Each Module “B” contains a function that assigns an algorithm for analyzing the conductivity data. The algorithm function occurs in two places in the deprotection module: once during initial deprotection and again in the monitored deprotection loop.
Initial deprotection
The first occurrence of the algorithm function generates the first deprotection peak for that cycle. With the Function 128 (Mon 1stPk-X) algorithm, the initial peak results from one deprotection only. After baseline correction, the value of this initial peak is compared to the subsequent peaks in the cycle. The subsequent deprotection peaks are generated by the second occurrence of Function 128 (Mon 1stPk-X), during the monitored loop.
The Function 130 (MonPrevPk) algorithm first occurs in a deprotection loop that is not monitored but is performed twice. During the first deprotection, most of the Fmoc protecting group is removed, so the first peak is much larger than the second peak. The second deprotection peak in the initial deprotection loop is much closer in value to the subsequent deprotection peaks in the monitored loop.
Subsequent deprotections in the monitored loop
In the FastMoc modules without monitoring, deprotection occurs with a set number of piperidine deliveries per cycle, usually two. The Basic Monitoring deprotection modules allow additional piperidine delivery steps as needed for difficult deprotections. To accomplish this, piperidine delivery steps are placed within “loops” (see page 8-32 for a discussion of loop functions). Monitored deprotection loops are introduced with Function 133 (MonBegLoop) and completed with Function 134 (MonEndLoop).
The differences between Function 128 and Function 130
With Function 128 (Mon 1stPk-X), there is one initial deprotection peak, followed by at least one deprotection in a monitored loop. With Function 130 (MonPrevPk), there are always two deprotections in the initial deprotection loop, followed by at least one deprotection in a monitored loop. It follows that there are minimally two deprotections completed with Function 128, but a minimum of three deprotection completed with Function 130. So, Function 128 (Mon 1stPk-X) requires less piperidine than Function 130 (MonPrevPk).
When you use Function 128 (Mon 1stPk-X), you must determine the conductivity baseline, and insert that number, divided by ten, wherever Function 128 occurs in every module. The conductivity baseline varies from
one instrument to the next and can change when you change reagent lot numbers. You do not have to know the conductivity baseline when you use Function 130 (MonPrevPk).
Two conditions that end the monitored deprotection loop
The value of “T” in two different monitoring functions determines the conditions that end the monitoring loop. In Function 133 (MonBegLoop), the value of “T” determines the maximum number of times the monitored loop is repeated. In Function 134 (MonEndLoop), the value of “T”
represents a percentage multiplied by ten. The percentage is applied to the data to compare two deprotection peaks.
Table 5-6 shows the values of “T” that have been written into the function in the pre-defined deprotection modules. You may change the values of “T” in Functions 128, 133, and 134 to customize the deprotection module.
Table 5-6. Values of “T” in Basic Monitoring Deprotection Modules
IMPORTANT In the monitoring functions, which include Function 128 through
Function 149, “T” does not represent time. Instead, “T” may represent a conductivity baseline, the maximum number of monitoring loops, a percentage, or a monitoring channel.
Function 133 (MonBegLoop) The value of “T” in Function 133 represents the maximum number of deprotection loops in module “B.” This value varies depending on:
• The algorithm used
• The anticipated difficulty of a deprotection • The value of “T” in Function 134 (MonEndLoop)
In general, when you use the Function 128 (Mon 1stPk-X) algorithm, the value of “T” in Function 133 is higher than that of Function 130
(MonPrevPk) algorithm. With Function 130, the initial deprotection loop generates two deprotection peaks. As a result, fewer deprotections are necessary in the subsequent deprotection monitoring loop. As a guideline, Applied Biosystems suggests you set the value of “T” in Function 133 (MonBegLoop) between 2 and 6 with either algorithm.
Module B Fxn 128* Fxn 130 Fxn 133 Fxn 134
Deprotection/ Mon 1st Pk - X, 0.10 mmol 110 — 4 100
Deprotection/ Mon 1st Pk - X, 0.25 mmol 110 — 4 100
Deprotection/ MonPrvPk, 0.10 mmol — 1 3 100
Deprotection/ MonPrvPk, 0.25 mmol — 1 3 100
Deprotection/ MonPrvPk, 1.0 mmol — 1 3 100
Note The total number of deprotections possible in any cycle equals the maximum number of deprotections in the monitored deprotection loop—defined by Function 133—plus the number of initial deprotections.
With Function 128, Mon 1st Pk-X), there is only one initial deprotection.
With Function 130, Mon PrevPk, there are two deprotections in the initial deprotection loop.
