high performance liquid chromatography (hplc) phc 213

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High Performance Liquid High Performance Liquid Chromatography Chromatography (HPLC) (HPLC) PHC 213

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Page 1: High Performance Liquid Chromatography (HPLC) PHC 213

High Performance Liquid High Performance Liquid ChromatographyChromatography

(HPLC)(HPLC)

PHC 213

Page 2: High Performance Liquid Chromatography (HPLC) PHC 213

Classification according to the technique used.

1.Columnar:

In this type the stationary phase is packed in a column, e.g. GC, LC and conventional column.

2.Planar:

In this type the stationary phase is spreaded as a thin layer on glass, plastic or aluminum plates; it is held in the network structure of paper (paper chromatography)

Page 3: High Performance Liquid Chromatography (HPLC) PHC 213

HPLC Separation Modes:HPLC Separation Modes:

Classified according to:Classified according to: PolarityPolarity Electrical chargeElectrical charge Molecular sizeMolecular size

Page 4: High Performance Liquid Chromatography (HPLC) PHC 213

Separations based on Separations based on polarity:polarity:

To design a chromatographic separation To design a chromatographic separation

system, we create competition for the system, we create competition for the

various compounds contained in the various compounds contained in the

sample by choosing a mobile phase and a sample by choosing a mobile phase and a

stationary phase with different polarities. stationary phase with different polarities.

Page 5: High Performance Liquid Chromatography (HPLC) PHC 213

Compounds in the sample that are Compounds in the sample that are similar in polarity to the stationary similar in polarity to the stationary phase will be delayed because they are phase will be delayed because they are more strongly attracted to it. more strongly attracted to it.

Compounds whose polarity is similar to Compounds whose polarity is similar to that of the mobile phase will be that of the mobile phase will be attracted to it and move faster.attracted to it and move faster.

Based upon differences in the relative Based upon differences in the relative attraction of each compound for each attraction of each compound for each phase, a separation is created by phase, a separation is created by changing the speeds of the analytes. changing the speeds of the analytes.

Page 6: High Performance Liquid Chromatography (HPLC) PHC 213

Normal Phase HPLC.

This is the same as in thin layer chromatography or column chromatography

Although it is described as "normal", it isn't the most commonly used in HPLC.

Polar compounds in the mixture being passed through the column will stick

longer to the polar silica than non-polar compounds will.

The non-polar ones will therefore pass more quickly through the column.

-Polar stationary phase Polar stationary phase and and non-polar solvent non-polar solvent .The column is .The column is filledfilled

with tiny silica particles, and the solvent is non-polar e.g. with tiny silica particles, and the solvent is non-polar e.g. hexane.hexane.

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Non-polar stationary phase and a polar mobile phase

• Reverse Phase.

The same column size , but the silica is modified to be non-polar by attaching

long hydrocarbon chains to its surface - typically with either C8 or C18 atoms . A polar solvent is used e.g. a mixture of water and methanol or acetonitril.

Polar molecules in the mixture having strong attraction with the polar

solvent (mobile phase) than hydrocarbon chains attached to the silica

(the stationary phase) therefore pass more quickly through the column.

Non-polar compounds in the mixture will tend to form attractions with the

hydrocarbon groups (stationary phase) and be less soluble in the solvent

(mobile phase) therefore pass slow down through the column.

Reversed phase HPLC is the most commonly used form of HPLC.

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Reversed phase HPLC is the most commonly used form of HPLC.

- “Like dissolves like”. In Normal Phase the analyte will be partitioned preferentially in the mobile phase and provide little interaction with the stationary phase. This is not desirable since selective retention on the column will be very hard to control. It can be controlled by modifying the stationary phase, a very time consuming and expensive proposition even if feasible.

In Reverse Phase the opposite is true. The analyte will be partitioned preferentially in the stationary phase (“Like dissolves like”). and by simply modifying the mobile phase, by adjusting the polarity, ionic strength or pH, selectivity can be virtually fully controlled.

 

Page 21: High Performance Liquid Chromatography (HPLC) PHC 213

Separations based on Separations based on charge: charge:

Ion-exchange chromatography:Ion-exchange chromatography: In ion-exchange chromatography, Likes In ion-exchange chromatography, Likes

repel, while opposites attract each other. repel, while opposites attract each other.

