Some Questions Regarding Reverse Phase HPLC

[LKguan]

Hi all,

I’m a student working on a purification project using HPLC. We are using C18 and C8 columns, and we tested two different elution buffers—ACN (gradient 0-100%) and EtOH (gradient 0-100%)—to determine which works better. Below is the method we followed. I'm a bit confused by some of the instructions from my senior, and I’d appreciate your help in clarifying them.

1.Equilibration Stage: The equilibration stage wasn’t set at the beginning of the method but was moved to the end instead. If I equilibrate the column before running the sequence, is that acceptable?

2.Sample and Standard Solvent: The extract samples were prepared using EtOH, but the standards I ran were prepared in ACN because the purified product couldn’t be fully dissolved in EtOH. Can I still use the standard for reference?

3.Blank Preparation: As I understand it, the blank should be prepared using the same solvent as the sample being analyzed. However, my senior suggested that Milli-Q water is sufficient. Is this correct?

4.Sequence Setup: Since I used two elution buffers, my sequence was divided into two groups, like this:
1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.
I separated the two groups with a single blank (6. Blank), but my senior added an extra blank, so there were two blanks between the groups. What was the reason for adding this additional blank?

5.Method Step: Below is the method we used with ACN as the elution buffer. The third step includes a transition to 100% EtOH to confirm that the cleaning was thorough. Was this step necessary?

Method:
0-20 mins: 0% to 100% ACN
20-27.5 mins: 100% ACN
27.5-35 mins: 100% EtOH
35-42.5 mins: 0% ACN

6.Abnormal Chromatogram: The chromatograms for the blanks look strange. Do you know any potential reasons for these abnormal baselines?

[Consumer Products Guy]

My input in red below

Hi all,

I’m a student working on a purification project using HPLC. We are using C18 and C8 columns, and we tested two different elution buffers—ACN (gradient 0-100%) and EtOH (gradient 0-100%)—to determine which works better. Below is the method we followed. I'm a bit confused by some of the instructions from my senior, and I’d appreciate your help in clarifying them.

1.Equilibration Stage: The equilibration stage wasn’t set at the beginning of the method but was moved to the end instead. If I equilibrate the column before running the sequence, is that acceptable?
I don't see why not. Remember: equilibration is column volumes of solvent, not really in "minutes".

2.Sample and Standard Solvent: The extract samples were prepared using EtOH, but the standards I ran were prepared in ACN because the purified product couldn’t be fully dissolved in EtOH. Can I still use the standard for reference?
We always used the same solvent for each.

3.Blank Preparation: As I understand it, the blank should be prepared using the same solvent as the sample being analyzed. However, my senior suggested that Milli-Q water is sufficient. Is this correct?
We always used the same solvent for each.

4.Sequence Setup: Since I used two elution buffers, my sequence was divided into two groups, like this:
1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.
I separated the two groups with a single blank (6. Blank), but my senior added an extra blank, so there were two blanks between the groups. What was the reason for adding this additional blank?

5.Method Step: Below is the method we used with ACN as the elution buffer. The third step includes a transition to 100% EtOH to confirm that the cleaning was thorough. Was this step necessary?

Method:
0-20 mins: 0% to 100% ACN
20-27.5 mins: 100% ACN
27.5-35 mins: 100% EtOH
35-42.5 mins: 0% ACN
Seems like overkill. And we never exceeded 95% organic in a gradient wash. Then we programmed down to initial conditions, and had the column equilibrate accordingly automatically before the subsequent injection.

6.Abnormal Chromatogram: The chromatograms for the blanks look strange. Do you know any potential reasons for these abnormal baselines?

[LKguan]

Thanks a lot for your reply, it's very helpful!

My input in red below

Hi all,

I’m a student working on a purification project using HPLC. We are using C18 and C8 columns, and we tested two different elution buffers—ACN (gradient 0-100%) and EtOH (gradient 0-100%)—to determine which works better. Below is the method we followed. I'm a bit confused by some of the instructions from my senior, and I’d appreciate your help in clarifying them.

