reverse-phase high-performance liquid chromatography ...jmlvll/lab-reports/hplc/hplc.pdf · uc...

UC Berkeley College of Chemistry

Chemistry 105

Instrumental Methods in Analytical Chemistry

Reverse-Phase High-PerformanceLiquid Chromatography Analysis of

BiomoleculesShort Report

Author:

Jonathan Melville

Lab Partners:

Jake Precht, Phuong Tran, and Jeremy Hsu

Graduate Student Instructors:

Richard Cooper & Daniel Mortensen

April 23, 2014

1 Theory

RP-HPLC, or Reverse-Phase High-Performance Liquid Chromatography is a type of chro-

matography the features a liquid mobile phase, higher resolving power than traditional

liquid chromatography, and a specific combination of stationary- and mobile-phases that

results in separations that are the opposite of a “traditional” HPLC. Chromatogra-

phy, in general, utilizes the flow of a “mobile-phase” containing the analyte through

a “stationary-phase” – depending on the intermolecular forces of attraction between the

individual analytes and the stationary- and mobile-phases, the various components of

the sample will exit the chromatographic “column” at different times[?]. A substance

that experiences greater intermolecular forces of attraction to the stationary-phase will

be slowed, and will exit the column later than a substance without such attraction. If

the mobile-phase is a liquid (as opposed to, say, a gas), the process is considered “liquid

chromatography”; HPLC improves upon this process by pumping a pressurized liquid

mobile-phase through an adsorbant stationary-phase, yielding far greater separation and

resolving power than regular gravitationally-run liquid chromatography. The labelling

“reverse-phase” is far more arbitrary in origin – historically, chromatographic columns

utilized a polar stationary-phase and a nonpolar mobile-phase; “reverse-phase chromatog-

raphy”, by contrast, uses a nonpolar stationary-phase and a polar mobile-phase, resulting

in an inverted separation from “normal-phase” chromatography: more polar analytes will

tend to elute earlier due to decreased interaction with the now-nonpolar stationary-phase.

Because of its versatility, ease-of-use, and superior separation, HPLC is one of the

most commonly-used classes of chromatography (itself one of the most common analytical

methods), along with other methods like gas chromatography and column chromatogra-

phy.

2 Results and Discussion

This section contains only tabulated results from the Appendix. Derivations can be found

in Appendix A on page 5. Graphs can be found in Appendix B on page 12. Raw data

1

Figure 1: Methionine, a nonpolar amino acid and one of our peptides

can be found in Appendix A.1 on page 5.

2.1 Results

Due to experimental failures that are detailed further in the appendix, we were unable

to absolutely and quantitatively determine the concentration of peptides in our unknown

sample. However, we were able to determine the ratios of peptides in our sample relative

to the concentration of our calibration samples:

Peak 1:40.63160.012312

+ 4.134980.051785

2= 1690. ppm (relative)

Peak 2:11.964110.03288

+ 15.3068−0.02443

2= −131.3 ppm (relative) (?!)

2.2 Accuracy and Error

Of course, our accuracy in this experiment was exceedingly low. Ignoring the fact that we

are missing a crucial piece of data to turn our relative concentration values into absolute

ones (due to a failure for the instrument to read our sample, no less), the data we do have

conspires to give us abysmal fits that lead to nonsensical data. A brief examination of

our calibration curves shows that our error bars are gigantic and our R2 values abysmal

(even, in one case, producing a negative slope that directly caused the apparently negative

concentration of peptide seen above). Because of the vast amounts of missing data points

in our tables (every N/A was a point not found on the instrument), we are simply not

able to calculate anything with any degree of confidence.

2

Figure 2: Bradykinin, a blood-vessel-dilating peptide that is comparatively polar

2.3 Discussion

While we cannot make many quantitative statements about the success of our experiment,

we can still state certain facts with a reasonable degree of certainty. We can, for instance,

determine the identity of the first and second peaks eluted by looking at the structures

of the two peptides analyzed.