Stationary phases for ion-exchange Stationary phases for ion-exchange separations are characterized by the separations are characterized by the nature and strength of the acidic or basic nature and strength of the acidic or basic functions on their surfaces and the types functions on their surfaces and the types of ions that they attract and retain.of ions that they attract and retain.

Page 22: High Performance Liquid Chromatography (HPLC) PHC 213

Cation exchange is used to retain and separate positively charged ions on a negative surface.

anion exchange is used to retain and separate negatively charged ions on a positive surface.

Page 23: High Performance Liquid Chromatography (HPLC) PHC 213

Types of Ion-ExchangerTypes of Ion-Exchanger

Page 24: High Performance Liquid Chromatography (HPLC) PHC 213

SO3-

SO3-

Na+

COO-

H3N+

Na+

COOHH3N

+

pH2

pH4.5

Ion-exchange Resin

MECHANISM OF ION-EXCHANGE MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS CHROMATOGRAPHY OF AMINO ACIDS

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Selecting an operating Selecting an operating modemode

Sample type Sample type LC LC ModeMode

• Moderate polarity molecules LSC or LLCLSC or LLC

• Compounds with similar functionality LSC or LLC

• Ionizable species IEC

• Compounds with differing solubility LLCLLC

• Mixture of varying sized molecules GPC GPC

Page 28: High Performance Liquid Chromatography (HPLC) PHC 213
Page 29: High Performance Liquid Chromatography (HPLC) PHC 213

Retention parametersRetention parameters

1. Retention volume (1. Retention volume (VVRR) )

The retention volume is the volume of mobile phase passed through the column between the injection point and the peak maximum

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2- Retention time (2- Retention time (ttRR) ) = the distance = the distance from the injection point to the peak from the injection point to the peak maxima in time units.maxima in time units.

It is used as an identifier for a given It is used as an identifier for a given analyte. analyte.

It depends on mobile phase It depends on mobile phase flow rate.flow rate.

VVRR = = FF x x ttRR

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Page 32: High Performance Liquid Chromatography (HPLC) PHC 213

3. Void volume (3. Void volume (VV00))

The volume of liquid phase in the The volume of liquid phase in the column.column.

VV00 = = F F x x tt00

Also called retention volume of Also called retention volume of unretained component.unretained component.

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4. 4. Retention factor (Retention factor (kk\\))

Sometimes called capacity factorSometimes called capacity factor

kk\\ = = VVRR – – VV00 / / VV00 = = ttRR – – tt00 / / tt00

Independent on mobile phase flow rate Independent on mobile phase flow rate and column dimensions.and column dimensions.

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KK\\

The optimum values for kThe optimum values for k\ \ are in the range 1- are in the range 1-10; very little can be gained by increasing k10; very little can be gained by increasing k\\ further.further.

High values of High values of kk\\ also mean long analysis times.also mean long analysis times.

The capacity factor is controlled largely by The capacity factor is controlled largely by adjustment of the mobile phase composition.adjustment of the mobile phase composition.

Increasing Increasing kk\\ improves resolution by causing the improves resolution by causing the solutes to spend more time in the stationary solutes to spend more time in the stationary phase( increase anal. Time), increasing phase( increase anal. Time), increasing selectivity moves one or both peaks relative to selectivity moves one or both peaks relative to the other.the other.

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55. Selectivity (. Selectivity ())

The ability of the chromatographic system to discriminate The ability of the chromatographic system to discriminate different analytes (distance between the peak maxima).different analytes (distance between the peak maxima).

= k= k22 /k /k11 = = ttR2R2 – – tt00 / / ttR1R1 – – tt00

It is always greater than It is always greater than unity.unity.

It depends primarily on the nature of the analyte and the It depends primarily on the nature of the analyte and the difference in its interaction with the stationary phase.difference in its interaction with the stationary phase.

It is not affected by the mobile phase composition unless It is not affected by the mobile phase composition unless this parameter modifies the analyte nature (ionization).this parameter modifies the analyte nature (ionization).

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for a desired degree of resolution, three conditions have to be met:

a- The peak must be separated from each other (α > 1 )

b- The peak must be retained on the column (k\ > 0 )

c- The column must develop some minimum number of plates.

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6. Efficiency6. Efficiency It is a property of the column.It is a property of the column.

The degree of band broadening (width of the The degree of band broadening (width of the chromatographic peak).chromatographic peak).

