Analytical Chem Week 10 - Chromatography^j Electrophoresis And Quality Control.pdf

Chromatography

Chromatography

• Chromatography is the separation of the components of a mixture based on the different degrees to which they interact with two separate material phases

Mobile phase – moving phase

Chromatography Stationary phase – does not move

Chromatography • The mixture (sample) is usually introduced into the mobile phase, which is then made to move through the stationary phase. • The components are attracted to and slowed by the stationary phase to varying degrees, and as a result, they move along with the mobile phase at varying rates, and are thus separated

Chromatography • The mobile phase can be either a gas or a liquid, while the stationary phase can be either a liquid or solid.

Chromatography

• All techniques which utilize a gas for the mobile phase come under the heading of gas chromatography. All techniques that utilize a liquid mobile phase come under the heading of liquid chromatography

Partition Chromatography • The mobile phase is a liquid that moves through a liquid stationary phase as the mixture components partition or distribute themselves between the two phases and become separated.

• The separation mechanism is thus one of the dissolving of the mixture components to different degrees in the two phases according to their individual solubilities in each.

Partition Chromatography • The stationary phase actually consists of a thin liquid film chemically bonded to the surface of finely divided solid particles • Such a stationary phase cannot be removed from the solid substrate by heat, reaction, or dissolving in the mobile phase.

The mobile phase for partition chromatography can also be a gas.

Partition Chromatography

In summary Partition chromatography is a type of chromatography in which the stationary phase is a liquid chemically bonded to the surface of a solid substrate, while the mobile phase is either a liquid or gas. The mixture components dissolve in and out of the mobile and stationary phases as the mobile phase moves through the stationary phase, and separation occurs as a result

Adsorption Chromatography The separation mechanism is adsorption

Adsorption Chromatography • The stationary phase consists of finely divided solid particles packed inside a tube, and the mixture components adsorb or stick to the surface of the solid

The nature of the adsorption involves the interaction of polar molecules, or molecules with polar groups, with a very polar solid stationary phase.

Adsorption Chromatography The mobile phase can be either a liquid or a gas

Ion Exchange Chromatography It is a method for separating mixtures of ions, both inorganic and organic.

Ion Exchange Chromatography • The stationary phase consists of very small polymer resin beads that have many ionic bonding sites on their surfaces. These sites selectively exchange ions with certain mobile phase compositions as the mobile phase moves. • Ions that bond to the charged site on the resin beads are thus separated from ions that do not bond

Ion Exchange Chromatography

The stationary phase material can be either an anion exchange resin, which possesses positively charged sites to exchange negative ions, or a cation exchange resin, which possesses negatively charged sites to exchange positive ions.

The mobile phase can only be a liquid.

Ion Exchange for Deionization of Water

• Wide columns packed with a mixture of an anion exchange resin that exchanges dissolved anions for hydroxide ions, and a cation exchange resin that exchanges dissolved cations for hydrogen ions are used because water that is passed through such a column becomes free of ions (deionized) since the hydrogen and hydroxide ions combine to form more water.

Ion Exchange for Deionization of Water

Size Exclusion Chromatography It is a technique for separating dissolved species on the basis of their size

Size Exclusion Chromatography • The stationary phase consists of porous polymer resin particles. The components to be separated can enter the pores of these particles and be slowed from progressing through this stationary phase as a result.

• The mobile phase for this type can only be a liquid

Chromatography Configurations Configurations can be broadly classified into two categories: Planar methods and the column methods

Planar Method

• The planar methods utilize a thin sheet of stationary phase material and the mobile phase moves across this sheet, either upward (ascending chromatography), downward (descending chromatography), or horizontally (radial chromatography).

Paper Chromatography

• Paper chromatography makes use of a sheet of paper having the consistency of filter paper (cellulose) for the stationary phase. • Since such paper is hydrophilic, the stationary phase is actually a thin film of water unintentionally adsorbed on the surface of the paper. • Thus, paper chromatography represents a form of partition chromatography only. • The mobile phase is always a liquid.

Thin Layer Chromatography • The stationary phase is a thin layer of material spread across a plastic sheet or glass or metal plate. • TLC can be any of the four types, including adsorption, partition, ion exchange, and size exclusion • The mobile phase for TLC is always a liquid.

