Forman Christian College University

(A Chartered University)

Name Roll Number Title Course Semester Submitted on Submitted to

Hadeel Naeem 12-10120 Analytical Techniques for Biomolecules Analytical Techniques in Biotechnology Fall 2011 20th January, 2012 Dr. Kanwar Shoaib

The study of biomolecules require efficient analytical techniques so that biomolecules complexes from cell cultures, fermentation broths etc. can be extracted, separated and analyzed thoroughly in laboratories. The techniques used for analyses keeps improving and developing for better and closer results that can help scientists draw accurate results from their research. Chromatography Chromatography is a technique used to isolate the various components of a mixture and this makes its application in analysis of biomolecules very important. It is used to separate and analyze the complex DNA sequences and other compounds and also the concentration of the samples. There are many types of chromatography used in the study of biomolecules which range from DNA/RNA to recombinant proteins and antibodies etc. Types of Analysis Liquid Chromatography Liquid chromatography is a type used in biomolecules analysis and the mobile phase in this type of chromatography is liquid. There are many types of liquid chromatography: High Performance Liquid Chromatography (HPLC) Small particles and High pressure is required to carry out this type of liquid chromatography. HPLC has many forms and its application revolves around drug analysis and other forensic applications. There are forms of HPLC which specifically deal with enzymology and purification of other biomolecules. The reversed phase chromatography has a larger application in bio industry. In this the stationary phase is non-polar, while the solvent or mobile phase used is polar which is opposite to normal chromatography where stationary phase is polar and the mobile phase is non-polar. The advantage of reverse phase liquid chromatography is that it allows the separation of a large

variety of samples, with a wide range of molecular weights and polarities involved. It is easy to use and results are attained rapidly. Fast Protein Liquid Chromatography FPLC is also a form of liquid chromatography and it specializes in separating proteins from complexes as the name suggests. FPLC is popularly used in enzymology, with a complete setup designed especially for separation of proteins and other biomolecules. Cross linked agarose beads are used the stationary phase in this type of liquid chromatography. Aqueous- Normal Phase Chromatography This type of chromatography has a special feature, it has a mobile has which is somewhere between polar and non-polar. The mobile phase is based on organic solvent with a small amount of water which results in it being semi polar and semi non-polar. Affinity Chromatography This type is again used in the purification of proteins which are bound to tags. The proteins being analyzed are marked or labeled with compounds like antigens or biotins. The reason of doing this is so that these proteins bind to the stationary phases specifically. To get pure protein in the end, the labels are removed; the labels are just there to provide accurate separation of proteins. The mechanism uses a property of biomolecules i.e. affinity for metals, hence various metals are used in the chromatography columns. Immobilized Metal Affinity Chromatography is an advanced and much refined version of affinity chromatography used in identification of biomolecules these days.

Ion-Exchange Chromatography Ion exchange chromatography relies on the ionic charges on the compounds to separate it and it used widely in identification and separation of biomolecules. The charges on the resin plotted and the charge on the proteins being analyzed make up the basis for ion chromatography. In most cases, the positively charged ions of ionized biomolecules bind with the negatively charged resin or vice versa. The solutes are bound, and then washed with eluents buffers, the pH of these eluents buffers are varied to give the pure separated form of protein that is required. Size Exclusion Chromatography This type of chromatography is also known as gel-filtration or gel-permeation chromatography, the technique used in this type of chromatography is porous particles, which help to separate molecules according to the sizes. It is important an important analytical technique for biomolecules as it not only separates biomolecules, it also determines molecular weight and molecular weight distribution of polymers. The total transit time for the particles to move through the column varies with respect to their size. Also, some part of the chromatogram is

selectively permeable and it allows only small molecules to pass, those which are smaller than the pores.

Hydrophilic Interaction Liquid Chromatography (HILIC) The mobile phase for this type of chromatography can be partially aqueous and it is a type of liquid chromatography. Its main application is to separate biomolecules, and it includes the normal separation of carbohydrates, histones, peptides, polar lipids, nucleic acids, many types of proteins and membranes. It separates all the biomolecules using the property of their polarity. HILIC is more sensitive than reverse phase chromatography and sample preparation for it is also simpler. Chiral Chromatography This type of chromatography is used to separate stereoisomers, and for this either the mobile phase or the stationary phase must be chiral. Chiral Chromatography is now widely being used in industries to separate chiral substances, and this type of chromatography is used in both form; chiral liquid chromatography as well as chiral gas chromatography. Chiral chromatography has a large scale application in pharmaceutical industry as it is cost effective and efficient. Many biomolecules are chiral and hence it is an important analytical technique, chiral substances cannot be separated by any other type of chromatography. The reason of its popularity is that it gives high optical purity and excellent yield.