Tow terms are used as quantitativeTow terms are used as quantitative

measures of column efficiency:measures of column efficiency:

Plate height(Plate height(HH) and Plate number() and Plate number(NN).).

N = L / HN = L / H

..

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The Theoretical Plate Model of The Theoretical Plate Model of ChromatographyChromatography

The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, NN (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better).If the length of the column is L, then the HETP is

HETP = L / N

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The number of theoretical plates that a real column possesses can be

found by examining a chromatographic peak after elution;

N = 16 (tN = 16 (tRR / W ) / W )22

where w w is the peak width ttR R is the retention time N N is the plate hight

As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture

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The efficiency of the column is increased as the number of theoretical plates (N) is increased,

Thus the movement of solutes and solvent is regarded as a series of stepwise transfer from one plate to the next.

At each plate of the stationary phase , continuous partitioning and equilibration of solutes (components molecules) occurs as the mobile phase moves down the column.

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Efficiency and selectivity are complementary Efficiency and selectivity are complementary chromatographic descriptors.chromatographic descriptors.

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66. Resolution . Resolution ((RR))

The resolution of a column provides a quantitative The resolution of a column provides a quantitative measure of its ability to separate two analytes.measure of its ability to separate two analytes.

The ratio of the distance between two peaks to the The ratio of the distance between two peaks to the

average width of these peaks (at baseline).average width of these peaks (at baseline).

RR = 2 ( = 2 (ttR2R2 – –ttR1)R1) / /ww22 + +ww1 1

1.5 1.5 is sufficient for the baseline separation of closely is sufficient for the baseline separation of closely eluted eluted

peaks.peaks.

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Page 44: High Performance Liquid Chromatography (HPLC) PHC 213

2. Parameters used in HPLC

Condition for good separation

Rs =4

1 - 1

1 + k’2

k’2N

A larger Rs value means a better separation.

1 + k’2

k’2: Capacity term

increases the retention time

- 1: Selectivity term

increases the time interval between peaks

N : Column efficiency termproduce narrow peaks

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Comparison of the variation of selectivity Comparison of the variation of selectivity and efficiency necessary to increase and efficiency necessary to increase

resolution from 1 to 1.5resolution from 1 to 1.5

Resolution Efficiency Selectivity

1 10000 1.04

1.5 22500 1.04

1.5 10000 1.06

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Page 47: High Performance Liquid Chromatography (HPLC) PHC 213

Optimization of column performance.

To optimize the column performance we have to change αand َK' to obtain Rs value of 1.5.

This can be achieved by:

1-Change the composition of mobile phase and /or its flow rate

2-The column must be perfectly packed with small regular spherical particles, without any cracks, gabs or spaces to minimize zone broadening and to maximize N.

3-If resolution is still less than 1, the stationary phase must be changed

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In regular gravity column the particle size of In regular gravity column the particle size of the stationary phase ranges from 150-250 the stationary phase ranges from 150-250 μm. Decreasing the particle size leads to μm. Decreasing the particle size leads to increase in surface area and consequently increase in surface area and consequently better separation is achieved. In HPLC the better separation is achieved. In HPLC the particle size used is ranging from 3-20 μm. particle size used is ranging from 3-20 μm. Particle of ≤ 5 μm would yield more than Particle of ≤ 5 μm would yield more than 10000 theoretical plates / meter.10000 theoretical plates / meter.

The capacity of HPLC column are usually low The capacity of HPLC column are usually low less than 20 μ g of sample so detector must less than 20 μ g of sample so detector must be very sensitive. be very sensitive.

Pump is necessary to push mobile phase Pump is necessary to push mobile phase through the finely divided stationary phase.through the finely divided stationary phase.

Page 49: High Performance Liquid Chromatography (HPLC) PHC 213

High Performance Liquid High Performance Liquid ChromatographyChromatography

(HPLC)(HPLC)

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Instrumentation: Instrumentation: 1- Solvent reservoir (S).1- Solvent reservoir (S).

2- High pressure pump.2- High pressure pump.

3- Injection.3- Injection.

4- Column.4- Column.

5- Detector.5- Detector.

6- Recorder.6- Recorder.

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1- Solvent reservoirs: May be one container in which the required solvent May be one container in which the required solvent system is added or may be two or more each containing system is added or may be two or more each containing one solvent and mixing is done in a solvent mixing unite. one solvent and mixing is done in a solvent mixing unite.

