Chemistry Reference
In-Depth Information
While the criteria in Table 5.7 seem straightforward, many of them do not com-
pletely account for recent advances in LC technology, and additional guidance is
forthcoming [23]. Some additional comments are also warranted from an LC method
perspective as summarized in the following subsections.
5.9.1
P
h A
djuStmentS
As shown in Table 5.7, the pH value of the buffer in the mobile phase can be adjusted
to ±0.2 pH units. Adjusting the pH should, however, take into account the p
K
values
of the compounds of interest; because near the p
K
, even a 0.1 unit change in the pH
can result in significant (>10%) changes in retention time [24]. Studies show that
for many compounds only operating at pH extremes (pH > 8 or pH < 4 for basic
compounds, pH < 3 or pH > 7 for acidic compounds), generally well away from the
compound p
K
, will accommodate the ±0.2 unit allowable change, due to the slope of
the pH-versus-retention curve [25]. Any adjustments to the buffer (if used) concen-
tration and temperature should also take into account the effect on pH. In the case of
LC column temperature, where a ±10ºC change is allowed, selectivity effects might
be encountered.
5.9.2 c
olumn
l
ength
, d
IAmeter
,
And
P
ArtIcle
S
Ize
A
djuStmentS
: S
cAlIng
the
S
ePArAtIon
Per Table 5.7, column internal diameter can be adjusted provided that a constant
linear velocity is maintained; length adjustments up to ±70% are allowed. It is pos-
sible to reduce flow rate by up to 50%; however, when column dimensions have been
modified, they should be adjusted using the following formula:
F
2
= F
1
× l
2
(d
2
)
2
/l
1
(d
1
)
2
where F
1
is the flow rate indicated in the monograph, in milliliters (mL) per minute;
F
2
is the adjusted flow rate, in milliliters per minute; l
1
is the length of the column
indicated in the monograph; l
2
is the length of the new column used; d
1
is the column
inner diameter indicated in the monograph; and d
2
is the internal diameter of the new
column used.
Column length, internal diameter, and particle size adjustments really must be
considered together, and when correctly scaled according to well-known theoreti-
cal principles, equivalent separations will result. For example, keeping the length-
to-particle-size ratio (L/d
p
) constant, an identical separation can be obtained for
a 5-cm, 1.7-µm column as for a 30-cm, 10-µm column (L/d
p
= 3 for both) as long
as an increase in the flow rate inversely proportional to the particle size is also
maintained.
Consider an example of converting or migrating a method from HPLC to newer
LC technology that uses sub-2-µm particle size chemistry, termed UHPLC [26-
30]. Chemistry (sub-2-µm particles) and instrumentation (systems capable of pres-
sures greater than 6,000 to 19,000 psi) necessary to take advantage of this new
