Chemistry Reference
In-Depth Information
0.50
0.40
0.30
0.20
0.10
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
Minutes
Peak Results
Name
RT
Height
GPV Percent
1
Full Scale
6.257
517135
100.00
2
A/B/C
12.159
51609
9.98
3
A/B/D
16.312
51216
9.90
FIgure 2.2
Example of a UV detector response for a gradient accuracy test on a qua-
ternary system. Solvent A is methanol; solvents B, C, and D contain 0.1% concentration of
propylparaben in methanol. The first “peak” is from a 50/50 mixture of the A and B solvents;
“peaks” two and three are due to adding 10% C solvent and 10% D solvent, respectively.
The values at each flow rate tested are recorded in a form similar to that shown in
Table 2.3.
2.3.3.2 gradient Accuracy
Most HPLC systems in use today are capable of delivering mobile-phase gradients
or a change in composition (e.g., % organic) versus time over the course of a chro-
matographic run. Gradient pumps are commonly binary (capable of delivering two
different solvents) or quaternary (capable of delivering up to four different solvents).
To test for gradient accuracy, one or more of the solvent lines can be primed with a
solvent containing an ultraviolet (UV) absorber (e.g., propylparaben at 254 nm) and a
UV detector used to monitor the response. Instrument gradient conditions are used to
deliver known proportions of the mobile phase, and the full-scale detector response
is measured against predetermined specifications to determine accuracy. Figure 2.2
is an example of a UV detector response for a gradient accuracy test for a system
capable of quaternary solvent delivery. The first “peak” is from a 50/50 mixture of
the A and B solvents; the subsequent “peaks” two and three are due to adding 10%
C solvent, and 10% D solvent, respectively. The results measured as peak heights of
9.98 and 9.90 are well within the stated manufacturer's specifications.
2.3.3.3 Injector Accuracy
Injector accuracy (volume) is typically performed by injecting a set amount of
sample, weighing the sample vial before and after. The average volume used per