The HPLC solvents must be of:The HPLC solvents must be of:

-High purity as HPLC grade solventsHPLC grade solvents

-Low viscosity

-limited flammability and toxicity

-low reactivity to avoid chemical interaction with solutes

-Compatibility with the detector

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Degassing is very important for the following reasons: is very important for the following reasons:

a- To get ride of dissolved gases especially oxygen that may a- To get ride of dissolved gases especially oxygen that may react with stationary or mobile phase.react with stationary or mobile phase.

b- Bubbles formation will disturb the detectors.b- Bubbles formation will disturb the detectors.

Degassing can be done by: can be done by:

1- Sonicator. 2- Passing helium in the 1- Sonicator. 2- Passing helium in the solventssolvents

The mobile phase may be, organic solvents-water-buffers.The mobile phase may be, organic solvents-water-buffers.

The solvents must be filtered and degassed. The solvents must be filtered and degassed.

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They are necessary to enable the flow of mobile phase They are necessary to enable the flow of mobile phase through the finely divided stationary phase.through the finely divided stationary phase.

2- Pumps:

Pumps Pumps should be able toshould be able to

-deliver up to 7000 psi (Pound/square inch).-deliver up to 7000 psi (Pound/square inch).-Have flow rates ranging from 0.1 to 10ml/min-have high resistance to corrosion by a variety of solvents.

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-Stepwise elution means the use of several eluting agents of gradually increasing strength for successive desorption of the

separated components.

-Continuous change of the desorption power of the mobile phase is achieved by adding stronger eluting agent (more polar solvent) gradually to less polar one which is currently used

i.e. we start with the less polar solvent then the more polar is added and mixed gradually according to a definite program.

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Page 56: High Performance Liquid Chromatography (HPLC) PHC 213

In order to introduce a sample onto

the column for analysis, a special

valve called the injector must be used to transfer the sample

into thepressurized system..

3- Injection.

Injectors mostly are 6-port rotary valves

Page 57: High Performance Liquid Chromatography (HPLC) PHC 213

Guard (or pre-) Column :Guard (or pre-) Column :

They are placed between injector They are placed between injector andand analytical columnItanalytical columnIt

-The job of the guard column is to to remove or retain sample and solvent impurities that could be irreversibly adsorbed onto the analytical column ,thus prevent contamination of the analytical column which decrease its performance.

-A precolumn is used to protect the column against high-pH mobile phases

--They are very short columns about 3 cm , packed with material They are very short columns about 3 cm , packed with material similar to that contained in the analytical column, for example similar to that contained in the analytical column, for example C18.C18.

Page 58: High Performance Liquid Chromatography (HPLC) PHC 213

Analytical column are typically 10-25 cm long and 5-10 Analytical column are typically 10-25 cm long and 5-10 mm internal diameter (id).mm internal diameter (id).

Columns are made of stainless steel lined with glass to Columns are made of stainless steel lined with glass to prevent metal interaction with solvent or solutes.prevent metal interaction with solvent or solutes.

Particle size from 3-20 μm. Particles ≤ 5 μm yield Particle size from 3-20 μm. Particles ≤ 5 μm yield >10,000 theoretical plates/ meter.>10,000 theoretical plates/ meter.

4- Analytical Columns:4- Analytical Columns:

Recently high speed and high performance microcolumns with 1-4mm(id) and length of 3 to 7.5 cm having the advantages of speed and minimal solvent consumption.

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Porous micro particles have diameter ranging from 3 to 10m, they are composed of silica, alumina, porous

polymer or ion exchange resin. Silica particles may be coated with thin organic film, which

is physically or chemically bonded to the surface.

