2. Glass ___ 3. Glass __ 4. Metal ___ 5. ___

Diatomaceous Earth Beads Powder Helices Teflon

Methods Used for Detection Colored Substances ● ___ Observation ● ___ Methods - Direct Observation are chemical methods using __ that produce some ____ as a visible color.

Direct Chemical reagents readily discernible reaction

Packing techniques ENUMERATE

Dry Packing Wet packing

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RETENTION TIME ○ Isocratic Elution - ___ of the mobile phase is ___ through the separation ○ Early Eluting Compounds - Have retention times close to ___ , producing ___ separation ○ Late Eluting Compounds - Have __ retention times, producing ____ resulting in:. ● ___ in sensitivity

Elution strength constant 0 poor long broad bands Poor/decrease

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS SAMPLE INJECTOR enumerate ● Fixed-loop injector ○ ___ method ○ Has the advantage of having higher ___ ○ Used at ___ pressures ● Syringe ○ Routinely used with a volume of ___ (microliters)

Fixed loop injector Syringe Best and most widely used reproducibility high high

GC-MS & MASS SPECTROMETERS SIMILARITIES ● ___ ● _____ Analyzer- Where ions are ___ according to their ___ Detector -With various___ Computer-___ data

Gas Chromatograph & Mass Spectrometer Interface Ionization Chamber or Ion Source separated -mass amplification systems Acquiring and processing

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS CHROMATOGRAPHY SYSTEM enumerate

Gas cylinder Sample injector Column Detector Sample

Following are materials used for support used to retain the solid phase at the bottom of the column ● ___ Plugs ● __ Paper ● ___ Glass Disks

Glass Wool Filter Sintered

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY TOXI LAB THIN LAYER CHROMATOGRAPHY (TLC) PARTITION SYSTEM OF TOXI LAB THIN LAYER CHROMATOGRAPHY ● Stationary Phase: ___ ● Mobile Phase: ___

Hydrated silica strips Nonpolar 10% methanol chloroform

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY ● This method was first developed by___ in ___ to separate ___ ● Same as ___ except that the mobile phase is ___ ● Separates mixtures of ___ compounds ● Can have a ____ stationary phase ● The principle is based on the __ of the substances to the ___ phase over the ___ phase so samples are ___ between the gaseous mobile phase and the liquid stationary phase

James and Martin 1952 fatty acids HPLC gas volatile solid or liquid affinity liquid stationary gaseous mobile partitioned

Chromatography terms Mobile Phase ___ that flows through a _ system Stationary Phase ____ phase of a chromatography system on which the materials to be separated are ___

Liquid or gas chromatography Solid or liquid selectively adsorbed

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' DETECTORS 2. Flame Ionization Detectors ● Widely used in the ___ ● More ___ than TC detectors ● __ is ___ to the concentration of ion formed

clinical laboratory sensitive Current , proportional

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-SOLID CHROMATOGRAPHY ● Involves a ___ packed with an ___ (such as __ ) ● Components of the mixture distribute themselves between the ___ phase and the ___ ● However, with the new advances in technology, this is now an __ method

column absorbent activated carbon gaseous surface of the solid obsolete

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PROCEDURE 4. As the solvent ___ fractions from the column, the material passes through a detection system where some properties such as ___ or other parameters are determined. 5. The change in __ is sent to a data processing system or recorder, which gives a read-out of__ for each constituent of the mixture. And finally 6. There will be ___ of materials separated

elutes light absorption, conductivity, fluorescence, signal elution time against peak intensity quantitation

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY ● Volatile compounds that are mainly present in the ___ phase will have ___ and will move ___ through the chromatography column ● The compounds with ___ boiling points will spend more time in the ____ phase and move ___ through the column. ● ___ differences affect the ___ of sample molecules through the gas chromatography column

gas low partition coefficients quickly higher stationary slowly Volatility and solubility rate of flow

CHROMATOGRAPHIC METHODS 1. COLUMN CHROMATOGRAPHY ● Chromatography column Should be made of __ to promote; ○ Observation of ___ of solid phase and solvent level; ○ Inspection of __ ; ○ Visualization of the ___ of colored bands ● Adsorbent material is packed ___ ● Length of column is at least ___ the internal diameter ● Size of column and amount of packing material are determined by the amount of __ to be separated

glass settling air bubbles Channeling and movement uniformly 10x solute

CHROMATOGRAPHIC METHODS 6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) ● The "___ " to confirm the results obtained using the screening methods such as ___ ● ____ ● It can detect ___ levels of drugs making this technique the___ reference method and the best ___ procedure available

gold standard EMIT or Enzyme Multiplied Immunoassay Technique Sensitive and reliable low ultimate available confirmatory testing

