lect. 1 intro (2-day)

8/2/2019 Lect. 1 Intro (2-Day)

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1

IntroductionHPLC Process

Lecture 1

Yuri KazakevichSeton Hall University



2

HPLC History

Chromatographywasdiscovered byM.S.Tswett in 1903.



3

Chromatographic Column



4

HPLC Retention

Major parameters,

• V R is retention volume, depends on the column type,

size, and the instrument parameters

• V o 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

k V V

V

R o

o

'

R R t FV



5

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

R R

w

V N

k

k

V

V V k

R Rt FV



6

Dead Volume

V o, 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

L DV

e



7

Retention Parameters



8

HPLC Selectivity



9

Efficiency Parameters



10

Efficiency

2

16

b

R

w

V N

N

L HETP

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

p d

HETP

h



11

Column Efficiency

• Column length is a compromise between the efficiency andbackpressure

• Column efficiency is proportional to the column length

• Specific efficiency (# of particles per one plate) decreaseswith an increase of column length

Length[cm]

ParticleDia. [um]

Efficiency,N

SpecificEfficiency, h

10 3 11111 310 5 10526 1.915 5 13636 2.225 5 15625 3.225 10 10000 2.5



12

Efficiency

N t R

total

total column inject cell connect

2

. .



13

Resolution



14

Parameters affecting efficiency:• Flow rate

• Column length

• Particle diameter

• Particle size distribution

Parameters affecting retention factor:• Eluent type

• Eluent composition

• Stationary phase type

• Analyte natureParameters affecting selectivity:

• Stationary phase type

• Analyte nature

• Eluent additives

• Temperature

• Eluent composition (ionizable analytes)

Factors Influencing HPLC Separation



15

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

R e s p o n s e

1 .

6 8

2 . 2

3

5 . 9

4

9 .

0 5

9 .

8 4



16

Retention Process (Surface Equilibria)

SK V V R 0

K V

S

V

V V

k

R

0

0

0'

RT

GG

RT

G K

eluent analyteexpexp

0

ln)'ln(V

S

RT

GG k

eluent analyte



17

Retention Factor, k’

Retention factor is a measure of the analyte

competitive interactions with the stationary phase

RT G

RT G

RT G k eluent analyte

expexp'

70%

80%

90%

100%

%MeCN



18

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



19

Selectivity

– Eluent composition

• Ideally does not have any effect on the selectivityfor 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

2

1 2

1 2

1 2



20

Eluent Composition Effect onSelectivity

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



21

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

1

2

3

4

5



22

Alkylpyridines vs. eluent

composition on Luna-C18

Eluent Composition EffectMeOH /Water



23

Eluent Composition EffectMeCN /Water

lect. 1 intro (2-day)

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