Column Packing Materials:

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Preparation of bonded phasePreparation of bonded phase

Si---oH + RoH → Si---oRSi---oH + RoH → Si---oR Thermaly un stable and easy hydrolysis.Thermaly un stable and easy hydrolysis. Si---oH + Cl-Si-(CH3)2 R →Si---oH + Cl-Si-(CH3)2 R →

Si OH + Cl Si R

CH3

CH3

Si O Si R

CH3

CH3

organochlorosilane(R: C4H17 or C18H37)

surface of a silicaparticles

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endcappingendcapping

In In chromatography, the replacement of , the replacement of accessible accessible silanol groups in a bonded groups in a bonded stationary phase by by trimethylsilyl groups is groups is referred to as referred to as endcappingendcapping

Si OH Si O Si CH3

CH3

CH3

Silanol "Active"

Doactlvated Silanol "inactive"

Example of deactivated Silanol a tnimethylsily (TMS)

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Endcapping technology prevents the tailing of a Endcapping technology prevents the tailing of a polar compound's peak adsorp polar comp.by 's peak adsorp polar comp.by silinol gpsilinol gp

Bonded phase must be used within Bonded phase must be used within pH 2-8pH 2-8 PH <2 bonded moity will be PH <2 bonded moity will be

removed.removed. Ph > 8 silica will dissolve Ph > 8 silica will dissolve

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Examples of the commonly used Examples of the commonly used detectors:detectors:

1- UV Detectors:1- UV Detectors:They detect solutes that can absorb They detect solutes that can absorb

UV light due to the presence of conjugated UV light due to the presence of conjugated system. These are the most commonly system. These are the most commonly used detectors.used detectors.2- 2- Fluorescence Detectors:Fluorescence Detectors:

Limited number of compounds can be Limited number of compounds can be detected by such detectors. detected by such detectors.

5- Detectors:5- Detectors:They are placed at the column exit and used to They are placed at the column exit and used to detect a specific property of solute materials in detect a specific property of solute materials in the column effluent. the column effluent.

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3- Infrared Detectors:3- Infrared Detectors:

It is more general. The solvent use It is more general. The solvent use must not absorb IR at the chosen must not absorb IR at the chosen wavelength for solute detection.wavelength for solute detection.

4- Radioactivity Detectors:4- Radioactivity Detectors:

Very specific for radioactive materials.Very specific for radioactive materials.

5- Refractive Index Detectors:5- Refractive Index Detectors:

Measure the change in the Refractive Measure the change in the Refractive Index of the mobile phase.Index of the mobile phase.

6. Mass Spectroscopy Detectors.6. Mass Spectroscopy Detectors.

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That means that conditions have to be carefully controlled

For a particular compound, the retention time will vary depending on:-the pressure used (because that affects the flow rate of the solvent)-the nature of the stationary phase (not only what material it is made of, but also particle size)-the exact composition of the solvent-the temperature of the column

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HPLC Column & efficiency

*If a column bed is stable and uniformly packed, its mechanical separation power is determined by the column length and the particle size.

*Mechanical separation power, also called efficiency, is often measured and compared by a plate number [symbol = N].

*Smaller-particle chromatographic beds have higher efficiency and higher backpressure. More mechanical separation power is gained by increasing column length.

Shorter column lengths minimize all these variables but also reduce mechanical separation power.

*However, the trade-offs are longer chromatographic run times, greater solvent consumption, and higher backpressure

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Effect of column length

Effect of particle size

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Effect of Increasing Efficiency (N)

The efficiency N can be doubled by halving the particle size (i.e. 10µm to 5µm). In the above example, resolution, Rs increased from 2.0 to 2.8. Column length can then be halved to decrease analysis time

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Importance of Peak Asymmetry (As)

These chromatograms illustrate the negative effect of increased peak asymmetry, As, on resolution, Rs.

When investigating the effect of bonded phase chain length on retention, it is important to maintain the use of a high purity silica so that peak asymmetry is minimized

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GENERAL FACTORS INCREASING RESOLUTION

1. Increase column length

2. Decrease column diameter

3. Decrease flow-rate

4. Pack column uniformly

5. Use uniform stationary phase (packing material)

6. Decrease sample size

7. Select proper stationary phase

8. Select proper mobile phase

9. Use proper pressure

10. Use gradient elution

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Atest mixture consisting of phenylmethyl ketone , nitrobenzene,benzene & hexane is separated on a C18 column with a mobile phase of CH3OH: H2O (60:40) . With these conditions, the ketone is eluted first.The order of the elution of the other solutes is

•Nitrobenzene, benzene & hexane•Nitrobenzene, hexane & benzene.•Benzene , hexane & nitrobenzene.• Hexane, benzene& nitrobenzene.

Quiz

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In the above question, If the mobile phase used is CH3OH: H2O (40:60) ,

the retention time of the other solutes will:•Increase

•Decrease.•Not changed.