Chromatographic Techniques WORD

8/10/2019 Chromatographic Techniques WORD

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Chromatographic Techniques

General principles

Distribution coefcients

The basis of all forms of chromatography is the partition or distribution coefficients (Kd),

which describes the way in which a compound distributes itself between immiscible

phases. For two such immiscible phases A and B, the value for coefficient is a constant

at a given temperature and is given by the epression

The term effective distribution coefficient is defined as the total amount! distinct from the

concentration, of substance present in "one phase divided total amount present in the

other phase. #t is in fact the distribution coeffient multiplied by the ratio of the volumes of

the two phases present.

Basically, all chromatographic systems consist

stationary phase, may be a solid, gel, li$uid or a solid%li$uid miture that is

immobili&ed,

mobile phase, which may be li$uid or gaseous and which flows over the

stationary phase.

Modes o chromatography

chromatographic separations may be achieved using three contrasting

modes:

Column chromatographyin which the stationary phase attached to a

suitable matrix (inert, insoluble support) is packed into a glass or metal

column and the mobile phase passed through the column either by gravity

feed or applied gas pressure.

Thin layer chromatography in which the stationary phase attached to a

suitable matrix is coated thinly onto a glass, plastic or metal foil plate. The mobile

liquid passes across the thin-layer plate, held either horiontally or vertically, by

capillary action. This mode of chromatography has the practical advantage over in

chromatography that a large number of samples can be studied simultaneously

Paper chromatography


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stationary liquid phase is supported by the cellulose !bres of a paper

sheet. "s in thin-layer chromatography, with which paper

chromatography has several similarities

the mobile phase passes along the paper sheet either by gravity feed

or by capillary action. This is one of the older forms of chromatographyand, although it is still used to demonstrate the principles of

chromatography,

Perormance o column chromatography

The principle of a column chromatographic separation may be depicted by

considering a column packed with a solid granular stationary phase to aheight of # cm, surrounded by the mobile liquid phase of which there is $ cm%

per cm of column, as shown.

&f %' g of a compound is added to the column in $ cm% of mobile phase,

then as this $ cm% moves on to the column to occupy position ", $ cm% of

mobile phase will leave the base of the column.

&f the compound added has an eective distribution coe*cient of $, it will

distribute itself equally between the solid and liquid phases (stage $).

&f a further $ cm% of mobile phase is introduced on to the column, the mobile

phase in section " will move down to +, taking $ g of the compound with it,

leaving $ g at " (stage ').

"t both " and + a redistribution of the compound will occur so that there is

g in the mobile phase and g in the solid phase.


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The addition of a further $ cm% of mobile phase to the column displaces the

mobile phase in " to + and that in + to giving the distribution of the

compound as shown in stage %(/$/).

"ddition of a further $ cm% of mobile phase leads to the distribution shown at

stage 0 (0/$'/$'/0), and a further $ cm% of mobile phase leads to thedistribution shown at stage # ('//$'/$'//').

&t is apparent that after a relatively small number of equilibrations the

compound distributes itself symmetrically within a band.

Column chromatographic components (gas or liquid mobile phase)


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1 " stationary phase2 hosen to be appropriate for the analytes to be

separated.

1 " column2 !lled with the matrix coated with the stationary phase, or

microbore type, which the stationary phase is coated directly on the inside

wall.

mobile phase and delivery system: hosen to complement the

stationary phase and to deliver a constant rate of 3ow into the column.

! n in"ector system2 To deliver test samples to the top of the column in a

reproducible manner.

! detector and chart recorder:To give a continuous record of the

presence of the analytes in the eluent as it emerges from the column.

4etection based on the measurement of parameter -visible or

ultraviolet absorption or 3uorescence. " peak on the chart recorder

represents each separated analyte.

! raction collector: 5or collecting the separated analytes for further

biochemical studies.

Column liquid chromatography can be subdivided according to

the bac# pressure generated $ithin the column during the

separation process.


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%o$ pressure liquid chromatography &%P%C' generates pressures of less

than # bar ($ bar 6 $0.# lbf 7n8' 6 9.$ :;a), since there is little resistance to

eluent 3ow owing to the physical nature of the stationary phase. ;= are often blurred and

their equipment and procedures are virtually identical.

+oth give excellent resolutions and hence the term high performance liquid

chromatography is preferred for both of them,

)election o stationary and mobile phases

)uccessul chromatographic separations depend upon the correct

choice stationary and mobile phases. This may be achieved by

setting up one o the ollo$ing:


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dsorption equilibrium:This is between a stationary solid phase and a

mobile liquid phase (adsorption chromatography? hydrophobic interaction

chromatography).

Partition equilibrium:This is between a stationary liquid phase and a

mobile liquid or gas phase (partition chromatography? perfusionchromatography? pair chromatography? chiral chromatography? gasliquid

chromatography).

*on+e,change equilibrium2 This is between a stationary, solid ion-

exchanger and mobile, liquid electrolyte phase (ion-exchange

chromatography? chromatofocusing? membrane chromatography).

-,clusion equilibrium:This is between a liquid phase trapped inside the of

a stationary porous structure and the same mobile liquid phase (molecular

exclusion or gel !ltration).

! inding equilibrium:This is between a stationary immobilised ligand and

a mobile liquid phase (a*nity chromatography? immunoa*nity

chromatography? lectin a*nity chromatography? metal chelate a*nity

chromatography? dye-ligand chromatography? covalent chromatography).

&n practice it is quite common for two or more of these equilibria to be

involved simultaneously in a particular chromatographic separation.

nalyte development and elution


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@eparation of the mixture of analytes applied to the stationary phase by the

mobile phase and their elution from the column.

olumn chromatographic techniques can be subdivided on the basis of thedevelopment and elution modes.

sample is dissolved in a suitable solvent and applied to the stationary phase

as a narrow? discrete band.

:obile phase, normally consisting of an organic solvent or a mixture of

solvents often incorporating a buered aqueous system, is then allowed to

3ow continuously over the stationary phase, resulting in the progressive

separation and elution of the sample analytes.