Range of Analytical Capability Chromatography targets separation and purification. Many biomolecules fall under the range of analytical capability of chromatography as an analytical technique. The main focus of chromatography is on analytical and preparative separation of proteins/amino acids. Other biomolecules include nucleic acids, carbohydrates, and vitamins etc. The most common application of chromatography when it comes to bio industry is the production of therapeutic molecules, which are proteins basically, purified and separated to be used in biopharmaceuticals. Some types of chromatograph make use of polarity of biomolecules for the separation mechanism and hence compound for example hydrophobic pigments, Lipid pigments, hydrophobic proteins, Chlorophyll pigments, Phycobilins proteins, all can be identified by chromatography. Advantages and Disadvantages of Chromatography as an Analytical Technique for Biomolecules Chromatography as an analytical technique has a number of advantages; the main advantage is that very minute amount of sample is required, only a few micrograms of any complex mixture are required to get the desired results. Analyses done by chromatography is sensitive and reliable, you can collected the separated components individually. The equipment is easy to use and takes less time. HPLC especially has a hand over all the former means of analyses, it show results in a matter of minutes and this is because of the high speed pumps which force compounds through the tubing. The results determined by HPLC have a great advantage of being easy to read, they are displayed in high resolution. This technique is effective for even very low concentration of samples.

The main disadvantage associated with it is that since it is so sensitive, a slight mistake, improper set up or contamination even in nanograms will affect the results and ruin the analysis. The samples used for chromatography make take a long time to prepare properly, it is time consuming as the interaction between the protein required and ligand has to be carefully determined. Another disadvantage of chromatography is that very small amount or concentration of sample is used, hence not enough separated compound is left for further reactions or analysis. On a commercial level, initial installation of equipment for chromatography is expensive. Also, compounds which leave the tubing at the same time are very difficult to detect and for such compounds chromatography is not the most suitable method of analysis. Application in Agriculture, Health and Environment Sectors Chromatography plays an important role on the commercial level especially in the pharmaceutical industry and clinical studies. In the agricultural sector chromatography plays a role as in analytical technique to analyze active compounds like fungicide in soil. Analysis of neutral carbohydrates and other inorganic compounds for better and controlled growth of crops is carried out. When it comes to the identification and quantification of pollutants in the environment, gas chromatography is the most powerful analytical tool used. Organic contaminants in air, water and soils are sampled and checked using art capillary gas chromatography for very accurate and precise result. In the public health sector, chromatography is applied for testing food and beverages for toxicity. Ion-exchange chromatography is preferred most by laboratories in food industries to detect bacterial contamination. Pharmaceutical industry used chromatography to prepare the most

suitable medicines using the appropriate compound separated from complex mixtures of biomolecules. Chiral chromatography holds a lot of importance in making medicines.

Centrifugation This technique makes use of the size and weight of biomolecules to separate them for analyses. The equipment used for centrifugation is called a centrifuge and inside the centrifuge the samples are rotated and the separation takes place due to varying sedimentation rate of different compounds. For results we first make sure that all other factors are constant, because when all other factors are constant the rate of sedimentation would be directly proportional to size of the molecules or its molecular weight. Types of Analysis There are a number of types of centrifugation used for analysis of biomolecules, namely ultracentrifugation, zone centrifugation, micro centrifugation, Isopycnic centrifugation and differential centrifugation. Micro centrifugation This type of centrifuge is used in laboratories to analyze very small biological mixtures like a cell or nuclei. The tubes inside micro centrifuge have a very low capacity of handling very minute amount of the sample. The tubes are rotated at an angular speed of 12000-13000 rmp.

Ultracentrifugation Ultracentrifugation is an analytical technique applied to analyze biomolecules by disassembling the biochemical complexes into subunits. Through this sedimentation process the hydrodynamic and thermodynamic information of any type of biomolecule can be analyzed. For ultracentrifugation very high centrifugal force is applied to the centrifuge, and the advantage of such high centrifugal force is that much smaller biomolecules can be separated and studied. Ultracentrifugation goes beyond the cell itself and helps isolate even smaller structures like

ribosomes and viruses. The angular speed of rotation of ultracentrifugation is around 70000 rmp. Ultracentrifugation is used in the characterization of macromolecule, their shape, mass etc.