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC 2.SEPARATION OF TRICYCLIC ANTIDEPRESSANTS ● The __ use of GC ● Used in excess as ___ in suicide patients ● Examples: ___ ● __ μg/ml causes toxicity and coma

greatest drugs of abuse Protriptyline, Desipramine, and Nortriptyline >10 to 55

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS GRADIENT ELUTION TECHNIQUE ●Is used in ____ ● ____ pumps with different compositions of the mobile phase are used to provide a continual ___ in the solvent strength ● This results in ___ separation because as the ___ of the solvent increases, peaks that have ___ will become ___ peaks

high performance liquid chromatography Two increase better concentration broadened sharp

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY PROCESS 1. Molecules containing ___ groups in an ___ solution are added to an ____ organic solvent 2. After ___ shaking, __ phases are allowed to separate. ● Once these 2 phases are allowed to separate, they are ___ 3. Polar molecules remain in the ____ . Nonpolar molecules are ____ in the____

polar and nonpolar aqueous immiscible vigorous 2 detected/visualized aqueous solvent extracted /dissolved - organic solvent.

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS SILICA ● Most common material used for ___ ● Very ___ ● Used as __ packing in___ chromatography ● Coated with ___ and is to be used as the __ phase in __ chromatography

column packing stable solid liquid-solid solvent stationary liquid-liquid

CHROMATOGRAPHY A group of techniques used to separate ___ mixtures on the basis of different ___ interactions between the ____ and the ___ phase of the system.

complex physical individual compounds stationary

CHROMATOGRAPHIC METHODS 6. GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) DETECTION ● Resulting electric currents are __ and ___ in a computer that analyzes the data ● __ patterns or ___ give rise to a "__ " pattern __ to the compound ● The patterns are stored in a computer so that __ patterns are compared with these patterns to identify compounds of interest

digitalized stored Decomposition fragmentogram fingerprint unique unknown

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PROCEDURE 1. A sample is ___ in an appropriate solvent ___ to ___ into the system. ___ must be removed in most cases to avoid ___ and the material used to dissolve the sample is selected to be ___ with the elution solvent used. 2. Then, a ___ moves the sample and elution solvent into the column. After which 3. the components of the mixture __ to the column packing by varying ___ , allowing constituents to ___

dissolved prior injection Proteins column damage, compatible high-pressure pump bind degrees of affinity separate

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY ADVANTAGES 1. No___ equipment required 2. ___ obtained with a fairly simple apparatus 3. Very __ method that detects few __ of a substance under suitable conditions

elaborate or expensive Good results or resolution sensitive - micrograms

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS Produce ___ proportional to the concentration of each separated component

electronic signals

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RETENTION VOLUME ● This is the method of __ the materials ___ in HPLC. ● Used to ___ compounds when compared with ___ retention volumes run under identical conditions

quantitating separated identify standard

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY PROCEDURE 1. Sample (≤___ uL) is introduced into the system and interacts with the material ___ inside the column. ● Take note that this solution containing the sample should be introduced as ___ as possible 2. Carrier gas such as ___ flows through the column to move the __ material down the system. 3. As temperature ___ in the temperature control column oven, more material is in the ___ phase to be swept through the column towards the detector.

≤ 1 coating quickly helium, argon, nitrogen, and hydrogen vaporized increases vapor

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RETENTION TIME If you're using retention time, you should take note of the following Keywords: ENUMERATE

○ Isocratic Elution ○ Early Eluting Compounds ○ Late Eluting Compounds

STRONG ADSORBENTS ENUMERATE

● Alumina ● Charcoal ● Silica gel ● Fuller's earth

MAIN CHROMATOGRAPHIC TYPES ENUMERATE

● Liquid-Solid ● Liquid-Liquid ● Gas-Solid ● Gas-Liquid

IMMEDIATE ADSORBENTS

● Magnesia ● Calcium Carbonate ● Calcium phosphate

WEAK ADSORBENTS

● Sucrose ● Inulin ● Talc ● Starch

USES OF ION-EXCHANGE CHROMATOGRAPHY Remove ____ ○ Ex: ___ wherein anion and cation resins are mixed together and are used to deionize water. ● ___ Ion Solutions ● Separate Mixtures of ___ ○ Can be used for the separation of mixtures of ___ and charged molecules. Ion-exchange chromatography can also be used for ___

Interfering Substances Concentrate Dilute water filtration Charged Molecules small organic and inorganic ions amino acid analysis and hemoglobin analysis.