Thin Layer Chromatography

• The visual examination of the chromatogram can reveal the identities of the components, especially if standards were spotted on the same paper or plate.

• Retardation factors (so-called Rf factors) can also be calculated and used for qualitative analysis.

Thin Layer Chromatography • These Retardation factors, which are fractions less than or equal to 1, are compared to those of standards to reveal the identities of the components.

Thin Layer Chromatography

• Quantitative analysis is also possible. The spot representing the component of interest can be cut (in the case of paper chromatography) or scraped from the surface (TLC), dissolved, and quantitated by some other technique, such as spectrophotometry.

Column Method

• Column methods utilize a cylindrical tube to contain the stationary phase, and the mobile phase moves through this tube either by gravity, with the use of a high pressure pump, or by gas pressure

Open Column Chromatography • A type of chromatography that consists of a vertically positioned glass tube in which the stationary phase is placed

• It is typical for this tube to be open at the top (hence the name open-column chromatography), to have an inner diameter on the order of a centimeter or more, and to have a stopcock at the bottom • All four types (adsorption, partition, ion exchange, and size exclusion) can be used with this technique.

Open Column Chromatography • The mixture to be separated is placed at the top of the column and allowed to pass onto the stationary phase by opening the stopcock. • The mobile phase is then added and continuously fed into the top of the column and flushed through by gravity flow. The mixture components separate on the stationary phase as they travel downward and are then collected as they elute from the column.

Open Column Chromatography • A typical fraction collector consists of a rotating carousel of test tubes positioned under the column such that fractions of eluate are collected over a period of time

Instrumental Chromatography A column along with a continuous mobile phase flow system (that does not use gravity for the flow), a device for introducing the mixture to the flowing mobile phase, and an electronic sensor at the end of the column incorporated into a single unit

These are Gas chromatography and high-performance liquid chromatography.

Add a degree of efficiency and speed to the chromatography concept.

Electrophoresis A separation technique that utilizes an electric field

Electrophoresis

An electric field is an electrically charged region of space, such as between a pair of electrodes connected to a power supply.

The technique utilizes the varied rates and direction with which different organic ions (or large molecules with charged sites) migrate while under the influence of the electric field.

Electrophoresis

• A power supply is needed for connection to a pair of electrodes to create the electric field. The medium (cellulose or gel) and sample to be separated are positioned between the electrodes

Electrophoresis Electrophoresis is for separating ions, since only ions will migrate under the influence of an electric field, negative ions to the positive electrode and positive ions to the negative electrode

Useful in biochemistry experiments

Principles of Separation 1. Ions of opposite charge will migrate in different directions and become separated on that basis

2. Ions of like charge, while migrating in the same direction, become separated due to different migration rates • Factors influencing migration rate are charge values (e.g., –1 as opposed to –2) and different mobilities. The mobility of an ion is dependent on the size and shape of the ion as well as the nature of the medium through which it must migrate

Paper Electrophoresis

• The soaked cellulose sheet is sandwiched between two horizontal glass plates with the ends dipped into vessels containing more electrolyte solution. • The electrodes are also dipped into these vessels. • The sample is spotted in the center of the sheet, and the oppositely charged ions then have room to migrate in opposite directions on the sheet

Paper Electrophoresis • Qualitative analysis is performed much as with paper chromatography, by comparing the distances the individual components have migrated to those for standards spotted on the same sheet.

Gel Electrophoresis • The thin gel slab is contained between two glass plates. The slab is held in a vertical position and has notches at the top where the samples to be separated are spotted or streaked. • Only downward movement takes place, and thus only one type of ion, cation or anion, can be separated, since there is only one direction to go from the notch.

Gel Electrophoresis A tracking dye can be added to the sample so that the analyst can know when the experiment is completed

The slab can be removed from the glass plates and a staining dye can be applied that binds to the components, rendering them visible

Components with different mobilities through the gel show up as different bands or streaks on the gel.