Isopycnic centrifugation This type of centrifugation separates molecules on the basic of equal density. The particle being centrifuged will sink until it reaches the level where its density is exactly equal to the density of the surrounding solution. The particle will stay there and wont migrate anymore and hence particles of the same density are easily isolated. It is important that the densities of the sample used must lie within the limits of the gradient densities and enough time must be given for this isolation to take place and settle completely. Zonal Centrifugation This is similar to Isopycnic centrifugation but zonal centrifugation uses the equal mass or size of the particle compared to the surrounding solution instead of similar density. It is important for those biomolecules which have similar densities but different masses, especially the antibodies which cannot be isolated using the Isopycnic centrifugation. For zonal centrifugation, time is an important factor and it cannot be ignored, if the process is allowed to run longer than appropriate, the particle will eventually start settling at the bottom. Range of Analytical Capabilities

Centrifugation is a technique widely used in microbiology and other analytical studies of macromolecules etc. The range of analytical capabilities of centrifugation as a whole are vast, it extends from the isolation and separation of cells and viruses to further separation of subcellular organelles and macromolecules. These subcellular organelles include mitochondria, lysosomes, cystol, ribosomes and also various microsomes. In macromolecules, analyses of DNA, RNA, proteins and lipids is common at a large scale. Various antibodies are isolated and categorized using centrifugal techniques. Advantages and Disadvantages of Centrifugation as an Analytical Technique for Biomolecules Centrifugation is an important technique which preserves the isolated form of biomolecules required so that they can be used further. This method is easy to use and handle the isolation of liquid fractions on basis of their density is way easier than other techniques, and hence it can be repeated over and over for many samples, without wasting a lot of time. In short sample preparation for this technique is easy and quick. Centrifugation is preferred for smaller particles, as small as the subcellular organelles, and if the goal is to just collect an isolated sample of a particular particle from a mixture and not to analyze the composition of the mixture itself, then this technique is preferred above all others. The smaller the particles, the more favorable centrifugation technique becomes for its analysis. Centrifugation has a number of drawbacks associated with it too. The main problem is that particles with identical densities or particle size cannot be separated by centrifugation. This puts this technique to a limit. Also cost of maintenance of centrifuges is also high. The heat produced during the process can cause denaturation of proteins being analyzed. Another disadvantage that

cannot be ignored is that centrifugation technique provides incomplete separation of the particles, the process has to be repeated to get purer yield but that reduces the yield considerably too. Application of Centrifugation in Agriculture, Health and Environment Sector Centrifugation has a lot of applications in the health, environment and agriculture sector. Recycling which is an important solution to environmental problems can be dealt with using centrifugation technique. In environmental and agriculture, the main application of centrifugation technique is in form of decanter centrifuge. This centrifuge is used to purify the ground water from various pollutants (organic and inorganic), which sediment at the bottom of the water so that it is safe to use. For better growth of plants, preparation of manure which an organic fertilizer is done using centrifugation. In health sector this centrifugation contributes in research and testing of food products for possible contamination. Centrifuged biomolecule particles, are used in new developing technologies like in vitro fertilization etc. Spectrophotometry Spectrophotometry is an analytical technique used to study and analyze chemicals with the help of an instrument called the spectrophotometer. A spectrophotometer measures the amount of light (of specified wavelength) that passes through a transparent medium. This method is effective in the study of molecular biology. A spectrophotometer can identify the particles present in a mixture, and also calculate in what amount it is present. Light is focused on the sample being analyzed, this light is reflected from the sample and it falls through the monochromator. The monochromator then splits the different wavelengths of light and focuses them individually on a photo detector.

Types of Analysis UV-visible spectroscopy This spectrometer as the name suggests works for those wavelengths which lie in the UV or visible range of the spectrum. Very sample samples of biomolecules, like RNA, DNA and proteins can be analyzed and quantified using the UV-visible spectrophotometer. Nano UV-visible spectrophotometry This technique is more popular in the area of biomolecules analysis. And the main advantage of spectrophotometry is that is not only identifies the biomolecules, it also tells us how much amount of it is present. This quantification of substances like nucleic acid and proteins is important for various experiments like protein labeling etc. Ultra sample volumes of samples are used in this analytical technique.

Range of Analytical Capabilities Small volumes of biomolecules can be analyzed using a spectrophotometer. Its analytical capability ranges mainly from labeled nucleic acids, protein labeling and DNA and RNA quality and quantity analyses. Experiments like DNA microarray are conducted using a spectrophotometer, and the experiments are as precise as to give information about the mRNA and the tRNA present.

Advantages and Disadvantages of Spectrophotometry as an Analytical Technique for Biomolecules

Spectrophotometry as an analytical technique is quite rapid and efficient. It has the advantage of actually determining the quality of DNA (level of degradation) and not only identifying its components. The equipment is relatively more cost effective and the maintenance cost isnt too high. Also spectrophotometer is automatable which makes it very easy to use. UV-visible spectrophotometer has a high sensitivity for detecting organic compounds. As for disadvantages, a spectrophotometer needs to be calibrated from time to time to get accurate and precise results. Stray light must be taken into account to make sure the results are accurate.