MODES OF SEPARATION 1. ADSORPTION CHROMATOGRAPHY ● ____ chromatography ● ___ form of chromatography ● Based on the competition between the ___ and the ___ phase for ___ sites on the stationary phase ● Experimentally, it is easier than ___ since the ___ can be ___ and __ ____ ● It is preferred as a ____ method used for separating ___ quantities and to separate mixtures of solutes with widely varying ____ ● Not widely used in the ____

Liquid-solid Oldest sample mobile adsorptive partition chromatography adsorbent - constant solvents- vary preparative larger polarity and structure laboratory

Basic Components of Chromatography ENUMERATE

Mobile Phase Stationary Phase Column Eluate

CHROMATOGRAPHIC METHODS 1. ___ Chromatography 2. ___ Chromatography 3. ___ Chromatography 4. HPLC (__ ) 5. ___ Chromatography 6. GC-MS (___ )

Paper Column Thin Layer High Performance Liquid Chromatography Gas Gas Chromatography-Mass spectrometry

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY DEVELOPMENT ___ is removed from the ___ and allowed to __ or in a ___ : dried at an ___ temperature

Paper closed chamber dry suitably designed oven elevated

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY SUPPORT MEDIUM ● ___ (a cellulose of ___ Filter Paper) ● ___ (commercially available

Paper - high purity/Whatman #1 KIMTEC Chromatography Paper

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY - ___ chromatography ● Separates mixtures using a solvent that carries a solute up a ___ ● Primarily used as a ___ ● This has been __ by other chromatographic methods such as ___

Partition strip of paper teaching tool replaced thin layer chromatography

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS TYPES OF PUMPS FOR HPLC enumerate

Pneumatic Pump Syringe pump Mechanical Reciporcating pump Hydraulic Amplifier pump

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY ADVANTAGES OF TLC 1. ____ Method: generally takes ___ minutes to perform 2. __ range of sample size can be handled from ___ 3. ___ layers are used as the sorbent, therefore, more __ reagents can be used to visualize spots in comparison to ___ . 4. ___ are needed; readily and easily ___

Quick , 2-40 Wide , 0.5 to 500 micrograms Inorganic , reactive , paper chromatography Simple equipment , available

CLASS ACCORDING TO POLAR FUNCTION GROUPS MOBILE PHASE ● __ solvent ● Mixture of ___ (depending on the analytes to be ___ )

Single 2 or more solvents desorbed

Chromatography terms Adsorbent __ material that takes up the ___ of the substance passing through it Support A ___ base which supports the ___ in ___ chromatography Development A process where ___ phase is ___ to flow __ the ____

Solid particles solid liquid film partition mobile allowed over adsorbent or support

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS DIFFERENT TYPES OF DETECTORS ENUMERATE

Spectrophotometer Photodiode Array (PDA) Rapid Screening Detectors Fluorescence Detectors Amperometric/ Electrochemical Detectors

MOST COMMONLY USED SOLID SUPPORTS 1. Silica gels ● ___ adsorptive properties ● ___ is usually used ● It is almost always used with ___ or ____ as the stationary phase 2. Cellulose Powder ● Alternative to the use of ___ in the form of __ ● It is used in ___ as an alternative to cellulose in the form of sheets in paper chromatography so only ___ cellulose powder is used as it permits the separation of ___ quantities of materials 3. Diatomaceous Earth ● Commercially available as____

Strong 28-200 mesh water buffered aqueous solution cellulose sheets columns ashless large kieselguhr or celite

RESULTS IN THE PARTITIONING OF THE SOLUTE MOLECULES INTO 2 SEPARATE PHASES Take note: Since two liquids are immiscible, the solute molecules are partitioned into these 2 phases as previously stated. This is not however an ___ separation but it is based on r____ . The organic molecules with ___ sharing of electron pairs are ___ and therefore are ___ in water. Examples are: alcohols, ethers, carbonyl groups, anionic groups, and organic molecules are predominantly made up of ___ bonds with electrons ___ shared, therefore are ___ . The solubility of solute molecules in the 2 liquids is determined by how closely the relative polarities ____ .

