Unit 3.2 Analytic Techniques: Electrophoresis, Chromatography

Analytic techniques

Scientific, laboratory methods used to measure the concentration of analytes in blood and other body fluids

electrical charge

Electrophoresis separates charged compounds based on their ___

cathode (-)

anode (+)

cations (+) move toward __

anions (-) move toward __

• paper

• cellulose

• acetate

• agarose gel

• polyacrylamide gel

• starch gel

[CAPPAGS]

Enumerate Support media for electrophoresis

PROTEINS in serum, urine, CSF, and other body fluids

Electrophoresis lab applications

Electroendosmosis/Endosmosis

movement of buffer ions and solvent relative to the fixed support

Iontophoresis

migration of charged small charged ions

zone electrophoresis

migration of charged macromolecules

Electrophoretogram

the result of zone electrophoresis

consists of sharply separated zones of macromolecules

• Power supply – provides electric current

• Support medium

• Buffer – to maintain pH

• Sample

• Detecting system – quantitates the separated fraction of particles

Enumerate Components of Electrophoresis

Densitometer

quantitate the separation of protein fractions in electrophoresis

Electrophoretogram

visual representation of separated protein fractions

1. Amido black

2. Ponceau S

3. Oil red O

4. Sudan Black B

5. Fat Red 7B

6. Coomassie Blue

7. Gold/Silver stain

[APOSFCG]

Enumerate Stains for visualization of fractions in electrophoresis

Gold/Silver stain

very sensitive electrophoresis stain;

even in nanogram quantities of proteins

Densitometric scan

___ gives a graph showing the concentration of different proteins (globulins) in an electrophoretogram.

• ALP isoenzyme analysis

• CK isoenzyme analysis

Electrophoresis Lab applications

ALP (alkaline phosphatase) isoenzyme analysis

Electrophoresis is considered the most useful single technique for ___

(cathode) Intestinal → Placental → Bone → Liver (anode)

Migration direction of ALP in isoenzyme analysis using electrophoresis

CK BB (CK1)

In CK isoenzyme analysis, what is the fastest migrating substance?

CK MB (CK2)

In CK isoenzyme analysis, what is the second fastest migrating substance?

CK MM (CK3)

In CK isoenzyme analysis, what is the slowest migrating substance?

CK Macro

In CK isoenzyme analysis, what is the atypical enzyme?

cathodal

In CK isoenzyme analysis, CK Mi is band ____ only (did not migrate at all)

Osmometry

the measurement of osmolality of an aq. solution such as serum, plasma, or urine

• Glucose

• urea nitrogen

• sodium

[GUNS]

Ex. of osmotically active particles

Osmometer

device that measure the freezing point depression or vapor pressure depression of samples

milliosmolal per kilogram (mOsm/kg)

Unit used in osmometry

Sodium

Main omsmolality contributor

Osmolality

the concentration of dissolved (osmotically active) particles per kilogram of solvent.

↑ Plasma/Serum Osmolality

↑ Sodium concentration in blood

↑ thirst sensation

↑ ADH secretion (activated)

Explain the effect of INCREASED plasma/serum osmolality on sodium concentration, thirst sensation, and ADH secretion

↓ Plasma/Serum Osmolality

↓ Sodium concentration in blood

↓ thirst sensation

↓ ADH secretion (suppressed)

Explain the effect of DECREASED plasma/serum osmolality on sodium concentration, thirst sensation, and ADH secretion

Thirst sensation

body’s mechanism to dilute the concentrated sodium ions in the blood

directly proportional; directly proportional

↑ Freezing point depression

↑ no. of solute particles

↑ osmolality

Freezing point depression is _____ to the No. of solute particles and ____ to osmolality

• Chamber

• Sample container

• Steering wire

• Thermistor

• Read out device

• Refrigerant inlet & outlet ports

Enumerate Components of a Freezing point osmometer

Stirring wire

Components of a Freezing point osmometer:

• Stirs the sample to ensure an even temperature and initiates (starts) freezing by creating tiny ice crystals.

Thermistor

Components of a Freezing point osmometer:

• A sensitive temperature sensor that detects the sample's freezing point.

Readout device

Components of a Freezing point osmometer:

• Calculates and displays the osmolality in mOsm/kg based on the freezing point.

Refrigerant inlet and outlet ports

Components of a Freezing point osmometer:

• Circulate the cooling fluid that rapidly cools the sample; regulates cooling of the spx

Rapid cooling → Supercooling → Stirring starts freezing → Thermistor measures freezing point → Readout converts freezing point depression to osmolality (mOsm/kg).

Explain how a Freezing Point Osmometer works (process)

Chromatography

a separation method based on different interactions of the specimen compounds with the mobile phase and with the stationary phase as the compounds travel through a support medium

• Mobile phase (gas or liquid)

• Stationary phase (solid or liquid)

• Column

• Eluate

[MECS]

Enumerate components of Chromatography

mobile phase

Components of Chromatography

• carries the sample

• can be GAS or LIQUID

Stationary phase

Components of Chromatography

• which mobile phase flows

• can be SOLID or LIQUID

Column

Components of Chromatography

• holds the stationary phase

eluate

Components of Chromatography

• separated components

• Adsorption

• Partition

• Steric exclusion

• Ion exchange chromatography

[SIPA]

Enumerate Modes of Separation for Chromatography

Adsorption

Modes of Separation for Chromatography:

• separation based on polar functional groups

Liquid-Solid Chromatography

Adsorption a.k.a. __

• Mobile Phase = liquid

• Stationary phase = solid (usually polar)

Adsorption is called Liquid-Solid Chromatography because?

