Determination of common failures of high performance liquid chromatography and solutions to common failures and solutions

possible causes of diagnosis
(i) change in retention time 1. Column temperature change column
stration
, if necessary, need to be configured
stration box Between gradients, the flow phase balance column
3. buffer capacity is not sufficient 25mmol/L buffer
Condition changes stable sample conditions, adjust the flow phase
column fast to reach the life of the protection column
(ii) retention time shortened 1. flow rate increase inspection pump, reset flow rate
Volume
3. The pH value of the key-phase loss flow phase is maintained in the direction of the 3 to 7.5 check bar
4. Changes in the composition of the flow phase prevent evaporation or precipitation of the flow phase

III) Extended retention time 1. Flow rate drop line leakage, replacement of pump seals, removal of bubbles in the pump
2. Changes in the active point on the silicone column with flow phase modifiers, such as triethylamine, or alkali to passivation column
3 . Key-phase loss with the front (ii) 3
4. Flow phase composition change with the front (ii) 4
5. Temperature decrease with the front (ii) 5
(iv) shoulder peak or fork 1. Sample Too large with flow matching samples, the total sample volume is less than 15% of the first peak
2. Sample solvent is too strong using a weaker sample solvent


Weak corrosive conditions
4. Sintering stainless steel in columns fail to replace sintered stainless steel, add in-line
filtering
, filter sample
5. sampler damage replacement sampler rotor
(v) ghost peak1. Sample valve residual peak after each use with a strong solvent cleaning valve, improve the valve and sample cleaning
2. Sample handling unknowns in the sample
spectrum)
4. TEFA oxidation (peptide spectrum) daily new distribution, with
antoxidant 5. Water pollution (inverse phase) by changing the balance time to check water quality, with HPLC Level of water(vi) baseline noise 1. Bubbles (sharp peaks) flow phase degassing, plus column back pressure
2. Pollution (random noise) cleaning column, purifying samples, with HPLC grade

Reagent 3. Detector lamp continuous noise replacement
thrug lamp4. Electrical interference (accidental noise) using a regulatory power supply, check the source of interference (e.g. water bath, etc.)
5. The detector has bubble flow phase degassing, add column back pressure
(seven) peak drag tail 1. column overload to reduce the sample volume, increase the column diameter using a higher capacity fixed phase
2. peak interference cleaning sample, adjust the flow phase
3. Silicon hydroxyl action plus triethylamine, increase the concentration of buffer or salt with alkali-caused passivation column to reduce the pH of the flow phase, passivation sample
4. same as the previous (iv) 4
4
same as the front (iv) 35. With the former (iv) 3
6. Minimize the dead volume or the external volume of the column is too large connection points, make appropriate adjustments to all connection points, as far as possible, use a thin inner diameter connection tube
Column, the use of protective column
(eight) peak spread width 1. The size of the sample is too large with (iv) 1
2. In the sample valve caused the peak expansion sample before and after the discharge of bubbles to reduce diffusion
3. Data system sampling rate is too slow set rate should be more than 10 points per peak
4. Detector time constant is too large set time constant is interested in the first peak half-width 10
%
With a small volume pool, remove the heat exchanger
7. Increase the solvent content when the retention time is too long isothermal and can also be exhumed by gradient
8. The outer volume of the column is too large to minimize the connection pipe diameter and connection tube length