&f the composition of the mobile phase is constant, the process is said to be

isocratic mobile phase may be gradually changed, for example with respect

to salt concentration or polarity - referred to as gradient elution. The

composition of the mobile phase may be changed continuously or in a

stepwise manner.

@uccessful onal development results in the elution of pure samples of all the

analytes.

&n displacement or a*nity development, the analytes in the sample are

separated on the basis of their a*nity for the stationary phase,

The analytes are selectively eluted by using a mobile phase containing aspeci!c solute that has a higher a*nity for the stationary phase than have

the analytes in the sample.

Thus, as the mobile phase is added, this agent displaces the analytes from

the stationary phase in a competitive fashion, resulting in their repetitive

binding and displacement along the stationary phase and eventual elution

from the column in the order of their a*nity for the stationary phase,


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The one with the lowest a*nity being eluted !rst.

&n frontal development, the sample is continuously added to the stationary

phase, thereby forcing the analytes along the stationary phase in the order of

their a*nity for it. The analyte with the lowest a*nity accumulates at the

front of the moving sample band

&n practice, the technique is eectively restricted to the analysis of a single

trace impurity in an otherwise pure sample.

C(/0MT0G/P(*C P-/10/M2C- P/M-T-/)

/etention time and elution volume

The !rst is the time it takes the analyte molecules to pass through the free spaces

between the particles of the matrix coated with the stationary phase. This time is

referred to as dead time, (t:)

Aolume of the free space is referred to as the column void volume, (Ao)

The value of t: will be the same for all analytes and can be measured by

using an analyte that does not interact with the stationary phase but simply

spends all of the elution time in the mobile phase travelling through the void

volume.

The second component is the time the stationary phase retains the analyte

referred to as the ad7ustered retention time. This time is characteristic of the

analyte and is the dierence between the observed retention time and the

dead time2

Capacity actor '

Bne of the most important parameters in chromatography

"lso called retention factor and capcity ratio


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&t is simply the additional time that the analyte takes to elute from the

column relative to an unretained or excluded analyte that does not interact

with the stationary phase and which, by de!nition, has a k8 value of 9. Thus2

where :s is the mass of analyte in the stationary phase, :m is the mass of

analyte in the mobile phase, As is the volume of stationary phase, and Am is

the volume of mobile phase.

Plate (eight and /esolution


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Plate height

Chromatography columns + consist of a number of ad7acent ones in each

of which there is su*cient space for an analyte to completely equilibrate

between the two phases. Cach one is called a theoretical plate.

The length of the column containing one theoretical plate is referred to as the

plate height >. The numerical value of both D and > for a particular column is

expressed by reference to a particular analyte.

;late height is simply related to the width of the analyte peak

Pea# roadening

" number of processes oppose the formation of a narrow analyte peak

thereby increasing the plate height2

! pplication o the sample to the column: &t takes a !nite time to apply

the analyte mixture to the column, so that the part of the sample applied !rstwill already be moving along the column by the time the !nal part is applied.

The part of the sample applied !rst will elute at the front of the peak.

! %ongitudinal di4usion. 5ick8s law of diusion states that an analyte will

diuse from a region of high concentration to one of low concentration at a

rate determined by the concentration gradient between the two regions and

the diusion coe*cient of the analyte. Thus the analyte within a narrow band


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will tend to diuse outwards from the centre of the band, resulting in band

broadening.

! Multiple path$ays2 The random packing of the particles in the column

results in many routes between the particles for both mobile phase and

analytes. The smaller the particle sie the less serious is this problem.

! -quilibration time bet$een the t$o phases: &t takes a !nite time for

each analyte in the test sample to equilibrate between the stationary and

mobile phases as it passes down the column

symmetric Pea#

&n some chromatographic separations, ideal shaped peaks are not obtained.

&n cases where there is a gradual rise at the front of the peak and a sharp fall

after the peak, the phenomenon is known as fronting ( because ofoverloading the column) - reducing the amount of mixture applied to the

column resolves the problem.

&n cases where the rise in the peak is normal but the tail is protracted, the

phenomenon is known as tailing. (because -retention of analyte by a few

active sites on the stationary phase, commonly on the inert support matrix.

@uch sites strongly adsorb molecules of the analyte and only slowly release

them).

This problem can be overcome by chemically removing the sites, frequently

hydroxyl groups, by treating the matrix with a silanising reagent such ashexamethyldisilaine.This process is sometimes referred to as capping.

/esolution


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The success of a chromatographic separation is 7udged by the ability of the

system to resolve one analyte peak from another.

Eesolution (Es) is de!ned as the ratio of the dierence in retention timebetween the two peaks to the mean of their base widths.

)ample preparation

)olvent e,traction

;reliminary lean up is essential, particularly if the test analyte(s) is in a

complex matrix such as plasma, urine, cell homogenate or microbiological

culture medium (especially in >;=)

The most common extraction, This is based on the extraction of the analytes

from aqueous mixtures using a low boiling water-immiscible solvent such as

diethylether or dichloromethane.

This solvent extraction procedure tends lack selectivity and is often

unsatisfactory for the >;= analysis of compounds in the range of ng cm-% or

less.


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)olid+phase e,traction

&ts advantage over simple solvent extraction is that it exhibits greater

selectivity, mainly because it is a form of chromatography.

The test solution is passed through a small (few millimetres in length)

disposable column (cartridge) packed with relatively large particles of a

bonded silica similar to those used for >;=.

These selectively adsorb the analyte(s) under investigation and ideally allow

interfering compounds to pass through.

@everal commercial forms of this solid-phase extraction technique are

available that facilitate the simultaneous treatment of a large number of test

samples.

Column s$itching

@uited to the analysis of analytes in very low concentartions in complex

mixtures especially in >;=

The test solution is applied to a preliminary short column similar to the type

used in solid-phase extraction.

Bnce the test analyte has been adsorbed and impurities washed through the

column,

The analyte is eluted with a suitable organic solvent and the column eluate

transferred directly to an analytical >;= column.