• furthest

• slowest

In adsorption:

• Most soluble in the mobile phase (polar) = move ___

• Least soluble in the mobile phase = move ___

Partition

Modes of Separation for Chromatography:

• based on relative solubility in an organic (nonpolar) solvent and aquaeous (polar) solvent

Liquid-Liquid Chromatography

Partition a.k.a. ___

• Polar molecules = Aqueous solvent

• Nonpolar molecules = Organic solvent

[PANOt]

In partition:

• Polar molecules = ___

• Nonpolar molecules = ___

Steric Exclusion

Modes of Separation for Chromatography:

• a variation of liquid-solid chromatogrpahy

• separate solute based on size and shape

• funnel-like

Column (packed with porous material)

In Steric exclusion, what component traps the small molecules while excluding large and intermediate-sized molecules

Ion-Exchange chromatography

Modes of Separation for Chromatography:

• separation based on the magnitude and charge of ionic species

Resin with charged functional groups

(cation-exchange & anion-exchange resins)

What is the stationary phase in Ion-Exchange Chromatography

• TLC (thin layer chromatography)

• HPLC (high-performance liquid chromatography

• GC (gas chromatography)

• Paper chromatography

Enumerate Chromatographic Procedures

• alumina

• silica gel

• cellulose

• cross-linked dextran

[CCAS]

sorbents used in TLC

glass/plastic plate

In TLC, a thin layer of sorbent is uniformly coated on a _______

spot ;

plate

In TLC, each sample to be analyzed is applied as a ___ near one edge of the ___

Retention factor (Rf)

the distance leading edge of components moves / total distance solvent front move

semi-quantitative drug screening test

TLC application

HPLC (high-performance liquid chromatography)

uses pressure for fast separations

• Sample Injector (loop injector, syringe)

• Chromatogram

Ex of HPLC

loop injector

best and most used HPLC

• Reservoir

• Pump

• Column

• Detector

[Role Play Cum Dick]

Enumerate components of HPLC

Reservoir

Components of HPLC:

• holds the mobile phase;

• where the sample is being placed

Pump

Components of HPLC:

• forces the mobile phase from the reservoir to the column

Column

Components of HPLC:

• stationary phase;

• where separation happens

Detector

Components of HPLC:

• records the differences in separation

Chromatogram

Components of HPLC:

• final result;

• a graph showing the differences in separation

GC (gas chromatography)

best for compounds that are naturally volatile or can be easily converted into a volatile form

can be gas-solid or gas-liquid chromatography

gas-solid ;

gas-liquid

GC can be a ___ (stationary phase is solid) or ___ (stationary phase is liquid) chromatography

carrier gas

It is called gas chromatography because we need a ___ to volatilize the sample

• cylinder

• gas regulator

• needle valve

• syringe

• septum (injector port) & heater

• column oven

• detector oven

• concentration detector

• recorder

[CGe Na SiS Calm Down Ca Right meow]

Enumerate Components of GC

Cylinder

Components of GC:

• mobile phase that supplies inert gas (helium, argon, nitrogen)

• volatilize the sample

Gas regulator & Needle valve

Components of GC:

• controls the pressure and flow rate of the carrier gas passing through

Syringe

Components of GC:

• introduces the sample to the septum

Septum (injector port) and heater

Components of GC:

• makes sure the sample is volatilized/vaporized

Column oven

Components of GC:

• where mobile phase flows

Detector oven

Components of GC:

• stabilizes the temperature that will go to the concentration detector

Concentration detector

Components of GC:

• Measures concentration of the analyte of interests

Recorder

Components of GC:

• creates the chromatogram

Paper chromatography

technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper

fractionation sugar and amino acids

Paper chromatography applications

Paper chromatography

Place filter paper onto a container > immerse one edge of the paper on the solvent > wait for the movement of solvent upwards the paper

T

T or F

Just like TLC, we can use the same materials for Paper Chromatography (i.e., silica gel, cellulose, cross linked dextran)

Mass Spectrophotometer (MS)

Definitive identification of samples eluting from GC or HPLC columns is possible when a ____ is used as a detector

Gas chromatography = GC/MS

Liquid chromatography = LC/MS

MS must be coupled with other analytic techniques such as what?

mass-to-charge (m/z) ratio

In MS, compounds are measured based on their ___

• Sample inlet

• Ionization source

• Mass analyzer

• Ionization detection

[oh SIMION!]

Enumerate Components of MS

sample inlet

Components of MS

• where spx is introduced

Ionization source

Components of MS

• spx is initially volatilized by a carrier gas and heat

• then ionized to form charged molecules and fragments

Mass analyzer

Components of MS

• where separation of fragments based on mass-to-charge ratio happens

Ionization detection

Components of MS

• measures the analyte of interest by looking at its m/z ratio

• GC-MS: Electron ionization (EI)

• LC-MS: Atmospheric pressure ionization (API)

  • Electrospray ionization (ESI)

  • Atmospheric Pressure Chemical Ionization (APCI)

  • Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF)

Enumerate ionization sources for MS

EI (electron ionization)

most common form of ionization in GC-MS

requires a source of electrons in the form of a filament to which an electrical potential is applied

70 eV (electron volts)

electrical potential generated by electronic ionization

Atmospheric pressure ionization (API)

ionization source for LC-MS