CHAPTER - II - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8292/15/15_chapter 2.pdf · consisting of a single central asymmetric carbon atom with a left pthalidimide ring

Acharya Nagarjuna University, Guntur 64

CHAPTER - II


COMPARATIVE LIQUID CHROMATOGRAPHIC

ENANTIOMER RESOLUTION OF LENALIDOMIDE AND

THALIDOMIDE ON IMMOBILISED POLYSACCHARIDE

DERIVED CHIRAL STATIONARY PHASES

Polysaccharide-derived chiral stationary phases (CSPs) are notorious to

illustrate high chiral recognition ability in HPLC and have been comprehensively

used to separate a wide range of racemic compounds (Yashima, E., 2001). These type

CSPs are usually prepared coating or adsorbing the polysaccharide derivatives on

silica gel. Therefore, the solvents such as chloroform, methylene chloride, and

tetrahydrofuran, which dissolve or swell the chiral selectors of the polysaccharide

derivatives, can’t be used as mobile phases (Daicel Chemical Industries). For

example, in case of Chiralpak AD prepared by coating amylose tris (3,5-

dimethylphenylcarbamate) derivative, one of the widely used polysaccharide type

CSPs, suitable mobile phases like n-hexane, 2-propanol and ethanol should be used

for the column safety (Franco, P., 2001). It may be damaged in case of even the use

of only a little amount of inappropriate solvents used as mobile phases and/or sample

solvents. These limitations represent a disadvantage for new applications using these

CSPs and, especially, preparative separation due to solubilization of analytes. To

overcome these problems, therefore, the development of polysaccharide-derived

covalently bonded CSPs has been of great interest and various results of diverse

attempts have been reported (Franco, P., 1998). Recently, Chiralpak IA prepared by

chemically bonding amylose tris (3,5-dimethylphenylcarbamate) derivative on silica

gel, Chiralpak IB prepared by chemically bonding cellulose tris (3,5-

dimethylphenylcarbamate) derivative on silica gel, and very lately, a new covalently

immobilized type CSP, Chiralpak IC based on cellulose tris (3,5-

chlorophenylcarbamate) has been introduced (Daicel Chemical Industries., 2007).

The chiral recognition ability of these polysaccharide stationary phases is influenced

considerably by the substitutions introduced on the phenyl group of the polymer

(Chankvetadze, B., 1997) and the introduction of an electron-donating methyl group


or an electron-withdrawing halogen at the meta- and / or para-position of the phenyl

ring a lot develop the chiral recognition capability of the CSPs (Okamoto, Y., 1987).

These modified polysaccharide CSPs have been successfully applied to

enantioseparation utilizing normal (Chankvetadze, B., 2001), polar (Chankvetadze,

B., 2002), and reversed-phase modes. In this study, we present the comparative liquid

chromatographic enantiomer resolution of thalidomide and lenalidomide on these

three polysaccharide derived CSPs.

Thalidomide (fig. 2.1) was first developed and introduced as a sedative to

reduce nausea during pregnancy in the 1950s, but withdrawn from the market in 1961

due to its notorious teratogenicity. Thalidomide, it is an a-N-pthalimidoglutarimide

consisting of a single central asymmetric carbon atom with a left pthalidimide ring

and a right glutarimide ring, a derivative of glutamic acid, is pharmacologically

classified as an immunomodulatory drug (IMD). It contains two amide rings and a

single chiral center. The interconversion between the enantiomers of thalidomide is

very rapid at physiological pH in aqueous medium and biological matrices such as

plasma, undergoing rapid spontaneous hydrolysis (Eriksson, T., 1992) and the present

clinical formulation is a racemic mixture of the optically active (S) -isomer has been

linked to the teratogenic effects of thalidomide, whereas the (R) -isomer is

responsible for its sedative properties. The two isomers rapidly interconvert at

physiological pH invivo, and efforts at formulating only the (R)- isomer have failed to

obviate the teratogenic potential of thalidomide.