absolute Relative polarity unequal less hydrophobic partially soluble covalent equally hydrophobic match

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY STATIONARY PHASE Layer of __ ● Generally called Adsorbent, even when it functions as a ___ for solvent in Partition Chromatography ___ types of adsorbents for Column Chromatography may be used to prepare thing layers, except that article sizes should be ___ ○ In a grain size of approximately____ MOBILE PHASE ● It may be a ____ or a____ ● It can also be an __ , which may be modified with ___ to adjust the pH ● This phase may also be a ___ organic solvent ○ Examples of which are: ■ ___ ■ ___ - Which are used to increase the ___ of components

adsorbent support All smaller 0-25 micra or 1.25 mm single organic solvent solvent mixture aqueous solvent ammonium hydroxide, or acetic acid water miscible Methanol Ethanol solubility

CHROMATOGRAPHIC METHODS 1. COLUMN CHROMATOGRAPHY ● Principle is based on ___ ● In its simplest form it . Consists of a solid medium packed into a ___ . After mixtures of solutes have been introduced in the column; ● ___ is passed over the column, the solutes are eluted, as the solvent __ through the column

adsorption column Solvent percolates

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY ELECTROMETER ●A device capable of __ the detector signals and presenting them to a __ showing the __ of the mixture. ● This would now be your ___ ● Electronic signals from the detector are used to produce __ information for both ___ calibrating techniques

amplifying strip chart recorder elution pattern chromatogram quantitative external and internal

Chromatography terms Eluate The ____ that emerges from the ___ Elution The process of ___ one material from another by __ with a __ Resolution ___ measure of how well ___ can be differentiated in a chromatographic separation

analyte material chromatograph extracting washing solvent Quantitative 2 elution peaks

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC 1. THERAPEUTIC DRUG MONITORING & SCREENING examples Barbiturates ● Derivative of ___ ● Frequently used for preventing __ ● Can also be used to___ in anesthesia ● A common drug that we use in anesthesiology has this effect, called __ ● ___ μg/ml causes __

barbituric acid seizures induce sleep phenobarbital >10 to 55 - toxicity and coma

MODES OF SEPARATION 4. ION-EXCHANGE CHROMATOGRAPHY Stationary Phase - Resin consists of large polymers of substituted ___ with charged functional groups that are ___ as ___ in the resin beads.

benzene, silicates, or cellulose derivatives, with charged functional groups immobilized side chains

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC 1. THERAPEUTIC DRUG MONITORING & SCREENING examples Chloramphenicol ● Toxic ____ ● Toxicity manifests as ____ (___ ) ● Reversible ___

broad spectrum antibiotic aplastic anemia -Gray Baby Syndrome bone marrow suppression

EARLY METHODS OF STERIC EXCLUSION 1. Gel Filtration Uses ____ beads of cross-linked ___ , ____ or____ , or ___ In gel filtration that uses sephadex, the beads of cross-linked ___ are used as stationary phase of a chromatographic system. Sephadex is a ____ , ____ material, and each granule has a complex system of ___ . In the case of Biogel P or ____ , they are made up of ____ and it is important to note that the above gels cannot withstand ___ pressure greater than __ psi. The materials that are resistant to pressures greater than 800 psi are ___ products and these are the most ___ size exclusion packing for use under pressure.

hydrophilic dextran or sephadex polyacrylamide gel Biogel P agarose gel dextran granular hydrophilic water-insoluble porous holes polyacrylamide sepharose agarose gels high 800 water silica-based common

CHARACTERISTICS OF A GOOD SUPPORT 1. It should be as ___ as possible to the substances to be separated 2. It should posses ___ 3. It should ___ and expose a ____ to the mobile phase 4. It should be ____ and should ____ solvent flow

inert adsorptive properties retain stationary phase large surface area mechanically stable impede

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' COLUMN Filled with ___ or particles (such as ____ ) coated with a thin layer of a ___(___) phase' ● The liquid stationary phase must be: ○ ____ ○ ___ stable, and ○ must not ___

inert solid diatomaceous earth or porous polymer or glass beads nonvolatile liquid , stationary nonvolatile thermally react chemically