Methionine, the first peptide, is a rather small essential amino acid and is largely

nonpolar in character due to the presence of a fairly lengthy alkyl chain. Compared

to the much larger bradykinin, enriched with numerous amines, amides, imines, and

aromatic groups, we can say with a reasonable amount of certainty that the first peak

is that of bradykinin and the second is methionine. Because we are performing reverse-

phase HPLC, our stationary phase is a rather waxy resin with long alkyl chains that

give it significant nonpolar character, whereas our mobile phases are comparatively polar

in nature. As a result, the more nonpolar methionine is more slowed by intermolecular

interactions with the stationary phase, allowing the more polar bradykinin to elute first.

Our data was analyzed using the HPLC’s built-in light source, an Agilent 1100/1200

Diode Array Detector that filters light from a tungsten-filament bulb through a coupling

lens, a deuterium lamp, an achromatic lens, a holmium oxide filter, a support lens, a

flow cell, and a spectro lens befor finally passing through a programmable slit and onto

a grating which projects a final spectrum[3]. Because our sample only called for UV

irradiation and not visible light (a detail noticeable in the ACQ.TXT data reports), only

the deuterium lamp is used.

Because of the data loss that plagued this entire experiment, we are unable to cal-

culate the niceties of our isocratic separations, such as the number of theoretical plates.

3

For the exact same reasons, we are unable to determine the amount of peptide in our

unknown sample. We were able to determine the relative concentrations (1690. ppm and

-131.3 ppm) by using the relative response factor, but even this approach is unhelpful due

to abysmal correlation coefficients (ranging from 0.65 to 0.05) and simply nonsensical an-

swers (negative concentration?!). While it is possible that our poor outputs are the result

of detection limits and exceeding the linear range of the instrument, it is unlikely that our

samples deviated so significantly from reasonable values that virtually none of them were

able to construct a decent fit. Changing the wavelength of the detector would change

the response factors of our analytes; methionine, containing little to no π-conjugation,

is likely not an exceptional absorber compared to bradykinin, which has large, extended

πsystems that likely absorb electrons of UV/Vis light readily. This also explains the

comparatively lower absorption values of methionine compared to bradykinin.

Nevertheless, the many flaws that riddled this experiment were likely a result of poor

experimental preparation and instrumental methods rather than shortcomings of the

HPLC instrument itself. Because our group was one of the last groups to complete this

lab, we were forced to make do with insufficient quantities of peptide starting material,

and whilst coordinating our sample preparation with another group we inadvertently

screwed up the preparation of several of our samples. We most likely failed to get all

of our results out of the HPLC due to a combination of this experimental error and

incorrect setup of the HPLC methods, causing several of our key analytical data points

to be dropped. Worst of all, we were unable to recreate and rerun our samples because

we had run out of lab time over the semester with which to attempt to fix our mistakes.

HPLC is a useful analytical method, even over other comparable separation methods

like electrophoresis or other forms of chromatography, because it combines the relative

ease-of-use of many chromatographic methods with the high-precision data that many

more qualitative chromatographic methods lack.

4

References

[1] SHU HPLC ; Yuri Kazakevich. http://hplc.chem.shu.edu/ (accessed April 22, 2014).

[2] Diode Array Detector Optimization [Online]; Agilent Technologies.

https://www.chem.agilent.com/Library/slidepresentation/Public/2%20Diode%20Array%20Detector%20Optimization.pdf

(accessed Apr 19, 2014).

A Calculations

A.1 Peak Resolution Data

RA,B = 2 ∗ tret,A−tret,BWA+WB

RA,B=peak resolution

tret=retention time

W=baseline width

A.1.1 1000 ppm std.

Trial Method Peak 1 (min) Peak 2 (min) Peak 1 width Peak 2 width Resolution

1 C 1.134 2.311 0.202 0.166 6.3967391304

2 C N/A N/A N/A N/A N/A

3 C N/A N/A N/A N/A N/A

A.1.2 500 ppm std.