)upercritical 5uid e,traction

@5C exploits the fact that gases such as carbon dioxide exist as a liquid under

certain critical conditions .

&n the case of carbon dioxide, these conditions are %$.$F and G.% :;a and

the resultant liquid carbon dioxide can be used as the extraction solvent,

behaving as a low polarity solvent comparable to hexane.

+y altering the physical conditions of the extract, the carbon dioxide can be

made to revert to a gas, thus simplifying the recovery of the extractedanalytes.

)ample Deriviti6ation

@ome functional groups, especially hydroxyl, present in a test analyte may

compromise the quality of its behavior in a chromatographic system.


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The technique of analyte pre- or post-column derivatisation may facilitate

better chromatographic separation and detection by masking these

functional groups.

Column Chromatography

Columns

The glass column used should have a means of supporting the stationary

phase as near to the base of the column as possible in order to minimise the

dead space below the column support in which post-column mixing of

separated analytes could occur.

ommercial columns possess either a porous glass plate fused on to the base

of the column or a suitable device for supporting a replaceable nylon net,which in turn supports the stationary phase.

" cheaper alternative is to use a small plug of glass wool together with a

minimal amount of quart sand or glass beads.

Matri, materials


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The matrix is the material used to support the stationary phase. The selection

of a matrix for a particular stationary phase is vital to the successful

chromatographic use of the phase.

Generally spea#ing7 a matri, needs to have:

>igh mechanical stability to encourage good 3ow rates and to

minimise pressure drop along the column?

igh capacity, i.e. density of functional groups to minimise bed

volume.

&t also needs to be available in a range of particle sies.

&n addition some forms of chromatography require a matrix with aporous structure, in which case the pores need to be of the correct sie

and shape.

5inally, the surface of a matrix needs to be inert to minimie the non-

selective adsorption of analytes.

Commonly used types o matrices

garose - a polysaccharide made up of 4-galactose and %,-anhydro- $

-galactose units. The unbranched polysaccharide chains are cross-linked with

agents such as ',%-dibromopropanol to give gels that are stable in the p>

range %-$0.)epharose and io+Gel .

Cellulose - a polysaccharide of +eta $-0-linked glucose units. 5or matrix use

it is cross-linked with epichlorohydrin, the extent of cross-linking dictating the

pore sie. &t is available in bead, microgranular and !brous forms, has good

p> stability and 3ow properties, and is highly hydrophilic.

De,tran - a polysaccharide consisting of alpha-$--linked glucose units. 5or

matrix use it is cross-linked with epichlorohydrin but is less stable to acid

hydrolysis than are cellulose matrices. &t is stable up to p> $' and is

hydrophilic. )ephade,.

Polyacrylamide - a polymer of acrylamide cross-linked with D,D8methylene-

bisacrylarnide &t is stable in the p> range '-$$.+io- ranges and are most

commonly used for exclusion and ion-exchange chromatography. They have

relatively low hydrophilicities.


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)ilica - " polymeric material produced from orthosilic acid. The numerous

silanol (@i-B>) groups make it hydrophilic. Hhen derivatised, excess silanol

groups can be removed by treatment with trichloromethylsilane. p> range %-

. &t is chemically inert but, like the silicas, tends to dissolve above p>.

)tationary phases

The chemical nature of the stationary phase depends upon the particular

form of chromatography to be carried out.

:ost stationary phases are available attached to the matrices in a range of

sies and shapes.

+oth properties are important because they in3uence the 3ow rate and

resolution characteristics.

The larger the particle, the faster the 3ow rate but, conversely, the smaller

the particle the larger the surface area-to-volume ratio and potentially the

greater their resolving power.

The best packing characteristics are given by spherical particles

;article sie is commonly expressed by a mesh sie, which is a measure of

the openings per inch in a sieve? hence the larger the mesh sie, the smaller

is the particle.


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$99-$'9 mesh is most common for routine use, whereas a '99-099 mesh is

used for higher resolution work.

Column pac#ing

:ost critical factors in achieving a successful separation by any form of

column chromatography.

;acking a column is normally carried out by gently pouring a slurry of the

stationary phase in the mobile phase into a column that has its outlet closed,

Cnsure that no air bubbles are trapped and that the packing settles evenly.


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;oor column packing gives rise to uneven 3ow (channeling) and reduced

resolution.

The slurry is added until the required height is obtained, Bnce the required

column height has been obtained, the 3ow of mobile phase through the

packed column is started by opening the outlet, and continued until thepacking has completely settled.

To prevent the surface of the packed material from being disturbed either by

the addition of mobile phase to the column or during the application of the

sample to the column, it is normal to place a suitable protection device, such

as a !lter paper disc or nylon gaue, on the surface of the column. @ome

commercial columns possess an adaptor and plunger, which serve the dual

purpose of protecting the surface of the column and providing an inlet.

Do part of it should be allowed to run dry? hence a layer of mobile phase

should always be maintained above the column surface.

&t is di*cult to generalie about the ideal column height-to-diameter ratio and

the total bed volume. 5or example, in exclusion chromatography, a height-to-

diameter ratio of $92 $ to '92 $ is normally suitable.

pplication o sample

)everal methods are available

" simple way is to remove most of the mobile phase from above the column

by suction and 7ust to drain the remainder into the column bed. The sample is

then carefully applied by pipette and it too is allowed 7ust to run into thecolumn. " small volume of mobile phase is then applied in a similar manner

to wash !nal traces of the sample into the bed. :ore mobile phase is then

carefully added to the column to a height of '-# cm. The column is then

connected to a suitable reservoir that contains more mobile phase so that the

height of the phase in the column can be maintained at '-# cm.

"n alternative procedure, which avoids the necessity to drain the column to

the surface of the bed, is to increase the density of the sample by addition of

sucrose to a concentration of about $I. Hhen this solution is layered on to

the liquid above the column bed, it will automatically @ink to the surface of

the column and hence be quickly passed into the column.This method of

sample application is satisfactory, provided that the presence of @ucrose in

no way interferes with the separation and subsequent analysis of the sample.