1.Equilibration Stage: The equilibration stage wasn’t set at the beginning of the method but was moved to the end instead. If I equilibrate the column before running the sequence, is that acceptable?
I don't see why not. Remember: equilibration is column volumes of solvent, not really in "minutes".

2.Sample and Standard Solvent: The extract samples were prepared using EtOH, but the standards I ran were prepared in ACN because the purified product couldn’t be fully dissolved in EtOH. Can I still use the standard for reference?
We always used the same solvent for each.

3.Blank Preparation: As I understand it, the blank should be prepared using the same solvent as the sample being analyzed. However, my senior suggested that Milli-Q water is sufficient. Is this correct?
We always used the same solvent for each.

4.Sequence Setup: Since I used two elution buffers, my sequence was divided into two groups, like this:
1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.
I separated the two groups with a single blank (6. Blank), but my senior added an extra blank, so there were two blanks between the groups. What was the reason for adding this additional blank?

5.Method Step: Below is the method we used with ACN as the elution buffer. The third step includes a transition to 100% EtOH to confirm that the cleaning was thorough. Was this step necessary?

Method:
0-20 mins: 0% to 100% ACN
20-27.5 mins: 100% ACN
27.5-35 mins: 100% EtOH
35-42.5 mins: 0% ACN
Seems like overkill. And we never exceeded 95% organic in a gradient wash. Then we programmed down to initial conditions, and had the column equilibrate accordingly automatically before the subsequent injection.

6.Abnormal Chromatogram: The chromatograms for the blanks look strange. Do you know any potential reasons for these abnormal baselines?

[vmu]

1.Equilibration Stage: The equilibration stage wasn’t set at the beginning of the method but was moved to the end instead. If I equilibrate the column before running the sequence, is that acceptable?

No doubt, the column must be equilibrated before the sequence (before the first chromatogram of interest in the sequence). However, the layout of the gradient method can be different in various software. Some software can add the equilibration stage before injection as a part of the method. Other software cannot do this, and the pump program starts with injection. In the latter case, "the equilibration stage set at the beginning of the method" is actually a stage of the isocratic elution of your sample injected to the non-equilibrated column, i.e. a mistake.

[Consumer Products Guy]

We had Agilent Chemstations. We always set up so the very first line of the sequence was a standard injection, and the second line was same standard injection set as "Replace" for the software, so it replaced any calibration in the memory of that Method.

[vmu]

4.Sequence Setup: Since I used two elution buffers, my sequence was divided into two groups, like this:
1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.
I separated the two groups with a single blank (6. Blank), but my senior added an extra blank, so there were two blanks between the groups. What was the reason for adding this additional blank?

1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6a. Blank | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.

If injections 1, 2, 3, 4, and 5 are with one solvent in the mobile phase and injections 6a, 6, 7, 8, 9, 10, and 11 are with the other solvent, then blank injection 6a is for the column equilibration with the second solvent, and blank injection 6 is for the actual acquisition of the blank chromatogram with the second solvent.

[LKguan]

Big thanks with your answers!

4.Sequence Setup: Since I used two elution buffers, my sequence was divided into two groups, like this:
1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.
I separated the two groups with a single blank (6. Blank), but my senior added an extra blank, so there were two blanks between the groups. What was the reason for adding this additional blank?

1. Blank | 2. Standard | 3. Sample A | 4. Sample B | 5. Sample C | 6a. Blank | 6. Blank | 7. Standard | 8. Sample A | 9. Sample B | 10. Sample C | 11. Blank.

If injections 1, 2, 3, 4, and 5 are with one solvent in the mobile phase and injections 6a, 6, 7, 8, 9, 10, and 11 are with the other solvent, then blank injection 6a is for the column equilibration with the second solvent, and blank injection 6 is for the actual acquisition of the blank chromatogram with the second solvent.