1 A 2.191 6.44 0.182 0.185 23.1553133515

2 A 2.022 6.06 0.207 0.179 20.9222797927

3 A 2.076 6.156 0.172 0.176 23.4482758621

5

A.1.3 100 ppm std.


1 A 2.195 6.386 0.169 0.184 23.7450424929

2 A 2.063 6.165 0.18 0.19 22.172972973

3 A 2.044 6.241 0.161 0.199 23.3166666667

4 B N/A 2.19 0.151 0.157 7.025974026

5 B N/A 2.011 0.172 0.177 3.0601719198

6 B N/A 2.125 0.148 0.161 4.2459546926

7 C 1.112 2.198 0.164 0.149 6.9392971246

8 C N/A 2.198 N/A 0.16 N/A

9 C N/A 2.341 N/A 0.187 N/A

A.1.4 10 ppm std.


1 A 1.158 2.202 0.21 0.188 5.2462311558

2 A 1.465 2.039 0.165 0.204 3.1111111111

3 A 1.457 2.062 0.189 0.212 3.0174563591

A.1.5 1 ppm std.


1 A 1.176 2.478 0.17 0.173 7.5918367347

2 A 1.518 2.129 0.193 0.201 3.1015228426

3 A 1.448 2.127 0.21 0.171 3.5643044619

A.1.6 0.1 ppm std.


1 A 1.168 2.204 0.217 0.181 5.2060301508

2 A 1.481 2.067 0.127 0.185 3.7564102564

3 A 1.452 2.1 0.184 0.186 3.5027027027

6

A.1.7 Unknown Solution


1 A 1.176 2.2 0.175 0.204 5.4036939314

2 A N/A 2.076 N/A 0.172 N/A

3 A 1.59 2.072 0.167 0.183 2.7542857143

A.1.8 Bradykinin std.


N/A N/A N/A N/A N/A N/A N/A

Bradykinin Error Log

443 41e0 53462317 0

Method Method started: line# 20 vial# 80 inj# 1 21:50:31 04/09/14

67 41e0 53462319 0

Method Instrument running sample Vial 80 21:50:33 04/09/14

fb3 1da7 53462349 500

ALS 1 Missing vial (80) 21:51:21 04/09/14

67 41e0 53462349 0

Method Missing Vial, no Run possible 21:51:21 04/09/14

70 41de 53462349 0

CP Macro Analyzing rawdata 080-2001.D 21:51:21 04/09/14

70 41de 53462349 0

CP Macro Data not available; Data Analysis not done 21:51:21 04/09/14

d5 1da7 5346234b 500

ALS 1 Error Method started 21:51:23 04/09/14

67 41e0 5346234b 0

Method Method completed 21:51:23 04/09/14

7

A.1.9 Methionine std.


N/A N/A N/A N/A N/A N/A N/A

Methionine Error Log

443 41e0 534625d5 0

Method Method started: line# 22 vial# 81 inj# 1 22:02:13 04/09/14

67 41e0 534625d7 0

Method Instrument running sample Vial 81 22:02:15 04/09/14

fb3 1da7 53462600 500

ALS 1 Missing vial (81) 22:02:56 04/09/14

67 41e0 53462601 0

Method Missing Vial, no Run possible 22:02:57 04/09/14

70 41de 53462601 0

CP Macro Analyzing rawdata 081-2201.D 22:02:57 04/09/14

70 41de 53462601 0

CP Macro Data not available; Data Analysis not done 22:02:57 04/09/14

d5 1da7 53462603 500

ALS 1 Error Method started 22:02:59 04/09/14

67 41e0 53462603 0

Method Method completed 22:02:59 04/09/14

A.2 Bradykinin and Methionine Calibration

These calibration curves could not be created, due to a lack of any data (whatsoever)

from their respective samples1. As a result, we are completely unable to create calibration

curves for these analytes, as well as calculating LoD or quantitative determination of the

unknown peptide mixture, due to the lack of a known standard to use as a reference.