J

" third method involves the use of capillary tubing and/or syringe or

;eristaltic pump to pass the sample directly to the column surface. This latter

method is the most satisfactory of the three.


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&n all cases, care must be taken to avoid overloading the column with sample,

Btherwise irregular separation will occur. &t is also advantageous to apply the

sample in as small a volume of mobile phase.

Column development and sample elution

The components of the applied sample are separated by the continuous

passage of the mobile phase through the column. This is known as elution

development.

4uring the elution process it is essential that the 3ow of mobile phase is

maintained at a stable rate and this is most simply achieved by gravity feed.

"n alternative and Kmore eective method of maintaining stable 3ow rates is

to use a peristaltic pump.

olumn development using a single liquid as the mobile phase is known as an

isocratic elution. >owever, in many cases in order to increase the resolvingpower of the mobile phase, it is necessary continuously to change its p>,

ionic concentration or polarity. This is known as gradient elution.

Detectors and raction collection

"s the resolved analytes emerge in the eLuent from the column it is

necessary detect their presence.

5or coloured analytes this can be achieved simply by visual

observation but for colourless compounds alternatives are necessary.

4etection may be based on ultraviolet absorption, 3uorescence spectroscopy,

changes in refractive index of the eLuent, the presence of a radioactive

emission atom or the ease of oxidation or reduction of the analytes as

measured by an electrochemical detector.

Mltraviolet detectors are probably the most common form of detector for

biochemical analysis.

5luorescent detectors are also available.

*on+e,change chromatography

This form of chromatography relies on the attraction between oppositely

charged particles.


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:any biological materials, e.g. amino acids and proteins, have ionisable

groups and the fact that they may carry a net positive or negative charge can

be utilied in separating mixtures of such compounds.

The net charge exhibited by these compounds is dependent on their (pNa and

on the p> of the solution.

&on-exchange separations are carried out mainly in columns packed with an

ion exchanger.

There are two types of ion-exchanger, mainly cation and anion exchangers.

The cation exchangers possess negatively charged groups and these will

attract positively charged cations.

These exchangers are also called acidic ion exchangers because their

negative charges result from ioniation of acidic groups.

"nion exchangers have positively charged groups that will attract negatively

charged anions. The term basic ion-exchange materials is also used to

describe these exchangers, from the association of protons with basic groups.

The ion+e,change mechanism is thought to be composed o 8ve

distinct steps:

(i) 4iusion of the ion to the exchanger surface. This occurs very quickly in

homogeneous solutions.

(ii) 4iusion of the ion through the matrix structure of the exchanger tothe

exchange site. This is dependent upon the degree of cross-linkage of the

exchanger and the concentration of the solution. This process is thought to

be the feature that controls the rate of the whole ion- exchange process.

(iii) Cxchange of ions at the exchange site. This is thought to occur

instantaneously and to be an equilibrium process.The more highly charged

the molecule to be exchanged, the tighter it binds to the exchanger and the

less readily it is displaced by other ions


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(iv) 4iusion of the exchanged ion through the exchanger to the surface.

(v) @elective desorption by the eluent and diusion of the molecule into th

external eluent.

The selective desorption of the bound ion is achieved by changes in p>

and/or ionic concentration or by a*nity elution, in which case an ion that has

greater a*nity for the exchanger than the bound ion introduced into the

system.

Materials and applications

%o$ pressure ion+e,change chromatography can be carried out using

a variety o matrices and ionic groups.

:atrices used include polystyrene, cellulose and agarose.

5unctional ionic groups include sulphonate (-@B0) and quaternary ammonium(-DE%), both of which are strong exchangers because they are totally ionised

at all normal working p> values, and

arboxylate (-BB ) and diethylammonium (->D(>'>%)'), both of which

are termed weak exchangers because they are ionised over only a narrow

range of p> values.

"ll exchangers are characteried by a total exchange capacity, which is

de!ned as the number of milli equivalents of exchangeable ions available,

either gram of dried exchanger or per unit volume of hydrated resin.

ompounds that are stable over a wide range of p> may be separated by

either type of exchanger.

The choice between a strong and weak exchanger all depends on sample

stability and the eect of p> on sample charge.

Heak electrolytes requiring a very low or high p> for ionisation can be

separated only on strong exchangers, as only they operate over a wide p>

range.

&n contrast, for strong electrolytes, weak exchangers are advantageous for a

number of reasons including a reduced tendency to cause sampledenaturation, their inability to bind weakly charged impurities and their

enhanced elution characteristics.

The p> of the buer used should be at least one p> unit above or below the

isoionic point of the compounds being separated.


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&n general, cationic buers such as Tris, pyridine and alkylamine are used in

con7unction with anion exchanger and anionic buers such as acetate,

barbiturate and phosphate are used with cationic exchangers.

The precise initial buer p> and ionic strength should be such as 7ust to

allow the binding of the sample components to the exchanger.

Cqually, a buer of the lowest ionic strength that eects elution should

initially be used for the subsequent elution of the components.

This ensures that, initially, the minimum number of undesired substancesbind to the exchanger.

The amount of sample that can be applied to a column is dependent upon the

sie of the column and the capacity of the exchanger.


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&f, however gradient elution is to be used, the initial conditions chosen are

such that the entire sample is bound by the exchanger at the top of the

column.

&n this case the sample volume is not important and large volumes of dilute

solution can be applied, thereby eectively introducing a concentrationstage.

and ionic strength gradients may be employed.

+ut continuous gradients tend to give better resolution with less peak tailing.

gradient decreases and the ionic

strength increases,

Hhereas for cation exchangers both the p> and ionic gradients increase.

-,clusion &permeation' chromatography

The separation of molecules on the basis of their molecular sie and shape.

Mtilies the molecular sieve properties of a variety of porous materials.

