Standard Operating Procedure (SOP)

Define the Development Question

1. State the intended separation objective and identify critical analytes or peak pairs.
2. Select independent variables supported by the development protocol, such as flow rate, temperature, pH, or organic-phase proportion.
3. Define experimentally safe ranges and pre-approved decision criteria before modelling.
4. Ensure original chromatograms, integration results, run conditions, and sample identifiers are available as attributable source records.

Output: a documented modelling plan specifying peaks, variables, ranges, baseline, and acceptance criteria.

Create and Identify the Project

1. Sign in with the assigned account and confirm the displayed identity.
2. Select New Project, enter Analgesic Separation Optimisation, and associate it with project ID MDP-2026-0012 in the laboratory record.
3. Confirm the project header and status as shown in Figure 1.
4. Save to session once so a recoverable working state exists.

Enter the First Experimental Dataset - Select an Entry Route

Route A - Quick Data Input (bulk entry)

Format every record as Peak Name, Condition Name, Condition Value, Retention Time. The following dataset reproduces the illustrated three-peak, three-variable model.

Acetaminophen,Flow Rate,0.8,4.70
Acetaminophen,Flow Rate,1.0,4.20
Acetaminophen,Flow Rate,1.2,3.72
Acetaminophen,Temperature,25,4.45
Acetaminophen,Temperature,35,4.20
Acetaminophen,Temperature,45,3.96
Acetaminophen,Organic Phase,35,4.80
Acetaminophen,Organic Phase,40,4.20
Acetaminophen,Organic Phase,45,3.62
Caffeine,Flow Rate,0.8,6.82
Caffeine,Flow Rate,1.0,6.20
Caffeine,Flow Rate,1.2,5.61
Caffeine,Temperature,25,6.55
Caffeine,Temperature,35,6.20
Caffeine,Temperature,45,5.87
Caffeine,Organic Phase,35,7.10
Caffeine,Organic Phase,40,6.20
Caffeine,Organic Phase,45,5.32
Ibuprofen,Flow Rate,0.8,10.42
Ibuprofen,Flow Rate,1.0,9.40
Ibuprofen,Flow Rate,1.2,8.41
Ibuprofen,Temperature,25,9.86
Ibuprofen,Temperature,35,9.40
Ibuprofen,Temperature,45,8.95
Ibuprofen,Organic Phase,35,10.92
Ibuprofen,Organic Phase,40,9.40
Ibuprofen,Organic Phase,45,7.91

1. Paste the records into Quick Data Input (Figure 2) and select Process Data.
2. Confirm that 27 points were processed and that three peaks and three conditions appear.
3. Verify representative values against source results. Investigate parsing warnings, negative values, missing combinations, or transcription discrepancies before proceeding.

Route B - Peaks Management (manual per-peak entry)

Use this route when results are being transcribed a point at a time or when the analyst wants to review each peak record as it is assembled.

1. In a new project, open Conditions & Data and select + Add Condition three times. Create Flow Rate with baseline 1.0, Temperature with baseline 35, and Organic Phase with baseline 40.
2. Open Peaks Management and select + Add Peak three times. Create Acetaminophen, Caffeine, and Ibuprofen.
3. Keep Show Details enabled. In each peak card, locate the required condition table, enter the condition value and measured retention time in the Add Point row, and select Add Point.
4. The card refreshes after each addition. Return to the relevant peak and condition before entering the next pair.
5. Use the following map to reproduce the same 27 records:

Route C - Quick Multi-Condition Entry (experiment-by-experiment entry)

Use this route when entering results in experimental order. Work by condition state: set the experimental conditions, then record the observed retention time for every peak at that state before moving to the next condition. This example uses a one-factor-at-a-time study, so each non-baseline state differs from baseline in exactly one variable.

1. Start from a fresh project and create the same three conditions and three peaks described in Route B.
2. Work down the condition-state table below. For each row, establish the displayed Flow Rate, Temperature, and Organic Phase values, then enter the three retention times from left to right.
3. At a condition state, select Acetaminophen, enter its RT, and select Add Data Point. After the form refreshes, restore the same condition state, select Caffeine, and add its RT. Repeat for Ibuprofen.
4. Complete all three peaks at the current condition state before advancing to the next row. The peak selector identifies which observed peak the RT belongs to; it does not define the order of the workflow.

5. For the baseline row, all inputs must show ✓ Default. Each of its three additions records that peak's baseline RT in all three condition models, producing nine underlying condition-specific points.
6. For every other row, change only the emphasized input. The other two inputs must show ✓ Default, and the mode message must identify a Single-variable experiment. Each addition is recorded only against the changed condition.
7. Enter the upper row before the lower row for each condition. While initially seeding the range, this keeps the intended baseline as the middle/default value.
8. After all seven condition states are complete, confirm that 21 Add Data Point actions have produced 27 condition-specific points: three values for every peak/condition combination.

Output: a populated model with three observations for every peak/condition combination.

Set the Baseline and Trial Target

1. Set Initial Conditions to Flow Rate 1.00, Temperature 35, and Organic Phase 40 (Figure 3).
2. Set Target Conditions to Flow Rate 1.10, Temperature 40, and Organic Phase 43 (Figure 4).
3. Confirm the application recalculates without an error and the target values fall within the studied ranges.

Output: a defined reference state and a scientifically plausible trial method.

Review Model Quality and the Prediction

1. Open Peaks Management and confirm each peak card contains three points for every condition (Figure 8).
2. Review slope direction, point count, and R². Inspect condition plots for unexpected trends or outliers (Figure 9).
3. Return to Overview and inspect predicted retention times and all resolution pairs (Figures 5 and 7).
4. Use Display Settings only to clarify presentation (Figure 6); do not treat visual amplification, noise, fill, or merged rendering as a change in model performance.
5. For the illustrated target, confirm predicted retention times of approximately 3.485, 5.203, and 7.777 minutes and resolution above the illustrative 1.5 threshold.

Output: a documented decision to accept the trial target for experimental testing or to continue refinement.

Refine with Additional Experiments When Required

1. If fit or separation is inadequate, identify the variable and range requiring more evidence.
2. Perform the new experiment under the approved protocol and retain its original record.
3. Add a baseline point or a single-variable point through Conditions & Data. Change only one condition from baseline when using Quick Multi-Condition Entry.
4. Re-check the affected regression, all predicted retention times, and resolution after the addition.
5. Never remove a valid but inconvenient point solely to improve R²; investigate and document the scientific basis for any exclusion.

Generate, Review, and Archive the Result

1. Complete report metadata using the project ID and analyst identity (Figure 10).
2. Select the report sections required by the protocol. For technical review, normally include target conditions, chromatogram, elution order, resolution, statistics, method-development conditions, and raw points.
3. Generate the report, review every page and optional section, and regenerate after any model change (Figure 11).
4. Save to session, download the .hplc project, and export the reviewed report according to the site record-retention procedure.
5. Record the conclusion, experimental follow-up, filename/location, and reviewer disposition in the laboratory record.