Lenalidomide, 3-(4-amino-1-oxo-3H-isoindol-2-yl) piperidine-2, 6-dione (fig.

2.2) is a synthetic derivative of glutamic acid and is structurally close to thalidomide

identical backbone but differs from thalidomide by removing oxygen from the

phthalyl ring and by adding an amine group.

Separation of enantiomers can be achieved using different chromatographic

techniques such as gas chromatography, liquid chromatography (Torok, G., 2005),

supercritical fluid chromatography (Maftouh, M., 2005) and capillary

electrochromatography (Mangelings, D., 2003). Among these methods, high-

performance liquid chromatography (HPLC) is well known as a important, fast,

selective and highly capable technique, successfully employed for separation and


determination of enantiomers of drugs. In this technique, chiral selectors can be used

as mobile phase additives or as part of the stationary phase. The most common HPLC

approach for resolving enantiomers involves the use of chiral stationary phases

(CSPs) (Subramanian, G., 1994).

Only low or non-polar solvents such as n-pentane, n-hexane, n-heptane ,

alcohols (methanol, ethanol, 2-propanol etc.) or, sometimes, acetonitrile can be used

as mobile phases due to the coated nature of these CSPs, which effect into their

limited applications, as mobile phase is the key optimizing parameter in chiral

chromatography. Therefore, some polar solvents such as tetrahydrofuran (THF),

chloroform, dichloromethane, acetone, ethylacetate and methyl tert-butyl ether are

banned with these CSPs and can not be used (Okamoto, Y., 1986) while these

solvents are helpful for resolving some racemates, which could not be resolved by

using non-polar solvents. Due to these drawbacks of coated CSPs, the need of

chemical bonding between derivatized polysaccharides and silica gels was realized,

which is called as immobilization. Some workers attempted to immobilize chiral

polysaccharide phases on silica gel (Francotte, E., 2002). Recently, Chiralpak IA,

Chiralpak IB, Chiralpak IC columns, having polysaccharides immobilized on silica

gel, were launched into the market, which can be used with a broad selection of

solvents (Chiral Technologies., 2004). In view of the significance of immobilized

phases, attempts are made to elucidate a state-of-art of polysaccharides

immobilization and method protocol in this chapter including their applications under

optimized conditions, enantioselectivity, efficiencies and a comparison of the chiral

recognition capabilities of coated Vs immobilized CSPs.

1. Experimental

1.1 Chemicals and reagents

Thalidomide and Lenalidomide were obtained from Natco Pharmaceuticals

Ltd (Hyderabad, India). HPLC-grade acetonitrile (ACN), analytical-grade n-hexane,

2-propanol and Diethyl Amine (DEA) were purchased from Merck Research

Laboratory (Mumbai, India, 1,3,5-tri-tert-butylbenzene (the void time marker) from

Sigma-Aldrich (India, Bangalore)


1.2 Sample preparation

Analytical solutions of racemic LLM and TLM of 5 mg/mL were prepared in

ACN by dissolving in a 5 mL volumetric flask, while the stock solutions were serially

diluted with acetonitrile to give working solutions at preferred concentrations. Stock

solutions were protected from light by covering aluminum foil and kept in a

refrigerator at 4 ºC.

1.3 Chromatographic conditions

HPLC was carried out on a SHIMADZU LC- 2010A system (Kyoto, Japan)

equipped with quaternary pump, UV-VIS detector, autosampler and column oven.

Three different columns were used: Column I - Chiralpak IA (250 Х 4.6 mm)

amylose tris (3,5- dimethylphenylcarbamate) (fig. 2.3) immobilized on 5 μm silica

gel; column II - Chiralpak IB (250 Х 4.6 mm) - cellulose tris (3,5-

dimethylphenylcarbamate) (fig. 2.4) immobilized on 5 μm silica gel; column III

Chiralcel IC (250 Х 4.6 mm) - cellulose tris (3,5-dichlorolphenylcarbamate) (fig. 2.5)

immobilized on 5 μm silica gel.