;robably the most commonly used of such materials is a group of polymeric

organic compounds that possess a three-dimensional network of pores that

confers gel properties upon them.

The term gel !ltration is used to describe the separation of molecules of

varying molecular sie utiliing these gel materials.

;orous glass granules have also been used as molecular sieves and the term

controlled-pore glass chromatography introduced to describe this separation

technique.

The terms exclusion or permeation chromatography describe all molecular

separation processes using molecular sieves.

The general principle of exclusion chromatography is quite simple. =arge

molecules that are completely excluded from the pores will pass through the

interstitial spaces and will appear in the eLuent !rst.

@maller molecules will be distributed between the mobile phase inside and

outside the molecular sieve and will then pass through the column at a

slower rate, hence appearing last in the eLuent.


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5or a given type of gel, the distribution coe*cient Nd, of a particular analyte

between the inner and outer mobile phase is a function of its molecular sie.

&f the analyte is large and completely excluded from the mobile phase within

the gel, Nd 6 9,

whereas if the analyte is su*ciently small to gain complete access to the

inner mobile phase, Nd 6 $.

4ue to variation in pore sie between individual gel particles, there is some

inner mobile phase that will be available and some that will not be available

to analytes of intermediate sie?

hence Nd values vary between 9 and $.

&t is this complete variation of Nd between these two limits that makes

possible the separation of analytes within a narrow molecular @ie range on a

given gel.


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pplications

Puri8cation. ;uri!cation of biological macromolecules by facilitating theirseparation from $arger and smaller molecules. Airuses, proteins, enymes,

hormones, antibodies, nucleic acids and polysaccharides.

/elative molecular mass determination:The elution volumes of globular

protein are determined largely by their relative molecular mass (:r). &t has

been stated that over a considerable range of relative molecular masses, the

elution volume is approximately linear function of the logarithm of :r.

)olution concentration. @olutions of high :r substances can be

concentrated by the addition of dry @ephadex


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more e*cient than dialysis. "pplications include removal of phenol from

nucleic acid preparation, protein preparations and salts from samples eluted

from ion exchange chromatography.

fnity chromatography

;uri!cation by a*nity chromatography does not rely on dierences in the

physical properties of the molecules to be separated.

&nstead, it exploits the unique property of extremely speci!c biological

interactions to achieve separation and puri!cation.

"s a consequence, a*nity chromatography is theoretically capable of giving

absolute puri!cation, even from crude mixtures in a single process.

The technique was originally developed for the puri!cation of enymes, but it

has since been extended to nucleotides, nucleic acids, immunoglobulins,

membrane receptors and even to whole cells and cell fragments.

Mnder the correct experimental conditions, when a complex mixture

containing the speci!c compound to be puri!ed is added to the immobilised

ligand.

only that compound will bind to the ligand. "ll other compounds can therefore

be washed away and


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The compound subsequently recovered by displacement from the ligand.

The method requires a detailed prior knowledge of the structure and

biological speci!city of the compound to be puri!ed.

&n the case of an enyme, the ligand may be the substrate, a reversible

inhibitor or an allosteric activator.

The conditions chosen would normally be those that are optimal for enyme-

ligand binding.

Matri,

n ideal matri, or afnity chromatography must possess the ollo$ing

characteristics:

(i) &t must contain suitable and su*cient chemical groups to which the ligand

may be covalently coupled and it must be stable under the conditions of theattachment.

(ii) &t must be stable during binding of the macromolecule and its subsequent

elution.

(iii) &t must at the most interact only weakly with other macromolecules to

minimie nonspeci!c adsorption.

(iv) &t should exhibit good 3ow properties.

&n practice, particles that are uniform, spherical and rigid are used. The most

common ones are the cross-linked agarose (@epharose, +io


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Practical procedure

The ligand-treated matrix is packed into a column in the normal way for the

particular type of support.

The buer used must contain any cofactors, such as metal ions, necessary for

ligand macromolecule interaction.

Bnce the sample has been applied and the macromolecule bound, the

column eluted with more buer to remove non-speci!cally bound

contaminants.

The puri!ed compound is recovered from the ligand by either speci!c or non-

speci!c elution.

Don-speci!c elution may be achieved by a change in either p> or ionic

strength.

&f elution is achieved by a p> change, the p> of the collected fractions must

be read7usted to the optimum value to minimise the opportunity for protein

denaturation.


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"*nity elution involves the addition of a high concentration of substrate, or

reversible inhibitor of the macromolecule if it is an enyme or the addtion of

the ligand for which macromolecule has a higher a*nity than it has for the

immobilised ligand.

The puri!ed material is eventually recovered in a buered solution that maybe contaminated with speci!c eluting agents or high concentrations of salt

and these must be removed by such techniques as exclusion chromatography

before the isolation is complete.

pplications

" wide range of enymes O other proteins, including receptor proteins and

immunoglobulins, has been puri!ed by a*nity chromatography.

The principles have been extended to nucleic acids2 mED" for example, is

routinely isolated by selective hybridisation on poly(M)-@epharose 0+ by

exploiting its poly(") tail.

" valuable development of a*nity chromatography is its use for the

separation of a mixture of cells into homogeneous populations. The technique

relies on the antigenic properties of the cell surface or the chemical nature of

exposed carbohydrates residues on the cell surface or on a speci!c

membrane receptor-ligand interaction.

The immobilised ligands used include ;rotein ", which binds to the 5c region

of &g


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Clution of the bound protein quite often requires forceful conditions because

of the very tight binding with the antibody which may lead to protein

denaturation.

Cxamples of elution procedures include the use of high salt concentrations

with or without the use of a detergent

The use of urea, @4@, guanidine hydrochloride, all of which cause

denaturation.

The use of chaotropic agents such as thiocyanate, perchlorate and

tri3uoracetate or lowering the p> to about % may avoid denaturation.

Metal chelate chromatography

"lso called as immobilised metal a*nity chromatography

This is a special form of a*nity chromatography in which an ion such as

u'P, Qn'P, >g'P or d'P or a transition metal ion such as Di'P, or :n'P

is used to bind proteins selectively by reaction with groups of histidine

residues, thiol groups in cysteine residues and indole groups of tryptophan

residues.