Qualitative analysis is performed as with paper electrophoresis—standards are applied alongside the samples and the components identified by their positions relative to the standards

Isoelectric Focusing a useful extension of basic gel electrophoresis in protein analysis

In this technique, a series of ampholytes is placed on the slab via electrophoresis

An ampholyte is a substance whose molecule contains both acidic and basic functional groups (solution of different ampholytes have different pH values) Different ampholyte molecules differ in size and therefore will have varying mobilities in the electrophoresis experiment

Isoelectric Focusing Thus, these molecules migrate into the slab, take up different positions along the height of the slab, and create a pH gradient through the height of the slab. Amino acid molecules have different mobilities in different pH environments and also have their charges neutralized at particular pH values, rendering them immobile at some position in the gel

Mixture components migrate to a particular location in the gel.

Isoelectric Focusing

• The pH at which the sample component is neutralized is called the isoelectric point, and this technique is called isoelectric focusing, since samples are separated according to their components’ isoelectric points.

Capillary Electrophoresis • It is electrophoresis that is made to occur inside a piece of smalldiameter capillary tubing • The tubing contains the electrolyte medium, and the ends of the tube are dipped into solvent reservoirs

• An electronic detector is on-line and allows detection and quantitative analysis of mixture components.

Quality Control

To check the stability of the machine

Objectives of Quality Control

To check the quality of reagents

To check technical (operator) errors

Quality Control Quality control is a system of ensuring accuracy and precision in the laboratory by including quality control reagents in every series of measurements

It is a process of ensuring analytical results are correct by testing known samples that resemble patient samples

Involves the process of monitoring the characteristics of the analytical processes and detects analytical errors during testing, and ultimately prevent the reporting of inaccurate patient test results

Parameters of Quality Control

Sensitivity

Specificity

Accuracy

Precision or Reproducibility

Parameters of Quality Control

Practicability – degree by which a method is easily repeated

Reliability – ability of an analytical method to maintain accuracy and precision over an extended period of time during which equipment, reagents and personnel may change

Diagnostic Sensitivity

Diagnostic Specificity

Diagnostic Sensitivity

• It is the ability of the analytical method to detect the proportion of individuals with the disease • It indicates the ability of the test to generate more true-positive results and few false-negative • Screening tests require high sensitivity so that no case is missed Sensitivity (%) =

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ 𝑎 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑡𝑒𝑠𝑡𝑒𝑑

x 100

Diagnostic Specificity

• It is the ability of the analytical method to detect proportion of individuals without the disease • Reflects the ability of the method to detect true negatives with very few false positive • Confirmatory tests require high specificity to be certain of the diagnosis Specificity (%) =

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒 𝑤𝑖𝑡ℎ 𝑎 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑡𝑒𝑠𝑡 x 100 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙𝑠 𝑡𝑒𝑠𝑡𝑒𝑑 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑡ℎ𝑒 𝑑𝑖𝑠𝑒𝑎𝑠𝑒

Diagnostic Specificity and Sensitivity

• 100% sensitivity and specificity indicate that the test or method DETECTS every patient WITH the disease and that the test is NEGATIVE for every patient WITHOUT the disease

Kinds of Quality Control

Intralab Quality Control (Internal QC)

Interlab Quality Control (External QC)

Involves the analyses of control samples together with patient specimens

Intralab Quality Control

Detects changes on the performance between the present operation and the “stable” operation

It is important for the daily monitoring of accuracy and precision of analytical methods

Resembles human sample

Inexpensive and stable for long periods

Characteristics of an Ideal QC material

No communicable diseases

No matrix effects

With known analyte concentration

Convenient packaging for easy dispensing and storage

Interlab Quality Control It involves proficiency testing programs that periodically provide samples of unknown concentrations to participating clinical laboratories

It is important in maintaining long-term accuracy of the analytical methods

Conduct of External QC Testing • A series of unknown samples are sent to the laboratory from the reference laboratory or authorized program provider

• Unknown samples must be tested by the laboratorians who regularly perform analysis of patient specimens using the same reagents and equipment for actual patient specimens, and the results are submitted to the program provider

Rationale of the External QC/Proficiency Testing • To ensure clinicians that patient results are accurate • Allows each laboratory to compare and evaluate test results or outcomes with those laboratories that used the same methods (reagents and equipment)