Chromatographic experiments were carried out under normal phase

conditions. The mobile phases were composed of n-hexane and polar modifier. The

mobile phase flow rate was studied from 0.4 to 1.2 mL/min on selectivity and

resolution, all other experiments were performed at 1.0 mL/min. The column

temperature experiments were carried out at 25 to 45 0C and UV detection was

performed at 240 nm. The working sample solution was prepared in acetonitrile at a

concentration of 10 µg/mL. The injection volume was 10 µL.

1.4 Chromatographic characteristics

The selectivity coefficient was expressed as: α = k2/k1 where k1, k2 are

retention factors for the first and second eluting enantiomers. The retention factors k1

and k2 were calculated as follows:

k1= (tr1- t0) / t0 and k2 = (tr2-tr0)/tr0

Where t0 is dead elution time and tr1 and tr2 are retention time of enantiomers

1 and 2. The stereochemical resolution factor (Rs) of the first and second eluting


enantiomers is calculated by the ratio of the difference between the retention times tr1

and tr2 to the arithmetic mean of the peak widths w1 and w2

Rs= (tr2-tr1) / 1/2(w1+w2)

The resolution (Rs) between two enantiomers was determined by the method

of mid-height of the peaks. Where tr1 and tr2 are respectively the retention times of

the first and second peaks, w1 and w2 are the corresponding width at the mid-height of

the peaks. Injecting 1,3,5-tri-tert-butylbenzene as a non-retained indicator projected

the dead time.

2. Results and discussion

2.1 Optimization of the chromatographic conditions

The individual chromatographic enantiomer separation characteristic of

Chiralpak IA, Chiralpak IB and Chiralpak IC have been discussed in various papers

(Zhang, T., 2006). Considering the structural and stereochemical differences in these

polymeric chiral selectors, it is rational to expect that each of these three immobilized

CSPs may be capable of recognizing specific classes of solutes, sometimes with a

common overlapping spot. In this case, the combined use of these three chiral

resolving materials could give rise to an improved success rate of enantiomer

resolution.

If a successful enantioselective method has to be establishing on Chiralpak IA,

Chiralpak IB and Chiralpak IC for recemic TLM and LLM, the most important aim

would be achieving an excellent resolution with a shorter run time. At analytical level

we can think that the approach of method development on the immobilized CSPs can

be based on similar approach to the one used for the coated type phases. Basically, all

miscible solvents can be used with an immobilized CSP. In practice, the choice of the

mobile phase is guided not only by the chiral recognition state, but also by

considerations in relation to the physical and chemical natures of the solute, the

reason and scale of the projected chromatographic procedure.

The strategies to attain enantiomer separation are attractive capricious from

one laboratory to another depending on the type of substances, the equipment

available, but motivation for the development of screening approaches remain


common. We search the easiest and the most reliable way to analyze TLM and LLM

with physicochemical properties in a limited time. This is the perspective for chiral

compound screening based on the three columns packed with the immobilized CSPs.

The key point would be setting the accurate conditions to start the screening,

leading to proper retention times and successful chiral recognition. Enantioseparation

with polysaccharide type CSPs in normal phase conditions are often increasing, due

to competing property of the highly polar mobile phase components with polar

interactions involved in chiral recognition (Tachibana, K., 2001).

Basically, all miscible solvents can be used with an immobilized CSP either in

their pure form or as mobile phase components. In practice, considering the solute

nature, the purpose of the chromatographic operation and the general enantioselective

properties of each solvent towards the CSP when they are used in the mobile phase

can get used to the selection of mobile phase. The mobile phase selected should have

a minimum solubility towards the analyte so that the liquid chromatography method

becomes possible.

A Mixture of n-hexane, 2-propanol (75: 25) was first used as the mobile

phase to optimize on Chiralpak IA, IB and IC columns for the separation of LLM and

TLM. However the peak shapes was poor, long retention time with broad peaks with

huge tailing were observed. Therefore, more basic conditions were needed to achieve

suitable elution time. Obviously, changes of pH have an effect on the ionization state

of both the analyte and the CSP. The slight basic or neutral the eluent the stronger are

the ionic interaction in normal phase mode (llisz, I., 2009; Berthod, A., 2009).