The immobilisation of the protein involves the formation of coordinate bonds

that must be su*ciently stable to allow retention during the elution of non-

binding contaminating material.

The subsequent elution of the protein can be achieved either by simply

lowering the p> or by the use of complexing agents such C4T".

;roteins puri!ed by this technique include !brinogen, superoxide dismutase

and the histone nuclear proteins.

;ractical

"nalytical +iochemistry &(+T2 #%$,#%')

Terminology

:ole2 "mount of a substance present in a 3ask irrespective of volume. $

mole6:H in gms6 .9'%x$9'%6avogadronumber


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;reparation of solution on the basis of density, wt/wtI i.e g of solute /$99g of

solution

4ensity6 wt / unit volume

@;B0(:H) and Da>';B0(:H) required to prepare

#99ml of 9.': solution.

'. alculate the volume of acetic acid required to prepare $99ml of 9.$:

solution. 4ensity6$.9#$g/ml, purity6RR.#I

%.alculate volume of concentrated >l required to prepare ': and 'D

solution.

9hat is color


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Clectromagnetic radiation between %9-G9 nm

olor is one aspect of appearance

olor 6 light source P ob7ect properties P eye P brain

The human eye is most sensitive at ### nm

Colorimetry

Two ob7ects may appear the same when viewed under one light source,

but dierent under another 6 metamerism

:etamerism is one of the ma7or industrial problems in color matching

olorimetry attempts to quantify the perception of color

)ources and illuminants


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@ource 6 physical entity that produces radiation

&lluminant 6 table of values of spectral power distribution

&lluminant 4# represents average daylight. 4#9 represents typical

indoor light

0b"ects

Bb7ects are characteried by the amount of light they emit and re3ect

or transmit at each wavelength of interest

Hhen light is incident on an ob7ect a part of it is absorbed, a part is

re3ected and a part may be trasmitted

The ob7ect may also emit light

"ll these characteristics contribute to the observed color


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Transmitted light

=ight is characteried by its frequency ( ), the number of times the

crest of the wave passes some point in space per second,

or by its wavelength (), the distance between two successive crests.

These two quantities are related by the speed of light, a fundamental

constant2 U6c6%V$9m/s.

;lanck related the frequency of light to its energy (C) according

to E6hU, where h is ;lanckWs constant, h6.'V$9X%0Y/s.


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" compound will absorb light when the radiation posesses the energy

needed to move an electron from its lowest energy (ground) state to

some excited state. The particular energies of radiation that a

substance absorbs dictate the colors that it exhibits. onversely the

color of a compound can help us to determine its electronic

con!guration.

Hhite light contains all wavelengths in this visible region. Hhen a

transparent sample (like most aqueous solutions) absorbs visible light,

the color we perceive is the sum of the remaining colors that are

transmitted by the ob7ect and strike our eyes.


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The incident light from a tungsten &visible light source) or

deuterium &;< light source) lamp is focused by a lens and passes


38/56


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The eer+%ambert %a$

>ebc

Hhere " 6 absorbance (no units, sinceA = log10P0/ P)

e6 molar absorbtivity with units of = mol-$ cm-$6absorbanse if $:

solution

b6 path length of the sample - that is, the path length of the cuvette

in which the sample is contained. centimetres.

c >concentration of the compound in solution, expressed in mol =-$

" \ at low concentation

;ath length / cm 9 9.' 9.0 9. 9. $.9

IT $99 #9 '# $'.# .'# %.$'#

"bsorbance 9 9.% 9. 9.R $.' $.#


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The =aw is not obeyed at high concentrations

&nharent property of molecules2 &f molecule absorbs more light- low

conc can be detected.

"bsorption of photon \ concentration of absorbing molecules

\ Thickness of sample

4etermination of max of ;otassium dichromate

Aeri!cation of +eer8s =aw

'etermination of ma

repare *+mg%++ ml.repare+ ml of *+ -g%ml solution of potassium dichromate in

+.+* K/0 by diluting + times.

1ead absorbance from 23+nm to 3*+nm at a difference of *nm. 'raw the graph

between absorbance vs wavelength

'etermine the wavelength at which shows maimum absorbance ie ma

4erification of Beer!s law

repare different dilutions (+ml each) of potassium dichromate * -g%ml(+.ml),+

-g%ml(+.5ml),5+ -g%ml(+.3ml),2+ -g%ml(+.6ml),3+ -g%ml(+.7ml),6+ -g%ml(.5ml),7+-g%ml(.6ml),++ -g%ml(5ml).1ead absorbance at wavelength(nm) of maimum

absorbance.. 'raw the graph between absorbance vs concentration. 'raw the

interpretation.

8stimation of haemoglobin in blood%verification of Beer!s law

1eagent +.++9 Ammonium hydroide ( 3 ml ammonia%l)


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'ilute blood ml : ;;ml +.++9 ammoinum hydoide

'ilute blood 03/0 A*97nm

.+ml ;.+ml

5.+ 7.+

3.+ 6.+

6.+ 3.+

7.+ 5.+

+.+ +

8stimation of haemoglobin 0bgopper sulfate)


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(5) eutrali&e 03:with a/0 to get 02

(2) @team distillation of 02and trap in boric acid.

(3) Titrate with hydrochloric acid.

Calculation

ram nitrogen% gram of sample ?

(ml of sample C ml of blanD) (normality) of standard acid +.+3g%me$

weight of

sample

ml of hydrochloric acid re$uired to titrate sample solution.

'isadvantages not all itrogen is protein.

urine

yrimidine 'A, 1A, etc.

Erea

any plant tissues have G *+< nonC

protein itrogen.

< itrogen

6.5* ? < rotein

$iuret %est


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eptide >hains Biuret >omplees( purplecolor)

$iuret %est

1eproduciple

4ery few interfering agents

(ammonium salts being one such agent )

Fewer deviations than with the Howry or ultraviolet absorption methods

1e$uires large amounts protein (C5+mg)

How sensitivity

. 'ilute samples to an estimated to + mg%ml with buffer. Add ml to each assay tube.

