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Introduction Introduction HPLC ProcessHPLC Process

Lecture 1

Yuri KazakevichSeton Hall University

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HPLC HistoryChromatography was discovered by M.S.Tswett in 1903.

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Chromatographic Column

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HPLC Retention

Major parameters, • VR is retention volume, depends on the column type,

size, and the instrument parameters

• Vo is dead volume, volume of the liquid phase inside the column

• k’ is retention factor (capacity factor),

independent of the column size and instrument setup

kV V

VR o

o'

RR tFV

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Retention Characteristics

• Retention factor, k’• Selectivity, a • Efficiency, N

General recommendations:• Optimize retention factor between 1 – 10• HPLC selectivity should be > 1.2• Maximize efficiency

2

'1

'2

0

0 16;;'

b

RR

w

VN

k

k

V

VVk

RR tFV

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Dead Volume

Vo, Dead volume is the volume of the liquid phase in the column

Simple rule: Column dead volume = 65% of the volume of empty column 4

65.02

0

LDV

e

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Retention Parameters

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HPLC Selectivity

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Efficiency Parameters

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Efficiency

2

16

b

R

w

VN

N

LHETP

Reduced HETP is a measure of how well the column is packed.

pd

HETPh

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Column Efficiency

• Column length is a compromise between the efficiency and backpressure

• Column efficiency is proportional to the column length• Specific efficiency (# of particles per one plate) decreases

with an increase of column length

Length [cm]

Particle Dia. [um]

Efficiency, N

Specific Efficiency, h

10 3 11111 3 10 5 10526 1.9 15 5 13636 2.2 25 5 15625 3.2 25 10 10000 2.5

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Efficiency

NtR

totaltotal column inject cell connect

2

. .

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Resolution

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Parameters affecting efficiency:• Flow rate• Column length• Particle diameter• Particle size distribution

Parameters affecting retention factor:• Eluent type• Eluent composition• Stationary phase type• Analyte nature

Parameters affecting selectivity:• Stationary phase type• Analyte nature• Eluent additives• Temperature• Eluent composition (ionizable analytes)

Factors Influencing HPLC Separation

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Reversed Phase Separation Principle

• Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4)

• Difference in analyte sorption affinities results in their separation

• More polar analytes retained less

• Analytes with larger hydrophobic part are retained longer

• Almost no separation of structural isomers

1211109876543210Retention Time (min)

50

100

150

200

250

300

350

400

Respo

nse

1.6

8

2.2

3

5.9

4

9.0

5

9.8

4

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Retention Process(Surface Equilibria)

SKVVR 0

KV

S

V

VVk R

0

0

0'

RT

GG

RT

GK eluentanalyteexpexp

0

ln)'ln(V

S

RT

GGk eluentanalyte

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Retention Factor, k’

Retention factor is a measure of the analyte competitive interactions with the stationary phase

RT

G

RT

G

RT

Gk eluentanalyteexpexp'

0

5

10

15

20

25

30

0 2 4 6

alkylbenzenes, nc

70

80

90

100

-2

-1

0

1

2

3

4

0 1 2 3 4 5 6

nc

70%

80%

90%

100%

%MeCN

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Reversed-Phase HPLC Retention: Neutral Analytes

Dependencies of retention of alkylbenzenes, alkylphenones, and alkylparabenes plotted against the number of carbon atoms in alkyl chain.

ln(k’)= m (#carbon atoms in alkyl chain) + b

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Selectivity

– Eluent composition• Ideally does not have any effect on the selectivity

for neutral compounds

k

kk k

G

RT

G

RT

G

RT

G

RT

G

RT

G

RT

eluent eluent

'

'exp ln( ' ) ln( ' )

exp

exp

1

21 2

1 2

1 2

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Eluent Composition Effect on Selectivity

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6 7

0

5

10

15

20

25

0 1 2 3 4 5 6 7

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6 7 8 9

0

2

4

6

8

10

12

0 2 4 6 8 10 12

90% MeCN

80% MeCN

70% MeCN

60% MeCN

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Eluent Composition Effect on Selectivity

c%

Peak numbers

  2,1 3,2 4,3

70 1.75 1.6 1.45

80 1.72 1.58 1.4

90 1.7 1.65 1.43

100 1.71 1.62 1.42

02468

101214161820

70 80 90 100

Eluent composition

1

2

345

22

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100

MeOH [v/v%]

Alkylpyridines vs. eluent composition on Luna-C18

Eluent Composition EffectMeOH/Water

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Eluent Composition EffectMeCN/Water