Nevertheless, we will attempt to answer as many of these questions as possible using

1See error logs on pages 7 and 8.

8

relative values.

Based on the error logs, it is likely that human error was responsible for this data

loss, as the samples simply failed to run. This could be caused by an incorrect method or

by physical misplacement of the samples into the HPLC chamber. Whatever the cause,

however, there is no way for us to make up for this lost information (by interpolation or

the like) because there is simply no data to interpolate from.

A.3 Response Factor Calculation

fi = Ai

Astdfstd

Because of the limitations detailed earlier regarding the peptide standards, we are

unable to calculate absolute response factor and must settle for relative response factor:

fri = fifstd

= Ai

Astd

We can calculate this value by plotting analyte concentration against response (in the

form of area or height) and taking the slope of the best-fit line.

Unfortunately, in this instance “best-fit” may be a misnomer. “Mediocre-fit” is prob-

ably more accurate.

9

Based on these graphs, we receive the following relative response factors (averaging

out over multiple injections; injections were plotted separately because the data points

were clearly stratified):

Peak fri,1 fri,2,3 fri,avg

Peak 1 .012312 .051785 .03205

Peak 2 .03288 -.02443 .00844

A.4 Relative Unknown Concentrations

Using the previously calculated relative response factors, it is possible to calculate “rela-

tive unknown concentrations” in terms of the calibration solutions we created for curve-

fitting. Because our “1000 ppm” standard does not actually contain 1000 ppm of both

peptides, these “relative concentrations” are ratios to the actual concentration of the

standards created, something we could determine quantitatively if our reference values

had not been lost.

Peak 1:40.63160.012312

+ 4.134980.051785

2= 1690. ppm (relative)

Peak 2:11.964110.03288

+ 15.3068−0.02443

2= −131.3 ppm (relative) (?!)

11

A.5 Detection Limits

Once again, without concrete values to pin our relative numbers to, we cannot quantita-

tively determine this value. Qualitatively, however, a cursory glance at the error bars of

our “calibration curves” assures us that hitting the detection limit is likely the least of

our worries.

B Chromatographs

12

=====================================================================Acq. Operator : Chem 105 Seq. Line : 2Acq. Instrument : 305 Lat HPLC Location : Vial 71Injection Date : 4/9/2014 2:59:51 PM Inj : 1 Inj Volume : 20.0 µlSequence File : C:\Chem32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105.SMethod : C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105C.MLast changed : 4/2/2014 2:18:05 PM by Chem 105

min0 1 2 3 4 5 6 7 8 9

mAU

0

10

20

30

40

50

60

DAD1 A, Sig=254,4 Ref=360,100 (071-0201.D)

1.1

34

2.1

68 2

.311

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.134 BB 0.1262 550.56732 65.42467 59.5121 2 2.168 BV 0.2501 100.07494 4.98669 10.8173 3 2.311 VB 0.1326 274.49249 33.14954 29.6705 Totals : 925.13476 103.56089 ===================================================================== *** End of Report ***

Data File C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\071-0201.DSample Name: 1000Standard

305 Lat HPLC 4/9/2014 3:10:05 PM Chem 105 Page 1 of 1


min0 1 2 3 4 5 6 7 8 9

mAU

-4

-3.5

-3

-2.5

-2

-1.5

DAD1 A, Sig=254,4 Ref=360,100 (071-0202.D)

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs No peaks found ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-2

-1.75

-1.5

-1.25

-1

-0.75

-0.5

-0.25

0

DAD1 A, Sig=254,4 Ref=360,100 (071-0203.D)

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs No peaks found ===================================================================== *** End of Report ***