'uplicate samples are recommended, and a range of dilutions should be used if the

actual concentration cannot be estimated.

5. repare a reference tube with ml buffer.

2. repare standards from + mg%ml bovine serum albumin, preferably calibrated using

absorbance at 57+ nm and the etinction coefficient. 1ange should be from to + mg

protein.

3. Add ; ml Biuret reagent to each tube, vorte immediately, and let stand 5+ min.

*. 1ead at **+ nm.

&olin"Ciocalteu ( 'ory ) Assay


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@ensitive over a wide range

>an be performed at room temperature

+C5+ times more sensitive than E4 detection.

>an be performed in a microplate format .

any substances interfere with the assay .

(strong acids, ammonium sulfate, buer, 8'TA, nonionic and cationic detergents,

carbohydrate, lipids and some salts. )

TaDes a considerable amount of time to perform

The assay is photosensitive, so illumination during the assay must be Dept consistent for

all samples

Amount of color varies with different proteins.

The incubation time is very critical for a reproducible assay.

The reaction is also dependent on p0 and a worDing range of p0 ; to+.* is essential.

eagents A) 5< sodium carbonate in +. a/0.

B) +.*< copper sulfate in 5opper reagent *+ml of A and 5ml of B (Freashly prepared


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Folins reagent commercial contains phophmolybdic and phosphotungstate in oC

phosphoric acid. 'ilute with 'I.

+. a/0 3g in H

@tandard B@A +. mg in +. a/0.

EnDnown *+ diluted serum

. Add standard B@A containing up to 5+,3+,6+,7+,++ ,3+,7+ Jg

( +.5,+.3,+.6,+.7,.+,.3,.7 ml )of protein. TaDe +. and +.5 ml of unDnown. For

blanD 5ml of +. a/0

5. Bring all tubes to 5 mH total volume with +. a/0.

2. repare the copper reagent and diluted FolinC>iocalteu reagent ( with 'I).

3. To each tube add * mH of copper reagent and thoroughly vorte.

*. #ncubate tubes at room temperature for + min.

6. Add +.* mH of diluted FolinC>iocalteu reagent. 4orte immediately.

9. #ncubate at room temperature for 2+ min.

7. 4orte the tubes, &ero the spectrophotometer with the blanD and measure

absorbance at *++C9*+ nm.

Ultraviolet Absorbance

#f you dont Dnow what the protein concentration of an unDnown sample is liDely to be,

the ultraviolet method might be a good starting point.

This is often used to estimate protein concentration prior to a more sensitive method

onitors the absorbance of aromatic amino acids, tyrosine and tryptophan

0igher orders of protein structure, many other cellular components, and particularly

nucleic acids, also may absorb E4 light

This method is the least sensitive of the methods

The real advantages of this method are that the sample is not destroyed and that it is

very rapid.

Any nonCprotein component of the solution that absorbs ultraviolet light will intefere with

the assay.


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recipitate with +A dissolve in phosphate buffer and used for protein estimation.

>ell and tissue fractionation samples often contain insoluble or colored components that

interfere.

The most common use for the absorbance assay is to monitor fractions from

chromatography columns, or any time a $uicD estimation is needed and error in protein

concentration is not a concern.

An absorbance assay is recommended for calibrating bovine serum albumin or other

pure protein solutions for use as standards.

roteins in solution absorb ultraviolet light with absorbance maima at 57+ and 5++ nm.

Amino acids with aromatic rings are the primary reason for the absorbance peaD at 57+

nm.

eptide bonds are primarily responsible for the peaD at 5++ nm.

@econdary, tertiary, and $uaternary structure all affect absorbance, therefore factors

such as p0, ionic strength, etc. can alter the absorbance spectrum

>oncentration (mg%ml) ? (.** A57+) C +.96 A56+)

>oncentration (mg%ml) ? Absorbance at 57+ nm divided by absorbance coefficient

Absorbance coefficients of some common protein standards

Bovine serum albumin (B@A) 62

Bovine, human, or rabbit #g 27

>hicDen ovalbumin 9+

Procedure*

Iarm up the E4 lamp (about * min.)

AdLust wavelength to 57+ nm

>alibrate to &ero absorbance with buffer solution only

easure absorbance of the protein solution ( 5* ug%ml, *+ ug%ml )

AdLust wavelength to 56+ nm

>alibrate to &ero absorbance with buffer solution only


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easure absorbance of the protein solution

>alculate the protein concentration by Absorbance coefficients and by formula.

$icinchoninic Acid ( $CA ) Assay

4ery sensitive and rapid if you use elevated temperatures

>ompatible with many detergents

IorDing reagent is stable

4ery little variation in response between different proteins

Broad linear worDing range

The reaction does not go to completion when performed at room temperature

. repare the re$uired amount of protein determination reagent by adding volume

copper sulfate solution to *+ volumes of bicinchoninic acid solution.


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5. @et up test tubes containing samples and Dnown amounts of bovine serum albumin in

the range of + to ++ micrograms. 8ach tube should contain +. mH total volume.

2. Add 5.+ mH of the protein determination reagent to each tube and vorte.

3. #ncubate the tubes at 6+o> for * min.

*. >ool the tubes to room temperature and determine the absorbance at *65 nm.

Dye"$inding ( $radford ) Assay

>BB primarily responds to arginine residues

(eight times as much as the other listed residues)

#f you have an arginine rich protein,

Mou may need to find a standard

that is arginine rich as well.

>BB binds to these residues in the anionic form

Absorbance maimum at *;* nm (blue)

The free dye in solution is in the cationic form,

Absorbance maimum at 39+ nm (red).