Therefore we assume, that strong ionic interaction caused the long retention of the

compounds on the CSP under neutral condition. Several solvents of higher elution

strength, such as MeOH, EtOH, THF, DEA, can be competently used as modifier in

n-hexane to get enhanced separation and decrease run time. Although the percentage

of a modifier is generally low (mostly 2-10% in n-hexane), its nature can very much

affect the enantioselectivity of a given compound. By adding 1% DEA to the n-

hexane, both the analytes and the CSP become deprotonated then stronger ionic

interactions are impossible and it allows elution of LLM and TLM with amine group

so that the analysis time by half and peak shapes are reasonably improved. In


contrast, Chirlpak IA column resulted the lowest resolution with negligible separation

factor for LLM (fig. 2.6). The other two columns were not provided the separation

for LLM with the same conditions. Indeed, no chiral separation of TLM was

achieved under these conditions. However, when the content of 2-propanol was

reduced from 25 to 10% by volume, separation of LLM isomers was observed (fig.

2.7) on Chiralpak IA column, this revealed that the polar carbamate moieties on the

phenylcarbamate derivatives of polysaccharides are the major interaction sites for

chiral recognition, LLM have electronegative atoms such as oxygen and nitrogen, a

single NH group at chiral center, and at least one aromatic ring. Therefore, the chiral

recognition at the chiral selector amylose carbamate should mainly result from

dipole-dipole interactions, hydrogen bonds, and π- π interactions (Aboul-Enein, H.

Y., 2000), and secondary structure of polysaccharide backbone is maintained by the

H-bonding between the N-H group and neighboring glucose unit. A similar effect

observed when using Chiralpak IB and Chiralpak IC in the same mobile phase, but

retention factors was higher on Chiralpak IB and IC (fig. 2.8, 2.9) than on Chiralpak

IA.

A reasonable enantioseparation performance was identified for TLM on

Chiralpak IC (fig. 2.10), keeping a mobile phase composed of n-hexane: 2-propanol

(90:10) and DEA (1% v/v), the enantiomers could not be resolved on Chiralpak IA

and Chiralpak IB. Attempts to optimize the resolution of TLM on Chiralpak IC by

modifying the composition of the mobile phase to n-hexane: 2-propanol (95:5) and

DEA (0.1% v/v) proved doing well, as it is clear from the chromatogram (fig. 2.11).

Nevertheless, an absolute resolution was not achieved on Chiralpak IA and IB under

exactly the same chromatographic conditions (fig. 2.12, 2.13).

The key point for the mobile phase selection was that, in order to take full

advantage of the interactions between the stationary phase and solutes, the polarity of

the mobile phase could be distinguishes itself to a adequate quantity from that of

stationary phase. By extrapolation, the interactions stationary phase-solute would fall

down if the stationary phase mobile phase has very similar properties in terms of

polarity. This may be case of DEA and Chiralpak IC. Even though the retention


mechanisms may be more complex and may not basically follow the polarity

commandment.

Although the chiral selectors are the same, the corresponding immobilized

CSPs generally show diverse enantioselectivity under identical conditions. This

indicates differences in the capability to access the chiral interactions sites in the

selector due to conformational changes of the polysaccharide chains upon the other.

Since prediction of the most excellent solvent for resolution of specific compounds is

very complicated, the immobilized phases have screened with different mobile phases

appropriate on all columns as well as the more non-conventional solvents. The

comprehensive range of solvents applicable on the immobilized polysaccharide CSPs

can increase the possibility to attain new or enhanced enantioselectivity and

resolution.