=====================================================================Acq. Operator : Chem 105 Seq. Line : 4Acq. Instrument : 305 Lat HPLC Location : Vial 72Injection Date : 4/9/2014 3:45:31 PM Inj : 1 Inj Volume : 20.0 µlSequence File : C:\Chem32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105.SMethod : C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105A.MLast changed : 3/18/2014 3:50:33 PM by Chem 105

min0 2 4 6 8

mAU

-10

0

10

20

30

40

50

DAD1 A, Sig=254,4 Ref=360,100 (072-0401.D)

1.2

09

2.1

13 2

.191

2.5

01

4.8

43

6.4

40

6.6

97

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.209 BB 0.1403 495.95142 53.40386 31.4287 2 2.113 BV 0.2614 103.35991 4.75820 6.5500 3 2.191 VV 0.0857 80.62334 12.10583 5.1091 4 2.501 VV 0.1670 65.41753 5.91853 4.1455 5 4.843 BB 0.1155 8.37328 1.09158 0.5306 6 6.440 BV 0.1376 533.22046 56.76577 33.7905 7 6.697 VV 0.1579 291.07367 26.97762 18.4455 Totals : 1578.01961 161.02139



===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-50

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (072-0402.D)

0.8

75

1.5

13

2.0

22 2

.122

6.0

60

6.3

32

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.875 BV 0.3477 1113.19348 39.63150 48.5831 2 1.513 VV 0.0415 15.75548 5.48666 0.6876 3 2.022 VV 0.2473 594.30695 30.24431 25.9373 4 2.122 VV 0.1684 382.46872 28.53759 16.6921 5 6.060 BV 0.2640 118.48067 6.28218 5.1709 6 6.332 VV 0.1934 67.11346 4.67787 2.9290 Totals : 2291.31877 114.86010



===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (072-0403.D)

0.8

73

1.4

50

1.8

93 1

.969

2.0

76 2

.149

6.1

56

6.4

85

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.873 BV 0.3515 1115.88684 39.03070 48.8070 2 1.450 VV 0.0659 5.07510 1.28902 0.2220 3 1.893 VV 0.1489 300.45795 26.36786 13.1415 4 1.969 VV 0.0733 139.63464 26.57147 6.1074 5 2.076 VV 0.0780 158.51370 28.01682 6.9331 6 2.149 VV 0.1589 333.29025 27.98861 14.5776 7 6.156 BV 0.3180 147.91566 6.25299 6.4696 8 6.485 VV 0.2101 85.54984 5.36173 3.7418 Totals : 2286.32399 160.87920



===================================================================== *** End of Report ***




min0 2 4 6 8

mAU

-10

0

10

20

30

40

DAD1 A, Sig=254,4 Ref=360,100 (073-0601.D)

1.1

46

2.1

10 2

.195

2.4

62

6.3

86

6.6

67

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.146 BB 0.1168 353.13898 47.47415 39.8047 2 2.110 BV 0.2295 97.19534 5.16706 10.9555 3 2.195 VV 0.0838 78.58721 12.42897 8.8581 4 2.462 VV 0.1476 74.40478 7.25996 8.3867 5 6.386 BV 0.1241 202.56482 23.59933 22.8325 6 6.667 VV 0.1476 81.28827 8.06572 9.1626 Totals : 887.17940 103.99518



===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (073-0602.D)

0.8

52

1.4

55

2.0

63 2

.138

6.1

65

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.852 BV 0.3558 1071.70447 38.37623 56.6523 2 1.455 VV 0.0866 10.96265 1.81107 0.5795 3 2.063 VV 0.2603 493.35745 24.36183 26.0798 4 2.138 VV 0.1800 244.35648 22.62403 12.9171 5 6.165 BV 0.2528 71.34130 3.80328 3.7712 Totals : 1891.72234 90.97643 =====================================================================




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (073-0603.D)

0.8

20

1.4

64 1.5

68

1.8

80 2.0

44

6.2

41

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.820 BV 0.3312 995.76886 37.81387 51.8885 2 1.464 VV 0.0901 27.89626 4.16753 1.4536 3 1.568 VV 0.0524 25.14947 7.63329 1.3105 4 1.880 VV 0.1588 264.83774 21.33795 13.8004 5 2.044 VV 0.2770 533.56476 23.49139 27.8035 6 6.241 BV 0.2922 71.83978 3.27133 3.7435 Totals : 1919.05686 97.71537



