MPharm Chromatography Lecture 3: Chromatogram Data Features and Quiz

Chromatography Lecture 3: Overview

UNIVERSITYOF
PORTSMOUTH
MPharm (Medicinal and Analytical Chemistry)
Topic: Chromatography Lecture 3
Dr Priyanka DEY
Senior Lecturer in Bioanalytical Chemistry
priyanka.dey@port.ac.uk

Dr Priyanka DEY
Chromatography Part 2

Chromatography Session Plan

1. Plan for Chromatography sessions
   · Lecture 1:
   · QA and QC,
   . Introduction, types and uses of chromatography
   . Lecture 2: Specialized chromatography
   · Lecture 3: Chromatogram data features
   · Lecture 4: Chromatogram analysis and applications

Dr Priyanka DEY
Chromatography Part 3

Chromatogram Definition and Features

1. Chromatogram
   " The chromatogram is a two-dimensional plot with the ordinate axis giving
   concentration in terms of the detector response, and the abscissa represents the
   time.
   " The detector gives a response as a peak whose height should be ideally
   dependent on the concentration of the particular component.
   Each peak represents a component present in the sample.
   detector signal
   intensity (mV)
   B
   A
   C
   0
   t.
   t
   t
   time (min)

Dr Priyanka DEY
Chromatography 3

Retention Time in Chromatography

1. Retention time tR
   Retention time (t,) is time interval between
   sample injection and the maximum of the
   peak.
   t0 , T0, T sub zero, T naught, T zero, air
   peak, dead time, tM, hold time, or void time
   all refer to the same concept. It is the time
   that it takes for an unretained (meaning
   no interaction with the stationary phase)
   molecule to make it from injection to
   detector.
   IR
   Detector signal intensity
   Peak
   -
   ER
   : Retention Time
   to
   h
   to
   : Dead Time
   A
   : Peak Area
   -
   h
   : Peak Hight
   Baseline
   Time
   Injection of the sample into the column - Start of analysis
   ❑
   Identity of the component can be confirmed by making injections of reference
   material under the same operational conditions.
   The matching of retention time of reference material and the component peak
   confirms the identity of the unknown sample component.

Dr Priyanka DEY
Chromatography 3

Analyzing Component Concentration

1. Analyzing concentration of the components
   However, due to analysis conditions peaks
   may deviate from ideal shape and peak
   height can no longer be a true measure of
   the concentration and instead the area
   under the peak is considered as a
   measure of component concentration.
   Now let us consider a sample which
   contains more than one sample component.
   Likewise, each component will be eluted at
   different retention times depending upon
   solute - stationary phase interactions and
   mobile phase flow characteristics.
   A
   U
   Detector
   Responce
   Time
   A% =
   Area of Peak A x 100
   Total Areas of Peaks (A + B + C + D)

Dr Priyanka DEY
Chromatography 3

HPLC Chromatogram Example: Vitamins A and E

1. The actual chromatogram printout of HPLC separation of a mixture of
   vitamins A and E in a food matrix and see what the chromatogram
   represents
   - VWD: Signal A, 284 nm
   Retention Time
   40 -1
   40
   -I 7.867
   Volts
   20
   -20
   Volts
   =8.493
   18:210
   >20.803
   -22:988
   0-F
   0
   -
   5
   10
   15
   20
   25
   30
   35
   40
   Minutes
   Vitamin A and E % in the mixture are different when analysed with Height and area
   VWD: Signal
   A, 284 nm
   Results
   Retention Time
   Area
   Area %
   Height
   Height % Name
   7.867
   3239619
   72.84
   329451
   86.67
   VIT A
   8.493
   15779
   0.35
   1727
   0.45
   19.410
   140173
   3.15
   5631
   1.48
   19.710
   64889
   ·1.46
   4338
   1.14
   20.803
   874700
   19.67
   33762
   8.88
   VIT E
   21.640
   39563
   0.89
   2093
   0.55
   22.100
   73086
   1.64
   3119
   0.82

Dr Priyanka DEY
Chromatography 3

Assessing Chromatographic Separation Quality

1. Determining how good the chromatographic separation is
   Chromatograms can be used for qualitative and quantitative
   analysis - but before this can be done you need to be able to say
   "how good" the chromatogram is.
   Often the following terms are used and calculated:
   0
   Resolution
   Efficiency