Application of Spectrophotometry in Agriculture, Health and Environment Sector The purpose of spectrophotometry is to determine the concentration of analyte in a solution, and this outcome is applied and used to improve the health, environment and agriculture sectors. For quality control of water, waste water, contaminated air and soil, spectrophotometric techniques are applied. It is used as a powerful tool in environmental measurement. Analysis and monitoring of waste, this includes the determination of quantity and types of bacteria present in water and air. These detections make it useful for both, environment and agriculture. A spectrophotometer is a key instrument used in biomedical laboratories these days, it can determine in minutes the amounts of any substance present in a certain food material. Hence, this technique is also used for food testing and ensuring the right amount every substance is present in it. Similarly it is used in pharmaceuticals for making medicines, and tests like detection of components of blood and other fluids in body.

ELISA ELIZA stands for Enzyme-linked immunosorbent assay and it is an analytical technique which detects the presence of substances in a wet sample. The wet sample is a condition necessary because of the enzyme immunoassay which basically detects the presence of substances in the sample. ELISA analyses substances quantitatively as well as qualitatively in a liquid environment. The analyte in ELISA undergo a certain number of biochemical reactions which then identify and measure the amount of certain particles in the analyte. However, the analyte,

even after quantification remains in the liquid environment in which the reaction takes place, this is why ELISA analytical technique is known as a wet-lab.

Types of Analysis There are 4 types of analysis: Direct ELISA It involves a layer of immobilized antibody specific for a particular antigen. This antigen in turn binds with another type of antibody that has an enzyme linked to it. When a chromogenic or fluorescent substrate is added to the plate the specific substrate binds to the enzyme and helps in analysis of the antigen.

Indirect ELISA This type of ELISA is use to determine the strength and amount of an antibody response. It involves a layer of antigen immobilized on the inner surface of the microtitre plate; then a layer of antibodies and another layer of secondary antibodies that are linked to enzymes. Sandwich ELISA

Sandwich ELISA measures the amount of antigen between a sandwich of two layers of antibodies. This increases the specificity and makes this technique more widely used and more accurate than the others.

Competitive ELISA The competitive ELISA is used to quantify antigen using competitive method. Briefly, the free antigen and antibody are incubated to form antigen-antibody complex and then the complex are bound to antigen-coated surface in the assay plate. The unbound antibody-antigen complex is washed off before adding enzyme-linked secondary antibody against the primary antibody. The substrate is then added and the antigen concentration can then be determined by the signal strength elicited by the enzyme-substrate reaction. In this assay, the enzyme-linked secondary antibody competes with the sample antigen which is associated with the primary antibody. Multiple and Portable ELISA As the name suggests, this can work on more than one type of antibody at the same time. These devices are patented right now and research is being done to make them more convenient and cost effective. All four types have a slightly different mechanism of the biochemical reaction of the enzyme with the wet environment and all of these works for the detection and analysis of antibodies and their components. Range of Analytical Capabilities ELISA is a technique which makes use of enzymes and antibodies to interact with colorless substances to produce color so that they can be identified and measured. This type of detecting capability makes it useful for detection of small discrete biomolecules like viruses. HIV virus is

detected in humans using this technique. Biomolecules like antigens and antibodies, hormones and primarily all types of proteins are detected using ELISA. Advantages and Disadvantages of ELISA as an Analytical Technique for Biomolecules ELISA as tests gives very accurate and precise results, and the reason is that ELISA tests are very highly sensitive and specific. The main advantage lies in the fact that since ELISA has been introduced, radioisotopes required for medicals tests have been dropped, which were dangerous to use. ELISA also eliminates the need of costly radiation apparatuses used for testing viruses. If multiple and portable ELISA is used it has the advantages of simultaneously detecting different antibodies and the sample volume can also be increased. A disadvantage of ELISA as an analytical technique is that the labeling of proteins with enzymes etc. may disrupt the immunereactivity of primary antibodies. Another disadvantage is that the enzyme immunoassay is not very selective; it might react to more than one compound which can be a problem. Application of ELIZA in Agriculture, Health and Environment Sector In the health sector its main application lies in the field of drug testing and for testing presence of viruses like HIV in humans etc. It is largely used in hospital laboratories to analyze blood samples. Also, it has been very famous lately in testing dengue fever. As ELISA technique is capable of detecting low levels of chemicals, it is an important detection technique for pollutants in water; hence environmental sampling is an important use of this technique. Pesticides can also be labeled by enzymes and detected, which makes its application important in the agricultural sectors too.

Bibliography

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