After exhaustive investigations it is concluded that a limited number of

solvents and their mixtures can lead to improved resolution, selectivity and shorter

run time. The selected solvents n-hexane: 2-propanol and DEA results better

enantioselectivity of polysaccharide CSPs is repeatedly observed under normal phase

conditions since H-bonding, which plays an tremendously significant role in chiral

recognition with polysaccharide CSPs, is greatly enhanced under these conditions

(Okamoto, Y., 1998). TLM oxygen group was replaced with an amine group on

benzene ring to become LLM, which exhibited a decrement in the retention factor and

enantioselectivity. However, in LLM with an alkyl group, the retention and separation

factor were both enhanced. The results suggested that the amine group likely to

increased the dipole-dipole interaction with CSP.

Wainer et al. in 1987 demonstrated that steric factor of an enantiomer played

a significant role in it’s fitting into chiral cavities of cellulose CSPs. The aromatic

portion of LLM may fit into the chiral cavity to form complex with CSP (Wainer, I.

W., 1987). Chiralpak IA column, which contains the same phenylcarbamate moiety

as Chiralpak IB column but attached to the amylose backbone instead of cellulose,

exhibited rather high enantiomer resolving ability compared to its cellulosic analog.


Since these three stationary phases should be complementary in recognizing

chirality, because of identical derivatization groups, the different retention behaviors

should be due to the different conformation of the polysaccharides. Therefore we

believe, that the enhanced chiral resolution of the TLM the amylose-based column is

caused by differences in the superamolecular structure of these two CSPs in a

comparative study of amylose and cellulose based in NP mode. The consequence of a

number of experimental parameters upon the chiral resolution of the TLM and LLM

is discussed further.

2.2 Effect of analyte structure

Small difference in the structure of the analytes can significantly have an

effect on their enantioseparation. Incase of TLM and LLM, both analytes have a

similar basic molecular skeleton. It is a synthetic derivative of glutamic acid and is

structurally close to thalidomide (identical backbone but differs from thalidomide by

removing an oxygen from the phthalyl ring and by adding an amine group). Although

it is chiral and possesses an asymmetric carbon, it has been developed as a recemic

mixture since it undergoes racemisation under physiological conditions. It is obtained

as a hemihydrate form and is non-hygroscopic.

From the chromatographic data at optimized conditions it becomes obvious

that the so-called chemical selectivity of the CSP for separating the pairs of

enantiomers of LLM and TLM is not sufficient. However, it seems that the position

of amine group in place of oxygen on phthalyl ring are playing a important role in the

largely enantioselectivity of the compounds. The position of the oxygen and polar

group of amine obviously has an effect on the π-π interaction and therefore the chiral

recognition to a large extent. The enantioselectivity for LLM is approximately the

same as TLM.

2.3 Effect of 2-propanol content on chiral selectivity and resolution

Retention factors are reduced when the mobile phase composition is enriched

with alcohol (Zhang, T., 2005). The influence of the 2-propanol composition on the

selectivity and resolution (Table. 2.1) of LLM and TLM and the enantioseparation

was studied on Chiralpak IA and IC; illustrate that up concentration of about 25 to


5% of 2-propanol (fig. 2.14a, 2.14b), the resolution and selectivity of the two

enantiomers rapidly decreased for TLM and selectivity was increase and resolution

was decreased for LLM. It seems to be obvious that alcohol ratio has a weak

influence on the selectivity on TLM, while more than 30% of 2-propanol

considerably prejudiced this factor on LLM. This indicates that alcohol competes

with the analyte for the formation of hydrogen bond with the stationary phase, which

maximizes the opportunity to form a diastereomeric complex between the analyte and

the CSP (Zhang, W., 2006).

This phenomenon can be explained by the competition of alcohol with the

analyte for the formation of hydrogen bonding with the active sites of the CSP, which

decreases retention factors and resolution. These active sites can be also

nonstereoselective such as residual silanol groups on silica surface, in this case,

reduce in retention may be observed without major change in the enantioselective

factor (Zhang, T., 2008). The 10 and 5 percent of the alcohol used as organic

modifier in the mobile phase for LLM and TLM respectively to play a significant role

in the enantiorecognition method.