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY THE DETECTION METHODS USED 1. REACTIONS WITH CHEMICAL AGENTS steps: 1. Involves exposing the plate to____ which 2. ___ areas containing components of the sample and 3. colors it____ 2. CHARRING THE SEPARATED SOLUTE Spraying the plate with a mixture of ___ and an __ which can be ___ 3. UV RADIATION Involves incorporation of ___ within the ___ layer and upon irradiation with a ___ ● The phosphor then ___ and the black spots against ____ is read.

iodine vapor dissolves brown or golden brown H2SO4 oxidizing agent potassium permanganate or potassium dichromate phosphor solvent UV lamp. glows fluorescing background

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) NORMAL PHASE HPLC Is done for ___ of___ ● Stationary Phase: Which is __ , an ___ substance. ● Mobile Phase: is Composed of ___ organic solvents such as: ○ __ ■ So this would include mixtures of ___ in varying proportions. ○ Hexane Please take note that the compounds of interest interact with the polar side chains of the column packing. So, as the polarity of the mobile phase increases, binding to the column packing ___

isolation isomers and biochemical molecules. Silica -acidic polar mildly polar Alcohol alcohol, acetic acid, and water decreases

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RETENTION VOLUME Low Retention Volume would indicate that: - Solute has ___ affinity for the stationary phase - sample is eluted ___ Greatest Retention Volume would indicate that -Solute has ___ affinity for the stationary phase - Sample is eluted __

least first highest last

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY PROCESS > MODERN PARTITION CHROMATOGRAPHY PARTITION SYSTEMS 1. Normal Phase - Mobile solvent is___ than the stationary phase 2. Reverse Phase - Mobile solvent is ____ than the stationary phase ○ Note: In the video she said mobile phase ● More useful in the separation of ____ 3. Support - ___ by the stationary phase ● ___ material that acts as support for the liquid

less polar more polar drugs and other nonpolar substances Coated Solid

CLASS ACCORDING TO POLAR FUNCTION GROUPS STATIONARY PHASE Acidic - ex.___ Basic - ex: ___ Non-Polar - ex- ____

lipid gel alumina charcoal

MODES OF SEPARATION 3. STERILE EXCLUSION Variation of____ chromatography ● Separates solute molecules based on their ____ ● Mobile phase ○ ___ solvent ● Stationary phase ○ ____ packed with gels that are in ___ containing a network of ___ in which___ may pass

liquid- solid size and shape Aqueous Chromatographic column beads pores small molecules

MODES OF SEPARATION 4. ION-EXCHANGE CHROMATOGRAPHY ● Separated by ____ of ionic species ● Separation is based on how the ___ and bind to the ____ stationary phase. It is a process wherein ions of the sample and the mobile phase exchange with the ions of the stationary phase. The ions with the greater charge are held most ___ and this bond can be controlled by ___ and/or the ion exchange ___ . It is important to note that there is a ___ exchange of ions. ● Reversible exchange of ions between solid and liquid without____ in the structure of the solid phase

magnitude and charge analytes are charged ionic exchange strongly changing the pH media reversible radical change

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY PROCEDURE 1. Drop of solution is placed on a __ position near one edge of a ___ 2. When the spot is dry, place paper in a __ with one end immersed in the ___ as the mobile phase. But not covering the spot where the ___ 3. By means of ___ action, __ percolates through the paper and moves the components of the mixture to __ extents in the direction of the flow.

marked strip or sheet of filter paper suitable closed container solvent chosen solution is applied capillary solvent different

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS PEEK HEIGHT RATIO ● Needed to __ the ___ and ___ ___ ● In the standard curve, the peak height ratio is plotted against the ___ of the analyte standard FORMULA ?

plot -standard curve analyze -unknown patient sample concentration Peak height ratio = height of analyte peak/ height of internal standard

MODES OF SEPARATION 2. PARTITION CHROMATOGRAPHY PROCESS > PARTITION COEFFICIENT The __ of the ___ of the solute in the 2 liquids ● Determines the ___ at which solute moves between the 2 phases ● Compounds most soluble in the mobile phase move through the column ___ , and are easily ____ . While compounds with high partition coefficients are ____ in the stationary phase. This means that a compound with high partition coefficient has a greater __ to this phase. ● Expressed as ___ ● K= ____

ratio concentration rate faster eluted retained affinity K solute in the stationary phase/ solute in the mobile phase