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Fast and inepensive

0ighly specific for protein

4ery sensitive NC5+ Jg (micro assay) 5+C5++ Jg (macro assay)O

>ompatible with a wide range of substances

8tinction coCefficient for the dyeCprotein comple is stable over + orders of magnitude

(assessed in albumin)

'ye reagent is comple is stable for approimately one hour

onClinear standard curve over wide ranges

1esponse to different proteins can vary widely, choice of standard is very important

Absorption spectra of anionic and cationic forms of the dye overlap.

@o the standard curve is nonClinear although all Dit providers of the Bradford assay insist

that the assay performs linearly.

The assay performs linearly over short concentration stretches.


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#f your sample is more than 5+ micrograms, a second order curve will fit much better than a

linear curve

. Iarm up the spectrophotometer for * min. before use


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5. 'ilute samples with buffer to an estimated concentration of to 5+ micrograms%milliliter

2. repare standards containing a range of to 5+ micrograms protein (albumin or gamma

globulin are recommended) to a volume of 5++ Jl (to a volume of ++ Jl if you are

adding a/0)

3. repare unDnowns to estimated amounts of to 5+ micrograms protein per tube to 5++

Jl (++ Jl if you are using a/0)

*. Add ++ Jl a/0 to each sample and vorte.

6. Add 7++ Jl dye reagent and incubate * min.

9. easure the absorbance at *;* nm.

Ultraviolet Absorbance

PuicD

@ample can be recovered

Eseful for estimation of protein before using a more accurate method

0ighly susceptible to contamination by buffers, biological materials and salts

rotein amino acid composition is etremely important, thus the choice of a standard is

very difficult, especially for purified proteins

Absorbance is heavily influence by p0 and ionic strength of the solution.

+stimation Procedure

. Qero spectrophotometer to water (or buffer)

5. TaDe the absorbance at 57+ nm in a $uart& cuvette

2. >hange wavelength to 56+ nm and &ero with water (or buffer)

3. TaDe absorption at 56+ nm in a $uart& cuvette

*. Ese the following e$uation to estimate the protein concentration

NroteinO (mg%mH) ? .**A57+R +.96A56+

$ioad DC Protein Assay

Based on Howry Assay with following improvements


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. 1eaches ;+< of its maimum color development within * minutes

5. The color changes not more than +< in 5 hours

repare * dilutions of samples and * dilutions of a protein standard containing from

+.5 mg%ml to about .* mg%ml protein. A standard curve should be prepared each time

the assay is performed. For best results, the standard should be prepared in the same

buffer as the sample.

B@A

mg%ml

+.53

(%6)

+.37

(5%6)

+.95

(2%6)

+.;6

(3%6)

.5+

(*%6)

.33

(6%6

f celletract

+ +.> +.5> +.2> +.*> >

ipet * ul of standards and samples into a microtiter plate

Add 5*ul of reagent A into each well

Add 5++ul reagent B into each well

Agitate the plate to mi the reagents

, - . / 0 1 2

A 3 34-/ 34/5 3461 ,4-3 ,4// empty

$ 3 34-/ 34/5 3461 ,4-3 ,4// empty

C 3 34-/ 34/5 3461 ,4-3 ,4// empty

D blan7 blan7 blan7 blan7 blan7 blan7 empty

+ 3 34,C 34-C 34.C 340C C empty

& 3 34,C 34-C 34.C 340C C empty

8 3 34,C 34-C 34.C 340C C empty

9 empty empty empty empty empty empty empty


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8mpty? Air

BlanD?1eagent A:B

+?1eagent A:B:*ul water

After * minutes, absorbance can be read at 9*+nm. The absorbance will be stable for

about hour

:tandard , - . / 0 1

A 3 343.2 343/6 34313 34306 34352

$ 3 343/- 3430, 34323 34316 34355

C 3 343.6 3430, 3432, 34350 34352


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Average 3 343.6. 34303. 343123 3432,3 34352.

Con4 3 34-/ 34/5 342- 3461 ,4//

lot >oncentration as M ais

Standard

y = 19.815x - 0.2861

R2= 0.9868

0

0.5

1

1.5

2

0 0.02 0.04 0.06 0.08 0.1

Absorbancd

Concentration

y ? ;.7* C +.576

NroteinO? ;.7*NAOC +.576

;s

lot >oncentration as = ais

Standard

y = 0.0498x + 0.015

R2= 0.9868

0

0.02

0.04

0.06

0.08

0.1

0 0.5 1 1.5 2

Concentration

Abs

orbance

y ? +.+3;7 : +.+*

NroteinO?(NAO C+.++*)%+.+3;7

>ould introduce errors into the calculation

NroteinO? ;.7*NAOC +.576

:tandard , - . / 0 1

+ 3 343-5 34303 3430. 34,-/ 34,1,

& 3 343-6 343/2 34321 34362 34,2.


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8 3 343,5 343/6 34312 34,3/ 34,05

Average 3 343-03 343/52 34310. 34,35. 34,1/3

Con4 3 34-, 3415 ,43, ,451 -465

&raction 3 34, 34- 34. 340 ,

protein concentration

y = .205x - 0.0644

R2= 0.9!41

-0.5

0

0.5

1

1.5

2

2.5

.5

0 0.2 0.4 0.6 0.8 1 1.2

"raction

co

ncentrati

rotein >oncentration? 2.66mg%ml

%ips

Ese clean glassware and supplies

aDe sure cuvettes are clean of all residues

rotein assays are strongly influenced by the composition of the proteins present in your

sample

Become familiar with spectrophotometry before proceeding

Always let a spectrophotometer warm up for *C5+ minutes before using

Know the limits of the spectrophotometer with which you are using

@tandard curves are not always linear

The protein used for your standard curve must maDe sense

aDe sure your standard curve covers the absorbance range of your unDnown with at

least two points on either side

aDe sure that your protein solution behaves in a reproducible manner to the assay

method by maDing a dilution curve


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Ese buffer and water blanDs to anchor down your standard curve

lace the protein concentration on the yCais of you standard curve plot so that you can

use the bestCfit e$uation directly for concentration determination

ALai ***

Chromatographic Techniques WORD

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