===================================================================== *** End of Report ***



=====================================================================Acq. Operator : Chem 105 Seq. Line : 8Acq. Instrument : 305 Lat HPLC Location : Vial 74Injection Date : 4/9/2014 5:17:05 PM Inj : 1 Inj Volume : 20.0 µlSequence File : C:\Chem32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105.SMethod : C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\CHEM105B.MLast changed : 4/2/2014 2:00:08 PM by Chem 105

min0 1 2 3 4 5 6 7 8 9

mAU

-10

0

10

20

30

40

DAD1 A, Sig=254,4 Ref=360,100 (074-0801.D)

1.1

08

2.1

04 2

.190

2.4

71

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.108 BB 0.1259 377.30368 46.85508 58.6071 2 2.104 BV 0.2398 110.27272 5.59915 17.1288 3 2.190 VV 0.0810 81.81421 13.08161 12.7083 4 2.471 VV 0.1572 74.39433 6.93048 11.5558 Totals : 643.78493 72.46632 ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-60

-50

-40

-30

-20

-10

0

DAD1 A, Sig=254,4 Ref=360,100 (074-0802.D) 1

.477

2.0

11 2

.089

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.477 BV 0.1058 29.45708 4.00656 4.0340 2 2.011 VV 0.2417 400.07516 21.64254 54.7883 3 2.089 VV 0.1815 300.68777 20.94538 41.1777 Totals : 730.22001 46.59448 ===================================================================== *** End of Report ***




min0 2 4 6 8

mAU

-50

-40

-30

-20

-10

0

DAD1 A, Sig=254,4 Ref=360,100 (074-0803.D) 1

.469

2.0

05 2.1

25

2.5

06 3.0

97

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.469 BV 0.0939 13.76783 1.96233 2.1276 2 2.005 VV 0.2091 291.84280 17.27595 45.0995 3 2.125 VV 0.1991 299.09973 19.93204 46.2209 4 2.506 VV 0.0738 8.66611 1.63504 1.3392 5 3.097 VV 0.2704 33.73269 1.53500 5.2128 Totals : 647.10917 42.34035 =====================================================================




min0 2 4 6 8

mAU

-10

0

10

20

30

DAD1 A, Sig=254,4 Ref=360,100 (075-1001.D)

1.1

12

2.1

12 2

.198

2.4

51





min0 1 2 3 4 5 6 7 8 9

mAU

-6

-4

-2

0

2

4

6

DAD1 A, Sig=254,4 Ref=360,100 (075-1002.D)

2.1

98

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 2.198 BV 0.0796 78.62127 12.80807 100.0000 Totals : 78.62127 12.80807 ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-6

-4

-2

0

2

4

6

DAD1 A, Sig=254,4 Ref=360,100 (075-1003.D)

2.3

41

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 2.341 BV 0.0872 87.91441 13.28189 100.0000 Totals : 87.91441 13.28189 ===================================================================== *** End of Report ***




min0 2 4 6 8

mAU

-10

0

10

20

30

DAD1 A, Sig=254,4 Ref=360,100 (076-1201.D)

1.1

58

2.1

14 2

.202

2.4

69





min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (076-1202.D)

0.8

12

1.4

65

2.0

39 2

.154

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.812 BV 0.3228 981.17834 37.55931 62.8206 2 1.465 VV 0.0885 16.10502 2.52281 1.0311 3 2.039 VV 0.2610 365.82404 17.41731 23.4221 4 2.154 VV 0.1558 198.76657 16.83013 12.7262 Totals : 1561.87397 74.32956 ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (076-1203.D)

