Chromatography Forum

Good afternoon everyone, I'm an internship student currently.

Recently my supervisor assigned me a project, which to run diphenylguanidine (DPG) compound using HPLC.
The model of HPLC that using is Agilent 1100 auto-injection, DAD detector, and Zorbax c18 reversed phase column (150 mm)

I used 95:5 Acetonitrile: Deionized water to run the elution. Some website suggested to add 0.1% trifluoroacetic acid to mobile phase. However, I dont have the TFA, so I used 0.1% acetic acid to replace it.

Here is the problem. I tried to run standard solution for calibration curve. For standard preparation, I dissolved DPG powder into DCM. However, I noticed that the retention time of my DPG compound inconsistent. The retention time is decreasing when the concentration of standard is increase. Here is my data:

Mobile phase: A - 1 L (0.1% acetic acid), B - 1 L Deionised water (0.1% acetic acid)
Flow rate: 3.0 mL/min
Thermostat: 35 celcius

80 ppm - 1.655 min
60 ppm - 1.678 min
40 ppm - 1.704 min
20 ppm - 1.735 min
(Solvent peak 0.498min ~ 0.500min)

R-square for my calibration curve = 0.99979

I also tried another set with no adding acid into mobile phase, the outcome still the same, but with longer elution time (approx. 15 min). Besides that, I changed the mobile phase ratio, change mobile phase to methanol ... it also don't give a consistent retention time. I even try to dissolve DPG in acetone, but the outcome still the same.

So, may I know what is the problem with this HPLC elution?
The pressure is almost the same, which means the pump is good in condition.
Is that possible the problem is with the column?

Hope HPLC expert can give me a solution ASAP, it is very urgent. Thank You.

Reduce the acetonitrile content of the mobile phase to about 80%, and make up the standards in mobile phase, not dichloromethane.

Why do you fell unable to ask your supervisor for advice ?

Peter

Reduce the acetonitrile content of the mobile phase to about 80%, and make up the standards in mobile phase, not dichloromethane.

Why do you fell unable to ask your supervisor for advice ?

Peter

My supervisor told me that it may because of the acid, but I don't think so.
Acid is to suppress ionizing and improve the peak shape. When I'm not adding acid. The peak comes out at 15 minutes.


By the way, may I know why must use ACN to dissolve my standard but can't use DCM?

You actually don’t have any retention.
As Peter mentioned – decrease the acetonitrile concentration etc.
And do some chromatography related reading – Fx. What K factor means in that context.

Best Regards

Btw, your superviser could do some of the same reading him/her- self

You actually don’t have any retention.
As Peter mentioned – decrease the acetonitrile concentration etc.
And do some chromatography related reading – Fx. What K factor means in that context.

Best Regards

Thanks for you opinion, danko.
I will try it tmr =)

Reduce the acetonitrile content of the mobile phase to about 80%, and make up the standards in mobile phase, not dichloromethane.

Why do you fell unable to ask your supervisor for advice ?

Peter

My supervisor told me that it may because of the acid, but I don't think so.
Acid is to suppress ionizing and improve the peak shape. When I'm not adding acid. The peak comes out at 15 minutes.


By the way, may I know why must use ACN to dissolve my standard but can't use DCM?

I did not say dissolve the standards in ACN, I said use mobile phase. Details are important. The reason is that if you inject something that is a stronger eluent than the mobile phase (ACN or DCM) your "peak" can get swept through at least a part of the column in the plug of injection solvent. This usually gives a distorted peak and if you use a very strong eluent (DCM) inconsistent retention times.

Do you really think that acid suppresses ionisation of diphenylguanidine ? If it suppresses ionisation, why does retention increase when you leave out the acid ?

Peter

I did not say dissolve the standards in ACN, I said use mobile phase. Details are important. Peter

Yes.

The reason is that if you inject something that is a stronger eluent than the mobile phase (ACN or DCM) your "peak" can get swept through at least a part of the column in the plug of injection solvent. This usually gives a distorted peak and if you use a very strong eluent (DCM) inconsistent retention times. Peter

Yes, again. And if you MUST inject from a stronger solvent, then keep theat injection volume very small to help avoid the above.

Also, post more detail about the column you are using. 3ml/minute is almost certainly not an optimal flow rate for it (unless it's packed w/ 10µ beads).

Also, post more detail about the column you are using. 3ml/minute is almost certainly not an optimal flow rate for it (unless it's packed w/ 10µ beads).

My column is Agilent eclipse XDB c18, 3.5 micrometer, 4.6 x 150 mm (Reversed phase)

I tried to run another set, flow rate: 2mL/min, mobile phase ratio ACN:water 8:2, and standard in mobile phase

100ppm - 2.105min
80ppm - 2.178min
60ppm - 2.246min
40ppm - 2.287min

But the peak is broader and shorter than previously.
Injection: 10 microL

Reduce the acetonitrile content of the mobile phase to about 80%, and make up the standards in mobile phase, not dichloromethane.

Why do you fell unable to ask your supervisor for advice ?

Peter

My supervisor told me that it may because of the acid, but I don't think so.
Acid is to suppress ionizing and improve the peak shape. When I'm not adding acid. The peak comes out at 15 minutes.