Function 134 (MonEndLoop) The value of “T” in Function 134 represents a percentage, multiplied by 10. With the Function 128 (Mon 1stPk-X) algorithm, the percentage in Function 134 represents the relation of the last deprotection peak generated to the initial deprotection peak. With the Function 130 (MonPrevPk) algorithm, the percentage is used to compare two sequential deprotection peaks. As a guideline, Applied Biosystems suggests you set the value of “T” in Function 134 (MonEndLoop) between 50 and 150.
IMPORTANT The value of “T” for Function 134 represents a percentage,
multiplied by ten.
Example:
• With Function 128 (Mon 1stPk-X), if you set the value of “T” in Function 134 (MonEndLoop) at 60, the algorithm compares the last deprotection peak to the first deprotection peak until the last peaks is ≤6% of the first peak or until the maximum number of monitored deprotection loops—the value of “T” in Function 133
(MonBegLoop)—have occurred.
• With Function 130 (MonPrevPk), if you set the value of “T” in Function 134 (MonEndLoop) at 85, the algorithm compares the last two sequential peaks until the difference between the two peaks is ≤8.5% or until the maximum number of deprotection loops—the value of “T” in Function 133 (MonBegLoop)—have occurred.
FastMoc Module “B” (Deprotection/MonPrevPeak)
Two Basic Monitoring files contain module “B” (Deprotection/ MonPrevPeak), which applies the Function 130 algorithm to the conductivity data:
• FastMoc 0.10 MonPrevPk
• FastMoc 0.25 MonPrevPk
Each time module “B” (Deprotection/Ω MonPrevPeak) occurs in these Basic Monitoring files, it contains the same monitoring functions, in the same order and in the same steps, regardless of scale.
This example describes how you can use the monitoring functions to define your criteria for ending deprotection.
Figure 5-3. Using monitoring functions for ending deprotection
Step 3: Function 135 (Mon Reset) eliminates any conductivity values that may be stored in the memory. In this function, “T” =1 tells the controller which channel is collecting conductivity data.
Step 27: Function 130 (MonPrevPk) is in a deprotection loop that is performed twice and then stops. This first deprotection loop does not contain Functions 133 and 134, so it is not a monitored loop. During the first deprotection, most of the Fmoc protecting group is removed, so the first peak is much larger than the second peak. The second peak in the initial
Step# Fxn# Fxn Name “T” 3 135 Mon Reset 1 27 130 MonPrevPk 1 29 131 Mon Stop 1 30 132 Save MonPk 1 32 133 MonBegLoop 3 55 130 MonPrevPk 1 57 131 Mon Stop 1 58 132 Save MonPk 1 59 134 MonEndLoop 50 Initial deprotection
(Resin solvation, first delivery of piperidine and NMP)
(Loop deliveries of piperidine and NMP)
(Rinse valve blocks and delivery lines with NMP, flush with gas)
T=maximum # loops T=% x 10 T=1 tells controller to perform the function Monitored deprotection loops
(Two deprotection loops)
T= Channel 1
T=1 tells controller to perform the function
deprotection loop is much closer in value to the subsequent deprotection peaks in the monitored loop. The value of “T” =1, which tells the controller to perform the function.
Step 29: Function 131 (Mon Stop) stops conductivity data collection. The value of “T” = 1 tells the controller to perform the function.
Step 30: Function 132 (Save MonPk) saves the largest data point collected as the peak. The value “T” = 1 tells the controller to perform the function. This function is in the initial deprotection loop that is only repeated twice.
Step 32: Function 133 (MonBegLoop) begins the monitored deprotection loops. The user-defined value of “T” in this example is 3. Here, “T” defines the maximum number of monitored deprotection loops that can be performed in this module. When this maximum number of loops have been performed, the deprotection module ends.
Step 55: Function 130 (MonPrevPk) is inside a monitored loop that generates deprotection peaks. The value “T” =1 tells the controller to perform the function.
Step 57: Function 131 stops conductivity data collection. The value “T” =1 tells the controller to perform the function.
Step 58: Function 132 saves the largest data point collected as the peak. the value “T” =1 tells the controller to perform the function.
Step 59: Function 134 (MonEndLoop) ends the monitored loop when the difference between two sequential peaks is equal to or less than a user- defined percentage. The value of “T” is this percentage multiplied by 10. In this example, deprotection ends when the difference between the last two sequential peaks is equal to or less than 5%. If the percentage difference is
not equal to or less than 5% before 3 monitored deprotection loops have occurred, the deprotection loop ends anyway. The number of monitored loops counted “feeds back” to module “F” to determine the number of coupling loops in the cycle.
IMPORTANT In the Basic Monitoring Chemistry files, the value of “T” in Function
133 (MonBegLoop) defines the maximum number of monitored loops that can be performed in any cycle. If the percentage difference defined by Function 134 (MonEndLoop) does not occur, deprotection ends when the maximum number of loops have been performed.