16 :

(HIGH PRESSURE LIQUID CHROMATOGRAPHY)

.. 1906

(impelling force)
(stationary phase)
(retention force)
(specificity) (adsorption) (solubility)

(retention time) 16.1
()

(impelling force)

(

)
(retention force)

16.1

352


2
(mobile phase)
1. Liquid-solid chromatography
4
1.1 Gel filtration chromatography

1.2 Ion exchange chromatography (counter ion)

1.3 Affinity chromatography
(biological specificity)
enzyme/substrste , antigen/antibody , hormone/receptor



1.4 Adsorption chromatography
(adsorbent)
MgO , Silicic acid , Magnesium silicate (polarity)
(polarity group)


2. Liquid-liquid chromatography (partition chromatography)




(supporter) silicic acid silica gel

353


.


normal phase chromatography
n-alkyl 8
18

reversed phase chromatography

tetramethyl ammonium chloride , trioctylamine, tetrabutyl ammonium
chloride perchloric acid, sodium alkyl
sulfonate methanesulfonic acid
ion-pair chromatography

( 16.2) High Performance Liquid

354


16.2
Chromatography (HPLC)

( 16.3)

(injector)

(Solvent) (Sample)
(pump)

(column)

(waste)
(detector) (recorder)

16.3

1. (pump)
0.5-10 ./
2 1,000-6,000
3
1.1 (syringe type)

1.2 (reciprocating piston type)

355


1.3 ( constant pressure pump)

2. (injector)
0.5-10 2
2.1
(septum)

2.2 (rotary type)

(sample loop)
(fixed loop injector)( 16.3 )
( 16.3 )

(syringe loop injector)

+
() ()

16.3 (wait)() (inject)()

3. (column)

356


10-150 .
1 . .
6,000

4. (detector)

(photo detector)
(fluorescence detector)
(refractive index detector)
(radioactivity detector)
16.1

16.1

(./.)
UV absorption 5x10 -5 254-280 nm
IR absorption 10 -6 -
Fluorometry 10 -10 -
Refractive index 5x10 -7 -

Conductivity 10 -8 1 0
-
Flame ionization 10 -8


Mass 10 -10 0.2-1.0 ng
spectrometry

( 16.2)
5.

357


16.2

(nm) (nm)
C N 170 (C=C)3 260
C C 180 NO2 ,ONO2, 270
C=C, C=N 190 C=O 280
COOH, CONH2 210 C=S 330
(C=C)2 220 N=N, ONO 370

(peak high) (peak area)

5.1 (column efficiency)

(number of theoretical plates, N)

N = 5.54( Tr /W )2
Tr = retention time
=
W = .

5.2 Capacity factor(K)

K = (Tr T0)/ T0
Tr = retention time
T0 = retention time

6.

358


7.
HPLC

1. (solvent)



2
1.1 Isocratic elution 1
1.2 Gradient elution 1


2.
2
2.1 (external standard)

2.2 (internal standard)

(retention time)
Y
X

359


3. ( stationary phase)
5-10 m
silica gel , alumina celite
(diatomaceous earth) pellicular particle
40 m alumina 1-3 m
(ion exchange resin)

1.
2.
3.
4. HPLC
5. 2-3

6.
7.

8. 2-7

9.
10.

360


10.1 methylene cyanide(MeCN) methylene

hydroxide(MeOH) reverse phase hexane heptane isopropyl
alcohol 97 :3 normal phase
10.2 polystyrene divinyl benzene copolymer
(sodium azide) 0.02%
11.
12.

13.

(scale factor, S)

S = ()2 / ()2

3 . 200 .
1.0 ./ 1 . 200 .
0.11 ./ S = (0.5) 2 / (1.5) 2 = 0.11
1 ./ x 0.11

1. (column regeneration)


normal phase hexane / methylene chloride / isopropanol / methylene
chloride
50 .
2.

3.

361


1.
2. 5
3.

4. (application manual)
5.
6.
7.

16.3

16.3

Peak -

-

-
-(retention time)
-
Peak (tailing) -
50 0 .
-

(poor -
recovery)

362


16.3 ()

spike peak -
-

ghost peak -
-
(poor sensitivity) -
-
-

-
-
-
-
-
Base line -
-
-
-
-
-
-
-

363


1. Ferris CD. Guide to medical laboratory instruments. Little Brown and Company,
Boston, 1980.
2. Hickers MR, Schenmken JR, Steinrauf MA, Mc Whorler CA. Laboratory
instrumentation 2 nd ed. Harper&Row Publishers. Philadelphia. 1980.
3. Jone RR. HPLC keeping grow away the separation sciences. R&D 1991;31:54-7.
4. Skoog D. Principle of instrumental analysis. Library of Congress Cataloging in
Publication Data. USA. 1986.
5. Studt T. HPLC column thin down , speed up, get simpler. R&D 1994;36:43-4.
6. Studt T. HPLC users want system their way. R&D 1996;38:48-51.
7. Wade-Clark C. HPLC quality control. Quality 1989;28:30-2.

364