2.4 Effect of column temperature and flow rate of the mobile phase

The effect of column temperature on retention and resolution was studied over

the range of 25-45 0C; results were summarized in (Table. 2.2). The results as shown

for both LLM and TLM in (Fig. 2.15) prove a common behavior with a decrease of

enantioselectivity as temperature increases, which specify an enthalpic control of the

separation. A working temperature of 30 °C was selected where the system gave

sufficient resolution values.

Flow rate variation on selectivity and resolution was studied of both LLM and

TIM, as depicts in (fig 2.16a, 2.16b) the consequence of flow rate variation on the

chiral resolution. We can see that selectivity does not depend on this parameter, while

resolution was better for a flow rate of 0.4 mL / min, but selectivity and resolution

was reasonable superior at 1.0 mL / min with out compromising on peak shapes and

run time. Indeed, a lower flow rate corresponds to a higher retention, this allows more

interaction between the solute and the CSP and lets enough time for chiral

interactions to occur. The improvement of separation efficiency with reduced flow


rate on this type of CSPs observed. As expected, the flow rate did not change the

overall enantioseparation characteristics (retention factors and enantioselectivity), but

the resolution and selectivity was significantly improved for both LLM and TLM at

lower rates.

3. Conclusion

The superior immobilization technologies offer an impetus to the modern

chiral stationary phases for their applicability and reliability in enantiomeric

separation. Chiralpak IA, IB and IC are the first in new cohort of immobilized

polysaccharide-based supports. They exhibit selectivity for diverse structural

enantiomers and have the improvement to be compatible with all inorganic solvents.

Both of these properties improve their potential analytical and preparative

applications in an assortment of chromatographic modes. They remarkably

complementary characteristics in enantiomer resolution, making them an efficient

column set for method development. Common method development strategies can be

useful on the three columns to attain high success rate. Owing to the specific structure

of its chiral selector as well as its immobilized nature, it exhibits both high

enantioselective performance and exceptional solvent compatibility.

The consequence of the column temperature on the retention and

enantioseparation were studied. The corresponding apparent thermodynamic

parameters were deduced from Van’t Hoff plots. When 2-propanol was used at low

percent ratio with by adding DEA to the n-hexane, the enantioseparation of both TLM

and LLM remarkably improved.


Fig 2.1: Thalidomide: 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1, 3 (2H)–dione

Fig 2.2: Lenalidomide: 3-(4-amino-1-oxo-3H-isoindol-2-yl) piperidine-2, 6-dione

Fig 2.3: Amylose tris (3,5- dimethylphenylcarbamate) - Chiralpak IA


Fig 2.4: Cellulose tris (3,5-dimethylphenylcarbamate) -Chiralpak IB

Fig 2.5: Cellulose tris (3,5-dichlorolphenylcarbamate)-Chiralpak IC


Fig 2.6: Resolution of lenalidomide on Chiralpak IA: n-hexane: 2-propanol: DEA 75:25:1%

Fig 2.7: Resolution of lenalidomide on Chiralpak IA: n-hexane: 2-propanol: DEA 90:10:1%


Fig 2.8: Resolution of lenalidomide on Chiralpak IB: n-hexane: 2-propanol: DEA 90:10:1%

Fig 2.9: Resolution of lenalidomide on Chiralpak IC: n-hexane: 2-propanol: DEA 90:10:1%


Fig 2.10: Resolution of thalidomide on Chiralpak IC: n-hexane: 2-propanol: DEA 90:10:1%

Fig 2.11: Resolution of thalidomide on Chiralpak IC: n-hexane: 2-propanol: DEA 95:5:1%


Fig 2.12: Resolution of thalidomide on Chiralpak IA: n-hexane: 2-propanol: DEA 95:5:1%

Fig 2.13: Resolution of thalidomide on Chiralpak IB: n-hexane: 2-propanol: DEA 95:5:1%


Effect of 2-propanol content on the resolution and the

selectivity of LLM

1.03

1.03

1.03

1.04

1.04

10 15 20 25

% 2-Propanol

Se

lec

tiv

ity

(α

)

0.40

0.55

0.70

0.85

1.00

Re

so

luti

on

(R

s)

