APPENDIX V

CHARACTERIZING THE DIFFERENCES AMONG C8 OR C REVERSED-PHASE COLUMNS FROM DIFFERENT SUPPLIERS

Columns of the same nominal type (e.g., C8 or Qg) from different companies will often exhibit important differences in retention. These differences can arise from the following column packing characteristics (Section 5.2.4):

Differences in the silica particle (e.g., acidity) (Table 5.4) Choice of silane and bonding process (e.g., monomeric vs. polymeric

phases) (Section 5.2.3.1) Concentration of bonded phase (fimoVm2, Section 5.2.3.1) Presence or absence of endcapping Particle surface area

As a result, a column from one supplier that is nominally equivalent to a column from a different source (e.g., Qg columns in both cases) may not be a suitable replacement in an HPLC assay. It is useful to be able to compare different Cg or C18 columns in terms of sample retention. This allows columns to be identified that can be expected to perform similarly or quite differently in a given assay. Similar columns may be interchangeable for a given assay, which gives the chromatographer a choice of usable columns. Quite different columns can be used to test the sensitivity of an assay to the column; if the assay "works" for two such columns, it is likely that a number of other columns will also be usable.

Figure 5.9 provides useful retention data for a large number of commercial columns. Relative retention data are provided for acid, base, and neutral sample compounds. If two columns show similar retention for each compound

740

Practical HPLC Method Development, Second Edition by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch

Copyright 1997 John Wiley & Sons, Inc.

APPENDIX V 741

in this test mixture, it is more likely that these same two columns will be interchangeable for some HPLC assay. If relative and absolute retention of the test mixture is quite different for two columns, these two columns can serve as a test of the sensitivity of the assay procedure to differences in the column.

There are two additional ways of comparing C8 and/or C18 columns. Column acidity has been noted as an important column characteristic, and several relatively non-acidic columns were listed in Table 5.4. A ranking of some other columns according to their relative acidity is given in Table V.l. Columns that are close together in this table can be expected to be similar in terms of acidity, and such columns should perform similarly for the separation of basic compounds. The data in support of Table V.l were collected between 1983 and 1987, which raises the question of whether these data are still representative of columns produced today. Since manufacturers try to maintain the same separation characteristics for their columns over time, it seems likely that the ranking of Table V.l is still useful. Several silicas claimed to be less acidic and highly purified have been made available by manufacturers in recent years (see Table 5.4). However, insufficient data are available to place these silicas in the ranking of Table V.l.

TABLE V.1 Ranking of C$ or Cu Columns According to Relative Acidity

Column

(less acidic) Zorbax RX Vydac Nucleosil Supelcosil DB /iBondapak Novapak Partisil RSil Polygosil Spherisorb Lichrosorb Chrompack Rainin IBM Hypersil Perkin-Elmer Supelcosil Zorbax

(more acidic) Micropak

Source: Ref. 1.

ft

TA

BL

E X

J.

Ran

king

of

CM

Col

umns

Acc

ordi

ng t

o Sh

ape

Sele

ctiv

ity"

0.5

< T

BN

BP

^

0.9

Col

umn

Bak

erbo

nd C

18

Wid

e-Po

re

Chr

omsp

her

PAH

B

io-R

ad R

P 31

8 Su

pelc

osil

LC

-PA

H

Vyd

ac 2

01T

P Sp

heri

sorb

PA

H

Erb

as

C18

H

Man

ufac

ture

r

J. T

. B

aker

Chr

ompa

ck

Bio

-Rad

Su

pelc

o

Sepa

ratio

ns G

roup

Ph

ase

Sepa

ratio

ns

Car

lo E

rba

1.0

<

TBN

B>P

<

1-7

Col

umn

ES

Indu

stri

es B

F-C

18

LiC

hros

pher

100

RP-

18

Bak

erbo

nd C

18

Erb

as

C18

M

LiC

hros

pher

60

RP-

sele

ct B

Pa

rtisi

J 5

OD

S-2

Parti

sil

5 O

DS

Sphe

riso

rb O

DS-

1 B

row

nlee

OD

S 5A

Se

pral

yte

C18

Sp

heri

sorb

OD

S-2

Man

ufac

ture

r

ES

Indu

stri

es

E. M

erck

J.

