149 / 413
Equation 1
Adjusted column
length can easily be calculated
when scaling from HPLC to UHPLC.
Example:
l S0mm.
1.9~m
L
C'
=
S~m
L
C'
=
S7mm
IL
c
=
ColumnLength
I
dp
=
Particle Size
Equation 2
Changing column
dimensions requires an adju sted
injection volume.
[de" . Lc'J
VI'
=
VI' •
de" • LCI
Example:
r
2.1mm' ·SOmm ]
VI'
=
1
O~L
·
.
4.6mm'. lS0mm
VI'
=
0.69~L
VI
=
Injection Volume
L
c
=
Column Length
de
=
Column Diameter
Equation 3
Changing column
internal diameter requires using an
adjusted flow rate.
[ '
J'
d
d
C
F
c'
=
FCI
•
c
l
Example:
2
.1
mm ')'
'1
.
F
c'
=
4.6mm •
L Ornl/min.
,....---- -----,
F
, =
0.20B ml/min. !F
c
=
Column Flow
I
(
de
=
Column Diameter
Equation 4
When scaling down a
gradient method, the time pro
gram needs to be adjusted.
[ FF
c' J [de"de" J [L
c
' J
t
g,
=
t
g
l
c' •
•
~
•
Example:
'1.0mL/min.) [2.1mm' 1 [somm
1
t
g ,
=
5
min .
· lo.2mL/min. • 4.6mm' • lS0mm
t
g ,
=
1.7 min.
's
=
GradientTime
F
=
Column Flow
L
=
ColumnLength
c
de
=
Column Diameter
Conclusion
After determining the equivalent conditions for scal
ing-down the analysis of sulfonamides, we can see that
the separatio ns are equivalent, while the analysis time
was greatly redu ced (Figure 3). By following the proce
dure described here to ensure that the columns are
equivalent, scaling analytical procedures from HPLC
to UHP LC can easily be accomplished usin g
Pinnacle" DB columns.
Pinnacle' DB Biphenyl Columns (USP L11)
Physical Characteristics:
particle size:
1.9/lm
or
5/lm,
endcap: yes
spherical
pH range: 2.5to 7.5
pore size: 140A
temperature limit: 80
a
C
carbon load: 8%
:l.9J1m
Column, 2.lmm
cat #
50mm
9409252
S/J.!!!-Column, 4.6mm
cat #
150mm
9409565
For other dimensions and guard cartridges for these columns, visit
our website at
www.restek.com.
Figure
3
Restek's
Pinnacle'
A. Pinnacle DB Biphenyl
1.9Ilm, 50 x 2.1mm
I
~ca
l
ing
down
methods saves
analysis time!
2
1
A
\.,
'-J '---0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
LCPH0462
DB
1.91lm columns can easily be scaled
from
HPLCto UHPLCand vice versa.
7
B. Pinnacle DB Biphenyl
5fjm, 150
x
4.6mm
'- '--2.4 2.6 2.8
1 2
4
Peak List:
Column:
A.
1.9/lm
Pinnacle™ DB Biphenyl
Cal.#:
9409252
Dimensions:
50mm x 2.1mm
l. sulfadiazine
2. sulfathiazole
Particle size:
1.91l
m
3. sulfamerazine
4. sulfamethazine
Column:
B.Pinnacle™ DB Biphenyl
5. sulfachlorpyridizine
Cal.#:
9409565
6. sulfamethoxazole
Dimensions: 150mm
x
4.6mm
7.
sulfamethoxine
Particle size:
51lrl),
Pore size:
140A
Sample:
Inj.:
10/lL
Conditions:
Cone.:
100/lg/ mL
Mobilephase: A: 0.1%formicacid inwater
Sample diluent: starting mobile
B: 0.1%formicacid inacetonitrile
phase
(80:20
A:B)
TIme(min.)
9&B
0.0
20
1.0
20
6.0
80
8.0
80
Flow:
1.0mLlmin.
Temp.:
30
0
e
1""" "'1"""""""" "'1""""'1""""'1' """"1""""'1"" ""'1"
Del.:
uv
@
254nm
l.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Time(min)
LC_PH0461
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Website :
www.chromtech.net.auE-mail :
info@chromatech.net.auTelNo : 03 9762 2034 . . . in AUSTRALIA