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY DISADVANTAGES OF TLC 1 Difficulty in ___ TLC chromatograms. 2 ___ of Rf (retention factor) values is not easily achieved with TLC. 3 Greater __ of plates over paper

recording and preserving Reproducibility cost

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS RECORDER ● The recorder in a high performance liquid chromatography system ___ the ___ versus the ___ through the instrument ● Peaks produced are ___ to the concentration of the compound that produced the peak

records detector signal time the mobile phase passed proportional

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY' IDENTIFICATION ● Done through ___ or __ at which the unknown solute ___ from a column ● If the unknown solute produces a ____ at the same ___ as that of a __ compound, then your ___ and reference compound are one and the same

retention time volume elutes peak, band or spot time, volume, or distance reference unknown solute

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY IDENTIFICATION formula ?

rf= dspot/s solvent

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PRINCIPLE OF HPLC (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY) ● It follows the concept of ___ of molecules onto a ___ phase followed by ___ with selected solvents. ● ___ phases are used to separate ___ compounds based on the ___ .

selective adsorption solid removal Selective liquid polar relative polarity

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS METHODS OF DETECTION 2. FLUORESCENCE MEASUREMENT ● Improves __ greatly thereby enhancing the ___ as much as a __ fold ● The principle behind this method is based on the fact that molecules ___ at___ wavelength and ___ light at a longer wavelength. ● More ___ than ___ methods

selectivity sensitivity 100 absorb light 1 re-emit sensitive photometric

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY ADVANTAGES OF TLC 5. Very ___ method (Lower limit of detection is ___ grams) or____ of compound 6. No ____ are required 7.Experimental parameters, such as ___ , are easily varied to affect ___ 8.Better ___ of substances

sensitive , 10-9 , 1 microgram per mL spectral manipulative skills sorbent systems and sorbent activity , separation resolution

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY TOXI LAB THIN LAYER CHROMATOGRAPHY (TLC) Has limited ___ -because the identification of each drug is dependent on the use of ___ or/and present of __ which is ____ to naked eye ● Used in confirming a ____ test result of basic and acidic drugs ● Guides ___ of a potential overdose patient ● Used for drug ____

sensitivity color changes fluorescence subjective positive immunoassay therapy screening and identification

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY APPLICATIONS OF GC 1. THERAPEUTIC DRUG MONITORING & SCREENING - It is used for measuring ___ of the ___ and general depressants like ___

serum concentrations antibiotic chloramphenicol barbiturates

4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS SLOW COMPOUNDS ● Great affinity for ___ ● Sometimes does not ___ with the use of ____ that separates the earlier bands ● The problem is then solved by using ____

stationary phase elude, solvent strength Gradient Elution Technique

CHROMATOGRAPHIC METHODS 3. THIN LAYER CHROMATOGRAPHY ADSORBENTS You may notice that these may usually act as ___ in Partition Chromatography, but when activated, thay have a higher absorption capability and can be used to separate ___ organic molecules. ○ Example of which: ___ ● ___ are used as supports in Partition Methods ● So these adsorbents must be ____ coated on a glass or plastic plate ● Alternative plates may be used, such as plates made up of __ materials ○ Generally, these contain a binder, either ___ , for the purpose of improving __ of the adsorbent to the plate

support less polar Alumina & Silica Gel Cellulose and Cross-Linked Dextran uniformly inert starch or calcium sulfate adhesion

CHROMATOGRAPHIC METHODS 2. PAPER CHROMATOGRAPHY SOLVENT REQUIREMENTS ● Avoid solvents that are ___ in composition (E.g., ___ ) ● ___ state of purity ● Not too ___ because of the need for more ___ equilibrium. Examples are ___ ● Avoid ___

variable - xylene and petroleum High volatile , meticulous - lower alcohols, ethers, and ketones. large temperature changes

CHROMATOGRAPHIC METHODS 4. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) BASIC COMPONENTS DETECTORS METHODS OF DETECTION 1. ABSORBANCE MEASUREMENT ● Remains to be the most ___ single test for HPLC ● ___ wavelengths allow identification of ___ compounds

versatile Multiple, different

CHROMATOGRAPHIC METHODS 5. GAS CHROMATOGRAPHY GAS-LIQUID CHROMATOGRAPHY Has evolved to become the most ___ analytical tool available ● Has the capacity of separating and measuring ____ amounts of volatile substances ● Separation of the components is due to distribution between the __ and the ___according to their ___

versatile and powerful nanogram and picogram gas phase stationary liquid partition coefficient (k)