By the way, may I know why must use ACN to dissolve my standard but can't use DCM?

I did not say dissolve the standards in ACN, I said use mobile phase. Details are important. The reason is that if you inject something that is a stronger eluent than the mobile phase (ACN or DCM) your "peak" can get swept through at least a part of the column in the plug of injection solvent. This usually gives a distorted peak and if you use a very strong eluent (DCM) inconsistent retention times.

Do you really think that acid suppresses ionisation of diphenylguanidine ? If it suppresses ionisation, why does retention increase when you leave out the acid ?

Peter

Hmm .. I'm not sure, since the peak takes longer time to elute when not adding acid, I suspect the uses of acid is to prevent interaction with the column.

TFA and HOAc are not interchangeable,

0.5-0.75mL/min is a more typical flow rate for a 3.5µ 150mmx4.6mm column. You must be getting some really high back pressure running at 2!

Check your void volume time (initial baseline disturbance after injection) and compare to your peak retention time. Is peak time/void time > 1.5? If not, you need to cut organic, run w/ TFA or make other changes so that you are getting some reasonable retention.

Also, monitor your pressure. When you run isocratically, it should not vary much. (<1%)

TFA and HOAc are not interchangeable,

0.5-0.75mL/min is a more typical flow rate for a 3.5µ 150mmx4.6mm column. You must be getting some really high back pressure running at 2!

Check your void volume time (initial baseline disturbance after injection) and compare to your peak retention time. Is peak time/void time > 1.5? If not, you need to cut organic, run w/ TFA or make other changes so that you are getting some reasonable retention.

Also, monitor your pressure. When you run isocratically, it should not vary much. (<1%)

I dun really know that, because some of the method normally used 2.0mL/min.

Yeah my peak time/void time is 3, which is above 1.5 as u mentioned.

Since I have only 1 compound, so I run isocratic. The pressure is ok, plus/minus 1 bar.

Is that possible the amount of DPG compound shift my retention time. Sound not possible, but I really have no idea.

TFA and HOAc are not interchangeable,

0.5-0.75mL/min is a more typical flow rate for a 3.5µ 150mmx4.6mm column. You must be getting some really high back pressure running at 2!

Check your void volume time (initial baseline disturbance after injection) and compare to your peak retention time. Is peak time/void time > 1.5? If not, you need to cut organic, run w/ TFA or make other changes so that you are getting some reasonable retention.

Also, monitor your pressure. When you run isocratically, it should not vary much. (<1%)

I dun really know that, because some of the method normally used 2.0mL/min.

Yeah my peak time/void time is 3, which is above 1.5 as u mentioned.

Since I have only 1 compound, so I run isocratic. The pressure is ok, plus/minus 1 bar.

Is that possible the amount of DPG compound shift my retention time. Sound not possible, but I really have no idea.

It sounds unlikely - but it fits the symptoms and the implausibly low pressure - are you sure that you column does not have a huge void through the packing from one end to the other ? Has it ever been used with a mobile phase with a high pH ?

Peter

TFA and HOAc are not interchangeable,

0.5-0.75mL/min is a more typical flow rate for a 3.5µ 150mmx4.6mm column. You must be getting some really high back pressure running at 2!

Check your void volume time (initial baseline disturbance after injection) and compare to your peak retention time. Is peak time/void time > 1.5? If not, you need to cut organic, run w/ TFA or make other changes so that you are getting some reasonable retention.

Also, monitor your pressure. When you run isocratically, it should not vary much. (<1%)

I dun really know that, because some of the method normally used 2.0mL/min.

Yeah my peak time/void time is 3, which is above 1.5 as u mentioned.

Since I have only 1 compound, so I run isocratic. The pressure is ok, plus/minus 1 bar.

Is that possible the amount of DPG compound shift my retention time. Sound not possible, but I really have no idea.

It sounds unlikely - but it fits the symptoms and the implausibly low pressure - are you sure that you column does not have a huge void through the packing from one end to the other ? Has it ever been used with a mobile phase with a high pH ?

Peter

Sorry to make u misunderstood.
My pressure is around 150bar when flow rate 3mL/min. plus/minus 1 bar means it sometimes 149 or 151bar.

Today I try to decrease the injection volume. (Previously is I injected 10µL)
When I try to inject 0.1 to 0.5µL, by using the same condition.
My result is as below:
Flow rate: 3mL/min
Injection: 0.2µL
Mobile phase: ACN:water 95:5 (0.1% acetic acid added)
100ppm - 1.721min
80ppm - 1.724min
60ppm - 1.727min
40ppm - 1.731min
r-square = 0.990

Flow rate: 3mL/min
Injection: 0.1µL
Mobile phase ACN:water 95:5 (0.1% acetic acid added)
100ppm - 1.726min
80ppm - 1.729min
60ppm - 1.732min
40ppm - 1.733min

It seems that the problem improve abit. But the retention time still can't be the same.

Besides that, that may be a problem for me to do LOD & LOQ, because if I inject 1 or 2ppm standard, the peak of DPG will be insignificant.
(Less injection volume, shorter peak height)

I also tried to run in low flow rate as DR mentioned. But it didn't help in improve retention time.