Fig 2.14a: Effect of 2-propanol content on the resolution and the selectivity of LLM

Effect of 2-propanol content on the resolution and the

selectivity of TLM

0.94

0.98

1.02

1.06

1.10

10 15 20 25

% 2-Propanol

Se

lec

tiv

ity

(α

)

0.10

0.40

0.70

1.00

1.30

Re

so

luti

on

(R

s)

Fig 2.14b: Effect of 2-propanol content on the resolution and the selectivity of TLM


-0.61

-0.46

-0.31

-0.16

-0.01

3.12 3.20 3.27 3.35 3.42

1/T(10-3 K-1)

ln k

2

Fig 2.15: The Van’t Hoff plots for the enantioseparation of LLM and TLM

Flow rate optimization-LLM

1.00

1.01

1.02

1.03

1.2 1.0 0.8 0.6 0.4

Flow rate (mL min-1)

Sele

cti

vit

y (

α)

0.20

0.50

0.80

1.10

Reso

luti

on

(R

s)

Fig 2.16a: Flow rate effect on the resolution of LLM

Flow rate optimization-TLM

1.00

1.03

1.05

1.08

1.10

1.2 1.0 0.8 0.6 0.4

Flow rate (mL min-1)

Se

lec

tiv

ity

(α

)

0.20

0.60

1.00

1.40

1.80

Re

so

luti

on

(R

s)

Fig 2.16b: Flow rate effect on the resolution of TLM


Mobile Phase

Chiralpak IA Chiralpak IC

LLM TLM

k1 k2 α Rs k1 k2 α Rs

n-hexane:% 2-Propanol (95: 5 v/v)

0.72 0.74 1.03 0.88 0.63 0.69 1.10 1.11


0.70 0.72 1.03 0.74 0.76 0.78 1.03 1.25


0.68 0.71 1.03 0.69 0.77 0.78 1.01 0.21


0.67 0.69 1.03 0.67 0.79 0.79 1.01 0.12

Table 2.1: Influence of the 2-propanol composition on the selectivity and resolution of LLM and TLM

Acharya Nagarjuna Univeristy, Guntur 85

Temp (°C)

Flow rate (mL/min)

Dead Voulme

Time (min)

Peak Start Time (min)

rt1 min)

Peak End Time (min)

Peak Start Time (min)

rt2

(min)

PeakEnd Time (min)

w1 w2 k1 k2 α Rs

LLM 25 1 3 11.02 11.62 12.35 12.24 12.73 13.32 1.33 1.08 0.74 0.76 1.03 1.34 30 1 3 10.58 10.97 11.61 11.49 11.91 12.46 1.03 0.97 0.73 0.75 1.03 0.94 35 1 3 10.00 10.56 11.00 11.00 11.44 12.00 1.00 1.00 0.72 0.74 1.03 0.88 40 1 3 9.43 9.89 10.51 10.47 10.78 11.21 1.08 0.74 0.70 0.72 1.04 0.81 45 1 3 8.51 8.96 9.24 9.24 9.52 9.97 0.73 0.73 0.67 0.68 1.03 0.41

TLM 25 1 3 8.73 9.01 9.31 10.09 10.51 10.95 0.58 0.86 0.67 0.71 1.07 1.08 30 1 3 8.39 8.67 8.92 10.06 10.41 10.73 0.53 0.67 0.65 0.71 1.09 1.04 35 1 3 7.80 8.05 8.20 9.10 9.63 10.10 0.40 1.00 0.63 0.69 1.10 1.11 40 1 3 7.61 7.82 7.96 8.89 9.12 9.37 0.35 0.48 0.62 0.67 1.09 0.54 45 1 3 7.29 7.39 7.54 7.94 8.09 8.26 0.25 0.32 0.59 0.63 1.06 0.20

Table 2.2: Column temperature effect on retention and resolution of LLM and TLM


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CHAPTER - II - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/8292/15/15_chapter 2.pdf · consisting of a single central asymmetric carbon atom with a left pthalidimide ring