21. What is the preferred solvent to store the column if the column need not be used for a longer time? Why?
Acetonitrile is a preferred solvent as it is stable for a longer period of time compared to methanol. Methanol has the property of hydrolysis.
22 What are the checkpoints before starting an HPLC analysis?
1. Ensure no electrical connection is loose.
2. Verify the capillaries for absence of leakages.
3. Check the tubing and solvent container for absence of air bubbles. If any air bubbles are observed, remove air from tubes by sucking solvent with the help of a syringe by opening the purge valve. Perform the purging and priming of the system.
4. Ensure that the solvent container is closed to prevent objects falling into it as well as evaporation of solvents.
5. Check the system flow by switching the pump and ensure that solvent is coming from mobile phase container/ solvent containers to the waste container.
6. Probable reasons of absence of flow are:
a. Air in pump
b. Verify the leakage by touching the seals. It should not be wet.
c. Crystals of buffered mobile phases.
d. Malfunctioning can be verified through unusual pump noise
7. Ensure that mobile phase is prepared using HPLC grade solvents
8. Ensure that the mobile phase is prepared by counting required number of injections, run time and flow rate.
9. Ensure that buffered mobile phases are filtered using a membrane filter, degas with helium or with degasser.
10. While using a manual injector ensure that the container is kept under the overflow.
11. Maintain the injection needles clean to avoid contamination when using manual injector.
12. Clean injector needle using suitable solvent if needed.
13. Use a washing solution while using an autosampler.
14. Check the UV detectors lamp energy when using the UV detector.
15. For RP-HPLC, use 10-20% methanol in the water to prevent microorganism growth.
16. Use a waste container to collect waste. Ensure the safety precautions to prevent spillage and evaporation in the lab.
17. Before starting the standard, verify the baseline.
18. Before starting the sample, verify the peak shape of standard, system suitability and area.
23. What is the meaning of Peak in chromatography?
Peak is a detector response represented as a chromatogram. One peak represents one component when separation is done adequately. When there is incomplete separation, i.e. two or more components’ peak gets merged because they eluted in overlapping manner and remain unresolved.
24. What is a Chromatogram?
Chromatogram a representation of detector response or a quantitative measurement value in a graphical form vs. the volume or time. Typical chromatograms have series of Gaussian peaks on a baseline.
25. What is Retention time (tR) in liquid chromatography?
Retention time, (tR) is the time between the injection of the compound and the maximum peak response of the eluted.
[ads id="ads1"]
26. What is Dwell volume (D) in chromatography?
The volume between the is the volume between the point at which the eluents (mobile phase/ solvent) meet and the inlet of the column (Or the top of the column).
Dwell volume is also known as “gradient delay volume”.
27.What is Hold-up time (tM) in chromatography?
The time required for elution of an unretained component (In following schematic, an air or unretained solvent peak, with the baseline scale in minutes).
Reference: Working document QAS/21.905 – February 2022 (draft), 1.14.1 CHROMATOGRAPHY
28. What is Hold-up volume (VM) in chromatography?
The volume of mobile phase or solvent required for elution of an unretained component.
It is calculated using hold-up time ((tM)) and the flow rate (F), mL/min.
Formula is VM = tM × F
29. What is Number of theoretical plates (N) OR Plate number (N) in chromatography?
Number of theoretical plates (N) OR Plate number (N) is a measure that indicates column efficiency.
In other words, it is a number that is indicative of performance of column OR column efficiency.
For Gaussian peaks, it is calculated by:
N = 16(tR/W)^2 (As per USP)
tR – retention time
W – peak width
For electronic integrators it can be determined using the equation:
N = 5.54 (tR/Wh)^2
tR – retention time
Wh – peak width at half-height (h/2).
30. What is Plate Height (H) in chromatography?
Theoretical plate height is height equivalent to a theoretical plate. It is a ratio of the column length (L), in micrometers, to the plate number (N): H = 𝐿/𝑁
It indicates the rate for the band broadening of peak broadening in HPLC equipment. The smaller the H value, the bigger the plate number.
When bigger the plate number, the better the column is packed and the smaller the dead volume of the instrument and, the sharper the peaks. It also means that the efficiency of the column is good.
[ads id="ads1"]
31. What are the factors affecting the Number of theoretical plates (N) or Plate Height (H)?
Substance being chromatographed Operating conditions i.e flow rate and temperature of the mobile phase or carrier gas
Quality of the packing material
The uniformity of the packing within the column
For capillary columns, the thickness of the stationary phase film
Internal diameter and length of the column
32. What is the meaning of Resolution (RS) in chromatography?
The resolution is the separation of two components in a mixture.
It is calculated using following formula:
RS = 2 × (tR2 − tR1)/(W1 + W2)
tR1 and tR2 are the retention times of the two components W1 and W2 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.
For electronic integrators it is determine using following formula:
RS = 1.18 × (tR2 − tR1)/(W1,h/2 + W2,h/2)
33. What is the meaning of Retention volume (VR) in chromatography?
Retention volume (VR) a volume of mobile phase or solvent required for elution of a component.
It is calculated using formula VR = tR (retention time) × F (flow rate in mL/min)
34. What is Peak-to-valley ratio (p/v) in liquid chromatography?
The peak-to-valley ratio can be considered as a system suitability criterion for related substances test in the condition of non achieving separation of two peak at the baseline level.
Schematic representation is given as follows.
35. What is Relative retention (r) in liquid chromatography?
Relative retention (r) is the ratio between the adjusted retention time of a component relative to that of another used as a reference obtained under same chromatographic conditions.
It can be calculated using formula r = (tR2 − tM)/(tR1 − tM)
tR2 – retention time measured from the point of injection of the compound of interest
tR1 is the retention time measured from the point of injection of the compound used as reference
tM is the retention time of a non retained marker defined in the procedure
[ads id="ads1"]
36. What is Relative retention time (RRT) in liquid chromatography?
Relative retention time (RRT) is also known as the “unadjusted relative retention”.
It is calculate using formula RRT = tR2/tR1
37. What is “Symmetry Factor” or “Tailing Factor” in liquid chromatography?
The Gaussian form of peak is generally completely symmetrical. When the rear is spread out to
more or less extent and forms a ‘tail’. This phenomenon is called ‘tailing’.
“Symmetry Factor” or “Tailing Factor” (AS) is calculated using formula: W0.05/2f
W0.05- width of the peak at 5% height
f is the distance from the peak maximum to the leading edge of the peak
Reference: USP
38. What is ‘fronting’ or ‘leading’ in liquid chromatography?
In opposite of tailing phenomenon; when the front is flatter than the back, it is called as ‘fronting’ or ‘leading’.
39. What is System repeatability in liquid chromatography? OR What is System Suitability?
The repeatability and reproducibility of signal/ area/ response that is represented as % Relative
Standard Deviation (% RSD) of consecutive measurements for Not Less Than (NLT) three standard solutions or applications of an applicable reference solution.
Total mobile phase time (tt) 279
In size-exclusion chromatography, the retention time of a component whose molecules 280 are
smaller than the smallest gel pores (Figure 5).
40. What is the total mobile phase volume (Vt)?
This phenomenon is applicable for size-exclusion chromatography. The retention volume of a compound for those molecules having smaller than the minimum gel pores.
It may be measured using the flow rate (F) in ml/min and total mobile phase time. The equation is 𝑉𝑡=𝑡𝑡×𝐹