Dr Priyanka DEY
Chromatography 3

Chromatogram Peak Features

1. Chromatogram features
   L
   (b) No peak
   (f) "Split" peak
   T
   FWHM= full width at
   half maxima
   Response
   peak width
   at half height
   Datapoints =
   -
   peak height t
   spectrum
   peak area
   baseline
   peak width
   to
   İR
   (d) Tailing peak
   (g) "Negative" peak

Dr Priyanka DEY
Chromatography 3

Peak Shapes and Retention Time

1. 
   (a) "Perfect" peak
   (e) Fronting peak
   (c) Broad peak
   (g) "Fuzzy" peak
   Retention timeResolution
   This is a measure of how well the peaks have separated.
   Resolution is needed to be able to use peak area quantitatively.
   By convention, resolution (R) or Rs = twice the distances
   between the two peak maxima 2(tp2-tp) divided by the sum
   of base width of the two peaks (wb +wb2).
   R (or, R$) =
   wb. +wb2
   2(tR2 -tR)
   The greater the value of R, the better the resolution of the two
   compounds.
   ]
   A value of approximately 1.5 = good separation.
   A value of approximately 1.0 = resolution not fully complete (only
   about 90%).
   A value less than 1.0, and the separation is really not good enough to
   allow the analysis to make any quantitative analysis of concentration.
   Peak Resolution
   (ER)B
   (53)A
   High Resolution
   A
   B
   W
   B
   Detector Signal
   Moderate Resolution
   A
   B
   A B
   Low Resolution
   0
   1
   2
   3
   4
   5
   6
   7 8
   9
   10
   Time (min)

Dr Priyanka DEY
Chromatography 3

Quiz on Peak Resolution

1. Quiz #1: In chromatography, what does a resolution value of 0.5
   between peaks tell you?
   A. Peaks are fully resolved
   B. Peaks are poorly resolved
   C. Peaks are completely eluting
   D. Efficiency of separation is very good
   E. Efficiency of separation is poor
   Answer: B

Dr Priyanka DEY
Chromatography 3

Resolution Calculation Example

1. R = 0.50
   R = 1.00
   R = 1.50
   detector's response
   · R~ 1.5 = good separation.
   . R~ 1.0 = resolution not fully complete (only
   about 90%)
   · R <1.0, and the separation is poor.
   time
   tp 27.2
   B
   26.3
   İR
   A
   İR 27.6
   C
   W = 0.56
   W = 0.56
   W = 0.56
   Let's calculate
   resolution between
   R =
   2(tR2-tR)
   wb. + wb2
   · A and B
   · Band C
   R (A and B)=
   2(27.2 - 26.3)
   = 1.61
   0.56 + 0.56
   R (B and C)=
   0.56 + 0.56
   The greater the value of R, the better
   the resolution of the two compounds.

Dr Priyanka DEY
Chromatography 3

Betamethasone Resolution Example

1. Example question #1:
   The BP assay of betamethasone 17-valerate states that it must be resolved from
   betamethasone 21-valerate so that the resolution factor is > 1.0. Which of the following
   ODS columns meet the specification?
   Retention time of
   betamethasone 21-
   valerate (min)
   Retention time of
   betamethasone 17-
   valerate (min)
   Width at base of
   bet. 21-valerate
   (min)
   Width at base of
   bet.17-valerate
   (min)
   1. 9.5
   8.5
   0.4
   0.5
   2. 9.3
   8.6
   0.4
   0.4
   1. R = 2(9.5-8.5) = 2.22
   0.4+0.5
   2. R$ = 2(9.3 - 8.6) = 1.75
   0.4 +0.4
   Both columns meet the specifications!

Dr Priyanka DEY
Chromatography 3

Alternative Resolution Equation

1. Alternative form of the resolution equation (may see it in your practical):
   When it is difficult to measure the peak base width
   Chromatogram
   RG =
   S
   1 .18x (tp2 -tR1)
   W.1 + W,
   h1
   h2
   Normal
   Problem
   Baseline
   Normal
   Problem
   Whi and V
   h2 are the FWHM values for
   component 1 and 2
   . This can be used if the base line is
   difficult to measure.