0.8

43

1.4

57

2.0

62

2.4

96





min0 1 2 3 4 5 6 7 8 9

mAU

-10

-5

0

5

10

15

20

25

30

DAD1 A, Sig=254,4 Ref=360,100 (077-1401.D)

1.1

76

2.2

11 2.4

78

6.2

65

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.176 BB 0.1264 351.41290 43.44933 62.2963 2 2.211 BV 0.2961 48.73400 2.07436 8.6393 3 2.478 VV 0.1485 42.87768 4.29428 7.6011 4 6.265 BB 0.1297 121.07437 14.16961 21.4633 Totals : 564.09895 63.98757 ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (077-1402.D)

0.8

16

1.5

18

1.9

41 2

.129





min0 2 4 6 8

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (077-1403.D)

0.8

48

1.4

48

2.1

27

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.848 BV 0.3432 1053.58057 38.02576 64.9938 2 1.448 VV 0.0727 10.03283 2.06460 0.6189 3 2.127 VV 0.3468 557.43555 19.65986 34.3873 Totals : 1621.04895 59.75022 ===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-10

0

10

20

30

DAD1 A, Sig=254,4 Ref=360,100 (078-1601.D)

1.1

68

2.1

18 2

.204

2.4

43

6.3

06 6.5

87

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.168 BB 0.1197 348.67422 44.36583 48.3616 2 2.118 BV 0.2066 81.84194 5.46956 11.3516 3 2.204 VV 0.0801 70.78206 11.78743 9.8176 4 2.443 VV 0.1408 81.71107 8.30485 11.3334 5 6.306 BV 0.1540 62.11704 5.75156 8.6157 6 6.587 VV 0.1565 75.84663 7.22601 10.5200 Totals : 720.97297 82.90524

Data File C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\078-1601.DSample Name: 0.1Standard


===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (078-1602.D)

0.8

17

1.4

81 1

.583

1.7

00

2.0

67

2.4

98

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.817 BV 0.3190 977.10211 37.37093 59.9091 2 1.481 VV 0.0985 28.41631 4.11997 1.7423 3 1.583 VV 0.0471 16.49159 5.47909 1.0111 4 1.700 VV 0.0782 53.69276 9.76090 3.2921 5 2.067 VV 0.3344 546.24152 19.64394 33.4917 6 2.498 VB 0.0809 9.03005 1.52827 0.5537 Totals : 1630.97435 77.90310



===================================================================== *** End of Report ***




min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (078-1603.D)

0.8

32

1.4

52 1

.542

2.1

00

2.5

00





min0 1 2 3 4 5 6 7 8 9

mAU

-10

-5

0

5

10

15

20

25

30

DAD1 A, Sig=254,4 Ref=360,100 (079-1801.D)

1.1

76

2.1

15 2

.200

2.4

95

6.2

04

===================================================================== Area Percent Report ===================================================================== Sorted By : SignalMultiplier: : 1.0000Dilution: : 1.0000Use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 A, Sig=254,4 Ref=360,100 Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.176 BB 0.1154 297.52802 40.63152 45.2417 2 2.115 BV 0.1941 88.30638 5.59623 13.4277 3 2.200 VV 0.0826 76.47616 11.96411 11.6289 4 2.495 VV 0.1685 72.60163 6.20496 11.0397 5 6.204 BB 0.2100 122.72923 7.86909 18.6620 Totals : 657.64141 72.26591 =====================================================================

Data File C:\CHEM32\1\DATA\9 APRIL 2014\CHEM105 2014-04-09 14-47-10\079-1801.DSample Name: Unknown



min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (079-1802.D)

0.8

62

1.9

65 2.0

76 2

.166





min0 1 2 3 4 5 6 7 8 9

mAU

-40

-30

-20

-10

0

10

DAD1 A, Sig=254,4 Ref=360,100 (079-1803.D)

0.8

42

1.5

90

1.9

19 2

.072

2.1

58




reverse-phase high-performance liquid chromatography ...jmlvll/lab-reports/hplc/hplc.pdf · uc...

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