T.

Bak

er

Car

lo E

rba

E. M

erck

Wha

tman

W

hatm

an

Phas

e Se

para

tions

A

BI

Ana

lytic

hem

Ph

ase

Sepa

ratio

ns

1.7

< (*

TB

N

Col

umn

Erb

asil

C18

L

Peco

sphe

r 5

Cr

C18

Pa

rtis

pher

e C

18

Zor

bax

OD

S Se

rva

C18

Pa

rtis

il 5

OD

S-3

Hyp

ersi

l O

DS

(HP

) M

icro

sorb

C18

J&

W A

ccup

hase

OD

S 2

Nov

apak

C18

U

ltras

pher

e O

DS

Cap

cell

C18

SG

120

Su

pelc

osil

LC

-18

IBM

OD

S B

row

nlee

Sph

eri

5 R

P-18

O

DS

Hyp

ersi

l C

osm

osil

C18

-P

J&W

Acc

upha

se O

DS

YM

C 1

20 A

"A

" A

dsor

bosp

here

C18

H5

Supe

lcos

il L

C-1

8-D

B

B.P

^

2.2

Man

ufac

ture

r

Car

lo E

rba

Perk

in-E

lmer

W

hatm

an

Mac

-Mod

(di

stri

buto

r)

Serv

a W

hatm

an

Shan

don

Rai

nin

J&W

Sci

entif

ic

Wat

ers

Bec

kman

Sh

isei

do

Supe

lco

IBM

A

BI

Shan

don

Nac

alai

Tes

que

J&W

Sci

entif

ic

YM

C

Allt

ech

Supe

lco

Sour

ce: R

ef.

2.

' Se

lect

ivity

coe

ffic

ient

s w

ere

dete

rmin

ed u

sing

85%

ace

toni

trile

/wat

er m

obile

pha

se a

t 2

mL

/min

at

ambi

ent

tem

pera

ture

(25

2

C).

With

in e

ach

cate

gory

, co

lum

ns a

re l

iste

d (f

rom

top

to

botto

m)

in o

rder

of

incr

easi

ng C

*TBN

BP

val

ues;

how

ever

, be

caus

e th

ese

valu

es m

ay v

ary

with

diff

eren

t co

lum

n lo

ts, i

ndiv

idua

l T

BN B

P v

alue

s ar

e no

t lis

ted.

The

ran

ges

show

n ab

ove

shou

ld n

ot b

e co

nstru

ed t

o re

flect

lot

-to-

lot

vari

abili

ty.

APPENDIX V 743

A second way of characterizing the column is in terms of shape selectivity [2]. It has been shown that the separation of certain polyaromatic hydrocarbons (PAHs) is quite sensitive to column source, and it has been suggested that this is caused by "slots" in the stationary phase that are sensitive to the shape of sample molecules. It is therefore reasonable that this column characteristic will affect the retention of other samples as well. Table V.2 summarizes the results from a large number of commercial columns. Three different groups are defined according to the value of a for a particular pair of PAHs. Columns in the same group are more likely to give similar results than columns in different groups. Polyfunctional-silane bonded phases (e.g., Vydac 201TP) have low values of the selectivity factor OTN/BSP. while monofunctional-silane bonded phases (e.g., Zorbax ODS) have high values. The classification of Table V.2 thus distinguishes these two types of packing.

The concentration of the bonded phase (/xmol/m2) can affect both absolute retention and selectivity. A higher concentration of a particular ligand (e.g., Cu) generally gives greater retention, other factors being equal. A higher bonded-phase concentration can also affect shape selectivity [3]. Endcapping mainly affects the retention of basic compounds, which are retained more strongly for non-endcapped phases. Values of k for all compounds increase in proportion to packing surface area, but selectivity is not affected by small differences in surface area.

APPENDIX V

1. M. A. Stadalius, J. S. Berus, and L. R. Snyder, LC/GC, 6 (1988) 495. 2. L. C. Sander and S. A. Wise, LC/GC, 8 (1990) 378. 3. L. C. Sander and S. A. Wise, Anal. Chem., 67 (1995) 3284.