Dr Priyanka DEY
Chromatography 3

Quiz on Resolution Calculation

1. Quiz #2: The retention times of the two peaks observed in the resulting
   chromatogram were 3.9 minutes and 4.1 minutes. The width of each peak at half
   height was 0.1 minutes and 0.05 minutes respectively. What is the resolution
   between the two peaks?
   A. 1.57
   B. 2.67
   C. 1.33
   D. 0.20
   E. 0.25
   Rs =
   S
   1 . 18x (tR2 -tRI)
   W.1 + W,
   h2
   Answer: A

Dr Priyanka DEY
Chromatography 3

Improving Chromatographic Resolution

1. Ways to improve resolution
   Increasing the column length leads to an increase in the number of
   theoretical plates (N), and gives better resolution. However this has a bad
   effect on efficiency since there is more time for diffusion to occur,
   broadening the bands.
   By altering the mobile and stationary phases you can change the
   selectivity of the column for different solute molecules and can therefore
   improve upon the resolution of compounds in a mixture.
   Anything that increases efficiency and lead to narrowing of peaks (such
   as low sample concentration) will improve resolution.

Dr Priyanka DEY
Chromatography 3

Chromatogram Efficiency

1. Efficiency- how narrow each peak can be?
   The efficiency of a chromatogram peak is a measure of the dispersion of
   the analyte band as it travels through the HPLC system and column.
   The plate number (N) is a measure of the peak dispersion on the HPLC
   column, which reflects the column performance.
   The broader the chromatographic peak is relative to its retention time, the
   less efficient the column is at eluting it.
   Each plate is the distance over which the sample components achieve
   equilibrium between the stationary and mobile phase in the column.
   The more theoretical plates (N) there are in within a set length of a
   column, the better the efficiency (n) of the column.

Dr Priyanka DEY
Chromatography 3

Determining Column Efficiency

1. Determination of Efficiency
   The more theoretical plates (N) there are in within a set length of a column,
   the better the efficiency (n) of the column.
   N = 16
   Wb
   tR
   2
   = 5.54
   2
   W1/2
   tR
   -
   İR
   W1/2
   Inject
   I
   Time
   W b
   Higher values for the Plate Number (N) are
   expected for subsequent peaks within a
   chromatogram. Later eluting peaks that
   look broad in comparison to early eluting
   peaks may have a higher plate count.
   If this is not the case then your system
   contains a large extra-column dead
   volume!
   n=
   5.54 x (tp)2
   Lx (Wh)2

Dr Priyanka DEY
Chromatography 3

Column Efficiency Example Question

1. Example question #2:
   A standard operating system procedure states that a column must have an
   efficiency of >30,000 theoretical plates/m. Which of these 15 cm columns
   meets the specifications?
   (a) Retention time of analyte 6.4 min, Wh 0.2 min.
   n = 37819.7 plates/m
   n=
   5.54 x (tp )2
   L x (W)2
   (b) Retention time of analyte 5.6 min, W. 0.2 min
   n = 28955.7 plates/m
   (c) Retention time of analyte 10.6 min, Wh 0.6 min.
   n = 11527.3 plates/m
   Therefore only column 1 meets the specifications. Answer a

Dr Priyanka DEY
Chromatography 3

Quiz on Column Efficiency Criteria

1. Quiz #3: A standard operating system states that a chromatography column
   must have an efficiency of >20,000 theoretical plates. Which of the following
   20 cm columns meets this criteria?
   A. Retention time of analyte
   12449 m-1
   10.6 mins, Wh 0.5 mins
   B. Retention time of analyte
   8.5 mins, W. 0.5 mins
   8005 m-1
   n=
   5.54 x (tp )2
   Lx (Wh)2
   C. Retention time of analyte
   15.6 mins, Wn 0.5 mins
   26964 m-1
   D. Retention time of analyte
   9.8 mins, Wn 0.5 mins
   10641 m-1
   E. None of the above
   Answer: C

Dr Priyanka DEY
Chromatography 3

Factors Affecting Column Efficiency and Band Broadening

1. Factors affecting efficiency of a column, and causing band broadening
   in HPLC.
   Chromatographic peaks have widths and this means that molecules of a
   single compound, despite having the same properties, take different lengths of
   time to travel through a column.
   The longer an analyte takes to travel through a column, the more individual
   molecules making up the sample spread out and the broader the band
   becomes.
   The more rapidly a peak broadens the less efficient the column is.
   So why does this occur and what factors effect it?