High Pressure Issue in HPLC

Pressure issue can be two types: High Pressure and Low Pressure

Let’s talk about High Pressure issue: 

HPLC system is having different modules or component like Pump, Sampler, Column Compartment, Detector etc. These modules are interconnected with some kind of metal or different material Tubing/Capillaries. 

These tubing is having very narrow id like 0.18 mm, some case 0.12 mm or 0.17 mm also. If some particles stuck up inside this tubing, it will create partial or complete blockage and resulting high pressure. Generally, Pressure measurement in HPLC is done in Pump. Pump will measure back pressure.

Due to blockage, flow will not move further and HPLC system will show high pressure due to blockage in Flow path. Sometime due to partial blockage, system pressure will not go beyond limit, but it will be more than normal condition.

If you are facing high pressure issue in system, following things need to be checked:

• Quickest way to identify is to open Purge Valve and if it is releasing pressure that means there is no issue till purge valve in pump module.

• Some HPLC is having in-built filter in Pump. If that is having blockage, it will generate high pressure.

• You need to remove tubing from flow path component one by one, by removing tubing and you can find out which portion of system is generating high pressure. 

• You can remove Column "In" tubing also to check whether Column is generating high pressure or different component.

• To avoid frequent high-pressure issue, make sure to filter mobile phase properly as per requirement given in specific hardware specification. Need to filter Sample also before filling Vial. Replace Mobile Phase Suction filter in Bottle timely.

• Once Analysis is completed make sure to flush System and Column with appropriate Solvent as per guideline/SOP. It is always good to flush system weekly also.

Retention Time (RT) Shifting or RT Variation in HPLC analysis

This is one of common problem seen in HPLC analysis. 

To find out root cause of any problem, you need to identify exact behavior. 

Need to identify whether RT is continuously varying or eluting earlier or later. 

Also need to check whether RT variation occurs in Isocratic Method or Gradient Method or in both. 

Moreover, you can check whether RT shifting happens for one specific compound peak in chromatogram or for all peaks. 

Let’s take an Example for one of Specific Product Analysis: 

For example: 

Case 1: For one of Product analysis, expected RT for Compound peak is 5.5 minute. For multiple Injections, if this expected Peak is eluting at 4.6 minute, in some injection 4.8, in some injections 5.8, 5.9, 6.0 like this. Looking at this different RT, we can say it is continuously varying.

Case 2: Now, if Peak RT is varying like this for multiple injections: 

If peak is eluting earlier gradually run by run, like first run 5.5, second, 5.4, third, 5.2 and so on or gradually delaying. This case, RT is eluting early or delayed.

Above two cases describe problem behavior, let’s see probable causes which is generating this problem.

• Peak Elution depends on Flow, Mobile Phase Chemistry, Column Chemistry, System equilibration done properly before starting analysis and Temperature etc. Check basic functionality of HPLC.
• Make sure Instrument Method setup is proper, Mobile Phase preparation, different solvent mixing, properly degassed Mobile Phase, Lab temperature is proper before jump to HPLC system Hardware. 
• If you are using Solvent Compressibility and/or Stroke Volume Parameter in HPLC Pump Parameter, make sure it is set correctly. 
• Sometime if Temperature is not proper or Column Temperature is not stable or varying, it might cause RT variation. 
• Check whether you have replaced or changed any Capillary in HPLC system, and you are facing RT issue after this activity. If you will not use Standard tubing’s/capillaries it will impact Delay volume. Change in Delay volume can cause RT variation in Gradient Methods. 
• If Pump Flow is not proper, it can cause RT shifting run by run depends on Mobile Phase and Gradient composition also. 
• If flow is continuously varying it will cause RT variation. Also, you can check Pressure plot for each run which will give you an idea of flow variation. Flow variation will cause pressure fluctuation also. Comparing Problematic Pressure Graph with earlier good analysis pressure graph will give you some clue. 
• Improper flow can be caused by HPLC Pump component functionality like Inlet/outlet valves, pump drives, pump other components. 
• Check that HPLC degasser is working properly. 
• Sometime, Gradient Valve, usually called GPV (Gradient Proportional Valve) or MCGV (Multichannel Gradient Valve). If This Valve is not working properly, it may cause RT issue. Gradient valve mul-functionality can give you stable pressure in some cases but still RT variation happens. 
• If all the things are working well, you can check whether this RT variation occurs specific to one product analysis only or for all products. 

All above steps are given to have an idea to find out probable or root cause. There can be different reason for RT variation issue apart from given above. Appropriate solution need to be applied once root cause is identified. Also it might need engineer visit to resolve instrument issue.

Why are wavelengths scanned from high range to low range in UV-Vis Spectrophotometer?

Generally we have seen that UV-Vis Spectrophotometer scans from high to low wavelength. So it will scan first high wavelength and will come to low wavelength. For example, if we want to scan from 200 nm to 350 nm wavelength, then it will start scan from 350 nm and will come to 200 nm. Why it is designed like this? Let us understand it.

One sentence answer is: This mechanism minimize degradation of UV sensitive samples. 

How?

When we set the wavelength, it will not immediately start scan without pressing start button to scan. First it will come to starting wavelength which you have set in method. Here this starting wavelength will be higher one. Higher wavelength has low energy and lower wavelength has more energy. Let's understand this by equation. 

We all know that Energy, E = hv, where h = Planck constant and v = frequency.

Now frequency = c / λ  ; where c = speed of light which is constant and λ = wavelength

So E = h c / λ; so if Wavelength is more, Energy is less and if Wavelength is less, Energy will be more.

Hope this will solve your query for the mechanism in which, wavelengths scanned from high range to low in UV-Vis Spectrophotometer.

Chromatography Guide

Here I have posted Basics of Chromatography, Chromatography Technique (HPLC), Spectrophotometer each on different pages. All things are explained from basic level with simple language. Hope this will help to understand chromatography.

My first Technical blog

This is my first technical blog. I have also another blog on a topic Way to Earn online and Source of Information.